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Sample records for lrrk2 g2019s mutant-induced

  1. Synaptic function is modulated by LRRK2 and glutamate release is increased in cortical neurons of G2019S LRRK2 knock-in mice.

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    Beccano-Kelly, Dayne A; Kuhlmann, Naila; Tatarnikov, Igor; Volta, Mattia; Munsie, Lise N; Chou, Patrick; Cao, Li-Ping; Han, Heather; Tapia, Lucia; Farrer, Matthew J; Milnerwood, Austen J

    2014-01-01

    Mutations in Leucine-Rich Repeat Kinase-2 (LRRK2) result in familial Parkinson's disease and the G2019S mutation alone accounts for up to 30% in some ethnicities. Despite this, the function of LRRK2 is largely undetermined although evidence suggests roles in phosphorylation, protein interactions, autophagy and endocytosis. Emerging reports link loss of LRRK2 to altered synaptic transmission, but the effects of the G2019S mutation upon synaptic release in mammalian neurons are unknown. To assess wild type and mutant LRRK2 in established neuronal networks, we conducted immunocytochemical, electrophysiological and biochemical characterization of >3 week old cortical cultures of LRRK2 knock-out, wild-type overexpressing and G2019S knock-in mice. Synaptic release and synapse numbers were grossly normal in LRRK2 knock-out cells, but discretely reduced glutamatergic activity and reduced synaptic protein levels were observed. Conversely, synapse density was modestly but significantly increased in wild-type LRRK2 overexpressing cultures although event frequency was not. In knock-in cultures, glutamate release was markedly elevated, in the absence of any change to synapse density, indicating that physiological levels of G2019S LRRK2 elevate probability of release. Several pre-synaptic regulatory proteins shown by others to interact with LRRK2 were expressed at normal levels in knock-in cultures; however, synapsin 1 phosphorylation was significantly reduced. Thus, perturbations to the pre-synaptic release machinery and elevated synaptic transmission are early neuronal effects of LRRK2 G2019S. Furthermore, the comparison of knock-in and overexpressing cultures suggests that one copy of the G2019S mutation has a more pronounced effect than an ~3-fold increase in LRRK2 protein. Mutant-induced increases in transmission may convey additional stressors to neuronal physiology that may eventually contribute to the pathogenesis of Parkinson's disease.

  2. New contribution on the LRRK2 G2019S mutation associated to ...

    African Journals Online (AJOL)

    ... generations ago. Conclusion: Our conclusion is that the G2019S mutation of the LRRK2 gene originates 3,840 (95% CI 3,210-5,400) years ago in parkinsonian Moroccan Berbers patients. Key words: Parkinson's disease (PD), Leucine-rich repeat kinase 2 (LRRK2) gene, G2019S mutation, Haplotype, Founding mutation.

  3. RNAi Mediated Silencing of LRRK2G2019S in Parkinson’s Disease

    Science.gov (United States)

    2013-08-01

    WT, but not LRRK2 mutant, protected 446 dopaminergic neurons against rotenone or paraquat toxicity, 447Q12 agents which compromise [52]. The...and animal models of G2019S-mediated neurotoxicity to establish a novel therapy for PD. To achieve this objective we proposed the following Specific...kinase domain (kinase dead mutants) diminishes neurotoxicity and basal kinase levels appear to be required for the toxicity of all LRRK2 mutants [6

  4. Motor and non-motor features of Parkinson's disease in LRRK2 G2019S carriers versus matched controls.

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    Gunzler, Steven A; Riley, David E; Chen, Shu G; Tatsuoka, Curtis M; Johnson, William M; Mieyal, John J; Walter, Ellen M; Whitney, Christina M; Feng, I Jung; Owusu-Dapaah, Harry; Mittal, Shivam O; Wilson-Delfosse, Amy L

    2018-05-15

    LRRK2 G2019S mutation carriers with Parkinson's disease (PD) have been generally indistinguishable from those with idiopathic PD, with the exception of variable differences in some motor and non-motor domains, including cognition, gait, and balance. LRRK2 G2019S is amongst the most common genetic etiologies for PD, particularly in Ashkenazi Jewish (AJ) populations. This cross-sectional data collection study sought to clarify the phenotype of LRRK2 G2019S mutation carriers with PD. Primary endpoints were the Movement Disorder Society Unified Parkinson Disease Rating Scale (MDS-UPDRS) and Montreal Cognitive Assessment (MoCA). Other motor and non-motor data were also assessed. The Mann-Whitney U Test was utilized to compare LRRK2 G2019S carriers with PD (LRRK2+) with non-carrier PD controls who were matched for age, gender, education, and PD duration. Survival analyses and log rank tests were utilized to compare interval from onset of PD to development of motor and non-motor complications. We screened 251 subjects and 231 completed the study, of whom 9 were LRRK2+, including 7 AJ subjects. 22.73% of AJ subjects with a family history of PD (FH) and 12.96% of AJ subjects without a FH were LRRK2+. There were no significant differences between the 9 LRRK2+ subjects and 19 matched PD controls in MDS-UPDRS, MoCA, or other motor and non-motor endpoints. Prevalence of the LRRK2 G2019S mutation in AJ and non-AJ subjects in our study population in Cleveland, Ohio was comparable to other clinical studies. There were no significant motor or non-motor differences between LRRK2+ PD and matched PD controls. Copyright © 2018 Elsevier B.V. All rights reserved.

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

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    David Ramonet

    2011-04-01

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

  6. Frequency of the LRRK2 G2019S mutation in late-onset sporadic patients with Parkinson’s disease

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    Hsin Fen Chien

    2014-05-01

    Full Text Available Mutations in the LRRK2 gene, predominantly G2019S, have been reported in individuals with autosomal dominant inheritance and sporadic Parkinson’s disease (PD. The G2019S mutation has an age-dependent penetrance and evidence shows common ancestry. The clinical manifestations are indistinguishable from idiopathic PD. Its prevalence varies according to the population studied ranging from less than 0.1% in Asians to 41% in North African Arabs. This study aimed to identify G2019S mutation in Brazilian idiopathic PD patients. Method: We sampled 100 PD patients and 100 age- and gender-matched controls. Genetical analysis was accomplished by polymerase chain reaction (PCR. Results: No G2019S mutations were found in both patients with sporadic PD and controls. Conclusions: Our results may be explained by the relatively small sample size.

  7. Parkinson's disease-related LRRK2 G2019S mutation results from independent mutational events in humans.

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    Lesage, Suzanne; Patin, Etienne; Condroyer, Christel; Leutenegger, Anne-Louise; Lohmann, Ebba; Giladi, Nir; Bar-Shira, Anat; Belarbi, Soraya; Hecham, Nassima; Pollak, Pierre; Ouvrard-Hernandez, Anne-Marie; Bardien, Soraya; Carr, Jonathan; Benhassine, Traki; Tomiyama, Hiroyuki; Pirkevi, Caroline; Hamadouche, Tarik; Cazeneuve, Cécile; Basak, A Nazli; Hattori, Nobutaka; Dürr, Alexandra; Tazir, Meriem; Orr-Urtreger, Avi; Quintana-Murci, Lluis; Brice, Alexis

    2010-05-15

    Mutations in the leucine-rich-repeat kinase 2 (LRRK2) gene have been identified in families with autosomal dominant Parkinson's disease (PD) and in sporadic cases; the G2019S mutation is the single most frequent. Intriguingly, the frequency of this mutation in PD patients varies greatly among ethnic groups and geographic origins: it is present at <0.1% in East Asia, approximately 2% in European-descent patients and can reach frequencies of up to 15-40% in PD Ashkenazi Jews and North African Arabs. To ascertain the evolutionary dynamics of the G2019S mutation in different populations, we genotyped 74 markers spanning a 16 Mb genomic region around G2019S, in 191 individuals carrying the mutation from 126 families of different origins. Sixty-seven families were of North-African Arab origin, 18 were of North/Western European descent, 37 were of Jewish origin, mostly from Eastern Europe, one was from Japan, one from Turkey and two were of mixed origins. We found the G2019S mutation on three different haplotypes. Network analyses of the three carrier haplotypes showed that G2019S arose independently at least twice in humans. In addition, the population distribution of the intra-allelic diversity of the most widespread carrier haplotype, together with estimations of the age of G2019S determined by two different methods, suggests that one of the founding G2019S mutational events occurred in the Near East at least 4000 years ago.

  8. LRRK2 G2019S mutation in Parkinson's disease: a neuropsychological and neuropsychiatric study in a large Algerian cohort.

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    Belarbi, Soreya; Hecham, Nassima; Lesage, Suzanne; Kediha, Mohamed I; Smail, Nourredine; Benhassine, Traki; Ysmail-Dahlouk, Farida; Lohman, Ebba; Benhabyles, Badia; Hamadouche, Tarik; Assami, Salima; Brice, Alexis; Tazir, Meriem

    2010-12-01

    A series of 106 patients with isolated or familial Parkinsonism underwent clinical evaluation and genetic testing for the LRRK2 G2019S mutation which was identified in 34/106 patients (32%). Seventy one of them accepted to be evaluated for neuropsychological and neuropsychiatric studies with the aim to compare mutation carriers with non-carriers. For neuropsychological testing, comparisons between LRRK2 G2019S carriers and non-carriers were made after stratification according to the level of education: median and high school versus low level. Memory was investigated with the five words test, 2 novel tests with verbalized visual material dedicated to illiterate patients, the TNI-93 (nine pictures test), The TMA-93 (associative memory test), and digit spans (forward/backward). Cognitive analyse did not show major differences between the two groups of patients. Nevertheless, behavioral abnormalities, mostly depression and hallucinations, were more frequent in the LRRK2 G2019S carriers, suggesting the presence of a greater involvement of the limbic system in these patients. Sleep disorders which were also more common amongst mutation carriers than non-carriers might be related to depression. Copyright © 2010 Elsevier Ltd. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

  10. DaT-SPECT assessment depicts dopamine depletion among asymptomatic G2019S LRRK2 mutation carriers.

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    Moran Artzi

    Full Text Available Identification of early changes in Dopamine-Transporter (DaT SPECT imaging expected in the prodromal phase of Parkinson's disease (PD, are usually overlooked. Carriers of the G2019S LRRK2 mutation are known to be at high risk for developing PD, compared to non-carriers. In this work we aimed to study early changes in Dopamine uptake in non-manifesting PD carriers (NMC of the G2019S LRRK2 mutation using quantitative DaT-SPECT analysis and to examine the potential for early prediction of PD. Eighty Ashkenazi-Jewish subjects were included in this study: eighteen patients with PD; thirty-one NMC and thirty-one non-manifesting non-carriers (NMNC. All subjects underwent a through clinical assessment including evaluation of motor, olfactory, affective and non-motor symptoms and DaT-SPECT imaging. A population based DaT-SPECT template was created based on the NMNC cohort, and data driven volumes-of-interest (VOIs were defined. Comparisons between groups were performed based on VOIs and voxel-wise analysis. The striatum area of all three cohorts was segmented into four VOIs, corresponding to the right/left dorsal and ventral striatum. Significant differences in clinical measures were found between patients with PD and non-manifesting subjects with no differences between NMC and NMNC. Significantly lower uptake (p<0.001 was detected in the right and left dorsal striatum in the PD group (2.2±0.3, 2.3±0.4 compared to the NMC (4.2±0.6, 4.3±0.5 and NMNC (4.5±0.6, 4.6±0.6, and significantly (p = 0.05 lower uptake in the right dorsal striatum in the NMC group compared to NMNC. Converging results were obtained using voxel-wise analysis. Two NMC participants, who later phenoconverted into PD, demonstrated reduced uptake mainly in the dorsal striatum. No significant correlations were found between the DaT-SPECT uptake in the different VOIs and clinical and behavioral assessments in the non-manifesting groups. This study shows the clinical value of

  11. Dopaminergic expression of the Parkinsonian gene LRRK2-G2019S leads to non-autonomous visual neurodegeneration, accelerated by increased neural demands for energy

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    Hindle, Samantha; Afsari, Farinaz; Stark, Meg; Middleton, C. Adam; Evans, Gareth J.O.; Sweeney, Sean T.; Elliott, Christopher J.H.

    2013-01-01

    Parkinson's disease (PD) is associated with loss of dopaminergic signalling, and affects not just movement, but also vision. As both mammalian and fly visual systems contain dopaminergic neurons, we investigated the effect of LRRK2 mutations (the most common cause of inherited PD) on Drosophila electroretinograms (ERGs). We reveal progressive loss of photoreceptor function in flies expressing LRRK2-G2019S in dopaminergic neurons. The photoreceptors showed elevated autophagy, apoptosis and mitochondrial disorganization. Head sections confirmed extensive neurodegeneration throughout the visual system, including regions not directly innervated by dopaminergic neurons. Other PD-related mutations did not affect photoreceptor function, and no loss of vision was seen with kinase-dead transgenics. Manipulations of the level of Drosophila dLRRK suggest G2019S is acting as a gain-of-function, rather than dominant negative mutation. Increasing activity of the visual system, or of just the dopaminergic neurons, accelerated the G2019S-induced deterioration of vision. The fly visual system provides an excellent, tractable model of a non-autonomous deficit reminiscent of that seen in PD, and suggests that increased energy demand may contribute to the mechanism by which LRRK2-G2019S causes neurodegeneration. PMID:23396536

  12. CRISPR/Cas9 and piggyBac-mediated footprint-free LRRK2-G2019S knock-in reveals neuronal complexity phenotypes and α-Synuclein modulation in dopaminergic neurons.

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    Qing, Xiaobing; Walter, Jonas; Jarazo, Javier; Arias-Fuenzalida, Jonathan; Hillje, Anna-Lena; Schwamborn, Jens C

    2017-10-01

    The p.G2019S mutation of the leucine-rich repeat kinase 2 (LRRK2) has been identified as the most prevalent genetic cause of familial and sporadic Parkinson's disease (PD). The Cre-LoxP recombination system has been used to correct the LRRK2-G2019S mutation in patient derived human induced pluripotent stem cells (hiPSCs) in order to generate isogenic controls. However, the remaining LoxP site can influence gene expression. In this study, we report the generation of a footprint-free LRRK2-G2019S isogenic hiPS cell line edited with the CRISPR/Cas9 and piggyBac technologies. We observed that the percentage of Tyrosine Hydroxylase (TH) positive neurons with a total neurite length of >2000μm was significantly reduced in LRRK2-G2019S dopaminergic (DA) neurons. The average branch number in LRRK2-G2019S DA neurons was also decreased. In addition, we have shown that in vitro TH positive neurons with a total neurite length of >2000μm were positive for Serine 129 phosphorylated (S129P) alpha-Synuclein (αS) and we hypothesize that S129P-αS plays a role in the maintenance or formation of long neurites. In summary, our footprint-free LRRK2-G2019S isogenic cell lines allow standardized, genetic background independent, in vitro PD modeling and provide new insights into the role of LRRK2-G2019S and S129P-αS in the pathogenesis of PD. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  13. A voxel-based morphometry and diffusion tensor imaging analysis of asymptomatic Parkinson's disease-related G2019S LRRK2 mutation carriers.

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    Thaler, Avner; Artzi, Moran; Mirelman, Anat; Jacob, Yael; Helmich, Rick C; van Nuenen, Bart F L; Gurevich, Tanya; Orr-Urtreger, Avi; Marder, Karen; Bressman, Susan; Bloem, Bastiaan R; Hendler, Talma; Giladi, Nir; Ben Bashat, Dafna

    2014-05-01

    Patients with Parkinson's disease have reduced gray matter volume and fractional anisotropy in both cortical and sub-cortical structures, yet changes in the pre-motor phase of the disease are unknown. A comprehensive imaging study using voxel-based morphometry and diffusion tensor imaging tract-based spatial statistics analysis was performed on 64 Ashkenazi Jewish asymptomatic first degree relatives of patients with Parkinson's disease (30 mutation carriers), who carry the G2019S mutation in the leucine-rich repeat kinase 2 (LRRK2) gene. No between-group differences in gray matter volume could be noted in either whole-brain or volume-of-interest analysis. Diffusion tensor imaging analysis did not identify group differences in white matter areas, and volume-of-interest analysis identified no differences in diffusivity parameters in Parkinson's disease-related structures. G2019S carriers do not manifest changes in gray matter volume or diffusivity parameters in Parkinson's disease-related structures prior to the appearance of motor symptoms. © 2014 International Parkinson and Movement Disorder Society.

  14. Spread of neuronal degeneration in a dopaminergic, Lrrk-G2019S model of Parkinson disease

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    Hindle, Samantha J.; Elliott, Christopher J.H.

    2013-01-01

    Flies expressing the most common Parkinson disease (PD)-related mutation, LRRK2-G2019S, in their dopaminergic neurons show loss of visual function and degeneration of the retina, including mitochondrial abnormalities, apoptosis and autophagy. Since the photoreceptors that degenerate are not dopaminergic, this demonstrates nonautonomous degeneration, and a spread of pathology. This provides a model consistent with Braak’s hypothesis on progressive PD. The loss of visual function is specific for the G2019S mutation, implying the cause is its increased kinase activity, and is enhanced by increased neuronal activity. These data suggest novel explanations for the variability in animal models of PD. The specificity of visual loss to G2019S, coupled with the differences in neural firing rate, provide an explanation for the variability between people with PD in visual tests. PMID:23529190

  15. Evidence for prehistoric origins of the G2019S mutation in the North African Berber population.

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    Ben El Haj, Rafiqua; Salmi, Ayyoub; Regragui, Wafa; Moussa, Ahmed; Bouslam, Naima; Tibar, Houyam; Benomar, Ali; Yahyaoui, Mohamed; Bouhouche, Ahmed

    2017-01-01

    The most common cause of the monogenic form of Parkinson's disease known so far is the G2019S mutation of the leucine-rich repeat kinase 2 (LRRK2) gene. Its frequency varies greatly among ethnic groups and geographic regions ranging from less than 0.1% in Asia to 40% in North Africa. This mutation has three distinct haplotypes; haplotype 1 being the oldest and most common. Recent studies have dated haplotype 1 of the G2019S mutation to about 4000 years ago, but it remains controversial whether the mutation has a Near-Eastern or Moroccan-Berber ancestral origin. To decipher this evolutionary history, we genotyped 10 microsatellite markers spanning a region of 11.27 Mb in a total of 57 unrelated Moroccan PD patients carrying the G2019S mutation for which the Berber or Arab origin was established over 3 generations based on spoken language. We estimated the age of the most recent common ancestor for the 36 Arab-speaking and the 15 Berber-speaking G2019S carriers using the likelihood-based method with a mutation rate of 10-4. Data analysis suggests that the shortest haplotype originated in a patient of Berber ethnicity. The common founder was estimated to have lived 159 generations ago (95% CI 116-224) for Arab patients, and 200 generations ago (95% CI 123-348) for Berber patients. Then, 29 native North African males carrying the mutation were assessed for specific uniparental markers by sequencing the Y-chromosome (E-M81, E-M78, and M-267) and mitochondrial DNA (mtDNA) hypervariable regions (HV1 and HV2) to examine paternal and maternal contributions, respectively. Results showed that the autochthonous genetic component reached 76% for mtDNA (Eurasian and north African haplogroups) and 59% for the Y-chromosome (E-M81 and E-M78), suggesting that the G2019S mutation may have arisen in an autochthonous DNA pool. Therefore, we conclude that LRRK2 G2019S mutation most likely originated in a Berber founder who lived at least 5000 years ago (95% CI 3075-8700).

  16. Intact working memory in non-manifesting LRRK2 carriers--an fMRI study

    NARCIS (Netherlands)

    Thaler, A.; Helmich, R.C.G.; Or-Borichev, A.; Nuenen, B.F. van; Shapira-Lichter, I.; Gurevich, T.; Orr-Urtreger, A.; Marder, K.; Bressman, S.; Bloem, B.R.; Giladi, N.; Hendler, T.; Mirelman, A.

    2016-01-01

    Cognitive impairments are prevalent in patients with Parkinson's disease. Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of genetic Parkinsonism. Non-manifesting carriers of the G2019S mutation in the LRRK2 gene were found to have lower executive functions as

  17. LRRK2 affects vesicle trafficking, neurotransmitter extracellular level and membrane receptor localization.

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    Rossana Migheli

    Full Text Available The leucine-rich repeat kinase 2 (LRRK2 gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson's disease (PD. LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2(G2019S pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2(G2019S shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells.

  18. LRRK2 regulates voltage-gated calcium channel function.

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    Cade eBedford

    2016-05-01

    Full Text Available Voltage-gated Ca2+ (CaV channels enable Ca2+ influx in response to membrane depolarization. CaV2.1 channels are localized to the presynaptic membrane of many types of neurons where they are involved in triggering neurotransmitter release. Several signaling proteins have been identified as important CaV2.1 regulators including protein kinases, G-proteins and Ca2+ binding proteins. Recently, we discovered that leucine rich repeat kinase 2 (LRRK2, a protein associated with inherited Parkinson’s disease, interacts with specific synaptic proteins and influences synaptic transmission. Since synaptic proteins functionally interact with CaV2.1 channels and synaptic transmission is triggered by Ca2+ entry via CaV2.1, we investigated whether LRRK2 could impact CaV2.1 channel function. CaV2.1 channel properties were measured using whole cell patch clamp electrophysiology in HEK293 cells transfected with CaV2.1 subunits and various LRRK2 constructs. Our results demonstrate that both wild type LRRK2 and the G2019S LRRK2 mutant caused a significant increase in whole cell Ca2+ current density compared to cells expressing only the CaV2.1 channel complex. In addition, LRRK2 expression caused a significant hyperpolarizing shift in voltage-dependent activation while having no significant effect on inactivation properties. These functional changes in CaV2.1 activity are likely due to a direct action of LRRK2 as we detected a physical interaction between LRRK2 and the β3 CaV channel subunit via coimmunoprecipitation. Furthermore, effects on CaV2.1 channel function are dependent on LRRK2 kinase activity as these could be reversed via treatment with a LRRK2 inhibitor. Interestingly, LRRK2 also augmented endogenous voltage-gated Ca2+ channel function in PC12 cells suggesting other CaV channels could also be regulated by LRRK2. Overall, our findings support a novel physiological role for LRRK2 in regulating CaV2.1 function that could have implications for how

  19. Roles of the Drosophila LRRK2 homolog in Rab7-dependent lysosomal positioning.

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    Dodson, Mark W; Zhang, Ting; Jiang, Changan; Chen, Shengdi; Guo, Ming

    2012-03-15

    LRRK2 (PARK8) is the most common genetic determinant of Parkinson's disease (PD), with dominant mutations in LRRK2 causing inherited PD and sequence variation at the LRRK2 locus associated with increased risk for sporadic PD. Although LRRK2 has been implicated in diverse cellular processes encompassing almost all cellular compartments, the precise functions of LRRK2 remain unclear. Here, we show that the Drosophila homolog of LRRK2 (Lrrk) localizes to the membranes of late endosomes and lysosomes, physically interacts with the crucial mediator of late endosomal transport Rab7 and negatively regulates rab7-dependent perinuclear localization of lysosomes. We also show that a mutant form of lrrk analogous to the pathogenic LRRK2(G2019S) allele behaves oppositely to wild-type lrrk in that it promotes rather than inhibits rab7-dependent perinuclear lysosome clustering, with these effects of mutant lrrk on lysosome position requiring both microtubules and dynein. These data suggest that LRRK2 normally functions in Rab7-dependent lysosomal positioning, and that this function is disrupted by the most common PD-causing LRRK2 mutation, linking endolysosomal dysfunction to the pathogenesis of LRRK2-mediated PD.

  20. Pathogenic LRRK2 mutations, through increased kinase activity, produce enlarged lysosomes with reduced degradative capacity and increase ATP13A2 expression.

