Evaluation of reproductive parameters of vas deferens sperms in Caucasian snake (Gloydius halys caucasicus

Directory of Open Access Journals (Sweden)

Sayedeh Zahra Mozafari

2012-06-01

Full Text Available Reproductive parameters evaluation is considered as helpful tool for gene bank formation in ecological and economically important animals species. Gloydius halys caucasicus is venomous, viviparous pit viper of northwest of Iran. In this research, the spermatic reproductive parameters of this taxon were studied. Twenty six male snakes were collected from Takht-e-Soleiman region between September and October, 2010. Findings revealed that male snakes with body length of 45.07 ± 2.83 cm and body weight of 51.50 ± 10.42 g, and right and left gonads volume of 0.12 ± 0.03 mL and 0.06 ± 0.01 mL are mature ones and sperms concentration in first, middle and final regions of vas deferens duct were, 22.30 ± 19.34 ×106 mL-1, 30.34 ± 11.55 ×106 mL-1, and 37.65 ± 16.46×106 mL-1, respectively. The sperms motility at three regions of duct were 60.53%, 62.07%, and 60.00% and percentage of immotile sperms in these regions were 39.46%, 37.92%, and 39.84%, respectively. Percentage of morphologically normal sperms was 69.23 ± 10.57% and abnormal sperms was 30.76 ± 10.57%; including 12.69 ± 5.25% spiral tailed, 7.33 ± 4.37% coiled tailed and 4.16 ± 2.51% folded tailed sperms. Percentage of live sperms in the first, middle and final regions of duct were 55.76 ± 10.77%, 58.84 ± 12.77%, and 57.69 ± 9.91%, respectively and percentage of dead sperm in these regions were 44.23 ± 10.77%, 41.15 ± 12.77%, and 42.30 ± 9.91%, respectively. Results suggested, mature sperms with acceptable reproductive quality could be collected from Gloydius halys caucasicus snake of Iran between September and October.

Efficacy of insecticide residues on adult Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) mortality and injury in apple and peach orchards.

Science.gov (United States)

Leskey, Tracy C; Short, Brent D; Lee, Doo-Hyung

2014-07-01

The primary threat from Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) originates from populations continuously dispersing from and among wild and cultivated hosts, so many individuals may not be directly sprayed with insecticides. Limited information exists regarding field-based residual activity of insecticides for management of H. halys in tree fruit. Thus, we conducted field-based bioassays in apple and peach orchards to evaluate residual activity of insecticides commonly applied against H. halys. Adults used in these trials were collected from wild and cultivated hosts less than one week prior to testing to more accurately reflect the susceptibility of wild H. halys populations in the field throughout the season. Significantly higher mortality rates of Halyomorpha halys were observed early in the growing season, when overwintered adults were prevalent, compared with populations present later in the growing season that included new generation adults. Significantly higher mortality was recorded for adults exposed to fresh insecticide applications compared with three- and seven-day old residues. Typically, the addition of an adjuvant did not enhance efficacy or residual activity of insecticides. Significantly fewer injury sites were recorded on apples treated with dinotefuran and fenpropathrin compared with the untreated apples for all residue ages. Overwintered Halyomorpha halys populations are easier to kill with insecticide applications than the first and second generation which are present in the field during the mid- to late-season. Residual activity of nearly all insecticides decreased significantly three days after application and adjuvants generally did not increase residual activity. These factors should be considered in developing season-long programs for management of this invasive species in tree fruit. © 2013 Society of Chemical Industry.

Haliç, the urban sea Landscape and transformation of the central areas of Istanbul

Directory of Open Access Journals (Sweden)

Gianluca Frediani

2014-01-01

Full Text Available Haliç (The Golden Horn is a mythical place that belongs not only to the history of Istanbul but to the whole of Europe. At Haliç land and sea merge: the natural harbour of ancient Constantinople, home to the naval arsenal and place of delights, it saw its natural and urban state change completely in the final phases of the Ottoman Empire. Its recent history has been marked by a process of intense industrialization, developing uncontrollably on its banks between the 19th and 20th centuries. Its importance as a production centre -the country’s most important industrial area – grew in time in parallel with pollution levels in the surrounding environment. The climax of this process of transformation took place in the first decades after World War II when, due to heavy industrial pollution and the saturation of coastal spaces, Haliç became the productive heart but also the most run-down and densely populated urban area of the city. The history of the subsequent redevelopment of Haliç is fairly well known, having been the subject of numerous essays describing its socio-economic, cultural and political development. Less attention, despite the many publications on the subject, has been devoted to the analysis of this extensive process of de-industrialization and renewal from the point of view of architectural and urban design. The purpose of this essay is to contribute to the debate from this point of view, briefly reconstructing the major changes taking place in the urban landscape and updating the overview of critical reflection on the current urban situation, analyzed through some of the most important interventions carried out in recent years and the changes they induced.

The Royal Book by Haly Abbas from the 10th century: one of the earliest illustrations of the surgical approach to skull fractures.

Science.gov (United States)

Aciduman, Ahmet; Arda, Berna; Kahya, Esin; Belen, Deniz

2010-12-01

Haly Abbas was one of the pioneering physicians and surgeons of the Eastern world in the 10th century who influenced the Western world by his monumental work, The Royal Book. The book was first partly translated into Latin by Constantinus Africanus in the 11th century without citing the author's name. Haly Abbas was recognized in Europe after full translation of The Royal Book by Stephen of Antioch in 1127. The Royal Book has been accepted as an early source of jerrah-names (surgical books) in the Eastern world. The chapters regarding cranial fractures in Haly Abbas' work include unique management strategies for his period with essential quotations from Paul of Aegina's work Epitome. Both authors preferred free bone flap craniotomy in cranial fractures. Although Paul of Aegina, a Byzantine physician and surgeon, was a connection between ancient traditions and Islamic interpretation, Haly Abbas seemed to play a bridging role between the Roman-Byzantine and the School of Salerno in Europe.

The importance of gut symbionts in the development of the brown marmorated stink bug, Halyomorpha halys (Stål.

Directory of Open Access Journals (Sweden)

Christopher M Taylor

Full Text Available The invasive brown marmorated stink bug, Halyomorpha halys (Stål, has become a severe agricultural pest and nuisance problem since its introduction in the U.S. Research is being conducted to understand its biology and to find management solutions. Its symbiotic relationship with gut symbionts is one aspect of its biology that is not understood. In the family Pentatomidae, the reliance on gut symbionts for successful development seems to vary depending on the species of stink bug. This research assessed the role of gut symbionts in the development, survivorship, and fecundity of H. halys. We compared various fitness parameters of nymphs and adults reared from surface sterilized and untreated egg masses during two consecutive generations under laboratory conditions. Results provided direct evidence that H. halys is negatively impacted by the prevention of vertical transmission of its gut symbionts and that this impact is significant in the first generation and manifests dramatically in the subsequent generation. Developmental time and survivorship of treated cohorts in the first generation were significantly affected during third instar development through to the adult stage. Adults from the sterilized treatment group exhibited longer pre-oviposition periods, produced fewer egg masses, had significantly smaller clutch sizes, and the hatch rate and survivorship of those eggs were significantly reduced. Observations following hatch of surface sterilized eggs also revealed significant effects on wandering behavior of the first instars. The second generation progeny from adults of the sterilized cohorts showed significantly lower survival to adulthood, averaging only 0.3% compared to 20.8% for the control cohorts. Taken together, results demonstrate that H. halys is heavily impacted by deprival of its gut symbionts. Given the economic status of this invasive pest, further investigations may lead to management tactics that disrupt this close symbiotic

Temporal and Directional Patterns of Nymphal Halyomorpha halys (Hemiptera: Pentatomidae) Movement on the Trunk of Selected Wild and Fruit Tree Hosts in the Mid-Atlantic Region.

Science.gov (United States)

Acebes-Doria, Angelita L; Leskey, Tracy C; Bergh, J Christopher

2017-04-01

Halyomorpha halys (Stål) (Hemiptera: Pentatomidae) is an invasive and polyphagous herbivore that has been problematic in Mid-Atlantic fruit orchards, many of which are adjacent to woodlands containing its wild hosts. Our tree census in woodlands bordering 15 Mid-Atlantic apple orchards revealed 47 species of deciduous trees and shrubs, 76.6% of which were recorded hosts of H. halys. Tree of heaven was most common and abundant overall. Halyomorpha halys nymphs have a substantial walking dispersal capacity, and their fitness is enhanced by feeding on multiple hosts. Directional and temporal patterns of nymphal H. halys movement on selected wild hosts and apple and peach trees at the orchard-woodland interface were monitored in 2014 and 2015 using passive traps to capture nymphs walking up and down tree trunks. Weekly captures from mid-May to late September or mid-October were compared among hosts across both seasons. Despite higher total nymphal captures in 2014 than 2015, the seasonal trends for both years were similar and indicated bivoltine H. halys populations. In both years, more nymphs were intercepted while walking up than down and captures of upward- and downward-walking nymphs varied significantly among the hosts. All instars were captured, but captures of second instars predominated. Captures reflected seasonal changes in instar distribution and consisted predominantly of younger and older nymphs, early and later in the season, respectively. Results are discussed in relation to host and seasonal effects on the movement of nymphs at the orchard-woodland interface, and the implications for H. halys management. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Socio-Economic Concerns and Essential Elements in Estuary Management Strategies; Haliç Case, Istanbul

Science.gov (United States)

Alpar, Bedri; Isil Cetin, Basak

2016-04-01

Estuaries are transitional areas between the land and sea and home to a large and growing proportion of the world's population. They are highly productive ecosystems which create jobs, and boosts local economic growth with a higher percentage of collective and private enterprises and a larger share of production. They serve many important socio-economic functions and therefore receive untreated urban wastes and riverine inputs and concentrate various pollutants coming from inland domestic, agricultural and industrial activities. Therefore such kinds of complex systems are highly vulnerable because they are usually the sink for the hinterlands. Due to serious environmental problems felt more intensively day by day, central and local governments must adopt an integrated policy and decision making process to promote a balance of uses. As surrounded by many historical attractions, heritage sites, buzzing cultural scenes and other natural resources, the Haliç (the Golden Horn estuary) offers great opportunities and has a vitality fed by widespread economic and cultural factors. The typical landscape of the estuary, its bridges, geomorphic features, oceanographic and hydrodynamic features of its waters, sea bottom characteristics, environmental pollution, make this estuary a critical marine environment which impacts to economy, environment and community. However, rapid urban growth and uncontrolled industrial development (1950-1985) led to a severe increase in pollution levels of its water and cohesive sediments. The siltation due to liquid and solid waste dumped by two streams caused anaerobic decomposition problems. In addition, the ecological processes occurring in the Haliç are rather complex as they are interacted with the socio-economic system. This study focuses on the essential elements of integrated coastal zone management for the Haliç, and its probable impacts to economy, environment and community. All objectives and probable impacts need to be integrated

Synergy of aggregation pheromone with methyl (E,E,Z)-2,4,6-decatrienoate in attraction of brown marmorated stink bug, Halyomorpha halys

Science.gov (United States)

The male-produced aggregation pheromone of the brown marmorated stink bug ((BMSB), Halyomorpha halys (Stål) (Hemiptera: Pentatomidae)), recently identified as a mixture of (3S,6S,7R,10S)-10,11-epoxy-1-bisabolen-3-ol and (3S,6S,7R,10R)-10,11-epoxy-1-bisabolen-3-ol, offers new opportunities for manage...

Allosteric Mutant IDH1 Inhibitors Reveal Mechanisms for IDH1 Mutant and Isoform Selectivity

Energy Technology Data Exchange (ETDEWEB)

Xie, Xiaoling; Baird, Daniel; Bowen, Kimberly; Capka, Vladimir; Chen, Jinyun; Chenail, Gregg; Cho, YoungShin; Dooley, Julia; Farsidjani, Ali; Fortin, Pascal; Kohls, Darcy; Kulathila, Raviraj; Lin, Fallon; McKay, Daniel; Rodrigues, Lindsey; Sage, David; Touré, B. Barry; van der Plas, Simon; Wright, Kirk; Xu, Ming; Yin, Hong; Levell, Julian; Pagliarini, Raymond A. (Novartis)

2017-03-01

Oncogenic IDH1 and IDH2 mutations contribute to cancer via production of R-2-hydroxyglutarate (2-HG). Here, we characterize two structurally distinct mutant- and isoform-selective IDH1 inhibitors that inhibit 2-HG production. Both bind to an allosteric pocket on IDH1, yet shape it differently, highlighting the plasticity of this site. Oncogenic IDH1R132H mutation destabilizes an IDH1 “regulatory segment,” which otherwise restricts compound access to the allosteric pocket. Regulatory segment destabilization in wild-type IDH1 promotes inhibitor binding, suggesting that destabilization is critical for mutant selectivity. We also report crystal structures of oncogenic IDH2 mutant isoforms, highlighting the fact that the analogous segment of IDH2 is not similarly destabilized. This intrinsic stability of IDH2 may contribute to observed inhibitor IDH1 isoform selectivity. Moreover, discrete residues in the IDH1 allosteric pocket that differ from IDH2 may also guide IDH1 isoform selectivity. These data provide a deeper understanding of how IDH1 inhibitors achieve mutant and isoform selectivity.

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

Directory of Open Access Journals (Sweden)

Jiou Wang

2009-01-01

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

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.

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.

TAF-4 is required for the life extension of isp-1, clk-1 and tpk-1 Mit mutants.

Science.gov (United States)

Khan, Maruf H; Ligon, Melissa; Hussey, Lauren R; Hufnal, Bryce; Farber, Robert; Munkácsy, Erin; Rodriguez, Amanda; Dillow, Andy; Kahlig, Erynn; Rea, Shane L

2013-10-01

While numerous life-extending manipulations have been discovered in the nematode Caenorhabditis elegans, one that remains most enigmatic is disruption of oxidative phosphorylation. In order to unravel how such an ostensibly deleterious manipulation can extend lifespan, we sought to identify the ensemble of nuclear transcription factors that are activated in response to defective mitochondrial electron transport chain (ETC) function. Using a feeding RNAi approach, we targeted over 400 transcription factors and identified 15 that, when reduced in function, reproducibly and differentially altered the development, stress response, and/or fecundity of isp-1(qm150) Mit mutants relative to wild-type animals. Seven of these transcription factors--AHA-1, CEH-18, HIF-1, JUN-1, NHR-27, NHR-49 and the CREB homolog-1 (CRH-1)-interacting protein TAF-4--were also essential for isp-1 life extension. When we tested the involvement of these seven transcription factors in the life extension of two other Mit mutants, namely clk-1(qm30) and tpk-1(qm162), TAF-4 and HIF-1 were consistently required. Our findings suggest that the Mit phenotype is under the control of multiple transcriptional responses, and that TAF-4 and HIF-1 may be part of a general signaling axis that specifies Mit mutant life extension.

Abnormal grooming activity in Dab1(scm) (scrambler) mutant mice.

Science.gov (United States)

Strazielle, C; Lefevre, A; Jacquelin, C; Lalonde, R

2012-07-15

Dab1(scm) mutant mice, characterized by cell ectopias and degeneration in cerebellum, hippocampus, and neocortex, were compared to non-ataxic controls for different facets of grooming caused by brief water immersions, as well as some non-grooming behaviors. Dab1(scm) mutants were strongly affected in their quantitative functional parameters, exhibiting higher starting latencies before grooming relative to non-ataxic littermates of the A/A strain, fewer grooming bouts, and grooming components of shorter duration, with an unequal regional distribution targeting almost totally the rostral part (head washing and forelimb licking) of the animal. Only bouts of a single grooming element were preserved. The cephalocaudal order of grooming elements appeared less disorganized, mutant and control mice initiating the grooming with head washing and forelimb licking prior to licking posterior parts. However, mutants differed from controls in that all their bouts were incomplete but uninterrupted, although intergroup difference for percentage of the incorrect transitions was not significant. In contrast to grooming, Dab1(scm) mice ambulated for a longer time. During walking episodes, they exhibited more body scratching than controls, possibly to compensate for the lack of licking different body parts. In conjunction with studies with other ataxic mice, these results indicate that the cerebellar cortex affects grooming activity and is consequently involved in executing various components, but not in its sequential organization, which requires other brain regions such as cerebral cortices or basal ganglia. Copyright © 2012 Elsevier B.V. All rights reserved.

Reproductive health profile and circumcision of females in the Hali semi-urban region, Saudi Arabia: A community-based cross-sectional survey.

Science.gov (United States)

Milaat, Waleed Abdullah; Ibrahim, Nahla Khamis; Albar, Hussain Mohammed

2018-01-01

Improving the reproductive health of females has be.come the focus of the developmental efforts of many nations. To identify the reproductive health style of married females, and to determine the prevalence and predictors of circumcision among girls aged less than or equal 18 years in Hali semi-urban region. A cross-sectional household survey SETTING: Houses in Hali, Al-Qunfudhah governorate, western Saudi Arabia during 2017. A multistage systematic cluster random sampling method was used to select participants. A validated questionnaire was used in interviewing the head of the selected houses. Reproductive health profile of women, and circumcision of girls. 365 households. Reproductive life starts early in the Hali region as 41.4% of women are married at or before 18 years of age. Consanguinity was recorded in 57.0% of houses. The prevalence of grand multiparity (GMP) was 54.7%; it was significantly associated with current maternal age, age at marriage, low educational levels of both parents and husbands with non-professional jobs. Current use of birth control methods was reported by 28.9% of families, and oral contraceptives (OCs) were the commonest method. Contraceptive use was significantly associated with higher educational levels of both parents and with women having professional work. The prevalence of circumcision was 80.3%. Circumcision was most frequent (59.4%) at age 7 years or less, and almost always done by doctors (91.4%). Hemorrhage (2.9%) and fever (2.3%) were the minimal recorded complications. Girls with higher parental education, enough income, no parental consanguinity, and whose mothers married at an older age had slightly lower rates of circumcision, but the difference was without statistical significance. The pattern of early female marriage, high consanguinity, GMP, low contraceptive use, and a high frequency of circumcision in girls was apparent in Hali. Public health education and legislative policies are needed. Recall bias may affect the

The Shift of the Intestinal Microbiome in the Innate Immunity-Deficient Mutant rde-1 Strain of C. elegans upon Orsay Virus Infection.

Science.gov (United States)

Guo, Yuanyuan; Xun, Zhe; Coffman, Stephanie R; Chen, Feng

2017-01-01

The status of intestinal microbiota is a determinant of host health. However, the alteration of the gut microbiota caused by the innate immune response to virus infection is unclear. Caenorhabditis elegans and its natural virus Orsay provide an excellent model of host-virus interactions. We evaluated the intestinal microbial community complexity of the wild-type N2 and the innate immunity-deficient mutant rde-1 ( ne219 ) strains of C. elegans upon Orsay virus infection. The gut microbiota diversity was decreased in rde-1 ( ne219 ) mutant animals, and a large number of genes were associated with the difference between infected and uninfected rde-1 ( ne219 ) mutant animals. Therefore, this study provides the first evaluation of the alterations caused by Orsay virus on intestinal microbiota in wildtype and innate immunity-deficient animals using C. elegans as the model species. Our findings indicate that virus infection may alters the microbiome in animals with defective immune response.

The Shift of the Intestinal Microbiome in the Innate Immunity-Deficient Mutant rde-1 Strain of C. elegans upon Orsay Virus Infection

Directory of Open Access Journals (Sweden)

Yuanyuan Guo

2017-05-01

Full Text Available The status of intestinal microbiota is a determinant of host health. However, the alteration of the gut microbiota caused by the innate immune response to virus infection is unclear. Caenorhabditis elegans and its natural virus Orsay provide an excellent model of host–virus interactions. We evaluated the intestinal microbial community complexity of the wild-type N2 and the innate immunity-deficient mutant rde-1 (ne219 strains of C. elegans upon Orsay virus infection. The gut microbiota diversity was decreased in rde-1 (ne219 mutant animals, and a large number of genes were associated with the difference between infected and uninfected rde-1 (ne219 mutant animals. Therefore, this study provides the first evaluation of the alterations caused by Orsay virus on intestinal microbiota in wildtype and innate immunity-deficient animals using C. elegans as the model species. Our findings indicate that virus infection may alters the microbiome in animals with defective immune response.

Comprehensive behavioral analysis of ENU-induced Disc1-Q31L and -L100P mutant mice

Directory of Open Access Journals (Sweden)

Shoji Hirotaka

2012-02-01

Full Text Available Abstract Background Disrupted-in-Schizophrenia 1 (DISC1 is considered to be a candidate susceptibility gene for psychiatric disorders, including schizophrenia, bipolar disorder, and major depression. A recent study reported that N-ethyl-N-nitrosourea (ENU-induced mutations in exon 2 of the mouse Disc1 gene, which resulted in the amino acid exchange of Q31L and L100P, caused an increase in depression-like behavior in 31 L mutant mice and schizophrenia-like behavior in 100P mutant mice; thus, these are potential animal models of psychiatric disorders. However, remaining heterozygous mutations that possibly occur in flanking genes other than Disc1 itself might induce behavioral abnormalities in the mutant mice. Here, to confirm the effects of Disc1-Q31L and Disc1-L100P mutations on behavioral phenotypes and to investigate the behaviors of the mutant mice in more detail, the mutant lines were backcrossed to C57BL/6JJcl through an additional two generations and the behaviors were analyzed using a comprehensive behavioral test battery. Results Contrary to expectations, 31 L mutant mice showed no significant behavioral differences when compared with wild-type control mice in any of the behavioral tests, including the Porsolt forced swim and tail suspension tests, commonly used tests for depression-like behavior. Also, 100P mutant mice exhibited no differences in almost all of the behavioral tests, including the prepulse inhibition test for measuring sensorimotor gating, which is known to be impaired in schizophrenia patients; however, 100P mutant mice showed higher locomotor activity compared with wild-type control mice in the light/dark transition test. Conclusions Although these results are partially consistent with the previous study in that there was hyperactivity in 100P mutant mice, the vast majority of the results are inconsistent with those of the previous study; this discrepancy may be explained by differences in the genetic background of the

The Arabidopsis thiamin-deficient mutant pale green1 lacks thiamin monophosphate phosphatase of the vitamin B1 biosynthesis pathway.

Science.gov (United States)

Hsieh, Wei-Yu; Liao, Jo-Chien; Wang, Hsin-Tzu; Hung, Tzu-Huan; Tseng, Ching-Chih; Chung, Tsui-Yun; Hsieh, Ming-Hsiun

2017-07-01

Thiamin diphosphate (TPP, vitamin B 1 ) is an essential coenzyme present in all organisms. Animals obtain TPP from their diets, but plants synthesize TPPde novo. We isolated and characterized an Arabidopsis pale green1 (pale1) mutant that contained higher concentrations of thiamin monophosphate (TMP) and less thiamin and TPP than the wild type. Supplementation with thiamin, but not the thiazole and pyrimidine precursors, rescued the mutant phenotype, indicating that the pale1 mutant is a thiamin-deficient mutant. Map-based cloning and whole-genome sequencing revealed that the pale1 mutant has a mutation in At5g32470 encoding a TMP phosphatase of the TPP biosynthesis pathway. We further confirmed that the mutation of At5g32470 is responsible for the mutant phenotypes by complementing the pale1 mutant with constructs overexpressing full-length At5g32470. Most plant TPP biosynthetic enzymes are located in the chloroplasts and cytosol, but At5g32470-GFP localized to the mitochondrion of the root, hypocotyl, mesophyll and guard cells of the 35S:At5g32470-GFP complemented plants. The subcellular localization of a functional TMP phosphatase suggests that the complete vitamin B1 biosynthesis pathway may involve the chloroplasts, mitochondria and cytosol in plants. Analysis of PALE1 promoter-uidA activity revealed that PALE1 is mainly expressed in vascular tissues of Arabidopsis seedlings. Quantitative RT-PCR analysis of TPP biosynthesis genes and genes encoding the TPP-dependent enzymes pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and transketolase revealed that the transcript levels of these genes were upregulated in the pale1 mutant. These results suggest that endogenous levels of TPP may affect the expression of genes involved in TPP biosynthesis and TPP-dependent enzymes. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

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.

Long-lived mitochondrial (Mit) mutants of Caenorhabditis elegans utilize a novel metabolism.

Science.gov (United States)

Butler, Jeffrey A; Ventura, Natascia; Johnson, Thomas E; Rea, Shane L

2010-12-01

The Caenorhabditis elegans mitochondrial (Mit) mutants have disrupted mitochondrial electron transport chain (ETC) functionality, yet, surprisingly, they are long lived. We have previously proposed that Mit mutants supplement their energy needs by exploiting alternate energy production pathways normally used by wild-type animals only when exposed to hypoxic conditions. We have also proposed that longevity in the Mit mutants arises as a property of their new metabolic state. If longevity does arise as a function of metabolic state, we would expect to find a common metabolic signature among these animals. To test these predictions, we established a novel approach monitoring the C. elegans exometabolism as a surrogate marker for internal metabolic events. Using HPLC-ultraviolet-based metabolomics and multivariate analyses, we show that long-lived clk-1(qm30) and isp-1(qm150) Mit mutants have a common metabolic profile that is distinct from that of aerobically cultured wild-type animals and, unexpectedly, wild-type animals cultured under severe oxygen deprivation. Moreover, we show that 2 short-lived mitochondrial ETC mutants, mev-1(kn1) and ucr-2.3(pk732), also share a common metabolic signature that is unique. We show that removal of soluble fumarate reductase unexpectedly increases health span in several genetically defined Mit mutants, identifying at least 1 alternate energy production pathway, malate dismutation, that is operative in these animals. Our study suggests long-lived, genetically specified Mit mutants employ a novel metabolism and that life span may well arise as a function of metabolic state.

Purification, crystallization and preliminary X-ray diffraction analysis of an acidic phospholipase A2 with vasoconstrictor activity from Agkistrodon halys pallas venom

International Nuclear Information System (INIS)

Zou, Zhisong; Zeng, Fuxing; Zhang, Lu; Niu, Liwen; Teng, Maikun; Li, Xu

2012-01-01

A vasoconstrictor PLA 2 was purified from Agkistrodon halys pallas venom and the preliminary X-ray diffraction analysis had been described. Phospholipases A 2 (PLA 2 s) are the major component of snake venoms and exert a variety of relevant toxic actions such as neurotoxicity and myotoxicity, amongst others. An acidic PLA 2 , here named AhV-aPA, was purified from Agkistrodon halys pallas venom by means of a three-step chromatographic procedure. AhV-aPA migrated as a single band on SDS–PAGE gels, with a molecular weight of about 14 kDa. Like other acidic aPLA 2 s, AhV-aPA has high enzymatic activity. Tension measurements of mouse thoracic aortic rings remarkably indicated that AhV-aPA could induce a further contractile response on the 60 mM K + -induced contraction, with an EC 50 of 369 nmol l −1 . Rod-shaped crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 2.30 Å. The crystals belonged to space group P222, with unit-cell parameters a = 44.27, b = 68.39, c = 81.54 Å

mPeriod2 Brdm1 and other single Period mutant mice have normal food anticipatory activity.

Science.gov (United States)

Pendergast, Julie S; Wendroth, Robert H; Stenner, Rio C; Keil, Charles D; Yamazaki, Shin

2017-11-14

Animals anticipate the timing of food availability via the food-entrainable oscillator (FEO). The anatomical location and timekeeping mechanism of the FEO are unknown. Several studies showed the circadian gene, Period 2, is critical for FEO timekeeping. However, other studies concluded that canonical circadian genes are not essential for FEO timekeeping. In this study, we re-examined the effects of the Per2 Brdm1 mutation on food entrainment using methods that have revealed robust food anticipatory activity in other mutant lines. We examined food anticipatory activity, which is the output of the FEO, in single Period mutant mice. Single Per1, Per2, and Per3 mutant mice had robust food anticipatory activity during restricted feeding. In addition, we found that two different lines of Per2 mutant mice (ldc and Brdm1) anticipated restricted food availability. To determine if FEO timekeeping persisted in the absence of the food cue, we assessed activity during fasting. Food anticipatory (wheel-running) activity in all Period mutant mice was also robust during food deprivation. Together, our studies demonstrate that the Period genes are not necessary for the expression of food anticipatory activity.

PET imaging of HSV1-tk mutants with acquired specificity toward pyrimidine- and acycloguanosine-based radiotracers

Energy Technology Data Exchange (ETDEWEB)

Likar, Yury; Dobrenkov, Konstantin; Olszewska, Malgorzata; Shenker, Larissa; Hricak, Hedvig; Ponomarev, Vladimir [Memorial Sloan-Kettering Cancer Center, Molecular Imaging Laboratory, Department of Radiology, New York, NY (United States); Cai, Shangde [Memorial Sloan-Kettering Cancer Center, Radiochemistry/Cyclotron Core Facility, New York, NY (United States)

2009-08-15

The aim of this study was to create an alternative mutant of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene with reduced phosphorylation capacity for acycloguanosine derivatives, but not pyrimidine-based compounds that will allow for successful PET imaging. A new mutant of HSV1-tk reporter gene, suitable for PET imaging using pyrimidine-based radiotracers, was developed. The HSV1-tk mutant contains an arginine-to-glutamine substitution at position 176 (HSV1-R176Qtk) of the nucleoside binding region of the enzyme. The mutant-gene product showed favorable enzymatic characteristics toward pyrimidine-based nucleosides, while exhibiting reduced activity with acycloguanosine derivatives. In order to enhance HSV1-R176Qtk reporter activity with pyrimidine-based radiotracers, we introduced the R176Q substitution into the more active HSV1-sr39tk mutant. U87 human glioma cells transduced with the HSV1-R176Qsr39tk double mutant reporter gene showed high {sup 3}H-FEAU pyrimidine nucleoside and low {sup 3}H-penciclovir acycloguanosine analog uptake in vitro. PET imaging also demonstrated high {sup 18}F-FEAU and low {sup 18}F-FHBG accumulation in HSV1-R176Qsr39tk+ xenografts. The feasibility of imaging two independent nucleoside-specific HSV1-tk mutants in the same animal with PET was demonstrated. Two opposite xenografts expressing the HSV1-R176Qsr39tk reporter gene and the previously described acycloguanosine-specific mutant of HSV1-tk, HSV1-A167Ysr39tk reporter gene, were imaged using a short-lived pyrimidine-based {sup 18}F-FEAU and an acycloguanosine-based {sup 18}F-FHBG radiotracer, respectively, administered on 2 consecutive days. We conclude that in combination with acycloguanosine-specific HSV1-A167Ysr39tk reporter gene, a HSV1-tk mutant containing the R176Q substitution could be used for PET imaging of two different cell populations or concurrent molecular biological processes in the same living subject. (orig.)

Hydrogen alleviates hyperoxic acute lung injury related endoplasmic reticulum stress in rats through upregulation of SIRT1.

Science.gov (United States)

Sun, Qiang; Han, Wenjie; Hu, Huijun; Fan, Danfeng; Li, Yanbo; Zhang, Yu; Lv, Yan; Li, Mingxin; Pan, Shuyi

2017-06-01

Hyperoxic acute lung injury (HALI) is a major clinical problem for patients undergoing supplemental oxygen therapy. Currently in clinical settings there exist no effective means of prevention or treatment methods. Our previous study found that: hydrogen could reduce HALI, as well as oxidative stress. This research will further explore the mechanism underlying the protective effect of hydrogen on oxygen toxicity. Rats were randomly assigned into three experimental groups and were exposed in a oxygen chamber for 60 continuous hours: 100% balanced air (control); 100% oxygen (HALI); 100% oxygen with hydrogen treatment (HALI + HRS). We examined lung function by wet to dry ratio of lung, lung pleural effusion and cell apoptosis. We also detected endoplasmic reticulum stress (ERS) by examining the expression of CHOP, GRP78 and XBP1. We further investigated the role of Sirtuin 1 (SIRT1) in HALI, which contributes to cellular regulation including ERS, by examining its expression after hydrogen treatment with SIRT1 inhibitor. Hydrogen could significantly reduce HALI by reducing lung edema and apoptosis, inhibiting the elevating of ERS and increased SIRT1 expression. By inhibition of SIRT1 expression, the effect of hydrogen on prevention of HALI is significantly weakened, the inhibition of the ERS was also reversed. Our findings indicate that hydrogen could reduce HALI related ERS and the mechanism of hydrogen may be associated with upregulation of SIRT1, this study reveals the molecular mechanisms underlying the protective effect of hydrogen, which provides a new theoretical basis for clinical application of hydrogen.

Frequency of the MDR1 mutant allele associated with multidrug sensitivity in dogs from Brazil

Directory of Open Access Journals (Sweden)

Monobe MM

2015-04-01

Full Text Available Marina M Monobe,1 João P Araujo Junior,2 Kari V Lunsford,3 Rodrigo C Silva,4 Camilo Bulla41Department of Veterinary Clinics, School of Veterinary Medicine and Animal Science, 2Department of Microbiology and Immunology, Biosciences Institute, Sao Paulo State University (UNESP, Botucatu, Brazil; 3Department of Clinical Sciences and Animal Health Center, 4Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi State University, Mississippi, MS, USAAbstract: To date, a 4-bp deletion in the MDR1 gene has been detected in more than ten dog breeds, as well as in mixed breed dogs, in several countries, however information regarding this mutation in dogs from Brazil is lacking. For this reason, 103 Collies, 77 Border Collies, 76 Shetland Sheepdogs, 20 Old English Sheepdogs, 55 German Shepherds, 16 Australian Shepherds, and 53 Whippets from Brazil were screened for the presence of the mutation. The heterozygous mutated genotype, MDR1 (+/−, frequency found for Collies, Australian Shepherd, and Shetland Sheepdog was 50.5% (95% CI =41.1%–59.9%, 31.3% (95% CI =8.6%–53.2%, and 15.8% (95% CI =7.7%–23.9%, respectively. Homozygous mutated genotype, MDR1 (−/−, was detected only in Collies 35.9%. The MDR1 allele mutant frequency found for Collies, Australian Shepherd, and Shetland Sheepdog was 61.2% (95% CI =54.8%–67.5%, 15.6% (95% CI =3.1%–28.2%, and 7.9% (95% CI =3.7%–12.1%, respectively. Additionally, even free of the mutant allele, the maximum mutant prevalence (MMP in that population, with 95% CI, was 3.8%, 5.2%, 5.4%, and 13.8% for Border Collies, German Shepherds, Whippets, and Old English Sheepdogs, respectively. In this way, this information is important, not only for MDR1 genotype-based breeding programs and international exchange of breeding animals of predisposed breeds, but also for modification of drug therapy for breeds at risk.Keywords: P-glycoprotein, MDR1 mutation, ivermectin, dog, drug

Predation and parasitism by native and exotic natural enemies of Halyomorpha halys (Hemiptera: Pentatomidae) eggs augmented with semiochemicals and differing host stimuli

Science.gov (United States)

The brown marmorated stink bug, or Halyomorpha halys, is an invasive species in the United States, and causes severe agricultural damage to a variety of crops. Prior research with other stink bugs has shown that various stimuli, including the aggregation pheromone of a pest can be an important kairo...

Virulence of BotaniGard® to Second Instar Brown Marmorated Stink Bug, Halyomorpha halys (Stål (Heteroptera: Pentatomidae

Directory of Open Access Journals (Sweden)

Bruce L. Parker

2015-04-01

Full Text Available The brown marmorated stink bug, Halyomorpha halys (Stål (BMSB is an exotic invasive insect originating in East Asia, currently causing significant damage to fruits, vegetables and other crops throughout most of the Mid-Atlantic states of the U.S. It also is a nuisance pest, entering homes in the fall in search of suitable overwintering sites. Two formulations of BotaniGard® with a strain of Beauveria bassiana (GHA as the active ingredient were tested against second instar BMSB. Both the wettable powder and the emulsifiable suspension formulations were efficacious at 1 × 107 conidia mL−1, causing 67%–80% mortality 9 days post treatment and 95%–100% after 12 days. The wettable powder formulation was slightly more efficacious.

Intramuscular Immunization of Mice with a Live-Attenuated Triple Mutant of Yersinia pestis CO92 Induces Robust Humoral and Cell-Mediated Immunity To Completely Protect Animals against Pneumonic Plague.

Science.gov (United States)

Tiner, Bethany L; Sha, Jian; Ponnusamy, Duraisamy; Baze, Wallace B; Fitts, Eric C; Popov, Vsevolod L; van Lier, Christina J; Erova, Tatiana E; Chopra, Ashok K

2015-12-01

Earlier, we showed that the Δlpp ΔmsbB Δail triple mutant of Yersinia pestis CO92 with deleted genes encoding Braun lipoprotein (Lpp), an acyltransferase (MsbB), and the attachment invasion locus (Ail), respectively, was avirulent in a mouse model of pneumonic plague. In this study, we further evaluated the immunogenic potential of the Δlpp ΔmsbB Δail triple mutant and its derivative by different routes of vaccination. Mice were immunized via the subcutaneous (s.c.) or the intramuscular (i.m.) route with two doses (2 × 10(6) CFU/dose) of the above-mentioned triple mutant with 100% survivability of the animals. Upon subsequent pneumonic challenge with 70 to 92 50% lethal doses (LD(50)) of wild-type (WT) strain CO92, all of the mice survived when immunization occurred by the i.m. route. Since Ail has virulence and immunogenic potential, a mutated version of Ail devoid of its virulence properties was created, and the genetically modified ail replaced the native ail gene on the chromosome of the Δlpp ΔmsbB double mutant, creating a Δlpp ΔmsbB::ailL2 vaccine strain. This newly generated mutant was attenuated similarly to the Δlpp ΔmsbB Δail triple mutant when administered by the i.m. route and provided 100% protection to animals against subsequent pneumonic challenge. Not only were the two above-mentioned mutants cleared rapidly from the initial i.m. site of injection in animals with no histopathological lesions, the immunized mice did not exhibit any disease symptoms during immunization or after subsequent exposure to WT CO92. These two mutants triggered balanced Th1- and Th2-based antibody responses and cell-mediated immunity. A substantial increase in interleukin-17 (IL-17) from the T cells of vaccinated mice, a cytokine of the Th17 cells, further augmented their vaccine potential. Thus, the Δlpp ΔmsbB Δail and Δlpp ΔmsbB::ailL2 mutants represent excellent vaccine candidates for plague, with the latter mutant still retaining Ail immunogenicity but

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

Directory of Open Access Journals (Sweden)

Khoa D Tran

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

Characterization of Halyomorpha halys (brown marmorated stink bug) biogenic volatile organic compound emissions and their role in secondary organic aerosol formation.

Science.gov (United States)

Solomon, Danielle; Dutcher, Dabrina; Raymond, Timothy

2013-11-01

The formation of aerosols is a key component in understanding cloud formation in the context of radiative forcings and global climate modeling. Biogenic volatile organic compounds (BVOCs) are a significant source of aerosols, yet there is still much to be learned about their structures, sources, and interactions. The aims of this project were to identify the BVOCs found in the defense chemicals of the brown marmorated stink bug Halymorpha halys and quantify them using gas chromatography-mass spectrometry (GC/MS) and test whether oxidation of these compounds by ozone-promoted aerosol and cloud seed formation. The bugs were tested under two conditions: agitation by asphyxiation and direct glandular exposure. Tridecane, 2(5H)-furanone 5-ethyl, and (E)-2-decenal were identified as the three most abundant compounds. H. halys were also tested in the agitated condition in a smog chamber. It was found that in the presence of 100-180 ppm ozone, secondary aerosols do form. A scanning mobility particle sizer (SMPS) and a cloud condensation nuclei counter (CCNC) were used to characterize the secondary aerosols that formed. This reaction resulted in 0.23 microg/ bug of particulate mass. It was also found that these secondary organic aerosol particles could act as cloud condensation nuclei. At a supersaturation of 1%, we found a kappa value of 0.09. Once regional populations of these stink bugs stablilize and the populations estimates can be made, the additional impacts of their contribution to regional air quality can be calculated.

Progression of Hepatic Adenoma to Carcinoma in Ogg1 Mutant Mice Induced by Phenobarbital

Directory of Open Access Journals (Sweden)

Anna Kakehashi

2017-01-01

Full Text Available The carcinogenic potential of phenobarbital (PB was assessed in a mouse line carrying a mutant Mmh allele of the Mmh/Ogg1 gene encoding the enzyme oxoguanine DNA glycosylase (Ogg1 responsible for the repair of 8-hydroxy-2′-deoxyguanosine (8-OHdG. Mmh homozygous mutant (Ogg1−/− and wild-type (Ogg1+/+ male and female, 10-week-old, mice were treated with 500 ppm PB in diet for 78 weeks. Hepatocellular carcinomas (HCCs were found in PB-treated Ogg1−/− mice, while Ogg1+/+ animals developed only hepatocellular adenomas (HCAs at the same rate. This was coordinated with PB-induced significant elevation of 8-OHdG formation in DNA and cell proliferation in adjacent liver of Ogg1−/− mice. Proteome analysis predicted activation of transcriptional factor Nrf2 in the livers and HCAs of PB-administered Ogg1+/+ mice; however, its activation was insufficient or absent in the livers and HCCs of Ogg1−/− mice, respectively. Significant elevation of phase I and II metabolizing enzymes was demonstrated in both Ogg1−/− and Ogg1+/+ animals. Treatment of Ogg1−/− mice with PB resulted in significant elevation of cell proliferation in the liver. These results indicate that PB induced progression from HCA to HCC in Ogg1−/− mice, due to persistent accumulation of DNA oxidative base modifications and suppression of Nrf2-mediated oxidative stress response, resulting in significant elevation of cell proliferation.

Radio-sensitivity of the cells from amyotrophic lateral sclerosis model mice transfected with human mutant SOD1

International Nuclear Information System (INIS)

Wate, Reika; Ito, Hidefumi; Kusaka, Hirofumi; Takahashi, Sentaro; Kubota, Yoshihisa; Suetomi, Katsutoshi; Sato, Hiroshi; Okayasu, Ryuichi

2005-01-01

In order to clarify the possible involvement of oxidative damage induced by ionizing radiation in the onset and/or progression of familial amyotrophic lateral sclerosis (ALS), we studied radio-sensitivity in primary cells derived from ALS model mice expressing human mutant Cu/Zn superoxide dismutase (SOD1). The primary mouse cells expressed both mouse and the mutant human SOD1. The cell survival of the transgenic mice (with mutant SOD1), determined by counting cell numbers at a scheduled time after X-irradiation, is very similar to that of cells from wild type animals. The induction and repair of DNA damage in the transgenic cells, measured by single cell gel electrophoresis and pulsed field gel electrophoresis, are also similar to those of wild type cells. These results indicate that the human mutant SOD1 gene does not seem to contribute to the alteration of radio-sensitivity, at least in the fibroblastic cells used here. Although it is necessary to consider the difference in cell types between fibroblastic and neuronal cells, the present results may suggest that ionizing radiation is not primarily responsible for the onset of familial ALS with the SOD1 mutation, and that the excess risks are probably not a concern for radiation diagnosis and therapy in familial ALS patients. (author)

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.

Homologous series of induced early mutants in indican rice. Pt.1. The production of homologous series of early mutants

International Nuclear Information System (INIS)

Chen Xiulan; Yang Hefeng; He Zhentian; Han Yuepeng; Liu Xueyu

1999-01-01

The percentage of homologous series of early mutants induced from the same Indican rice variety were almost the same (1.37%∼1.64%) in 1983∼1993, but the ones from the different eco-typical varieties were different. The early variety was 0.73%, the mid variety was 1.51%, and the late variety was 1.97%. The percentage of homologous series of early mutants from the varieties with the same pedigree and relationship were similar, but the one from the cog nation were lower than those from distant varieties. There are basic laws and characters in the homologous series of early mutants: 1. The inhibited phenotype is the basic of the homologous series of early mutants; 2. The production of the homologous series of early mutants is closely related with the growing period of the parent; 3. The parallel mutation of the stem and leaves are simultaneously happened with the variation of early or late maturing; 4. The occurrence of the homologous series of early mutants is in a state of imbalance. According to the law of parallel variability, the production of homologous series of early mutants can be predicted as long as the parents' classification of plant, pedigree and ecological type are identified. Therefore, the early breeding can be guided by the law of homologous series of early mutants

Montovaná konstrukce haly ve Vysokém Mýtě

OpenAIRE

Jarmara, Pavel

2017-01-01

Tato diplomová práce se zabývá návrhem a statickým výpočtem montované, skladovací haly. V rámci práce jsem navrhnul a posoudil předpjatý vazník, železobetonový vazník, vazničku, sloup a kalichovou patku. Prvky jsou posouzeny na mezní stav únosnosti. Řešení vnitřních sil vazníků bylo provedeno ručním výpočtem. Vnitřní síly sloupu a patky byly určeny v programu Scia Engenner. Uvedené výpočty jsou provedeny dle Eurokódu 2. Ke všem navrženým prvků jsem také narýsoval výkresy tvaru a výztuže. T...

Neuwirth RJ, Svetlicinii A and De Castro Halis D (eds) The BRICS-Lawyers' Guide to Global Cooperation (Cambridge University Press 2017)

OpenAIRE

Sucker, F

2018-01-01

This contribution reviews the book The BRICS-Lawyers' Guide to Global Cooperation edited by Rostam J Neuwirth, Alexandr Svetlicinii and Denis De Castro Halis. It was published by Cambridge University Press in 2017, and deals with aspects of international trade and development involving the BRICS area -Brazil, Russia, India, China and South Africa. It is described as a unique reference book for academics, governmental officials, legal professionals, business executives, researchers and students.

A novel Arabidopsis CHITIN ELICITOR RECEPTOR KINASE 1 (CERK1) mutant with enhanced pathogen-induced cell death and altered receptor processing.

Science.gov (United States)

Petutschnig, Elena K; Stolze, Marnie; Lipka, Ulrike; Kopischke, Michaela; Horlacher, Juliane; Valerius, Oliver; Rozhon, Wilfried; Gust, Andrea A; Kemmerling, Birgit; Poppenberger, Brigitte; Braus, Gerhard H; Nürnberger, Thorsten; Lipka, Volker

2014-12-01

Plants detect pathogens by sensing microbe-associated molecular patterns (MAMPs) through pattern recognition receptors. Pattern recognition receptor complexes also have roles in cell death control, but the underlying mechanisms are poorly understood. Here, we report isolation of cerk1-4, a novel mutant allele of the Arabidopsis chitin receptor CERK1 with enhanced defense responses. We identified cerk1-4 in a forward genetic screen with barley powdery mildew and consequently characterized it by pathogen assays, mutant crosses and analysis of defense pathways. CERK1 and CERK1-4 proteins were analyzed biochemically. The cerk1-4 mutation causes an amino acid exchange in the CERK1 ectodomain. Mutant plants maintain chitin signaling capacity but exhibit hyper-inducible salicylic acid concentrations and deregulated cell death upon pathogen challenge. In contrast to chitin signaling, the cerk1-4 phenotype does not require kinase activity and is conferred by the N-terminal part of the receptor. CERK1 undergoes ectodomain shedding, a well-known process in animal cell surface proteins. Wild-type plants contain the full-length CERK1 receptor protein as well as a soluble form of the CERK1 ectodomain, whereas cerk1-4 plants lack the N-terminal shedding product. Our work suggests that CERK1 may have a chitin-independent role in cell death control and is the first report of ectodomain shedding in plants. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

Sensorimotor learning in Dab1(scm) (scrambler) mutant mice.

Science.gov (United States)

Lalonde, R; Strazielle, C

2011-04-15

Homozygous Dab1(scm) mouse mutants with cell ectopias in cerebellar cortex and neocortex were compared with non-ataxic controls on two tests of motor coordination: rotorod and grid climbing. Even at the minimal speed of 4 rpm and unlike controls, none of the Dab1(scm) mutants reached criterion on the constant speed rotorod. In contrast, Dab1(scm) mutants improved their performances on the vertical grid over the course of the same number of trials. Thus, despite massive cerebellar degeneration, sensorimotor learning for equilibrium is still possible, indicating the potential usefulness of the grid-climbing test in determining residual functions in mice with massive cerebellar damage. Copyright © 2010. Published by Elsevier B.V.

Use of Transgenic and Mutant Animal Models in the Study of Heterocyclic Amine-induced Mutagenesis and Carcinogenesis

Science.gov (United States)

Dashwood, Roderick H.

2008-01-01

Heterocyclic amines (HCAs) are potent mutagens generated during the cooking of meat and fish, and several of these compounds produce tumors in conventional experimental animals. During the past 5 years or so, HCAs have been tested in a number of novel in vivo murine models, including the following: lacZ, lacI, cII, c-myc/lacZ, rpsL, and gptΔ transgenics, XPA−/−, XPC−/−, Msh2+/−, Msh2−/− and p53+/− knock-outs, Apc mutant mice (ApcΔ716, Apc1638N, Apcmin), and A33ΔNβ-cat knock-in mice. Several of these models have provided insights into the mutation spectra induced in vivo by HCAs in target and non-target organs for tumorigenesis, as well as demonstrating enhanced susceptibility to HCA-induced tumors and preneoplastic lesions. This review describes several of the more recent reports in which novel animal models were used to examine HCA-induced mutagenesis and carcinogenesis in vivo, including a number of studies which assessed the inhibitory activities of chemopreventive agents such as 1,2-dithiole-3-thione, conjugated linoleic acids, tea, curcumin, chlorophyllin-chitosan, and sulindac. PMID:12542973

Differences in morphometrics and reproductive physiology between two populations of Trissolcus japonicus, a promising biological control agent candidate for brown marmorated stink bug (Halyomorpha halys Stal) in the US

Science.gov (United States)

Trissolcus japonicus (Ashmead), a solitary egg parasitoid of Pentatomidae native to Southeast Asia, has been undergoing host-range testing in U.S. quarantine facilities since 2009 as a candidate for the biological control of brown marmorated stink bug (Halyomorpha halys Stål)(BMSB), an invasive agri...

The yeast complex I equivalent NADH dehydrogenase rescues pink1 mutants.

Directory of Open Access Journals (Sweden)

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.

Gamma-radiation Mutagenesis in Genetically Unstable Barley Mutants. Pt. 1. Chlorophyll Mutations in Allelic tw Mutants and Their Revertants

International Nuclear Information System (INIS)

Vaitkuniene, V.

1995-01-01

Genotypical environment is an essential factor determining the mutability of mutants of the same type. Decreased chlorophyll mutant frequency was a common characteristic of all tested tw type (tw, tw 1 , tw 2 ) mutants induced in barley c. 'Auksiniai II'. The mutability of all the tested revertants was close to that of the initial c. 'Auksiniai II'. (author). 9 refs., 2 tabs

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.

Clear Plaque Mutants of Lactococcal Phage TP901-1

DEFF Research Database (Denmark)

Kot, Witold; Kilstrup, Mogens; Vogensen, Finn K.

2016-01-01

We report a method for obtaining turbid plaques of the lactococcal bacteriophage TP901-1 and its derivative TP901-BC1034. We have further used the method to isolate clear plaque mutants of this phage. Analysis of 8 such mutants that were unable to lysogenize the host included whole genome...

WYKORZYSTANIE ODNAWIALNYCH ŹRÓDEŁ ENERGII I CIEPŁA ODPADOWEGO W NISKOTEMPERATUROWYM SYSTEMIE OGRZEWCZO-CHŁODZĄCYM HALI DRUKARNI

Directory of Open Access Journals (Sweden)

Wojciech BIALIK

Full Text Available W pracy przedstawiono nowoczesny system ogrzewania hali drukarni oraz pomieszczeń biurowych i socjalnych. Został on zaprezentowany na dwóch schematach oraz omówiony w pierwszej części artykułu. Układ wykorzystuje pompy ciepła ciecz-ciecz oraz powietrze ciecz. Dla pomp ciecz-ciecz dolnym źródłem jest pionowy wymiennik gruntowy składający się z 13 otworów o głębokości 100 m. W opisywanym rozwiązaniu zastosowano także poziomy gruntowy wymiennik ciepła do ogrzewania czerpanego z otoczenia powietrza wentylacyjnego płynącego następnie do rekuperatorów pomieszczeń biurowych i socjalnych. Gruntowe wymienniki ciepła w trakcie ich budowy zostały zaprezentowane na jednym z rysunków. Pomieszczenia biurowe i socjalne ogrzewane lub schładzane są medium płynącym w matach umieszczonych w ścianach oraz w wybranych częściach podłogi. Dla ograniczenia strat ciepła związanych z wentylacją w instalacji zastosowano trzy układy rekuperacji (osobno dla pomieszczeń biurowych, socjalnych i hali drukarskiej. Poprawne działanie systemu zobrazowano w postaci wykresu zmian temperatur we wspomnianych pomieszczeniach. Dla największej centrali wentylacyjnej zamieszczono też przebieg temperatur strumieni powietrza dla wybranych dni. W podsumowaniu zawarto wnioski płynące z dotychczasowej eksploatacji oraz plany dalszego rozwoju systemu.

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.

Ectopic Mineralization and Conductive Hearing Loss in Enpp1asj Mutant Mice, a New Model for Otitis Media and Tympanosclerosis.

Science.gov (United States)

Tian, Cong; Harris, Belinda S; Johnson, Kenneth R

2016-01-01

Otitis media (OM), inflammation of the middle ear, is a common cause of hearing loss in children and in patients with many different syndromic diseases. Studies of the human population and mouse models have revealed that OM is a multifactorial disease with many environmental and genetic contributing factors. Here, we report on otitis media-related hearing loss in asj (ages with stiffened joints) mutant mice, which bear a point mutation in the Enpp1 gene. Auditory-evoked brainstem response (ABR) measurements revealed that around 90% of the mutant mice (Enpp1asj/asj) tested had moderate to severe hearing impairment in at least one ear. The ABR thresholds were variable and generally elevated with age. We found otitis media with effusion (OME) in all of the hearing-impaired Enpp1asj/asj mice by anatomic and histological examinations. The volume and inflammatory cell content of the effusion varied among the asj mutant mice, but all mutants exhibited a thickened middle ear epithelium with fibrous polyps and more mucin-secreting goblet cells than controls. Other abnormalities observed in the Enpp1 mutant mice include over-ossification at the round window ridge, thickened and over-calcified stapedial artery, fusion of malleus and incus, and white patches on the inside of tympanic membrane, some of which are typical symptoms of tympanosclerosis. An excessive yellow discharge was detected in the outer ear canal of older asj mutant mice, with 100% penetrance by 5 months of age, and contributes to the progressive nature of the hearing loss. This is the first report of hearing loss and ear pathology associated with an Enpp1 mutation in mice. The Enpp1asj mutant mouse provides a new animal model for studying tympanosclerotic otitis and otitis media with effusion, and also provides a specific model for the hearing loss recently reported to be associated with human ENPP1 mutations causing generalized arterial calcification of infancy and hypophosphatemic rickets.

Histological Characterization of the Dicer1 Mutant Zebrafish Retina

Directory of Open Access Journals (Sweden)

Saeed Akhtar

2015-01-01

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

Conserved role of unc-79 in ethanol responses in lightweight mutant mice.

Directory of Open Access Journals (Sweden)

David J Speca

2010-08-01

Full Text Available The mechanisms by which ethanol and inhaled anesthetics influence the nervous system are poorly understood. Here we describe the positional cloning and characterization of a new mouse mutation isolated in an N-ethyl-N-nitrosourea (ENU forward mutagenesis screen for animals with enhanced locomotor activity. This allele, Lightweight (Lwt, disrupts the homolog of the Caenorhabditis elegans (C. elegans unc-79 gene. While Lwt/Lwt homozygotes are perinatal lethal, Lightweight heterozygotes are dramatically hypersensitive to acute ethanol exposure. Experiments in C. elegans demonstrate a conserved hypersensitivity to ethanol in unc-79 mutants and extend this observation to the related unc-80 mutant and nca-1;nca-2 double mutants. Lightweight heterozygotes also exhibit an altered response to the anesthetic isoflurane, reminiscent of unc-79 invertebrate mutant phenotypes. Consistent with our initial mapping results, Lightweight heterozygotes are mildly hyperactive when exposed to a novel environment and are smaller than wild-type animals. In addition, Lightweight heterozygotes exhibit increased food consumption yet have a leaner body composition. Interestingly, Lightweight heterozygotes voluntarily consume more ethanol than wild-type littermates. The acute hypersensitivity to and increased voluntary consumption of ethanol observed in Lightweight heterozygous mice in combination with the observed hypersensitivity to ethanol in C. elegans unc-79, unc-80, and nca-1;nca-2 double mutants suggests a novel conserved pathway that might influence alcohol-related behaviors in humans.

A mutation in the dynein heavy chain gene compensates for energy deficit of mutant SOD1 mice and increases potentially neuroprotective IGF-1

Directory of Open Access Journals (Sweden)

Larmet Yves

2011-04-01

Full Text Available Abstract Background Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons. ALS patients, as well as animal models such as mice overexpressing mutant SOD1s, are characterized by increased energy expenditure. In mice, this hypermetabolism leads to energy deficit and precipitates motor neuron degeneration. Recent studies have shown that mutations in the gene encoding the dynein heavy chain protein are able to extend lifespan of mutant SOD1 mice. It remains unknown whether the protection offered by these dynein mutations relies on a compensation of energy metabolism defects. Results SOD1(G93A mice were crossbred with mice harboring the dynein mutant Cramping allele (Cra/+ mice. Dynein mutation increased adipose stores in compound transgenic mice through increasing carbohydrate oxidation and sparing lipids. Metabolic changes that occurred in double transgenic mice were accompanied by the normalization of the expression of key mRNAs in the white adipose tissue and liver. Furthermore, Dynein Cra mutation rescued decreased post-prandial plasma triglycerides and decreased non esterified fatty acids upon fasting. In SOD1(G93A mice, the dynein Cra mutation led to increased expression of IGF-1 in the liver, increased systemic IGF-1 and, most importantly, to increased spinal IGF-1 levels that are potentially neuroprotective. Conclusions These findings suggest that the protection against SOD1(G93A offered by the Cramping mutation in the dynein gene is, at least partially, mediated by a reversal in energy deficit and increased IGF-1 availability to motor neurons.

AGO1, QDE-2, and RDE-1 are related proteins required for post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference in animals.

Science.gov (United States)

Fagard, M; Boutet, S; Morel, J B; Bellini, C; Vaucheret, H

2000-10-10

Introduction of transgene DNA may lead to specific degradation of RNAs that are homologous to the transgene transcribed sequence through phenomena named post-transcriptional gene silencing (PTGS) in plants, quelling in fungi, and RNA interference (RNAi) in animals. It was shown previously that PTGS, quelling, and RNAi require a set of related proteins (SGS2, QDE-1, and EGO-1, respectively). Here we report the isolation of Arabidopsis mutants impaired in PTGS which are affected at the Argonaute1 (AGO1) locus. AGO1 is similar to QDE-2 required for quelling and RDE-1 required for RNAi. Sequencing of ago1 mutants revealed one amino acid essential for PTGS that is also present in QDE-2 and RDE-1 in a highly conserved motif. Taken together, these results confirm the hypothesis that these processes derive from a common ancestral mechanism that controls expression of invading nucleic acid molecules at the post-transcriptional level. As opposed to rde-1 and qde-2 mutants, which are viable, ago1 mutants display several developmental abnormalities, including sterility. These results raise the possibility that PTGS, or at least some of its elements, could participate in the regulation of gene expression during development in plants.

Deletion map of CYC1 mutants and its correspondence to mutationally altered iso-1-cytochromes c of yeast

International Nuclear Information System (INIS)

Sherman, F.; Jackson, M.; Liebman, S.W.; Schweingruber, A.M.; Stewart, J.W.

1975-01-01

Mutants arising spontaneously from sporulated cultures of certain strains of yeast, Saccharomyces cerevisiae, contained deletions of the CYC1 gene which controls the primary structure of iso-1-cytochrome c. At least 60 different kinds of deletions were uncovered among the 104 deletions examined and these ranged in length from those encompassing only two adjacent point mutants to those encompassing at least the entire CYC1 gene. X-ray-induced recombination rates of crosses involving these deletions and cyc1 point mutants resulted in the assignment of 211 point mutants to 47 mutational sites and made it possible to unambiguously order 40 of these 47 sites. Except for one mutant, cyc1-15, there was a strict colinear relationship between the deletion map and the positions of 13 sites that were previously determined by amino acid alterations in iso-1-cytochromes c from intragenic revertants

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

Science.gov (United States)

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

2018-02-01

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

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

Directory of Open Access Journals (Sweden)

Luke W Meredith

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

Modeling the dual pacemaker system of the tau mutant hamster.

Science.gov (United States)

Oda, G A; Menaker, M; Friesen, W O

2000-06-01

Circadian pacemakers in many animals are compound. In rodents, a two-oscillator model of the pacemaker composed of an evening (E) and a morning (M) oscillator has been proposed based on the phenomenon of "splitting" and bimodal activity peaks. The authors describe computer simulations of the pacemaker in tau mutant hamsters viewed as a system of mutually coupled E and M oscillators. These mutant animals exhibit normal type 1 PRCs when released into DD but make a transition to a type 0 PRC when held for many weeks in DD. The two-oscillator model describes particularly well some recent behavioral experiments on these hamsters. The authors sought to determine the relationships between oscillator amplitude, period, PRC, and activity duration through computer simulations. Two complementary approaches proved useful for analyzing weakly coupled oscillator systems. The authors adopted a "distinct oscillators" view when considering the component E and M oscillators and a "system" view when considering the system as a whole. For strongly coupled systems, only the system view is appropriate. The simulations lead the authors to two primary conjectures: (1) the total amplitude of the pacemaker system in tau mutant hamsters is less than in the wild-type animals, and (2) the coupling between the unit E and M oscillators is weakened during continuous exposure of hamsters to DD. As coupling strength decreases, activity duration (alpha) increases due to a greater phase difference between E and M. At the same time, the total amplitude of the system decreases, causing an increase in observable PRC amplitudes. Reduced coupling also increases the relative autonomy of the unit oscillators. The relatively autonomous phase shifts of E and M oscillators can account for both immediate compression and expansion of activity bands in tau mutant and wild-type hamsters subjected to light pulses.

The deoxyhypusine synthase mutant dys1-1 reveals the association of eIF5A and Asc1 with cell wall integrity.

Directory of Open Access Journals (Sweden)

Fabio Carrilho Galvão

Full Text Available The putative eukaryotic translation initiation factor 5A (eIF5A is a highly conserved protein among archaea and eukaryotes that has recently been implicated in the elongation step of translation. eIF5A undergoes an essential and conserved posttranslational modification at a specific lysine to generate the residue hypusine. The enzymes deoxyhypusine synthase (Dys1 and deoxyhypusine hydroxylase (Lia1 catalyze this two-step modification process. Although several Saccharomyces cerevisiae eIF5A mutants have importantly contributed to the study of eIF5A function, no conditional mutant of Dys1 has been described so far. In this study, we generated and characterized the dys1-1 mutant, which showed a strong depletion of mutated Dys1 protein, resulting in more than 2-fold decrease in hypusine levels relative to the wild type. The dys1-1 mutant demonstrated a defect in total protein synthesis, a defect in polysome profile indicative of a translation elongation defect and a reduced association of eIF5A with polysomes. The growth phenotype of dys1-1 mutant is severe, growing only in the presence of 1 M sorbitol, an osmotic stabilizer. Although this phenotype is characteristic of Pkc1 cell wall integrity mutants, the sorbitol requirement from dys1-1 is not associated with cell lysis. We observed that the dys1-1 genetically interacts with the sole yeast protein kinase C (Pkc1 and Asc1, a component of the 40S ribosomal subunit. The dys1-1 mutant was synthetically lethal in combination with asc1Δ and overexpression of TIF51A (eIF5A or DYS1 is toxic for an asc1Δ strain. Moreover, eIF5A is more associated with translating ribosomes in the absence of Asc1 in the cell. Finally, analysis of the sensitivity to cell wall-perturbing compounds revealed a more similar behavior of the dys1-1 and asc1Δ mutants in comparison with the pkc1Δ mutant. These data suggest a correlated role for eIF5A and Asc1 in coordinating the translational control of a subset of m

RNA surveillance via nonsense-mediated mRNA decay is crucial for longevity in daf-2/insulin/IGF-1 mutant C. elegans.

Science.gov (United States)

Son, Heehwa G; Seo, Mihwa; Ham, Seokjin; Hwang, Wooseon; Lee, Dongyeop; An, Seon Woo A; Artan, Murat; Seo, Keunhee; Kaletsky, Rachel; Arey, Rachel N; Ryu, Youngjae; Ha, Chang Man; Kim, Yoon Ki; Murphy, Coleen T; Roh, Tae-Young; Nam, Hong Gil; Lee, Seung-Jae V

2017-03-09

Long-lived organisms often feature more stringent protein and DNA quality control. However, whether RNA quality control mechanisms, such as nonsense-mediated mRNA decay (NMD), which degrades both abnormal as well as some normal transcripts, have a role in organismal aging remains unexplored. Here we show that NMD mediates longevity in C. elegans strains with mutations in daf-2/insulin/insulin-like growth factor 1 receptor. We find that daf-2 mutants display enhanced NMD activity and reduced levels of potentially aberrant transcripts. NMD components, including smg-2/UPF1, are required to achieve the longevity of several long-lived mutants, including daf-2 mutant worms. NMD in the nervous system of the animals is particularly important for RNA quality control to promote longevity. Furthermore, we find that downregulation of yars-2/tyrosyl-tRNA synthetase, an NMD target transcript, by daf-2 mutations contributes to longevity. We propose that NMD-mediated RNA surveillance is a crucial quality control process that contributes to longevity conferred by daf-2 mutations.

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

Data supporting mitochondrial morphological changes by SPG13-associated HSPD1 mutants

Directory of Open Access Journals (Sweden)

Yuki Miyamoto

2016-03-01

Full Text Available The data is related to the research article entitled “Hypomyelinating leukodystrophy-associated missense mutation in HSPD1 blunts mitochondrial dynamics” [1]. In addition to hypomyelinating leukodystrophy (HLD 4 (OMIM no. 612233, it is known that spastic paraplegia (SPG 13 (OMIM no. 605280 is caused by HSPD1’s amino acid mutation. Two amino acid mutations Val-98-to-Ile (V98I and Gln-461-to-Glu (Q461E are associated with SPG13 [2]. In order to investigate the effects of HSPD1’s V98I or Q461E mutant on mitochondrial morphological changes, we transfected each of the respective mutant-encoding genes into Cos-7 cells. Either of V98I or Q461E mutant exhibited increased number of mitochondria and short length mitochondrial morphologies. Using MitoTracker dye-incorporating assay, decreased mitochondrial membrane potential was also observed in both cases. The data described here supports that SPG13-associated HSPD1 mutant participates in causing aberrant mitochondrial morphological changes with decreased activities. Keywords: SPG13, HSPD1, Mitochondrion, Morphological change

Trehalose, glycogen and ethanol metabolism in the gcr1 mutant of Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Seker, Tamay; Hamamci, H.

2003-01-01

Since Gcr1p is pivotal in controlling the transcription of glycolytic enzymes and trehalose metabolism seems to be one of the control points of glycolysis, we examined trehalose and glycogen synthesis in response to 2 % glucose pulse during batch growth in gcr1 (glucose regulation-1) mutant lacking...... fully functional glycolytic pathway and in the wild-type strain. An increase in both trehalose and glycogen stores was observed 1 and 2 h after the pulse followed by a steady decrease in both the wild-type and the gcr1 mutant. The accumulation was faster while the following degradation was slower in gcr......1 cells compared to wild-type ones. Although there was no distinct glucose consumption in the mutant cells it seemed that the glucose repression mechanism is similar in gcr1 mutant and in wild-type strain at least with respect to trehalose and glycogen metabolism....

Mutant IDH1 Promotes Glioma Formation In Vivo

Directory of Open Access Journals (Sweden)

Beatrice Philip

2018-05-01

Full Text Available Summary: Isocitrate dehydrogenase 1 (IDH1 is the most commonly mutated gene in grade II–III glioma and secondary glioblastoma (GBM. A causal role for IDH1R132H in gliomagenesis has been proposed, but functional validation in vivo has not been demonstrated. In this study, we assessed the role of IDH1R132H in glioma development in the context of clinically relevant cooperating genetic alterations in vitro and in vivo. Immortal astrocytes expressing IDH1R132H exhibited elevated (R-2-hydroxyglutarate levels, reduced NADPH, increased proliferation, and anchorage-independent growth. Although not sufficient on its own, IDH1R132H cooperated with PDGFA and loss of Cdkn2a, Atrx, and Pten to promote glioma development in vivo. These tumors resembled proneural human mutant IDH1 GBM genetically, histologically, and functionally. Our findings support the hypothesis that IDH1R132H promotes glioma development. This model enhances our understanding of the biology of IDH1R132H-driven gliomas and facilitates testing of therapeutic strategies designed to combat this deadly disease. : Philip et al. show that mutant IDH1 cooperates with PDGFA and loss of Cdkn2a, Atrx, and Pten to promote gliomagenesis in vivo in a mouse model of glioma. These tumors resemble proneural human mutant IDH1 glioblastoma and exhibit enhanced sensitivity to PARP inhibition in combination with chemotherapy. Keywords: IDH1, Cdkn2a, Atrx, Pten, glioma, mouse model, RCAS/TVA

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.

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.

Neurospora crassa ncs-1, mid-1 and nca-2 double-mutant ...

Indian Academy of Sciences (India)

logue of Neuronal Calcium Sensor-1 has a role in growth, cal- cium stress tolerance, and ultraviolet survival. Genetica 139,. 885–894. Lew R. R., Abbas Z., Anderca M. I. and Free S. J. 2008 Phe- notype of a mechanosensitive channel mutant, mid-1, in a fil- amentous fungus, Neurospora crassa. Eukaryot. Cell. 7, 647–. 655.

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

Science.gov (United States)

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

2017-08-15

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

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

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

OpenAIRE

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

2017-01-01

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

Transgenic animal models for study of the pathogenesis of Huntington’s disease and therapy

Directory of Open Access Journals (Sweden)

Chang RB

2015-04-01

Full Text Available Renbao Chang,1 Xudong Liu,1 Shihua Li,2 Xiao-Jiang Li1,2 1State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, People’s Republic of China; 2Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA Abstract: Huntington’s disease (HD is caused by a genetic mutation that results in polyglutamine expansion in the N-terminal regions of huntingtin. As a result, this polyQ expansion leads to the misfolding and aggregation of mutant huntingtin as well as age-dependent neurodegeneration. The genetic mutation in HD allows for generating a variety of animal models that express different forms of mutant huntingtin and show differential pathology. Studies of these animal models have provided an important insight into the pathogenesis of HD. Mouse models of HD include transgenic mice, which express N-terminal or full-length mutant huntingtin ubiquitously or selectively in different cell types, and knock-in mice that express full-length mutant Htt at the endogenous level. Large animals, such as pig, sheep, and monkeys, have also been used to generate animal HD models. This review focuses on the different features of commonly used transgenic HD mouse models as well as transgenic large animal models of HD, and also discusses how to use them to identify potential therapeutics. Since HD shares many pathological features with other neurodegenerative diseases, identification of therapies for HD would also help to develop effective treatment for different neurodegenerative diseases that are also caused by protein misfolding and occur in an age-dependent manner. Keywords: transgenic animal models, Huntington’s disease, pathogenesis, therapy

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

Science.gov (United States)

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

2014-03-01

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

Construction of insertion and deletion mxa mutants of Methylobacterium extorquens AM1 by electroporation.

Science.gov (United States)

Toyama, H; Anthony, C; Lidstrom, M E

1998-09-01

Methylobacterium extorquens AM1 is a pink-pigmented facultative methylotroph which is widely used for analyzing pathways of C1 metabolism with biochemical and molecular biological techniques. To facilitate this approach, we have applied a new method to construct insertion or disruption mutants with drug resistance genes by electroporation. By using this method, mutants were obtained in four genes present in the mxa methylotrophy gene cluster for which the functions were unknown, mxaR, mxaS, mxaC and mxaD. These mutants were unable to grow on methanol except the mutant of mxaD, which showed reduced growth on methanol.

Isolation and characteristics of minute plaque forming mutant of cyanophage AS-1

International Nuclear Information System (INIS)

Amla, D.V.

1981-01-01

Minute plaque forming mutant (m) of cyanophage AS-1 infecting unicellular blue-green algae was isolated spontaneously and after mutagenic treatment. Compared to wild type m mutant formed small plaques, adsorption rate was slow and the burst-size was significantly decreased with prolonged eclipse and latent period. The plaque forming ability of mutant phage was sensitive to pH, heat, EDTA shock, distilled water and photosensitisation with acriflavine whereas ultraviolet sensitivity of free and intracellular phage was identical to the parent. The spontaneous reversion frequencies of mutant phage to wild type were between 10 -5 to 10 -3 and appeared to be clonal property. Reversion studies suggested possibilities of frame-shift or base-pair substitution for m mutation. (author)

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.

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

Energy Technology Data Exchange (ETDEWEB)

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

2016-04-22

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

Effect of CCS on the accumulation of FALS SOD1 mutant-containing aggregates and on mitochondrial translocation of SOD1 mutants: implication of a free radical hypothesis.

Science.gov (United States)

Kim, Ha Kun; Chung, Youn Wook; Chock, P Boon; Yim, Moon B

2011-05-15

Missense mutations of SOD1 are linked to familial amyotrophic lateral sclerosis (FALS) through a yet-to-be identified toxic-gain-of-function. One of the proposed mechanisms involves enhanced aggregate formation. However, a recent study showed that dual transgenic mice overexpressing both G93A and CCS copper chaperone (G93A/CCS) exhibit no SOD1-positive aggregates yet show accelerated FALS symptoms with enhanced mitochondrial pathology compared to G93A mice. Using a dicistronic mRNA to simultaneously generate hSOD1 mutants, G93A, A4V and G85R, and hCCS in AAV293 cells, we revealed: (i) CCS is degraded primarily via a macroautophagy pathway. It forms a stable heterodimer with inactive G85R, and via its novel copper chaperone-independent molecular chaperone activity facilitates G85R degradation via a macroautophagy-mediated pathway. For active G93A and A4V, CCS catalyzes their maturation to form active and soluble homodimers. (ii) CCS reduces, under non-oxidative conditions, yet facilitates in the presence of H(2)O(2), mitochondrial translocation of inactive SOD1 mutants. These results, together with previous reports showing FALS SOD1 mutants enhanced free radical-generating activity, provide a mechanistic explanation for the observations with G93A/CCS dual transgenic mice and suggest that free radical generation by FALS SOD1, enhanced by CCS, may, in part, be responsible for the FALS SOD1 mutant-linked aggregation, mitochondrial translocation, and degradation. Published by Elsevier Inc.

Differential disease resistance response in the barley necrotic mutant nec1

Directory of Open Access Journals (Sweden)

Kunga Laura

2011-04-01

Full Text Available Abstract Background Although ion fluxes are considered to be an integral part of signal transduction during responses to pathogens, only a few ion channels are known to participate in the plant response to infection. CNGC4 is a disease resistance-related cyclic nucleotide-gated ion channel. Arabidopsis thaliana CNGC4 mutants hlm1 and dnd2 display an impaired hypersensitive response (HR, retarded growth, a constitutively active salicylic acid (SA-mediated pathogenesis-related response and elevated resistance against bacterial pathogens. Barley CNGC4 shares 67% aa identity with AtCNGC4. The barley mutant nec1 comprising of a frame-shift mutation of CNGC4 displays a necrotic phenotype and constitutively over-expresses PR-1, yet it is not known what effect the nec1 mutation has on barley resistance against different types of pathogens. Results nec1 mutant accumulated high amount of SA and hydrogen peroxide compared to parental cv. Parkland. Experiments investigating nec1 disease resistance demonstrated positive effect of nec1 mutation on non-host resistance against Pseudomonas syringae pv. tomato (Pst at high inoculum density, whereas at normal Pst inoculum concentration nec1 resistance did not differ from wt. In contrast to augmented P. syringae resistance, penetration resistance against biotrophic fungus Blumeria graminis f. sp. hordei (Bgh, the causal agent of powdery mildew, was not altered in nec1. The nec1 mutant significantly over-expressed race non-specific Bgh resistance-related genes BI-1 and MLO. Induction of BI-1 and MLO suggested putative involvement of nec1 in race non-specific Bgh resistance, therefore the effect of nec1on mlo-5-mediated Bgh resistance was assessed. The nec1/mlo-5 double mutant was as resistant to Bgh as Nec1/mlo-5 plants, suggesting that nec1 did not impair mlo-5 race non-specific Bgh resistance. Conclusions Together, the results suggest that nec1 mutation alters activation of systemic acquired resistance

Characterization and fine mapping of a light-dependent leaf lesion mimic mutant 1 in rice.

Science.gov (United States)

Wang, Jing; Ye, Bangquan; Yin, Junjie; Yuan, Can; Zhou, Xiaogang; Li, Weitao; He, Min; Wang, Jichun; Chen, Weilan; Qin, Peng; Ma, Bintian; Wang, Yuping; Li, Shigui; Chen, Xuewei

2015-12-01

Plants that spontaneously produce lesion mimics or spots, without any signs of obvious adversity, such as pesticide and mechanical damage, or pathogen infection, are so-called lesion mimic mutants (lmms). In rice, many lmms exhibit enhanced resistance to pathogens, which provides a unique opportunity to uncover the molecular mechanism underlying lmms. We isolated a rice light-dependent leaf lesion mimic mutant 1 (llm1). Lesion spots appeared in the leaves of the llm1 mutant at the tillering stage. Furthermore, the mutant llm1 had similar agronomic traits to wild type rice. Trypan blue and diamiobenzidine staining analyses revealed that the lesion spot formation on the llm1 mutant was due to programmed cell death and reactive oxygen species. The chloroplasts were severely damaged in the llm1 mutant, suggesting that chloroplast damage was associated with the formation of lesion spots in llm1. More importantly, llm1 exhibited enhanced resistance to bacterial blight pathogens within increased expression of pathogenesis related genes (PRs). Using a map-based cloning approach, we delimited the LLM1 locus to a 121-kb interval between two simple sequence repeat markers, RM17470 and RM17473, on chromosome 4. We sequenced the candidate genes on the interval and found that a base mutation had substituted adenine phosphate for thymine in the last exon of LOC_Os04g52130, which led to an amino acid change (Asp(388) to Val) in the llm1 mutant. Our investigation showed that the putative coproporphyrinogen III oxidase (CPOX) encoded by LOC_Os04g52130 was produced by LLM1 and that amino acid Asp(388) was essential for CPOX function. Our study provides the basis for further investigations into the mechanism underlying lesion mimic initiation associated with LLM1. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

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.

Dissecting the Mechanisms of Drug Resistance in BRCA1/2-Mutant Breast Cancers

Science.gov (United States)

2017-10-01

AWARD NUMBER: W81XWH-16-1-0600 TITLE: Dissecting the Mechanisms of Drug Resistance in BRCA1/2-Mutant Breast Cancers PRINCIPAL INVESTIGATOR: Dr...2017 4. TITLE AND SUBTITLE Dissecting the Mechanisms of Drug Resistance in BRCA1/2- Mutant Breast Cancers 5a. CONTRACT NUMBER W81XWH-16-1-0600 5b...therapeutic modality for targeting homologous recombination (HR) deficient tumors such as BRCA1 and BRCA2-mutated triple negative breast cancers

RNAi-mediated Gene Silencing of Mutant Myotilin Improves Myopathy in LGMD1A Mice

Directory of Open Access Journals (Sweden)

Jian Liu

2014-01-01

Full Text Available Recent progress suggests gene therapy may one day be an option for treating some forms of limb girdle muscular dystrophy (LGMD. Nevertheless, approaches targeting LGMD have so far focused on gene replacement strategies for recessive forms of the disease. In contrast, no attempts have been made to develop molecular therapies for any of the eight dominantly inherited forms of LGMD. Importantly, the emergence of RNA interference (RNAi therapeutics in the last decade provided new tools to combat dominantly inherited LGMDs with molecular therapy. In this study, we describe the first RNAi-based, preclinical gene therapy approach for silencing a gene associated with dominant LGMD. To do this, we developed adeno-associated viral vectors (AAV6 carrying designed therapeutic microRNAs targeting mutant myotilin (MYOT, which is the underlying cause of LGMD type 1A (LGMD1A. Our best MYOT-targeted microRNA vector (called miMYOT significantly reduced mutant myotilin mRNA and soluble protein expression in muscles of LGMD1A mice (the TgT57I model both 3 and 9 months after delivery, demonstrating short- and long-term silencing effects. This MYOT gene silencing subsequently decreased deposition of MYOT-seeded intramuscular protein aggregates, which is the hallmark feature of LGMD1A. Histological improvements were accompanied by significant functional correction, as miMYOT-treated animals showed increased muscle weight and improved specific force in the gastrocnemius, which is one of the most severely affected muscles in TgT57I mice and patients with dominant myotilin mutations. These promising results in a preclinical model of LGMD1A support the further development of RNAi-based molecular therapy as a prospective treatment for LGMD1A. Furthermore, this study sets a foundation that may be refined and adapted to treat other dominant LGMD and related disorders.

Coupling Traditional Monitoring and Citizen Science to Disentangle the Invasion of Halyomorpha halys

Directory of Open Access Journals (Sweden)

Robert Malek

2018-05-01

Full Text Available The brown marmorated stink bug, Halyomorpha halys Stål (Hemiptera: Pentatomidae, is an invasive pest that has expanded its range outside of its original confinements in Eastern Asia, spreading through the United States, Canada and most of the European and Eurasian countries. The invasiveness of this agricultural and public nuisance pest is facilitated by the availability of an array of suitable hosts, an r-selected life history and the release from natural enemies in the invaded zones. Traditional monitoring methods are usually impeded by the lack of time and resources to sufficiently cover large geographical ranges. Therefore, the citizen science initiative “BugMap” was conceived to complement and assist researchers in breaking down the behavior of this invasive pest via a user-friendly, freely available mobile application. The collected data were employed to forecast its predicted distribution and to identify the areas at risk in Trentino, Northern Italy. Moreover, they permitted the uncovering of the seasonal invasion dynamics of this insect, besides providing insight into its phenological patterns, life cycle and potential management methods. Hence, the outcomes of this work emphasize the need to further integrate citizens in scientific endeavors to resolve ecological complications and reduce the gap between the public and science.

Assessment of TS-1, a thick cane mutant

International Nuclear Information System (INIS)

Shama Rao, H.K.

1979-01-01

A true breeding thick cane mutant TS-1, induced by radiations, was obtained in variety Co-419. TS-1 was found to be superior to Co-419 with respect to cane size, weight, yield and juice quality. The thick canes of TS-1 were found to be solid even at 14 months age and so also their ratoons. The tillering habit of TS-1 has a definite advantage over other varieties with respect to easy intercultural field operations. TS-1 is now being tested under various agroclimatic zones in Karnataka, Maharashtra and U.P. (auth.)

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

Forward and reverse genetics: The LORE1 retrotransposon insertion mutants

DEFF Research Database (Denmark)

Fukai, Eigo; Malolepszy, Anna; Sandal, Niels Nørgaard

2014-01-01

The endogenous Lotus retrotransposon 1 (LORE1) transposes in the germ line of Lotus japonicus plants that carry an active element. This feature of LORE1 has been exploited for generation of a large non-transgenic insertion mutant population, where insertions have been annotated using next-generat...