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    Henry, Anastasia G; Aghamohammadzadeh, Soheil; Samaroo, Harry; Chen, Yi; Mou, Kewa; Needle, Elie; Hirst, Warren D

    2015-11-01

    Lysosomal dysfunction plays a central role in the pathogenesis of several neurodegenerative disorders, including Parkinson's disease (PD). Several genes linked to genetic forms of PD, including leucine-rich repeat kinase 2 (LRRK2), functionally converge on the lysosomal system. While mutations in LRRK2 are commonly associated with autosomal-dominant PD, the physiological and pathological functions of this kinase remain poorly understood. Here, we demonstrate that LRRK2 regulates lysosome size, number and function in astrocytes, which endogenously express high levels of LRRK2. Expression of LRRK2 G2019S, the most common pathological mutation, produces enlarged lysosomes and diminishes the lysosomal capacity of these cells. Enlarged lysosomes appears to be a common phenotype associated with pathogenic LRRK2 mutations, as we also observed this effect in cells expressing other LRRK2 mutations; R1441C or Y1699C. The lysosomal defects associated with these mutations are dependent on both the catalytic activity of the kinase and autophosphorylation of LRRK2 at serine 1292. Further, we demonstrate that blocking LRRK2's kinase activity, with the potent and selective inhibitor PF-06447475, rescues the observed defects in lysosomal morphology and function. The present study also establishes that G2019S mutation leads to a reduction in lysosomal pH and increased expression of the lysosomal ATPase ATP13A2, a gene linked to a parkinsonian syndrome (Kufor-Rakeb syndrome), in brain samples from mouse and human LRRK2 G2019S carriers. Together, these results demonstrate that PD-associated LRRK2 mutations perturb lysosome function in a kinase-dependent manner, highlighting the therapeutic promise of LRRK2 kinase inhibitors in the treatment of PD. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. Clinical Correlations With Lewy Body Pathology in LRRK2-Related Parkinson Disease

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    Kalia, Lorraine V.; Lang, Anthony E.; Hazrati, Lili-Naz; Fujioka, Shinsuke; Wszolek, Zbigniew K.; Dickson, Dennis W.; Ross, Owen A.; Van Deerlin, Vivianna M.; Trojanowski, John Q.; Hurtig, Howard I.; Alcalay, Roy N.; Marder, Karen S.; Clark, Lorraine N.; Gaig, Carles; Tolosa, Eduardo; Ruiz-Martínez, Javier; Marti-Masso, Jose F.; Ferrer, Isidre; de Munain, Adolfo López; Goldman, Samuel M.; Schüle, Birgitt; Langston, J. William; Aasly, Jan O.; Giordana, Maria T.; Bonifati, Vincenzo; Puschmann, Andreas; Canesi, Margherita; Pezzoli, Gianni; De Paula, Andre Maues; Hasegawa, Kazuko; Duyckaerts, Charles; Brice, Alexis; Stoessl, A. Jon; Marras, Connie

    2015-01-01

    IMPORTANCE Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of genetic Parkinson disease (PD) known to date. The clinical features of manifesting LRRK2 mutation carriers are generally indistinguishable from those of patients with sporadic PD. However, some PD cases associated with LRRK2 mutations lack Lewy bodies (LBs), a neuropathological hallmark of PD. We investigated whether the presence or absence of LBs correlates with different clinical features in LRRK2-related PD. OBSERVATIONS We describe genetic, clinical, and neuropathological findings of 37 cases of LRRK2-related PD including 33 published and 4 unpublished cases through October 2013. Among the different mutations, the LRRK2 p.G2019S mutation was most frequently associated with LB pathology. Nonmotor features of cognitive impairment/dementia, anxiety, and orthostatic hypotension were correlated with the presence of LBs. In contrast, a primarily motor phenotype was associated with a lack of LBs. CONCLUSIONS AND RELEVANCE To our knowledge, this is the first report of clinicopathological correlations in a series of LRRK2-related PD cases. Findings from this selected group of patients with PD demonstrated that parkinsonian motor features can occur in the absence of LBs. However, LB pathology in LRRK2-related PD may be a marker for a broader parkinsonian symptom complex including cognitive impairment. PMID:25401511

  2. Role of LRRK2 in the regulation of dopamine receptor trafficking.

    Directory of Open Access Journals (Sweden)

    Mauro Rassu

    Full Text Available Mutations in LRRK2 play a critical role in both familial and sporadic Parkinson's disease (PD. Up to date, the role of LRRK2 in PD onset and progression remains largely unknown. However, experimental evidence highlights a critical role of LRRK2 in the control of vesicle trafficking that in turn may regulate different aspects of neuronal physiology. We have analyzed the role of LRRK2 in regulating dopamine receptor D1 (DRD1 and D2 (DRD2 trafficking. DRD1 and DRD2 are the most abundant dopamine receptors in the brain. They differ in structural, pharmacological and biochemical properties, as well as in localization and internalization mechanisms. Our results indicate that disease-associated mutant G2019S LRRK2 impairs DRD1 internalization, leading to an alteration in signal transduction. Moreover, the mutant forms of LRRK2 affect receptor turnover by decreasing the rate of DRD2 trafficking from the Golgi complex to the cell membrane. Collectively, our findings are consistent with the conclusion that LRRK2 influences the motility of neuronal vesicles and the neuronal receptor trafficking. These findings have important implications for the complex role that LRRK2 plays in neuronal physiology and the possible pathological mechanisms that may lead to neuronal death in PD.

  3. The Gly2019Ser mutation in LRRK2 is not fully penetrant in familial Parkinson's disease: the GenePD study

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    Corbett Alastair

    2008-11-01

    Full Text Available Abstract Background We report age-dependent penetrance estimates for leucine-rich repeat kinase 2 (LRRK2-related Parkinson's disease (PD in a large sample of familial PD. The most frequently seen LRRK2 mutation, Gly2019Ser (G2019S, is associated with approximately 5 to 6% of familial PD cases and 1 to 2% of idiopathic cases, making it the most common known genetic cause of PD. Studies of the penetrance of LRRK2 mutations have produced a wide range of estimates, possibly due to differences in study design and recruitment, including in particular differences between samples of familial PD versus sporadic PD. Methods A sample, including 903 affected and 58 unaffected members from 509 families ascertained for having two or more PD-affected members, 126 randomly ascertained PD patients and 197 controls, was screened for five different LRRK2 mutations. Penetrance was estimated in families of LRRK2 carriers with consideration of the inherent bias towards increased penetrance in a familial sample. Results Thirty-one out of 509 families with multiple cases of PD (6.1% were found to have 58 LRRK2 mutation carriers (6.4%. Twenty-nine of the 31 families had G2019S mutations while two had R1441C mutations. No mutations were identified among controls or unaffected relatives of PD cases. Nine PD-affected relatives of G2019S carriers did not carry the LRRK2 mutation themselves. At the maximum observed age range of 90 to 94 years, the unbiased estimated penetrance was 67% for G2019S families, compared with a baseline PD risk of 17% seen in the non-LRRK2-related PD families. Conclusion Lifetime penetrance of LRRK2 estimated in the unascertained relatives of multiplex PD families is greater than that reported in studies of sporadically ascertained LRRK2 cases, suggesting that inherited susceptibility factors may modify the penetrance of LRRK2 mutations. In addition, the presence of nine PD phenocopies in the LRRK2 families suggests that these susceptibility

  4. Caracterización clínica, bioquímica y de neuroimagen de la enfermedad de Parkinson asociada a mutaciones del gen LRRK2 y de su fase prodrómica

    OpenAIRE

    Vilas Rolán, Dolores

    2016-01-01

    La presente memoria se basa en cuatro trabajos que pertenecen a una misma línea de estudio: el estudio de la enfermedad de Parkinson asociada a mutaciones del gen LRRK2 (EP-LRRK2) . En primer lugar, se ha estudiado la EP-LRRK2 desde un punto de vista clínico, centrándonos en los síntomas no motores. En segundo lugar se ha realizado un estudio a través de la sonografía transcraneal en portadores de la mutación G2019S del gen LRRK2 , tanto pacientes con enfermedad de Parkinson como portadores a...

  5. LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate.

    Science.gov (United States)

    Belluzzi, Elisa; Gonnelli, Adriano; Cirnaru, Maria-Daniela; Marte, Antonella; Plotegher, Nicoletta; Russo, Isabella; Civiero, Laura; Cogo, Susanna; Carrion, Maria Perèz; Franchin, Cinzia; Arrigoni, Giorgio; Beltramini, Mariano; Bubacco, Luigi; Onofri, Franco; Piccoli, Giovanni; Greggio, Elisa

    2016-01-13

    Lrrk2, a gene linked to Parkinson's disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins. Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity. Given that the most common Parkinson's disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF.

  6. PAK6 Phosphorylates 14-3-3γ to Regulate Steady State Phosphorylation of LRRK2

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    Laura Civiero

    2017-12-01

    Full Text Available Mutations in Leucine-rich repeat kinase 2 (LRRK2 are associated with Parkinson's disease (PD and, as such, LRRK2 is considered a promising therapeutic target for age-related neurodegeneration. Although the cellular functions of LRRK2 in health and disease are incompletely understood, robust evidence indicates that PD-associated mutations alter LRRK2 kinase and GTPase activities with consequent deregulation of the downstream signaling pathways. We have previously demonstrated that one LRRK2 binding partner is P21 (RAC1 Activated Kinase 6 (PAK6. Here, we interrogate the PAK6 interactome and find that PAK6 binds a subset of 14-3-3 proteins in a kinase dependent manner. Furthermore, PAK6 efficiently phosphorylates 14-3-3γ at Ser59 and this phosphorylation serves as a switch to dissociate the chaperone from client proteins including LRRK2, a well-established 14-3-3 binding partner. We found that 14-3-3γ phosphorylated by PAK6 is no longer competent to bind LRRK2 at phospho-Ser935, causing LRRK2 dephosphorylation. To address whether these interactions are relevant in a neuronal context, we demonstrate that a constitutively active form of PAK6 rescues the G2019S LRRK2-associated neurite shortening through phosphorylation of 14-3-3γ. Our results identify PAK6 as the kinase for 14-3-3γ and reveal a novel regulatory mechanism of 14-3-3/LRRK2 complex in the brain.

  7. A voxel-based morphometry and diffusion tensor imaging analysis of asymptomatic Parkinson's disease-related G2019S LRRK2 mutation carriers

    NARCIS (Netherlands)

    Thaler, A.; Artzi, M.; Mirelman, A.; Jacob, Y.; Helmich, R.C.G.; Nuenen, B.F.L. van; Gurevich, T.; Orr-Urtreger, A.; Marder, K.; Bressman, S.; Bloem, B.R.; Hendler, T.; Giladi, N.; Bashat, D. Ben; et al.,

    2014-01-01

    BACKGROUND: Patients with Parkinson's disease have reduced gray matter volume and fractional anisotropy in both cortical and sub-cortical structures, yet changes in the pre-motor phase of the disease are unknown. METHODS: A comprehensive imaging study using voxel-based morphometry and diffusion

  8. Impaired intracortical transmission in G2019S leucine rich-repeat kinase Parkinson patients.

    Science.gov (United States)

    Ponzo, Viviana; Di Lorenzo, Francesco; Brusa, Livia; Schirinzi, Tommaso; Battistini, Stefania; Ricci, Claudia; Sambucci, Manolo; Caltagirone, Carlo; Koch, Giacomo

    2017-05-01

    A mutation in leucine-rich repeat kinase 2 is the most common cause of hereditary Parkinson's disease (PD), yet the neural mechanisms and the circuitry potentially involved are poorly understood. We used different transcranial magnetic stimulation protocols to explore in the primary motor cortex the activity of intracortical circuits and cortical plasticity (long-term potentiation) in patients with the G2019S leucine-rich repeat kinase 2 gene mutation when compared with idiopathic PD patients and age-matched healthy subjects. Paired pulse transcranial magnetic stimulation was used to investigate short intracortical inhibition and facilitation and short afferent inhibition. Intermittent theta burst stimulation, a form of repetitive transcranial magnetic stimulation, was used to test long-term potentiation-like cortical plasticity. Leucine-rich repeat kinase 2 and idiopathic PD were tested both in ON and in OFF l-dopa therapy. When compared with idiopathic PD and healthy subjects, leucine-rich repeat kinase 2 PD patients showed a remarkable reduction of short intracortical inhibition in both ON and in OFF l-dopa therapy. This reduction was paralleled by an increase of intracortical facilitation in OFF l-dopa therapy. Leucine-rich repeat kinase 2 PD showed abnormal long-term potentiation-like cortical plasticity in ON l-dopa therapy. The motor cortex in leucine-rich repeat kinase 2 mutated PD patients is strongly disinhibited and hyperexcitable. These abnormalities could be a result of an impairment of inhibitory (gamma-Aminobutyric acid) transmission eventually related to altered neurotransmitter release. © 2017 International Parkinson and Movement Disorder Society. © 2017 International Parkinson and Movement Disorder Society.

  9. Functional variants in the LRRK2 gene confer shared effects on risk for Crohn's disease and Parkinson's disease.

    Science.gov (United States)

    Hui, Ken Y; Fernandez-Hernandez, Heriberto; Hu, Jianzhong; Schaffner, Adam; Pankratz, Nathan; Hsu, Nai-Yun; Chuang, Ling-Shiang; Carmi, Shai; Villaverde, Nicole; Li, Xianting; Rivas, Manual; Levine, Adam P; Bao, Xiuliang; Labrias, Philippe R; Haritunians, Talin; Ruane, Darren; Gettler, Kyle; Chen, Ernie; Li, Dalin; Schiff, Elena R; Pontikos, Nikolas; Barzilai, Nir; Brant, Steven R; Bressman, Susan; Cheifetz, Adam S; Clark, Lorraine N; Daly, Mark J; Desnick, Robert J; Duerr, Richard H; Katz, Seymour; Lencz, Todd; Myers, Richard H; Ostrer, Harry; Ozelius, Laurie; Payami, Haydeh; Peter, Yakov; Rioux, John D; Segal, Anthony W; Scott, William K; Silverberg, Mark S; Vance, Jeffery M; Ubarretxena-Belandia, Iban; Foroud, Tatiana; Atzmon, Gil; Pe'er, Itsik; Ioannou, Yiannis; McGovern, Dermot P B; Yue, Zhenyu; Schadt, Eric E; Cho, Judy H; Peter, Inga

    2018-01-10

    Crohn's disease (CD), a form of inflammatory bowel disease, has a higher prevalence in Ashkenazi Jewish than in non-Jewish European populations. To define the role of nonsynonymous mutations, we performed exome sequencing of Ashkenazi Jewish patients with CD, followed by array-based genotyping and association analysis in 2066 CD cases and 3633 healthy controls. We detected association signals in the LRRK2 gene that conferred risk for CD (N2081D variant, P = 9.5 × 10 -10 ) or protection from CD (N551K variant, tagging R1398H-associated haplotype, P = 3.3 × 10 -8 ). These variants affected CD age of onset, disease location, LRRK2 activity, and autophagy. Bayesian network analysis of CD patient intestinal tissue further implicated LRRK2 in CD pathogenesis. Analysis of the extended LRRK2 locus in 24,570 CD cases, patients with Parkinson's disease (PD), and healthy controls revealed extensive pleiotropy, with shared genetic effects between CD and PD in both Ashkenazi Jewish and non-Jewish cohorts. The LRRK2 N2081D CD risk allele is located in the same kinase domain as G2019S, a mutation that is the major genetic cause of familial and sporadic PD. Like the G2019S mutation, the N2081D variant was associated with increased kinase activity, whereas neither N551K nor R1398H variants on the protective haplotype altered kinase activity. We also confirmed that R1398H, but not N551K, increased guanosine triphosphate binding and hydrolyzing enzyme (GTPase) activity, thereby deactivating LRRK2. The presence of shared LRRK2 alleles in CD and PD provides refined insight into disease mechanisms and may have major implications for the treatment of these two seemingly unrelated diseases. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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

    Science.gov (United States)

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

    2017-11-15

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

  11. Dysregulation of lysosomal morphology by pathogenic LRRK2 is corrected by TPC2 inhibition.

    Science.gov (United States)

    Hockey, Leanne N; Kilpatrick, Bethan S; Eden, Emily R; Lin-Moshier, Yaping; Brailoiu, G Cristina; Brailoiu, Eugen; Futter, Clare E; Schapira, Anthony H; Marchant, Jonathan S; Patel, Sandip

    2015-01-15

    Two-pore channels (TPCs) are endolysosomal ion channels implicated in Ca(2+) signalling from acidic organelles. The relevance of these ubiquitous proteins for human disease, however, is unclear. Here, we report that lysosomes are enlarged and aggregated in fibroblasts from Parkinson disease patients with the common G2019S mutation in LRRK2. Defects were corrected by molecular silencing of TPC2, pharmacological inhibition of TPC regulators [Rab7, NAADP and PtdIns(3,5)P2] and buffering local Ca(2+) increases. NAADP-evoked Ca(2+) signals were exaggerated in diseased cells. TPC2 is thus a potential drug target within a pathogenic LRRK2 cascade that disrupts Ca(2+)-dependent trafficking in Parkinson disease. © 2015. Published by The Company of Biologists Ltd.

  12. Parkinson’s disease and low frequency alleles found together throughout LRRK2

    Science.gov (United States)

    Paisán-Ruiz, Coro; Washecka, Nicole; Nath, Priti; Singleton, Andrew B.; Corder, Elizabeth H.

    2016-01-01

    Mutations within LRRK2, most notably p.G2019S, cause Parkinson’s disease (PD) in rare monogenic families, and sporadic occurrences in diverse populations. We investigated variation throughout LRRK2 (84 SNPs; genotype or diplotype found for 49 LD blocks) for 275 cases (European ancestry, onset at age 60 or older) and 275 neurologically healthy control subjects (NINDS Neurogenetics Repository). Three grade-of-membership groups, i.e. genetic risk sets, were identified that exactly matched many subjects (cases: 46, 4, 137; controls: 0, 178, 0), and distinguished 94% of the subjects (i.e. > 50% likeness to one set). Set I, affected, carried certain low frequency alleles located in multiple functional domains. Set II was unaffected. Set III, also affected, resembled II except for slightly elevated frequencies of minor alleles not defining set I. We conclude that certain low frequency alleles distributed throughout LRRK2 are a genetic background to a third of cases, defining a distinct subset. PMID:19489756

  13. LRRK2 kinase activity is dependent on LRRK2 GTP binding capacity but independent of LRRK2 GTP binding.

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    Jean-Marc Taymans

    Full Text Available Leucine rich repeat kinase 2 (LRRK2 is a Parkinson's disease (PD gene that encodes a large multidomain protein including both a GTPase and a kinase domain. GTPases often regulate kinases within signal transduction cascades, where GTPases act as molecular switches cycling between a GTP bound "on" state and a GDP bound "off" state. It has been proposed that LRRK2 kinase activity may be increased upon GTP binding at the LRRK2 Ras of complex proteins (ROC GTPase domain. Here we extensively test this hypothesis by measuring LRRK2 phosphorylation activity under influence of GDP, GTP or non-hydrolyzable GTP analogues GTPγS or GMPPCP. We show that autophosphorylation and lrrktide phosphorylation activity of recombinant LRRK2 protein is unaltered by guanine nucleotides, when co-incubated with LRRK2 during phosphorylation reactions. Also phosphorylation activity of LRRK2 is unchanged when the LRRK2 guanine nucleotide binding pocket is previously saturated with various nucleotides, in contrast to the greatly reduced activity measured for the guanine nucleotide binding site mutant T1348N. Interestingly, when nucleotides were incubated with cell lysates prior to purification of LRRK2, kinase activity was slightly enhanced by GTPγS or GMPPCP compared to GDP, pointing to an upstream guanine nucleotide binding protein that may activate LRRK2 in a GTP-dependent manner. Using metabolic labeling, we also found that cellular phosphorylation of LRRK2 was not significantly modulated by nucleotides, although labeling is significantly reduced by guanine nucleotide binding site mutants. We conclude that while kinase activity of LRRK2 requires an intact ROC-GTPase domain, it is independent of GDP or GTP binding to ROC.

  14. Structural Characterization of LRRK2 Inhibitors

    NARCIS (Netherlands)

    Gilsbach, Bernd K; Messias, Ana C; Ito, Genta; Sattler, Michael; Alessi, Dario R; Wittinghofer, Alfred; Kortholt, Arjan

    2015-01-01

    Kinase inhibition is considered to be an important therapeutic target for LRRK2 mediated Parkinson's disease (PD). Many LRRK2 kinase inhibitors have been reported but have yet to be optimized in order to qualify as drug candidates for the treatment of the disease. In order to start a

  15. Sequence conservation between porcine and human LRRK2

    DEFF Research Database (Denmark)

    Larsen, Knud; Madsen, Lone Bruhn

    2009-01-01

     Leucine-rich repeat kinase 2 (LRRK2) is a member of the ROCO protein superfamily (Ras of complex proteins (Roc) with a C-terminal Roc domain). Mutations in the LRRK2 gene lead to autosomal dominant Parkinsonism. We have cloned the porcine LRRK2 cDNA in an attempt to characterize conserved...... and expression patterns are conserved across species. The porcine LRRK2 gene was mapped to chromosome 5q25. The results obtained suggest that the LRRK2 gene might be of particular interest in our attempt to generate a transgenic porcine model for Parkinson's disease...

  16. Interaction of LRRK2 with kinase and GTPase signaling cascades

    Directory of Open Access Journals (Sweden)

    Joon Y Boon

    2014-07-01

    Full Text Available LRRK2 is a protein that interacts with a plethora of signaling molecules, but the complexity of LRRK2 function presents a challenge for understanding the role of LRRK2 in the pathophysiology of Parkinson’s disease. Studies of LRRK2 using over-expression in transgenic mice have been disappointing, however studies using invertebrate systems have yielded a much clearer picture, with clear effects of LRRK2 expression, knockdown or deletion in C. elegans and Drosophila on modulation of survival of dopaminergic neurons. Recent studies have begun to focus attention on particular signaling cascades that are a target of LRRK2 function. LRRK2 interacts with members of the MAPK pathway and might regulate the pathway action by acting as a scaffold that directs the location of MAPK pathway activity, without strongly affecting the amount of MAPK pathway activity. Binding to GTPases, GAPs and GEFs are another strong theme in LRRK2 biology, with LRRK2 binding to Rac1, cdc42, rab5, rab7L1, endoA, RGS2, ArfGAP1 and ArhGEF7. All of these molecules appear to feed into a function output for LRRK2 that modulates cytoskeletal outgrowth and vesicular dynamics, including autophagy. These functions likely impact modulation of α-synuclein aggregation and associated toxicity eliciting the disease processes that we term Parkinson’s disease.

  17. LRRK2 G2385R and R1628P Mutations Are Associated with an Increased Risk of Parkinson's Disease in the Malaysian Population

    Science.gov (United States)

    Chua, Jing Yi; Lim, Thien Thien; Mohamed Ibrahim, Norlinah; Tan, Ai Huey; Eow, Gaik Bee; Abdul Aziz, Zariah; Puvanarajah, Santhi Datuk; Viswanathan, Shanthi; Lim, Soo Kun; Tan, Li Ping; Chong, Yip Boon; Tan, Chong Tin; Zhao, Yi; Tan, E. K.

    2014-01-01

    The LRRK2 gene has been associated with both familial and sporadic forms of Parkinson's disease (PD). The G2019S variant is commonly found in North African Arab and Caucasian PD patients, but this locus is monomorphic in Asians. The G2385R and R1628P variants are associated with a higher risk of developing PD in certain Asian populations but have not been studied in the Malaysian population. Therefore, we screened the G2385R and R1628P variants in 1,202 Malaysian subjects consisting of 695 cases and 507 controls. The G2385R and R1628P variants were associated with a 2.2-fold (P = 0.019) and 1.2-fold (P = 0.054) increased risk of PD, respectively. Our data concur with other reported findings in Chinese, Taiwanese, Singaporean, and Korean studies. PMID:25243190

  18. LRRK2 G2385R and R1628P Mutations Are Associated with an Increased Risk of Parkinson’s Disease in the Malaysian Population

    Directory of Open Access Journals (Sweden)

    Aroma Agape Gopalai

    2014-01-01

    Full Text Available The LRRK2 gene has been associated with both familial and sporadic forms of Parkinson’s disease (PD. The G2019S variant is commonly found in North African Arab and Caucasian PD patients, but this locus is monomorphic in Asians. The G2385R and R1628P variants are associated with a higher risk of developing PD in certain Asian populations but have not been studied in the Malaysian population. Therefore, we screened the G2385R and R1628P variants in 1,202 Malaysian subjects consisting of 695 cases and 507 controls. The G2385R and R1628P variants were associated with a 2.2-fold (P=0.019 and 1.2-fold (P=0.054 increased risk of PD, respectively. Our data concur with other reported findings in Chinese, Taiwanese, Singaporean, and Korean studies.

  19. Mutant LRRK2 Toxicity in Neurons Depends on LRRK2 Levels and Synuclein But Not Kinase Activity or Inclusion Bodies

    Science.gov (United States)

    Skibinski, Gaia; Nakamura, Ken; Cookson, Mark R.