Interaction of a non-peptide agonist with angiotensin II AT1 receptor mutants

DEFF Research Database (Denmark)

Costa-Neto, Claudio M; Miyakawa, Ayumi A; Pesquero, João B

2002-01-01

and inositol phosphate turnover assays in COS-7 cells transiently transfected with the wild-type and mutant forms of the receptor. Mutant receptors bore modifications in the extracellular region: T88H, Y92H, G1961, G196W, and D278E. Compound L-162,313 displaced [125I]-Sar1,Leu8-AngII from the mutants G196I...... and G196W with IC50 values similar to that of the wild-type. The affinity was, however, slightly affected by the D278E mutation and more significantly by the T88H and Y92H mutations. In inositol phosphate turnover assays, the ability of L-162,313 to trigger the activation cascade was compared...... with that of angiotensin II. These assays showed that the G196W mutant reached a relative maximum activation exceeding that of the wild-type receptor; the efficacy was slightly reduced in the G1961 mutant and further reduced in the T88H, Y92H, and D278E mutants. Our data suggest that residues of the extracellular domain...

Respiration responses of a polA1 and a tif-1 mutant of Escherichia coli to far-ultraviolet irradiation

International Nuclear Information System (INIS)

Swenson, P.A.

1981-01-01

Cessation of respiration in Escherichia coli 60 min after far - ultra-violet (254 nm) irradiation is dependent upon the recA and lexA gene products and is regulated by cyclic 3', 5'-adenosine monophosphate (cAMP) and its receptor protein. Two E. coli B/r mutants were studied, polA1 and tif-1, both of which express other rec/lex functions after UV irradiation. After receiving a relatively high UV fluence, the polA1 mutant, deficient in DNA polymerase 1, showed a respiration shutoff response like the wild type cells. 5-Fluorouracil and rifampin, an RNA synthesis inhibitor, did not prevent respiration shutoff in the mutant cells as they did in the wild type cells. At lower fluences which did not shut off respiration of polA1 cells, cAMP did not cause a more complete shutoff as it did for the wild type cells. The tif-1 mutant has a modified recA protein, and when unirradiated cells are incubated at 42 0 C they form filaments, mutate, and show other rec/lex responses. This mutant did not shut off its respiration at either 30 or 42 0 C, and the response was not modified by cAMP. In an E. coli K12 strain, W3110, 52 J/m 2 UV did not shut off respiration and cAMP had no effect. (author)

ARID1B is a specific vulnerability in ARID1A-mutant cancers.

Science.gov (United States)

Helming, Katherine C; Wang, Xiaofeng; Wilson, Boris G; Vazquez, Francisca; Haswell, Jeffrey R; Manchester, Haley E; Kim, Youngha; Kryukov, Gregory V; Ghandi, Mahmoud; Aguirre, Andrew J; Jagani, Zainab; Wang, Zhong; Garraway, Levi A; Hahn, William C; Roberts, Charles W M

2014-03-01

Recent studies have revealed that ARID1A, encoding AT-rich interactive domain 1A (SWI-like), is frequently mutated across a variety of human cancers and also has bona fide tumor suppressor properties. Consequently, identification of vulnerabilities conferred by ARID1A mutation would have major relevance for human cancer. Here, using a broad screening approach, we identify ARID1B, an ARID1A homolog whose gene product is mutually exclusive with ARID1A in SWI/SNF complexes, as the number 1 gene preferentially required for the survival of ARID1A-mutant cancer cell lines. We show that loss of ARID1B in ARID1A-deficient backgrounds destabilizes SWI/SNF and impairs proliferation in both cancer cells and primary cells. We also find that ARID1A and ARID1B are frequently co-mutated in cancer but that ARID1A-deficient cancers retain at least one functional ARID1B allele. These results suggest that loss of ARID1A and ARID1B alleles cooperatively promotes cancer formation but also results in a unique functional dependence. The results further identify ARID1B as a potential therapeutic target for ARID1A-mutant cancers.

Meiotic gene conversion mutants in Saccharomyces cerevisiae. I. Isolation and characterization of PMS1-1 and PMS1-2

International Nuclear Information System (INIS)

Williamson, M.S.; Game, J.C.; Fogel, S.

1985-01-01

The PMS1 mutants, isolated on the basis of sharply elevated meiotic prototroph frequencies for two closely linked HIS4 alleles, display pleiotropic phenotypes in meiotic and mitotic cells. Two isolates carrying recessive mutations in PMS1 were characterized. They identify a function required to maintain low postmeiotic segregation (PMS) frequencies at many heterozygous sites. In addition, they are mitotic mutators. In mutant diploids, spore viability is reduced, and among survivors, gene conversion and postmeiotic segregation frequencies are increased, but reciprocal exchange frequencies are not affected. The conversion event pattern is also dramatically changed in multiply marked regions in PMS1 homozygotes. The PMS1 locus maps near MET4 on chromosome XIV. The PMS1 gene may identify an excision-resynthesis long patch mismatch correction function or a function that facilitates correction tract elongation. The PMS1 gene product may also play an important role in spontaneous mitotic mutation avoidance and correction of mismatches in heteroduplex DNA formed during spontaneous and UV-induced mitotic recombination. Based on meiotic recombination models emphasizing mismatch correction in heteroduplex DNA intermediates, this interpretation is favored, but alternative interpretations involving longer recombination intermediates in the mutants are also considered

Characterization of Brucella abortus mutant strain Δ22915, a potential vaccine candidate.

Science.gov (United States)

Bao, Yanqing; Tian, Mingxing; Li, Peng; Liu, Jiameng; Ding, Chan; Yu, Shengqing

2017-04-04

Brucellosis, caused by Brucella spp., is an important zoonosis worldwide. Vaccination is an effective strategy for protection against Brucella infection in livestock in developing countries and in wildlife in developed countries. However, current vaccine strains including S19 and RB51 are pathogenic to humans and pregnant animals, limiting their use. In this study, we constructed the Brucella abortus (B. abortus) S2308 mutant strain Δ22915, in which the putative lytic transglycosylase gene BAB_RS22915 was deleted. The biological properties of mutant strain Δ22915 were characterized and protection of mice against virulent S2308 challenge was evaluated. The mutant strain Δ22915 showed reduced survival within RAW264.7 cells and survival in vivo in mice. In addition, the mutant strain Δ22915 failed to escape fusion with lysosomes within host cells, and caused no observable pathological damage. RNA-seq analysis indicated that four genes associated with amino acid/nucleotide transport and metabolism were significantly upregulated in mutant strain Δ22915. Furthermore, inoculation of ∆22915 at 10 5 colony forming units induced effective host immune responses and long-term protection of BALB/c mice. Therefore, mutant strain ∆22915 could be used as a novel vaccine candidate in the future to protect animals against B. abortus infection.

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.

Purification and characterization of mutant miniPlasmin for thrombolytic therapy

Directory of Open Access Journals (Sweden)

Lin Xiaotao

2013-01-01

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

36 CFR 10.1 - Animals available.

Science.gov (United States)

2010-07-01

... 36 Parks, Forests, and Public Property 1 2010-07-01 2010-07-01 false Animals available. 10.1 Section 10.1 Parks, Forests, and Public Property NATIONAL PARK SERVICE, DEPARTMENT OF THE INTERIOR DISPOSAL OF CERTAIN WILD ANIMALS § 10.1 Animals available. From time to time there are surplus live elk...

Immuogenicity and safety of a natural rough mutant of Brucella suis as a vaccine for swine

Science.gov (United States)

The objective of the current study was to evaluate the safety, immunogenicity and clearance of the natural rough mutant of Brucella suis strain 353-1 (353-1) as a vaccine in domestic swine. In three studies encompassing 155 animals, pigs were inoculated with 353-1 by conjunctival (5 x 10**7 CFU), p...

Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1

Directory of Open Access Journals (Sweden)

Meijia Yang

2017-09-01

Full Text Available BRAF-V600E (1799T > A is one of the most frequently reported driver mutations in multiple types of cancers, and patients with such mutations could benefit from selectively inactivating the mutant allele. Near this mutation site, there are two TTTN and one NGG protospacer-adjacent motifs (PAMs for Cpf1 and Cas9 CRISPR nucleases, respectively. The 1799T > A substitution also leads to the occurrence of a novel NGNG PAM for the EQR variant of Cas9. We examined the editing efficacy and selectivity of Cpf1, Cas9, and EQR variant to this mutation site. Only Cpf1 demonstrated robust activity to induce specific disruption of only mutant BRAF, not wild-type sequence. Cas9 recognized and cut both normal and mutant alleles, and no obvious gene editing events were observed using EQR variant. Our results support the potential applicability of Cpf1 in precision medicine through highly specific inactivation of many other gain-of-function mutations. Keywords: Cpf1, targeted therapy, BRAF V600E

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

Science.gov (United States)

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

2002-07-01

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

Characteristics of the repair - deficient mutants 1435 plague microbe strain

International Nuclear Information System (INIS)

Temiralieva, G.A.

1977-01-01

Repair-deficient mutants 1435 A uvr - hcr - , 1435-17 uvr - hcr + and 1435-35 lon have been obtained from 1435 plague microbe strain, isolated from a large gerbil living in the Central Asian desert region. The mutants have the same cultural-morphological and enzymatic characteristics, the same need in growth factors and similar virulence determinants as the original strain, but they do not cause death of the experimental animals

Gamma ray induced mutants in Coleus

Energy Technology Data Exchange (ETDEWEB)

Vasudevan, K; Jos, J S [Central Tuber Crops Research Institute, Trivandrum, Kerala (India)

1988-07-01

The germplasm collection of Chinese potato (Coleus parviflorus Benth) contains almost no variation for yield contributing traits. The crop does not produce seeds. Treatment of underground tubers with 1 kR, 2 kR, 3 kR and 4 kR gamma rays resulted in 50 morphologically different mutants which are maintained as mutant clones. In the M{sub 1}V{sub 1} generation, suspected mutant sprouts, were carefully removed and grown separately. The most interesting mutant types are the following: (i) erect mutant with spoon shaped light green leaves, 30 cm long inflorescences against 20 cm in the control, cylindrical tubers measuring ca. 7.0 cm long and 3 cm girth against 4 cm and 2.5 cm in the control (ii) early mutants 1 and 2, one having less leaf serration, the other having light green small leaves and dwarf type (iii) fleshy leaf mutant, dark green, thick and smooth leaves. Control plants spread almost in 1 m{sup 2} area and bear tubers from the nodes of branches. In the early mutants tuber formation is mainly restricted to the base of the plant, which makes harvest easier. The crop usually matures within 150 - 160 days, the early mutants are ready for harvest 100 days after planting. As the mutants are less spreading, the yield could be increased by closer spacing.

Gamma ray induced mutants in Coleus

International Nuclear Information System (INIS)

Vasudevan, K.; Jos, J.S.

1988-01-01

The germplasm collection of Chinese potato (Coleus parviflorus Benth) contains almost no variation for yield contributing traits. The crop does not produce seeds. Treatment of underground tubers with 1 kR, 2 kR, 3 kR and 4 kR gamma rays resulted in 50 morphologically different mutants which are maintained as mutant clones. In the M 1 V 1 generation, suspected mutant sprouts, were carefully removed and grown separately. The most interesting mutant types are the following: (i) erect mutant with spoon shaped light green leaves, 30 cm long inflorescences against 20 cm in the control, cylindrical tubers measuring ca. 7.0 cm long and 3 cm girth against 4 cm and 2.5 cm in the control (ii) early mutants 1 and 2, one having less leaf serration, the other having light green small leaves and dwarf type (iii) fleshy leaf mutant, dark green, thick and smooth leaves. Control plants spread almost in 1 m 2 area and bear tubers from the nodes of branches. In the early mutants tuber formation is mainly restricted to the base of the plant, which makes harvest easier. The crop usually matures within 150 - 160 days, the early mutants are ready for harvest 100 days after planting. As the mutants are less spreading, the yield could be increased by closer spacing

A new mutant gene su-1 in corn obtained by irradiation with low doses of gamma rays

International Nuclear Information System (INIS)

Diaconu, P.

1993-01-01

This paper provides a description of a sugar corn mutant obtained by irradiation of wetted kernels of Romanesc de Studina variety with low doses of gamma rays (300 R). This mutant influences the structure of the endosperm similarly to the su-1 genes developed spontaneously which resulted in the corn variety Zea mays saccharata thousands of years ago. Although the mutant is a multiple allele of the su-1 locus in chromosome IV it differs widely from the spontaneous mutant. The length of the ears is much reduced, varying between 4 and 6 cm, with numbers of kernels per ear varying between 45 and 72. Attempts to improve the cob size and the number of kernels by breeding and propagation in an insulated area led to no result. Crossing the mutants with the sugar hybrid Delicious resulted in sugar type progeny which confirms the common position of the mutant gene induced by irradiation and the spontaneous su-1 gene. The progenies of sugar mutant x Delicious are 38-43 % lower in cob vigor and 36-46% lower in kernel number. (author). 2 figs, 2 tab., 16 refs

50 CFR 30.1 - Surplus range animals.

Science.gov (United States)

2010-10-01

... 50 Wildlife and Fisheries 6 2010-10-01 2010-10-01 false Surplus range animals. 30.1 Section 30.1... NATIONAL WILDLIFE REFUGE SYSTEM RANGE AND FERAL ANIMAL MANAGEMENT Range Animals § 30.1 Surplus range animals. Range animals on fenced wildlife refuge areas, including buffalo and longhorn cattle, determined...

Genetics of leaf rust-resistant mutant WH 147-LM-1 in hexaploid wheat variety WH 147

International Nuclear Information System (INIS)

Reddy, V.R.K.; Viswanathan, P.

1999-01-01

By applying gamma rays, EMS and their combination in hexaploid wheat variety WH 147, a total of 20 mutants (0.0226%) exhibiting complete leaf rust resistance were isolated from segregating M2 rows.When one of the rust-resistant mutants, WH 147-LM-1 was crossed with the universally susceptible, suggesting that the mutant character is controlled by one dominant gene and one recessive gene.The F2 plants derived by crossing the mutant WH 147-LM with seven near-isogenic wheat lines showed segregation for susceptibility, indicating that the mutant character was indeed generated through induced mutations

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

Science.gov (United States)

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

2017-01-01

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

ALS-causing profilin-1-mutant forms a non-native helical structure in membrane environments.

Science.gov (United States)

Lim, Liangzhong; Kang, Jian; Song, Jianxing

2017-11-01

Despite having physiological functions completely different from superoxide dismutase 1 (SOD1), profilin 1 (PFN1) also carries mutations causing amyotrophic lateral sclerosis (ALS) with a striking similarity to that triggered by SOD1 mutants. Very recently, the C71G-PFN1 has been demonstrated to cause ALS by a gain of toxicity and the acceleration of motor neuron degeneration preceded the accumulation of its aggregates. Here by atomic-resolution NMR determination of conformations and dynamics of WT-PFN1 and C71G-PFN1 in aqueous buffers and in membrane mimetics DMPC/DHPC bicelle and DPC micelle, we deciphered that: 1) the thermodynamic destabilization by C71G transforms PFN1 into coexistence with the unfolded state, which is lacking of any stable tertiary/secondary structures as well as restricted ps-ns backbone motions, thus fundamentally indistinguishable from ALS-causing SOD1 mutants. 2) Most strikingly, while WT-PFN1 only weakly interacts with DMPC/DHPC bicelle without altering the native structure, C71G-PFN1 acquires abnormal capacity in strongly interacting with DMPC/DHPC bicelle and DPC micelle, energetically driven by transforming the highly disordered unfolded state into a non-native helical structure, similar to what has been previously observed on ALS-causing SOD1 mutants. Our results imply that one potential mechanism for C71G-PFN1 to initiate ALS might be the abnormal interaction with membranes as recently established for SOD1 mutants. Copyright © 2017 Elsevier B.V. All rights reserved.

Multiple antibiotic susceptibility of polyphosphate kinase mutants (ppk1 and ppk2 from Pseudomonas aeruginosa PAO1 as revealed by global phenotypic analysis

Directory of Open Access Journals (Sweden)

Javiera Ortiz-Severín

2015-01-01

Full Text Available BACKGROUND: Pseudomonas aeruginosa is known to be a multidrug resistant opportunistic pathogen. Particularly, P. aeruginosa PAO1 polyphosphate kinase mutant (ppk1 is deficient in motility, quorum sensing, biofilm formation and virulence FINDINGS: By using Phenotypic Microarrays (PM we analyzed near 2000 phenotypes of P. aeruginosa PAO1 polyP kinase mutants (ppk1 and ppk2. We found that both ppk mutants shared most of the phenotypic changes and interestingly many of them related to susceptibility toward numerous and different type of antibiotics such as Ciprofloxacin, Chloramphenicol and Rifampicin CONCLUSIONS: Combining the fact that ppk1 mutants have reduced virulence and are more susceptible to antibiotics, polyP synthesis and particularly PPK1, is a good target for the design of molecules with anti-virulence and anti-persistence properties.

A Medicago truncatula Tobacco Retrotransposon Insertion Mutant Collection with Defects in Nodule Development and Symbiotic Nitrogen Fixation1[W][OA

Science.gov (United States)

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

2012-01-01

A Tnt1-insertion mutant population of Medicago truncatula ecotype R108 was screened for defects in nodulation and symbiotic nitrogen fixation. Primary screening of 9,300 mutant lines yielded 317 lines with putative defects in nodule development and/or nitrogen fixation. Of these, 230 lines were rescreened, and 156 lines were confirmed with defective symbiotic nitrogen fixation. Mutants were sorted into six distinct phenotypic categories: 72 nonnodulating mutants (Nod−), 51 mutants with totally ineffective nodules (Nod+ Fix−), 17 mutants with partially ineffective nodules (Nod+ Fix+/−), 27 mutants defective in nodule emergence, elongation, and nitrogen fixation (Nod+/− Fix−), one mutant with delayed and reduced nodulation but effective in nitrogen fixation (dNod+/− Fix+), and 11 supernodulating mutants (Nod++Fix+/−). A total of 2,801 flanking sequence tags were generated from the 156 symbiotic mutant lines. Analysis of flanking sequence tags revealed 14 insertion alleles of the following known symbiotic genes: NODULE INCEPTION (NIN), DOESN’T MAKE INFECTIONS3 (DMI3/CCaMK), ERF REQUIRED FOR NODULATION, and SUPERNUMERARY NODULES (SUNN). In parallel, a polymerase chain reaction-based strategy was used to identify Tnt1 insertions in known symbiotic genes, which revealed 25 additional insertion alleles in the following genes: DMI1, DMI2, DMI3, NIN, NODULATION SIGNALING PATHWAY1 (NSP1), NSP2, SUNN, and SICKLE. Thirty-nine Nod− lines were also screened for arbuscular mycorrhizal symbiosis phenotypes, and 30 mutants exhibited defects in arbuscular mycorrhizal symbiosis. Morphological and developmental features of several new symbiotic mutants are reported. The collection of mutants described here is a source of novel alleles of known symbiotic genes and a resource for cloning novel symbiotic genes via Tnt1 tagging. PMID:22679222

Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia

Directory of Open Access Journals (Sweden)

Chao Liu

2015-08-01

Full Text Available Apolipoprotein C-II (APOC2 is an obligatory activator of lipoprotein lipase. Human patients with APOC2 deficiency display severe hypertriglyceridemia while consuming a normal diet, often manifesting xanthomas, lipemia retinalis and pancreatitis. Hypertriglyceridemia is also an important risk factor for development of cardiovascular disease. Animal models to study hypertriglyceridemia are limited, with no Apoc2-knockout mouse reported. To develop a genetic model of hypertriglyceridemia, we generated an apoc2 mutant zebrafish characterized by the loss of Apoc2 function. apoc2 mutants show decreased plasma lipase activity and display chylomicronemia and severe hypertriglyceridemia, which closely resemble the phenotype observed in human patients with APOC2 deficiency. The hypertriglyceridemia in apoc2 mutants is rescued by injection of plasma from wild-type zebrafish or by injection of a human APOC2 mimetic peptide. Consistent with a previous report of a transient apoc2 knockdown, apoc2 mutant larvae have a minor delay in yolk consumption and angiogenesis. Furthermore, apoc2 mutants fed a normal diet accumulate lipid and lipid-laden macrophages in the vasculature, which resemble early events in the development of human atherosclerotic lesions. In addition, apoc2 mutant embryos show ectopic overgrowth of pancreas. Taken together, our data suggest that the apoc2 mutant zebrafish is a robust and versatile animal model to study hypertriglyceridemia and the mechanisms involved in the pathogenesis of associated human diseases.

Genetic analysis of rice semidwarf mutant Tad-M-1

International Nuclear Information System (INIS)

Wang Naiyuan; Yang Rencui

1995-01-01

This paper dealed with the inheritance of the rice semidwarf of Tad-M-,a mutant line bred from traditional indica rice Variety Tadukan by radiation. The results indicated that semidwarf of Tad-M-1 was controlled by one pair of recessive gene, which was nonallelic to sd-1 gene of variety Aijiaonante and sd-g gene of variety Xinguiai and allelic to the semidwarf gene of Yunnan japonica variety Xueheaizao and Sichuan indica variety Yizila.The possible uses of Tad-M-1 in rice breeding was also discussed

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

Science.gov (United States)

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

2013-05-10

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

Fitness ranking of individual mutants drives patterns of epistatic interactions in HIV-1.

Directory of Open Access Journals (Sweden)

Javier P Martínez

Full Text Available Fitness interactions between mutations, referred to as epistasis, can strongly impact evolution. For RNA viruses and retroviruses with their high mutation rates, epistasis may be particularly important to overcome fitness losses due to the accumulation of deleterious mutations and thus could influence the frequency of mutants in a viral population. As human immunodeficiency virus type 1 (HIV-1 resistance to azidothymidine (AZT requires selection of sequential mutations, it is a good system to study the impact of epistasis. Here we present a thorough analysis of a classical AZT-resistance pathway (the 41-215 cluster of HIV-1 variants by fitness measurements in single round infection assays covering physiological drug concentrations ex vivo. The sign and value of epistasis varied and did not predict the epistatic effect on the mutant frequency. This complex behavior is explained by the fitness ranking of the variants that strongly depends on environmental factors, i.e., the presence and absence of drugs and the host cells used. Although some interactions compensate fitness losses, the observed small effect on the relative mutant frequencies suggests that epistasis might be inefficient as a buffering mechanism for fitness losses in vivo. While the use of epistasis-based hypotheses to make general assumptions on the evolutionary dynamics of viral populations is appealing, our data caution their interpretation without further knowledge on the characteristics of the viral mutant spectrum under different environmental conditions.

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

Science.gov (United States)

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

2010-03-01

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

Waiting times for the appearance of cytotoxic T-lymphocyte escape mutants in chronic HIV-1 infection

International Nuclear Information System (INIS)

Liu Yi; Mullins, James I.; Mittler, John E.

2006-01-01

The failure of HIV-1 to escape at some cytotoxic T-lymphocyte (CTL) epitopes has generally been explained in terms of viral fitness costs or ineffective or attenuated CTL responses. Relatively little attention has been paid to the evolutionary time required for escape mutants to be detected. This time is significantly affected by selection, mutation rates, the presence of other advantageous mutations, and the effective population size of HIV-1 in vivo (typically estimated to be ∼10 3 in chronically infected patients, though one study has estimated it to be ∼10 5 ). Here, we use a forward simulator with experimentally estimated HIV-1 parameters to show that these delays can be substantial. For an effective population size of 10 3 , even highly advantageous mutants (s = 0.5) may not be detected for a couple of years in chronically infected patients, while moderately advantageous escape mutants (s = 0.1) may not be detected for up to 10 years. Even with an effective population size of 10 5 , a moderately advantageous escape mutant (s = 0.1) may not be detected in the population within 2 years if it has to compete with other selectively advantageous mutants. Stochastic evolutionary forces, therefore, in addition to viral fitness costs and ineffective or attenuated CTL responses, must be taken into account when assessing the selection of CTL escape mutations

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

Pilot study of large-scale production of mutant pigs by ENU mutagenesis.

Science.gov (United States)

Hai, Tang; Cao, Chunwei; Shang, Haitao; Guo, Weiwei; Mu, Yanshuang; Yang, Shulin; Zhang, Ying; Zheng, Qiantao; Zhang, Tao; Wang, Xianlong; Liu, Yu; Kong, Qingran; Li, Kui; Wang, Dayu; Qi, Meng; Hong, Qianlong; Zhang, Rui; Wang, Xiupeng; Jia, Qitao; Wang, Xiao; Qin, Guosong; Li, Yongshun; Luo, Ailing; Jin, Weiwu; Yao, Jing; Huang, Jiaojiao; Zhang, Hongyong; Li, Menghua; Xie, Xiangmo; Zheng, Xuejuan; Guo, Kenan; Wang, Qinghua; Zhang, Shibin; Li, Liang; Xie, Fei; Zhang, Yu; Weng, Xiaogang; Yin, Zhi; Hu, Kui; Cong, Yimei; Zheng, Peng; Zou, Hailong; Xin, Leilei; Xia, Jihan; Ruan, Jinxue; Li, Hegang; Zhao, Weiming; Yuan, Jing; Liu, Zizhan; Gu, Weiwang; Li, Ming; Wang, Yong; Wang, Hongmei; Yang, Shiming; Liu, Zhonghua; Wei, Hong; Zhao, Jianguo; Zhou, Qi; Meng, Anming

2017-06-22

N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research.

Differential calcium sensitivity in NaV 1.5 mixed syndrome mutants.

Science.gov (United States)

Abdelsayed, Mena; Baruteau, Alban-Elouen; Gibbs, Karen; Sanatani, Shubhayan; Krahn, Andrew D; Probst, Vincent; Ruben, Peter C

2017-09-15

SCN5a mutations may express gain-of-function (Long QT Syndrome-3), loss-of-function (Brugada Syndrome 1) or both (mixed syndromes), depending on the mutation and environmental triggers. One such trigger may be an increase in cytosolic calcium, accompanying exercise. Many mixed syndromes mutants, including ∆KPQ, E1784K, 1795insD and Q1909R, are found in calcium-sensitive regions. Elevated cytosolic calcium attenuates gain-of-function properties in ∆KPQ, 1795insD and Q1909R, but not in E1784K. By contrast, elevated cytosolic calcium further exacerbates gain-of-function in E1784K by destabilizing slow inactivation. Action potential modelling, using a modified O'Hara Rudy model, suggests that elevated heart rate rescues action potential duration in ∆KPQ, 1795insD and Q1909R, but not in E1784K. Action potential simulations suggest that E1784K carriers have an increased intracellular sodium-to-calcium ratio under bradycardia and tachycardia conditions. Elevated cytosolic calcium, which is common during high heart rates, ameliorates or exacerbates the mixed syndrome phenotype depending on the genetic signature. Inherited arrhythmias may arise from mutations in the gene for SCN5a, which encodes the cardiac voltage-gated sodium channel, Na V 1.5. Mutants in Na V 1.5 result in Brugada Syndrome (BrS1), Long-QT Syndrome (LQT3) or mixed syndromes (an overlap of BrS1/LQT3). Exercise is a potential arrhythmogenic trigger in mixed syndromes. We aimed to determine the effects of elevated cytosolic calcium, which is common during exercise, in mixed syndrome Na V 1.5 mutants. We used whole-cell patch clamp to assess the biophysical properties of Na V 1.5 wild-type (WT), ∆KPQ, E1784K, 1795insD and Q1909R mutants in human embryonic kidney 293 cells transiently transfected with the Na V 1.5 α subunit (WT or mutants), β1 subunit and enhanced green fluorescent protein. Voltage-dependence and kinetics were measured at cytosolic calcium levels of approximately 0, 500 and 2500�

[Biofilm Formation by the Nonflagellated flhB1 Mutant of Azospirillum brasilense Sp245].

Science.gov (United States)

Shelud'ko, A V; Filip'echeva, Yu A; Shumiliva, E M; Khlebtsov, B N; Burov, A M; Petrova, L P; Katsy, E I

2015-01-01

Azospirillum brasilense Sp245 with mixed flagellation are able to form biofilms on various surfaces. A nonflagellated mutant of this strain with inactivated chromosomal copy of the flhB gene (flhB1) was shown to exhibit specific traits at the later stages of biofilm formation on a hydrophilic (glass) surface. Mature biofilms of the flhB1::Omegon-Km mutant Sp245.1063 were considerably thinner than those of the parent strain Sp245. The biofilms of the mutant were more susceptible to the forces of hydrodynamic shear. A. brasilense Sp245 cells in biofilms were not found to possess lateral flagella. Cells with polar flagella were, however, revealed by atomic force microscopy of mature native biofilms of strain Sp245. Preservation of a polar flagellum (probably nonmotile) on the cells of A. brasilense Sp245 may enhance the biofilm stability.

Isolation of two independent allyl alcohol resistant Adh-1 null mutants following selection of pollen and seeds.

NARCIS (Netherlands)

Wisman, E.; Ramanna, M.S.; Zabel, P.

1993-01-01

The Adh-1 null mutant (B15-1-8) isolated previously was used to establish conditions that allow the selection of ADH-deficient pollen grains and seeds of tomato. New Adh-1 null mutants were then selected among the progenies derived from crosses between the genetically unstable tomato lines Yvms,

Isolation of transformation-defective, replication-nondefective early region 1B mutants of adenovirus 12

International Nuclear Information System (INIS)

Fukui, Y.; Saito, I.; Shiroki, K.; Shimojo, H.

1984-01-01

The authors isolated three adenovirus 12 early region 1B mutants (in205B, in205C, and dl205) by ligation of the cleaved DNA-protein complex and transfection of human embryo kidney cells with the ligation products. These mutants could replicate efficiently in human embryo kidney or KB cells but showed markedly reduced transforming capacities both in vitro and in vivo. In cells infected with the mutants, the early region 1B gene was transcribed efficiently. In cells infected with in205B, the products corresponding to the early region 1B-coded 19,000-molecular-weight polypeptide was detected by in vitro translation but not immunoprecipitated extract of labeled cells. In cells infected with in205C or dl205, the products corresponding to the same polypeptide were not detected by either in vitro translation or immunoprecipitation of labeled cell extracts. The results suggest that the 19,000-molecular-weight polypeptide encoded by early region 1B is required for cell transformation but not for viral propagation

Bivalent vaccination against pneumonic pasteurellosis in domestic sheep and goats with modified-live in-frame lktA deletion mutants of Mannheimia haemolytica.

Science.gov (United States)

Briggs, Robert E; Hauglund, Melissa J; Maheswaran, Samuel K; Tatum, Fred M

2013-11-01

A temperature-sensitive shuttle vector, pBB80C, was utilized to generate in-frame deletion mutants of the leukotoxin structural gene (lktA) of Mannheimia haemolytica serotypes 1, 2, 5, 6, 7, 8, 9, and 12. Culture supernatants from the mutants contained a truncated protein with an approximate molecular weight of 66 kDa which was reactive to anti-leukotoxin monoclonal antibody. No protein reactive to anti-LktA monoclonal antibody was detected at the molecular weight 100-105 kDa of native LktA. Sheep and goats vaccinated intramuscularly with a mixture of serotypes 5 and 6 mutants were resistant to virulent challenge with a mixture of the wild-type parent strains. These vaccinates responded serologically to both vaccine serotypes and exhibited markedly-reduced lung lesion volume and pulmonary infectious load compared to control animals. Control animals yielded a mixture of serotypes from lung lobes, but the proportion even within an individual animal varied widely from 95% serotype 5-95% serotype 6. Cultures recovered from liver were homogeneous, but two animals yielded serotype 5 and the other two yielded serotype 6 in pure culture. Published by Elsevier Ltd.

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

Science.gov (United States)

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

2014-02-10

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

Effect of Potent γ-Secretase Modulator in Human Neurons Derived From Multiple Presenilin 1–Induced Pluripotent Stem Cell Mutant Carriers

Science.gov (United States)

Liu, Qing; Waltz, Shannon; Woodruff, Grace; Ouyang, Joe; Israel, Mason A.; Herrera, Cheryl; Sarsoza, Floyd; Tanzi, Rudolph E.; Koo, Edward H.; Ringman, John M.; Goldstein, Lawrence S. B.; Wagner, Steven L.; Yuan, Shauna H.

2015-01-01

Importance Although considerable effort has been expended developing drug candidates for Alzheimer disease, none have yet succeeded owing to the lack of efficacy or to safety concerns. One potential shortcoming of current approaches to Alzheimer disease drug discovery and development is that they rely primarily on transformed cell lines and animal models that substantially overexpress wild-type or mutant proteins. It is possible that drug development failures thus far are caused in part by the limits of these approaches, which do not accurately reveal how drug candidates will behave in naive human neuronal cells. Objective To analyze purified neurons derived from human induced pluripotent stem cells from patients carrying 3 different presenilin 1 (PS1) mutations and nondemented control individuals in the absence of any overexpression. We tested the efficacy of γ-secretase inhibitor and γ-secretase modulator (GSM) in neurons derived from both normal control and 3 PS1 mutations (A246E, H163R, and M146L). Design, Setting, and Participants Adult human skin biopsies were obtained from volunteers at the Alzheimer Disease Research Center, University of California, San Diego. Cell cultures were treated with γ-secretase inhibitor or GSM. Comparisons of total β-amyloid (Aβ) and Aβ peptides 38, 40, and 42 in the media were made between vehicle- vs drug-treated cultures. Main Outcomes and Measures Soluble Aβ levels in the media were measured by enzyme-linked immunosorbent assay. Results As predicted, mutant PS1 neurons exhibited an elevated Aβ42:Aβ40 ratio (P <.05) at the basal state as compared with the nondemented control neurons. Treatment with a potent non–nonsteroidal anti-inflammatory druglike GSM revealed a new biomarker signature that differs from all previous cell types and animals tested. This new signature was the same in both the mutant and control neurons and consisted of a reduction in Aβ42, Aβ40, and Aβ38 and in the Aβ42:Aβ40 ratio, with no

Comparative metabolic profiling of mce1 operon mutant vs wild-type Mycobacterium tuberculosis strains.

Science.gov (United States)

Queiroz, Adriano; Medina-Cleghorn, Daniel; Marjanovic, Olivera; Nomura, Daniel K; Riley, Lee W

2015-11-01

Mycobacterium tuberculosis disrupted in a 13-gene operon (mce1) accumulates free mycolic acids (FM) in its cell wall and causes accelerated death in mice. Here, to more comprehensively analyze differences in their cell wall lipid composition, we used an untargeted metabolomics approach to compare the lipid profiles of wild-type and mce1 operon mutant strains. By liquid chromatography-mass spectrometry, we identified >400 distinct lipids significantly altered in the mce1 mutant compared to wild type. These lipids included decreased levels of saccharolipids and glycerophospholipids, and increased levels of alpha-, methoxy- and keto mycolic acids (MA), and hydroxyphthioceranic acid. The mutant showed reduced expression of mmpL8, mmpL10, stf0, pks2 and papA2 genes involved in transport and metabolism of lipids recognized to induce proinflammatory response; these lipids were found to be decreased in the mutant. In contrast, the transcripts of mmpL3, fasI, kasA, kasB, acpM and RV3451 involved in MA transport and metabolism increased; MA inhibits inflammatory response in macrophages. Since the mce1 operon is known to be regulated in intracellular M. tuberculosis, we speculate that the differences we observed in cell wall lipid metabolism and composition may affect host response to M. tuberculosis infection and determine the clinical outcome of such an infection. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Discovery of AG-120 (Ivosidenib): A First-in-Class Mutant IDH1 Inhibitor for the Treatment of IDH1 Mutant Cancers.

Science.gov (United States)

Popovici-Muller, Janeta; Lemieux, René M; Artin, Erin; Saunders, Jeffrey O; Salituro, Francesco G; Travins, Jeremy; Cianchetta, Giovanni; Cai, Zhenwei; Zhou, Ding; Cui, Dawei; Chen, Ping; Straley, Kimberly; Tobin, Erica; Wang, Fang; David, Muriel D; Penard-Lacronique, Virginie; Quivoron, Cyril; Saada, Véronique; de Botton, Stéphane; Gross, Stefan; Dang, Lenny; Yang, Hua; Utley, Luke; Chen, Yue; Kim, Hyeryun; Jin, Shengfang; Gu, Zhiwei; Yao, Gui; Luo, Zhiyong; Lv, Xiaobing; Fang, Cheng; Yan, Liping; Olaharski, Andrew; Silverman, Lee; Biller, Scott; Su, Shin-San M; Yen, Katharine

2018-04-12

Somatic point mutations at a key arginine residue (R132) within the active site of the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) confer a novel gain of function in cancer cells, resulting in the production of d-2-hydroxyglutarate (2-HG), an oncometabolite. Elevated 2-HG levels are implicated in epigenetic alterations and impaired cellular differentiation. IDH1 mutations have been described in an array of hematologic malignancies and solid tumors. Here, we report the discovery of AG-120 (ivosidenib), an inhibitor of the IDH1 mutant enzyme that exhibits profound 2-HG lowering in tumor models and the ability to effect differentiation of primary patient AML samples ex vivo. Preliminary data from phase 1 clinical trials enrolling patients with cancers harboring an IDH1 mutation indicate that AG-120 has an acceptable safety profile and clinical activity.

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

Science.gov (United States)

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

2018-06-01

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

Early Postnatal but Not Late Adult Neurogenesis Is Impaired in the Pitx3-Mutant Animal Model of Parkinson's Disease

Directory of Open Access Journals (Sweden)

Moritz D. Brandt

2017-08-01

Full Text Available The generation of new neurons in the adult dentate gyrus has functional implications for hippocampal formation. Reduced hippocampal neurogenesis has been described in various animal models of hippocampal dysfunction such as dementia and depression, which are both common non-motor-symptoms of Parkinson's disease (PD. As dopamine plays an important role in regulating precursor cell proliferation, the loss of dopaminergic neurons in the substantia nigra (SN in PD may be related to the reduced neurogenesis observed in the neurogenic regions of the adult brain: subventricular zone (SVZ and dentate gyrus (DG. Here we examined adult hippocampal neurogenesis in the Pitx3-mutant mouse model of PD (aphakia mice, which phenotypically shows a selective embryonic degeneration of dopamine neurons within the SN and to a smaller extent in the ventral tegmental area (VTA. Proliferating cells were labeled with BrdU in aphakia mice and healthy controls from 3 to 42 weeks of age. Three weeks old mutant mice showed an 18% reduction of proliferating cells in the DG and of 26% in the SVZ. Not only proliferation but also the number of new neurons was impaired in young aphakia mice resulting in 33% less newborn cells 4 weeks after BrdU-labeling. Remarkably, however, the decline in the number of proliferating cells in the neurogenic regions vanished in older animals (8–42 weeks indicating that aging masks the effect of dopamine depletion on adult neurogenesis. Region specific reduction in precursor cells proliferation correlated with the extent of dopaminergic degeneration in mesencephalic subregions (VTA and SN, which supports the theory of age- and region-dependent regulatory effects of dopaminergic projections. Physiological stimulation of adult neurogenesis by physical activity (wheel running almost doubled the number of proliferating cells in the dentate gyrus of 8 weeks old aphakia mice to a number comparable to that of wild-type mice, abolishing the slight

Conservation of Salmonella infection mechanisms in plants and animals.

Directory of Open Access Journals (Sweden)

Adam Schikora

Full Text Available Salmonella virulence in animals depends on effectors injected by Type III Secretion Systems (T3SSs. In this report we demonstrate that Salmonella mutants that are unable to deliver effectors are also compromised in infection of Arabidopsis thaliana plants. Transcriptome analysis revealed that in contrast to wild type bacteria, T3SS mutants of Salmonella are compromised in suppressing highly conserved Arabidopsis genes that play a prominent role during Salmonella infection of animals. We also found that Salmonella originating from infected plants are equally virulent for human cells and mice. These results indicate a high degree of conservation in the defense and infection mechanism of animal and plant hosts during Salmonella infection.

Fine Mapping and Cloning of Leafy Head Mutant Gene pla1-5 in Rice

Directory of Open Access Journals (Sweden)

Gong-neng FENG

2013-09-01

Full Text Available We identified a leafy head mutant pla1-5 (plastochron 1-5 from the progeny of japonica rice cultivar Taipei 309 treated with 60Co-γ ray irradiation. The pla1-5 mutant has a dwarf phenotype and small leaves. Compared with its wild type, pla1-5 has more leaves and fewer tillers, and it fails to produce normal panicles at the maturity stage. Genetic analysis showed that the pla1-5 phenotype is controlled by a single recessive nuclear gene. Using the map-based cloning strategy, we narrowed down the location of the target gene to a 58-kb region between simple sequence repeat markers CHR1027 and CHR1030 on the long arm of chromosome 10. The target gene cosegregated with molecular markers CHR1028 and CHR1029. There were five predicted genes in the mapped region. The results from sequencing analysis revealed that there was one base deletion in the first exon of LOC_Os10g26340 encoding cytochrome P450 CYP78A11 in the pla1-5 mutant, which might result in a downstream frame shift and premature termination. These results suggest that the P450 CYP78A11 gene is the candidate gene of PLA1-5.

Root hair mutants of barley

International Nuclear Information System (INIS)

Engvild, K.C.; Rasmussen, K.

2005-01-01

Barley mutants without root hairs or with short or reduced root hairs were isolated among M 2 seeds of 'Lux' barley (Hordeum vulgare L.) after acidified sodium azide mutagenesis. Root hair mutants are investigated intensively in Arabidopsis where about 40 genes are known. A few root hair mutants are known in maize, rice, barley and tomato. Many plants without root hairs grow quite well with good plant nutrition, and mutants have been used for investigations of uptake of strongly bound nutrients like phosphorus, iron, zinc and silicon. Seed of 'Lux' barley (Sejet Plant Breeding, Denmark) were soaked overnight, and then treated with 1.5-millimolarsodium azide in 0.1 molar sodium phosphate buffer, pH 3, for 2.5 hours according to the IAEA Manual on Mutation Breeding (2nd Ed.). After rinsing in tap water and air-drying, the M 2 seeds were sown in the field the same day. Spikes, 4-6 per M 1 plant, were harvested. The mutation frequency was similar to that obtained with other barley cultivars from which low-phytate mutants were isolated [5]. Seeds were germinated on black filter paper in tap water for 3 or 4 days before scoring for root hair mutants

Gr and hp-1 tomato mutants unveil unprecedented interactions between arbuscular mycorrhizal symbiosis and fruit ripening.

Science.gov (United States)

Chialva, Matteo; Zouari, Inès; Salvioli, Alessandra; Novero, Mara; Vrebalov, Julia; Giovannoni, James J; Bonfante, Paola

2016-07-01

Systemic responses to an arbuscular mycorrhizal fungus reveal opposite phenological patterns in two tomato ripening mutants depending whether ethylene or light reception is involved. The availability of tomato ripening mutants has revealed many aspects of the genetics behind fleshy fruit ripening, plant hormones and light signal reception. Since previous analyses revealed that arbuscular mycorrhizal symbiosis influences tomato berry ripening, we wanted to test the hypothesis that an interplay might occur between root symbiosis and fruit ripening. With this aim, we screened seven tomato mutants affected in the ripening process for their responsiveness to the arbuscular mycorrhizal fungus Funneliformis mosseae. Following their phenological responses we selected two mutants for a deeper analysis: Green ripe (Gr), deficient in fruit ethylene perception and high-pigment-1 (hp-1), displaying enhanced light signal perception throughout the plant. We investigated the putative interactions between ripening processes, mycorrhizal establishment and systemic effects using biochemical and gene expression tools. Our experiments showed that both mutants, notwithstanding a normal mycorrhizal phenotype at root level, exhibit altered arbuscule functionality. Furthermore, in contrast to wild type, mycorrhization did not lead to a higher phosphate concentration in berries of both mutants. These results suggest that the mutations considered interfere with arbuscular mycorrhiza inducing systemic changes in plant phenology and fruits metabolism. We hypothesize a cross talk mechanism between AM and ripening processes that involves genes related to ethylene and light signaling.

Structure prediction and activity analysis of human heme oxygenase-1 and its mutant.

Science.gov (United States)

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

2004-08-15

To predict wild human heme oxygenase-1 (whHO-1) and hHO-1 His25Ala mutant (delta hHO-1) structures, to clone and express them and analyze their activities. Swiss-PdbViewer and Antheprot 5.0 were used for the prediction of structure diversity and physical-chemical changes between wild and mutant hHO-1. hHO-1 His25Ala mutant cDNA was constructed by site-directed mutagenesis in two plasmids of E. coli DH5alpha. Expression products were purified by ammonium sulphate precipitation and Q-Sepharose Fast Flow column chromatography, and their activities were measured. rHO-1 had the structure of a helical fold with the heme sandwiched between heme-heme oxygenase-1 helices. Bond angle, dihedral angle and chemical bond in the active pocket changed after Ala25 was replaced by His25, but Ala25 was still contacting the surface and the electrostatic potential of the active pocket was negative. The mutated enzyme kept binding activity to heme. Two vectors pBHO-1 and pBHO-1(M) were constructed and expressed. Ammonium sulphate precipitation and column chromatography yielded 3.6-fold and 30-fold higher purities of whHO-1, respectively. The activity of delta hHO-1 was reduced 91.21% after mutation compared with whHO-1. Proximal His25 ligand is crucial for normal hHO-1 catalytic activity. delta hHO-1 is deactivated by mutation but keeps the same binding site as whHO-1. delta hHO-1 might be a potential inhibitor of whHO-1 for preventing neonatal hyperbilirubinemia.

An ALS-Associated Mutant SOD1 Rapidly Suppresses KCNT1 (Slack) Na+-Activated K+ Channels in Aplysia Neurons.

Science.gov (United States)

Zhang, Yalan; Ni, Weiming; Horwich, Arthur L; Kaczmarek, Leonard K

2017-02-22

Mutations that alter levels of Slack (KCNT1) Na + -activated K + current produce devastating effects on neuronal development and neuronal function. We now find that Slack currents are rapidly suppressed by oligomers of mutant human Cu/Zn superoxide dismutase 1 (SOD1), which are associated with motor neuron toxicity in an inherited form of amyotrophic lateral sclerosis (ALS). We recorded from bag cell neurons of Aplysia californica , a model system to study neuronal excitability. We found that injection of fluorescent wild-type SOD1 (wt SOD1YFP) or monomeric mutant G85R SOD1YFP had no effect on net ionic currents measured under voltage clamp. In contrast, outward potassium currents were significantly reduced by microinjection of mutant G85R SOD1YFP that had been preincubated at 37°C or of cross-linked dimers of G85R SOD1YFP. Reduction of potassium current was also seen with multimeric G85R SOD1YFP of ∼300 kDa or >300 kDa that had been cross-linked. In current clamp recordings, microinjection of cross-linked 300 kDa increased excitability by depolarizing the resting membrane potential, and decreasing the latency of action potentials triggered by depolarization. The effect of cross-linked 300 kDa on potassium current was reduced by removing Na + from the bath solution, or by knocking down levels of Slack using siRNA. It was also prevented by pharmacological inhibition of ASK1 (apoptosis signal-regulating kinase 1) or of c-Jun N-terminal kinase, but not by an inhibitor of p38 mitogen-activated protein kinase. These results suggest that soluble mutant SOD1 oligomers rapidly trigger a kinase pathway that regulates the activity of Na + -activated K + channels in neurons. SIGNIFICANCE STATEMENT Slack Na + -activated K + channels (KCNT1, K Na 1.1) regulate neuronal excitability but are also linked to cytoplasmic signaling pathways that control neuronal protein translation. Mutations that alter the amplitude of these currents have devastating effects on neuronal

Genetic fingerprinting of mutant rose cultivars

Energy Technology Data Exchange (ETDEWEB)

Kumar, S; Prasad, K V; Singh, K P; Singh, A.P. [Division of Floriculture and Landscaping, Indian Agricultural Research Institute, Pusa, New Delhi (India)], E-mail: kvprasad66@gmail.com

2008-07-01

Six rose mutants evolved at the Indian Agricultural Research Institute, New Delhi from four parent cultivars were characterized based on RAPD markers. Contrary to the earlier findings our effort has conclusively proven that the RAPD markers are indeed robust tools to discern the mutants from their parents. Among 40 primers screened, 7 primers produced inconsistent banding pattern. The number of polymorphic bands varied between 4 (OPA 14) and 10 (OPA1) with an average of 6.5 bands per primer. The percentage polymorphism ranged from 62.5 (OPM 9) to 100 percent (OPA 1). Most of the primers produced monomorphic bands between parent and mutant rose cultivars. When primer OPA 2 was used a specific band of 2.5 kb was noticed in mutant cv. Pusa Urmil and cv. Pusa Abhishek but was absent in parent cv. Jantar Mantar. A polymorphic band of 750 bp was noticed in the parent Kiss of Fire and helped in differentiating the parent from its mutant when amplified with OPK 3. Primer OPS 16 produced discriminatory band of 800 bp in mutant cv. Pink Sport of Montezuma while it was absent in its parent cv. Montezuma. Another specific band of 650 bp was present in parent cv. Montezuma and absent in its mutant cv. Pink Sport of Montezuma signifying the uniqueness of the mutant. Primer OPM 5 brought out distinct polymorphism among the parent Jantar Mantar and its three mutants with absence of a specific band of 1.5 kb in the parent. The four parents and 6 mutants were divided into four distinct groups in the Dendogram constructed by UPGMA method. The most genetically similar cultivar among the 10 cultivars analyzed are Montezuma and its pink sport of Montezuma whereas Abhisarika a mutant of cv. Kiss of Fire was distinctly different and formed a separate cluster. (author)

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

Science.gov (United States)

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

2016-01-01

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

Improvement of heavy metal stress and toxicity assays by coupling a transgenic reporter in a mutant nematode strain

Energy Technology Data Exchange (ETDEWEB)

Chu, K.-W. [Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China); Chan, Shirley K.W. [Atmospheric, Marine and Coastal Environment Program, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China); Chow, King L. [Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China) and Atmospheric, Marine and Coastal Environment Program, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China)]. E-mail: bokchow@ust.hk

2005-09-30

Previous studies have demonstrated that wild type Caenorhabditis elegans displays high sensitivity to heavy metals in a lethality test at a level comparable to that of other bioindicator organisms. Taking advantage of the genetics of this model organism, we have tested a number of mutant strains for enhanced sensitivity in heavy metal induced lethality and stress response. These mutants are defective in genes controlling dauer formation, longevity or response to reactive oxygen species (ROS). Among the tested mutants, a double mutant daf-16 unc-75 strain was identified to have superior sensitivity. It has a 6-, 3- and 2-fold increase in sensitivity to cadmium, copper and zinc, respectively, as compared with that of wild type animals. When a fluorescent reporter transgene was coupled with this double mutant for stress detection, a 10-fold enhancement of sensitivity to cadmium over the wild type strain was observed. These transgenic animals, superior to most of the model organisms currently used in bioassays for environmental pollutants, offer a fast and economic approach to reveal the bioavailability of toxic substance in field samples. This study also demonstrates that combination of genetic mutations and transgenesis is a viable approach to identify sensitive indicator animals for environmental monitoring.

Improvement of heavy metal stress and toxicity assays by coupling a transgenic reporter in a mutant nematode strain

International Nuclear Information System (INIS)

Chu, K.-W.; Chan, Shirley K.W.; Chow, King L.

2005-01-01

Previous studies have demonstrated that wild type Caenorhabditis elegans displays high sensitivity to heavy metals in a lethality test at a level comparable to that of other bioindicator organisms. Taking advantage of the genetics of this model organism, we have tested a number of mutant strains for enhanced sensitivity in heavy metal induced lethality and stress response. These mutants are defective in genes controlling dauer formation, longevity or response to reactive oxygen species (ROS). Among the tested mutants, a double mutant daf-16 unc-75 strain was identified to have superior sensitivity. It has a 6-, 3- and 2-fold increase in sensitivity to cadmium, copper and zinc, respectively, as compared with that of wild type animals. When a fluorescent reporter transgene was coupled with this double mutant for stress detection, a 10-fold enhancement of sensitivity to cadmium over the wild type strain was observed. These transgenic animals, superior to most of the model organisms currently used in bioassays for environmental pollutants, offer a fast and economic approach to reveal the bioavailability of toxic substance in field samples. This study also demonstrates that combination of genetic mutations and transgenesis is a viable approach to identify sensitive indicator animals for environmental monitoring

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

Science.gov (United States)

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

2004-05-01

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

Enhanced transpiration rate in the high pigment 1 tomato mutant and its physiological significance.

Science.gov (United States)

Carvalho, R F; Aidar, S T; Azevedo, R A; Dodd, I C; Peres, L E P

2011-05-01

Tomato high pigment (hp) mutants represent an interesting horticultural resource due to their enhanced accumulation of carotenoids, flavonoids and vitamin C. Since hp mutants are known for their exaggerated light responses, the molecules accumulated are likely to be antioxidants, recruited to deal with light and others stresses. Further phenotypes displayed by hp mutations are reduced growth and an apparent disturbance in water loss. Here, we examined the impact of the hp1 mutation and its near isogenic line cv Micro-Tom (MT) on stomatal conductance (gs), transpiration (E), CO(2) assimilation (A) and water use efficiency (WUE). Detached hp1 leaves lost water more rapidly than control leaves, but this behaviour was reversed by exogenous abscisic acid (ABA), indicating the ability of hp1 to respond to this hormone. Although attached hp1 leaves had enhanced gs, E and A compared to control leaves, genotypic differences were lost when water was withheld. Both instantaneous leaf-level WUE and long-term whole plant WUE did not differ between hp1 and MT. Our results indicate a link between exaggerated light response and water loss in hp1, which has important implications for the use of this mutant in both basic and horticultural research. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

Transgenic animal models for study of the pathogenesis of Huntington's disease and therapy.

Science.gov (United States)

Chang, Renbao; Liu, Xudong; Li, Shihua; Li, Xiao-Jiang

2015-01-01

Huntington's disease (HD) is caused by a genetic mutation that results in polyglutamine expansion in the N-terminal regions of huntingtin. As a result, this polyQ expansion leads to the misfolding and aggregation of mutant huntingtin as well as age-dependent neurodegeneration. The genetic mutation in HD allows for generating a variety of animal models that express different forms of mutant huntingtin and show differential pathology. Studies of these animal models have provided an important insight into the pathogenesis of HD. Mouse models of HD include transgenic mice, which express N-terminal or full-length mutant huntingtin ubiquitously or selectively in different cell types, and knock-in mice that express full-length mutant Htt at the endogenous level. Large animals, such as pig, sheep, and monkeys, have also been used to generate animal HD models. This review focuses on the different features of commonly used transgenic HD mouse models as well as transgenic large animal models of HD, and also discusses how to use them to identify potential therapeutics. Since HD shares many pathological features with other neurodegenerative diseases, identification of therapies for HD would also help to develop effective treatment for different neurodegenerative diseases that are also caused by protein misfolding and occur in an age-dependent manner.

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.

Transcriptional regulation of male-sterility in 7B-1 male-sterile tomato mutant.

Directory of Open Access Journals (Sweden)

Vahid Omidvar

Full Text Available The 7B-1 tomato (Solanum lycopersicum L. cv Rutgers is a male-sterile mutant with enhanced tolerance to abiotic stress, which makes it a potential candidate for hybrid seed breeding and stress engineering. To underline the molecular mechanism regulating the male-sterility in 7B-1, transcriptomic profiles of the 7B-1 male-sterile and wild type (WT anthers were studied using mRNA sequencing (RNA-Seq. In total, 768 differentially expressed genes (DEGs were identified, including 132 up-regulated and 636 down-regulated transcripts. Gene ontology (GO enrichment analysis of DEGs suggested a general impact of the 7B-1 mutation on metabolic processes, such as proteolysis and carbohydrate catabolic process. Sixteen candidates with key roles in regulation of anther development were subjected to further analysis using qRT-PCR and in situ hybridization. Cytological studies showed several defects associated with anther development in the 7B-1 mutant, including unsynchronized anther maturation, dysfunctional meiosis, arrested microspores, defect in callose degradation and abnormal tapetum development. TUNEL assay showed a defect in programmed cell death (PCD of tapetal cells in 7B-1 anthers. The present study provides insights into the transcriptome of the 7B-1 mutant. We identified several genes with altered expression level in 7B-1 (including beta-1,3 glucanase, GA2oxs, cystatin, cysteine protease, pectinesterase, TA29, and actin that could potentially regulate anther developmental processes, such as meiosis, tapetum development, and cell-wall formation/degradation.

Leishmania infantum HSP70-II null mutant as candidate vaccine against leishmaniasis: a preliminary evaluation

Directory of Open Access Journals (Sweden)

Fresno Manuel

2011-07-01

Full Text Available Abstract Background Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy. Results In this study, we tested the ability of a Leishmania infantum deletion mutant, lacking both HSP70-II alleles (ΔHSP70-II, to provide protection against Leishmania infection in the L. major-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-Leishmania IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (Mesocricetus auratus, infected with mutant parasites did not develop any sign of pathology. Conclusions The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis.

Low Ascorbic Acid in the vtc-1 Mutant of Arabidopsis Is Associated with Decreased Growth and Intracellular Redistribution of the Antioxidant System1

Science.gov (United States)

Veljovic-Jovanovic, Sonja D.; Pignocchi, Cristina; Noctor, Graham; Foyer, Christine H.

2001-01-01

Ascorbic acid has numerous and diverse roles in plant metabolism. We have used the vtc-1 mutant of Arabidopsis, which is deficient in ascorbate biosynthesis, to investigate the role of ascorbate concentration in growth, regulation of photosynthesis, and control of the partitioning of antioxidative enyzmes. The mutant possessed 70% less ascorbate in the leaves compared with the wild type. This lesion was associated with a slight increase in total glutathione but no change in the redox state of either ascorbate or glutathione. In vtc-1, total ascorbate in the apoplast was decreased to 23% of the wild-type value. The mutant displayed much slower shoot growth than the wild type when grown in air or at high CO2 (3 mL L−1), where oxidative stress is diminished. Leaves were smaller, and shoot fresh weight and dry weight were lower in the mutant. No significant differences in the light saturation curves for CO2 assimilation were found in air or at high CO2, suggesting that the effect on growth was not due to decreased photosynthetic capacity in the mutant. Analysis of chlorophyll a fluorescence quenching revealed only a slight effect on non-photochemical energy dissipation. Hydrogen peroxide contents were similar in the leaves of the vtc-1 mutant and the wild type. Total leaf peroxidase activity was increased in the mutant and compartment-specific differences in ascorbate peroxidase (APX) activity were observed. In agreement with the measurements of enzyme activity, the expression of cytosolic APX was increased, whereas that for chloroplast APX isoforms was either unchanged or slightly decreased. These data implicate ascorbate concentration in the regulation of the compartmentalization of the antioxidant system in Arabidopsis. PMID:11598218

Possible role for plasmalogens in protecting animal cells against photosensitized killing

International Nuclear Information System (INIS)

Zoeller, R.A.; Morand, O.H.; Raetz, C.R.

1988-01-01

Chinese hamster ovary (CHO) cells incorporate 12-(1'-pyrene) dodecanoic acid (P12) into membrane lipids. Exposure of P12-labeled cells to long wavelength ultraviolet light causes cell killing, presumably because excitation of the pyrene moiety (a photosensitizer) leads to the generation of reactive oxygen species. Cytotoxicity is dependent upon the concentration of P12 used to label the cells, and time of UV exposure, and the presence of oxygen during irradiation. CHO mutant cells deficient in plasmalogen biosynthesis and peroxisome assembly are several orders of magnitude more sensitive to P12/UV treatment than wild-type cells, permitting direct selection of one wild-type cell in 1 X 10(4) mutant cells. A major factor responsible for the P12/UV hypersensitivity of these mutants appears to be the absence of plasmalogens. Supplementation of the mutants with 1-O-hexadecyl-sn-glycerol restores plasmalogen levels and nearly normal resistance to P12/UV treatment, whereas the biogenesis of peroxisomes is not restored. The P12/UV hypersensitivity of the plasmalogen-deficient mutants, together with the selective, P12/UV-induced decomposition of plasmalogens in wild-type cells, documented in the accompanying manuscript, suggest that the vinyl ether linkage of plasmalogens plays a direct role in protecting animal cell membranes against certain oxidative stresses

The Arabidopsis thaliana mutant air1 implicates SOS3 in the regulation of anthocyanins under salt stress

KAUST Repository

Van Oosten, Michael James

2013-08-08

The accumulation of anthocyanins in plants exposed to salt stress has been largely documented. However, the functional link and regulatory components underlying the biosynthesis of these molecules during exposure to stress are largely unknown. In a screen of second site suppressors of the salt overly sensitive3-1 (sos3-1) mutant, we isolated the anthocyanin-impaired-response-1 (air1) mutant. air1 is unable to accumulate anthocyanins under salt stress, a key phenotype of sos3-1 under high NaCl levels (120 mM). The air1 mutant showed a defect in anthocyanin production in response to salt stress but not to other stresses such as high light, low phosphorous, high temperature or drought stress. This specificity indicated that air1 mutation did not affect anthocyanin biosynthesis but rather its regulation in response to salt stress. Analysis of this mutant revealed a T-DNA insertion at the first exon of an Arabidopsis thaliana gene encoding for a basic region-leucine zipper transcription factor. air1 mutants displayed higher survival rates compared to wild-type in oxidative stress conditions, and presented an altered expression of anthocyanin biosynthetic genes such as F3H, F3′H and LDOX in salt stress conditions. The results presented here indicate that AIR1 is involved in the regulation of various steps of the flavonoid and anthocyanin accumulation pathways and is itself regulated by the salt-stress response signalling machinery. The discovery and characterization of AIR1 opens avenues to dissect the connections between abiotic stress and accumulation of antioxidants in the form of flavonoids and anthocyanins. © 2013 Springer Science+Business Media Dordrecht.

Determining mutant spectra of three RNA viral samples using ultra-deep sequencing

Energy Technology Data Exchange (ETDEWEB)

Chen, H

2012-06-06

RNA viruses have extremely high mutation rates that enable the virus to adapt to new host environments and even jump from one species to another. As part of a viral transmission study, three viral samples collected from naturally infected animals were sequenced using Illumina paired-end technology at ultra-deep coverage. In order to determine the mutant spectra within the viral quasispecies, it is critical to understand the sequencing error rates and control for false positive calls of viral variants (point mutantations). I will estimate the sequencing error rate from two control sequences and characterize the mutant spectra in the natural samples with this error rate.

The animal model determines the results of Aeromonas virulence factors

Directory of Open Access Journals (Sweden)

Alejandro Romero

2016-10-01

Full Text Available The selection of an experimental animal model is of great importance in the study of bacterial virulence factors. Here, a bath infection of zebrafish larvae is proposed as an alternative model to study the virulence factors of A. hydrophila. Intraperitoneal infections in mice and trout were compared with bath infections in zebrafish larvae using specific mutants. The great advantage of this model is that bath immersion mimics the natural route of infection, and injury to the tail also provides a natural portal of entry for the bacteria. The implication of T3SS in the virulence of A. hydrophila was analysed using the AH-1::aopB mutant. This mutant was less virulent than the wild-type strain when inoculated into zebrafish larvae, as described in other vertebrates. However, the zebrafish model exhibited slight differences in mortality kinetics only observed using invertebrate models. Infections using the mutant AH-1∆vapA lacking the gene coding for the surface S-layer suggested that this protein was not totally necessary to the bacteria once it was inside the host, but it contributed to the inflammatory response. Only when healthy zebrafish larvae were infected did the mutant produce less mortality than the wild type. Variations between models were evidenced using the AH-1∆rmlB, which lacks the O-antigen lipopolysaccharide (LPS, and the AH-1∆wahD, which lacks the O-antigen LPS and part of the LPS outer-core. Both mutants showed decreased mortality in all of the animal models, but the differences between them were only observed in injured zebrafish larvae, suggesting that residues from the LPS outer core must be important for virulence. The greatest differences were observed using the AH-1ΔFlaB-J (lacking polar flagella and unable to swim and the AH-1::motX (non-motile but producing flagella. They were as pathogenic as the wild-type strain when injected into mice and trout, but no mortalities were registered in zebrafish larvae. This study

Novel siRNA formulation to effectively knockdown mutant p53 in osteosarcoma.

Science.gov (United States)

Kundu, Anup K; Iyer, Swathi V; Chandra, Sruti; Adhikari, Amit S; Iwakuma, Tomoo; Mandal, Tarun K

2017-01-01

The tumor suppressor p53 plays a crucial role in the development of osteosarcoma. The primary objective of this study is to develop and optimize lipid based nanoparticle formulations that can carry siRNA and effectively silence mutant p53 in 318-1, a murine osteosarcoma cell line. The nanoparticles were composed of a mixture of two lipids (cholesterol and DOTAP) and either PLGA or PLGA-PEG and prepared by using an EmulsiFlex-B3 high pressure homogenizer. A series of studies that include using different nanoparticles, different amount of siRNAs, cell numbers, incubation time, transfection media volume, and storage temperature was performed to optimize the gene silencing efficiency. Replacement of lipids by PLGA or PLGA-PEG decreased the particle size and overall cytotoxicity. Among all lipid-polymer nanoformulations, nanoparticles with 10% PLGA showed highest mutant p53 knockdown efficiency while maintaining higher cell viability when a nanoparticle to siRNA ratio equal to 6.8:0.66 and 75 nM siRNA was used. With long term storage the mutant p53 knockdown efficiency decreased to a greater extent. This study warrants a future evaluation of this formulation for gene silencing efficiency of mutant p53 in tissue culture and animal models for the treatment of osteosarcoma.

Novel siRNA formulation to effectively knockdown mutant p53 in osteosarcoma.

Directory of Open Access Journals (Sweden)

Anup K Kundu

Full Text Available The tumor suppressor p53 plays a crucial role in the development of osteosarcoma. The primary objective of this study is to develop and optimize lipid based nanoparticle formulations that can carry siRNA and effectively silence mutant p53 in 318-1, a murine osteosarcoma cell line.The nanoparticles were composed of a mixture of two lipids (cholesterol and DOTAP and either PLGA or PLGA-PEG and prepared by using an EmulsiFlex-B3 high pressure homogenizer. A series of studies that include using different nanoparticles, different amount of siRNAs, cell numbers, incubation time, transfection media volume, and storage temperature was performed to optimize the gene silencing efficiency.Replacement of lipids by PLGA or PLGA-PEG decreased the particle size and overall cytotoxicity. Among all lipid-polymer nanoformulations, nanoparticles with 10% PLGA showed highest mutant p53 knockdown efficiency while maintaining higher cell viability when a nanoparticle to siRNA ratio equal to 6.8:0.66 and 75 nM siRNA was used. With long term storage the mutant p53 knockdown efficiency decreased to a greater extent.This study warrants a future evaluation of this formulation for gene silencing efficiency of mutant p53 in tissue culture and animal models for the treatment of osteosarcoma.

Increased BRAF Heterodimerization Is the Common Pathogenic Mechanism for Noonan Syndrome-Associated RAF1 Mutants

Science.gov (United States)

Wu, Xue; Yin, Jiani; Simpson, Jeremy; Kim, Kyoung-Han; Gu, Shengqing; Hong, Jenny H.; Bayliss, Peter; Backx, Peter H.

2012-01-01

Noonan syndrome (NS) is a relatively common autosomal dominant disorder characterized by congenital heart defects, short stature, and facial dysmorphia. NS is caused by germ line mutations in several components of the RAS–RAF–MEK–extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway, including both kinase-activating and kinase-impaired alleles of RAF1 (∼3 to 5%), which encodes a serine-threonine kinase for MEK1/2. To investigate how kinase-impaired RAF1 mutants cause NS, we generated knock-in mice expressing Raf1D486N. Raf1D486N/+ (here D486N/+) female mice exhibited a mild growth defect. Male and female D486N/D486N mice developed concentric cardiac hypertrophy and incompletely penetrant, but severe, growth defects. Remarkably, Mek/Erk activation was enhanced in Raf1D486N-expressing cells compared with controls. RAF1D486N, as well as other kinase-impaired RAF1 mutants, showed increased heterodimerization with BRAF, which was necessary and sufficient to promote increased MEK/ERK activation. Furthermore, kinase-activating RAF1 mutants also required heterodimerization to enhance MEK/ERK activation. Our results suggest that an increased heterodimerization ability is the common pathogenic mechanism for NS-associated RAF1 mutations. PMID:22826437

Phenotypic, genetic and molecular characterization of a maize low phytic acid mutant (lpa241)

DEFF Research Database (Denmark)

Pilu, R.; Panzeri, D.; Gavazzi, G.

2003-01-01

-nutritional factor for animals, and isolation of maize low phytic acid (lpa) mutants provides a novel approach to study its biochemical pathway and to tackle the nutritional problems associated with it. Following chemical mutagenesis of pollen, we have isolated a viable recessive mutant named lpa 241 showing about...... 90% reduction of phytic acid and about a tenfold increase in seed-free phosphate content. Although germination rate was decreased by about 30% compared to wild-type, developement of mutant plants was apparentely unaffected. The results of the genetic, biochemical and molecular characterization...

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

Directory of Open Access Journals (Sweden)

Meredith E Judy

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

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

Energy Technology Data Exchange (ETDEWEB)

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

1990-01-01

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

Neurologic function during developmental and adult stages in Dab1(scm) (scrambler) mutant mice.

Science.gov (United States)

Jacquelin, C; Strazielle, C; Lalonde, R

2012-01-01

Homozygous Dab1(scm) mouse mutants with cell ectopias in cerebellar cortex, hippocampus, and neocortex were compared to non-ataxic controls on the SHIRPA primary screening battery on postnatal days 8, 15, and 22, as well as in the adult period. Dab1(scm) mutants were distinguished from non-ataxic controls as early as postnatal day 8 based on body tremor, gait anomalies, and body weight. On postnatal day 15, motor coordination deficits were evident on horizontal bar and inclined or vertical grid tests in association with a weaker grip strength. Likewise, mutants were distinguished from controls on drop righting and hindpaw clasping tests. Further differences were detected on postnatal day 22 in the form of fewer visual placing, touch escape, trunk curl, freezing, and vocalization responses, as well as squares traversed in the open-field. Evaluation at the adult age demonstrated similar impairments, indicative of permanent motor alterations. Neuronal metabolic activity was estimated by cytochrome oxidase histochemistry on cerebellar sections. Cerebellar cortical layers and efferent deep nuclei of Dab1(scm) mice appeared hypometabolic relative to non-ataxic mice despite normal metabolism in both regular and ectopic Purkinje cells. Copyright © 2011 Elsevier B.V. All rights reserved.

Lateral root development in the maize (Zea mays) lateral rootless1 mutant.

Science.gov (United States)

Husakova, Eva; Hochholdinger, Frank; Soukup, Ales

2013-07-01

The maize lrt1 (lateral rootless1) mutant is impaired in its development of lateral roots during early post-embryonic development. The aim of this study was to characterize, in detail, the influences that the mutation exerts on lateral root initiation and the subsequent developments, as well as to describe the behaviour of the entire plant under variable environmental conditions. Mutant lrt1 plants were cultivated under different conditions of hydroponics, and in between sheets of moist paper. Cleared whole mounts and anatomical sections were used in combination with both selected staining procedures and histochemical tests to follow root development. Root surface permeability tests and the biochemical quantification of lignin were performed to complement the structural data. The data presented suggest a redefinition of lrt1 function in lateral roots as a promoter of later development; however, neither the complete absence of lateral roots nor the frequency of their initiation is linked to lrt1 function. The developmental effects of lrt1 are under strong environmental influences. Mutant primordia are affected in structure, growth and emergence; and the majority of primordia terminate their growth during this last step, or shortly thereafter. The lateral roots are impaired in the maintenance of the root apical meristem. The primary root shows disturbances in the organization of both epidermal and subepidermal layers. The lrt1-related cell-wall modifications include: lignification in peripheral layers, the deposition of polyphenolic substances and a higher activity of peroxidase. The present study provides novel insights into the function of the lrt1 gene in root system development. The lrt1 gene participates in the spatial distribution of initiation, but not in its frequency. Later, the development of lateral roots is strongly affected. The effect of the lrt1 mutation is not as obvious in the primary root, with no influences observed on the root apical meristem

Lateral root development in the maize (Zea mays) lateral rootless1 mutant

Science.gov (United States)

Husakova, Eva; Hochholdinger, Frank; Soukup, Ales

2013-01-01

Background and Aims The maize lrt1 (lateral rootless1) mutant is impaired in its development of lateral roots during early post-embryonic development. The aim of this study was to characterize, in detail, the influences that the mutation exerts on lateral root initiation and the subsequent developments, as well as to describe the behaviour of the entire plant under variable environmental conditions. Methods Mutant lrt1 plants were cultivated under different conditions of hydroponics, and in between sheets of moist paper. Cleared whole mounts and anatomical sections were used in combination with both selected staining procedures and histochemical tests to follow root development. Root surface permeability tests and the biochemical quantification of lignin were performed to complement the structural data. Key Results The data presented suggest a redefinition of lrt1 function in lateral roots as a promoter of later development; however, neither the complete absence of lateral roots nor the frequency of their initiation is linked to lrt1 function. The developmental effects of lrt1 are under strong environmental influences. Mutant primordia are affected in structure, growth and emergence; and the majority of primordia terminate their growth during this last step, or shortly thereafter. The lateral roots are impaired in the maintenance of the root apical meristem. The primary root shows disturbances in the organization of both epidermal and subepidermal layers. The lrt1-related cell-wall modifications include: lignification in peripheral layers, the deposition of polyphenolic substances and a higher activity of peroxidase. Conclusions The present study provides novel insights into the function of the lrt1 gene in root system development. The lrt1 gene participates in the spatial distribution of initiation, but not in its frequency. Later, the development of lateral roots is strongly affected. The effect of the lrt1 mutation is not as obvious in the primary root, with no

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

Science.gov (United States)

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

1998-01-01

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

Simulation of Fungal-Mediated Cell Death by Fumonisin B1 and Selection of Fumonisin B1–Resistant (fbr) Arabidopsis Mutants

Science.gov (United States)

Stone, Julie M.; Heard, Jacqueline E.; Asai, Tsuneaki; Ausubel, Frederick M.

2000-01-01

Fumonisin B1 (FB1), a programmed cell death–eliciting toxin produced by the necrotrophic fungal plant pathogen Fusarium moniliforme, was used to simulate pathogen infection in Arabidopsis. Plants infiltrated with 10 μM FB1 and seedlings transferred to agar media containing 1 μM FB1 develop lesions reminiscent of the hypersensitive response, including generation of reactive oxygen intermediates, deposition of phenolic compounds and callose, accumulation of phytoalexin, and expression of pathogenesis-related (PR) genes. Arabidopsis FB1-resistant (fbr) mutants were selected directly by sowing seeds on agar containing 1 μM FB1, on which wild-type seedlings fail to develop. Two mutants chosen for further analyses, fbr1 and fbr2, had altered PR gene expression in response to FB1. fbr1 and fbr2 do not exhibit differential resistance to the avirulent bacterial pathogen Pseudomonas syringae pv maculicola (ES4326) expressing the avirulence gene avrRpt2 but do display enhanced resistance to a virulent isogenic strain that lacks the avirulence gene. Our results demonstrate the utility of FB1 for high-throughput isolation of Arabidopsis defense-related mutants and suggest that pathogen-elicited programmed cell death of host cells may be an important feature of compatible plant–pathogen interactions. PMID:11041878

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

Directory of Open Access Journals (Sweden)

Luca eTadini

2012-12-01

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

Characterisation of prostate cancer lesions in heterozygous Men1 mutant mice

International Nuclear Information System (INIS)

Seigne, Christelle; Fontanière, Sandra; Carreira, Christine; Lu, Jieli; Tong, Wei-Ming; Fontanière, Bernard; Wang, Zhao-Qi; Zhang, Chang Xian; Frappart, Lucien

2010-01-01

Mutations of the MEN1 gene predispose to multiple endocrine neoplasia type 1 (MEN1) syndrome. Our group and others have shown that Men1 disruption in mice recapitulates MEN1 pathology. Intriguingly, rare lesions in hormone-dependent tissues, such as prostate and mammary glands, were also observed in the Men1 mutant mice. To study the occurrence of prostate lesions, we followed a male mouse cohort of 47 Men1 +/- mice and 23 age-matched control littermates, starting at 18 months of age, and analysed the prostate glands from the cohort. Six Men1 +/- mice (12.8%) developed prostate cancer, including two adenocarcinomas and four in situ carcinomas, while none of the control mice developed cancerous lesions. The expression of menin encoded by the Men1 gene was found to be drastically reduced in all carcinomas, and partial LOH of the wild-type Men1 allele was detected in three of the five analysed lesions. Using immunostaining for the androgen receptor and p63, a basal epithelial cell marker, we demonstrated that the menin-negative prostate cancer cells did not display p63 expression and that the androgen receptor was expressed but more heterogeneous in these lesions. Furthermore, our data showed that the expression of the cyclin-dependent kinase inhibitor CDKN1B (p27), a Men1 target gene known to be inactivated during prostate cell tumorigenesis, was notably decreased in the prostate cancers that developed in the mutant mice. Our work suggests the possible involvement of Men1 inactivation in the tumorigenesis of the prostate gland

Blood-CNS Barrier Impairment in ALS Patients versus an Animal Model

Directory of Open Access Journals (Sweden)

Svitlana eGarbuzova-Davis

2014-02-01

Full Text Available Amyotrophic lateral sclerosis (ALS is a severe neurodegenerative disease with a compli-cated and poorly understood pathogenesis. Recently, alterations in the blood-Central Nervous System barrier (B-CNS-B have been recognized as a key factor possibly aggravating motor neuron damage. The majority of findings on ALS microvascular pathology have been deter-mined in mutant SOD1 rodent models, identifying barrier damage during disease develop-ment which might similarly occur in familial ALS patients carrying the SOD1 mutation. However, our knowledge of B-CNS-B competence in sporadic ALS (SALS has been limited. We recently showed structural and functional impairment in postmortem gray and white mat-ter microvessels of medulla and spinal cord tissue from SALS patients, suggesting pervasive barrier damage. Although numerous signs of barrier impairment (endothelial cell degenera-tion, capillary leakage, perivascular edema, downregulation of tight junction proteins, and microhemorrhages are indicated in both mutant SOD1 animal models of ALS and SALS pa-tients, other pathogenic barrier alterations have as yet only been identified in SALS patients. Pericyte degeneration, perivascular collagen IV expansion, and white matter capillary abnor-malities in SALS patients are significant barrier related pathologies yet to be noted in ALS SOD1 animal models. In the current review, these important differences in blood-CNS barrier damage between ALS patients and animal models, which may signify altered barrier transport mechanisms, are discussed. Understanding discrepancies in barrier condition between ALS patients and animal models may be crucial for developing effective therapies.

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

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

Science.gov (United States)

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

2017-01-01

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

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

Science.gov (United States)

Swain, Swadhin; Roy, Shweta; Shah, Jyoti; Van Wees, Saskia; Pieterse, Corné M; Nandi, Ashis K

2011-12-01

Arabidopsis genotypes with a hyperactive salicylic acid-mediated signalling pathway exhibit enhanced disease resistance, which is often coupled with growth and developmental defects, such as dwarfing and spontaneous necrotic lesions on the leaves, resulting in reduced biomass yield. In this article, we report a novel recessive mutant of Arabidopsis, cdd1 (constitutive defence without defect in growth and development1), that exhibits enhanced disease resistance associated with constitutive salicylic acid signalling, but without any observable pleiotropic phenotype. Both NPR1 (NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1)-dependent and NPR1-independent salicylic acid-regulated defence pathways are hyperactivated in cdd1 mutant plants, conferring enhanced resistance against bacterial pathogens. However, a functional NPR1 allele is required for the cdd1-conferred heightened resistance against the oomycete pathogen Hyaloperonospora arabidopsidis. Salicylic acid accumulates at elevated levels in cdd1 and cdd1 npr1 mutant plants and is necessary for cdd1-mediated PR1 expression and disease resistance phenotypes. In addition, we provide data which indicate that the cdd1 mutation negatively regulates the npr1 mutation-induced hyperactivation of ethylene/jasmonic acid signalling. © 2011 The Authors. Molecular Plant Pathology © 2011 BSPP and Blackwell Publishing Ltd.

Comparative Analysis of Light-Harvesting Antennae and State Transition in chlorina and cpSRP Mutants1[OPEN

Science.gov (United States)

Wang, Peng

2016-01-01

State transitions in photosynthesis provide for the dynamic allocation of a mobile fraction of light-harvesting complex II (LHCII) to photosystem II (PSII) in state I and to photosystem I (PSI) in state II. In the state I-to-state II transition, LHCII is phosphorylated by STN7 and associates with PSI to favor absorption cross-section of PSI. Here, we used Arabidopsis (Arabidopsis thaliana) mutants with defects in chlorophyll (Chl) b biosynthesis or in the chloroplast signal recognition particle (cpSRP) machinery to study the flexible formation of PS-LHC supercomplexes. Intriguingly, we found that impaired Chl b biosynthesis in chlorina1-2 (ch1-2) led to preferentially stabilized LHCI rather than LHCII, while the contents of both LHCI and LHCII were equally depressed in the cpSRP43-deficient mutant (chaos). In view of recent findings on the modified state transitions in LHCI-deficient mutants (Benson et al., 2015), the ch1-2 and chaos mutants were used to assess the influence of varying LHCI/LHCII antenna size on state transitions. Under state II conditions, LHCII-PSI supercomplexes were not formed in both ch1-2 and chaos plants. LHCII phosphorylation was drastically reduced in ch1-2, and the inactivation of STN7 correlates with the lack of state transitions. In contrast, phosphorylated LHCII in chaos was observed to be exclusively associated with PSII complexes, indicating a lack of mobile LHCII in chaos. Thus, the comparative analysis of ch1-2 and chaos mutants provides new evidence for the flexible organization of LHCs and enhances our understanding of the reversible allocation of LHCII to the two photosystems. PMID:27663408

Potential relationships between morphological differentiation and mutants with high nuclease P1 production of Penicillium citrinum

Energy Technology Data Exchange (ETDEWEB)

Xinle, Liang; Qian, Shou; Hong, Zhang; Min, Chen [Department of Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang (China); Xuan, Liu [Beihai Institute of Environmental Science, Beihai, Guangxi (China)

2009-08-15

Diversification of colony characteristics of mutants derived from Penicillium citrinum CICC 4011 treated with {sup 60}Co {gamma}-irradiation and protoplast fusion were analyzed. There were distinct differences among mutants with different nuclease P1 activity, especially in pigment productivity. Color of colony was changed from the original green to white, grey-green, or yellow-green etc., while the nuclease P1 activity would be fluctuated with the color change. The hypothesis was suggested that there would be a relationship between pigments and nuclease P1 production. Mutants with grey-green colony would give out high nuclease P1 outputs in a high probability such as mutant J1Y6 (nuclease P1 activity, 167.3U/ml) and fusant F-13 (nuclease P1 activity, 568.7U/ml), while others with deep-green colony observed low nuclease outputs. Four variation strains didn't show any significant difference in growth rate. Broom branches of conidiophore stem in J1Y6 and F-13 were obviously reduced, conidiophores productivity reduced, but hyphae growth haled. These suggested that nuclease P1 production was associated with growth phase, but pigment synthesis course wasn't. RAPD from 6 randomly selected primers was used to analyze the polymorphic rich of the four strains, the results showed that there were 70 percent polymorphism detection rate among those. UPGMA cluster analysis and genetic map constructed by NTSYS-PC software, which showed that J1Y6 and F-14 were clustered as one group at similar coefficient 0.9, where there was an appear distance from the group of 4011 and F-R-33 strains (similar coefficient 0.8). (authors)

Potential relationships between morphological differentiation and mutants with high nuclease P1 production of Penicillium citrinum

International Nuclear Information System (INIS)

Liang Xinle; Shou Qian; Zhang Hong; Chen Min; Liu Xuan

2009-01-01

Diversification of colony characteristics of mutants derived from Penicillium citrinum CICC 4011 treated with 60 Co γ-irradiation and protoplast fusion were analyzed. There were distinct differences among mutants with different nuclease P1 activity, especially in pigment productivity. Color of colony was changed from the original green to white, grey-green, or yellow-green etc., while the nuclease P1 activity would be fluctuated with the color change. The hypothesis was suggested that there would be a relationship between pigments and nuclease P1 production. Mutants with grey-green colony would give out high nuclease P1 outputs in a high probability such as mutant J1Y6( nuclease P1 activity, 167.3U/ml) and fusant F-13 (nuclease P1 activity, 568.7U/ml), while others with deep-green colony observed low nuclease outputs. Four variation strains didn't show any significant difference in growth rate. Broom branches of conidiophore stem in J1Y6 and F-13 were obviously reduced, conidiophores productivity reduced, but hyphae growth haled. These suggested that nuclease P1 production was associated with growth phase, but pigment synthesis course wasn't. RAPD from 6 randomly selected primers was used to analyze the polymorphic rich of the four strains, the results showed that there were 70 percent polymorphism detection rate among those. UPGMA cluster analysis and genetic map constructed by NTSYS-PC software, which showed that J1Y6 and F-14 were clustered as one group at similar coefficient 0.9, where there was an appear distance from the group of 4011 and F-R-33 strains (similar coefficient 0.8). (authors)

Semi-dwarf mutants in triticale and wheat breeding

International Nuclear Information System (INIS)

Driscoll, C.J.