    2014-01-01

    By combining experimental neuron models and mathematical tools, we developed a “systems” approach to deconvolve cellular mechanisms of neurodegeneration underlying the most common known cause of Parkinson's disease (PD), mutations in leucine-rich repeat kinase 2 (LRRK2). Neurons ectopically expressing mutant LRRK2 formed inclusion bodies (IBs), retracted neurites, accumulated synuclein, and died prematurely, recapitulating key features of PD. Degeneration was predicted from the levels of diffuse mutant LRRK2 that each neuron contained, but IB formation was neither necessary nor sufficient for death. Genetic or pharmacological blockade of its kinase activity destabilized LRRK2 and lowered its levels enough to account for the moderate reduction in LRRK2 toxicity that ensued. By contrast, targeting synuclein, including neurons made from PD patient-derived induced pluripotent cells, dramatically reduced LRRK2-dependent neurodegeneration and LRRK2 levels. These findings suggest that LRRK2 levels are more important than kinase activity per se in predicting toxicity and implicate synuclein as a major mediator of LRRK2-induced neurodegeneration. PMID:24403142

  20. Activation Mechanism of LRRK2 and Its Cellular Functions in Parkinson's Disease

    NARCIS (Netherlands)

    Rosenbusch, Katharina E.; Kortholt, Arjan

    2016-01-01

    Human LRRK2 (Leucine-Rich Repeat Kinase 2) has been associated with both familial and idiopathic Parkinson's disease (PD). Although several LRRK2 mediated pathways and interaction partners have been identified, the cellular functions of LRRK2 and LRRK2 mediated progression of PD are still only

  1. GTPase activity plays a key role in the pathobiology of LRRK2.

    Directory of Open Access Journals (Sweden)

    Yulan Xiong

    2010-04-01

    Full Text Available Mutations in the leucine-rich repeat kinase 2 (LRRK2 gene are associated with late-onset, autosomal-dominant, familial Parkinson's disease (PD and also contribute to sporadic disease. The LRRK2 gene encodes a large protein with multiple domains, including functional Roc GTPase and protein kinase domains. Mutations in LRRK2 most likely cause disease through a toxic gain-of-function mechanism. The expression of human LRRK2 variants in cultured primary neurons induces toxicity that is dependent on intact GTP binding or kinase activities. However, the mechanism(s underlying LRRK2-induced neuronal toxicity is poorly understood, and the contribution of GTPase and/or kinase activity to LRRK2 pathobiology is not well defined. To explore the pathobiology of LRRK2, we have developed a model of LRRK2 cytotoxicity in the baker's yeast Saccharomyces cerevisiae. Protein domain analysis in this model reveals that expression of GTPase domain-containing fragments of human LRRK2 are toxic. LRRK2 toxicity in yeast can be modulated by altering GTPase activity and is closely associated with defects in endocytic vesicular trafficking and autophagy. These truncated LRRK2 variants induce similar toxicity in both yeast and primary neuronal models and cause similar vesicular defects in yeast as full-length LRRK2 causes in primary neurons. The toxicity induced by truncated LRRK2 variants in yeast acts through a mechanism distinct from toxicity induced by human alpha-synuclein. A genome-wide genetic screen identified modifiers of LRRK2-induced toxicity in yeast including components of vesicular trafficking pathways, which can also modulate the trafficking defects caused by expression of truncated LRRK2 variants. Our results provide insight into the basic pathobiology of LRRK2 and suggest that the GTPase domain may contribute to the toxicity of LRRK2. These findings may guide future therapeutic strategies aimed at attenuating LRRK2-mediated neurodegeneration.

  2. Parkinson-Related LRRK2 Mutation R1628P Enables Cdk5 Phosphorylation of LRRK2 and Upregulates Its Kinase Activity.

    Directory of Open Access Journals (Sweden)

    Yang Shu

    Full Text Available Recent studies have linked certain single nucleotide polymorphisms in the leucine-rich repeat kinase 2 (LRRK2 gene with Parkinson's disease (PD. Among the mutations, LRRK2 c.4883G>C (R1628P variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown.Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn't alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5. Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+ phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner.Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death.

  3. The Role of LRRK2 in Parkinson's Disease

    NARCIS (Netherlands)

    A. Di Fonzo (Alessio)

    2009-01-01

    textabstractThis thesis focuses on the role of the leucine rich repeat kinase 2 (LRRK2) gene in Parkinson’s disease (PD). PD is the second most frequent human neurodegenerative disorder after Alzheimer’s disease. The etiology of PD remains unknown in most cases, but several

  4. The LRRK2 Variant E193K Prevents Mitochondrial Fission Upon MPP+ Treatment by Altering LRRK2 Binding to DRP1

    Directory of Open Access Journals (Sweden)

    Maria Perez Carrion

    2018-02-01

    Full Text Available Mutations in leucine-rich repeat kinase 2 gene (LRRK2 are associated with familial and sporadic Parkinson’s disease (PD. LRRK2 is a complex protein that consists of multiple domains, including 13 putative armadillo-type repeats at the N-terminus. In this study, we analyzed the functional and molecular consequences of a novel variant, E193K, identified in an Italian family. E193K substitution does not influence LRRK2 kinase activity. Instead it affects LRRK2 biochemical properties, such as phosphorylation at Ser935 and affinity for 14-3-3ε. Primary fibroblasts obtained from an E193K carrier demonstrated increased cellular toxicity and abnormal mitochondrial fission upon 1-methyl-4-phenylpyridinium treatment. We found that E193K alters LRRK2 binding to DRP1, a crucial mediator of mitochondrial fission. Our data support a role for LRRK2 as a scaffolding protein influencing mitochondrial fission.

  5. LRRK2 mediated Rab8a phosphorylation promotes lipid storage.

    Science.gov (United States)

    Yu, Miao; Arshad, Muhammad; Wang, Wenmin; Zhao, Dongyu; Xu, Li; Zhou, Linkang

    2018-02-27

    Several mutations in leucine rich repeat kinase 2 (LRRK2) gene have been associated with pathogenesis of Parkinson's disease (PD), a neurodegenerative disorder marked by resting tremors, and rigidity, leading to Postural instability. It has been revealed that mutations that lead to an increase of kinase activity of LRRK2 protein are significantly associated with PD pathogenesis. Recent studies have shown that some Rab GTPases, especially Rab8, serve as substrates of LRRK2 and undergo phosphorylation in its switch II domain upon interaction. Current study was performed in order to find out the effects of the phosphorylation of Rab8 and its mutants on lipid metabolism and lipid droplets growth. The phosphorylation status of Rab8a was checked by phos-tag gel. Point mutant construct were generated to investigate the function of Rab8a. 3T3L1 cells were transfected with indicated plasmids and the lipid droplets were stained with Bodipy. Fluorescent microscopy experiments were performed to examine the sizes of lipid droplets. The interactions between Rab8a and Optineurin were determined by immunoprecipitation and western blot. Our assays demonstrated that Rab8a was phosphorylated by mutated LRRK2 that exhibits high kinase activity. Phosphorylation of Rab8a on amino acid residue T72 promoted the formation of large lipid droplets. T72D mutant of Rab8a had higher activity to promote the formation of large lipid droplets compared with wild type Rab8a, with increase in average diameter of lipid droplets from 2.10 μm to 2.46 μm. Moreover, phosphorylation of Rab8a weakened the interaction with its effector Optineurin. Y1699C mutated LRRK2 was able to phosphorylate Rab8a and phosphorylation of Rab8a on site 72 plays important role in the fusion and enlargement of lipid droplets. Taken together, our study suggests an indirect relationship between enhanced lipid storage capacity and PD pathogenesis.

  6. Gene and MicroRNA transcriptome analysis of Parkinson's related LRRK2 mouse models.

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    Véronique Dorval

    Full Text Available Mutations in leucine-rich repeat kinase 2 (LRRK2 are the most frequent cause of genetic Parkinson's disease (PD. The biological function of LRRK2 and how mutations lead to disease remain poorly defined. It has been proposed that LRRK2 could function in gene transcription regulation; however, this issue remains controversial. Here, we investigated in parallel gene and microRNA (miRNA transcriptome profiles of three different LRRK2 mouse models. Striatal tissue was isolated from adult LRRK2 knockout (KO mice, as well as mice expressing human LRRK2 wildtype (hLRRK2-WT or the PD-associated R1441G mutation (hLRRK2-R1441G. We identified a total of 761 genes and 24 miRNAs that were misregulated in the absence of LRRK2 when a false discovery rate of 0.2 was applied. Notably, most changes in gene expression were modest (i.e., <2 fold. By real-time quantitative RT-PCR, we confirmed the variations of selected genes (e.g., adra2, syt2, opalin and miRNAs (e.g., miR-16, miR-25. Surprisingly, little or no changes in gene expression were observed in mice expressing hLRRK2-WT or hLRRK2-R1441G when compared to non-transgenic controls. Nevertheless, a number of miRNAs were misexpressed in these models. Bioinformatics analysis identified several miRNA-dependent and independent networks dysregulated in LRRK2-deficient mice, including PD-related pathways. These results suggest that brain LRRK2 plays an overall modest role in gene transcription regulation in mammals; however, these effects seem context and RNA type-dependent. Our data thus set the stage for future investigations regarding LRRK2 function in PD development.

  7. LRRK2 kinase inhibition prevents pathological microglial phagocytosis in response to HIV-1 Tat protein

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    Marker Daniel F

    2012-11-01

    Full Text Available Abstract Background Human Immunodeficiency Virus-1 (HIV-1 associated neurocognitive disorders (HANDs are accompanied by significant morbidity, which persists despite the use of combined antiretroviral therapy (cART. While activated microglia play a role in pathogenesis, changes in their immune effector functions, including phagocytosis and proinflammatory signaling pathways, are not well understood. We have identified leucine-rich repeat kinase 2 (LRRK2 as a novel regulator of microglial phagocytosis and activation in an in vitro model of HANDs, and hypothesize that LRRK2 kinase inhibition will attenuate microglial activation during HANDs. Methods We treated BV-2 immortalized mouse microglia cells with the HIV-1 trans activator of transcription (Tat protein in the absence or presence of LRRK2 kinase inhibitor (LRRK2i. We used Western blot, qRT-PCR, immunocytochemistry and latex bead engulfment assays to analyze LRRK2 protein levels, proinflammatory cytokine and phagocytosis receptor expression, LRRK2 cellular distribution and phagocytosis, respectively. Finally, we utilized ex vivo microfluidic chambers containing primary hippocampal neurons and BV-2 microglia cells to investigate microglial phagocytosis of neuronal axons. Results We found that Tat-treatment of BV-2 cells induced kinase activity associated phosphorylation of serine 935 on LRRK2 and caused the formation of cytoplasmic LRRK2 inclusions. LRRK2i decreased Tat-induced phosphorylation of serine 935 on LRRK2 and inhibited the formation of Tat-induced cytoplasmic LRRK2 inclusions. LRRK2i also decreased Tat-induced process extension in BV-2 cells. Furthermore, LRRK2i attenuated Tat-induced cytokine expression and latex bead engulfment. We examined relevant cellular targets in microfluidic chambers and found that Tat-treated BV-2 microglia cells cleared axonal arbor and engulfed neuronal elements, whereas saline treated controls did not. LRRK2i was found to protect axons in the presence

  8. LRRK2: an éminence grise of Wnt-mediated neurogenesis?

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    Daniel C Berwick

    2013-05-01

    Full Text Available The importance of Leucine-Rich Repeat Kinase 2 (LRRK2 to mature neurons is well-established, since mutations in PARK8, the gene encoding LRRK2, are the most common known cause of Parkinson’s disease. Nonetheless, despite the LRRK2 knockout mouse having no overt neurodevelopmental defect, numerous lines of in vitro data point towards a central role for this protein in neurogenesis. Roles for LRRK2 have been described in many key processes, including neurite outgrowth and the regulation of microtubule dynamics. Moreover, LRRK2 has been implicated in cell cycle control, suggesting additional roles in neurogenesis that precede terminal differentiation. However, we contend that the suggested function of LRRK2 as a scaffolding protein at the heart of numerous Wnt signaling cascades provides the most tantalizing link to neurogenesis in the developing brain. Numerous lines of evidence show a critical requirement for multiple Wnt pathways in the development of certain brain regions, not least the dopaminergic neurons of the ventral mid-brain. In conclusion, these observations indicate a function of LRRK2 as a subtle yet critical mediator of the action of Wnt ligands on developing neurons. We suggest that LRRK2 loss- or gain-of-function are likely modifiers of developmental phenotypes seen in animal models of Wnt signaling deregulation, a hypothesis that can be tested by cross-breeding relevant genetically modified experimental strains.

  9. Mutations in the LRRK2 Roc-COR tandem domain link Parkinson's disease to Wnt signalling pathways.

    Science.gov (United States)

    Sancho, Rosa M; Law, Bernard M H; Harvey, Kirsten

    2009-10-15

    Mutations in PARK8, encoding LRRK2, are the most common known cause of Parkinson's disease. The LRRK2 Roc-COR tandem domain exhibits GTPase activity controlling LRRK2 kinase activity via an intramolecular process. We report the interaction of LRRK2 with the dishevelled family of phosphoproteins (DVL1-3), key regulators of Wnt (Wingless/Int) signalling pathways important for axon guidance, synapse formation and neuronal maintenance. Interestingly, DVLs can interact with and mediate the activation of small GTPases with structural similarity to the LRRK2 Roc domain. The LRRK2 Roc-COR domain and the DVL1 DEP domain were necessary and sufficient for LRRK2-DVL1 interaction. Co-expression of DVL1 increased LRRK2 steady-state protein levels, an effect that was dependent on the DEP domain. Strikingly, LRRK2-DVL1-3 interactions were disrupted by the familial PARK8 mutation Y1699C, whereas pathogenic mutations at residues R1441 and R1728 strengthened LRRK2-DVL1 interactions. Co-expression of DVL1 with LRRK2 in mammalian cells resulted in the redistribution of LRRK2 to typical cytoplasmic DVL1 aggregates in HEK293 and SH-SY5Y cells and co-localization in neurites and growth cones of differentiated dopaminergic SH-SY5Y cells. This is the first report of the modulation of a key LRRK2-accessory protein interaction by PARK8 Roc-COR domain mutations segregating with Parkinson's disease. Since the DVL1 DEP domain is known to be involved in the regulation of small GTPases, we propose that: (i) DVLs may influence LRRK2 GTPase activity, and (ii) Roc-COR domain mutations modulating LRRK2-DVL interactions indirectly influence kinase activity. Our findings also link LRRK2 to Wnt signalling pathways, suggesting novel pathogenic mechanisms and new targets for genetic analysis in Parkinson's disease.

  10. Phenotype, genotype, and worldwide genetic penetrance of LRRK2-associated Parkinson's disease: a case-control study

    NARCIS (Netherlands)

    D.G. Healy (Daniel); M. Falchi (Mario); S.S. O'Sullivan (Sean); V. Bonifati (Vincenzo); A. Durr; S. Bressman (Susan); A. Brice; J.O. Aasly (Jan); C.P. Zabetian (Cyrus); S. Goldwurm (Stefano); J.J. Ferreira (Joaquim); E. Tolosa; D.M. Kay (Denise); C. Klein (Christoph); D.R. Williams (David); C. Marras (Connie); A.E. Lang; Z.K. Wszolek (Zbigniew); J. Berciano (José); A.H.V. Schapira (Anthony); T. Lynch (Tim); K.P. Bhatia (Kailash); T. Gasser (Thomas); A.J. Lees (Andrew); N.W. Wood (Nicholas)

    2008-01-01

    textabstractBackground: Mutations in LRRK2, the gene that encodes leucine-rich repeat kinase 2, are a cause of Parkinson's disease (PD). The International LRRK2 Consortium was established to answer three key clinical questions: can LRRK2-associated PD be distinguished from idiopathic PD; which

  11. LRRK2 interacts with ATM and regulates Mdm2-p53 cell proliferation axis in response to genotoxic stress.

    Science.gov (United States)

    Chen, Zhongcan; Cao, Zhen; Zhang, Wei; Gu, Minxia; Zhou, Zhi Dong; Li, Baojie; Li, Jing; Tan, Eng King; Zeng, Li

    2017-11-15

    Pathogenic leucine-rich repeat kinase 2 (LRRK2) mutations are recognized as the most common cause of familial Parkinson's disease in certain populations. Recently, LRRK2 mutations were shown to be associated with a higher risk of hormone-related cancers. However, how LRRK2 itself contributes to cancer risk remains unknown. DNA damage causes cancer, and DNA damage responses are among the most important pathways in cancer biology. To understand the role of LRRK2 in DNA damage response pathway, we induced DNA damage by applying genotoxic stress to the cells with Adriamycin. We found that DNA damage enhances LRRK2 phosphorylation at Serine 910, Serine 935 and Serine 1292. We further showed that LRRK2 phosphorylation is abolished in the absence of ATM, suggesting that LRRK2 phosphorylation requires ATM. It should also be noted that LRRK2 interacts with ATM. In contrast, overexpression or knockdown of LRRK2 does not affect ATM phosphorylation, indicating that LRRK2 is the downstream target of ATM in response to DNA damage. Moreover, we demonstrated that LRRK2 increases the expression of p53 and p21 by increasing the Mdm2 phosphorylation in response to DNA damage. Loss-of-function in LRRK2 has the opposite effect to that of LRRK2. In addition, FACS analysis revealed that LRRK2 enhances cell cycle progression into S phase in response to DNA damage, a finding that was confirmed by 5-bromo-2'-deoxyuridine immunostaining. Taken together, our findings demonstrate that LRRK2 plays an important role in the ATM-Mdm2-p53 pathway that regulates cell proliferation in response to DNA damage. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  12. Emerging role of LRRK2 in human neural progenitor cell cycle progression, survival and differentiation

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    Meyer Anne K

    2009-06-01

    Full Text Available Abstract Despite a comprehensive mapping of the Parkinson's disease (PD-related mRNA and protein leucine-rich repeat kinase 2 (LRRK2 in the mammalian brain, its physiological function in healthy individuals remains enigmatic. Based on its structural features and kinase properties, LRRK2 may interact with other proteins involved in signalling pathways. Here, we show a widespread LRRK2 mRNA and/or protein expression in expanded or differentiated human mesencephalic neural progenitor cells (hmNPCs and in post-mortem substantia nigra PD patients. Using small interfering RNA duplexes targeting LRRK2 in hmNPCs following their differentiation into glia and neurons, we observed a reduced number of dopaminergic neurons due to apoptosis in LRRK2 knockdown samples. LRRK2-deficient hmNPCs exhibited elevated cell cycle- and cell death-related markers. In conclusion, a reduction of LRRK2 expression in hmNPCs severely impaired dopaminergic differentiation and/or survival of dopaminergic neurons most likely via preserving or reactivating the cell cycle.

  13. Cognitive Impairments in LRRK2-Related Parkinson’s Disease: A Study in Chinese Individuals

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    Yifan Zheng

    2015-01-01

    Full Text Available Background. LRRK2 S1647T has been identified as a polymorphic risk variant for Parkinson’s disease (PD in Chinese individuals. As LRRK2 is the most common genetic cause for PD, it has drawn great interest regarding whether cognitive impairments in PD are related with LRRK2. Purpose. This study aimed to explore the effects of LRRK2 S1647T polymorphism on cognitive function in PD. Method. 90 PD patients were randomly recruited. They underwent a series of clinical evaluations and genetic testing for the LRRK2 S1647T polymorphism. Global intellect and five cognitive domains (language fluency, visuospatial function, attention, memory, and executive function were compared between S1647T carriers and noncarriers. Results. No differences in motor features were found between two groups, but the executive function evaluation showed that Stroop word colour test time (SWCT-TIME scores were lower in LRRK2 S1647T carriers than in noncarriers (P=0.017. However, multiple linear regression analysis indicated that the correlation between S1647T polymorphism and SWCT-TIME scores did not reach significant level (P=0.051. Conclusion. Our findings suggest that cognitive impairments are not correlated with different LRRK2 S1647T polymorphisms in Chinese PD individuals.

  14. A Missense LRRK2 Variant Is a Risk Factor for Excessive Inflammatory Responses in Leprosy.

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    Vinicius M Fava

    2016-02-01

    Full Text Available Depending on the epidemiological setting, a variable proportion of leprosy patients will suffer from excessive pro-inflammatory responses, termed type-1 reactions (T1R. The LRRK2 gene encodes a multi-functional protein that has been shown to modulate pro-inflammatory responses. Variants near the LRRK2 gene have been associated with leprosy in some but not in other studies. We hypothesized that LRRK2 was a T1R susceptibility gene and that inconsistent association results might reflect different proportions of patients with T1R in the different sample settings. Hence, we evaluated the association of LRRK2 variants with T1R susceptibility.An association scan of the LRRK2 locus was performed using 156 single-nucleotide polymorphisms (SNPs. Evidence of association was evaluated in two family-based samples: A set of T1R-affected and a second set of T1R-free families. Only SNPs significant for T1R-affected families with significant evidence of heterogeneity relative to T1R-free families were considered T1R-specific. An expression quantitative trait locus (eQTL analysis was applied to evaluate the impact of T1R-specific SNPs on LRRK2 gene transcriptional levels.A total of 18 T1R-specific variants organized in four bins were detected. The core SNP capturing the T1R association was the LRRK2 missense variant M2397T (rs3761863 that affects LRRK2 protein turnover. Additionally, a bin of nine SNPs associated with T1R were eQTLs for LRRK2 in unstimulated whole blood cells but not after exposure to Mycobacterium leprae antigen.The results support a preferential association of LRRK2 variants with T1R. LRRK2 involvement in T1R is likely due to a pathological pro-inflammatory loop modulated by LRRK2 availability. Interestingly, the M2397T variant was reported in association with Crohn's disease with the same risk allele as in T1R suggesting common inflammatory mechanism in these two distinct diseases.

  15. A comparative study of LRRK2, PINK1 and genetically undefined familial Parkinson's disease.

    Science.gov (United States)

    Nishioka, Kenya; Kefi, Mounir; Jasinska-Myga, Barbara; Wider, Christian; Vilariño-Güell, Carles; Ross, Owen A; Heckman, Michael G; Middleton, Lefkos T; Ishihara-Paul, Lianna; Gibson, Rachel A; Amouri, Rim; Ben Yahmed, Samia; Ben Sassi, Samia; Zouari, Mourad; El Euch, Ghada; Farrer, Matthew J; Hentati, Faycal

    2010-04-01

    Genetic classification of Parkinson's disease (PD) subtypes may become the preferred diagnostic tool for neurologists. Herein we compare clinical features from a large cohort of patients with familial PD of unknown aetiology or attributable to distinct genetic forms. Comprehensive neurological examinations were performed in 231 familial PD patients from Tunisia. Analysis was previously performed to screen for mutations in leucine rich repeat kinase 2 (LRRK2), PTEN induced kinase 1 (PINK1) and parkin (PRKN). Clinical features were compared between patients with genetically undefined PD (n=107) and those with LRRK2 (n=73) and PINK1 (n=42) mutations using regression analyses adjusted for gender, age of onset and disease duration. PRKN cases (n=9) were too few for meaningful statistical analysis. In comparison with genetically undefined patients, LRRK2 mutation carriers had more severe motor symptoms (median Unified Parkinson's Disease Rating Scale scores approximately 1.6 times higher, pundefined patients. As expected, PINK1 patients had younger ages and ages at disease onset, and a longer disease duration compared with LRRK2 mutation carriers and genetically undefined patients. Clinical differences between LRRK2, PINK1 and genetically undefined familial PD appear more pronounced than previously appreciated, and may prove useful in clinical practice. As future therapies are targeted to specific protein abnormalities, identifying the genetic causes and associated clinical and pathological features will determine diagnosis, preventative medicine and drug intervention strategies.

  16. Familial knockin mutation of LRRK2 causes lysosomal dysfunction and accumulation of endogenous insoluble α-synuclein in neurons.