1984-01-01

The triticale lines Beagle and DR-IRA have been subjected to ionizing irradiation and chemical mutagenesis in order to produce semi-dwarf mutants. Beagle is 100 cm tall and DR-IRA 80 cm under average field conditions. A bulk then pedigree method is currently represented by 158 single plots of M 6 (or in some cases M 7 ) mutants that are from 5 to 35 cm shorter than the control variety. The shortest mutants are 65 cm in height. Forty of these mutants are also earlier flowering than the control varieties. Replicated yield testing will be conducted on confirmed mutants in 1983. Response to gibberellic acid of these mutants will also be determined. The Cornerstone male-sterility mutant (ms1c) on chromosome arm 4Aα has been combined with the GA-insensitive/reduced height gene Gai/Rht1 which is also on chromosome arm 4Aα. The ms1c mutant has also been combined with Gai/Rht2 on chromosome 4D and with both Gai/Rht1 and Gai/Rht2. The combination ms1c and Gai/Rht1 has been chosen as the basis of a composite cross. Thirteen varieties were tested with GA 3 and seven (Warigal, Aroona, Oxley, Banks, Avocet, Matipo and Toquifen) which contain Gai/Rht1 were crossed with ms1c Gai/Rht1 and entered into an interpollinating F 2 . The entire composite is homozygous for this semi-dwarf allele and selection will be practiced for increased height on a GA-insensitive background. (author)

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.

Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1

Science.gov (United States)

Ng, Jason Liang Pin; Hassan, Samira; Truong, Thy T.; Hocart, Charles H.; Laffont, Carole; Frugier, Florian; Mathesius, Ulrike

2015-01-01

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation. PMID:26253705

Hypoxic resistance of KRAS mutant tumor cells to 3-Bromopyruvate is counteracted by Prima-1 and reversed by N-acetylcysteine.

Science.gov (United States)

Orue, Andrea; Chavez, Valery; Strasberg-Rieber, Mary; Rieber, Manuel

2016-11-18

The metabolic inhibitor 3-bromopyruvate (3-BrPA) is a promising anti-cancer alkylating agent, shown to inhibit growth of some colorectal carcinoma with KRAS mutation. Recently, we demonstrated increased resistance to 3-BrPA in wt p53 tumor cells compared to those with p53 silencing or mutation. Since hypoxic microenvironments select for tumor cells with diminished therapeutic response, we investigated whether hypoxia unequally increases resistance to 3-BrPA in wt p53 MelJuso melanoma harbouring (Q61L)-mutant NRAS and wt BRAF, C8161 melanoma with (G12D)-mutant KRAS (G464E)-mutant BRAF, and A549 lung carcinoma with a KRAS (G12S)-mutation. Since hypoxia increases the toxicity of the p53 activator, Prima-1 against breast cancer cells irrespective of their p53 status, we also investigated whether Prima-1 reversed hypoxic resistance to 3-BrPA. In contrast to the high susceptibility of hypoxic mutant NRAS MelJuso cells to 3-BrPA or Prima-1, KRAS mutant C8161 and A549 cells revealed hypoxic resistance to 3-BrPA counteracted by Prima-1. In A549 cells, Prima-1 increased p21CDKN1mRNA, and reciprocally inhibited mRNA expression of the SLC2A1-GLUT1 glucose transporter-1 and ALDH1A1, gene linked to detoxification and stem cell properties. 3-BrPA lowered CAIX and VEGF mRNA expression. Death from joint Prima-1 and 3-BrPA treatment in KRAS mutant A549 and C8161 cells seemed mediated by potentiating oxidative stress, since it was antagonized by the anti-oxidant and glutathione precursor N-acetylcysteine. This report is the first to show that Prima-1 kills hypoxic wt p53 KRAS-mutant cells resistant to 3-BrPA, partly by decreasing GLUT-1 expression and exacerbating pro-oxidant stress.

Hyperpolarized 13C MR imaging detects no lactate production in mutant IDH1 gliomas: Implications for diagnosis and response monitoring

Directory of Open Access Journals (Sweden)

Myriam M. Chaumeil

2016-01-01

Full Text Available Metabolic imaging of brain tumors using 13C Magnetic Resonance Spectroscopy (MRS of hyperpolarized [1-13C] pyruvate is a promising neuroimaging strategy which, after a decade of preclinical success in glioblastoma (GBM models, is now entering clinical trials in multiple centers. Typically, the presence of GBM has been associated with elevated hyperpolarized [1-13C] lactate produced from [1-13C] pyruvate, and response to therapy has been associated with a drop in hyperpolarized [1-13C] lactate. However, to date, lower grade gliomas had not been investigated using this approach. The most prevalent mutation in lower grade gliomas is the isocitrate dehydrogenase 1 (IDH1 mutation, which, in addition to initiating tumor development, also induces metabolic reprogramming. In particular, mutant IDH1 gliomas are associated with low levels of lactate dehydrogenase A (LDHA and monocarboxylate transporters 1 and 4 (MCT1, MCT4, three proteins involved in pyruvate metabolism to lactate. We therefore investigated the potential of 13C MRS of hyperpolarized [1-13C] pyruvate for detection of mutant IDH1 gliomas and for monitoring of their therapeutic response. We studied patient-derived mutant IDH1 glioma cells that underexpress LDHA, MCT1 and MCT4, and wild-type IDH1 GBM cells that express high levels of these proteins. Mutant IDH1 cells and tumors produced significantly less hyperpolarized [1-13C] lactate compared to GBM, consistent with their metabolic reprogramming. Furthermore, hyperpolarized [1-13C] lactate production was not affected by chemotherapeutic treatment with temozolomide (TMZ in mutant IDH1 tumors, in contrast to previous reports in GBM. Our results demonstrate the unusual metabolic imaging profile of mutant IDH1 gliomas, which, when combined with other clinically available imaging methods, could be used to detect the presence of the IDH1 mutation in vivo.

Energetic basis for drug resistance of HIV-1 protease mutants against amprenavir

Science.gov (United States)

Kar, Parimal; Knecht, Volker

2012-02-01

Amprenavir (APV) is a high affinity (0.15 nM) HIV-1 protease (PR) inhibitor. However, the affinities of the drug resistant protease variants V32I, I50V, I54V, I54M, I84V and L90M to amprenavir are decreased 3 to 30-fold compared to the wild-type. In this work, the popular molecular mechanics Poisson-Boltzmann surface area method has been used to investigate the effectiveness of amprenavir against the wild-type and these mutated protease variants. Our results reveal that the protonation state of Asp25/Asp25' strongly affects the dynamics, the overall affinity and the interactions of the inhibitor with individual residues. We emphasize that, in contrast to what is often assumed, the protonation state may not be inferred from the affinities but requires pKa calculations. At neutral pH, Asp25 and Asp25' are ionized or protonated, respectively, as suggested from pKa calculations. This protonation state was thus mainly considered in our study. Mutation induced changes in binding affinities are in agreement with the experimental findings. The decomposition of the binding free energy reveals the mechanisms underlying binding and drug resistance. Drug resistance arises from an increase in the energetic contribution from the van der Waals interactions between APV and PR (V32I, I50V, and I84V mutant) or a rise in the energetic contribution from the electrostatic interactions between the inhibitor and its target (I54M and I54V mutant). For the V32I mutant, also an increased free energy for the polar solvation contributes to the drug resistance. For the L90M mutant, a rise in the van der Waals energy for APV-PR interactions is compensated by a decrease in the polar solvation free energy such that the net binding affinity remains unchanged. Detailed understanding of the molecular forces governing binding and drug resistance might assist in the design of new inhibitors against HIV-1 PR variants that are resistant against current drugs.

Engineered disulfide bonds restore chaperone-like function of DJ-1 mutants linked to familial Parkinson's disease.

Science.gov (United States)

Logan, Todd; Clark, Lindsay; Ray, Soumya S

2010-07-13

Loss-of-function mutations such as L166P, A104T, and M26I in the DJ-1 gene (PARK7) have been linked to autosomal-recessive early onset Parkinson's disease (PD). Cellular and structural studies of the familial mutants suggest that these mutations may destabilize the dimeric structure. To look for common dynamical signatures among the DJ-1 mutants, short MD simulations of up to 1000 ps were conducted to identify the weakest region of the protein (residues 38-70). In an attempt to stabilize the protein, we mutated residue Val 51 to cysteine (V51C) to make a symmetry-related disulfide bridge with the preexisting Cys 53 on the opposite subunit. We found that the introduction of this disulfide linkage stabilized the mutants A104T and M26I against thermal denaturation, improved their ability to scavenge reactive oxygen species (ROS), and restored a chaperone-like function of blocking alpha-synuclein aggregation. The L166P mutant was far too unstable to be rescued by introduction of the V51C mutation. The results presented here point to the possible development of pharmacological chaperones, which may eventually lead to PD therapeutics.

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.

Sequential super-stereotypy of an instinctive fixed action pattern in hyper-dopaminergic mutant mice: a model of obsessive compulsive disorder and Tourette's

Directory of Open Access Journals (Sweden)

Houchard Kimberly R

2005-02-01

Full Text Available Abstract Background Excessive sequential stereotypy of behavioral patterns (sequential super-stereotypy in Tourette's syndrome and obsessive compulsive disorder (OCD is thought to involve dysfunction in nigrostriatal dopamine systems. In sequential super-stereotypy, patients become trapped in overly rigid sequential patterns of action, language, or thought. Some instinctive behavioral patterns of animals, such as the syntactic grooming chain pattern of rodents, have sufficiently complex and stereotyped serial structure to detect potential production of overly-rigid sequential patterns. A syntactic grooming chain is a fixed action pattern that serially links up to 25 grooming movements into 4 predictable phases that follow 1 syntactic rule. New mutant mouse models allow gene-based manipulation of brain function relevant to sequential patterns, but no current animal model of spontaneous OCD-like behaviors has so far been reported to exhibit sequential super-stereotypy in the sense of a whole complex serial pattern that becomes stronger and excessively rigid. Here we used a hyper-dopaminergic mutant mouse to examine whether an OCD-like behavioral sequence in animals shows sequential super-stereotypy. Knockdown mutation of the dopamine transporter gene (DAT causes extracellular dopamine levels in the neostriatum of these adult mutant mice to rise to 170% of wild-type control levels. Results We found that the serial pattern of this instinctive behavioral sequence becomes strengthened as an entire entity in hyper-dopaminergic mutants, and more resistant to interruption. Hyper-dopaminergic mutant mice have stronger and more rigid syntactic grooming chain patterns than wild-type control mice. Mutants showed sequential super-stereotypy in the sense of having more stereotyped and predictable syntactic grooming sequences, and were also more likely to resist disruption of the pattern en route, by returning after a disruption to complete the pattern from the

Specific Silencing of L392V PSEN1 Mutant Allele by RNA Interference

Directory of Open Access Journals (Sweden)

Malgorzata Sierant

2011-01-01

Full Text Available RNA interference (RNAi technology provides a powerful molecular tool to reduce an expression of selected genes in eukaryotic cells. Short interfering RNAs (siRNAs are the effector molecules that trigger RNAi. Here, we describe siRNAs that discriminate between the wild type and mutant (1174 C→G alleles of human Presenilin1 gene (PSEN1. This mutation, resulting in L392V PSEN1 variant, contributes to early onset familial Alzheimer's disease. Using the dual fluorescence assay, flow cytometry and fluorescent microscopy we identified positions 8th–11th, within the central part of the antisense strand, as the most sensitive to mismatches. 2-Thiouridine chemical modification introduced at the 3′-end of the antisense strand improved the allele discrimination, but wobble base pairing adjacent to the mutation site abolished the siRNA activity. Our data indicate that siRNAs can be designed to discriminate between the wild type and mutant alleles of genes that differ by just a single nucleotide.

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.

IDH1-mutant cancer cells are sensitive to cisplatin and an IDH1-mutant inhibitor counteracts this sensitivity.

Science.gov (United States)

Khurshed, Mohammed; Aarnoudse, Niels; Hulsbos, Renske; Hira, Vashendriya V V; van Laarhoven, Hanneke W M; Wilmink, Johanna W; Molenaar, Remco J; van Noorden, Cornelis J F

2018-06-07

Isocitrate dehydrogenase ( IDH1)-1 is mutated in various types of human cancer, and the presence of this mutation is associated with improved responses to irradiation and chemotherapy in solid tumor cells. Mutated IDH1 (IDH1 MUT ) enzymes consume NADPH to produce d-2-hydroxyglutarate (d-2HG) resulting in the decreased reducing power needed for detoxification of reactive oxygen species (ROS), for example. The objective of the current study was to investigate the mechanism behind the chemosensitivity of the widely-used anticancer agent cisplatin in IDH1 MUT cancer cells. Oxidative stress, DNA damage, and mitochondrial dysfunction caused by cisplatin treatment were monitored in IDH1 MUT HCT116 colorectal cancer cells and U251 glioma cells. We found that exposure to cisplatin induced higher levels of ROS, DNA double-strand breaks (DSBs), and cell death in IDH1 MUT cancer cells, as compared with IDH1 wild-type ( IDH1 WT ) cells. Mechanistic investigations revealed that cisplatin treatment dose dependently reduced oxidative respiration in IDH1 MUT cells, which was accompanied by disturbed mitochondrial proteostasis, indicative of impaired mitochondrial activity. These effects were abolished by the IDH1 MUT inhibitor AGI-5198 and were restored by treatment with d-2HG. Thus, our study shows that altered oxidative stress responses and a vulnerable oxidative metabolism underlie the sensitivity of IDH1 MUT cancer cells to cisplatin.-Khurshed, M., Aarnoudse, N., Hulsbos, R., Hira, V. V. V., van Laarhoven, H. W. M., Wilmink, J. W., Molenaar, R. J., van Noorden, C. J. F. IDH1-mutated cancer cells are sensitive to cisplatin and an IDH1-mutant inhibitor counteracts this sensitivity.

Twist1 suppresses senescence programs and thereby accelerates and maintains mutant Kras-induced lung tumorigenesis.

Directory of Open Access Journals (Sweden)

Phuoc T Tran

Full Text Available KRAS mutant lung cancers are generally refractory to chemotherapy as well targeted agents. To date, the identification of drugs to therapeutically inhibit K-RAS have been unsuccessful, suggesting that other approaches are required. We demonstrate in both a novel transgenic mutant Kras lung cancer mouse model and in human lung tumors that the inhibition of Twist1 restores a senescence program inducing the loss of a neoplastic phenotype. The Twist1 gene encodes for a transcription factor that is essential during embryogenesis. Twist1 has been suggested to play an important role during tumor progression. However, there is no in vivo evidence that Twist1 plays a role in autochthonous tumorigenesis. Through two novel transgenic mouse models, we show that Twist1 cooperates with Kras(G12D to markedly accelerate lung tumorigenesis by abrogating cellular senescence programs and promoting the progression from benign adenomas to adenocarcinomas. Moreover, the suppression of Twist1 to physiological levels is sufficient to cause Kras mutant lung tumors to undergo senescence and lose their neoplastic features. Finally, we analyzed more than 500 human tumors to demonstrate that TWIST1 is frequently overexpressed in primary human lung tumors. The suppression of TWIST1 in human lung cancer cells also induced cellular senescence. Hence, TWIST1 is a critical regulator of cellular senescence programs, and the suppression of TWIST1 in human tumors may be an effective example of pro-senescence therapy.

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

Directory of Open Access Journals (Sweden)

Dan Jiang

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

E4orf1 Limits the Oncolytic Potential of the E1B-55K Deletion Mutant Adenovirus▿

Science.gov (United States)

Thomas, Michael A.; Broughton, Robin S.; Goodrum, Felicia D.; Ornelles, David A.

2009-01-01

Clinical trials have shown oncolytic adenoviruses to be tumor selective with minimal toxicity toward normal tissue. The virus ONYX-015, in which the gene encoding the early region 1B 55-kDa (E1B-55K) protein is deleted, has been most effective when used in combination with either chemotherapy or radiation therapy. Therefore, improving the oncolytic nature of tumor-selective adenoviruses remains an important objective for improving this form of cancer therapy. Cells infected during the G1 phase of the cell cycle with the E1B-55K deletion mutant virus exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less efficiently killed than cells infected during the S phase. Here we demonstrate that the G1 restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1). E4orf1 has been reported to signal through the phosphatidylinositol 3′-kinase pathway leading to the activation of Akt, mTOR, and p70 S6K. Evidence presented here shows that E4orf1 may also induce the phosphorylation of Akt and p70 S6K in a manner that depends on Rac1 and its guanine nucleotide exchange factor Tiam1. Accordingly, agents that have been reported to disrupt the Tiam1-Rac1 interaction or to prevent phosphorylation of the ribosomal S6 kinase partially alleviated the E4orf1 restriction to late viral protein synthesis and enhanced tumor cell killing by the E1B-55K mutant virus. These results demonstrate that E4orf1 limits the oncolytic nature of a conditionally replicating adenovirus such as ONYX-015. The therapeutic value of similar oncolytic adenoviruses may be improved by abrogating E4orf1 function. PMID:19129452

E4orf1 limits the oncolytic potential of the E1B-55K deletion mutant adenovirus.

Science.gov (United States)

Thomas, Michael A; Broughton, Robin S; Goodrum, Felicia D; Ornelles, David A

2009-03-01

Clinical trials have shown oncolytic adenoviruses to be tumor selective with minimal toxicity toward normal tissue. The virus ONYX-015, in which the gene encoding the early region 1B 55-kDa (E1B-55K) protein is deleted, has been most effective when used in combination with either chemotherapy or radiation therapy. Therefore, improving the oncolytic nature of tumor-selective adenoviruses remains an important objective for improving this form of cancer therapy. Cells infected during the G(1) phase of the cell cycle with the E1B-55K deletion mutant virus exhibit a reduced rate of viral late protein synthesis, produce fewer viral progeny, and are less efficiently killed than cells infected during the S phase. Here we demonstrate that the G(1) restriction imposed on the E1B-55K deletion mutant virus is due to the viral oncogene encoded by open reading frame 1 of early region 4 (E4orf1). E4orf1 has been reported to signal through the phosphatidylinositol 3'-kinase pathway leading to the activation of Akt, mTOR, and p70 S6K. Evidence presented here shows that E4orf1 may also induce the phosphorylation of Akt and p70 S6K in a manner that depends on Rac1 and its guanine nucleotide exchange factor Tiam1. Accordingly, agents that have been reported to disrupt the Tiam1-Rac1 interaction or to prevent phosphorylation of the ribosomal S6 kinase partially alleviated the E4orf1 restriction to late viral protein synthesis and enhanced tumor cell killing by the E1B-55K mutant virus. These results demonstrate that E4orf1 limits the oncolytic nature of a conditionally replicating adenovirus such as ONYX-015. The therapeutic value of similar oncolytic adenoviruses may be improved by abrogating E4orf1 function.

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

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

Science.gov (United States)

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

2015-05-20

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

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

DEFF Research Database (Denmark)

Jensen, Helle Lone; Norrild, Bodil

2003-01-01

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

An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis

Science.gov (United States)

Lin, Ching-Jung; Smibert, Peter; Zhao, Xiaoyu; Hu, Jennifer F.; Ramroop, Johnny; Kellner, Stefanie M.; Benton, Matthew A.; Govind, Shubha; Dedon, Peter C.; Sternglanz, Rolf; Lai, Eric C.

2015-01-01

N6-threonylcarbamoyl-adenosine (t6A) is one of the few RNA modifications that is universally present in life. This modification occurs at high frequency at position 37 of most tRNAs that decode ANN codons, and stabilizes cognate anticodon–codon interactions. Nearly all genetic studies of the t6A pathway have focused on single-celled organisms. In this study, we report the isolation of an extensive allelic series in the Drosophila ortholog of the core t6A biosynthesis factor Kae1. kae1 hemizygous larvae exhibit decreases in t6A that correlate with allele strength; however, we still detect substantial t6A-modified tRNAs even during the extended larval phase of null alleles. Nevertheless, complementation of Drosophila Kae1 and other t6A factors in corresponding yeast null mutants demonstrates that these metazoan genes execute t6A synthesis. Turning to the biological consequences of t6A loss, we characterize prominent kae1 melanotic masses and show that they are associated with lymph gland overgrowth and ectopic generation of lamellocytes. On the other hand, kae1 mutants exhibit other phenotypes that reflect insufficient tissue growth. Interestingly, whole-tissue and clonal analyses show that strongly mitotic tissues such as imaginal discs are exquisitely sensitive to loss of kae1, whereas nonproliferating tissues are less affected. Indeed, despite overt requirements of t6A for growth of many tissues, certain strong kae1 alleles achieve and sustain enlarged body size during their extended larval phase. Our studies highlight tissue-specific requirements of the t6A pathway in a metazoan context and provide insights into the diverse biological roles of this fundamental RNA modification during animal development and disease. PMID:26516084

Dopamine modulation of avoidance behavior in Caenorhabditis elegans requires the NMDA receptor NMR-1.

Directory of Open Access Journals (Sweden)

Melvin Baidya

Full Text Available The nematode C. elegans utilizes a relatively simple neural circuit to mediate avoidance responses to noxious stimuli such as the volatile odorant octanol. This avoidance behavior is modulated by dopamine. cat-2 mutant animals that are deficient in dopamine biosynthesis have an increased response latency to octanol compared to wild type animals, and this defect can be fully restored with the application of exogenous dopamine. Because this avoidance behavior is mediated by glutamatergic signaling between sensory neurons and premotor interneurons, we investigated the genetic interactions between dopaminergic signaling and ionotropic glutamate receptors. cat-2 mutant animals lacking either the GLR-1 or GLR-2 AMPA/kainate receptors displayed an increased response latency to octanol, which could be restored via exogenous dopamine. However, whereas cat-2 mutant animals lacking the NMR-1 NMDA receptor had increased response latency to octanol they were insensitive to exogenous dopamine. Mutants that lacked both AMPA/kainate and NMDA receptors were also insensitive to exogenous dopamine. Our results indicate that dopamine modulation of octanol avoidance requires NMR-1, consistent with NMR-1 as a potential downstream signaling target for dopamine.

Identification of genes potentially involved in solute stress response in Sphingomonas wittichii RW1 by transposon mutant recovery

Directory of Open Access Journals (Sweden)

Edith eCoronado

2014-11-01

Full Text Available The term water stress refers to the effects of low water availability on microbial growth and physiology. Water availability has been proposed as a major constraint for the use of microorganisms in contaminated sites with the purpose of bioremediation. Sphingomonas wittichii RW1 is a bacterium capable of degrading the xenobiotic compounds dibenzofuran and dibenzo-p-dioxin, and has potential to be used for targeted bioremediation. The aim of the current work was to identify genes implicated in water stress in RW1 by means of transposon mutagenesis and mutant growth experiments. Conditions of low water potential were mimicked by adding NaCl to the growth media. Three different mutant selection or separation method were tested, which, however recovered different mutants. Recovered transposon mutants with poorer growth under salt-induced water stress carried insertions in genes involved in proline and glutamate biosynthesis, and further in a gene putatively involved in aromatic compound catabolism. Transposon mutants growing poorer on medium with lowered water potential also included ones that had insertions in genes involved in more general functions such as transcriptional regulation, elongation factor, cell division protein, RNA polymerase β or an aconitase.

Plant vegetative and animal cytoplasmic actins share functional competence for spatial development with protists.

Science.gov (United States)

Kandasamy, Muthugapatti K; McKinney, Elizabeth C; Roy, Eileen; Meagher, Richard B

2012-05-01

Actin is an essential multifunctional protein encoded by two distinct ancient classes of genes in animals (cytoplasmic and muscle) and plants (vegetative and reproductive). The prevailing view is that each class of actin variants is functionally distinct. However, we propose that the vegetative plant and cytoplasmic animal variants have conserved functional competence for spatial development inherited from an ancestral protist actin sequence. To test this idea, we ectopically expressed animal and protist actins in Arabidopsis thaliana double vegetative actin mutants that are dramatically altered in cell and organ morphologies. We found that expression of cytoplasmic actins from humans and even a highly divergent invertebrate Ciona intestinalis qualitatively and quantitatively suppressed the root cell polarity and organ defects of act8 act7 mutants and moderately suppressed the root-hairless phenotype of act2 act8 mutants. By contrast, human muscle actins were unable to support prominently any aspect of plant development. Furthermore, actins from three protists representing Choanozoa, Archamoeba, and green algae efficiently suppressed all the phenotypes of both the plant mutants. Remarkably, these data imply that actin's competence to carry out a complex suite of processes essential for multicellular development was already fully developed in single-celled protists and evolved nonprogressively from protists to plants and animals.

Mechanical touch responses of Arabidopsis TCH1-3 mutant roots on inclined hard-agar surface

Science.gov (United States)

Zha, Guodong; Wang, Bochu; Liu, Junyu; Yan, Jie; Zhu, Liqing; Yang, Xingyan

2016-01-01

The gravity-induced mechanical touch stimulus can affect plant root architecture. Mechanical touch responses of plant roots are an important aspect of plant root growth and development. Previous studies have reported that Arabidopsis TCH1-3 genes are involved in mechano-related events, how-ever, the physiological functions of TCH1-3 genes in Arabidopsis root mechanoresponses remain unclear. In the present study, we applied an inclined hard agar plate method to produce mechanical touch stimulus, and provided evidence that altered mechanical environment could influence root growth. Furthermore, tch1-3 Arabidopsis mutants were investigated on inclined agar surfaces to explore the functions of TCH1-3 genes on Arabidopsis root mechanoresponses. The results showed that two tch2 mutants, cml24-2 and cml24-4, exhibited significantly reduced root length, biased skewing, and decreased density of lateral root. In addition, primary root length and density of lateral root of tch3 (cml12-2) was significantly decreased on inclined agar surfaces. This study indicates that the tch2 and tch3 mutants are hypersensitive to mechanical touch stimulus, and TCH2 (CML24-2 and CML24-4) and TCH3 (CML12-2) genes may participate in the mechanical touch response of Arabidopsis roots.

Characterization of overwintering sites of the invasive brown marmorated stink bug in natural landscapes using human surveyors and detector canines.

Directory of Open Access Journals (Sweden)

Doo-Hyung Lee

Full Text Available Halyomorpha halys is an invasive species from Asia causing major economic losses in agricultural production in the mid-Atlantic region of the United States. Unlike other crop pests, H. halys is also well-known for nuisance problems in urban, suburban, and rural areas, as massive numbers of adults often invade human-made structures to overwinter inside protected environments. Research efforts have focused on populations in human-made structures while overwintering ecology of H. halys in natural landscapes is virtually unknown. We explored forested landscapes in the mid-Atlantic region to locate and characterize natural overwintering structures used by H. halys. We also evaluated the use of detector canines to locate overwintering H. halys to enhance the accuracy and efficiency of surveys. From these studies, we indentified shared characteristics of overwintering sites used by H. halys in natural landscapes. Overwintering H. halys were recovered from dry crevices in dead, standing trees with thick bark, particularly oak (Quercus spp. and locust (Robinia spp.; these characteristics were shared by 11.8% of all dead trees in surveyed landscapes. For trees with favorable characteristics, we sampled ∼20% of the total above-ground tree area and recovered 5.9 adults per tree from the trees with H. halys present. Two detector canines were successfully trained to recognize and detect the odor of adult H. halys yielding >84% accuracy in laboratory and semi-field trials. Detector canines also found overwintering H. halys under field conditions. In particular, overwintering H. halys were recovered only from dead trees that yielded positive indications from the canines and shared key tree characteristics established by human surveyors. The identified characteristics of natural overwintering sites of H. halys will serve as baseline information to establish crop economic risk levels posed by overwintering populations, and accordingly develop sustainable

Bacterio-opsin mutants of Halobacterium halobium

Science.gov (United States)

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

1983-01-01

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

Uvm mutants of Escherichia coli K 12 deficient in UV mutagenesis. Pt. 1

International Nuclear Information System (INIS)

Steinborn, G.

1978-01-01

Selection for defective reversion induction, after UV treatment of E. coli K 12, yielded uvm mutants. These mutants exhibited highly reduced or no UV mutability for all loci tested although they were moderately and normally mutable by X-rays and EMS, respectively. Uvm mutations confer only a slight sensitivity to killing by UV and X-rays and no clear sensitivity to the lethal effect of HN2, EMS or MMS. Growth and viability of untreated uvm cells were normal. The properties of uvm mutants are discussed in relation to those of other relevant mutant types and to some actual problems of induced mutagenesis. (orig.) 891 AJ [de

On the structural affinity of macromolecules with different biological properties: Molecular dynamics simulations of a series of TEM-1 mutants

Energy Technology Data Exchange (ETDEWEB)

Giampaolo, Alessia Di [Dipartimento di Scienze Fisiche e Chimiche, Universita’ degli Studi di l’Aquila, Via Vetoio snc, 67100 Coppito (AQ) (Italy); Mazza, Fernando [Department of Health Sciences, Univ. of L’Aquila, 67010 L’Aquila (Italy); Daidone, Isabella [Dipartimento di Scienze Fisiche e Chimiche, Universita’ degli Studi di l’Aquila, Via Vetoio snc, 67100 Coppito (AQ) (Italy); Amicosante, Gianfranco; Perilli, Mariagrazia [Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Università degli Studi di l’Aquila, Via Vetoio snc, 67100 Coppito (AQ) (Italy); Aschi, Massimiliano, E-mail: massimiliano.aschi@univaq.it [Dipartimento di Scienze Fisiche e Chimiche, Universita’ degli Studi di l’Aquila, Via Vetoio snc, 67100 Coppito (AQ) (Italy)

2013-07-12

Highlights: •We have performed molecular dynamics simulations of TEM-1 mutants. •Mutations effects on the mechanical properties are considered. •Mutants do not significantly alter the average enzymes structure. •Mutants produce sharp alterations in enzyme conformational repertoire. •Mutants also produce changes in the active site volume. -- Abstract: Molecular Dynamics simulations have been carried out in order to provide a molecular rationalization of the biological and thermodynamic differences observed for a class of TEM β-lactamases. In particular we have considered the TEM-1(wt), the single point mutants TEM-40 and TEM-19 representative of IRT and ESBL classes respectively, and TEM-1 mutant M182T, TEM-32 and TEM-20 which differ from the first three for the additional of M182T mutation. Results indicate that most of the thermodynamic, and probably biological behaviour of these systems arise from subtle effects which, starting from the alterations of the local interactions, produce drastic modifications of the conformational space spanned by the enzymes. The present study suggests that systems showing essentially the same secondary and tertiary structure may differentiate their chemical–biological activity essentially (and probably exclusively) on the basis of the thermal fluctuations occurring in their physiological environment.

On the structural affinity of macromolecules with different biological properties: Molecular dynamics simulations of a series of TEM-1 mutants

International Nuclear Information System (INIS)

Giampaolo, Alessia Di; Mazza, Fernando; Daidone, Isabella; Amicosante, Gianfranco; Perilli, Mariagrazia; Aschi, Massimiliano

2013-01-01

Highlights: •We have performed molecular dynamics simulations of TEM-1 mutants. •Mutations effects on the mechanical properties are considered. •Mutants do not significantly alter the average enzymes structure. •Mutants produce sharp alterations in enzyme conformational repertoire. •Mutants also produce changes in the active site volume. -- Abstract: Molecular Dynamics simulations have been carried out in order to provide a molecular rationalization of the biological and thermodynamic differences observed for a class of TEM β-lactamases. In particular we have considered the TEM-1(wt), the single point mutants TEM-40 and TEM-19 representative of IRT and ESBL classes respectively, and TEM-1 mutant M182T, TEM-32 and TEM-20 which differ from the first three for the additional of M182T mutation. Results indicate that most of the thermodynamic, and probably biological behaviour of these systems arise from subtle effects which, starting from the alterations of the local interactions, produce drastic modifications of the conformational space spanned by the enzymes. The present study suggests that systems showing essentially the same secondary and tertiary structure may differentiate their chemical–biological activity essentially (and probably exclusively) on the basis of the thermal fluctuations occurring in their physiological environment

Drastic anthocyanin increase in response to PAP1 overexpression in fls1 knockout mutant confers enhanced osmotic stress tolerance in Arabidopsis thaliana.

Science.gov (United States)

Lee, Won Je; Jeong, Chan Young; Kwon, Jaeyoung; Van Kien, Vu; Lee, Dongho; Hong, Suk-Whan; Lee, Hojoung

2016-11-01

KEY MESSAGE : pap1 - D/fls1ko double mutant plants that produce substantial amounts of anthocyanin show tolerance to abiotic stress. Anthocyanins are flavonoids that are abundant in various plants and have beneficial effects on both plants and humans. Many genes in flavonoid biosynthetic pathways have been identified, including those in the MYB-bHLH-WD40 (MBW) complex. The MYB gene Production of Anthocyanin Pigment 1 (PAP1) plays a particularly important role in anthocyanin accumulation. PAP1 expression in many plant systems strongly increases anthocyanin levels, resulting in a dark purple color in many plant organs. In this study, we generated double mutant plants that harbor fls1ko in the pap1-D background (i.e., pap1-D/fls1ko plants), to examine whether anthocyanins can be further enhanced by blocking flavonol biosynthesis under PAP1 overexpression. We also wanted to examine whether the increased anthocyanin levels contribute to defense against osmotic stresses. The pap1-D/fls1ko mutants accumulated higher anthocyanin levels than pap1-D plants in both control and sucrose-treated conditions. However, flavonoid biosynthesis genes were slightly down-regulated in the pap1-D/fls1ko seedlings as compared to their expression in pap1-D seedlings. We also report the performance of pap1-D/fls1ko seedlings in response to plant osmotic stresses.

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)

Identification and Map-Based Cloning of the Light-Induced Lesion Mimic Mutant 1 (LIL1) Gene in Rice.

Science.gov (United States)

Zhou, Qian; Zhang, Zhifei; Liu, Tiantian; Gao, Bida; Xiong, Xingyao

2017-01-01

The hypersensitive response (HR) is a mechanism by which plants prevent the spread of pathogen. Despite extensive study, the molecular mechanisms underlying HR remain poorly understood. Lesion mimic mutants (LMMs), such as LIL1 that was identified in an ethylmethane sulfonate mutagenized population of Indica rice ( Oryza sativa L. ssp. Indica ) 93-11, can be used to study the HR. Under natural field conditions, the leaves of LIL1 mutant plants exhibited light-induced, small, rust-red lesions that first appeared at the leaf tips and subsequently expanded throughout the entire leaf blade to the leaf sheath. Histochemical staining indicated that LIL1 lesions displayed an abnormal accumulation of reactive oxygen species (ROS) and resulted from programmed cell death (PCD). The LIL1 mutants also displayed increased expression of defense-related genes and enhanced resistance to rice blast fungus ( Magnaporthe grisea ). Genetic analysis showed that mutation of LIL1 created a semi-dominant allele. Using 1,758 individuals in the F 2 population, LIL1 was mapped in a 222.3 kb region on the long arm of chromosome 7. That contains 12 predicted open reading frames (ORFs). Sequence analysis of these 12 candidate genes revealed a G to A base substitution in the fourth exon of LOC_Os07g30510, a putative cysteine-rich receptor-like kinase (CRK), which led to an amino acid change (Val 429 to Ile) in the LIL1 protein. Comparison of the transcript accumulation of the 12 candidate genes between LIL1 and 93-11 revealed that LOC_Os07g30510 was up-regulated significantly in LIL1 . Overexpression of the LOC_Os07g30510 gene from LIL1 induced a LIL1 -like lesion phenotype in Nipponbare. Thus, LIL1 is a novel LMM in rice that will facilitate the further study of the molecular mechanisms of HR and the rice blast resistance.

Reduced starch granule number per chloroplast in the dpe2/phs1 mutant is dependent on initiation of starch degradation.

Science.gov (United States)

Malinova, Irina; Fettke, Joerg

2017-01-01

An Arabidopsis double knock-out mutant lacking cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) revealed a dwarf-growth phenotype, reduced starch content, an uneven distribution of starch within the plant rosette, and a reduced number of starch granules per chloroplast under standard growth conditions. In contrast, the wild type contained 5-7 starch granules per chloroplast. Mature and old leaves of the double mutant were essentially starch free and showed plastidial disintegration. Several analyses revealed that the number of starch granules per chloroplast was affected by the dark phase. So far, it was unclear if it was the dark phase per se or starch degradation in the dark that was connected to the observed decrease in the number of starch granules per chloroplast. Therefore, in the background of the double mutant dpe2/phs1, a triple mutant was generated lacking the initial starch degrading enzyme glucan, water dikinase (GWD). The triple mutant showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to wild type. However, starch granule morphology was only slightly affected by the lack of GWD as in the triple mutant and, like in dpe2/phs1, more spherical starch granules were observed. The characterized triple mutant was discussed in the context of the generation of starch granules and the formation of starch granule morphology.

Isoenzymes performance of some rice varieties and their mutants

International Nuclear Information System (INIS)

Winarno, Ermin; Suliwarno, Ambyah; Ismachin, M.

1992-01-01

Isoenzymes performance of some rice varieties and their mutants. Genetics studies on alcohol dehydrogenase, malic enzyme, peroxidase, acid phosphase, and aminopeptidase isoenzymes were carried out on several groups of rice varieties and their mutant lines. The first groups consisted of Atomita I, Pelita I/1, A227/5, Mudgo, TN-1, and IR-26. The second group was Cisadane variety and its five mutants, namely OBS 18, OBS 208, OBS 297, OBS 306, and OBS 330. The third group was mutants line 627-10-3 and its mutants, namely 1063, 1066, 1067, 1076, and 1090. Isoenzymes extracts of the rice leaves were fractionated using polyacrylamide gel disc electrophoresis. The pattern of acid phosphate isoenzyme shows the specific character of rice mutants susceptible to brown plant hopper biotype 1. The gene(s) controlling malic enzyme in Cisadane's mutants is (are) estimated more resistant toward gamma irradiation than gene(s) responsible for controlling the other enzymes. Generally, the isoenzymes zymograms show that gene(s) controlling the mutants enzyme have undergone mutation. This case is shown by the changes of Rm value, as well as the amount and intensity of mutants bands. (authors). 7 refs., 7 figs

Zebrafish model of tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of mutant tuberin

Directory of Open Access Journals (Sweden)

Seok-Hyung Kim

2011-03-01

Tuberous sclerosis complex (TSC is an autosomal dominant disease caused by mutations in either the TSC1 (encodes hamartin or TSC2 (encodes tuberin genes. Patients with TSC have hamartomas in various organs throughout the whole body, most notably in the brain, skin, eye, heart, kidney and lung. To study the development of hamartomas, we generated a zebrafish model of TSC featuring a nonsense mutation (vu242 in the tsc2 gene. This tsc2vu242 allele encodes a truncated Tuberin protein lacking the GAP domain, which is required for inhibition of Rheb and of the TOR kinase within TORC1. We show that tsc2vu242 is a recessive larval-lethal mutation that causes increased cell size in the brain and liver. Greatly elevated TORC1 signaling is observed in tsc2vu242/vu242 homozygous zebrafish, and is moderately increased in tsc2vu242/+ heterozygotes. Forebrain neurons are poorly organized in tsc2vu242/vu242 homozygous mutants, which have extensive gray and white matter disorganization and ectopically positioned cells. Genetic mosaic analyses demonstrate that tsc2 limits TORC1 signaling in a cell-autonomous manner. However, in chimeric animals, tsc2vu242/vu242 mutant cells also mislocalize wild-type host cells in the forebrain in a non-cell-autonomous manner. These results demonstrate a highly conserved role of tsc2 in zebrafish and establish a new animal model for studies of TSC. The finding of a non-cell-autonomous function of mutant cells might help explain the formation of brain hamartomas and cortical malformations in human TSC.

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

Cytogenetic characteristics of soft wheat mutants under x-irradiation

International Nuclear Information System (INIS)

Shakaryan, Zh.O.; Avakyan, V.A.; Amirbekyan, V.A.