    Science.gov (United States)

    Schapansky, Jason; Khasnavis, Saurabh; DeAndrade, Mark P; Nardozzi, Jonathan D; Falkson, Samuel R; Boyd, Justin D; Sanderson, John B; Bartels, Tim; Melrose, Heather L; LaVoie, Matthew J

    2018-03-01

    Missense mutations in the multi-domain kinase LRRK2 cause late onset familial Parkinson's disease. They most commonly with classic proteinopathy in the form of Lewy bodies and Lewy neurites comprised of insoluble α-synuclein, but in rare cases can also manifest tauopathy. The normal function of LRRK2 has remained elusive, as have the cellular consequences of its mutation. Data from LRRK2 null model organisms and LRRK2-inhibitor treated animals support a physiological role for LRRK2 in regulating lysosome function. Since idiopathic and LRRK2-linked PD are associated with the intraneuronal accumulation of protein aggregates, a series of critical questions emerge. First, how do pathogenic mutations that increase LRRK2 kinase activity affect lysosome biology in neurons? Second, are mutation-induced changes in lysosome function sufficient to alter the metabolism of α-synuclein? Lastly, are changes caused by pathogenic mutation sensitive to reversal with LRRK2 kinase inhibitors? Here, we report that mutation of LRRK2 induces modest but significant changes in lysosomal morphology and acidification, and decreased basal autophagic flux when compared to WT neurons. These changes were associated with an accumulation of detergent-insoluble α-synuclein and increased neuronal release of α-synuclein and were reversed by pharmacologic inhibition of LRRK2 kinase activity. These data demonstrate a critical and disease-relevant influence of native neuronal LRRK2 kinase activity on lysosome function and α-synuclein homeostasis. Furthermore, they also suggest that lysosome dysfunction, altered neuronal α-synuclein metabolism, and the insidious accumulation of aggregated protein over decades may contribute to pathogenesis in this late-onset form of familial PD. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. LRRK2 in Parkinson's disease ? drawing the curtain of penetrance: a commentary

    OpenAIRE

    Kr?ger, Rejko

    2008-01-01

    Abstract Parkinson's disease is the most common neurodegenerative movement disorder and affects about 2% of the population over the age of 60 years. In 2004, mutations in the LRRK2 gene were first described and turned out to be the most frequent genetic cause of familial and sporadic Parkinson's disease and may account for up to 40% of patients in distinct populations. Based on these findings, Latourelle and colleagues show that the penetrance of the most common LRRK2 mutation is higher in pa...

  18. High nigral iron deposition in LRRK2 and Parkin mutation carriers using R2* relaxometry

    DEFF Research Database (Denmark)

    Pyatigorskaya, Nadya; Sharman, Michael; Corvol, Jean-Christophe

    2015-01-01

    symptomatic and two asymptomatic Parkin subjects, nine symptomatic and five asymptomatic LRRK2 subjects) were compared with 20 patients with idiopathic PD (IPD) and 20 healthy subjects. Images were obtained at 3 teslas, using multi-echo T2 and T2* sequences. R2 and R2* values were calculated in the substantia...

  19. A cognitive fMRI study in non-manifesting LRRK2 and GBA carriers

    NARCIS (Netherlands)

    Bregman, N.; Thaler, A.; Mirelman, A.; Helmich, R.C.G.; Gurevich, T.; Orr-Urtreger, A.; Marder, K.; Bressman, S.; Bloem, B.R.; Giladi, N.

    2017-01-01

    Mutations in the GBA and LRRK2 genes account for one-third of the prevalence of Parkinson's disease (PD) in Ashkenazi Jews. Non-manifesting carriers (NMC) of these mutations represent a population at risk for future development of PD. PD patient who carry mutations in the GBA gene demonstrates more

  20. Phenotype, genotype, and worldwide genetic penetrance of LRRK2-associated Parkinson's disease: a case-control study

    Science.gov (United States)

    Healy, Daniel G; Falchi, Mario; O'Sullivan, Sean S; Bonifati, Vincenzo; Durr, Alexandra; Bressman, Susan; Brice, Alexis; Aasly, Jan; Zabetian, Cyrus P; Goldwurm, Stefano; Ferreira, Joaquim J; Tolosa, Eduardo; Kay, Denise M; Klein, Christine; Williams, David R; Marras, Connie; Lang, Anthony E; Wszolek, Zbigniew K; Berciano, Jose; Schapira, Anthony HV; Lynch, Timothy; Bhatia, Kailash P; Gasser, Thomas; Lees, Andrew J; Wood, Nicholas W

    2008-01-01

    Summary Background Mutations in LRRK2, the gene that encodes leucine-rich repeat kinase 2, are a cause of Parkinson's disease (PD). The International LRRK2 Consortium was established to answer three key clinical questions: can LRRK2-associated PD be distinguished from idiopathic PD; which mutations in LRRK2 are pathogenic; and what is the age-specific cumulative risk of PD for individuals who inherit or are at risk of inheriting a deleterious mutation in LRRK2? Methods Researchers from 21 centres across the world collaborated on this study. The frequency of the common LRRK2 Gly2019Ser mutation was estimated on the basis of data from 24 populations worldwide, and the penetrance of the mutation was defined in 1045 people with mutations in LRRK2 from 133 families. The LRRK2 phenotype was defined on the basis of 59 motor and non-motor symptoms in 356 patients with LRRK2-associated PD and compared with the symptoms of 543 patients with pathologically proven idiopathic PD. Findings Six mutations met the consortium's criteria for being proven pathogenic. The frequency of the common LRRK2 Gly2019Ser mutation was 1% of patients with sporadic PD and 4% of patients with hereditary PD; the frequency was highest in the middle east and higher in southern Europe than in northern Europe. The risk of PD for a person who inherits the LRRK2 Gly2019Ser mutation was 28% at age 59 years, 51% at 69 years, and 74% at 79 years. The motor symptoms (eg, disease severity, rate of progression, occurrence of falls, and dyskinesia) and non-motor symptoms (eg, cognition and olfaction) of LRRK2-associated PD were more benign than those of idiopathic PD. Interpretation Mutations in LRRK2 are a clinically relevant cause of PD that merit testing in patients with hereditary PD and in subgroups of patients with PD. However, this knowledge should be applied with caution in the diagnosis and counselling of patients. Funding UK Medical Research Council; UK Parkinson's Disease Society; UK Brain Research

  1. Non-motor and motor features in LRRK2 transgenic mice.

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    Zoë Bichler

    Full Text Available Non-motor symptoms are increasingly recognized as important features of Parkinson's disease (PD. LRRK2 mutations are common causes of familial and sporadic PD. Non-motor features have not been yet comprehensively evaluated in LRRK2 transgenic mouse models.Using a transgenic mouse model overexpressing the R1441G mutation of the human LRRK2 gene, we have investigated the longitudinal correlation between motor and non-motor symptoms and determined if specific non-motor phenotypes precede motor symptoms.We investigated the onset of motor and non-motor phenotypes on the LRRK2(R1441G BAC transgenic mice and their littermate controls from 4 to 21 month-old using a battery of behavioral tests. The transgenic mutant mice displayed mild hypokinesia in the open field from 16 months old, with gastrointestinal dysfunctions beginning at 6 months old. Non-motor features such as depression and anxiety-like behaviors, sensorial functions (pain sensitivity and olfaction, and learning and memory abilities in the passive avoidance test were similar in the transgenic animals compared to littermate controls.LRRK2(R1441G BAC transgenic mice displayed gastrointestinal dysfunction at an early stage but did not have abnormalities in fine behaviors, olfaction, pain sensitivity, mood disorders and learning and memory compared to non-transgenic littermate controls. The observations on olfaction and gastrointestinal dysfunction in this model validate findings in human carriers. These mice did recapitulate mild Parkinsonian motor features at late stages but compensatory mechanisms modulating the progression of PD in these models should be further evaluated.

  2. LRRK2 knockout mice have an intact dopaminergic system but display alterations in exploratory and motor co-ordination behaviors

    Science.gov (United States)

    2012-01-01

    Mutations in the LRRK2 gene are the most common cause of genetic Parkinson’s disease. Although the mechanisms behind the pathogenic effects of LRRK2 mutations are still not clear, data emerging from in vitro and in vivo models suggests roles in regulating neuronal polarity, neurotransmission, membrane and cytoskeletal dynamics and protein degradation. We created mice lacking exon 41 that encodes the activation hinge of the kinase domain of LRRK2. We have performed a comprehensive analysis of these mice up to 20 months of age, including evaluation of dopamine storage, release, uptake and synthesis, behavioral testing, dendritic spine and proliferation/neurogenesis analysis. Our results show that the dopaminergic system was not functionally comprised in LRRK2 knockout mice. However, LRRK2 knockout mice displayed abnormal exploratory activity in the open-field test. Moreover, LRRK2 knockout mice stayed longer than their wild type littermates on the accelerated rod during rotarod testing. Finally, we confirm that loss of LRRK2 caused degeneration in the kidney, accompanied by a progressive enhancement of autophagic activity and accumulation of autofluorescent material, but without evidence of biphasic changes. PMID:22647713

  3. A proteomic analysis of LRRK2 binding partners reveals interactions with multiple signaling components of the WNT/PCP pathway.

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    Salašová, Alena; Yokota, Chika; Potěšil, David; Zdráhal, Zbyněk; Bryja, Vítězslav; Arenas, Ernest

    2017-07-11

    Autosomal-dominant mutations in the Park8 gene encoding Leucine-rich repeat kinase 2 (LRRK2) have been identified to cause up to 40% of the genetic forms of Parkinson's disease. However, the function and molecular pathways regulated by LRRK2 are largely unknown. It has been shown that LRRK2 serves as a scaffold during activation of WNT/β-catenin signaling via its interaction with the β-catenin destruction complex, DVL1-3 and LRP6. In this study, we examine whether LRRK2 also interacts with signaling components of the WNT/Planar Cell Polarity (WNT/PCP) pathway, which controls the maturation of substantia nigra dopaminergic neurons, the main cell type lost in Parkinson's disease patients. Co-immunoprecipitation and tandem mass spectrometry was performed in a mouse substantia nigra cell line (SN4741) and human HEK293T cell line in order to identify novel LRRK2 binding partners. Inhibition of the WNT/β-catenin reporter, TOPFlash, was used as a read-out of WNT/PCP pathway activation. The capacity of LRRK2 to regulate WNT/PCP signaling in vivo was tested in Xenopus laevis' early development. Our proteomic analysis identified that LRRK2 interacts with proteins involved in WNT/PCP signaling such as the PDZ domain-containing protein GIPC1 and Integrin-linked kinase (ILK) in dopaminergic cells in vitro and in the mouse ventral midbrain in vivo. Moreover, co-immunoprecipitation analysis revealed that LRRK2 binds to two core components of the WNT/PCP signaling pathway, PRICKLE1 and CELSR1, as well as to FLOTILLIN-2 and CULLIN-3, which regulate WNT secretion and inhibit WNT/β-catenin signaling, respectively. We also found that PRICKLE1 and LRRK2 localize in signalosomes and act as dual regulators of WNT/PCP and β-catenin signaling. Accordingly, analysis of the function of LRRK2 in vivo, in X. laevis revelaed that LRKK2 not only inhibits WNT/β-catenin pathway, but induces a classical WNT/PCP phenotype in vivo. Our study shows for the first time that LRRK2 activates the WNT

  4. Pathogenic Parkinson's disease mutations across the functional domains of LRRK2 alter the autophagic/lysosomal response to starvation.

    Science.gov (United States)

    Manzoni, Claudia; Mamais, Adamantios; Dihanich, Sybille; McGoldrick, Phillip; Devine, Michael J; Zerle, Julia; Kara, Eleanna; Taanman, Jan-Willem; Healy, Daniel G; Marti-Masso, Jose-Felix; Schapira, Anthony H; Plun-Favreau, Helene; Tooze, Sharon; Hardy, John; Bandopadhyay, Rina; Lewis, Patrick A

    2013-11-29

    LRRK2 is one of the most important genetic contributors to Parkinson's disease (PD). Point mutations in this gene cause an autosomal dominant form of PD, but to date no cellular phenotype has been consistently linked with mutations in each of the functional domains (ROC, COR and Kinase) of the protein product of this gene. In this study, primary fibroblasts from individuals carrying pathogenic mutations in the three central domains of LRRK2 were assessed for alterations in the autophagy/lysosomal pathway using a combination of biochemical and cellular approaches. Mutations in all three domains resulted in alterations in markers for autophagy/lysosomal function compared to wild type cells. These data highlight the autophagy and lysosomal pathways as read outs for pathogenic LRRK2 function and as a marker for disease, and provide insight into the mechanisms linking LRRK2 function and mutations. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  5. LRRK2 Kinase Activity and Biology are Not Uniformly Predicted by its Autophosphorylation and Cellular Phosphorylation Site Status

    Directory of Open Access Journals (Sweden)

    April eReynolds

    2014-06-01

    Full Text Available Missense mutations in the Leucine Rich Repeat protein Kinase 2 (LRRK2 gene are the most common genetic predisposition to develop Parkinson’s disease (PD LRRK2 is a large multi-domain phosphoprotein with a GTPase domain and a serine/threonine protein kinase domain whose activity is implicated in neuronal toxicity; however the precise mechanism is unknown. LRRK2 autophosphorylates on several serine/threonine residues across the enzyme and is found constitutively phosphorylated on Ser910, Ser935, Ser955 and Ser973, which are proposed to be regulated by upstream kinases. Here we investigate the phosphoregulation at these sites by analyzing the effects of disease-associated mutations Arg1441Cys, Arg1441Gly, Ala1442Pro, Tyr1699Cys, Ile2012Thr, Gly2019Ser, and Ile2020Thr. We also studied alanine substitutions of phosphosite serines 910, 935, 955 and 973 and specific LRRK2 inhibition on autophosphorylation of LRRK2 Ser1292, Thr1491, Thr2483 and phosphorylation at the cellular sites. We found that mutants in the Roc-COR domains, including Arg1441Cys, Arg1441His, Ala1442Pro and Tyr1699Cys, can positively enhance LRRK2 kinase activity while concomitantly inducing the dephosphorylation of the cellular sites. Mutation of the cellular sites individually did not affect LRRK2 intrinsic kinase activity; however, Ser910/935/955/973Ala mutations trended toward increased kinase activity of LRRK2. Increased cAMP levels did not lead to increased LRRK2 cellular site phosphorylation, 14-3-3 binding or kinase activity. In cells, inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser1292 by Calyculin A and okadaic acid sensitive phosphatases, while the cellular sites are dephosphorylated by Calyculin A sensitive phosphatases. These findings indicate that comparative analysis of both Ser1292 and Ser910/935/955/973 phosphorylation sites will provide important and distinct measures of LRRK2 kinase and biological activity in vitro and in vivo.

  6. LRRK2 in Parkinson's disease – drawing the curtain of penetrance: a commentary

    Directory of Open Access Journals (Sweden)

    Krüger Rejko

    2008-11-01

    Full Text Available Abstract Parkinson's disease is the most common neurodegenerative movement disorder and affects about 2% of the population over the age of 60 years. In 2004, mutations in the LRRK2 gene were first described and turned out to be the most frequent genetic cause of familial and sporadic Parkinson's disease and may account for up to 40% of patients in distinct populations. Based on these findings, Latourelle and colleagues show that the penetrance of the most common LRRK2 mutation is higher in patients with familial compared with sporadic Parkinson's disease and identified a substantial number of affected relatives of mutation carriers not presenting with a LRRK2 mutation themselves. This commentary discusses the role of genetic and/or environmental susceptibility factors modulating the expressivity of the disease trait, how these factors may contribute to the phenomenon of phenocopies in genetically defined Parkinson's disease pedigrees, and how the findings of Latourelle and colleagues, published this month in BMC Medicine, relate to current concepts of genetic counselling.

  7. The IkappaB kinase family phosphorylates the Parkinson's disease kinase LRRK2 at Ser935 and Ser910 during Toll-like receptor signaling.

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    Nicolas Dzamko

    Full Text Available Mutations in leucine-rich repeat kinase 2 (LRRK2 are strongly associated with late-onset autosomal dominant Parkinson's disease. LRRK2 is highly expressed in immune cells and recent work points towards a link between LRRK2 and innate immunity. Here we demonstrate that stimulation of the Toll-Like Receptor (TLR pathway by MyD88-dependent agonists in bone marrow-derived macrophages (BMDMs or RAW264.7 macrophages induces marked phosphorylation of LRRK2 at Ser910 and Ser935, the phosphorylation sites that regulate the binding of 14-3-3 to LRRK2. Phosphorylation of these residues is prevented by knock-out of MyD88 in BMDMs, but not the alternative TLR adaptor protein TRIF. Utilising both pharmacological inhibitors, including a new TAK1 inhibitor, NG25, and genetic models, we provide evidence that both the canonical (IKKα and IKKβ and IKK-related (IKKε and TBK1 kinases mediate TLR agonist induced phosphorylation of LRRK2 in vivo. Moreover, all four IKK members directly phosphorylate LRRK2 at Ser910 and Ser935 in vitro. Consistent with previous work describing Ser910 and Ser935 as pharmacodynamic biomarkers of LRRK2 activity, we find that the TLR independent basal phosphorylation of LRRK2 at Ser910 and Ser935 is abolished following treatment of macrophages with LRRK2 kinase inhibitors. However, the increased phosphorylation of Ser910 and Ser935 induced by activation of the MyD88 pathway is insensitive to LRRK2 kinase inhibitors. Finally, employing LRRK2-deficient BMDMs, we present data indicating that LRRK2 does not play a major role in regulating the secretion of inflammatory cytokines induced by activation of the MyD88 pathway. Our findings provide the first direct link between LRRK2 and the IKKs that mediate many immune responses. Further work is required to uncover the physiological roles that phosphorylation of LRRK2 by IKKs play in controlling macrophage biology and to determine how phosphorylation of LRRK2 by IKKs impacts upon the use of Ser

  8. Inflexible ethanol intake: A putative link with the Lrrk2 pathway.

    Science.gov (United States)

    da Silva E Silva, Daniel Almeida; Frozino Ribeiro, Andrea; Damasceno, Samara; Rocha, Cristiane S; Berenguer de Matos, Alexandre H; Boerngen-Lacerda, Roseli; Correia, Diego; Brunialti Godard, Ana Lúcia

    2016-10-15

    Alcoholism is a complex multifactorial disorder with a strong genetic influence. Although several studies have shown the impact of high ethanol intake on the striatal gene expression, few have addressed the relationship between the patterns of gene expression underlying the compulsive behaviour associated with the two major concerns in addiction: the excessive drug consumption and relapsing. In this study, we used a chronic three-bottle free-choice murine model to address striatal transcript regulation among animals with different ethanol intakes and preferences: Light Drinkers (preference for water throughout the experiment), Heavy Drinkers (preference for ethanol with a non-compulsive intake) and Inflexible Drinkers (preference for ethanol and simultaneous loss of control over the drug intake). Our aim was to correlate the intake patterns observed in this model with gene expression changes in the striatum, a brain region critical for the development of alcohol addiction. We found that the transcripts of the Lrrk2 gene, which encodes a multifunctional protein with kinase and GTPase activities, is upregulated only in Inflexible Drinkers suggesting, for the first time, that the Lrrk2 pathway plays a major role in the compulsive ethanol intake behaviour of addicted subjects. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Actigraphy Detects Greater Intra-Individual Variability During Gait in Non-Manifesting LRRK2 Mutation Carriers.

    Science.gov (United States)

    van den Heuvel, Lieneke; Lim, Andrew S; Visanji, Naomi P; Huang, Jana; Ghate, Taneera; Mestre, Tiago A; AlDakheel, Amaal; Connolly, Barbara S; Gasca-Salas, Carmen; Kern, Drew S; Jain, Jennifer; Slow, Elizabeth J; Pondal, Margarita; Faust-Socher, Achinoam; Rogaeva, Ekaterina; Tomlinson, George; Lang, Anthony E; Marras, Connie

    2018-01-01

    With recent advances in the search for disease-modifying therapies for Parkinson's disease (PD) the importance of identifying prodromal markers becomes greater. Non-manifesting LRRK2 mutation carriers (NMC) are at risk for developing PD, and provide a population in which to identify possible markers. The aim of this study was to test the hypothesis that NMC have differences in daily activity, fragmentation of sleep, arm swing asymmetry, and movement variability during walking, detectable by actigraphy, as compared to matched control subjects. Eleven NMC, fourteen PD patients (4 LRRK2-PD, 10 idiopathic PD (iPD)), and twenty-nine controls wore wristbands containing an accelerometer for seven days, and performed a daily walking task. Outcome measures included daily activity, fragmentation of activity, fragmentation of sleep, arm swing asymmetry during walking, and intra-individual variability. Compared to healthy controls, both NMC and LRRK2/iPD showed higher intra-individual variability in activity during walking compared to healthy controls. Individuals with LRRK2-PD/iPD, but not NMC, tend to have lower activity levels, more arm swing asymmetry and less increase of arm swing with transition from slow to faster walking speed compared to healthy controls. Higher intra-individual variability of gait-associated movements might be a useful biomarker of prodromal PD. These results encourage replication in a larger sample and longitudinal analysis is warranted.

  10. LRRK2 A419V is not associated with Parkinson's disease in different Chinese populations.

    Directory of Open Access Journals (Sweden)

    Yih Ru Wu

    Full Text Available It has been suggested that a common LRRK2 polymorphic variant (A419V (rs34594498 C >T may be a risk factor among Asians (especially in Taiwan. In this study, we examined this variant in a larger and independent Taiwan cohort. We found the frequency of the variant (A419V to be very rare in our Taiwan PD and controls (?0.6%. Further studies were conducted in two other Chinese populations (Singapore and China, comprising of a total of 3004 subjects including 1517 PD patients and 1487 control subjects. However, our multi-center Chinese study revealed that the frequency of the variant was rare (?0.4% and was not associated with risk of PD, suggesting that the variant is not a major risk factor for PD among Chinese, at least in our study population.

  11. Leucine-rich repeat kinase 2 (LRRK2-deficient rats exhibit renal tubule injury and perturbations in metabolic and immunological homeostasis.

    Directory of Open Access Journals (Sweden)

    Daniel Ness

    Full Text Available Genetic evidence links mutations in the LRRK2 gene with an increased risk of Parkinson's disease, for which no neuroprotective or neurorestorative therapies currently exist. While the role of LRRK2 in normal cellular function has yet to be fully described, evidence suggests involvement with immune and kidney functions. A comparative study of LRRK2-deficient and wild type rats investigated the influence that this gene has on the phenotype of these rats. Significant weight gain in the LRRK2 null rats was observed and was accompanied by significant increases in insulin and insulin-like growth factors. Additionally, LRRK2-deficient rats displayed kidney morphological and histopathological alterations in the renal tubule epithelial cells of all animals assessed. These perturbations in renal morphology were accompanied by significant decreases of lipocalin-2, in both the urine and plasma of knockout animals. Significant alterations in the cellular composition of the spleen between LRRK2 knockout and wild type animals were identified by immunophenotyping and were associated with subtle differences in response to dual infection with rat-adapted influenza virus (RAIV and Streptococcus pneumoniae. Ontological pathway analysis of LRRK2 across metabolic and kidney processes and pathological categories suggested that the thioredoxin network may play a role in perturbing these organ systems. The phenotype of the LRRK2 null rat is suggestive of a complex biology influencing metabolism, immune function and kidney homeostasis. These data need to be extended to better understand the role of the kinase domain or other biological functions of the gene to better inform the development of pharmacological inhibitors.

  12. The LRRK2 G2385R variant is a partial loss-of-function mutation that affects synaptic vesicle trafficking through altered protein interactions.

    Science.gov (United States)

    Carrion, Maria Dolores Perez; Marsicano, Silvia; Daniele, Federica; Marte, Antonella; Pischedda, Francesca; Di Cairano, Eliana; Piovesana, Ester; von Zweydorf, Felix; Kremmer, Elisabeth; Gloeckner, Christian Johannes; Onofri, Franco; Perego, Carla; Piccoli, Giovanni

    2017-07-14

    Mutations in the Leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial Parkinson's disease (PD). LRRK2 protein contains several functional domains, including protein-protein interaction domains at its N- and C-termini. In this study, we analyzed the functional features attributed to LRRK2 by its N- and C-terminal domains. We combined TIRF microscopy and synaptopHluorin assay to visualize synaptic vesicle trafficking. We found that N- and C-terminal domains have opposite impact on synaptic vesicle dynamics. Biochemical analysis demonstrated that different proteins are bound at the two extremities, namely β3-Cav2.1 at N-terminus part and β-Actin and Synapsin I at C-terminus domain. A sequence variant (G2385R) harboured within the C-terminal WD40 domain increases the risk for PD. Complementary biochemical and imaging approaches revealed that the G2385R variant alters strength and quality of LRRK2 interactions and increases fusion of synaptic vesicles. Our data suggest that the G2385R variant behaves like a loss-of-function mutation that mimics activity-driven events. Impaired scaffolding capabilities of mutant LRRK2 resulting in perturbed vesicular trafficking may arise as a common pathophysiological denominator through which different LRRK2 pathological mutations cause disease.