1981-01-01

Radiosensitivity of induced mutants of soft wheat is studied by criteria of frequency and character of changes in 1 and 2 divisions of meiosis. Two constant induced mutant forms of soft wheat were investigated. Mutant lines of squareheads with red ear (re) and erectoids 37/1 were obtained by X-ray irradiating hydride seeds F 1 of hybride combination of Alty-Agach Awnless 1. Seeds of mutants and initial kinds were exposed to X-rays at a dose of 10 kR. A conclusion may be drawn on the basis of studying the meiosis process in mutants and initial kinds of soft wheat on X-ray radiation that the mutants are more radiosensitive. This testifies to that that the induced mutants of soft wheat represent new genotypes in comparison with the initial kinds and differ from the latter not only in morphological characters but in the reaction norm with respect to external medium factors, i.e. the limit of possible changeability of the genotype has been extended [ru

9 CFR 107.1 - Veterinary practitioners and animal owners.

Science.gov (United States)

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Veterinary practitioners and animal... FROM PREPARATION PURSUANT TO AN UNSUSPENDED AND UNREVOKED LICENSE § 107.1 Veterinary practitioners and...)(1) Products prepared by a veterinary practitioner (veterinarian) solely for administration to...

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)

Intermediate rough Brucella abortus S19Δper mutant is DIVA enable, safe to pregnant guinea pigs and confers protection to mice.

Science.gov (United States)

Lalsiamthara, Jonathan; Gogia, Neha; Goswami, Tapas K; Singh, R K; Chaudhuri, Pallab

2015-05-21

Brucella abortus S19 is a smooth strain used as live vaccine against bovine brucellosis. Smooth lipopolysaccharide (LPS) is responsible for its residual virulence and serological interference. Rough mutants defective of LPS are more attenuated but confers lower level of protection. We describe a modified B. abortus S19 strain, named as S19Δper, which exhibits intermediate rough phenotype with residual O-polysaccharide (OPS). Deletion of perosamine synthetase gene resulted in substantial attenuation of S19Δper mutant without affecting immunogenic properties. It mounted strong immune response in Swiss albino mice and conferred protection similar to S19 vaccine. Immunized mice produced higher levels of IFN-γ, IgG2a and thus has immune response inclined towards Th1 cell mediated immunity. Sera from immunized animals did not show agglutination reaction with RBPT antigen and thus could serve as DIVA (Differentiating Infected from Vaccinated Animals) vaccine. S19Δper mutant displayed more susceptibility to serum complement mediated killing and sensitivity to polymyxin B. Pregnant guinea pigs injected with S19Δper mutant completed full term of pregnancy and did not cause abortion, still birth or birth of weak offspring. S19Δper mutant with intermediate rough phenotype displayed remarkable resemblance to S19 vaccine strain with improved properties of safety, immunogenicity and DIVA capability for control of bovine brucellosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

IDH1 mutant negative ganglioglioma progression to gliosarcoma

Directory of Open Access Journals (Sweden)

Liming Qiu

2017-09-01

Full Text Available Gangliogliomas comprise of less than 1% of all primary brain tumours. Malignant transformation is rare. Correspondingly, secondary gliosarcomas, especially in the absence of previous irradiation exposure is too, exceedingly rare. Recent evidence supports the use of mutant IDH1 testing in suspected adult gangliogliomas as a distinguishing feature between ganglioglioma and early diffuse astrocytoma. The authors report a case of a ganglioglioma (WHO I in the cingulate gyrus that underwent malignant transformation and propagated into a multi-lesional gliosarcoma (WHO IV glioblastoma variant. A previously well 59-year-old lady presented with first-onset focal seizures secondary to a left cingulate gyrus lesion. Stereotactic biopsy of the lesion demonstrated an IDH1 (R132H ganglioglioma. The patient remained well until a routine follow-up MRI performed 36 months later showed significant progression of the initial lesion and a new, separate lesion in the left temporoparietal lobe. Surgical excision was performed. The final histology reported a gliosarcoma. In view of the unusual findings, the case is reviewed corroboratively with current disease understanding.

Characterization of developmental immature fiber (im) mutant and Texas Marker-1 (TM-1) cotton fibers by Attenuated Total Reflection Fourier Transform Infrared (ATR FT-IR) spectroscopy

Science.gov (United States)

The immature fiber (im) mutant is one type of cotton fiber mutants with unique characteristics of non-fluffy cotton bolls. Compared to its near-isogenic wild type Texas Marker-1 (TM-1), im fiber has thin secondary cell wall and is less mature. In this work, we applied the previously proposed princip...

Lipase production from a wild (LPF-5) and a mutant (HN1) strain of ...

African Journals Online (AJOL)

Lipase production from a wild (LPF-5) and a mutant (HN1) strain of Aspergillus niger. ... Several physical parameters (carbon source, nitrogen source, pH, ... for the development of industrial biotechnology for production of extracellular lipase.

Phenotypic effects of repeated psychosocial stress during adolescence in mice mutant for the schizophrenia risk gene neuregulin-1: a putative model of gene × environment interaction.

Science.gov (United States)

Desbonnet, Lieve; O'Tuathaigh, Colm; Clarke, Gerard; O'Leary, Claire; Petit, Emilie; Clarke, Niamh; Tighe, Orna; Lai, Donna; Harvey, Richard; Cryan, John F; Dinan, Timothy G; Waddington, John L

2012-05-01

There is a paucity of animal models by which the contributions of environmental and genetic factors to the pathobiology of psychosis can be investigated. This study examined the individual and combined effects of chronic social stress during adolescence and deletion of the schizophrenia risk gene neuregulin-1 (NRG1) on adult mouse phenotype. Mice were exposed to repeated social defeat stress during adolescence and assessed for exploratory behaviour, working memory, sucrose preference, social behaviour and prepulse inhibition in adulthood. Thereafter, in vitro cytokine responses to mitogen stimulation and corticosterone inhibition were assayed in spleen cells, with measurement of cytokine and brain-derived neurotrophic factor (BDNF) mRNA in frontal cortex, hippocampus and striatum. NRG1 mutants exhibited hyperactivity, decreased anxiety, impaired sensorimotor gating and reduced preference for social novelty. The effects of stress on exploratory/anxiety-related parameters, spatial working memory, sucrose preference and basal cytokine levels were modified by NRG1 deletion. Stress also exerted varied effect on spleen cytokine response to concanavalin A and brain cytokine and BDNF mRNA expression in NRG1 mutants. The experience of psychosocial stress during adolescence may trigger further pathobiological features that contribute to the development of schizophrenia, particularly in those with underlying NRG1 gene abnormalities. This model elaborates the importance of gene × environment interactions in the etiology of schizophrenia. Copyright © 2012 Elsevier Inc. All rights reserved.

The Swedish mutant barley collection

Energy Technology Data Exchange (ETDEWEB)

NONE

1989-07-01

Full text: The Swedish mutation research programme in barley began about 50 years ago and has mainly been carried out at Svaloev in co-operation with the institute of Genetics at the University of Lund. The collection has been produced from different Swedish high-yielding spring barley varieties, using the following mutagens: X-rays, neutrons, several organic chemical compounds such as ethyleneimine, several sulfonate derivatives and the inorganic chemical mutagen sodium azide. Nearly 10,000 barley mutants are stored in the Nordic Gene Bank and documented in databases developed by Udda Lundquist, Svaloev AB. The collection consists of the following nine categories with 94 different types of mutants: 1. Mutants with changes in the spike and spikelets; 2. Changes in culm length and culm composition; 3. Changes in growth types; 4. Physiological mutants; 5. Changes in awns; 6. Changes in seed size and shape; 7. Changes in leaf blades; 8. Changes in anthocyanin and colour; 9. Resistance to barley powdery mildew. Barley is one of the most thoroughly investigated crops in terms of induction of mutations and mutation genetics. So far, about half of the mutants stored at the Nordic Gene Bank, have been analysed genetically; They constitute, however, only a minority of the 94 different mutant types. The genetic analyses have given valuable insights into the mutation process but also into the genetic architecture of various characters. A number of mutants of two-row barley have been registered and commercially released. One of the earliest released, Mari, an early maturing, daylength neutral, straw stiff mutant, is still grown in Iceland. The Swedish mutation material has been used in Sweden, but also in other countries, such as Denmark, Germany, and USA, for various studies providing a better understanding of the barley genome. The collection will be immensely valuable for future molecular genetical analyses of clone mutant genes. (author)

The Swedish mutant barley collection

International Nuclear Information System (INIS)

1989-01-01

Full text: The Swedish mutation research programme in barley began about 50 years ago and has mainly been carried out at Svaloev in co-operation with the institute of Genetics at the University of Lund. The collection has been produced from different Swedish high-yielding spring barley varieties, using the following mutagens: X-rays, neutrons, several organic chemical compounds such as ethyleneimine, several sulfonate derivatives and the inorganic chemical mutagen sodium azide. Nearly 10,000 barley mutants are stored in the Nordic Gene Bank and documented in databases developed by Udda Lundquist, Svaloev AB. The collection consists of the following nine categories with 94 different types of mutants: 1. Mutants with changes in the spike and spikelets; 2. Changes in culm length and culm composition; 3. Changes in growth types; 4. Physiological mutants; 5. Changes in awns; 6. Changes in seed size and shape; 7. Changes in leaf blades; 8. Changes in anthocyanin and colour; 9. Resistance to barley powdery mildew. Barley is one of the most thoroughly investigated crops in terms of induction of mutations and mutation genetics. So far, about half of the mutants stored at the Nordic Gene Bank, have been analysed genetically; They constitute, however, only a minority of the 94 different mutant types. The genetic analyses have given valuable insights into the mutation process but also into the genetic architecture of various characters. A number of mutants of two-row barley have been registered and commercially released. One of the earliest released, Mari, an early maturing, daylength neutral, straw stiff mutant, is still grown in Iceland. The Swedish mutation material has been used in Sweden, but also in other countries, such as Denmark, Germany, and USA, for various studies providing a better understanding of the barley genome. The collection will be immensely valuable for future molecular genetical analyses of clone mutant genes. (author)

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.

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

Directory of Open Access Journals (Sweden)

Marcelo R Vargas

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

Isolation of New Gravitropic Mutants under Hypergravity Conditions.

Science.gov (United States)

Mori, Akiko; Toyota, Masatsugu; Shimada, Masayoshi; Mekata, Mika; Kurata, Tetsuya; Tasaka, Masao; Morita, Miyo T

2016-01-01

Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upward. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in Arabidopsis stems and hypocotyls are capable of sensing gravity (i.e., statocytes). In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using next-generation sequencing (NGS) and single nucleotide polymorphism (SNP)-based markers. Using the endodermal-amyloplast less 1 ( eal1 ) mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g) restored the reduced gravity responsiveness in eal1 hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the eal1 background. Using the new screening system, we successfully isolated six ene ( enhancer of eal1 ) mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in Arabidopsis .

Effect of Bacillus subtilis mutants on growth performance of piglets fed tryptophan- and valine-deficient diets

DEFF Research Database (Denmark)

Nørgaard, Jan Værum; Canibe, Nuria; Assadi Soumeh, Elham

2016-01-01

The objective was to determine the concentration of l-Trp and l-Val to be substituted by feeding piglets Bacillus subtilis strains developed to overproduce Trp (B. subtilis Trp mutant [BsTrp]) and Val (B. subtilis Val mutant [BsVal]) and by using equations obtained in 3 dose–response studies......-Val per kilogram feed using curvilinear plateau and broken-line equations obtained by modeling the 6 AA levels. Bacillus subtilis Val mutant increased animal performance corresponding to 0.88 and 0.39 g l-Leu and 0.17 and 0.44 g l-Val per kilogram feed for 10x and 100x doses, respectively. Bacillus...... subtilis Trp mutant was equivalent to 0.02 and 0.11 g l-Trp/kg feed for 10x and 100x doses, respectively. Bacillus subtilis Val mutant (10x dose) increased (P Bacillus subtilis Trp mutant tended (P = 0.06) to increase Trp plasma concentrations...

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.

Indolyl aryl sulfones (IASs): development of highly potent NNRTIs active against wt-HIV-1 and clinically relevant drug resistant mutants.

Science.gov (United States)

Silvestri, Romano; Artico, Marino

2005-01-01

Indolyl aryl sulfones (IASs) are a potent class of NNRTIs developed from L-737,126, a lead agent discovered by Merck AG. IAS derivatives are endowed with inhibitory activities against wt HIV-1 in the low nanomolar concentration range. Introduction of two methyl groups at positions 3 and 5 of the phenyl ring of the aryl sulfonyl moiety furnished IAS derivatives such as 5-chloro- or 5-bromo-3-[(3,5-dimethylphenyl)sulfonyl]indole-2-carboxyamide, which showed very potent and selective anti-HIV-1 activity against some mutants carrying NNRTI resistant mutations at positions 103 and 181 of the reverse transcriptase. IAS derivatives bearing 2-hydroxyethylcarboxyamide or 2-hydroxyethylcarboxyhydrazide groups at position 2 of the indole nucleus were more active than L-737,126 against the K103N-Y181C double mutant. A great improvement of antiviral activity against wt HIV-1 and resistant mutants was obtained by coupling 1-3 simple amino acids, such as glycine and alanine, in sequence, with the 3-[(3,5-dimethylphenyl)sulfonyl]-1H-indole-2-carbonyl moiety. The transformation of the chain terminus into amide or hydrazide, produced short peptides with high selectivity and potent activity against wt HIV-1, and the viral mutants Y181C, K103N-Y181C and EFV(R). IAS having two halogen atoms at the indole showed potent inhibitory activity against the Y181C and the EFV(R) resistant mutant strains. In particular, the introduction of a fluorine atom at position 4 of the indole ring notably contributed to improve the antiviral activities against both wt and the related resistant mutants. 5-Nitro-IASs were highly active against wt HIV-1 and exhibited low cytotoxicity. Experimental data highlighted the class IAS derivatives as promising candidates for clinical trials.

Selenolate complexes of CYP101 and the heme-bound hHO-1/H25A proximal cavity mutant.

Science.gov (United States)

Jiang, Yongying; Ortiz de Montellano, Paul R

2008-05-05

Thiolate and selenolate complexes of CYP101 (P450cam) and the H25A proximal cavity mutant of heme-bound human heme oxygenase-1 (hHO-1) have been examined by UV-vis spectroscopy. Both thiolate and selenolate ligands bound to the heme distal side in CYP101 and gave rise to characteristic hyperporphyrin spectra. Thiolate ligands also bound to the proximal side of the heme in the cavity created by the H25A mutation in hHO-1, giving a Soret absorption similar to that of the H25C hHO-1 mutant. Selenolate ligands also bound to this cavity mutant under anaerobic conditions but reduced the heme iron to the ferrous state, as shown by the formation of a ferrous CO complex. Under aerobic conditions, the selenolate ligand but not the thiolate ligand was rapidly oxidized. These results indicate that selenocysteine-coordinated heme proteins will not be stable species in the absence of a redox potential stabilizing effect.

Structural Basis for a Switch in Receptor Binding Specificity of Two H5N1 Hemagglutinin Mutants

Directory of Open Access Journals (Sweden)

Xueyong Zhu

2015-11-01

Full Text Available Avian H5N1 influenza viruses continue to spread in wild birds and domestic poultry with sporadic infection in humans. Receptor binding specificity changes are a prerequisite for H5N1 viruses and other zoonotic viruses to be transmitted among humans. Previous reported hemagglutinin (HA mutants from ferret-transmissible H5N1 viruses of A/Vietnam/1203/2004 and A/Indonesia/5/2005 showed slightly increased, but still very weak, binding to human receptors. From mutagenesis and glycan array studies, we previously identified two H5N1 HA mutants that could more effectively switch receptor specificity to human-like α2-6-linked sialosides with avidity comparable to wild-type H5 HA binding to avian-like α2-3-linked sialosides. Here, crystal structures of these two H5 HA mutants free and in complex with human and avian glycan receptor analogs reveal the structural basis for their preferential binding to human receptors. These findings suggest continuous surveillance should be maintained to monitor and assess human-to-human transmission potential of H5N1 viruses.

Spectrum of induced floral mutants in Petunia

International Nuclear Information System (INIS)

Padmaja, V.; Sudhakar, P.

1987-01-01

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

An Inactive Geminin Mutant That Binds Cdt1

Directory of Open Access Journals (Sweden)

Marissa Suchyta

2015-05-01

Full Text Available The initiation of DNA replication is tightly regulated in order to ensure that the genome duplicates only once per cell cycle. In vertebrate cells, the unstable regulatory protein Geminin prevents a second round of DNA replication by inhibiting the essential replication factor Cdt1. Cdt1 recruits mini-chromosome maintenance complex (MCM2-7, the replication helicase, into the pre-replication complex (pre-RC at origins of DNA replication. The mechanism by which Geminin inhibits MCM2-7 loading by Cdt1 is incompletely understood. The conventional model is that Geminin sterically hinders a direct physical interaction between Cdt1 and MCM2-7. Here, we describe an inactive missense mutant of Geminin, GemininAWA, which binds to Cdt1 with normal affinity yet is completely inactive as a replication inhibitor even when added in vast excess. In fact, GemininAWA can compete with GemininWT for binding to Cdt1 and prevent it from inhibiting DNA replication. GemininAWA does not inhibit the loading of MCM2-7 onto DNA in vivo, and in the presence of GemininAWA, nuclear DNA is massively over-replicated within a single S phase. We conclude that Geminin does not inhibit MCM loading by simple steric interference with a Cdt1-MCM2-7 interaction but instead works by a non-steric mechanism, possibly by inhibiting the histone acetyltransferase HBO1.

Bending patterns of chlamydomonas flagella: III. A radial spoke head deficient mutant and a central pair deficient mutant.

Science.gov (United States)

Brokaw, C J; Luck, D J

1985-01-01

Flash photomicrography at frequencies up to 300 Hz and computer-assisted image analysis have been used to obtain parameters describing the flagellar bending patterns of mutants of Chlamydomonas reinhardtii. All strains contained the uni1 mutation, to facilitate photography. The radial spoke head deficient mutant pf17, and the central pair deficient mutant, pf15, in combination with suppressor mutations that restore motility without restoring the ultrastructural or biochemical deficiencies, both generate forward mode bending patterns with increased shear amplitude and decreased asymmetry relative to the "wild-type" uni1 flagella described previously. In the reverse beating mode, the suppressed pf17 mutants generate reverse bending patterns with large shear amplitudes. Reverse beating of the suppressed pf15 mutants is rare. There is a reciprocal relationship between increased shear amplitude and decreased beat frequency, so that the velocity of sliding between flagellar microtubules is not increased by an increase in shear amplitude. The suppressor mutations alone cause decreased frequency and sliding velocity in both forward and reverse mode beating, with little change in shear amplitude or symmetry.

Developmental delay in a Streptomyces venezuelae glgE null mutant is associated with the accumulation of α-maltose 1-phosphate.

Science.gov (United States)

Miah, Farzana; Bibb, Maureen J; Barclay, J Elaine; Findlay, Kim C; Bornemann, Stephen

2016-07-01

The GlgE pathway is thought to be responsible for the conversion of trehalose into a glycogen-like α-glucan polymer in bacteria. Trehalose is first converted to maltose, which is phosphorylated by maltose kinase Pep2 to give α-maltose 1-phosphate. This is the donor substrate of the maltosyl transferase GlgE that is known to extend α-1,4-linked maltooligosaccharides, which are thought to be branched with α-1,6 linkages. The genome of Streptomyces venezuelae contains all the genes coding for the GlgE pathway enzymes but none of those of related pathways, including glgC and glgA of the glycogen pathway. This provides an opportunity to study the GlgE pathway in isolation. The genes of the GlgE pathway were upregulated at the onset of sporulation, consistent with the known timing of α-glucan deposition. A constructed ΔglgE null mutant strain was viable but showed a delayed developmental phenotype when grown on maltose, giving less cell mass and delayed sporulation. Pre-spore cells and spores of the mutant were frequently double the length of those of the wild-type, implying impaired cross-wall formation, and spores showed reduced tolerance to stress. The mutant accumulated α-maltose 1-phosphate and maltose but no α-glucan. Therefore, the GlgE pathway is necessary and sufficient for polymer biosynthesis. Growth of the ΔglgE mutant on galactose and that of a Δpep2 mutant on maltose were analysed. In both cases, neither accumulation of α-maltose 1-phosphate/α-glucan nor a developmental delay was observed. Thus, high levels of α-maltose 1-phosphate are responsible for the developmental phenotype of the ΔglgE mutant, rather than the lack of α-glucan.

Cerebrovascular defects in Foxc1 mutants correlate with aberrant WNT and VEGF-A pathways downstream of retinoic acid from the meninges.

Science.gov (United States)

Mishra, Swati; Choe, Youngshik; Pleasure, Samuel J; Siegenthaler, Julie A

2016-12-01

Growth and maturation of the cerebrovasculature is a vital event in neocortical development however mechanisms that control cerebrovascular development remain poorly understood. Mutations in or deletions that include the FOXC1 gene are associated with congenital cerebrovascular anomalies and increased stroke risk in patients. Foxc1 mutant mice display severe cerebrovascular hemorrhage at late gestational ages. While these data demonstrate Foxc1 is required for cerebrovascular development, its broad expression in the brain vasculature combined with Foxc1 mutant's complex developmental defects have made it difficult to pinpoint its function(s). Using global and conditional Foxc1 mutants, we find 1) significant cerebrovascular growth defects precede cerebral hemorrhage and 2) expression of Foxc1 in neural crest-derived meninges and brain pericytes, though not endothelial cells, is required for normal cerebrovascular development. We provide evidence that reduced levels of meninges-derived retinoic acid (RA), caused by defects in meninges formation in Foxc1 mutants, is a major contributing factor to the cerebrovascular growth defects in Foxc1 mutants. We provide data that suggests that meninges-derived RA ensures adequate growth of the neocortical vasculature via regulating expression of WNT pathway proteins and neural progenitor derived-VEGF-A. Our findings offer the first evidence for a role of the meninges in brain vascular development and provide new insight into potential causes of cerebrovascular defects in patients with FOXC1 mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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

The Arabidopsis cax1 mutant exhibits impaired ion homeostasis, development, and hormonal responses and reveals interplay among vacuolar transporters.

Science.gov (United States)

Cheng, Ning-Hui; Pittman, Jon K; Barkla, Bronwyn J; Shigaki, Toshiro; Hirschi, Kendal D

2003-02-01

The Arabidopsis Ca(2+)/H(+) transporter CAX1 (Cation Exchanger1) may be an important regulator of intracellular Ca(2+) levels. Here, we describe the preliminary localization of CAX1 to the tonoplast and the molecular and biochemical characterization of cax1 mutants. We show that these mutants exhibit a 50% reduction in tonoplast Ca(2+)/H(+) antiport activity, a 40% reduction in tonoplast V-type H(+)-translocating ATPase activity, a 36% increase in tonoplast Ca(2+)-ATPase activity, and increased expression of the putative vacuolar Ca(2+)/H(+) antiporters CAX3 and CAX4. Enhanced growth was displayed by the cax1 lines under Mn(2+) and Mg(2+) stress conditions. The mutants exhibited altered plant development, perturbed hormone sensitivities, and altered expression of an auxin-regulated promoter-reporter gene fusion. We propose that CAX1 regulates myriad plant processes and discuss the observed phenotypes with regard to the compensatory alterations in other transporters.

The Arabidopsis cax1 Mutant Exhibits Impaired Ion Homeostasis, Development, and Hormonal Responses and Reveals Interplay among Vacuolar Transporters

Science.gov (United States)

Cheng, Ning-Hui; Pittman, Jon K.; Barkla, Bronwyn J.; Shigaki, Toshiro; Hirschi, Kendal D.

2003-01-01

The Arabidopsis Ca2+/H+ transporter CAX1 (Cation Exchanger1) may be an important regulator of intracellular Ca2+ levels. Here, we describe the preliminary localization of CAX1 to the tonoplast and the molecular and biochemical characterization of cax1 mutants. We show that these mutants exhibit a 50% reduction in tonoplast Ca2+/H+ antiport activity, a 40% reduction in tonoplast V-type H+-translocating ATPase activity, a 36% increase in tonoplast Ca2+-ATPase activity, and increased expression of the putative vacuolar Ca2+/H+ antiporters CAX3 and CAX4. Enhanced growth was displayed by the cax1 lines under Mn2+ and Mg2+ stress conditions. The mutants exhibited altered plant development, perturbed hormone sensitivities, and altered expression of an auxin-regulated promoter-reporter gene fusion. We propose that CAX1 regulates myriad plant processes and discuss the observed phenotypes with regard to the compensatory alterations in other transporters. PMID:12566577

Comparison of the crystal structure and function to wild-type and His25Ala mutant human heme oxygenase-1.

Science.gov (United States)

Zhou, Wen-Pu; Zhong, Wen-Wei; Zhang, Xue-Hong; Ding, Jian-Ping; Zhang, Zi-Li; Xia, Zhen-Wei

2009-03-01

Human heme oxygenase-1 (hHO-1) is a rate-limiting enzyme in heme metabolism. It regulates serum bilirubin level. Site-directed mutagenesis studies indicate that the proximal residue histidine 25 (His25) plays a key role in hHO-1 activity. A highly purified hHO-1 His25Ala mutant was generated and crystallized with a new expression system. The crystal structure of the mutant was determined by X-ray diffraction technology and molecular replacement at the resolution of 2.8 A, and the model of hHO-1 His25Ala mutant was refined. The final crystallographic and free R factors were 0.245 and 0.283, respectively. The standard bond length deviation was 0.007 A, and the standard bond angle deviation was 1.3 degrees . The mutation of His25 to Ala led to an empty pocket underneath the ferric ion in the heme, leading to loss of binding iron ligand. Although this did not cause an overall structural change, the enzymatic activity of the mutant hHO-1 was reduced by 90%. By supplementing imidazole, the HO-1 activity was restored approximately 90% to its normal level. These data suggest that Ala25 remains unchanged in the structure compared to His25, but the important catalytic function of hHO-1 is lost. Thus, it appears that His25 is a crucial residue for proper hHO-1 catalysis.

Isolation of new gravitropic mutants under hypergravity conditions

Directory of Open Access Journals (Sweden)

Akiko Mori

2016-09-01

Full Text Available Forward genetics is a powerful approach used to link genotypes and phenotypes, and mutant screening/analysis has provided deep insights into many aspects of plant physiology. Gravitropism is a tropistic response in plants, in which hypocotyls and stems sense the direction of gravity and grow upwards. Previous studies of gravitropic mutants have suggested that shoot endodermal cells in Arabidopsis stems and hypocotyls are capable of sensing gravity (i.e., statocytes. In the present study, we report a new screening system using hypergravity conditions to isolate enhancers of gravitropism mutants, and we also describe a rapid and efficient genome mapping method, using Next-Generation Sequencing (NGS and Single Nucleotide Polymorphism (SNP-based markers. Using the endodermal-amyloplast less 1 (eal1 mutant, which exhibits defective development of endodermal cells and gravitropism, we found that hypergravity (10 g restored the reduced gravity responsiveness in eal1 hypocotyls and could, therefore, be used to obtain mutants with further reduction in gravitropism in the eal1 background. Using the new screening system, we successfully isolated six ene (enhancer of eal1 mutants that exhibited little or no gravitropism under hypergravity conditions, and using NGS and map-based cloning with SNP markers, we narrowed down the potential causative genes, which revealed a new genetic network for shoot gravitropism in Arabidopsis.

Plant Vegetative and Animal Cytoplasmic Actins Share Functional Competence for Spatial Development with Protists[W][OA

Science.gov (United States)

Kandasamy, Muthugapatti K.; McKinney, Elizabeth C.; Roy, Eileen; Meagher, Richard B.

2012-01-01

Actin is an essential multifunctional protein encoded by two distinct ancient classes of genes in animals (cytoplasmic and muscle) and plants (vegetative and reproductive). The prevailing view is that each class of actin variants is functionally distinct. However, we propose that the vegetative plant and cytoplasmic animal variants have conserved functional competence for spatial development inherited from an ancestral protist actin sequence. To test this idea, we ectopically expressed animal and protist actins in Arabidopsis thaliana double vegetative actin mutants that are dramatically altered in cell and organ morphologies. We found that expression of cytoplasmic actins from humans and even a highly divergent invertebrate Ciona intestinalis qualitatively and quantitatively suppressed the root cell polarity and organ defects of act8 act7 mutants and moderately suppressed the root-hairless phenotype of act2 act8 mutants. By contrast, human muscle actins were unable to support prominently any aspect of plant development. Furthermore, actins from three protists representing Choanozoa, Archamoeba, and green algae efficiently suppressed all the phenotypes of both the plant mutants. Remarkably, these data imply that actin’s competence to carry out a complex suite of processes essential for multicellular development was already fully developed in single-celled protists and evolved nonprogressively from protists to plants and animals. PMID:22589468

DAF-16 and Δ9 desaturase genes promote cold tolerance in long-lived Caenorhabditis elegans age-1 mutants.

Directory of Open Access Journals (Sweden)

Fiona R Savory

Full Text Available In Caenorhabditis elegans, mutants of the conserved insulin/IGF-1 signalling (IIS pathway are long-lived and stress resistant due to the altered expression of DAF-16 target genes such as those involved in cellular defence and metabolism. The three Δ(9 desaturase genes, fat-5, fat-6 and fat-7, are included amongst these DAF-16 targets, and it is well established that Δ(9 desaturase enzymes play an important role in survival at low temperatures. However, no assessment of cold tolerance has previously been reported for IIS mutants. We demonstrate that long-lived age-1(hx546 mutants are remarkably resilient to low temperature stress relative to wild type worms, and that this is dependent upon daf-16. We also show that cold tolerance following direct transfer to low temperatures is increased in wild type worms during the facultative, daf-16 dependent, dauer stage. Although the cold tolerant phenotype of age-1(hx546 mutants is predominantly due to the Δ(9 desaturase genes, additional transcriptional targets of DAF-16 are also involved. Surprisingly, survival of wild type adults following a rapid temperature decline is not dependent upon functional daf-16, and cellular distributions of a DAF-16::GFP fusion protein indicate that DAF-16 is not activated during low temperature stress. This suggests that cold-induced physiological defences are not specifically regulated by the IIS pathway and DAF-16, but expression of DAF-16 target genes in IIS mutants and dauers is sufficient to promote cross tolerance to low temperatures in addition to other forms of stress.

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.

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.

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

Directory of Open Access Journals (Sweden)

Huai-Wei Huang

2018-02-01

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

A molecular chaperone activity of CCS restores the maturation of SOD1 fALS mutants.

Science.gov (United States)

Luchinat, Enrico; Barbieri, Letizia; Banci, Lucia

2017-12-12

Superoxide dismutase 1 (SOD1) is an important metalloprotein for cellular oxidative stress defence, that is mutated in familiar variants of Amyotrophic Lateral Sclerosis (fALS). Some mutations destabilize the apo protein, leading to the formation of misfolded, toxic species. The Copper Chaperone for SOD1 (CCS) transiently interacts with SOD1 and promotes its correct maturation by transferring copper and catalyzing disulfide bond formation. By in vitro and in-cell NMR, we investigated the role of the SOD-like domain of CCS (CCS-D2). We showed that CCS-D2 forms a stable complex with zinc-bound SOD1 in human cells, that has a twofold stabilizing effect: it both prevents the accumulation of unstructured mutant SOD1 and promotes zinc binding. We further showed that CCS-D2 interacts with apo-SOD1 in vitro, suggesting that in cells CCS stabilizes mutant apo-SOD1 prior to zinc binding. Such molecular chaperone function of CCS-D2 is novel and its implications in SOD-linked fALS deserve further investigation.

Production and characterization of radiation-sensitive meiotic mutants of Coprinus cinereus

International Nuclear Information System (INIS)

Zolan, M.E.; Tremel, C.J.; Pukkila, P.J.

1988-01-01

We have isolated four gamma-sensitive mutants of the basidiomycete Coprinus cinereus. When homozygous, two of these (rad 3-1 and rad 9-1) produce fruiting bodies with very few viable basidiospores, the products of meiosis in this organism. A less radiation-sensitive allele of RAD 3, rad 3-2, causes no apparent meiotic defect in homozygous strains. Quantitative measurements of oidial survival of rad 3-1;rad 9-1 double mutants compared to the single mutants indicated that rad 3-1 and rad 9-1 mutants are defective in the same DNA repair pathway. In the pew viable basidiospores that are produced by these two strains, essentially normal levels of meiotic recombination can be detected. None of the mutants exhibits increased sensitivity to UV radiation. Cytological examination of meiotic chromosomes from mutant and wild-type fruiting bodies showed that rad 3-1 homozygous strains fail to condense and pair homologous chromosomes during prophase I. Although rad 9-1 strains are successful at chromosome pairing, meiosis is usually not completed in these mutants

Productive mutants in lemongrass induced by gamma rays

International Nuclear Information System (INIS)

Gopinathan Nair, V.

1980-01-01

Seeds of the lemongrass variety O.D. 19 were irradiated with gamma rays at a dose range of 5 to 30 krad. M 1 plants with one or a few tillers differing from the standard plants of O.D. 19 were selected, split into single slips and planted as clonal progenies. Mutants were isolated in M 1 V 1 and carried forward. Forty two M 1 V 2 mutant clones differing from O.D. 19 in morphological characters such as vigour, plant height, growth habit, pigmentation and number of tillers have been established. These were evaluated for tiller number, grass yield and oil content. Six clones gave higher grass yield, the highest being 556 gm per plant per cutting as against 360 gm in the standard. Five clones gave higher oil yield, the highest being 0.42% as against 0.23% in the standard. Isolation of viable mutants with high grass yield and essential oil content indicate the scope for evolving productive mutant varieties in this perennial aromatic grass. The eleven M 1 V 2 mutant clones are being critically evaluated by estimating oil yield per hectare per year. (author)

Delayed Disease Onset and Extended Survival in the SOD1G93A Rat Model of Amyotrophic Lateral Sclerosis after Suppression of Mutant SOD1 in the Motor Cortex

Science.gov (United States)

Thomsen, Gretchen M.; Gowing, Genevieve; Latter, Jessica; Chen, Maximus; Vit, Jean-Philippe; Staggenborg, Kevin; Avalos, Pablo; Alkaslasi, Mor; Ferraiuolo, Laura; Likhite, Shibi; Kaspar, Brian K.

2014-01-01

Sporadic amyotrophic lateral sclerosis (ALS) is a fatal disease with unknown etiology, characterized by a progressive loss of motor neurons leading to paralysis and death typically within 3–5 years of onset. Recently, there has been remarkable progress in understanding inherited forms of ALS in which well defined mutations are known to cause the disease. Rodent models in which the superoxide dismutase-1 (SOD1) mutation is overexpressed recapitulate hallmark signs of ALS in patients. Early anatomical changes in mouse models of fALS are seen in the neuromuscular junctions (NMJs) and lower motor neurons, and selective reduction of toxic mutant SOD1 in the spinal cord and muscle of these models has beneficial effects. Therefore, much of ALS research has focused on spinal motor neuron and NMJ aspects of the disease. Here we show that, in the SOD1G93A rat model of ALS, spinal motor neuron loss occurs presymptomatically and before degeneration of ventral root axons and denervation of NMJs. Although overt cell death of corticospinal motor neurons does not occur until disease endpoint, we wanted to establish whether the upper motor neuron might still play a critical role in disease progression. Surprisingly, the knockdown of mutant SOD1 in only the motor cortex of presymptomatic SOD1G93A rats through targeted delivery of AAV9–SOD1–shRNA resulted in a significant delay of disease onset, expansion of lifespan, enhanced survival of spinal motor neurons, and maintenance of NMJs. This datum suggests an early dysfunction and thus an important role of the upper motor neuron in this animal model of ALS and perhaps patients with the disease. PMID:25411487

Regulation of 1, 4, 5-triphosphate receptor channel gating dynamics by mutant presenilin in Alzheimer's disease cells

Science.gov (United States)

Wei, Fang; Li, Xiang; Cai, Meichun; Liu, Yanping; Jung, Peter; Shuai, Jianwei

2017-06-01

In neurons of patients with Alzheimer's disease, the intracellular Ca2+ concentration is increased by its release from the endoplasmic reticulum via the inositol 1, 4, 5-triphosphate receptor (IP3R). In this paper, we discuss the IP3R gating dynamics in familial Alzheimer's disease (FAD) cells induced with presenilin mutation PS1. By fitting the parameters of an IP3R channel model to experimental data of the open probability, the mean open time and the mean closed time of IP3R channels, in control cells and FAD mutant cells, we suggest that the interaction of presenilin mutation PS1 with IP3R channels leads the decrease in the unbinding rates of IP3 and the activating Ca2+ from IP3Rs. As a result, the increased affinities of IP3 and activating Ca2+ for IP3R channels induce the increase in the Ca2+ signal in FAD mutant cells. Specifically, the PS1 mutation decreases the IP3 dissociation rate of IP3R channels significantly in FAD mutant cells. Our results suggest possible novel targets for FAD therapeutic intervention.

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

International Nuclear Information System (INIS)

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

1980-01-01

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

MicroRNA Regulation of Abiotic Stress Response in 7B-1 Male-Sterile Tomato Mutant

Czech Academy of Sciences Publication Activity Database

Omidvar, Vahid; Mohorianu, I.; Dalmay, T.; Fellner, Martin

2015-01-01

Roč. 8, č. 3 (2015), s. 1-13 ISSN 1940-3372 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : 7B-1 mutant * abiotic stress * miRNAs Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.509, year: 2015

Isolation of uvh1, an Arabidopsis mutant hypersensitive to ultraviolet light and ionizing radiation

International Nuclear Information System (INIS)

Harlow, G.R.; Jenkins, M.E.; Pittalwala, T.S.; Mount, D.W.

1994-01-01

A genetic screen for mutants of Arabidopsis that are hypersensitive to UV light was developed and used to isolate a new mutant designated uvh1. UV hypersensitivity in uvh1 was due to a single recessive trait that is probably located on chromosome 3. Although isolated as hypersensitive to an acute exposure to UV-C light, uvh1 was also hypersensitive to UV-B wavelengths, which are present in sunlight that reaches the earth's surface. UV-B damage to both wild-type and uvh1 plants could be significantly reduced by subsequent exposure of UV-irradiated plants to photoreactivating light, showing that photoreactivation of UV-B damage is important for plant viability and that uvh1 plants are not defective in photoreactivation. A new assay for DNA damage, the Dral assay, was developed and used to show that exposure of wild-type and uvh1 plants to a given dose of UV light induces the same amount of damage in chloroplast and nuclear DNA. Thus, uvh1 is not defective in a UV protective mechanism. uvh1 plants were also found to be hypersensitive to ionizing radiation. These results suggest that uvh1 is defective in a repair or tolerance mechanism that normally provides plants with resistance to several types of DNA damage

Isolation of uvh1, an Arabidopsis mutant hypersensitive to ultraviolet light and ionizing radiation

Energy Technology Data Exchange (ETDEWEB)

Harlow, G. R.; Jenkins, M. E.; Pittalwala, T. S.; Mount, D. W.

1994-02-15

A genetic screen for mutants of Arabidopsis that are hypersensitive to UV light was developed and used to isolate a new mutant designated uvh1. UV hypersensitivity in uvh1 was due to a single recessive trait that is probably located on chromosome 3. Although isolated as hypersensitive to an acute exposure to UV-C light, uvh1 was also hypersensitive to UV-B wavelengths, which are present in sunlight that reaches the earth's surface. UV-B damage to both wild-type and uvh1 plants could be significantly reduced by subsequent exposure of UV-irradiated plants to photoreactivating light, showing that photoreactivation of UV-B damage is important for plant viability and that uvh1 plants are not defective in photoreactivation. A new assay for DNA damage, the Dral assay, was developed and used to show that exposure of wild-type and uvh1 plants to a given dose of UV light induces the same amount of damage in chloroplast and nuclear DNA. Thus, uvh1 is not defective in a UV protective mechanism. uvh1 plants were also found to be hypersensitive to ionizing radiation. These results suggest that uvh1 is defective in a repair or tolerance mechanism that normally provides plants with resistance to several types of DNA damage.

Inhibition of Early Stages of HIV-1 Assembly by INI1/hSNF5 Transdominant Negative Mutant S6 ▿

Science.gov (United States)

Cano, Jennifer; Kalpana, Ganjam V.

2011-01-01

INI1/hSNF5 is an HIV-1 integrase (IN) binding protein specifically incorporated into virions. A truncated mutant of INI1 (S6, amino acids 183 to 294) harboring the minimal IN binding Rpt1 domain potently inhibits HIV-1 particle production in a transdominant manner. The inhibition requires interaction of S6 with IN within Gag-Pol. While INI1 is a nuclear protein and harbors a masked nuclear export signal (NES), the transdominant negative mutant S6 is cytoplasmic, due to the unmasking of NES. Here, we examined the effects of subcellular localization of S6 on HIV-1 inhibition and further investigated the stages of assembly that are affected. We found that targeting a nuclear localization signal-containing S6 variant [NLS-S6(Rpt1)] to the nucleoplasm (but not to the nucleolus) resulted in complete reversal of inhibition of particle production. Electron microscopy indicated that although no electron-dense particles at any stage of assembly were seen in cells expressing S6, virions were produced in cells expressing the rescue mutant NLS-S6(Rpt1) to wild-type levels. Immunofluorescence analysis revealed that p24 exhibited a diffuse pattern of localization within the cytoplasm in cells expressing S6 in contrast to accumulation along the membrane in controls. Pulse-chase analysis indicated that in S6-expressing cells, although Gag(Pr55gag) protein translation was unaffected, processing and release of p24 were defective. Together, these results indicate that expression of S6 in the cytoplasm interferes with trafficking of Gag-Pol/Gag to the membrane and causes a defective processing leading to inhibition of assembly at an early stage prior to particle formation and budding. PMID:21159874

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

Science.gov (United States)

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

2012-01-01

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

miR-155 Controls Lymphoproliferation in LAT Mutant Mice by Restraining T-Cell Apoptosis via SHIP-1/mTOR and PAK1/FOXO3/BIM Pathways.