  13. Hypothesis: Do miRNAs Targeting the Leucine-Rich Repeat Kinase 2 Gene (LRRK2) Influence Parkinson's Disease Susceptibility?

    Science.gov (United States)

    Yılmaz, Şenay Görücü; Geyik, Sırma; Neyal, Ayşe Münife; Soko, Nyarai D; Bozkurt, Hakan; Dandara, Collet

    2016-04-01

    Parkinson's disease (PD) is a frequently occurring neurodegenerative motor disorder adversely impacting global health. There is a paucity of biomarkers and diagnostics that can forecast susceptibility to PD. A new research frontier for PD pathophysiology is the study of variations in microRNA (miRNA) expression whereby miRNAs serve as "upstream regulators" of gene expression in relation to functioning of the dopamine neuronal pathways. Leucine-Rich Repeat Kinase 2 (LRRK2) is a frequently studied gene in PD. Little is known about the ways in which expression of miRNAs targeting LRKK2 impact PD susceptibility. In a sample of 204 unrelated subjects (102 persons with PD and 102 healthy controls), we report here candidate miRNA expression in whole blood samples as measured by real-time PCR (hsa-miR-4671-3p, hsa-miR-335-3p, hsa-miR-561-3p, hsa-miR-579-3p, and hsa-miR-3143) that target LRRK2. Using step-wise logistic regression, and controlling for covariates such as age, gender, PD disease severity, concomitant medications, and co-morbidity, we found that the combination of has-miR-335-3p, has-miR-561-3p, and has-miR-579-3p account for 50% of the variation in regards to PD susceptibility (p<0.0001). Notably, the hsa-miR-561-3p expression was the most robust predictor of PD in both univariate and multivariate analyses (p<0.001). Moreover, the biological direction (polarity) of the association was plausible in that the candidate miRNAs displayed a diminished expression in patients. This is consistent with the hypothesis that decreased levels of miRNAs targeting LRRK2 might result in a gain of function for LRRK2, and by extension, loss of neuronal viability. To the best of our knowledge, this is the first clinical association study of the above candidate miRNAs' expression in PD using peripheral samples. These observations may guide future clinical diagnostics research on PD.

  14. Novel ethyl methanesulfonate (EMS-induced null alleles of the Drosophila homolog of LRRK2 reveal a crucial role in endolysosomal functions and autophagy in vivo

    Directory of Open Access Journals (Sweden)

    Mark W. Dodson

    2014-12-01

    Full Text Available Mutations in LRRK2 cause a dominantly inherited form of Parkinson’s disease (PD and are the most common known genetic determinant of PD. Inhibitor-based therapies targeting LRRK2 have emerged as a key therapeutic strategy in PD; thus, understanding the consequences of inhibiting the normal cellular functions of this protein is vital. Despite much interest, the physiological functions of LRRK2 remain unclear. Several recent studies have linked the toxicity caused by overexpression of pathogenic mutant forms of LRRK2 to defects in the endolysosomal and autophagy pathways, raising the question of whether endogenous LRRK2 might play a role in these processes. Here, we report the characterization of multiple novel ethyl methanesulfonate (EMS-induced nonsense alleles in the Drosophila LRRK2 homolog, lrrk. Using these alleles, we show that lrrk loss-of-function causes striking defects in the endolysosomal and autophagy pathways, including the accumulation of markedly enlarged lysosomes that are laden with undigested contents, consistent with a defect in lysosomal degradation. lrrk loss-of-function also results in the accumulation of autophagosomes, as well as the presence of enlarged early endosomes laden with mono-ubiquitylated cargo proteins, suggesting an additional defect in lysosomal substrate delivery. Interestingly, the lysosomal abnormalities in these lrrk mutants can be suppressed by a constitutively active form of the small GTPase rab9, which promotes retromer-dependent recycling from late endosomes to the Golgi. Collectively, our data provides compelling evidence of a vital role for lrrk in lysosomal function and endolysosomal membrane transport in vivo, and suggests a link between lrrk and retromer-mediated endosomal recycling.

  15. Novel ethyl methanesulfonate (EMS)-induced null alleles of the Drosophila homolog of LRRK2 reveal a crucial role in endolysosomal functions and autophagy in vivo.

    Science.gov (United States)

    Dodson, Mark W; Leung, Lok K; Lone, Mohiddin; Lizzio, Michael A; Guo, Ming

    2014-12-01

    Mutations in LRRK2 cause a dominantly inherited form of Parkinson's disease (PD) and are the most common known genetic determinant of PD. Inhibitor-based therapies targeting LRRK2 have emerged as a key therapeutic strategy in PD; thus, understanding the consequences of inhibiting the normal cellular functions of this protein is vital. Despite much interest, the physiological functions of LRRK2 remain unclear. Several recent studies have linked the toxicity caused by overexpression of pathogenic mutant forms of LRRK2 to defects in the endolysosomal and autophagy pathways, raising the question of whether endogenous LRRK2 might play a role in these processes. Here, we report the characterization of multiple novel ethyl methanesulfonate (EMS)-induced nonsense alleles in the Drosophila LRRK2 homolog, lrrk. Using these alleles, we show that lrrk loss-of-function causes striking defects in the endolysosomal and autophagy pathways, including the accumulation of markedly enlarged lysosomes that are laden with undigested contents, consistent with a defect in lysosomal degradation. lrrk loss-of-function also results in the accumulation of autophagosomes, as well as the presence of enlarged early endosomes laden with mono-ubiquitylated cargo proteins, suggesting an additional defect in lysosomal substrate delivery. Interestingly, the lysosomal abnormalities in these lrrk mutants can be suppressed by a constitutively active form of the small GTPase rab9, which promotes retromer-dependent recycling from late endosomes to the Golgi. Collectively, our data provides compelling evidence of a vital role for lrrk in lysosomal function and endolysosomal membrane transport in vivo, and suggests a link between lrrk and retromer-mediated endosomal recycling. © 2014. Published by The Company of Biologists Ltd.

  16. LRRK2 and RIPK2 variants in the NOD 2-mediated signaling pathway are associated with susceptibility to Mycobacterium leprae in Indian populations.

    Directory of Open Access Journals (Sweden)

    Patrick Marcinek

    Full Text Available In recent years, genome wide association studies have discovered a large number of gene loci that play a functional role in innate and adaptive immune pathways associated with leprosy susceptibility. The immunological control of intracellular bacteria M. leprae is modulated by NOD2-mediated signaling of Th1 responses. In this study, we investigated 211 clinically classified leprosy patients and 230 ethnically matched controls in Indian population by genotyping four variants in NOD2 (rs9302752A/G, LRRK2 (rs1873613A/G, RIPK2 (rs40457A/G and rs42490G/A. The LRRK2 locus is associated with leprosy outcome. The LRRK2 rs1873613A minor allele and respective rs1873613AA genotypes were significantly associated with an increased risk whereas the LRRK2 rs1873613G major allele and rs1873613GG genotypes confer protection in paucibacillary and leprosy patients. The reconstructed GA haplotypes from RIPK2 rs40457A/G and rs42490G/A variants was observed to contribute towards increased risk whereas haplotypes AA was observed to confer protective role. Our results indicate that a possible shared mechanisms underlying the development of these two clinical forms of the disease as hypothesized. Our findings confirm and validates the role of gene variants involved in NOD2-mediated signalling pathways that play a role in immunological control of intracellular bacteria M. leprae.

  17. Derivation, Characterization, and Neural Differentiation of Integration-Free Induced Pluripotent Stem Cell Lines from Parkinson's Disease Patients Carrying SNCA, LRRK2, PARK2, and GBA Mutations

    DEFF Research Database (Denmark)

    Momcilovic, Olga; Sivapatham, Renuka; Oron, Tal Ronnen

    2016-01-01

    We report generation of induced pluripotent stem cell (iPSC) lines from ten Parkinson's disease (PD) patients carrying SNCA, PARK2, LRRK2, and GBA mutations, and one age-matched control. After validation of pluripotency, long-term genome stability, and integration-free reprogramming, eight...... not be sufficient to determine the cause or mechanism of the disease, and highlights the need to use more focused strategies for large-scale data analysis........ We further examined gene expression in a stress model (MPTP-induced dopaminergic neuronal death) using two clones from the SNCA triplication line, and detected changes in genes associated with mitophagy. Our data suggested that even a well-characterized line of a monogenic disease may...

  18. Stress-tolerant mutants induced by heavy-ion beams

    Energy Technology Data Exchange (ETDEWEB)

    Abe, Tomoko; Yoshida, Shigeo [Institute of Physical and Chemical Research, Wako, Saitama (Japan); Bae, Chang-Hyu [Sunchon National University, Sunchon (Korea); Ozaki, Takuo [Japan Atomic Energy Research Inst., Tokai, Ibaraki (Japan). Tokai Research Establishment; Wang, Jing Ming [Akita Prefectural Univ. (Japan)

    2000-07-01

    Comparative study was made on mutagenesis in tobacco embryo induced by exposure to EMS (ethyl methane-sulfonate) ion beams during the fertilization cycle. Tobacco embryo cells immediately after pollination were exposed to heavy ion beam and the sensitivity to the irradiation was assessed in each developmental stage and compared with the effects of EMS, a chemical mutagen. Morphologically abnormality such as chlorophyll deficiency was used as a marker. A total of 17 salt-tolerant plants were selected from 3447 M{sub 1} seeds. A cell line showed salt resistance. The cell growth and chlorophyll content were each two times higher than that of WT cells in the medium containing 154 mM NaCl. Seven strains of M{sub 3} progeny of 17 salt-tolerant plants, showed strong resistance, but no salt tolerant progeny were obtained from Xanthi or Ne-ion irradiation. This shows that the sensitivity of plant embryo to this irradiation technique may vary among species. When exposed to {sup 14}N ion beam for 24-108 hours after pollination, various morphological mutants appeared at 18% in M{sub 1} progeny and herbicide tolerant and salt tolerant mutants were obtained. A strong Co-tolerant strain was obtained in two of 17 salt-tolerant strains and a total of 46 tolerant strains (0.2%) were obtained from 22,272 grains of M{sub 1} seeds. In these tolerant strains, the absorption of Co was slightly decreased, but those of Mg and Mn were increased. Mutants induced with ion-beam irradiation have potential not only for practical use in the breeding of stress-tolerant plants but also for gene analysis that will surely facilitate the molecular understanding of the tolerance mechanisms. (M.N.)

  19. Stress-tolerant mutants induced by heavy-ion beams

    International Nuclear Information System (INIS)

    Abe, Tomoko; Yoshida, Shigeo; Bae, Chang-Hyu; Ozaki, Takuo

    2000-01-01

    Comparative study was made on mutagenesis in tobacco embryo induced by exposure to EMS (ethyl methane-sulfonate) ion beams during the fertilization cycle. Tobacco embryo cells immediately after pollination were exposed to heavy ion beam and the sensitivity to the irradiation was assessed in each developmental stage and compared with the effects of EMS, a chemical mutagen. Morphologically abnormality such as chlorophyll deficiency was used as a marker. A total of 17 salt-tolerant plants were selected from 3447 M 1 seeds. A cell line showed salt resistance. The cell growth and chlorophyll content were each two times higher than that of WT cells in the medium containing 154 mM NaCl. Seven strains of M 3 progeny of 17 salt-tolerant plants, showed strong resistance, but no salt tolerant progeny were obtained from Xanthi or Ne-ion irradiation. This shows that the sensitivity of plant embryo to this irradiation technique may vary among species. When exposed to 14 N ion beam for 24-108 hours after pollination, various morphological mutants appeared at 18% in M 1 progeny and herbicide tolerant and salt tolerant mutants were obtained. A strong Co-tolerant strain was obtained in two of 17 salt-tolerant strains and a total of 46 tolerant strains (0.2%) were obtained from 22,272 grains of M 1 seeds. In these tolerant strains, the absorption of Co was slightly decreased, but those of Mg and Mn were increased. Mutants induced with ion-beam irradiation have potential not only for practical use in the breeding of stress-tolerant plants but also for gene analysis that will surely facilitate the molecular understanding of the tolerance mechanisms. (M.N.)

  20. Yield and its components and some agronomic characters of four mungbean mutants induces by gamma ray

    International Nuclear Information System (INIS)

    Al-Mosawi, A. R. A.; Ibrahim, A. F.; Azzo, F. Z.; Khadim, A. A.; Ibrahim, W. H.; Abdul-Al-majed, A.

    2012-12-01

    A field experiment was carried out at the Tuwaitha research station / Iraqi Atomic Energy Commission (past) Ministry of science and Technology (now) in Agricultural seasons 2000 - 2001 to evaluate four mungbean mutants induced by gamma ray compared with local variety and Australian pedigree. The results showed that mutants AMU-45 was superiority on anther mutants and the local in date of flowering (41.50 days), maturity (71.25 days), yield (1609.88 kg/ha) and its components. (Author)

  1. Isozymes variability in rice mutants induced by fast neutrons and gamma rays

    International Nuclear Information System (INIS)

    Fuentes, J.L.; Alvarez, A.; Gutierrez, L.; Deus, J.E.

    2001-01-01

    The isozyme variability of a group of rice mutants induced through gamma and fast neutron (14 MeV) irradiation was studied. Polymorphisms were detected using esterase, peroxidase, polyphenol oxidase and alcohol dehydrogenase systems. The mean value of genetic similarity among the different cultivars, which arose from isozymes, was 0.75. The dendrogram was constructed based on genetic similarity matrices, designed with isozyme data using the unweighed pair group method arithmetic average (UPGMA) method. The efficiency of the UPGMA model for the estimation of genetic relationship among cultivars was supported by cophenetic correlation coefficients. Such values indicate that the distortion degree for the estimated similarities was minimal. It was found that both gamma rays and fast neutrons generated a wide range of variability which can be detected by means of isozyme patterns, even in closely related cultivars. (author)

  2. Isozymes variability in rice mutants induced by fast neutrons and gamma rays

    Energy Technology Data Exchange (ETDEWEB)

    Fuentes, J L; Alvarez, A [Centro de Estudios Aplicados al Desarrollo Nuclear (CEADEN), Miramar, Playa, Havana (Cuba); Gutierrez, L; Deus, J E [Instituto de Investigaciones del Arroz, Bauta, Havana (Cuba)

    2001-05-01

    The isozyme variability of a group of rice mutants induced through gamma and fast neutron (14 MeV) irradiation was studied. Polymorphisms were detected using esterase, peroxidase, polyphenol oxidase and alcohol dehydrogenase systems. The mean value of genetic similarity among the different cultivars, which arose from isozymes, was 0.75. The dendrogram was constructed based on genetic similarity matrices, designed with isozyme data using the unweighed pair group method arithmetic average (UPGMA) method. The efficiency of the UPGMA model for the estimation of genetic relationship among cultivars was supported by cophenetic correlation coefficients. Such values indicate that the distortion degree for the estimated similarities was minimal. It was found that both gamma rays and fast neutrons generated a wide range of variability which can be detected by means of isozyme patterns, even in closely related cultivars. (author)

  3. Integrating Gene Correction in the Reprogramming and Transdifferentiation Processes: A One-Step Strategy to Overcome Stem Cell-Based Gene Therapy Limitations

    Directory of Open Access Journals (Sweden)

    Seo-Young Lee

    2016-01-01

    Full Text Available The recent advent of induced pluripotent stem cells (iPSCs and gene therapy tools has raised the possibility of autologous cell therapy for rare genetic diseases. However, cellular reprogramming is inefficient in certain diseases such as ataxia telangiectasia, Fanconi anemia, LIG4 syndrome, and fibrodysplasia ossificans progressiva syndrome, owing to interference of the disease-related genes. To overcome these therapeutic limitations, it is necessary to fundamentally correct the abnormal gene during or prior to the reprogramming process. In addition, as genetic etiology of Parkinson’s disease, it has been well known that induced neural stem cells (iNSCs were progressively depleted by LRRK2 gene mutation, LRRK2 (G2019S. Thus, to maintain the induced NSCs directly derived from PD patient cells harboring LRRK2 (G2019S, it would be ideal to simultaneously treat the LRRK2 (G2019S fibroblast during the process of TD. Therefore, simultaneous reprogramming (or TD and gene therapy would provide the solution for therapeutic limitation caused by vulnerability of reprogramming or TD, in addition to being suitable for general application to the generation of autologous cell-therapy products for patients with genetic defects, thereby obviating the need for the arduous processes currently required.

  4. SRAP analysis of M3 lotus mutants induced by Fe+ ion implantation

    International Nuclear Information System (INIS)

    Jia Yanyan; Deng Chuanliang; Gao Jun; Ren Yingxue; Wang Ningna; Gao Wujun; Lu Longdou; Zhang Tao; Li Pengfei

    2011-01-01

    To examine and determine the lotus mutants induced by the same Fe + ion implantation at the molecular level, the SRAP technique was used, and the non denatured polyacrylamide gel electrophoresis was undertaken to analyze the PCR products. At the optimized SRAP reaction condition, of the 121 primer pairs tested, 10 primer pairs could amplify stable and remarkable specific bands, with primer polymorphism of 8.26%. This 10 primer pairs amplified a total of 215 bands, 83 of which were polymorphic, and the percent of polymorphic bands was 38.6%. A total of 141 bands were amplified for the mutant 1, 22 of which were different from the control with the variation ratio of 15.6%. However, the variation ratios of mutant 2 to 6 were 16.4%, 17.1%, 16.9%, 18.2% and 20.5% respectively. The results indicated that Fe + ion implantation into the seeds of Baiyangdian red lotus could induce random genetic DNA variations. (authors)

  5. Evaluation of Chemical Changes in Some Soybean Mutants Induced by Gamma Irradiation

    International Nuclear Information System (INIS)

    Abd-Elkalik, K.; Mekkawy, S.H.; El-Demerdash, H.M.

    2010-01-01

    The Egyptian soybean cultivar Giza-22 was used to induce genetic variability by using gamma rays at dose levels of 100, 150 and 200 Gy. Sixteen mutants (including parental cultivar) were evaluated in M3 generation for their agronomic traits and chemical analysis was done in seeds of M3 generation. Four mutants A21 (150 Gy), A22, A23 and A24 (200 Gy) showed superiority in their agronomic traits compared with parental cultivar. The results of chemical analysis of seeds of M3 generation showed that, oil and energy contents were unaffected by irradiation treatments while protein contents were significantly increased at doses 150 and 200 Gy. Phenolic and tannin contents in seeds of M3 generation showed no significant changes in their percentages due to irradiation treatments. Gamma irradiation significantly increased in linoleic acid content in most of the mutants compared with the control (Giza-22), whereas, there were decreases in linolenic acid content. Investigation of amino acid composition of mutants of M3 generation revealed significant increases in the essential amino acids in most mutants induced by gamma irradiation at 150 and 200 Gy. It could be suggested that the use of gamma irradiation can induce an improvement of the oil and protein composition in soybean

  6. Acetylome in Human Fibroblasts From Parkinson's Disease Patients

    Directory of Open Access Journals (Sweden)

    Sokhna M. S. Yakhine-Diop

    2018-04-01

    Full Text Available Parkinson's disease (PD is a multifactorial neurodegenerative disorder. The pathogenesis of this disease is associated with gene and environmental factors. Mutations in leucine-rich repeat kinase 2 (LRRK2 are the most frequent genetic cause of familial and sporadic PD. Moreover, posttranslational modifications, including protein acetylation, are involved in the molecular mechanism of PD. Acetylation of lysine proteins is a dynamic process that is modulated in PD. In this descriptive study, we characterized the acetylated proteins and peptides in primary fibroblasts from idiopathic PD (IPD and genetic PD harboring G2019S or R1441G LRRK2 mutations. Identified acetylated peptides are modulated between individuals' groups. Although acetylated nuclear proteins are the most represented in cells, they are hypoacetylated in IPD. Results display that the level of hyperacetylated and hypoacetylated peptides are, respectively, enhanced in genetic PD and in IPD cells.

  7. Distinctive genomic signature of neural and intestinal organoids from familial Parkinson's disease patient-derived induced pluripotent stem cells.

    Science.gov (United States)

    Son, M-Y; Sim, H; Son, Y S; Jung, K B; Lee, M-O; Oh, J-H; Chung, S-K; Jung, C-R; Kim, J

    2017-12-01

    The leucine-rich repeat kinase 2 (LRRK2) G2019S mutation is the most common genetic cause of Parkinson's disease (PD). There is compelling evidence that PD is not only a brain disease but also a gastrointestinal disorder; nonetheless, its pathogenesis remains unclear. We aimed to develop human neural and intestinal tissue models of PD patients harbouring an LRRK2 mutation to understand the link between LRRK2 and PD pathology by investigating the gene expression signature. We generated PD patient-specific induced pluripotent stem cells (iPSCs) carrying an LRRK2 G2019S mutation (LK2GS) and then differentiated into three-dimensional (3D) human neuroectodermal spheres (hNESs) and human intestinal organoids (hIOs). To unravel the gene and signalling networks associated with LK2GS, we analysed differentially expressed genes in the microarray data by functional clustering, gene ontology (GO) and pathway analyses. The expression profiles of LK2GS were distinct from those of wild-type controls in hNESs and hIOs. The most represented GO biological process in hNESs and hIOs was synaptic transmission, specifically synaptic vesicle trafficking, some defects of which are known to be related to PD. The results were further validated in four independent PD-specific hNESs and hIOs by microarray and qRT-PCR analysis. We provide the first evidence that LK2GS also causes significant changes in gene expression in the intestinal cells. These hNES and hIO models from the same genetic background of PD patients could be invaluable resources for understanding PD pathophysiology and for advancing the complexity of in vitro models with 3D expandable organoids. © 2017 British Neuropathological Society.

  8. 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

  9. 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

  10. Transcranial sonography and functional imaging in glucocerebrosidase mutation Parkinson disease.

    Science.gov (United States)

    Barrett, M J; Hagenah, J; Dhawan, V; Peng, S; Stanley, K; Raymond, D; Deik, A; Gross, S J; Schreiber-Agus, N; Mirelman, A; Marder, K; Ozelius, L J; Eidelberg, D; Bressman, S B; Saunders-Pullman, R

    2013-02-01

    Heterozygous glucocerebrosidase (GBA) mutations are the leading genetic risk factor for Parkinson disease, yet imaging correlates, particularly transcranial sonography, have not been extensively described. To determine whether GBA mutation heterozygotes with Parkinson disease demonstrate hyperechogenicity of the substantia nigra, transcranial sonography was performed in Ashkenazi Jewish Parkinson disease subjects, tested for the eight most common Gaucher disease mutations and the LRRK2 G2019S mutation, and in controls. [(18)F]-fluorodeoxyglucose or [(18)F]-fluorodopa positron emission tomography is also reported from a subset of Parkinson disease subjects with heterozygous GBA mutations. Parkinson disease subjects with heterozygous GBA mutations (n = 23) had a greater median maximal area of substantia nigral echogenicity compared to controls (n = 34, aSNmax = 0.30 vs. 0.18, p = 0.007). There was no difference in median maximal area of nigral echogenicity between Parkinson disease groups defined by GBA and LRRK2 genotype: GBA heterozygotes; GBA homozygotes/compound heterozygotes (n = 4, aSNmax = 0.27); subjects without LRRK2 or GBA mutations (n = 32, aSNmax = 0.27); LRRK2 heterozygotes/homozygotes without GBA mutations (n = 27, aSNmax = 0.28); and GBA heterozygotes/LRRK2 heterozygotes (n = 4, aSNmax = 0.32, overall p = 0.63). In secondary analyses among Parkinson disease subjects with GBA mutations, maximal area of nigral echogenicity did not differ based on GBA mutation severity or mutation number. [(18)F]-fluorodeoxyglucose (n = 3) and [(18)F]-fluorodopa (n = 2) positron emission tomography in Parkinson disease subjects with heterozygous GBA mutations was consistent with findings in idiopathic Parkinson disease. Both transcranial sonography and positron emission tomography are abnormal in GBA mutation associated Parkinson disease, similar to other Parkinson disease subjects. Copyright © 2012 Elsevier Ltd. All rights reserved.