Directory of Open Access Journals (Sweden)

Alexandre K Rouquette-Jazdanian

Full Text Available Linker for Activation of T cells (LAT is an adapter protein that is essential for T cell function. Knock-in mice with a LAT mutation impairing calcium flux develop a fatal CD4+ lymphoproliferative disease. miR-155 is a microRNA that is correlated with hyperproliferation in a number of cancers including lymphomas and leukemias and is overexpressed in mutant LAT T cells. To test whether miR-155 was merely indicative of T cell activation or whether it contributes to lymphoproliferative disease in mutant LAT mice, we interbred LAT mutant and miR-155-deficient mice. miR-155 deficiency markedly inhibited lymphoproliferative disease by stimulating BIM-dependent CD4+ T cell apoptosis, even though ERK activation and T cell proliferation were increased in double mutant CD4+ T cells. Bim/Bcl2l11 expression is activated by the forkhead transcription factor FOXO3. Using miR-155-deficient, LAT mutant T cells as a discovery tool, we found two connected pathways that impact the nuclear translocation and activation of FOXO3 in T cells. One pathway is mediated by the inositide phosphatase SHIP-1 and the serine/threonine kinases AKT and PDK1. The other pathway involves PAK1 and JNK kinase activation. We define crosstalk between the two pathways via the kinase mTOR, which stabilizes PAK1. This study establishes a role for PAK1 in T cell apoptosis, which contrasts to its previously identified role in T cell proliferation. Furthermore, miR-155 regulates the delicate balance between PAK1-mediated proliferation and apoptosis in T cells impacting lymphoid organ size and function.

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

The functions of grainy head-like proteins in animals and fungi and the evolution of apical extracellular barriers.

Directory of Open Access Journals (Sweden)

Adam Paré

Full Text Available The Grainy head (GRH family of transcription factors are crucial for the development and repair of epidermal barriers in all animals in which they have been studied. This is a high-level functional conservation, as the known structural and enzymatic genes regulated by GRH proteins differ between species depending on the type of epidermal barrier being formed. Interestingly, members of the CP2 superfamily of transcription factors, which encompasses the GRH and LSF families in animals, are also found in fungi--organisms that lack epidermal tissues. To shed light on CP2 protein function in fungi, we characterized a Neurospora crassa mutant lacking the CP2 member we refer to as grainy head-like (grhl. We show that Neurospora GRHL has a DNA-binding specificity similar to that of animal GRH proteins and dissimilar to that of animal LSF proteins. Neurospora grhl mutants are defective in conidial-spore dispersal due to an inability to remodel the cell wall, and we show that grhl mutants and the long-known conidial separation-2 (csp-2 mutants are allelic. We then characterized the transcriptomes of both Neurospora grhl mutants and Drosophila grh mutant embryos to look for similarities in the affected genes. Neurospora grhl appears to play a role in the development and remodeling of the cell wall, as well as in the activation of genes involved in defense and virulence. Drosophila GRH is required to activate the expression of many genes involved in cuticular/epidermal-barrier formation. We also present evidence that GRH plays a role in adult antimicrobial defense. These results, along with previous studies of animal GRH proteins, suggest the fascinating possibility that the apical extracellular barriers of some animals and fungi might share an evolutionary connection, and that the formation of physical barriers in the last common ancestor was under the control of a transcriptional code that included GRH-like proteins.

Mutants of Escherichia coli K-12 with enhanced resistance to ionizing radiation. 4. Peculiarities of recombination in Gamsup(r) mutants

International Nuclear Information System (INIS)

Bresler, S.E.; Kalinin, V.L.; Laneeva, N.I.

1984-01-01

Radioresistant mutant Gam sup(r) 444 differs from a wild type and from Gam sup(r) 445 mutant in decreased frequency of long episome heritage ORF 1 (pur E + -tsx + -proC + -lac + ) and F 14 (ilv + -argE + ), containing hot points of RecRecF - depending recombination and in increased frequency of chromosome mobilization and integrative suppression of temperature sensitive dna A46 mutation by sexual factor F. In this respect Gam sup(r) 444 mutant resembles rec BC sbs B mutant with RecF - recombination type

Biodistribution patterns of native and mutant 99mTc-labelled annexin V in mice

International Nuclear Information System (INIS)

Mariani, G.; Erba, P.; Pellegrino, D.; Volterrani, D.; Lazzeri, E.; Freer, G.; Bevilacqua, G.; Blankenberg, F.G.; Tait, J.F.; Strauss, H.W.

2003-01-01

Full text: Annexin is a 36 kD protein with high binding affinity to phosphatidylserine (PS), a phospholipid exposed on the membrane surface of cells upon activation of the enzyme caspase, the first step of apoptosis. Radiolabeled annexin V could thus be used for imaging apoptosis in-vivo. When the 319 amino acid protein is made by recombinant techniques and expressed as the human material, it can be radiolabeled with 99mTc after derivatization with a bifunctional agent such as HYNIC. Alternatively, the amino acid structure of the protein can be modified by producing annexin V mutants with an endogenous chelation site for 99mTc, the NH2 residue Ala-Gly-Gly-Cys-Gly-His-Met. Mutant annexin has similar affinity for membrane-bound PS as unmodified annexin. This study was performed to compare the biodistribution of 99mTc-labeled HYNIC annexin (HyA) to mutant annexin (MuA). 99mTc-labeling efficiency of the two annexin preparations was >99% by gel chromatography on Sephadex G10 columns. Groups of adult male mice (n 10, body weight 18-25 grams) were injected iv with either HyA or MuA (1-3 MBq, 3-9 μg/animal). Animals were sacrificed one hour later and dissected for organ biodistribution. Similar biodistribution was performed after pretreatment with cyclophosphamide (150 mg/kg ip 6-15 hr prior to the study). The results of the biodistribution study showed significantly reduced (p<0.05 to p<0.01) uptake of MuA versus HyA in the kidneys (Δ- 81.4%), spleen (Δ- 58.2%), liver (Δ- 56.2%), and bone marrow (Δ- 33.7%), while it was increased in lymph nodes (Δ+ 131%, p<0.001). Pretreatment with the pro-apoptotic agent cyclophosphamide induced significantly increased uptake of MuA (p<0.05) versus baseline in the heart (Δ+ 34.7%), spleen (Δ+ 30.1%) and bowel (Δ+ 44.5%), while uptake of HyA was increased only in the spleen (Δ+ 44.1%). The marked reduction in the renal, splenic, liver, and bone marrow localization of MuA compared to HyA in control animals outlines a pattern of

Deletion mutants of region E1 a of AD12 E1 plasmids: Effect on oncogenic transformation

NARCIS (Netherlands)

Bos, J.L.; Jochemsen, A.G.; Bernards, R.A.; Schrier, P.I.; Ormondt, H. van; Eb, A.J. van der

1983-01-01

Plasmids containing the El region of Ad12 DNA can transform certain rodent cells into oncogenic cells. To study the role of the Ela subregion in the process of oncogenic transformation, Ad12 region El mutants carrying deletions in the Ela region were constructed. Deletion mutants pR7 and pR8 affect

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

Science.gov (United States)

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

2004-10-01

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

Fisetin Exerts Antioxidant and Neuroprotective Effects in Multiple Mutant hSOD1 Models of Amyotrophic Lateral Sclerosis by Activating ERK.

Science.gov (United States)

Wang, T H; Wang, S Y; Wang, X D; Jiang, H Q; Yang, Y Q; Wang, Y; Cheng, J L; Zhang, C T; Liang, W W; Feng, H L

2018-05-21

Oxidative stress exhibits a central role in the course of amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease commonly found to include a copper/zinc superoxide dismutase (SOD1) gene mutation. Fisetin, a natural antioxidant, has shown benefits in varied neurodegenerative diseases. The possible effect of fisetin in ALS has not been clarified as of yet. We investigated whether fisetin affected mutant hSOD1 ALS models. Three different hSOD1-related mutant models were used: Drosophila expressing mutant hSOD1 G85R , hSOD1 G93A NSC34 cells, and transgenic mice. Fisetin treatment provided neuroprotection as demonstrated by an improved survival rate, attenuated motor impairment, reduced ROS damage and regulated redox homeostasis compared with those in controls. Furthermore, fisetin increased the expression of phosphorylated ERK and upregulated antioxidant factors, which were reversed by MEK/ERK inhibition. Finally, fisetin reduced the levels of both mutant and wild-type hSOD1 in vivo and in vitro, as well as the levels of detergent-insoluble hSOD1 proteins. The results indicate that fisetin protects cells from ROS damage and improves the pathological behaviors caused by oxidative stress in disease models related to SOD1 gene mutations probably by activating ERK, thereby providing a potential treatment for ALS. Copyright © 2018 IBRO. Published by Elsevier Ltd. All rights reserved.

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)

Distinct neurobehavioural effects of cannabidiol in transmembrane domain neuregulin 1 mutant mice.

Directory of Open Access Journals (Sweden)

Leonora E Long

Full Text Available The cannabis constituent cannabidiol (CBD possesses anxiolytic and antipsychotic properties. We have previously shown that transmembrane domain neuregulin 1 mutant (Nrg1 TM HET mice display altered neurobehavioural responses to the main psychoactive constituent of cannabis, Δ(9-tetrahydrocannabinol. Here we investigated whether Nrg1 TM HET mice respond differently to CBD and whether CBD reverses schizophrenia-related phenotypes expressed by these mice. Adult male Nrg1 TM HET and wild type-like littermates (WT received vehicle or CBD (1, 50 or 100 mg/kg i.p. for 21 days. During treatment and 48 h after withdrawal we measured behaviour, whole blood CBD concentrations and autoradiographic receptor binding. Nrg1 HET mice displayed locomotor hyperactivity, PPI deficits and reduced 5-HT(2A receptor binding density in the substantia nigra, but these phenotypes were not reversed by CBD. However, long-term CBD (50 and 100 mg/kg selectively enhanced social interaction in Nrg1 TM HET mice. Furthermore, acute CBD (100 mg/kg selectively increased PPI in Nrg1 TM HET mice, although tolerance to this effect was manifest upon repeated CBD administration. Long-term CBD (50 mg/kg also selectively increased GABA(A receptor binding in the granular retrosplenial cortex in Nrg1 TM HET mice and reduced 5-HT(2A binding in the substantia nigra in WT mice. Nrg1 appears necessary for CBD-induced anxiolysis since only WT mice developed decreased anxiety-related behaviour with repeated CBD treatment. Altered pharmacokinetics in mutant mice could not explain our findings since no genotype differences existed in CBD blood concentrations. Here we demonstrate that Nrg1 modulates acute and long-term neurobehavioural effects of CBD, which does not reverse the schizophrenia-relevant phenotypes.

The Effects of Bee Venom Acupuncture on the Central Nervous System and Muscle in an Animal hSOD1G93A Mutant

Directory of Open Access Journals (Sweden)

MuDan Cai

2015-03-01

Full Text Available Amyotrophic lateral sclerosis (ALS is caused by the degeneration of lower and upper motor neurons, leading to muscle paralysis and respiratory failure. However, there is no effective drug or therapy to treat ALS. Complementary and alternative medicine (CAM, including acupuncture, pharmacopuncture, herbal medicine, and massage is popular due to the significant limitations of conventional therapy. Bee venom acupuncture (BVA, also known as one of pharmacopunctures, has been used in Oriental medicine to treat inflammatory diseases. The purpose of this study is to investigate the effect of BVA on the central nervous system (CNS and muscle in symptomatic hSOD1G93A transgenic mice, an animal model of ALS. Our findings show that BVA at ST36 enhanced motor function and decreased motor neuron death in the spinal cord compared to that observed in hSOD1G93A transgenic mice injected intraperitoneally (i.p. with BV. Furthermore, BV treatment at ST36 eliminated signaling downstream of inflammatory proteins such as TLR4 in the spinal cords of symptomatic hSOD1G93A transgenic mice. However, i.p. treatment with BV reduced the levels of TNF-α and Bcl-2 expression in the muscle hSOD1G93A transgenic mice. Taken together, our findings suggest that BV pharmacopuncture into certain acupoints may act as a chemical stimulant to activate those acupoints and subsequently engage the endogenous immune modulatory system in the CNS in an animal model of ALS.

Sequential emergence and clinical implications of viral mutants with K70E and K65R mutation in reverse transcriptase during prolonged tenofovir monotherapy in rhesus macaques with chronic RT-SHIV infection

Directory of Open Access Journals (Sweden)

Pedersen Niels C

2007-04-01

Full Text Available Abstract Background We reported previously on the emergence and clinical implications of simian immunodeficiency virus (SIVmac251 mutants with a K65R mutation in reverse transcriptase (RT, and the role of CD8+ cell-mediated immune responses in suppressing viremia during tenofovir therapy. Because of significant sequence differences between SIV and HIV-1 RT that affect drug susceptibilities and mutational patterns, it is unclear to what extent findings with SIV can be extrapolated to HIV-1 RT. Accordingly, to model HIV-1 RT responses, 12 macaques were inoculated with RT-SHIV, a chimeric SIV containing HIV-1 RT, and started on prolonged tenofovir therapy 5 months later. Results The early virologic response to tenofovir correlated with baseline viral RNA levels and expression of the MHC class I allele Mamu-A*01. For all animals, sensitive real-time PCR assays detected the transient emergence of K70E RT mutants within 4 weeks of therapy, which were then replaced by K65R mutants within 12 weeks of therapy. For most animals, the occurrence of these mutations preceded a partial rebound of plasma viremia to levels that remained on average 10-fold below baseline values. One animal eventually suppressed K65R viremia to undetectable levels for more than 4 years; sequential experiments using CD8+ cell depletion and tenofovir interruption demonstrated that both CD8+ cells and continued tenofovir therapy were required for sustained suppression of viremia. Conclusion This is the first evidence that tenofovir therapy can select directly for K70E viral mutants in vivo. The observations on the clinical implications of the K65R RT-SHIV mutants were consistent with those of SIVmac251, and suggest that for persons infected with K65R HIV-1 both immune-mediated and drug-dependent antiviral activities play a role in controlling viremia. These findings suggest also that even in the presence of K65R virus, continuation of tenofovir treatment as part of HAART may be

Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans with the high oleic acid seed oil trait

Directory of Open Access Journals (Sweden)

Pham Anh-Tung

2010-09-01

Full Text Available Abstract Background The alteration of fatty acid profiles in soybean [Glycine max (L. Merr.] to improve soybean oil quality is an important and evolving theme in soybean research to meet nutritional needs and industrial criteria in the modern market. Soybean oil with elevated oleic acid is desirable because this monounsaturated fatty acid improves the nutrition and oxidative stability of the oil. Commodity soybean oil typically contains 20% oleic acid and the target for high oleic acid soybean oil is approximately 80% of the oil; previous conventional plant breeding research to raise the oleic acid level to just 50-60% of the oil was hindered by the genetic complexity and environmental instability of the trait. The objective of this work was to create the high oleic acid trait in soybeans by identifying and combining mutations in two delta-twelve fatty acid desaturase genes, FAD2-1A and FAD2-1B. Results Three polymorphisms found in the FAD2-1B alleles of two soybean lines resulted in missense mutations. For each of the two soybean lines, there was one unique amino acid change within a highly conserved region of the protein. The mutant FAD2-1B alleles were associated with an increase in oleic acid levels, although the FAD2-1B mutant alleles alone were not capable of producing a high oleic acid phenotype. When existing FAD2-1A mutations were combined with the novel mutant FAD2-1B alleles, a high oleic acid phenotype was recovered only for those lines which were homozygous for both of the mutant alleles. Conclusions We were able to produce conventional soybean lines with 80% oleic acid in the oil in two different ways, each requiring the contribution of only two genes. The high oleic acid soybean germplasm developed contained a desirable fatty acid profile, and it was stable in two production environments. The presumed causative sequence polymorphisms in the FAD2-1B alleles were developed into highly efficient molecular markers for tracking the

Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans with the high oleic acid seed oil trait.

Science.gov (United States)

Pham, Anh-Tung; Lee, Jeong-Dong; Shannon, J Grover; Bilyeu, Kristin D

2010-09-09

The alteration of fatty acid profiles in soybean [Glycine max (L.) Merr.] to improve soybean oil quality is an important and evolving theme in soybean research to meet nutritional needs and industrial criteria in the modern market. Soybean oil with elevated oleic acid is desirable because this monounsaturated fatty acid improves the nutrition and oxidative stability of the oil. Commodity soybean oil typically contains 20% oleic acid and the target for high oleic acid soybean oil is approximately 80% of the oil; previous conventional plant breeding research to raise the oleic acid level to just 50-60% of the oil was hindered by the genetic complexity and environmental instability of the trait. The objective of this work was to create the high oleic acid trait in soybeans by identifying and combining mutations in two delta-twelve fatty acid desaturase genes, FAD2-1A and FAD2-1B. Three polymorphisms found in the FAD2-1B alleles of two soybean lines resulted in missense mutations. For each of the two soybean lines, there was one unique amino acid change within a highly conserved region of the protein. The mutant FAD2-1B alleles were associated with an increase in oleic acid levels, although the FAD2-1B mutant alleles alone were not capable of producing a high oleic acid phenotype. When existing FAD2-1A mutations were combined with the novel mutant FAD2-1B alleles, a high oleic acid phenotype was recovered only for those lines which were homozygous for both of the mutant alleles. We were able to produce conventional soybean lines with 80% oleic acid in the oil in two different ways, each requiring the contribution of only two genes. The high oleic acid soybean germplasm developed contained a desirable fatty acid profile, and it was stable in two production environments. The presumed causative sequence polymorphisms in the FAD2-1B alleles were developed into highly efficient molecular markers for tracking the mutant alleles. The resources described here for the creation

Mutant PrP Suppresses Glutamatergic Neurotransmission in Cerebellar Granule Neurons by Impairing Membrane Delivery of VGCC α2δ-1 Subunit

Science.gov (United States)

Senatore, Assunta; Colleoni, Simona; Verderio, Claudia; Restelli, Elena; Morini, Raffaella; Condliffe, Steven B.; Bertani, Ilaria; Mantovani, Susanna; Canovi, Mara; Micotti, Edoardo; Forloni, Gianluigi; Dolphin, Annette C.; Matteoli, Michela; Gobbi, Marco; Chiesa, Roberto

2012-01-01

Summary How mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α2δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α2δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies. PMID:22542184

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)

Analysis of STAT1 activation by six FGFR3 mutants associated with skeletal dysplasia undermines dominant role of STAT1 in FGFR3 signaling in cartilage.

Directory of Open Access Journals (Sweden)

Pavel Krejci

Full Text Available Activating mutations in FGFR3 tyrosine kinase cause several forms of human skeletal dysplasia. Although the mechanisms of FGFR3 action in cartilage are not completely understood, it is believed that the STAT1 transcription factor plays a central role in pathogenic FGFR3 signaling. Here, we analyzed STAT1 activation by the N540K, G380R, R248C, Y373C, K650M and K650E-FGFR3 mutants associated with skeletal dysplasias. In a cell-free kinase assay, only K650M and K650E-FGFR3 caused activatory STAT1(Y701 phosphorylation. Similarly, in RCS chondrocytes, HeLa, and 293T cellular environments, only K650M and K650E-FGFR3 caused strong STAT1 activation. Other FGFR3 mutants caused weak (HeLa or no activation (293T and RCS. This contrasted with ERK MAP kinase activation, which was strongly induced by all six mutants and correlated with the inhibition of proliferation in RCS chondrocytes. Thus the ability to activate STAT1 appears restricted to the K650M and K650E-FGFR3 mutants, which however account for only a small minority of the FGFR3-related skeletal dysplasia cases. Other pathways such as ERK should therefore be considered as central to pathological FGFR3 signaling in cartilage.

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

Directory of Open Access Journals (Sweden)

Yangrae Cho

2014-07-01

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

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

Pharmacokinetic properties of 2nd-generation fibroblast growth factor-1 mutants for therapeutic application.

Directory of Open Access Journals (Sweden)

Xue Xia

Full Text Available Fibroblast growth factor-1 (FGF-1 is an angiogenic factor with therapeutic potential for the treatment of ischemic disease. FGF-1 has low intrinsic thermostability and is characteristically formulated with heparin as a stabilizing agent. Heparin, however, adds a number of undesirable properties that negatively impact safety and cost. Mutations that increase the thermostability of FGF-1 may obviate the need for heparin in formulation and may prove to be useful "2nd-generation" forms for therapeutic use. We report a pharmacokinetic (PK study in rabbits of human FGF-1 in the presence and absence of heparin, as well as three mutant forms having differential effects upon thermostability, buried reactive thiols, and heparin affinity. The results support the hypothesis that heparan sulfate proteoglycan (HSPG in the vasculature of liver, kidney and spleen serves as the principle peripheral compartment in the distribution kinetics. The addition of heparin to FGF-1 is shown to increase endocrine-like properties of distribution. Mutant forms of FGF-1 that enhance thermostability or eliminate buried reactive thiols demonstrate a shorter distribution half-life, a longer elimination half-life, and a longer mean residence time (MRT in comparison to wild-type FGF-1. The results show how such mutations can produce useful 2nd-generation forms with tailored PK profiles for specific therapeutic application.

Isolation and characterization of MMS-sensitive mutants of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Prakash, L.; Prakash, S.

1977-01-01

We have isolated mutants sensitive to methyl methanesulfonate (MMS) in Saccharomyces cerevisiae. Alleles of rad1, rad4, rad6, rad52, rad55 and rad57 were found among these mms mutants. Twenty-nine of the mms mutants which complement the existing radiation-sensitive (rad and rev) mutants belong to 22 new complementation groups. Mutants from five complementation groups are sensitive only to MMS. Mutants of 11 complementation groups are sensitive to uv or x rays in addition to MMS, mutants of six complementation groups are sensitive to all three agents. The cross-sensitivities of these mms mutants to uv and x rays are discussed in terms of their possible involvement in DNA repair. Sporulation is reduced or absent in homozygous diploids of mms mutants from nine complementation groups

A METABOLIC SIGNATURE FOR LONG-LIFE IN THE C. ELEGANS MIT MUTANTS

Science.gov (United States)

Butler, Jeffrey A.; Mishur, Robert J.; Bhaskaran, Shylesh; Rea, Shane L.

2012-01-01

SUMMARY Mit mutations that disrupt function of the mitochondrial electron transport chain can, inexplicably, prolong Caenorhabditis elegans lifespan. In this study we use a metabolomics approach to identify an ensemble of mitochondrial-derived α-ketoacids and α-hydroxyacids that are produced by long-lived Mit mutants but not by other long-lived mutants or by short-lived mitochondrial mutants. We show that accumulation of these compounds is dependent upon concerted inhibition of three α-ketoacid dehydrogenases that share dihydrolipoamide dehydrogenase (DLD) as a common subunit, a protein previously linked in humans with increased risk of Alzheimer’s disease. When the expression of DLD in wild type animals was reduced using RNA interference we observed an unprecedented effect on lifespan - as RNAi dosage was increased lifespan was significantly shortened but, at higher doses, it was significantly lengthened, suggesting DLD plays a unique role in modulating length of life. Our findings provide novel insight into the origin of the Mit phenotype. PMID:23173729

Deletion of Slc26a1 and Slc26a7 Delays Enamel Mineralization in Mice

Directory of Open Access Journals (Sweden)

Kaifeng Yin

2017-05-01

Full Text Available Amelogenesis features two major developmental stages—secretory and maturation. During maturation stage, hydroxyapatite deposition and matrix turnover require delicate pH regulatory mechanisms mediated by multiple ion transporters. Several members of the Slc26 gene family (Slc26a1, Slc26a3, Slc26a4, Slc26a6, and Slc26a7, which exhibit bicarbonate transport activities, have been suggested by previous studies to be involved in maturation-stage amelogenesis, especially the key process of pH regulation. However, details regarding the functional role of these genes in enamel formation are yet to be clarified, as none of the separate mutant animal lines demonstrates any discernible enamel defects. Continuing with our previous investigation of Slc26a1−/− and Slc26a7−/− animal models, we generated a double-mutant animal line with the absence of both Slc26a1 and Slc26a7. We showed in the present study that the double-mutant enamel density was significantly lower in the regions that represent late maturation-, maturation- and secretory-stage enamel development in wild-type mandibular incisors. However, the “maturation” and “secretory” enamel microstructures in double-mutant animals resembled those observed in wild-type secretory and/or pre-secretory stages. Elemental composition analysis revealed a lack of mineral deposition and an accumulation of carbon and chloride in double-mutant enamel. Deletion of Slc26a1 and Slc26a7 did not affect the stage-specific morphology of the enamel organ. Finally, compensatory expression of pH regulator genes and ion transporters was detected in maturation-stage enamel organs of double-mutant animals when compared to wild-type. Combined with the findings from our previous study, these data indicate the involvement of SLC26A1and SLC26A7 as key ion transporters in the pH regulatory network during enamel maturation.

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

NARCIS (Netherlands)

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

2003-01-01

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

Genetic Characterization of the Carotenoid Biosynthetic Pathway in Methylobacterium extorquens AM1 and Isolation of a Colorless Mutant

OpenAIRE

Van Dien, Stephen J.; Marx, Christopher J.; O'Brien, Brooke N.; Lidstrom, Mary E.

2003-01-01

Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, r...

Phage Pl mutants with altered transducing abilities for Escherichia coli

International Nuclear Information System (INIS)

Wall, J.D.; Harriman, P.D.

1974-01-01

A search was made for mutants of the coliphage P1 with altered transducing frequencies. A method was developed for the rapid assay of transducing frequencies in single plaques using prophage lambda as the transduced bacterial marker. This procedure selects for mutants altered in their ability to package host DNA. Mutants with 5 to 10 times higher or 10 to 20 times lower frequencies than those of wild-type P1 were found. Not only are the markers used for the detection of the mutants affected, but all other markers are similarly affected (not always to the same extent). One of the high transducing frequency mutants is a suppressible amber, indicating that loss of a function increases P1's ability to package host DNA preferentially. (U.S.)

The Arabidopsis aba4-1 mutant reveals a specific function for neoxanthin in protection against photooxidative stress.

Science.gov (United States)

Dall'Osto, Luca; Cazzaniga, Stefano; North, Helen; Marion-Poll, Annie; Bassi, Roberto

2007-03-01

The aba4-1 mutant completely lacks neoxanthin but retains all other xanthophyll species. The missing neoxanthin in light-harvesting complex (Lhc) proteins is compensated for by higher levels of violaxanthin, albeit with lower capacity for photoprotection compared with proteins with wild-type levels of neoxanthin. Detached leaves of aba4-1 were more sensitive to oxidative stress than the wild type when exposed to high light and incubated in a solution of photosensitizer agents. Both treatments caused more rapid pigment bleaching and lipid oxidation in aba4-1 than wild-type plants, suggesting that neoxanthin acts as an antioxidant within the photosystem II (PSII) supercomplex in thylakoids. While neoxanthin-depleted Lhc proteins and leaves had similar sensitivity as the wild type to hydrogen peroxide and singlet oxygen, they were more sensitive to superoxide anions. aba4-1 intact plants were not more sensitive than the wild type to high-light stress, indicating the existence of compensatory mechanisms of photoprotection involving the accumulation of zeaxanthin. However, the aba4-1 npq1 double mutant, lacking zeaxanthin and neoxanthin, underwent stronger PSII photoinhibition and more extensive oxidation of pigments than the npq1 mutant, which still contains neoxanthin. We conclude that neoxanthin preserves PSII from photoinactivation and protects membrane lipids from photooxidation by reactive oxygen species. Neoxanthin appears particularly active against superoxide anions produced by the Mehler's reaction, whose rate is known to be enhanced in abiotic stress conditions.

Automated high-content live animal drug screening using C. elegans expressing the aggregation prone serpin α1-antitrypsin Z.

Directory of Open Access Journals (Sweden)

Sager J Gosai

2010-11-01

Full Text Available The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.

A quadruple mutant of Arabidopsis reveals a β-carotene hydroxylation activity for LUT1/CYP97C1 and a regulatory role of xanthophylls on determination of the PSI/PSII ratio.

Science.gov (United States)

Fiore, Alessia; Dall'Osto, Luca; Cazzaniga, Stefano; Diretto, Gianfranco; Giuliano, Giovanni; Bassi, Roberto

2012-04-18

Xanthophylls are oxygenated carotenoids playing an essential role as structural components of the photosynthetic apparatus. Xanthophylls contribute to the assembly and stability of light-harvesting complex, to light absorbance and to photoprotection. The first step in xanthophyll biosynthesis from α- and β-carotene is the hydroxylation of ε- and β-rings, performed by both non-heme iron oxygenases (CHY1, CHY2) and P450 cytochromes (LUT1/CYP97C1, LUT5/CYP97A3). The Arabidopsis triple chy1chy2lut5 mutant is almost completely depleted in β-xanthophylls. Here we report on the quadruple chy1chy2lut2lut5 mutant, additionally carrying the lut2 mutation (affecting lycopene ε-cyclase). This genotype lacks lutein and yet it shows a compensatory increase in β-xanthophylls with respect to chy1chy2lut5 mutant. Mutant plants show an even stronger photosensitivity than chy1chy2lut5, a complete lack of qE, the rapidly reversible component of non-photochemical quenching, and a peculiar organization of the pigment binding complexes into thylakoids. Biochemical analysis reveals that the chy1chy2lut2lut5 mutant is depleted in Lhcb subunits and is specifically affected in Photosystem I function, showing a deficiency in PSI-LHCI supercomplexes. Moreover, by analyzing a series of single, double, triple and quadruple Arabidopsis mutants in xanthophyll biosynthesis, we show a hitherto undescribed correlation between xanthophyll levels and the PSI-PSII ratio. The decrease in the xanthophyll/carotenoid ratio causes a proportional decrease in the LHCII and PSI core levels with respect to PSII. The physiological and biochemical phenotype of the chy1chy2lut2lut5 mutant shows that (i) LUT1/CYP97C1 protein reveals a major β-carotene hydroxylase activity in vivo when depleted in its preferred substrate α-carotene; (ii) xanthophylls are needed for normal level of Photosystem I and LHCII accumulation.

A quadruple mutant of Arabidopsis reveals a β-carotene hydroxylation activity for LUT1/CYP97C1 and a regulatory role of xanthophylls on determination of the PSI/PSII ratio

Directory of Open Access Journals (Sweden)

Fiore Alessia

2012-04-01

Full Text Available Abstract Background Xanthophylls are oxygenated carotenoids playing an essential role as structural components of the photosynthetic apparatus. Xanthophylls contribute to the assembly and stability of light-harvesting complex, to light absorbance and to photoprotection. The first step in xanthophyll biosynthesis from α- and β-carotene is the hydroxylation of ε- and β-rings, performed by both non-heme iron oxygenases (CHY1, CHY2 and P450 cytochromes (LUT1/CYP97C1, LUT5/CYP97A3. The Arabidopsis triple chy1chy2lut5 mutant is almost completely depleted in β-xanthophylls. Results Here we report on the quadruple chy1chy2lut2lut5 mutant, additionally carrying the lut2 mutation (affecting lycopene ε-cyclase. This genotype lacks lutein and yet it shows a compensatory increase in β-xanthophylls with respect to chy1chy2lut5 mutant. Mutant plants show an even stronger photosensitivity than chy1chy2lut5, a complete lack of qE, the rapidly reversible component of non-photochemical quenching, and a peculiar organization of the pigment binding complexes into thylakoids. Biochemical analysis reveals that the chy1chy2lut2lut5 mutant is depleted in Lhcb subunits and is specifically affected in Photosystem I function, showing a deficiency in PSI-LHCI supercomplexes. Moreover, by analyzing a series of single, double, triple and quadruple Arabidopsis mutants in xanthophyll biosynthesis, we show a hitherto undescribed correlation between xanthophyll levels and the PSI-PSII ratio. The decrease in the xanthophyll/carotenoid ratio causes a proportional decrease in the LHCII and PSI core levels with respect to PSII. Conclusions The physiological and biochemical phenotype of the chy1chy2lut2lut5 mutant shows that (i LUT1/CYP97C1 protein reveals a major β-carotene hydroxylase activity in vivo when depleted in its preferred substrate α-carotene; (ii xanthophylls are needed for normal level of Photosystem I and LHCII accumulation.

Tau passive immunotherapy in mutant P301L mice: antibody affinity versus specificity.

Directory of Open Access Journals (Sweden)

Cristina d'Abramo

Full Text Available The use of antibodies to treat neurodegenerative diseases has undergone rapid development in the past decade. To date, immunotherapeutic approaches to Alzheimer's disease have mostly targeted amyloid beta as it is a secreted protein that can be found in plasma and CSF and is consequently accessible to circulating antibodies. Few recent publications have suggested the utility of treatment of tau pathology with monoclonal antibodies to tau. Our laboratory has begun a systematic study of different classes of tau monoclonal antibodies using mutant P301L mice. Three or seven months old mutant tau mice were inoculated weekly with tau monoclonal antibodies at a dose of 10 mg/Kg, until seven or ten months of age were reached respectively. Our data strongly support the notion that in P301L animals treated with MC1, a conformational monoclonal antibody specific for PHF-tau, the rate of development of tau pathology is effectively reduced, while injecting DA31, a high affinity tau sequence antibody, does not exert such benefit. MC1 appears superior to DA31 in overall effects, suggesting that specificity is more important than affinity in therapeutic applications. Unfortunately the survival rate of the P301L treated mice was not improved when immunizing either with MC1 or PHF1, a high affinity phospho-tau antibody previously reported to be efficacious in reducing pathological tau. These data demonstrate that passive immunotherapy in mutant tau models may be efficacious in reducing the development of tau pathology, but a great deal of work remains to be done to carefully select the tau epitopes to target.

Radiation studies in Cajanus cajan: meiotic behaviour in some M/sub 2/ mutants

Energy Technology Data Exchange (ETDEWEB)

Sinha, S.S.N.; Akhaury, S.B. (Ranchi Univ. (India). Dept. of Botany)

1982-01-01

A qualitative study of the mutants produced in M/sub 2/ generation has been made. The mutants were classified as: (1) chlorophyll mutant, (2) morphological mutant, (3) pollen mutant, (4) semi-sterile and (5) sterile mutant. Cytological investigations of pollen mutants, sterile and semi-sterile mutants have revealed that these mutants generally arise at higher dose levels (20 Kr and 25 Kr).

Characterization of Cell Surface and EPS Remodeling of Azospirillum brasilense Chemotaxis-like 1 Signal Transduction Pathway mutants by Atomic Force Microscopy

Energy Technology Data Exchange (ETDEWEB)

Billings, Amanda N [ORNL; Siuti, Piro [ORNL; Bible, Amber [University of Tennessee, Knoxville (UTK); Alexandre, Gladys [University of Tennessee, Knoxville (UTK); Retterer, Scott T [ORNL; Doktycz, Mitchel John [ORNL; Morrell-Falvey, Jennifer L [ORNL

2011-01-01

To compete in complex microbial communities, bacteria must quickly sense environmental changes and adjust cellular functions for optimal growth. Chemotaxis-like signal transduction pathways are implicated in the modulation of multiple cellular responses, including motility, EPS production, and cell-to-cell interactions. Recently, the Che1 chemotaxis-like pathway from Azospirillum brasilense was shown to modulate flocculation. In A. brasilense, cell surface properties, including EPS production, are thought to play a direct role in promoting flocculation. Using atomic force microscopy (AFM), we have detected distinct changes in the surface morphology of flocculating A. brasilense Che1 mutant strains that are absent in the wild type strain. Whereas the wild type strain produces a smooth mucosal extracellular matrix, the flocculating Che1 mutant strains produce distinctive extracellular fibril structures. Further analyses using flocculation inhibition and lectin-binding assays suggest that the composition of EPS components in the extracellular matrix differs between the cheA1 and cheY1 mutants, despite an apparent similarity in the macroscopic floc structures. Collectively, these data indicate that mutations in the Che1 pathway that result in increased flocculation are correlated with distinctive changes in the extracellular matrix structure produced by the mutants, including likely changes in the EPS structure and/or composition.

Phenotype of transgenic mice carrying a very low copy number of the mutant human G93A superoxide dismutase-1 gene associated with amyotrophic lateral sclerosis.

Directory of Open Access Journals (Sweden)

Jeffrey S Deitch

Full Text Available Amyotrophic lateral sclerosis (ALS is a progressive neurodegenerative disease of the motor neuron. While most cases of ALS are sporadic, 10% are familial (FALS with 20% of FALS caused by a mutation in the gene that codes for the enzyme Cu/Zn superoxide dismutase (SOD1. There is variability in sporadic ALS as well as FALS where even within the same family some siblings with the same mutation do not manifest disease. A transgenic (Tg mouse model of FALS containing 25 copies of the mutant human SOD1 gene demonstrates motor neuron pathology and progressive weakness similar to ALS patients, leading to death at approximately 130 days. The onset of symptoms and survival of these transgenic mice are directly related to the number of copies of the mutant gene. We report the phenotype of a very low expressing (VLE G93A SOD1 Tg carrying only 4 copies of the mutant G93ASOD1 gene. While weakness can start at 9 months, only 74% of mice 18 months or older demonstrate disease. The VLE mice show decreased motor neurons compared to wild-type mice as well as increased cytoplasmic translocation of TDP-43. In contrast to the standard G93A SOD1 Tg mouse which always develops motor weakness leading to death, not all VLE animals manifested clinical disease or shortened life span. In fact, approximately 20% of mice older than 24 months had no motor symptoms and only 18% of VLE mice older than 22 months reached end stage. Given the variable penetrance of clinical phenotype, prolonged survival, and protracted loss of motor neurons the VLE mouse provides a new tool that closely mimics human ALS. This tool will allow the study of pathologic events over time as well as the study of genetic and environmental modifiers that may not be causative, but can exacerbate or accelerate motor neuron disease.

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...

Strain improvement in dye decolourising mutants of Mucor mucedo ...

African Journals Online (AJOL)

STORAGESEVER

2009-12-15

Dec 15, 2009 ... M. mucedo {MMM1-U.V. irradiated mutant and MMM2-EMS (ethyl methyl sulfonate) treated ... tions were induced and two positive mutants (MMM1, .... yeast biofilter for the treatment of a Nigerian fertilizer plant effluent. World J.

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

Science.gov (United States)

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

2002-01-01

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

Pollen irradiation method to obtain mutants in cucumber

International Nuclear Information System (INIS)

Iida, S.; Amano, E.

1988-01-01

Seed irradiation for mutation induction in dioecious crops like cucumber is not very useful because chimerism of the mutated tissues makes the segregation of mutants in the M 2 generation nearly impossible. This problem does not exist with pollen irradiation. Cucumber (Cucumis sativus L. var. Nishikisuyo) was used for a model experiment. The petals of male and female flowers were closed by pinching with binding wire before flowering to prevent pollination by insects. On the flowering day, the male flowers were collected and irradiated with 1kR to 10 kR of acute gamma rays (137-Cs), then used to pollinate the female flowers. The M 1 seeds thus obtained are not chimeric but heterozygous for induced mutations. When planted, no mutant phenotype appeared. Selfing within a plant lead to segregation of mutants in the M 2 generation. Seedling examination revealed eight mutants. One mutant line, in which the shape of leaves changed from pentagonal to round heart shape, was found under field conditions. The optimal dose for pollen irradiation seems to be between 2 kR and 4kR

Genetic analyses of nonfluorescent root mutants induced by mutagenesis in soybean

International Nuclear Information System (INIS)

Sawada, S.; Palmer, R.G.

1987-01-01

Nonfluorescent root mutants in soybean [Glycine max (L.) Merr.] are useful as markers in genetic studies and in tissue culture research. Our objective was to obtain mutagen-induced nonfluorescent root mutants and to conduct genetic studies with them. Thirteen nonfluorescent mutants were detected among 154016 seedlings derived from soybean lines treated with six mutagens. One of these mutants, derived from Williams treated with 20 kR gamma rays, did not correspond to any of the known (standard) nonfluorescent spontaneous mutants. This is the first mutagen-induced nonfluorescent root mutant in soybean. It was assigned Genetic Type Collection no. T285 and the gene symbol fr5 fr5. The fr5 allele was not located on trisomics A, B, or C and was not linked to five chlorophyll-deficient mutants (y9, y11, y12, y13, and y20-k2) or flower color mutant w1. The remaining nonfluorescent root mutants were at the same loci as known spontaneous mutants; i.e., four had the fr1 allele, five had the fr2 allele, and three had the fr4 allele

Hyperoxia exposure induced hormesis decreases mitochondrial superoxide radical levels via Ins/IGF-1 signaling pathway in a long-lived age-1 mutant of Caenorhabditis elegans

International Nuclear Information System (INIS)

Yanase, Sumino; Ishii, Naoaki

2008-01-01

The hormetic effect, which extends the lifespan by various stressors, has been confirmed in Caenorhabditis elegans (C. elegans). We have previously reported that oxidative stress resistance in a long-lived mutant age-1 is associated with the hormesis. In the age-1 allele, which activates an insulin/insulin-like growth factor-1 (Ins/IGF-1) signaling pathway, the superoxide dismutase (SOD) and catalase activities increased during normal aging. We now demonstrate changes in the mitochondrial superoxide radical (O 2 - ) levels of the hormetic conditioned age-related strains. The O 2 - levels in age-1 strain significantly decreased after intermittent hyperoxia exposure. On the other hand, this phenomenon was not observed in a daf-16 null mutant. This hormesis-dependent reduction of the O 2 - levels was observed even if the mitochondrial Mn-SOD was experimentally reduced. Therefore, it is indicated that the hormesis is mediated by events that suppress the mitochondrial O 2 - production. Moreover, some SOD gene expressions in the hormetic conditioned age-1 mutant were induced over steady state messenger ribonucleic acid (mRNA) levels. These data suggest that oxidative stress-inducible hormesis is associated with a reduction of the mitochondrial O 2 - production by activation of the antioxidant system via the Ins/IGF-1 signaling pathway. (author)

Alopecia in a viable phospholipase C delta 1 and phospholipase C delta 3 double mutant.

Directory of Open Access Journals (Sweden)

Fabian Runkel

Full Text Available BACKGROUND: Inositol 1,4,5trisphosphate (IP(3 and diacylglycerol (DAG are important intracellular signalling molecules in various tissues. They are generated by the phospholipase C family of enzymes, of which phospholipase C delta (PLCD forms one class. Studies with functional inactivation of Plcd isozyme encoding genes in mice have revealed that loss of both Plcd1 and Plcd3 causes early embryonic death. Inactivation of Plcd1 alone causes loss of hair (alopecia, whereas inactivation of Plcd3 alone has no apparent phenotypic effect. To investigate a possible synergy of Plcd1 and Plcd3 in postnatal mice, novel mutations of these genes compatible with life after birth need to be found. METHODOLOGY/PRINCIPAL FINDINGS: We characterise a novel mouse mutant with a spontaneously arisen mutation in Plcd3 (Plcd3(mNab that resulted from the insertion of an intracisternal A particle (IAP into intron 2 of the Plcd3 gene. This mutation leads to the predominant expression of a truncated PLCD3 protein lacking the N-terminal PH domain. C3H mice that carry one or two mutant Plcd3(mNab alleles are phenotypically normal. However, the presence of one Plcd3(mNab allele exacerbates the alopecia caused by the loss of functional Plcd1 in Del(9olt1Pas mutant mice with respect to the number of hair follicles affected and the body region involved. Mice double homozygous for both the Del(9olt1Pas and the Plcd3(mNab mutations survive for several weeks and exhibit total alopecia associated with fragile hair shafts showing altered expression of some structural genes and shortened phases of proliferation in hair follicle matrix cells. CONCLUSIONS/SIGNIFICANCE: The Plcd3(mNab mutation is a novel hypomorphic mutation of Plcd3. Our investigations suggest that Plcd1 and Plcd3 have synergistic effects on the murine hair follicle in specific regions of the body surface.

Genetic and agronomic evaluation of induced semi-dwarf mutants of rice

International Nuclear Information System (INIS)

Rutger, J.N.

1984-01-01

Induced semi-dwarf mutants have played an important role in California's rapid shift from nearly all tall rice varieties in 1978 to nearly all semi-dwarf varieties at present. In 1981 over half of the California rice area was planted with semi-dwarf varieties carrying the induced mutant semi-dwarfing gene sd 1 , while much of the other half was planted to a variety deriving its semi-dwarfism from IR8. The sd 1 mutant is allelic to the major semi-dwarfing gene in DGWG and IR8. Current objectives are to determine the inheritance of new semi-dwarf mutants, including allelism tests with sd 1 , and to evaluate the agronomic potential of nonallelic sources and of double-dwarfs. To date semi-dwarf mutants from 10 varieties have been partially or completely evaluated. At least three nonallelic semi-dwarfing genes, sd 1 , sd 2 , and sd 4 , have been described. Rather than attempt to determine all possible allelic relationships of new mutants, crosses are being made only to the reference sd 1 source, since sd 1 , still seems to be the most productive semi-dwarfing gene source. However, nonallelic semi-dwarf mutants in the varieties M5 and Labelle may be useful if genetic vulnerability from widespread usage of the sd 1 source becomes a problem. (author)

Enhanced Abscisic Acid-Mediated Responses in nia1nia2noa1-2 Triple Mutant Impaired in NIA/NR- and AtNOA1-Dependent Nitric Oxide Biosynthesis in Arabidopsis1[W

Science.gov (United States)

Lozano-Juste, Jorge; León, José

2010-01-01

Nitric oxide (NO) regulates a wide range of plant processes from development to environmental adaptation. Despite its reported regulatory functions, it remains unclear how NO is synthesized in plants. We have generated a triple nia1nia2noa1-2 mutant that is impaired in nitrate reductase (NIA/NR)- and Nitric Oxide-Associated1 (AtNOA1)-mediated NO biosynthetic pathways. NO content in roots of nia1nia2 and noa1-2 plants was lower than in wild-type plants and below the detection limit in nia1nia2noa1-2 plants. NIA/NR- and AtNOA1-mediated biosynthesis of NO were thus active and responsible for most of the NO production in Arabidopsis (Arabidopsis thaliana). The nia1nia2noa1-2 plants displayed reduced size, fertility, and seed germination potential but increased dormancy and resistance to water deficit. The increasing deficiency in NO of nia1nia2, noa1-2, and nia1nia2noa1-2 plants correlated with increased seed dormancy, hypersensitivity to abscisic acid (ABA) in seed germination and establishment, as well as dehydration resistance. In nia1nia2noa1-2 plants, enhanced drought tolerance was due to a very efficient stomata closure and inhibition of opening by ABA, thus uncoupling NO from ABA-triggered responses in NO-deficient guard cells. The NO-deficient mutants in NIA/NR- and AtNOA1-mediated pathways in combination with the triple mutant will be useful tools to functionally characterize the role of NO and the contribution of both biosynthetic pathways in regulating plant development and defense. PMID:20007448

A comparative glycoproteome study of developing endosperm in the hexose-deficient miniature1 (mn1 seed mutant and its wild type Mn1 in maize

Directory of Open Access Journals (Sweden)

Cecilia eSilva-Sanchez

2014-02-01

Full Text Available In maize developing seeds, transfer cells are prominently located at the basal endosperm transfer layer (BETL. As the first filial cell layer, BETL is a gateway to sugars, nutrients and water from mother plant; and anchor of numerous functions such as sucrose turnover, auxin and cytokinin biosynthesis/accumulation, energy metabolism, defense response, and signaling between maternal and filial generations. Previous studies showed that basal developing endosperms of miniature1 (mn1 mutant seeds lacking the Mn1-encoded cell wall invertase II, are also deficient for hexose. Given the role of glucose as one of the key sugars in protein glycosylation and proper protein folding; we performed a comparative large scale glycoproteome profiling of total proteins of these two genotypes (mn1 mutant vs Mn1 wild type using 2D gel electrophoresis and glycosylation/total protein staining, followed by image analysis. Protein identification was done by LC-MS/MS. A total of 413 spots were detected; from which, 113 spots matched between the two genotypes. Of these, 45 showed > 20% decrease/increase in glycosylation level and were selected for protein identification. A large number of identified proteins showed decreased glycosylation levels in mn1 developing endosperms as compared to the Mn1. Functional classification of proteins, showed mainly of post-translational modification, protein turnover, chaperone activities, carbohydrate and amino acid biosynthesis / transport, and cell wall biosynthesis. These proteins and activities were related to endoplasmic reticulum (ER stress and unfolded protein response (UPR as a result of the low glycolsylation levels of the mutant proteins. Overall, these results provide for the first time a global glycoproteome profile of maize BETL-enriched basal endosperm to better understand their role in seed development in maize.

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

Energy Technology Data Exchange (ETDEWEB)

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

1994-09-01

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

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.

Mutant DD genotype of NFKB1 gene is associated with the susceptibility and severity of coronary artery disease.

Science.gov (United States)

Luo, Jun-Yi; Li, Xiao-Mei; Zhou, Yun; Zhao, Qiang; Chen, Bang-Dang; Liu, Fen; Chen, Xiao-Cui; Zheng, Hong; Ma, Yi-Tong; Gao, Xiao-Ming; Yang, Yi-Ning

2017-02-01

Nuclear factor κappa B (NF-κB) is an important transcription factor in the development and progression of coronary artery disease (CAD). Recent evidence suggests that -94 ATTG ins/del mutant in the promoter of NFKB1 gene is an essential functional mutant. The present study demonstrated the frequencies of the del/del (DD) genotype and del (D) allele were significantly higher in CAD patients than in controls. CAD patients carrying mutant DD genotype had worse stenosis of diseased coronary arteries compared to those carrying ins/ins (II) or ins/del (ID) genotype. Plasma levels of endothelial nitric oxide synthase (eNOS) were lower, while inflammatory cytokine incnterlukin-6 (IL-6) was higher in CAD patients with DD genotype than those with II or ID genotype (both PDD genotype HUVECs) were more susceptible to H 2 O 2 -induced apoptosis, which was accompanied with a decreased Bcl-2 expression. Further, mutant HUVECs had lower eNOS but higher IL-6 mRNA levels and decreased phosphorylation of eNOS under H 2 O 2 -stimulation (both PDD genotype of NFKB1 gene is associated with the risk and severity of CAD. Dwonregulation of NF-κB p50 subunit leads to exacerbated endothelial dysfunction and apoptosis and enhanced inflammatory response that is the potential underlying mechanism. Copyright © 2017 Elsevier Ltd. All rights reserved.

An extra early mutant of pigeonpea

International Nuclear Information System (INIS)

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

2001-01-01

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

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

Science.gov (United States)

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

2005-01-01

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

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.

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.

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.

Indy mutants: live long and prosper

Directory of Open Access Journals (Sweden)

Stewart eFrankel

2012-02-01

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

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)

Characterization of the Escherichia coli prsA1-encoded mutant phosphoribosylpyrophosphate synthetase identifies a divalent cation-nucleotide binding site

DEFF Research Database (Denmark)

Bower, Stanley G.; Harlow, Kenneth W.; Switzer, Robert L.

1989-01-01

: DLHAXQIQGFFDI/VPI/VD. There was little alteration in the Km for ribose 5-phosphate. The Km for ATP of the mutant enzyme was increased 27-fold when Mg2+ was the activating cation but only 5-fold when Mn2+ was used. Maximal velocities of the wild type and mutant enzymes were the same. The mutant enzyme has a 6......-fold lower affinity for Ca2+, as judged by the ability of Ca2+ to inhibit the reaction in the presence of 10 mM Mg2+. Wild type PRPP synthetase is subject to product inhibition by AMP, but AMP inhibition of the prsA1 mutant enzyme could not be detected. It has been previously proposed that a divalent...

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

Isolation and characterization of X-linked mutants of Drosophila melanogaster which are sensitive to mutagens

International Nuclear Information System (INIS)

Boyd, J.B.; Golino, M.D.; Nguyen, T.D.; Green, M.M.

1976-01-01

Thirteen X-linked mutants have been isolated in Drosophila melanogaster which render male and homozygous female larvae sensitive to the mutagen methyl methanesulfonate. Their characterization and preliminary assignment to functional groups is described. Four of these mutants are alleles of mei-41. Like previously isolated alleles of this locus, these mutants reduce fertility and increase loss and nondisjunction of the X-chromosome in homozygous females. The remaining mutants have been tentatively assigned to six functional groups (two mutants to the mus(1)101 locus, two to mus(1)102, two to mus(1)103, and one each to mus(1)104, mus(1)105, and mus(1)106. Several of the complementation groups can be distinguished on the basis of nondisjunction and cross sensitivity to mutagens. Females homozygous for the mei-41, mus(1)101 and mus(1)102 mutants exhibit elevated levels of nondisjunction. Mutants belonging to complementation groups mei-41, mus(1)101, and mus(1)104 are sensitive to nitrogen mustard (HN2) in addition to their MMS sensitivity. Among these mutants there is currently a direct correlation between sensitivity to HN2, sensitivity to 2-acetylaminofluorene and a deficiency in post-replication repair. Only the mei-41 mutants are hypersensitive to uv radiation, although several of the mutants exhibit sensitivity to γ-rays. Semidominance is observed in female larvae of the mei-41, mus(1)104, and mus(1)103 mutants after exposure to high concentrations of MMS. The properties of the mutants generally conform to a pattern which has been established for related mutants in yeast

Characterisation of the Aspergillus nidulans frA1 mutant: hexose phosphorylation and apparent lack of involvement of hexokinase in glucose repression.

NARCIS (Netherlands)

Ruijter, G.J.G.; Panneman, H.; Broeck, van den H.C.; Bennett, J.M.; Visser, J.

1996-01-01

Hexose phosphorylation was studied in Aspergillus nidulans wild-type and in a fructose non-utilising mutant (frA). The data indicate the presence of at least one hexokinase and one glucokinase in wild-type A. nidulans, while the frA1 mutant lacks hexokinase activity. The A. nidulans gene encoding

Radiation induced mutants in cassava (Manihot esculenta Crantz)

International Nuclear Information System (INIS)

Nayar, G.G.; Rajendran, P.G.

1987-01-01

Full text: Stem cuttings and true seeds of three promising cultivars of cassava were exposed respectively to 1 to 5 kR and 10 to 50 kR acute gamma rays from a 60 Co source. Treatments of stem cuttings beyond 5 kR and seeds beyond 50 kR were lethal. One mutant each in the cultivars M4, H-165 and H-2304 was obtained from the stem irradiated populations. Another mutant was found in the seed irradiated progeny of H-2304. The mutant of M4 is characterised by light green (chlorina) leaves. The mutant of H-165 shows significantly shorter petiole (22,5 against 35.2 cm) and narrow leaf lobes, while the H-2304 mutant shows speckled leaves, branching and early flowering. The mutant found in the seed irradiated progeny of H-2304 is having yellow tuber flesh indicating the presence of carotene. The mutants may be useful in studies related to basic information as well as in practical breeding. The chlorina mutant in M4 showed slow growth and high HCN content in leaves. Late branching may be a useful trait in the traditionally non-branching clones of cassava to maintain the desirable leaf area index during high leaf fall period. Early flowering could be useful in a recombinant breeding programme. The tuber yield of the short petiole mutant in H-165 increased by 20% - 25% through closer planting. The narrow leaf lobes of this mutant permit better light penetration to lower leaves. (author)

The Arabidopsis mutant cev1 has constitutively active jasmonate and ethylene signal pathways and enhanced resistance to pathogens.

Science.gov (United States)

Ellis, C; Turner, J G

2001-05-01

Jasmonates (JAs) inhibit plant growth and induce plant defense responses. To define genes in the Arabidopsis JA signal pathway, we screened for mutants with constitutive expression of a luciferase reporter for the JA-responsive promoter from the vegetative storage protein gene VSP1. One mutant, named constitutive expression of VSP1 (cev1), produced plants that were smaller than wild type, had stunted roots with long root hairs, accumulated anthocyanin, had constitutive expression of the defense-related genes VSP1, VSP2, Thi2.1, PDF1.2, and CHI-B, and had enhanced resistance to powdery mildew diseases. Genetic evidence indicated that the cev1 phenotype required both COI1, an essential component of the JA signal pathway, and ETR1, which encodes the ethylene receptor. We conclude that cev1 stimulates both the JA and the ethylene signal pathways and that CEV1 regulates an early step in an Arabidopsis defense pathway.

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

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

Science.gov (United States)

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

2015-01-01

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

A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development.

Science.gov (United States)

Covassin, L D; Siekmann, A F; Kacergis, M C; Laver, E; Moore, J C; Villefranc, J A; Weinstein, B M; Lawson, N D

2009-05-15

In this work we describe a forward genetic approach to identify mutations that affect blood vessel development in the zebrafish. By applying a haploid screening strategy in a transgenic background that allows direct visualization of blood vessels, it was possible to identify several classes of mutant vascular phenotypes. Subsequent characterization of mutant lines revealed that defects in Vascular endothelial growth factor (Vegf) signaling specifically affected artery development. Comparison of phenotypes associated with different mutations within a functional zebrafish Vegf receptor-2 ortholog (referred to as kdr-like, kdrl) revealed surprisingly varied effects on vascular development. In parallel, we identified an allelic series of mutations in phospholipase c gamma 1 (plcg1). Together with in vivo structure-function analysis, our results suggest a requirement for Plcg1 catalytic activity downstream of receptor tyrosine kinases. We further find that embryos lacking both maternal and zygotic plcg1 display more severe defects in artery differentiation but are otherwise similar to zygotic mutants. Finally, we demonstrate through mosaic analysis that plcg1 functions autonomously in endothelial cells. Together our genetic analyses suggest that Vegf/Plcg1 signaling acts at multiple time points and in different signaling contexts to mediate distinct aspects of artery development.

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

International Nuclear Information System (INIS)

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

1987-01-01

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

Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells

Science.gov (United States)

Li, Luyuan; Paz, Ana C.; Wilky, Breelyn A.; Johnson, Britt; Galoian, Karina; Rosenberg, Andrew; Hu, Guozhi; Tinoco, Gabriel; Bodamer, Olaf; Trent, Jonathan C.

2015-01-01

Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2) were recently described in several cancers including chondrosarcomas. The IDH1 inhibitor AGI-5198 abrogates the ability of mutant IDH1 to produce the oncometabolite D-2 hydroxyglutarate (D-2HG) in gliomas. We sought to determine if treatment with AGI-5198 would similarly inhibit tumorigenic activity and D-2HG production in IDH1-mutant human chondrosarcoma cells. Two human chondrosarcoma cell lines, JJ012 and HT1080 with endogenous IDH1 mutations and a human chondrocyte cell line C28 with wild type IDH1 were employed in our study. Mutation analysis of IDH was performed by PCR-based DNA sequencing, and D-2HG was detected using tandem mass spectrometry. We confirmed that JJ012 and HT1080 harbor IDH1 R132G and R132C mutation, respectively, while C28 has no mutation. D-2HG was detectable in cell pellets and media of JJ012 and HT1080 cells, as well as plasma and urine from an IDH-mutant chondrosarcoma patient, which decreased after tumor resection. AGI-5198 treatment decreased D-2HG levels in JJ012 and HT1080 cells in a dose-dependent manner, and dramatically inhibited colony formation and migration, interrupted cell cycling, and induced apoptosis. In conclusion, our study demonstrates anti-tumor activity of a mutant IDH1 inhibitor in human chondrosarcoma cell lines, and suggests that D-2HG is a potential biomarker for IDH mutations in chondrosarcoma cells. Thus, clinical trials of mutant IDH inhibitors are warranted for patients with IDH-mutant chondrosarcomas. PMID:26368816

Treatment with a Small Molecule Mutant IDH1 Inhibitor Suppresses Tumorigenic Activity and Decreases Production of the Oncometabolite 2-Hydroxyglutarate in Human Chondrosarcoma Cells.

Directory of Open Access Journals (Sweden)

Luyuan Li

Full Text Available Chondrosarcomas are malignant bone tumors that produce cartilaginous matrix. Mutations in isocitrate dehydrogenase enzymes (IDH1/2 were recently described in several cancers including chondrosarcomas. The IDH1 inhibitor AGI-5198 abrogates the ability of mutant IDH1 to produce the oncometabolite D-2 hydroxyglutarate (D-2HG in gliomas. We sought to determine if treatment with AGI-5198 would similarly inhibit tumorigenic activity and D-2HG production in IDH1-mutant human chondrosarcoma cells. Two human chondrosarcoma cell lines, JJ012 and HT1080 with endogenous IDH1 mutations and a human chondrocyte cell line C28 with wild type IDH1 were employed in our study. Mutation analysis of IDH was performed by PCR-based DNA sequencing, and D-2HG was detected using tandem mass spectrometry. We confirmed that JJ012 and HT1080 harbor IDH1 R132G and R132C mutation, respectively, while C28 has no mutation. D-2HG was detectable in cell pellets and media of JJ012 and HT1080 cells, as well as plasma and urine from an IDH-mutant chondrosarcoma patient, which decreased after tumor resection. AGI-5198 treatment decreased D-2HG levels in JJ012 and HT1080 cells in a dose-dependent manner, and dramatically inhibited colony formation and migration, interrupted cell cycling, and induced apoptosis. In conclusion, our study demonstrates anti-tumor activity of a mutant IDH1 inhibitor in human chondrosarcoma cell lines, and suggests that D-2HG is a potential biomarker for IDH mutations in chondrosarcoma cells. Thus, clinical trials of mutant IDH inhibitors are warranted for patients with IDH-mutant chondrosarcomas.

Biochemical characteristics of mutant lines of currant tomato

International Nuclear Information System (INIS)

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

1988-01-01

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

Functional phenotypic rescue of Caenorhabditis elegans neuroligin-deficient mutants by the human and rat NLGN1 genes.

Directory of Open Access Journals (Sweden)

Fernando Calahorro

Full Text Available Neuroligins are cell adhesion proteins that interact with neurexins at the synapse. This interaction may contribute to differentiation, plasticity and specificity of synapses. In humans, single mutations in neuroligin encoding genes lead to autism spectrum disorder and/or mental retardation. Caenorhabditis elegans mutants deficient in nlg-1, an orthologue of human neuroligin genes, have defects in different behaviors. Here we show that the expression of human NLGN1 or rat Nlgn1 cDNAs in C. elegans nlg-1 mutants rescues the fructose osmotic strength avoidance and gentle touch response phenotypes. Two specific point mutations in NLGN3 and NLGN4 genes, involved in autistic spectrum disorder, were further characterized in this experimental system. The R451C allele described in NLGN3, was analyzed with both human NLGN1 (R453C and worm NLG-1 (R437C proteins, and both were not functional in rescuing the osmotic avoidance behavior and the gentle touch response phenotype. The D396X allele described in NLGN4, which produces a truncated protein, was studied with human NLGN1 (D432X and they did not rescue any of the behavioral phenotypes analyzed. In addition, RNAi feeding experiments measuring gentle touch response in wild type strain and worms expressing SID-1 in neurons (which increases the response to dsRNA, both fed with bacteria expressing dsRNA for nlg-1, provided evidence for a postsynaptic in vivo function of neuroligins both in muscle cells and neurons, equivalent to that proposed in mammals. This finding was further confirmed generating transgenic nlg-1 deficient mutants expressing NLG-1 under pan-neuronal (nrx-1 or pan-muscular (myo-3 specific promoters. All these results suggest that the nematode could be used as an in vivo model for studying particular synaptic mechanisms with proteins orthologues of humans involved in pervasive developmental disorders.

Forgetting in C. elegans Is Accelerated by Neuronal Communication via the TIR-1/JNK-1 Pathway

Directory of Open Access Journals (Sweden)

Akitoshi Inoue

2013-03-01

Full Text Available The control of memory retention is important for proper responses to constantly changing environments, but the regulatory mechanisms underlying forgetting have not been fully elucidated. Our genetic analyses in C. elegans revealed that mutants of the TIR-1/JNK-1 pathway exhibited prolonged retention of olfactory adaptation and salt chemotaxis learning. In olfactory adaptation, conditioning induces attenuation of odor-evoked Ca2+ responses in olfactory neurons, and this attenuation is prolonged in the TIR-1/JNK-1-pathway mutant animals. We also found that a pair of neurons in which the pathway functions is required for the acceleration of forgetting, but not for sensation or adaptation, in wild-type animals. In addition, the neurosecretion from these cells is important for the acceleration of forgetting. Therefore, we propose that these neurons accelerate forgetting through the TIR-1/JNK-1 pathway by sending signals that directly or indirectly stimulate forgetting.

The Arabidopsis aba4-1 Mutant Reveals a Specific Function for Neoxanthin in Protection against Photooxidative Stress[W

Science.gov (United States)

Dall'Osto, Luca; Cazzaniga, Stefano; North, Helen; Marion-Poll, Annie; Bassi, Roberto

2007-01-01

The aba4-1 mutant completely lacks neoxanthin but retains all other xanthophyll species. The missing neoxanthin in light-harvesting complex (Lhc) proteins is compensated for by higher levels of violaxanthin, albeit with lower capacity for photoprotection compared with proteins with wild-type levels of neoxanthin. Detached leaves of aba4-1 were more sensitive to oxidative stress than the wild type when exposed to high light and incubated in a solution of photosensitizer agents. Both treatments caused more rapid pigment bleaching and lipid oxidation in aba4-1 than wild-type plants, suggesting that neoxanthin acts as an antioxidant within the photosystem II (PSII) supercomplex in thylakoids. While neoxanthin-depleted Lhc proteins and leaves had similar sensitivity as the wild type to hydrogen peroxide and singlet oxygen, they were more sensitive to superoxide anions. aba4-1 intact plants were not more sensitive than the wild type to high-light stress, indicating the existence of compensatory mechanisms of photoprotection involving the accumulation of zeaxanthin. However, the aba4-1 npq1 double mutant, lacking zeaxanthin and neoxanthin, underwent stronger PSII photoinhibition and more extensive oxidation of pigments than the npq1 mutant, which still contains neoxanthin. We conclude that neoxanthin preserves PSII from photoinactivation and protects membrane lipids from photooxidation by reactive oxygen species. Neoxanthin appears particularly active against superoxide anions produced by the Mehler's reaction, whose rate is known to be enhanced in abiotic stress conditions. PMID:17351115

A zebrafish screen for craniofacial mutants identifies wdr68 as a highly conserved gene required for endothelin-1 expression

Directory of Open Access Journals (Sweden)

Amsterdam Adam

2006-06-01

Full Text Available Abstract Background Craniofacial birth defects result from defects in cranial neural crest (NC patterning and morphogenesis. The vertebrate craniofacial skeleton is derived from cranial NC cells and the patterning of these cells occurs within the pharyngeal arches. Substantial efforts have led to the identification of several genes required for craniofacial skeletal development such as the endothelin-1 (edn1 signaling pathway that is required for lower jaw formation. However, many essential genes required for craniofacial development remain to be identified. Results Through screening a collection of insertional zebrafish mutants containing approximately 25% of the genes essential for embryonic development, we present the identification of 15 essential genes that are required for craniofacial development. We identified 3 genes required for hyomandibular development. We also identified zebrafish models for Campomelic Dysplasia and Ehlers-Danlos syndrome. To further demonstrate the utility of this method, we include a characterization of the wdr68 gene. We show that wdr68 acts upstream of the edn1 pathway and is also required for formation of the upper jaw equivalent, the palatoquadrate. We also present evidence that the level of wdr68 activity required for edn1 pathway function differs between the 1st and 2nd arches. Wdr68 interacts with two minibrain-related kinases, Dyrk1a and Dyrk1b, required for embryonic growth and myotube differentiation, respectively. We show that a GFP-Wdr68 fusion protein localizes to the nucleus with Dyrk1a in contrast to an engineered loss of function mutation Wdr68-T284F that no longer accumulated in the cell nucleus and failed to rescue wdr68 mutant animals. Wdr68 homologs appear to exist in all eukaryotic genomes. Notably, we found that the Drosophila wdr68 homolog CG14614 could substitute for the vertebrate wdr68 gene even though insects lack the NC cell lineage. Conclusion This work represents a systematic

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

Science.gov (United States)

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

2011-04-06

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

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.

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

Science.gov (United States)

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

2017-01-01

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

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

Directory of Open Access Journals (Sweden)

Peng Li

2017-09-01

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

Characterization of a Weak Allele of Zebrafish cloche Mutant

Science.gov (United States)

Ma, Ning; Huang, Zhibin; Chen, Xiaohui; He, Fei; Wang, Kun; Liu, Wei; Zhao, Linfeng; Xu, Xiangmin; Liao, Wangjun; Ruan, Hua; Luo, Shenqiu; Zhang, Wenqing

2011-01-01

Hematopoiesis is a complicated and dynamic process about which the molecular mechanisms remain poorly understood. Danio rerio (zebrafish) is an excellent vertebrate system for studying hematopoiesis and developmental mechanisms. In the previous study, we isolated and identified a cloche 172 (clo 172) mutant, a novel allele compared to the original cloche (clo) mutant, through using complementation test and initial mapping. Here, according to whole mount in-situ hybridization, we report that the endothelial cells in clo 172 mutant embryos, although initially developed, failed to form the functional vascular system eventually. In addition, further characterization indicates that the clo 172 mutant exhibited weaker defects instead of completely lost in primitive erythroid cells and definitive hematopoietic cells compared with the clo s5 mutant. In contrast, primitive myeloid cells were totally lost in clo 172 mutant. Furthermore, these reappeared definitive myeloid cells were demonstrated to initiate from the remaining hematopoietic stem cells (HSCs) in clo 172 mutant, confirmed by the dramatic decrease of lyc in clo 172 runx1w84x double mutant. Collectively, the clo 172 mutant is a weak allele compared to the clo s5 mutant, therefore providing a model for studying the early development of hematopoietic and vascular system, as well as an opportunity to further understand the function of the cloche gene. PMID:22132109

Evolution of inhibitor-resistant natural mutant forms of HIV-1 protease probed by pre-steady state kinetic analysis.

Science.gov (United States)

Zakharova, Maria Yu; Kuznetsova, Alexandra A; Kaliberda, Elena N; Dronina, Maria A; Kolesnikov, Alexander V; Kozyr, Arina V; Smirnov, Ivan V; Rumsh, Lev D; Fedorova, Olga S; Knorre, Dmitry G; Gabibov, Alexander G; Kuznetsov, Nikita A

2017-11-01

Pre-steady state kinetic analysis of mechanistic features of substrate binding and processing is crucial for insight into the evolution of inhibitor-resistant forms of HIV-1 protease. These data may provide a correct vector for rational drug design assuming possible intrinsic dynamic effects. These data should also give some clues to the molecular mechanism of protease action and resistance to inhibitors. Here we report pre-steady state kinetics of the interaction of wild type or mutant forms of HIV-1 protease with a FRET-labeled peptide. The three-stage "minimal" kinetic scheme with first and second reversible steps of substrate binding and with following irreversible peptide cleavage step adequately described experimental data. For the first time, a set of "elementary" kinetic parameters of wild type HIV-1 protease and its natural mutant inhibitor-resistant forms MDR-HM, ANAM-11 and prDRV4 were compared. Inhibitors of the first and second generation were used to estimate the inhibitory effects on HIV-1 protease activity. The resulting set of kinetic data supported that the mutant forms are kinetically unaffected by inhibitors of the first generation, proving their functional resistance to these compounds. The second generation inhibitor darunavir inhibited mutant forms MDR-HM and ANAM-11, but was ineffective against prDRV4. Our kinetic data revealed that these inhibitors induced different conformational changes in the enzyme and, thereby they have different mode of binding in the enzyme active site. These data confirmed hypothesis that the driving force of the inhibitor-resistance evolution is disruption of enzyme-inhibitor complex by changing of the contact network in the inhibitor binding site. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Increased spontaneous mitotic segregation in MMS-sensitive mutants of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Prakash, S.; Prakash, L.

1977-01-01

Methyl methanesulfonate (MMS)-sensitive mutants of Saccharomyces cerevisiae belonging to four different complementation groups, when homozygous, increase the rate of spontaneous mitotic segregation to canavanine resistance from heterozygous sensitive (can/sup r//+) diploids by 13- to 170-fold. The mms8-1 mutant is MMS and x-ray sensitive and increases the rate of spontaneous mitotic segregation 170-fold. The mms9-1 and mms13-1 mutants are sensitive to x rays and uv, respectively, in addition to MMS, and increase the rate of spontaneous mitotic segregation by 13-fold and 85-fold, respectively. The mutant mms21-1 is sensitive to MMS, x rays and uv and increases the rate of spontaneous mitotic segregation 23-fold

Study on the early and late mutants of radiation induced rice

International Nuclear Information System (INIS)

Yang Hefeng; Chen Xiulan; He Zhentian; Gu Shiliang; Xu Chenwu

1990-12-01

After three years of consecutive experiments for the irradiated M 2 generations of 53 different varieties of rice, the following results have been obtained: (1) The average of early mutant plant rate is 1.4%. The rate in the early-maturing varieties is lower than that in the late-maturing varieties. It is in proportion to the length of growing period of these varieties tested. The shortened days of growing period of early mutants are 3 to 32 days (the average was 9.5 days), and it is increasing as the growing period increases. (2) In the irradiated M 2 generation of same variety, the early mutants and late mutants could be simultaneously happened, but the rate of the late mutants is 2.67%, which is higher than the rate of early mutants (1.39%). The shortened and prolonged days of growing period are 11.5 and 10.5 days respectively. These early and late mutants have some changes, both good and bad, in agronomical traits such as plant height, weight per kilo-grains and grains number per tassel. In some extent these changes are significant

A metabolic signature for long life in the Caenorhabditis elegans Mit mutants.

Science.gov (United States)

Butler, Jeffrey A; Mishur, Robert J; Bhaskaran, Shylesh; Rea, Shane L

2013-02-01

Mit mutations that disrupt function of the mitochondrial electron transport chain can, inexplicably, prolong Caenorhabditis elegans lifespan. In this study we use a metabolomics approach to identify an ensemble of mitochondrial-derived α-ketoacids and α-hydroxyacids that are produced by long-lived Mit mutants but not by other long-lived mutants or by short-lived mitochondrial mutants. We show that accumulation of these compounds is dependent on concerted inhibition of three α-ketoacid dehydrogenases that share dihydrolipoamide dehydrogenase (DLD) as a common subunit, a protein previously linked in humans with increased risk of Alzheimer's disease. When the expression of DLD in wild-type animals was reduced using RNA interference we observed an unprecedented effect on lifespan - as RNAi dosage was increased lifespan was significantly shortened, but, at higher doses, it was significantly lengthened, suggesting that DLD plays a unique role in modulating length of life. Our findings provide novel insight into the origin of the Mit phenotype. © 2012 The Authors Aging Cell © 2012 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.

Purification and characterization of naturally occurring HIV-1 (South African subtype C) protease mutants from inclusion bodies.

Science.gov (United States)

Maseko, Sibusiso B; Natarajan, Satheesh; Sharma, Vikas; Bhattacharyya, Neelakshi; Govender, Thavendran; Sayed, Yasien; Maguire, Glenn E M; Lin, Johnson; Kruger, Hendrik G

2016-06-01

Human immunodeficiency virus (HIV) infections in sub-Saharan Africa represent about 56% of global infections. Many studies have targeted HIV-1 protease for the development of drugs against AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. Along with the wild type (C-SA) we also over-expressed and characterized two mutant forms from patients that had shown resistance to protease inhibitors. Using recombinant DNA technology, we constructed three recombinant plasmids in pGEX-6P-1 and expressed them containing a sequence encoding wild type HIV protease and two mutants (I36T↑T contains 100 amino acids and L38L↑N↑L contains 101 amino acids). These recombinant proteins were isolated from inclusion bodies by using QFF anion exchange and GST trap columns. In SDS-PAGE, we obtained these HIV proteases as single bands of approximately 11.5, 11.6 and 11.7 kDa for the wild type, I36T↑Tand L38L↑N↑L mutants, respectively. The enzyme was recovered efficiently (0.25 mg protein/L of Escherichia coli culture) and had high specific activity of 2.02, 2.20 and 1.33 μmol min(-1) mg(-1) at an optimal pH of 5 and temperature of 37 °C for the wild type, I36T↑T and L38L↑N↑L, respectively. The method employed here provides an easy and rapid purification of the HIV-1(C-SA) protease from the inclusion bodies, with high yield and high specific activities. Copyright © 2016 Elsevier Inc. All rights reserved.

UV-induced lethal sectoring and pure mutant clones in yeast.

Science.gov (United States)

Hannan, M A; Duck, P; Nasim, A

1976-08-01

The induction of lethal sectoring and pure mutant clones by ultraviolet light has been studied in a homogeneous G1 population of Saccharomyces cerevisiae grown in a normal growth medium. At the lowest UV dose of 250 ergs, which corresponds to a shoulder in the survival curve, all mutants appeared as pure clones. At higher doses the frequency of mosaic mutants progressively increased. These results indicate a relationship between the highest frequency of complete mutants and the maximum repair activity. In addition, the frequency of lethal sectoring at all doses tested was too low to account for the origin of pure mutant clones.

PtAUREO1a and PtAUREO1b knockout mutants of the diatom Phaeodactylum tricornutum are blocked in photoacclimation to blue light.

Science.gov (United States)

Mann, Marcus; Serif, Manuel; Jakob, Torsten; Kroth, Peter G; Wilhelm, Christian

2017-10-01

Aureochromes are blue light receptors specifically found in photosynthetic Stramenopiles (algae). Four different Aureochromes have been identified in the marine diatom Phaeodactylum tricornutum (PtAUREO 1a, 1b, 1c, and 2). Since blue light is necessary for high light acclimation in diatoms, it has been hypothesized that Aureochromes might play an important role in the light acclimation capacity of diatoms. This hypothesis was supported by an RNAi knockdown line of PtAUREO1a, which showed a phenotype different from wild type cells when grown in either blue or red light. Here, we show for the first time the phenotype and the photoacclimation reaction of TALEN-mediated knockout mutants of PtAUREO1a and PtAUREO1b, clearly proving the necessity of Aureochromes for light acclimation under blue light. However, both mutants do also show specific differences in their respective phenotypes. Hence, PtAUREO1a and 1b are not functionally redundant in photoacclimation to blue light, and their specific contribution needs to be clarified further. Copyright © 2017 Elsevier GmbH. All rights reserved.

Genetic Characterization of the Carotenoid Biosynthetic Pathway in Methylobacterium extorquens AM1 and Isolation of a Colorless Mutant

Science.gov (United States)

Van Dien, Stephen J.; Marx, Christopher J.; O'Brien, Brooke N.; Lidstrom, Mary E.

2003-01-01

Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments. PMID:14660416

Genetic characterization of the carotenoid biosynthetic pathway in Methylobacterium extorquens AM1 and isolation of a colorless mutant.

Science.gov (United States)

Van Dien, Stephen J; Marx, Christopher J; O'Brien, Brooke N; Lidstrom, Mary E

2003-12-01

Genomic searches were used to reconstruct the putative carotenoid biosynthesis pathway in the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. Four genes for putative phytoene desaturases were identified. A colorless mutant was obtained by transposon mutagenesis, and the insertion was shown to be in one of the putative phytoene desaturase genes. Mutations in the other three did not affect color. The tetracycline marker was removed from the original transposon mutant, resulting in a pigment-free strain with wild-type growth properties useful as a tool for future experiments.

PARP-1 inhibition alleviates diabetic cardiac complications in experimental animals.

Science.gov (United States)

Zakaria, Esraa M; El-Bassossy, Hany M; El-Maraghy, Nabila N; Ahmed, Ahmed F; Ali, Abdelmoneim A

2016-11-15

Cardiovascular complications are the major causes of mortality among diabetic population. Poly(ADP-ribose) polymerase-1 enzyme (PARP-1) is activated by oxidative stress leading to cellular damage. We investigated the implication of PARP-1 in diabetic cardiac complications. Type 2 diabetes was induced in rats by high fructose-high fat diet and low streptozotocin dose. PARP inhibitor 4-aminobenzamide (4-AB) was administered daily for ten weeks after diabetes induction. At the end of study, surface ECG, blood pressure and vascular reactivity were studied. PARP-1 activity, reduced glutathione (GSH) and nitrite contents were assessed in heart muscle. Fasting glucose, fructosamine, insulin, and tumor necrosis factor alpha (TNF-α) levels were measured in serum. Finally, histological examination and collagen deposition detection in rat ventricular and aortic sections were carried out. Hearts isolated from diabetic animals showed increased PARP-1 enzyme activity compared to control animals while significantly reduced by 4-AB administration. PARP-1 inhibition by 4-AB alleviated cardiac ischemia in diabetic animals as indicated by ECG changes. PARP-1 inhibition also reduced cardiac inflammation in diabetic animals as evidenced by histopathological changes. In addition, 4-AB administration improved the elevated blood pressure and the associated exaggerated vascular contractility, endothelial destruction and vascular inflammation seen in diabetic animals. Moreover, PARP-1 inhibition decreased serum levels of TNF-α and cardiac nitrite but increased cardiac GSH contents in diabetic animals. However, PARP-1 inhibition did not significantly affect the developed hyperglycemia. Our findings prove that PARP-1 enzyme plays an important role in diabetic cardiac complications through combining inflammation, oxidative stress, and fibrosis mechanisms. Copyright © 2016. Published by Elsevier B.V.

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

Directory of Open Access Journals (Sweden)

Seok Joo Park

2014-09-01

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

UV and gamma-ray sensitivity of meiosis-deficient mutants in Podospora anserina

International Nuclear Information System (INIS)

Simonet, J.M.

1976-01-01

Two mutants, mei1 and mei2, were isolated by screening for deficiencies occurring in the meiotic process. The sensitivity of mei1 and mei2 mutant strains to UV irradiation showed a significant increase as compared with that of the wild-type stock, hwhereas the sensitivity to γ-rays remained unchanged. The double-mutant strains were no more sensitive than each single mutant. The data indicate that both mei1 and mei2 loci are probably involved in the same pathway of excision-repair of UV-induced lesions

Towards the Identification of New Genes Involved in ABA-Dependent Abiotic Stresses Using Arabidopsis Suppressor Mutants of abh1 Hypersensitivity to ABA during Seed Germination

Directory of Open Access Journals (Sweden)

Iwona Szarejko

2013-06-01

Full Text Available Abscisic acid plays a pivotal role in the abiotic stress response in plants. Although great progress has been achieved explaining the complexity of the stress and ABA signaling cascade, there are still many questions to answer. Mutants are a valuable tool in the identification of new genes or new alleles of already known genes and in elucidating their role in signaling pathways. We applied a suppressor mutation approach in order to find new components of ABA and abiotic stress signaling in Arabidopsis. Using the abh1 (ABA hypersensitive 1 insertional mutant as a parental line for EMS mutagenesis, we selected several mutants with suppressed hypersensitivity to ABA during seed germination. Here, we present the response to ABA and a wide range of abiotic stresses during the seed germination and young seedling development of two suppressor mutants—soa2 (suppressor of abh1 hypersensitivity to ABA 2 and soa3 (suppressor of abh1 hypersensitivity to ABA 3. Generally, both mutants displayed a suppression of the hypersensitivity of abh1 to ABA, NaCl and mannitol during germination. Both mutants showed a higher level of tolerance than Columbia-0 (Col-0—the parental line of abh1 in high concentrations of glucose. Additionally, soa2 exhibited better root growth than Col-0 in the presence of high ABA concentrations. soa2 and soa3 were drought tolerant and both had about 50% fewer stomata per mm2 than the wild-type but the same number as their parental line—abh1. Taking into account that suppressor mutants had the same genetic background as their parental line—abh1, it was necessary to backcross abh1 with Landsberg erecta four times for the map-based cloning approach. Mapping populations, derived from the cross of abh1 in the Landsberg erecta background with each suppressor mutant, were created. Map based cloning in order to identify the suppressor genes is in progress.