  11. PREDICT-PD: An online approach to prospectively identify risk indicators of Parkinson's disease.

    Science.gov (United States)

    Noyce, Alastair J; R'Bibo, Lea; Peress, Luisa; Bestwick, Jonathan P; Adams-Carr, Kerala L; Mencacci, Niccolo E; Hawkes, Christopher H; Masters, Joseph M; Wood, Nicholas; Hardy, John; Giovannoni, Gavin; Lees, Andrew J; Schrag, Anette

    2017-02-01

    A number of early features can precede the diagnosis of Parkinson's disease (PD). To test an online, evidence-based algorithm to identify risk indicators of PD in the UK population. Participants aged 60 to 80 years without PD completed an online survey and keyboard-tapping task annually over 3 years, and underwent smell tests and genotyping for glucocerebrosidase (GBA) and leucine-rich repeat kinase 2 (LRRK2) mutations. Risk scores were calculated based on the results of a systematic review of risk factors and early features of PD, and individuals were grouped into higher (above 15th centile), medium, and lower risk groups (below 85th centile). Previously defined indicators of increased risk of PD ("intermediate markers"), including smell loss, rapid eye movement-sleep behavior disorder, and finger-tapping speed, and incident PD were used as outcomes. The correlation of risk scores with intermediate markers and movement of individuals between risk groups was assessed each year and prospectively. Exploratory Cox regression analyses with incident PD as the dependent variable were performed. A total of 1323 participants were recruited at baseline and >79% completed assessments each year. Annual risk scores were correlated with intermediate markers of PD each year and baseline scores were correlated with intermediate markers during follow-up (all P values < 0.001). Incident PD diagnoses during follow-up were significantly associated with baseline risk score (hazard ratio = 4.39, P = .045). GBA variants or G2019S LRRK2 mutations were found in 47 participants, and the predictive power for incident PD was improved by the addition of genetic variants to risk scores. The online PREDICT-PD algorithm is a unique and simple method to identify indicators of PD risk. © 2017 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society. © 2016 International Parkinson and Movement Disorder

  12. AF-6 Protects Against Dopaminergic Dysfunction and Mitochondrial Abnormalities in Drosophila Models of Parkinson’s Disease

    Directory of Open Access Journals (Sweden)

    Adeline H. Basil

    2017-08-01

    Full Text Available Afadin 6 (AF-6 is an F-actin binding multidomain-containing scaffolding protein that is known for its function in cell-cell adhesion. Interestingly, besides this well documented role, we recently found that AF-6 is a Parkin-interacting protein that augments Parkin/PINK1-mediated mitophagy. Notably, mutations in Parkin and PINK1 are causative of recessively inherited forms of Parkinson’s disease (PD and aberrant mitochondrial homeostasis is thought to underlie PD pathogenesis. Given the novel role of AF-6 in mitochondrial quality control (QC, we hypothesized that AF-6 overexpression may be beneficial to PD. Using the Drosophila melanogaster as a model system, we demonstrate in this study that transgenic overexpression of human AF-6 in parkin and also pink1 null flies rescues their mitochondrial pathology and associated locomotion deficit, which results in their improved survival over time. Similarly, AF-6 overexpression also ameliorates the pathological phenotypes in flies expressing the Leucine Rich Repeat Kinase 2 (LRRK2 G2019S mutant, a mutation that is associated with dominantly-inherited PD cases in humans. Conversely, when endogenous AF-6 expression is silenced, it aggravates the disease phenotypes of LRRK2 mutant flies. Aside from these genetic models, we also found that AF-6 overexpression is protective against the loss of dopaminergic neurons in flies treated with rotenone, a mitochondrial complex I inhibitor commonly used to generate animal models of PD. Taken together, our results demonstrate that AF-6 protects against dopaminergic dysfunction and mitochondrial abnormalities in multiple Drosophila models of PD, and suggest the therapeutic value of AF-6-related pathways in mitigating PD pathogenesis.

  13. Mildew resistant and less lodging wheat mutants induced in Iran

    International Nuclear Information System (INIS)

    Naghedi-Ahmadi, I.

    1989-01-01

    ''Tabassi'' is a lodging and mildew susceptible cultivar. To induce mutations, seeds were gamma irradiated (50 to 150 Gy) in 1982 and selection for lodging resistance was carried out in M 2 . During field experiments with the mutant lines in 1985/86 there has been a heavy mildew epidemic under which mutant 63-5-I (derived from 50 Gy treatment) exhibited considerable resistance and as a consequence, higher yield. The control was 100% infected, the mutant only 40%. The mutant yielded 31% more grain, 7.5% less straw and 4.5% more protein than the control. Lodging of 63-5-I was only 60% in an experiment under rainfed conditions in the same season, resulting in a relative yield increase of about 11%. In 1986/87 there was no mildew epidemic and the mutant yielded the same as ''Tabassi''

  14. Breeding value of wheat mutants induced by gamma irradiation

    International Nuclear Information System (INIS)

    Szilagyi, Gy.

    1979-01-01

    The combined use of the irradiation techniques available in Hungary and a number of other breeding methods has resulted in the production at Martonvasar of a new wheat variety, Martonvasari 8. It has valuable agronomic characteristics for commercial production and it will no doubt be of good service in the coming years in the development of Hungarian agricultural production. (author)

  15. Efficient Generation of Orthologous Point Mutations in Pigs via CRISPR-assisted ssODN-mediated Homology-directed Repair

    Directory of Open Access Journals (Sweden)

    Kankan Wang

    2016-01-01

    Full Text Available Precise genome editing in livestock is of great value for the fundamental investigation of disease modeling. However, genetically modified pigs carrying subtle point mutations were still seldom reported despite the rapid development of programmable endonucleases. Here, we attempt to investigate single-stranded oligonucleotides (ssODN mediated knockin by introducing two orthologous pathogenic mutations, p.E693G for Alzheimer's disease and p.G2019S for Parkinson's disease, into porcine APP and LRRK2 loci, respectively. Desirable homology-directed repair (HDR efficiency was achieved in porcine fetal fibroblasts (PFFs by optimizing the dosage and length of ssODN templates. Interestingly, incomplete HDR alleles harboring partial point mutations were observed in single-cell colonies, which indicate the complex mechanism of ssODN-mediated HDR. The effect of mutation-to-cut distance on incorporation rate was further analyzed by deep sequencing. We demonstrated that a mutation-to-cut distance of 11 bp resulted in a remarkable difference in HDR efficiency between two point mutations. Finally, we successfully obtained one cloned piglet harboring the orthologous p.C313Y mutation at the MSTN locus via somatic cell nuclear transfer (SCNT. Our proof-of-concept study demonstrated efficient ssODN-mediated incorporation of pathogenic point mutations in porcine somatic cells, thus facilitating further development of disease modeling and genetic breeding in pigs.

  16. Improving Dual-Task Walking Paradigms to Detect Prodromal Parkinson’s and Alzheimer’s Diseases

    Directory of Open Access Journals (Sweden)

    Maroua Belghali

    2017-05-01

    Full Text Available Gait control is a complex movement, relying on spinal, subcortical, and cortical structures. The presence of deficits in one or more of these structures will result in changes in gait automaticity and control, as is the case in several neurodegenerative diseases, such as Alzheimer’s disease (AD and Parkinson’s disease (PD. By reviewing recent findings in this field of research, current studies have shown that gait performance assessment under dual-task conditions could contribute to predict both of these diseases. Such suggestions are relevant mainly for people at putatively high risk of developing AD (i.e., older adults with mild cognitive impairment subtypes or PD (i.e., older adults with either Mild Parkinsonian signs or LRRK2 G2019S mutation. Despite the major importance of these results, the type of cognitive task that should be used as a concurrent secondary task has to be selected among the plurality of tasks proposed in the literature. Furthermore, the key aspects of gait control that represent sensitive and specific “gait signatures” for prodromal AD or PD need to be determined. In the present perspective article, we suggest the use of a Stroop interference task requiring inhibitory attentional control and a set-shifting task requiring reactive flexibility as being particularly relevant secondary tasks for challenging gait in prodromal AD and PD, respectively. Investigating how inhibition and cognitive flexibility interfere with gait control is a promising avenue for future research aimed at enhancing early detection of AD and PD, respectively.

  17. Yellow hilum color mutants induced by X-ray irradiation in the soybean variety Fukuyutaka

    International Nuclear Information System (INIS)

    Takagi, Y.; Nakamura, D.; Yokoo, H.; Kishikawa, H.

    1990-01-01

    A total of 33 000 seeds with a light brown hilum were X-irradiated with 20 and 24 kr in 1982 and 1983, respectively. Seven yellow hilum mutants were selected from the M 2 (3 in 1983 and 4 in 1984) and compared for yield components and 2 quality traits with the original variety Fukuyutaka. Line 85-1 was selected from the M 6 in 1987. It was similar to Fukuyutaka for all characters and showed better tofu quality with regard to appearance and lustre. It was recommended for release under the name Murayutaka in 1988

  18. Tolerance of some Potato Mutants Induced with Gamma Irradiation to Drought in Vitro

    International Nuclear Information System (INIS)

    Al-Safadi, B.; Al-Ayyoubi, Z.

    2007-01-01

    An in vitro selection program was conducted in order to improve potato (Solanum tuberosum,L.) tolerance to drought. Potato mutant plants were obtained through a previously conducted mutation breeding program on three potato cultivars (Draga, Spunta, and Diamant) aimed to improve potato tolerance to salinity and resistance to late blight disease. In order to apply selection pressure, growth media (MS based) were prepared with the addition of 1%, 2%, 3% concentrations of Poly Ethylene Glycol (PEG). As a result, three mutants were selected that were tolerant to water stress (i.e. drought tolerant), two of them were derived from the cultivar Draga and one came from Spunta. Physiological growth parameters (plant length, leaf number, branch number, roots number, leaf area, stomata number, and chlorophyll concentration content) were determined on the growing plantlets. The selected mutants were distinguished based on some characteristics which being associated with in their tolerance to drought. Such as an increases in leaf number, root number, and a decrease in stomata number. However a reduction in chlorophyll content was observed as compared with the control. This is considered a negative parameter which may result in a decrease in number and size of tubers. Thus it is important to continue selection for higher chlorophyll content. Also, these mutant lines will need further selection in the field for plants with larger tubers before they can be considered as certified lines.

  19. Genetic analysis and molecular detection of the corn endosperm mutants induced by space flight

    International Nuclear Information System (INIS)

    Zhang Caibo; Zhou Yuanyuan; Wang Hanyu; Wang Hongwei; Wang Shengqing; Rong Tingzhao; Cao Moju

    2013-01-01

    In this study, two maize inbred lines 08-641 and 18-599 were carried into cosmic space by recoverable satellite 'Shijian 8', grain shrunken transparently and opaquely mutants were selected as experimental materials and their soluble sugar content in kernel were measured by annthrone colorimetry. The content of soluble sugar in mutant st1 kernels began to rise in 10 days after pollination, to reach the peak in 25 days and significantly higher than the contrast 08-641, while in mutant sol kernels it began to rise in 10 days after pollination, to reach the peak in 20 days and significantly higher than the contrast 18-599. The results of genetic analysis and allelism test showed that the trait in both mutants was all controlled by a single recessive gene, the mutant st1 was allelic to the su1 and the mutant sol was allelic to the sh2. DNA sequence alignment found 2 single-base mutations in 2 and 13 exon of su1 gene in the mutant st1 and 3 single-base mutations in 2, 5 and 16 exon of sh2 gene in mutant so1 leading to the change in amino acid sequences. So it is inferred that starch biosynthesis in the mutants may be blocked by these mutations, which lead to the increase of soluble sugar content in kernel. (authors)

  20. Evaluation of Yield and Chemical Characteristics of some Peanut Mutants Induced by Gamma Irradiation

    International Nuclear Information System (INIS)

    Abd El-daem, G.A.; Anwar, M.M.

    2013-01-01

    This study was conducted to evaluate some promising mutants in peanut for yielding ability over three generation (M5, M6 and M7) and to evaluate yield attributes as will as chemical characteristics of these mutants in M7 generation induced by 100 Gy gamma radiation. The obtained results showed that the increase of yield / plot over three generation as a percentage of control was 5% for mutant 7, 10.2 % for mutant 10; 22% for mutant 9 and 22.9% for mutant 8. This increase in yield may be due to increase of one or more of yield attributes for most mutant lines. The significant increase for. No .of pods and seeds/ plant, weight of pods and seeds/ plant and 100- seed weight in M7 as compared to the control. For saturated fatty acid composition, results revealed that total saturated fatty acids ranged from 17.79% for mutant 8 to 21.75 for mutant 2 compared to 24.21% for control. Reduction of total saturated fatty acid was noticed for different mutants compared to that of the original variety. However, for total unsaturated fatty acids, results indicated that total unsaturated fatty acid composition ranged from 77.95% for mutant 9 to 82.09% for mutant 8 compared to 75.49% for control. Higher total unsaturated fatty acids for all mutant lines were obtained than that of the control, however, total saturated (TS)/ total unsaturated (TU) ratio was decreased for all mutants compared to control. The physical and chemical contents of Peanut oils showed that the refractive indices were ranged from 1.4620 to 1.4718 specific gravity were in range of 0.9146 to 0.9177. Acid value was range from 0.54 to 0.89% lodine value was ranged from 94.56 to 101.85. Saponification value was ranged from 185.2 to 190.7 and unsaponifiable matter was ranged from 0.98 to 1.33. The peroxide values ranged from 1.15 to 2.33 meq/kg oil. Also, fortified yoghurt made with replaced mutant peanut oil by 50% as milk fat substitute. Data showed that chemical composition and organolyptic properties had the nearly scores. The produced yoghurt of low milk fat by replacing peanut by 50% of milk fat avoid health problems associated with milk fat such as diabetes and heart disease. It can be concluded that peanut oil can be used as milk fat substitute at 50% according to its nutritional point of view where it contained a high percent of total unsaturated fatty acids 82% compared with milk fat 50%.

  1. Valuable fenugreek (Trigonella foenum-graecum L.) mutants induced by gamma rays and alkylating agents

    Energy Technology Data Exchange (ETDEWEB)

    Floria, F [Biotechnology and Molecular Genetics Group, ' Stejarul' Research Centre for Biological Sciences, Piatra Neamt (Romania); Ichim, M C [Biotechnology and Molecular Genetics Group, ' Stejarul' Research Centre for Biological Sciences, Piatra Neamt (Romania)

    2006-12-15

    Seeds of fenugreek (Trigonella foenum-graecum L.) were treated with either gamma rays (25-15 kR) or ethyl methanesulphonate (EMS 0.1-0.5%, 2h) or ethylene imine (EI, 0.01- 0.1%, 2h). After repeated selection for better morphological performance from M{sub 2} to M{sub 4} generation, 11 mutant lines were identified with better yield potential and higher diosgenin content than their parent. (author)

  2. Cadmium Bio sorption by Some Bacterial Isolates and Their Mutants Induced by gamma Radiation

    International Nuclear Information System (INIS)

    Tawfik, Z.S.; Elsonbaty, S.M.; Abdalla, N.M.

    1999-01-01

    Cadmium bio sorption by bacterial cells is recognized as a potential alternative to existing recovery technologies. Bacterial strains under investigation were isolated from air surrounding gamma industrial facility Co 60 source of the NCRRT, Cairo. The effect of different concentrations of cadmium on the growth was determined for the spore forming bacteria B.coagulans, B.megaterium, B.pumilus, B.pantothenticus, and also for Staphylo coccus aureus, the reference standard strain used in these study for comparison was B.subtilis MERK 10646. The results indicated that, B.pantothenticus was the most tolerant isolate, and it can resist up to 400 ppm. Cadmium capacity for B.subtilis parent strain was increased through the influence of different doses of gamma radiation, selected mutant of B.subtilis show enhanced level of cadmium accumulation. The effect of environmental parameters as ph, temperature and also the effect of biomass factor on cadmium uptake by B.pantothenticus and B.subtilis (m) was traced

  3. 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

  4. Evaluation of Some Chemical Characteristics of barley Mutants induced by Gamma Irradiation

    International Nuclear Information System (INIS)

    Abdeldaiem, M.H.; Ali, H.G.M.

    2011-01-01

    This study aims to evaluate the antioxidant activity of acetonic extract from some barley mutations (P1, P2 and P3 varieties) induced by gamma irradiation as compared with local barley variety (Hordeum vulgare L.) as control. Barley samples were obtained from Plant Breeding Unit, Plant Research Department, Nuclear Research Centre, Atomic Energy Authority, Egypt. The measurements of the antioxidant activity using a radical scavenging capacity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ?-carotene bleaching assay were assessed in the barley acetonic extract. Furthermore, amino acids composition of barley mutant samples was determined. The results indicated that the acetonic extract of barley varieties under investigation possess marked antioxidant and anti radical capacities. The data showed that the acetonic extract of barley mutant P1 possessed the higher antioxidant activity as compared with the antioxidant activities of acetonic extract from control and other barley mutant samples. Meanwhile, the flour of barley mutations under investigation contained trace elements of iron, copper and manganese. GC and mass analyses were used to identify the active compound of extract of control and mutant barley samples. The results illustrated that the main components of the control sample of barely extract was pentane, 3 methyl (47.73%) while gamma irradiation caused noticeable change in the relative percentage of some components of acetonic extract from barley mutant samples. Moreover, the results presented that changes were disappeared, and some compounds of the acetonic extract from mutant barley samples were appeared. Furthermore, the results exhibited that barley flour supplemented with wheat flour at 30% level produced acceptable cookies. Accordingly, the phenolic constituents of barley acetonic extract induced by gamma irradiation, especially samples of P1 mutant, may have a future role as ingredients in the development of functional foods.

  5. Enhanced lipid productivity and photosynthesis efficiency in a Desmodesmus sp. mutant induced by heavy carbon ions.

    Science.gov (United States)

    Hu, Guangrong; Fan, Yong; Zhang, Lei; Yuan, Cheng; Wang, Jufang; Li, Wenjian; Hu, Qiang; Li, Fuli

    2013-01-01

    The unicellular green microalga Desmodesmus sp. S1 can produce more than 50% total lipid of cell dry weight under high light and nitrogen-limitation conditions. After irradiation by heavy (12)C(6+) ion beam of 10, 30, 60, 90 or 120 Gy, followed by screening of resulting mutants on 24-well microplates, more than 500 mutants were obtained. One of those, named D90G-19, exhibited lipid productivity of 0.298 g L(-1)⋅d(-1), 20.6% higher than wild type, likely owing to an improved maximum quantum efficiency (Fv/Fm) of photosynthesis under stress. This work demonstrated that heavy-ion irradiation combined with high-throughput screening is an effective means for trait improvement. The resulting mutant D90G-19 may be used for enhanced lipid production.

  6. The stem and leaf super green mutant induced by 60Co γ-rays irradiation

    International Nuclear Information System (INIS)

    Wei Yubo; Liang Naiting; Buhaliqiem; Zhang Yinbao

    2003-01-01

    Super green gene mutant was developed from population of M 2 generation after the dry seeds of rice Huazhiwu from Japan with good quality and resistance to cold had been irradiated with 50 Gy 60 Co γ-ray. The leaf, sheath, panicle axis and petiole of mutant was characterized by deeply green, and did not turn yellow after maturing date. The chlorophyll content in straw is 2.2 times higher than that in common straw. The results of raising livestock showed that horse, donkey and sheep had evident selectivity to the green straw

  7. In vitro selection of mutants: Inducible gene regulation for salt tolerance

    International Nuclear Information System (INIS)

    Winicov, I.; Bastola, D.R.; Deutch, C.E.; Pethe, V.V.; Petrusa, L.

    2001-01-01

    Regulation of differentially expressed genes in plants may be involved in inducing tolerance to stress. Isogenic salt-sensitive and salt-tolerant alfalfa lines were investigated for molecular differences in their response to salt. The genes, which are differentially induced by salt in the salt-tolerant alfalfa cells and are also regulated by salt at the whole plant level, were cloned. Both transcriptional and post- transcriptional mechanisms influenced salt-induced product accumulation in the salt-tolerant alfalfa. The salt-tolerant plants doubled proline concentration rapidly in roots, while salt-sensitive plants showed a delayed response. To understand the regulatory system in the salt-tolerant alfalfa, two genes that are expressed in roots were studied. Alfin1 encodes a zinc-finger type putative DNA transcription factor conserved in alfalfa, rice and Arabidopsis, and MsPRP2 encodes a protein that serves as a cell wall- membrane linker in roots. Recombinant Alfin1 protein was selected, amplified, cloned and its consensus sequence was identified. The recombinant Alfin1 also bound specifically to fragments of the MsPRP2 promoter in vitro, containing the Alfin1 binding consensus sequence. The results show unambiguously binding specificity of Alfin1 DNA, supporting its role in gene regulation. Alfin1 function was tested in transformed alfalfa in vivo by over-expressing Alfin1 from 35S CaMV promoter. The transgenic plants appeared normal. However, plants harboring the anti-sense construct did not grow well in soil, indicating that Alfin1 expression was essential. Alfin1 over-expression in transgenic alfalfa led to enhanced levels of MsPRP2 transcript accumulation, demonstrating that Alfin1 functioned in vivo in gene regulation. Since MsPRP2 gene is also induced by salt, it is likely that Alfin1 is an important transcription factor for gene regulation in salt-tolerant alfalfa, and an excellent target for manipulation to improve salt tolerance. (author)

  8. A new Arabidopsis mutant induced by ion beams affects flavonoid synthesis with spotted pigmentation in testa

    International Nuclear Information System (INIS)

    Tanaka, A.; Tano, S.; Chantes, T.; Yokota, Y.; Shikazono, N.; Watanabe, H.

    1997-01-01

    A new stable mutant of Arabidopsis thaliana with a spotted pigment in the seed coat, named anthocyanin spotted testa (ast), was induced by carbon ion irradiation. The spotted pigmentation of ast mutant was observed in immature seeds from 1-2 days after flowering (DAF), at the integument of the ovule, and spread as the seed coat formed. Anthocyanin accumulation was about 6 times higher in ast mutant than in the wild-type at 6 DAF of the immature seeds, but was almost the same in mature dry seeds. A higher anthocyanin accumulation was not observed in the seedlings, leaves or floral buds of ast mutant compared with the wild-type, which suggests that a high accumulation of anthocyanins is specific to the seed coat of the immature ast seeds. Reciprocal crosses between ast mutant and the wild-type indicated that ast is a single recessive gene mutation and segregates as a delayed inheritance. The results of crossing with tt7 and ttg mutants also confirmed that the AST gene is probably a regulatory locus that controls flavonoid biosynthesis. A mapping analysis revealed that the gene is located on chromosome I and is closely linked to the SSLP DNA marker nga280 with a distance of 3.2 cM. AST has been registered as a new mutant of Arabidopsis

  9. The breeding of Japonica Yanjing 10 rice mutant induced by space mutation

    International Nuclear Information System (INIS)

    Wang Jianhua; Chen Xiulan; Zhang Rong; Wang Jinrong; Liu Jian; Jiao Juan; He Zhentian; Wang Lin

    2011-01-01

    The dry seed of mid-maturing Japonica rice Yanjing 10 was used for space mutation breeding which was carried by a satellite for 15 days in 2006. Through three generations of breeding, a group of mutants were obtained. In the article, we reported in detail the breeding procedures, proposed the breeding technical method for space mutation for rice improvement. Planting multiple seedlings per hill to prohibit tillering at SP 1 generation, and bulked selection in combination with directional selection at the SP 2 ∼ SP 3 generation were the two key points of the breeding methods. (authors)

  10. Analysis on the DNA Fingerprinting of Aspergillus Oryzae Mutant Induced by High Hydrostatic Pressure

    International Nuclear Information System (INIS)

    Wang Hua; Zhang Jian; Wang Kai; Liu Bing-Bing; Zou Bo; Zou Guang-Tian; Yang Fan; Shen Si-Le

    2011-01-01

    The mutant strains of aspergillus oryzae (HP300a) are screened under 300 MPa for 20 min. Compared with the control strains, the screened mutant strains have unique properties such as genetic stability, rapid growth, lots of spores, and high protease activity. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) are used to analyze the DNA fingerprinting of HP300a and the control strains. There are 67.9% and 51.3% polymorphic bands obtained by these two markers, respectively, indicating significant genetic variations between HP300a and the control strains. In addition, comparison of HP300a and the control strains, the genetic distances of random sequence and simple sequence repeat of DNA are 0.51 and 0.34, respectively. (general)

  11. Analysis on the DNA Fingerprinting of Aspergillus Oryzae Mutant Induced by High Hydrostatic Pressure

    Science.gov (United States)

    Wang, Hua; Zhang, Jian; Yang, Fan; Wang, Kai; Shen, Si-Le; Liu, Bing-Bing; Zou, Bo; Zou, Guang-Tian

    2011-01-01

    The mutant strains of aspergillus oryzae (HP300a) are screened under 300 MPa for 20 min. Compared with the control strains, the screened mutant strains have unique properties such as genetic stability, rapid growth, lots of spores, and high protease activity. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR) are used to analyze the DNA fingerprinting of HP300a and the control strains. There are 67.9% and 51.3% polymorphic bands obtained by these two markers, respectively, indicating significant genetic variations between HP300a and the control strains. In addition, comparison of HP300a and the control strains, the genetic distances of random sequence and simple sequence repeat of DNA are 0.51 and 0.34, respectively.

  12. Tolerance of some potato mutants induced with gamma irradiation to drought in vitro

    International Nuclear Information System (INIS)

    Al-Safadi, B.; Ayyoubi, Z.

    2006-04-01

    An in vitro selection program was conducted in order to improve potato (Solanum tuberosum) tolerance to drought. Potato mutant plants were obtained through a previously conducted mutation breeding program on three potato cultivars (Draga, Spunta, and Diamant) aimed at improving potato tolerance to salinity and resistance to late blight disease. In order to apply selection pressure, growth media (MS based) were prepared with the addition of 1%, 2%, 3% concentrations of Poly Ethylene Glycol (PEG). As a result, three mutants were selected that were tolerant to water stress (i.e. drought tolerant) two of which came from the cultivar Draga and one from Spunta. Physiological growth parameters (plant length, leaf number, branch number, roots number, leaf area, stomata number, and chlorophyll concentration content) were taken on the growing plantlets. The selected mutants were distinguished with some characteristics which can help in their tolerance to drought. Some of these characteristics were an increase in leaf number, root number, and a decrease in stomata number. However a reduction in chlorophyll content was observed as compared with the control. These mutant lines will need further selection in the field for plants with larger tubers before they can be considered as certified lines. (author)

  13. Production and partial characterization of alkaline protease from bacillus subtilis mutant induced by gamma radiation

    International Nuclear Information System (INIS)

    Ibrahim, H.M.M.; Bashandy, A.S.

    2010-01-01

    Fourteen bacterial isolates belonging to B.subtilis were locally isolated from soil and screened for alkaline protease production. Only one strain, the highly potent one, was selected as alkaline protease producer and subjected to further studies to optimize its production. Alkaline protease production was maximum at 35 degree C after 72 h of incubation and at ph 10.0. molasses as a carbon source and combination of peptone and yeast extract as a nitrogen source enhanced greatly alkaline protease production. The mutant strain induced by gamma radiation showed higher alkaline protease production by 1.97 fold as compared with the parent strain. The alkaline protease enzyme was active at 40 degree C and ph 10. It was compatible with many commercial detergents and showed high stability (84 %) of its original activity with Ariel detergent. Moreover, alkaline protease enhanced the washing performance, and retained 95 % of its activity in the formulated dry powder.

  14. Mutation Analysis of Consanguineous Moroccan Patients with Parkinson’s Disease Combining Microarray and Gene Panel

    Directory of Open Access Journals (Sweden)

    Ahmed Bouhouche

    2017-10-01

    Full Text Available During the last two decades, 15 different genes have been reported to be responsible for the monogenic form of Parkinson’s disease (PD, representing a worldwide frequency of 5–10%. Among them, 10 genes have been associated with autosomal recessive PD, with PRKN and PINK1 being the most frequent. In a cohort of 145 unrelated Moroccan PD patients enrolled since 2013, 19 patients were born from a consanguineous marriage, of which 15 were isolated cases and 4 familial. One patient was homozygous for the common LRRK2 G2019S mutation and the 18 others who did not carry this mutation were screened for exon rearrangements in the PRKN gene using Affymetrix Cytoscan HD microarray. Two patients were determined homozygous for PRKN exon-deletions, while another patient presented with compound heterozygous inheritance (3/18, 17%. Two other patients showed a region of homozygosity covering the 1p36.12 locus and were sequenced for the candidate PINK1 gene, which revealed two homozygous point mutations: the known Q456X mutation in exon 7 and a novel L539F variation in exon 8. The 13 remaining patients were subjected to next-generation sequencing (NGS that targeted a panel of 22 PD-causing genes and overlapping phenotypes. NGS data showed that two unrelated consanguineous patients with juvenile-onset PD (12 and 13 years carried the same homozygous stop mutation W258X in the ATP13A2 gene, possibly resulting from a founder effect; and one patient with late onset (76 years carried a novel heterozygous frameshift mutation in SYNJ1. Clinical analysis showed that patients with the ATP13A2 mutation developed juvenile-onset PD with a severe phenotype, whereas patients having either PRKN or PINK1 mutations displayed early-onset PD with a relatively mild phenotype. By identifying pathogenic mutations in 45% (8/18 of our consanguineous Moroccan PD series, we demonstrate that the combination of chromosomal microarray analysis and NGS is a powerful approach to

  15. ARHGEF7 (Beta-PIX acts as guanine nucleotide exchange factor for leucine-rich repeat kinase 2.

    Directory of Open Access Journals (Sweden)

    Karina Haebig

    Full Text Available BACKGROUND: Mutations within the leucine-rich repeat kinase 2 (LRRK2 gene are a common cause of familial and sporadic Parkinson's disease. The multidomain protein LRRK2 exhibits overall low GTPase and kinase activity in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that the rho guanine nucleotide exchange factor ARHGEF7 and the small GTPase CDC42 are interacting with LRRK2 in vitro and in vivo. GTPase activity of full-length LRRK2 increases in the presence of recombinant ARHGEF7. Interestingly, LRRK2 phosphorylates ARHGEF7 in vitro at previously unknown phosphorylation sites. We provide evidence that ARHGEF7 might act as a guanine nucleotide exchange factor for LRRK2 and that R1441C mutant LRRK2 with reduced GTP hydrolysis activity also shows reduced binding to ARHGEF7. CONCLUSIONS/SIGNIFICANCE: Downstream effects of phosphorylation of ARHGEF7 through LRRK2 could be (i a feedback control mechanism for LRRK2 activity as well as (ii an impact of LRRK2 on actin cytoskeleton regulation. A newly identified familial mutation N1437S, localized within the GTPase domain of LRRK2, further underlines the importance of the GTPase domain of LRRK2 in Parkinson's disease pathogenesis.

  16. Nature of mutants induced by ionizing radiation in cultured hamster cells. II. Antigenic response and reverse mutation of HPRT-deficient mutants induced by. gamma. -rays or ethyl methanesulphonate

    Energy Technology Data Exchange (ETDEWEB)

    Brown, R; Stretch, A; Thacker, J

    1986-04-01

    A large series of independent mutants deficient in HPRT enzyme activity, isolated from V79-4 hamster cells, were assessed for properties which reflect the nature of the genetic changes induced. A total of 88 mutants were screened, 43 isolated from ..gamma..-ray-treated cultures and 45 induced by ethyl methanesulphonate (EMS). Firstly, each mutant was assayed for the presence of protein with the antigenic response of HPRT. In a competitive inhibition assay, 31% of EMS-induced mutants were CRM-positive compared to 7% of the ..gamma..-ray series. Secondly, each mutant was tested for ability to revert to HPRT proficiency. All except 2 of the EMS-induced mutants reverted with ethyl nitrosourea ENU, and many reverted spontaneously, under the given conditions. However reversion was not detected in about 80% of ..gamma..-ray-induced mutants, suggesting that the types of forward mutation caused by ionizing radiation differ qualitatively from those caused by EMS. (Auth.). 30 refs.; 6 figs.; 2 tabs.

  17. Isolation and characterization of useful mutants induced by gamma irradiation in 'Kranti' indian mustard (Brassica juncea)

    International Nuclear Information System (INIS)

    Rai, B.; Kumar, H.

    1998-01-01

    Genetic variability was induced, in the 'Kranti' Indian mustard [Brassica/juncea (L.) Czemj. & Cosson]. utilizing 30,40 and 50 kr doses of gamma-ray Irradiation. 'In M 3 generation, compared with the econtrol, substantial decrease was observed in the mean value of various quantitative characters in the mutagen-treated populations. More variation was induced at 30 kr compared with that at 40 or 50 kr dose. Four mutants were identified ar 30 kr dose, which besides being early in maturity, gave better yield also-compared with the control. The better seed yield of the transmutated plants was due either to increase in seed weight or to increase in number of siliquae/plant

  18. PATHOLOGICAL AND MOLECULAR GENETIC STUDIES ON SOME SOYBEAN MUTANTS INDUCED BY GAMMA RAYS IN RELATION TO CHARCOAL ROT DISEASE

    International Nuclear Information System (INIS)

    ASHRY, N.A.; EL-DEMERDASH, H.M.; ABD EL-RAHMAN, S.S.

    2008-01-01

    The Egyptian soybean cultivar Giza-22 was used to induce resistant mutants for charcoal rot disease using gamma rays. Sixteen mutants and their parental cultivar were evaluated in M3 generation for their agronomic traits and for resistance to charcoal rot disease. Four mutants showed superiority in their agronomic traits as compared with their parental cultivar. Three mutants were significantly resistant to the disease than their parental cultivar (Giza-22). These three resistant mutants showed non-significant improvement in their agronomic traits as compared with Giza-22 cultivar. DNA extractions from the three resistant mutants and their parent were used to test the differences on the molecular level. Seven random amplified polymorphic DNA (RAPD) primers were used to detect RAPD markers related to charcoal rot resistance in soybean, and to differentiate these mutants. Six RAPD-primers showed molecular markers associated with resistance to charcoal rot in soybean, where five RAPD-primers could differentiate each of the three mutants from each other and from their parental cultivar

  19. 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)

  20. Quantification of Arabinose content and batter volume in elite black gram mutants induced by gamma rays and electron beam

    International Nuclear Information System (INIS)

    Vanniarajan, C.; Sri Subalakhshmi, V.K.I.; Se Shamyugtha; Rajeswaran, G.; Monica, R.; Veni, K.

    2017-01-01

    Black gram (Vignamungo (L) Hepper) is one among the mostly preferred sources of protein, especially in vegetarian diet. One of the most notable biochemical attributes of black gram is Arabinose. As it plays a vital role in the yeast metabolism, it has a direct correlation with the battering quality of Black gram. MDU 1 variety has the highest Arabinose content of 7.5 per cent and VBN(Bg)4yields higher. Both the varieties have indeterminate growth habit. Hence, these two varieties were chosen and treated with Gamma rays and Electron beam of 100–500 Gy and 200–600 Gy respectively. Twenty desirable mutants were selected in M6 generation based on determinate growth habit and high yield with short duration. These mutants were tested for the Arabinose content along with MDU 1 and VBN (Bg)4, using Bial (1902) method. The results of the selected M6 generation mutants revealed that only three mutants excelled in Arabinose content than MDU 1. Two mutants were found to retain the same Arabinose content as that of MDU 1.Three mutants containing higher Arabinose content, one with lower Arabinose content and the parents were further analysed for batter quantity. The unveiled fact is that the increase in Arabinose content increases the batter volume to a certain extent (Arabinose = 10 %). Eventually, with a due course of increase in Arabinose content, the batter volume decreases. The results were tested for significance and were found to be significant. In this present investigation, it was found that the optimum Arabinose content is 10 per cent, which showed an increased batter volume. It has to be further confirmed by using advanced biochemical and molecular methods. (author)

  1. Release to farmers of ''Carioca Arbustivo Precoce 1070'' (CAP-1070), a bushy bean mutant induced by gamma rays in Brazil

    International Nuclear Information System (INIS)

    Tulmann Neto, A.; Albertini, J.

    1989-01-01

    Full text: Seeds of the indeterminate growth type bean cultivar ''Carioca'' have been treated with 32 krad gamma rays. In M 2 , a mutant showing bushy growth type has been selected. The mutant also shows earlier maturity (5-14 days) and therefore was called ''Carioca Arbustivo Precoce 1070'' (CAP-1070). The determinate (bushy) growth habit is due to one recessive gene and earliness is associated with this habit. CAP-1070 maintained the same response to diseases as the original cultivar. In trials carried out in several states of Brazil, yield was lower, similar or greater than ''Carioca'' depending on conditions. The short flowering period of CAP-1070, resulting from the bushy growth habit may reduce grain yields but under favourable circumstances, CAP-1070 may yield more than other varieties. CAP-1070 raised great interest among farmers visiting experimental fields of F.T. Pesquisa e Sementes, a private plant breeding firm at Ponta Grossa, Parana. Therefore, the firm decided to multiply the seeds and distribute them to farmers, who have now been cultivating CAP-1070 since 1986 between coffee rows. The CAP-1070 is the first induced bean mutant cultivated by farmers in Brazil. However, like the original cultivar ''Carioca'', CAP-1070 became susceptible to diseases. Therefore, we crossed the mutant and have obtained promising lines with bushy habits, disease resistance and higher yield. CAP-1070 is also used in cross-breeding programmes of Government research institutes in Brazil. Research was supported by IAEA under Research Contract No. 2195/SD, EMBRAPA, CNEN and CNPQ. (author)

  2. Comparative proteomic analysis of a high-tillering dwarf mutant induced by spaceflight at different tillering stages

    International Nuclear Information System (INIS)

    Wang Wei; Wei Lijun; Wang Junmin; Xu Jianlong; Sun Yeqing

    2011-01-01

    To investigate changes of proteins during rice tiller development, a comparative proteomic analysis between a high-tillering dwarf mutant R955 induced by spaceflight and its ground control was performed at three developmental stages during the vegetative growth, i. e. at days 14 (no tiller appearance), 21 (initial tillering) and 55 (maximum tillering) after sowing. Analysis of the protein spots on two-dimensional fluorescence difference gel electrophoresis 2-D DIGE images revealed 97 proteins that were differentially expressed at the three stages. Among them, 59 unique proteins were successfully identified by mass spectrometry. Through functional and quantitative analysis of proteomic data, it was found that biological processes including energy pathway, photosynthesis, protein metabolism, nitrogen assimilation, amino acid metabolism and stimulus response were mainly involved in tiller development in mutant plant. K-means clustering revealed that proteins regulated at different stages tended to be involved in different biological processes. Two-way analysis of variance (Two-way ANOVA) showed that S-like was directly correlated RNase with the high-tillering ability. (authors)

  3. Nullbasic, a potent anti-HIV tat mutant, induces CRM1-dependent disruption of HIV rev trafficking.

    Directory of Open Access Journals (Sweden)

    Min-Hsuan Lin

    Full Text Available Nullbasic, a mutant of the HIV-1 Tat protein, has anti-HIV-1 activity through mechanisms that include inhibition of Rev function and redistribution of the HIV-1 Rev protein from the nucleolus to the nucleoplasm and cytoplasm. Here we investigate the mechanism of this effect for the first time, establishing that redistribution of Rev by Nullbasic is not due to direct interaction between the two proteins. Rather, Nullbasic affects subcellular localization of cellular proteins that regulate Rev trafficking. In particular, Nullbasic induced redistribution of exportin 1 (CRM1, nucleophosmin (B23 and nucleolin (C23 from the nucleolus to the nucleus when Rev was coexpressed, but never in its absence. Inhibition of the Rev:CRM1 interaction by leptomycin B or a non-interacting RevM10 mutant completely blocked redistribution of Rev by Nullbasic. Finally, Nullbasic did not inhibit importin β- or transportin 1-mediated nuclear import, suggesting that cytoplasmic accumulation of Rev was due to increased export by CRM1. Overall, our data support the conclusion that CRM1-dependent subcellular redistribution of Rev from the nucleolus by Nullbasic is not through general perturbation of either nuclear import or export. Rather, Nullbasic appears to interact with and disrupt specific components of a Rev trafficking complex required for its nucleocytoplasmic shuttling and, in particular, its nucleolar accumulation.

  4. Genetic variation for germination and physiological traits in sunflower mutants induced by gamma rays [Helianthus annuus L.

    International Nuclear Information System (INIS)

    Alejo-Jaimes, A.; Jardinaud, M.F.; Maury, P.; Alibert, G.; Gentzbittel, L.; Sarrafi, A.; Grieu, P.; Petiprez, M.

    2004-01-01

    Seeds of sunflower line AS-613 were irradiated with gamma rays and 1,559 M4 progenies were studied for their germination characteristics and the following traits were studied: thousand seed weight, seed size, time before emergence, percentage of emerged seedlings, hypocotyl length and diameter, number of cotyledons and cotyledons pigmentation intensity. A high genetic variability was observed for all the studied traits. Through M4 progenies, 9 mutants presenting the most differences with the original genotype (AS-613) were planted in a randomized blocks design with 8 replications in a controlled greenhouse and some morphological and physiological traits were studied, which are: plant height, number of leaves, total leaf area, net photosynthesis, transpiration, stomatal conductance, water use efficiency and net carbon assimilation. When harvesting, flower head diameter, head weight, stem weight, leaves weight, total number of seeds per plant and thousand seed weight were measured. The differences between mutants and also non irradiated genotype (AS-613) were significant for most of studied traits suggesting that several developmental processes have been modified [it

  5. A direct interaction between leucine-rich repeat kinase 2 and specific β-tubulin isoforms regulates tubulin acetylation.

    Science.gov (United States)

    Law, Bernard M H; Spain, Victoria A; Leinster, Veronica H L; Chia, Ruth; Beilina, Alexandra; Cho, Hyun J; Taymans, Jean-Marc; Urban, Mary K; Sancho, Rosa M; Blanca Ramírez, Marian; Biskup, Saskia; Baekelandt, Veerle; Cai, Huaibin; Cookson, Mark R; Berwick, Daniel C; Harvey, Kirsten

    2014-01-10

    Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease.

  6. Up-regulation of granzyme B and perforin by staphylococcal enterotoxin C2 mutant induces enhanced cytotoxicity in Hepa1–6 cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Guojun [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); University of Chinese Academy of Sciences, Beijing (China); Xu, Mingkai, E-mail: mkxu@iae.ac.cn [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); Zhang, Huiwen [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); Song, Yubo [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China); University of Chinese Academy of Sciences, Beijing (China); Wang, Jian; Zhang, Chenggang [Institute of Applied Ecology, Chinese Academy of Sciences, No.72 Wenhua Road Shenhe Dis., Shenyang, Liaoning (China)

    2016-12-15

    Staphylococcal enterotoxin C2 (SEC2), a member of bacterial superantigen, is one of the most potent known activators of T lymphocytes. With this property, SEC2 has already been used in clinic as a tumor immunotherapy agent in China. To increase the antitumor activity, a SEC2 mutant named ST-4 (GKVTG102-106WWH) with amino acid substitutions in T cell receptor (TCR)-binding domain was generated by site-directed mutagenesis, and the molecular mechanism of the enhanced antitumor activity was investigated. Results showed that ST-4 could activate much more Vβ 8.2 and 8.3 T cells and NK cells compared with SEC2, and exhibited significantly enhanced immunocyte stimulation and antitumor activity in vitro. The synthetic peptide sequencing the residues of mutant TCR-binding domain could competitively inhibit the immunocyte stimulation activity of ST-4. Most importantly, ST-4 up-regulated granzyme B and perforin at both mRNA and protein levels. We also found that expression of proapoptotic proteins cytochrome c, BAX and activation of caspase-3, 9 was up-regulated, and antiapoptotic protein Bcl-xL was down-regulated in the treatment with either ST-4 or SEC2. When granzyme B inhibitor or perforin inhibitor is presented, tumor cell viability was significantly rescued. Taken together, we demonstrate that increased ST-4-TCR recognition contributed to massive T cells and NK cells activation. These activated cells released up-regulated granzyme B and perforin, which induced the enhanced tumor cells apoptosis by mitochondrial apoptotic pathway, and ultimately led to enhanced tumor cell growth inhibition. ST-4 may be a promising candidate for antitumor clinic usage in future. - Highlights: • We obtained a SEC2 mutant ST-4 with enhanced superantigen and antitumor activity. • Increased ST-4-TCR recognition contributed to massive T cells and NK cells activation. • Up-regulated GzmB and PRF1 in T cell by ST-4 induced enhanced tumor cells apoptosis. • Enhanced tumor cell apoptosis induced by ST-4 via mitochondrial apoptotic pathway.

  7. Evaluation of some field bean mutants induced by using gamma rays and Ethyl methane sulphonate in M9 and M10 generations

    International Nuclear Information System (INIS)

    Atia, Z.M.A.

    2008-01-01

    Selection was practices within and between, M2,M3 and M4 field been populations derived from gamma irradiation treatments (30 and 60 Gy) and EMS treatments (0.15-0.3%) ten mutants were isolated and evaluated for yield and yield components and chemical contents of seeds in M 5 generation. The evaluation was done until M 8 generation. In this generation (M 8 ) we isolated seven mutants, in M 9 and M 1 0 generations, a comparison was done between these mutants and some local varieties; Sakha1, Sakha2, Masr1 and Giza3 in addition the mother variety Giza 2. The results indicated that:1-All faba bean mutants increased significantly Number of branches / plant in Comparison with the local commercial varieties. 2-Mutant lines No 3,4,5,6 and 7 increased number of pods per plant weight pods per plant number and weight seeds per plant and protein percentage of seeds in comparison with the local varieties in the two generations only . 3-Mutant No. 8 increased significantly No. and weight of pods, No. and weight of seeds / plant in M 9 generation . 4-Mutants No 3,4,5,6,7 and 8 increased significantly number of seeds/ pod and shelling percentage in comparison with local varieties Sakha1 and Sakha2 in the two generations on the contrast decreased significantly seed index (100 seeds weight) and shedding percentages of followers and pods

  8. Analysis of drought-tolerant sugar beet (Beta vulgaris L.) mutants induced with gamma radiation using SDS-PAGE and ISSR markers.

    Science.gov (United States)

    Sen, Ayse; Alikamanoglu, Sema

    2012-01-01

    Drought is one of the major environmental stresses which greatly affect the plant growth and productivity. In the present study, various doses (0-75Gy) of gamma rays were applied to investigate the effect of radiation on shoot tip explants. It was observed that the regeneration rates and plant fresh weights decreased significantly with an increase in radiation dose. The optimal irradiation doses for mutation induction were determined at 15 and 20Gy. Afterwards, the induction of somatic mutation in sugar beet (Beta vulgaris L.) was investigated by irradiation of shoot tips with 15 and 20Gy gamma rays. Irradiated shoot tips were sub-cultured and M(1)V(1)-M(1)V(3) generations were obtained. Mutants tolerant to drought stress were selected on MS medium, supplemented with 10 and 20gl(-1) PEG6000. Of the M(1)V(3) plantlets, drought-tolerant mutants were selected. Leaf soluble proteins obtained from the control and drought-tolerant mutants were analyzed by SDS-PAGE. A total of 22 protein bands were determined and 2 of them were observed to be drought-tolerant mutants except the control. Polymorphism was also detected among the control and drought-tolerant mutants by DNA fingerprinting using ISSR markers. A total of 106 PCR fragments were amplified with 19 ISSR primers and 91 of them were polymorphic. The dendrograms were separated into two main clusters. First cluster included M8 mutant plant, which was applied 20Gy gamma radiation and regenerated on selective culture media containing 10gl(-1) PEG6000 concentration, and the second cluster was further divided into five sub-clusters. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Analysis of drought-tolerant sugar beet (Beta vulgaris L.) mutants induced with gamma radiation using SDS-PAGE and ISSR markers

    International Nuclear Information System (INIS)

    Sen, Ayse; Alikamanoglu, Sema

    2012-01-01

    Drought is one of the major environmental stresses which greatly affect the plant growth and productivity. In the present study, various doses (0–75 Gy) of gamma rays were applied to investigate the effect of radiation on shoot tip explants. It was observed that the regeneration rates and plant fresh weights decreased significantly with an increase in radiation dose. The optimal irradiation doses for mutation induction were determined at 15 and 20 Gy. Afterwards, the induction of somatic mutation in sugar beet (Beta vulgaris L.) was investigated by irradiation of shoot tips with 15 and 20 Gy gamma rays. Irradiated shoot tips were sub-cultured and M 1 V 1 –M 1 V 3 generations were obtained. Mutants tolerant to drought stress were selected on MS medium, supplemented with 10 and 20 gl −1 PEG6000. Of the M 1 V 3 plantlets, drought-tolerant mutants were selected. Leaf soluble proteins obtained from the control and drought-tolerant mutants were analyzed by SDS-PAGE. A total of 22 protein bands were determined and 2 of them were observed to be drought-tolerant mutants except the control. Polymorphism was also detected among the control and drought-tolerant mutants by DNA fingerprinting using ISSR markers. A total of 106 PCR fragments were amplified with 19 ISSR primers and 91 of them were polymorphic. The dendrograms were separated into two main clusters. First cluster included M8 mutant plant, which was applied 20 Gy gamma radiation and regenerated on selective culture media containing 10 g l −1 PEG6000 concentration, and the second cluster was further divided into five sub-clusters.

  10. Analysis of drought-tolerant sugar beet (Beta vulgaris L.) mutants induced with gamma radiation using SDS-PAGE and ISSR markers

    Energy Technology Data Exchange (ETDEWEB)

    Sen, Ayse, E-mail: senayse@istanbul.edu.tr [Istanbul University, Faculty of Science, Department of Biology, 34459 Vezneciler, Istanbul (Turkey); Alikamanoglu, Sema [Istanbul University, Faculty of Science, Department of Biology, 34459 Vezneciler, Istanbul (Turkey)

    2012-10-15

    Drought is one of the major environmental stresses which greatly affect the plant growth and productivity. In the present study, various doses (0-75 Gy) of gamma rays were applied to investigate the effect of radiation on shoot tip explants. It was observed that the regeneration rates and plant fresh weights decreased significantly with an increase in radiation dose. The optimal irradiation doses for mutation induction were determined at 15 and 20 Gy. Afterwards, the induction of somatic mutation in sugar beet (Beta vulgaris L.) was investigated by irradiation of shoot tips with 15 and 20 Gy gamma rays. Irradiated shoot tips were sub-cultured and M{sub 1}V{sub 1}-M{sub 1}V{sub 3} generations were obtained. Mutants tolerant to drought stress were selected on MS medium, supplemented with 10 and 20 gl{sup -1} PEG6000. Of the M{sub 1}V{sub 3} plantlets, drought-tolerant mutants were selected. Leaf soluble proteins obtained from the control and drought-tolerant mutants were analyzed by SDS-PAGE. A total of 22 protein bands were determined and 2 of them were observed to be drought-tolerant mutants except the control. Polymorphism was also detected among the control and drought-tolerant mutants by DNA fingerprinting using ISSR markers. A total of 106 PCR fragments were amplified with 19 ISSR primers and 91 of them were polymorphic. The dendrograms were separated into two main clusters. First cluster included M8 mutant plant, which was applied 20 Gy gamma radiation and regenerated on selective culture media containing 10 g l{sup -1} PEG6000 concentration, and the second cluster was further divided into five sub-clusters.

  11. Mutant-inducing effect of γ-ray irradiation for hybrid rice F1 derived from cross of black glutinous rice x wild rice

    International Nuclear Information System (INIS)

    Mao Dezhi; Tang Yilan

    1998-01-01

    The hybrid rice F 1 plant derived from the back crossing of glutinous rice x wild rice was irradiated with γ-ray. The result of investigation to the induced mutant showed that through the selection and backcross, a black glutinous rice strain with the short stem, cold tolerance and high yield was developed. The analysis of the ability of heredity variance showed that the selection was effective for the husk colour, black glutinous and black Indica rice, but ineffective for the white Indica rice and seed setting

  12. Vaccination with a HSV-2 UL24 mutant induces a protective immune response in murine and guinea pig vaginal infection models.

    Science.gov (United States)

    Visalli, Robert J; Natuk, Robert J; Kowalski, Jacek; Guo, Min; Blakeney, Susan; Gangolli, Seema; Cooper, David

    2014-03-10

    The rational design and development of genetically attenuated HSV-2 mutant viruses represent an attractive approach for developing both prophylactic and therapeutic vaccines for genital herpes. Previously, HSV-2 UL24 was shown to be a virulence determinant in both murine and guinea pig vaginal infection models. An UL24-βgluc insertion mutant produced syncytial plaques and replicated to nearly wild type levels in tissue culture, but induced little or no pathological effects in recipient mice or guinea pigs following vaginal infection. Here we report that immunization of mice or guinea pigs with high or low doses of UL24-βgluc elicited a highly protective immune response. UL24-βgluc immunization via the vaginal or intramuscular routes was demonstrated to protect mice from a lethal vaginal challenge with wild type HSV-2. Moreover, antigen re-stimulated splenic lymphocytes harvested from immunized mice exhibited both HSV-2 specific CTL activity and IFN-γ expression. Humoral anti-HSV-2 responses in serum were Th1-polarized (IgG2a>IgG1) and contained high-titer anti-HSV-2 neutralizing activity. Guinea pigs vaccinated subcutaneously with UL24-βgluc or the more virulent parental strain (186) were challenged with a heterologous HSV-2 strain (MS). Acute disease scores were nearly indistinguishable in guinea pigs immunized with either virus. Recurrent disease scores were reduced in UL24-βgluc immunized animals but not to the same extent as those immunized with strain 186. In addition, challenge virus was not detected in 75% of guinea pigs subcutaneously immunized with UL24-βgluc. In conclusion, disruption of the UL24 gene is a prime target for the development of a genetically attenuated live HSV-2 vaccine. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Evaluation Of Yield And Chemical Properties Of Some Faba Bean (Vicia FABA L.) Mutants Induced By GAMMA Radiation And Ethyl Methane Sulphonate

    International Nuclear Information System (INIS)

    Sallam, E.M.; Nasr, E.H.; Attia, Z.M.; Shawki, H.A.

    2013-01-01

    This investigation aims to study the effect of physical and chemical mutagens on the yield and chemical properties as well as amino acids composition of defatted faba bean (Vicia faba L.) seeds meal as a control (Giza 2) compared with mutants produced by gamma radiation and ethyl methane sulphonate (EMS). Also, the functional properties of these samples were determined. The results indicated high differences between mutation for seed yield and its components than the untreated samples. In addition, radiation mutation of faba bean seeds showed slight increase in protein content as the main constituent of faba bean seeds as well as total oil percentages in some mutant of these seeds in return of decreasing in total carbohydrate. Furthermore, radiation mutation had detectable effects on the total amino acids contents of faba bean seeds meal which had a higher percentages on essential amino acids (EAA) and non-essential amino acids (NEAA) and mutant 3 was the highest values of EAA and NEAA as compared to the control. On the other hand, radiation mutation improved the protein functional properties of some mutant of faba bean meal flour than the other mutant samples as compared to local commercial variety

  14. Study on homologous series of induced early mutants in Indica rice Ⅱ. the relationship between the homologous series of early mutants induced and the ecotype in Indica rice

    International Nuclear Information System (INIS)

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

    2001-01-01

    The induced mutation in light sensitivity of the Indica rice leads to induction of the homologous series of early mutants along with the variation of ecological character and the ecoclimate. The induction of mutants was closely related to the ecotype of Indica rice, the homologous series of early mutants in different level were derived from the different ecotype of the Indica rice, otherwise, the similar homologous series of early mutants were derived from the same ecotypic variety. The induction of the early ecotypic variety derived from the homologous series of early mutants provides the basis and possibility for accelerating the development of the new cultivars. (authors)

  15. Expression, purification and preliminary biochemical and structural characterization of the leucine rich repeat namesake domain of leucine rich repeat kinase 2.

    Science.gov (United States)

    Vancraenenbroeck, Renée; Lobbestael, Evy; Weeks, Stephen D; Strelkov, Sergei V; Baekelandt, Veerle; Taymans, Jean-Marc; De Maeyer, Marc

    2012-03-01

    Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease. Much research effort has been directed towards the catalytic core region of LRRK2 composed of GTPase (ROC, Ras of complex proteins) and kinase domains and a connecting COR (C-terminus of ROC) domain. In contrast, the precise functions of the protein-protein interaction domains, such as the leucine-rich repeat (LRR) domain, are not known. In the present study, we modeled the LRRK2 LRR domain (LRR(LRRK2)) using a template assembly approach, revealing the presence of 14 LRRs. Next, we focused on the expression and purification of LRR(LRRK2) in Escherichia coli. Buffer optimization revealed that the protein requires the presence of a zwitterionic detergent, namely Empigen BB, during solubilization and the subsequent purification and characterization steps. This indicates that the detergent captures the hydrophobic surface patches of LRR(LRRK2) thereby suppressing its aggregation. Circular dichroism (CD) spectroscopy measured 18% α-helices and 21% β-sheets, consistent with predictions from the homology model. Size exclusion chromatography (SEC) and dynamic light scattering measurements showed the presence of a single species, with a Stokes radius corresponding to the model dimensions of a protein monomer. Furthermore, no obvious LRR(LRRK2) multimerization was detected via cross-linking studies. Finally, the LRR(LRRK2) clinical mutations did not influence LRR(LRRK2) secondary, tertiary or quaternary structure as determined via SEC and CD spectroscopy. We therefore conclude that these mutations are likely to affect putative LRR(LRRK2) inter- and intramolecular interactions. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Author Details

    African Journals Online (AJOL)

    Original Synthetic Report: Origin of the G2019S mutation associated to Parkinson's disease in Europeans and in North Africans Abstract PDF · Vol 1, No 2 (2009) - Articles Original Synthetic Report: Carrier frequencies of the common GJB2 (connexin-26) 35delG mutation in the Greek-Turkish area: predominance of the ...

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

    Energy Technology Data Exchange (ETDEWEB)

    Thacker, J

    1986-05-01

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

  18. A graph-clustering approach to search important molecular markers ...

    African Journals Online (AJOL)

    use

    2011-11-07

    Nov 7, 2011 ... inclusions and a movement disorder (Chen and Feany,. 2005). LRRK2 ... The gene ontology (Ashburner et al., 2000) project is a major bioinformatics initiative ... et al. 15657 particular organisms (http://www.genome.jp/kegg/).

  19. The I2020T Leucine-rich repeat kinase 2 transgenic mouse exhibits impaired locomotive ability accompanied by dopaminergic neuron abnormalities

    Directory of Open Access Journals (Sweden)

    Maekawa Tatsunori

    2012-04-01

    Full Text Available Abstract Background Leucine-rich repeat kinase 2 (LRRK2 is the gene responsible for autosomal-dominant Parkinson’s disease (PD, PARK8, but the mechanism by which LRRK2 mutations cause neuronal dysfunction remains unknown. In the present study, we investigated for the first time a transgenic (TG mouse strain expressing human LRRK2 with an I2020T mutation in the kinase domain, which had been detected in the patients of the original PARK8 family. Results The TG mouse expressed I2020T LRRK2 in dopaminergic (DA neurons of the substantia nigra, ventral tegmental area, and olfactory bulb. In both the beam test and rotarod test, the TG mice exhibited impaired locomotive ability in comparison with their non-transgenic (NTG littermates. Although there was no obvious loss of DA neurons in either the substantia nigra or striatum, the TG brain showed several neurological abnormalities such as a reduced striatal dopamine content, fragmentation of the Golgi apparatus in DA neurons, and an increased degree of microtubule polymerization. Furthermore, the tyrosine hydroxylase-positive primary neurons derived from the TG mouse showed an increased frequency of apoptosis and had neurites with fewer branches and decreased outgrowth in comparison with those derived from the NTG controls. Conclusions The I2020T LRRK2 TG mouse exhibited impaired locomotive ability accompanied by several dopaminergic neuron abnormalities. The TG mouse should provide valuable clues to the etiology of PD caused by the LRRK2 mutation.

  20. Stemcell Information: SKIP000292 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available stem.2013.01.008 Genetic correction of a LRRK2 mutation in human iPSCs links parkinsonian neurodegeneration ... SKIP000292 ... Diseased L1-2Mut L1-2Mut ... パーキンソン病 G20 Parkinson's disease 607060

  1. Stemcell Information: SKIP000300 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ... 23472874 10.1016/j.stem.2013.01.008 Genetic correction of a LRRK2 mutation in human iPSCs links parkinson... SKIP000300 ... Diseased L2-1GC L2-1GC ... パーキンソン病 G20 Parkinson's disease 607060 ...

  2. Stemcell Information: SKIP000297 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available j.stem.2013.01.008 Genetic correction of a LRRK2 mutation in human iPSCs links parkinsonian neurodegeneratio... SKIP000297 ... Diseased L2-1Mut L2-1Mut ... パーキンソン病 G20 Parkinson's disease 607060

  3. Stemcell Information: SKIP000293 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ... 23472874 10.1016/j.stem.2013.01.008 Genetic correction of a LRRK2 mutation in human iPSCs links parkinson... SKIP000293 ... Diseased L1-1GC2 L1-1GC2 ... パーキンソン病 G20 Parkinson's disease 607060

  4. Stemcell Information: SKIP000290 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available 1016/j.stem.2013.01.008 Genetic correction of a LRRK2 mutation in human iPSCs links parkinsonian neurodegene... SKIP000290 ... Diseased L1-1Mut L1-1Mut ... パーキンソン病 G20 Parkinson's disease 607060

  5. Stemcell Information: SKIP000299 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ... 23472874 10.1016/j.stem.2013.01.008 Genetic correction of a LRRK2 mutation in human iPSCs links parkinsoni... SKIP000299 ... Diseased L2-3Mut L2-3Mut ... パーキンソン病 G20 Parkinson's disease 607060

  6. Stemcell Information: SKIP000298 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ... 23472874 10.1016/j.stem.2013.01.008 Genetic correction of a LRRK2 mutation in human iPSCs links parkinsoni... SKIP000298 ... Diseased L2-2Mut L2-2Mut ... パーキンソン病 G20 Parkinson's disease 607060

  7. Stemcell Information: SKIP000302 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ... Information Only ... 23472874 10.1016/j.stem.2013.01.008 Genetic correction of a LRRK2 mutation in human iPSCs links parkinso... SKIP000302 ... Diseased L2-3GC L2-3GC ... パーキンソン病 G20 Parkinson's disease 607060 ...

  8. Stemcell Information: SKIP000301 [SKIP Stemcell Database[Archive

    Lifescience Database Archive (English)

    Full Text Available ... Information Only ... 23472874 10.1016/j.stem.2013.01.008 Genetic correction of a LRRK2 mutation in human iPSCs links parkinson... SKIP000301 ... Diseased L2-2GC L2-2GC ... パーキンソン病 G20 Parkinson's disease 607060 ...

  9. 76 FR 36549 - Government-Owned Inventions; Availability for Licensing

    Science.gov (United States)

    2011-06-22

    ... measured information in real-time. Applications: Transarterial chemoembolization Drug eluting bead Intravenous drug delivery Drug distribution monitoring Real-time imaging Inventors: Matthew Dreher, Elliot... a potential contribution of normal LRRK2 protein to the etiology of sporadic PD cases. Micro-RNAs...

  10. Levetiracetam Affects Differentially Presynaptic Proteins in Rat Cerebral Cortex

    Directory of Open Access Journals (Sweden)

    Daniele Marcotulli

    2017-12-01

    Full Text Available Presynaptic proteins are potential therapeutic targets for epilepsy and other neurological diseases. We tested the hypothesis that chronic treatment with the SV2A ligand levetiracetam affects the expression of other presynaptic proteins. Results showed that in rat neocortex no significant difference was detected in SV2A protein levels in levetiracetam treated animals compared to controls, whereas levetiracetam post-transcriptionally decreased several vesicular proteins and increased LRRK2, without any change in mRNA levels. Analysis of SV2A interactome indicates that the presynaptic proteins regulation induced by levetiracetam reported here is mediated by this interactome, and suggests that LRRK2 plays a role in forging the pattern of effects.

  11. Genetic perspective on the role of the autophagy-lysosome pathway in Parkinson disease

    OpenAIRE

    Gan-Or, Ziv; Dion, Patrick A; Rouleau, Guy A

    2015-01-01

    Parkinson disease (PD), once considered as a prototype of a sporadic disease, is now known to be considerably affected by various genetic factors, which interact with environmental factors and the normal process of aging, leading to PD. Large studies determined that the hereditary component of PD is at least 27%, and in some populations, single genetic factors are responsible for more than 33% of PD patients. Interestingly, many of these genetic factors, such as LRRK2, GBA, SMPD1, SNCA, PARK2...

  12. Diversity of Mitochondrial Pathology in a Mouse Model of Axonal Degeneration in Synucleinopathies

    Directory of Open Access Journals (Sweden)

    Akio Sekigawa

    2013-01-01

    Full Text Available There is mounting evidence for a role of mitochondrial dysfunction in the pathogenesis of α-synucleinopathies such as Parkinson's disease (PD and dementia with Lewy bodies (DLB. In particular, recent studies have demonstrated that failure of mitochondrial quality control caused by loss of function of the PTEN-induced kinase 1 (PINK1, PARK6 Parkin (PARK2 pathway may be causative in some familial PD. In sporadic PD, α-synuclein aggregation may interfere with mitochondrial function, and this might be further exacerbated by leucine-rich repeat kinase 2 (LRRK2. The majority of these findings have been obtained in Drosophila and cell cultures, whereas the objective of this paper is to discuss our recent results on the axonal pathology of brains derived from transgenic mice expressing α-synuclein or DLB-linked P123H β-synuclein. In line with the current view of the pathogenesis of sporadic PD, mitochondria abnormally accumulated in α-synuclein/LRRK2-immunopositive axonal swellings in mice expressing α-synuclein. Curiously, neither mitochondria nor LRRK2 was present in the swellings of mice expressing P123H β-synuclein, suggesting that α- and β-synuclein might play differential roles in the mitochondrial pathology of α-synucleinopathies.

  13. Structural imaging in the presymptomatic stage of genetically determined parkinsonism

    DEFF Research Database (Denmark)

    Reetz, Kathrin; Tadic, Vera; Kasten, Meike

    2010-01-01

    Several genes associated with monogenic forms of Parkinson's disease (PD) have been discovered, opening up new avenues for the investigation of presymptomatic stages of PD. Using voxel-based morphometry in 30 asymptomatic mutation carriers (MC) with mutations in four different genes for PD and 100....... The observed striatal GMV increase might be the common structural correlate of compensatory mechanisms due to the latent dopaminergic deficit, reflecting the different, but probably interrelated pathogenic pathways resulting in nigral cell death. Asymptomatic PINK1 and LRRK2 MC also revealed smaller GMV...

  14. Pharmacological rescue of mitochondrial deficits in iPSC-derived neural cells from patients with familial Parkinson's disease

    DEFF Research Database (Denmark)

    Cooper, Oliver; Seo, Hyemyung; Andrabi, Shaida

    2012-01-01

    , or the LRRK2 kinase inhibitor GW5074. Analysis of mitochondrial responses in iPSC-derived neural cells from PD patients carrying different mutations provides insight into convergence of cellular disease mechanisms between different familial forms of PD and highlights the importance of oxidative stress...... of reactive oxygen species, mitochondrial respiration, proton leakage, and intraneuronal movement of mitochondria. Cellular vulnerability associated with mitochondrial dysfunction in iPSC-derived neural cells from familial PD patients and at-risk individuals could be rescued with coenzyme Q(10), rapamycin...

  15. Genetic susceptibility loci, environmental exposures, and Parkinson's disease: a case-control study of gene-environment interactions.

    Science.gov (United States)

    Chung, Sun Ju; Armasu, Sebastian M; Anderson, Kari J; Biernacka, Joanna M; Lesnick, Timothy G; Rider, David N; Cunningham, Julie M; Ahlskog, J Eric; Frigerio, Roberta; Maraganore, Demetrius M

    2013-06-01

    Prior studies causally linked mutations in SNCA, MAPT, and LRRK2 genes with familial Parkinsonism. Genome-wide association studies have demonstrated association of single nucleotide polymorphisms (SNPs) in those three genes with sporadic Parkinson's disease (PD) susceptibility worldwide. Here we investigated the interactions between SNPs in those three susceptibility genes and environmental exposures (pesticides application, tobacco smoking, coffee drinking, and alcohol drinking) also associated with PD susceptibility. Pairwise interactions between environmental exposures and 18 variants (16 SNPs and two variable number tandem repeats, or "VNTRs") in SNCA, MAPT and LRRK2, were investigated using data from 1098 PD cases from the upper Midwest, USA and 1098 matched controls. Environmental exposures were assessed using a validated telephone interview script. Five pairwise interactions had uncorrected P-values coffee drinking × MAPT H1/H2 haplotype or MAPT rs16940806, and alcohol drinking × MAPT rs2435211. None of these interactions remained significant after Bonferroni correction. Secondary analyses in strata defined by type of control (sibling or unrelated), sex, or age at onset of the case also did not identify significant interactions after Bonferroni correction. This study documented limited pairwise interactions between established genetic and environmental risk factors for PD; however, the associations were not significant after correction for multiple testing. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Structural genomic variations and Parkinson's disease.

    Science.gov (United States)

    Bandrés-Ciga, Sara; Ruz, Clara; Barrero, Francisco J; Escamilla-Sevilla, Francisco; Pelegrina, Javier; Vives, Francisco; Duran, Raquel

    2017-10-01

    Parkinson's disease (PD) is the second most common neurodegenerative disease, whose prevalence is projected to be between 8.7 and 9.3 million by 2030. Until about 20 years ago, PD was considered to be the textbook example of a "non-genetic" disorder. Nowadays, PD is generally considered a multifactorial disorder that arises from the combination and complex interaction of genes and environmental factors. To date, a total of 7 genes including SNCA, LRRK2, PARK2, DJ-1, PINK 1, VPS35 and ATP13A2 have been seen to cause unequivocally Mendelian PD. Also, variants with incomplete penetrance in the genes LRRK2 and GBA are considered to be strong risk factors for PD worldwide. Although genetic studies have provided valuable insights into the pathogenic mechanisms underlying PD, the role of structural variation in PD has been understudied in comparison with other genomic variations. Structural genomic variations might substantially account for such genetic substrates yet to be discovered. The present review aims to provide an overview of the structural genomic variants implicated in the pathogenesis of PD.

  17. Regulation of the autophagy protein LC3 by phosphorylation

    Science.gov (United States)

    Cherra, Salvatore J.; Kulich, Scott M.; Uechi, Guy; Balasubramani, Manimalha; Mountzouris, John; Day, Billy W.

    2010-01-01

    Macroautophagy is a major catabolic pathway that impacts cell survival, differentiation, tumorigenesis, and neurodegeneration. Although bulk degradation sustains carbon sources during starvation, autophagy contributes to shrinkage of differentiated neuronal processes. Identification of autophagy-related genes has spurred rapid advances in understanding the recruitment of microtubule-associated protein 1 light chain 3 (LC3) in autophagy induction, although braking mechanisms remain less understood. Using mass spectrometry, we identified a direct protein kinase A (PKA) phosphorylation site on LC3 that regulates its participation in autophagy. Both metabolic (rapamycin) and pathological (MPP+) inducers of autophagy caused dephosphorylation of endogenous LC3. The pseudophosphorylated LC3 mutant showed reduced recruitment to autophagosomes, whereas the nonphosphorylatable mutant exhibited enhanced puncta formation. Finally, autophagy-dependent neurite shortening induced by expression of a Parkinson disease–associated G2019S mutation in leucine-rich repeat kinase 2 was inhibited by dibutyryl–cyclic adenosine monophosphate, cytoplasmic expression of the PKA catalytic subunit, or the LC3 phosphorylation mimic. These data demonstrate a role for phosphorylation in regulating LC3 activity. PMID:20713600

  18. Mutagenic effect of radionuclides incorporated into DNA of Drosophila melanogaster. Progress report, July 16, 1983-June 30, 1984

    International Nuclear Information System (INIS)

    Lee, W.R.

    1984-01-01

    Progress is reported in the development of methods for economically analyzing mutants at the molecular level so that mutants induced by x-rays, N-ethyl-N-nitrosourea (ENU) and tritium could be classified

  19. Cellular and Molecular Basis of Neurodegeneration in Parkinson Disease

    Directory of Open Access Journals (Sweden)

    Xian-Si Zeng

    2018-04-01

    Full Text Available It has been 200 years since Parkinson disease (PD was described by Dr. Parkinson in 1817. The disease is the second most common neurodegenerative disease characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Although the pathogenesis of PD is still unknown, the research findings from scientists are conducive to understand the pathological mechanisms. It is well accepted that both genetic and environmental factors contribute to the onset of PD. In this review, we summarize the mutations of main seven genes (α-synuclein, LRRK2, PINK1, Parkin, DJ-1, VPS35 and GBA1 linked to PD, discuss the potential mechanisms for the loss of dopaminergic neurons (dopamine metabolism, mitochondrial dysfunction, endoplasmic reticulum stress, impaired autophagy, and deregulation of immunity in PD, and expect the development direction for treatment of PD.

  20. Imaging the impact of genes on Parkinson's disease

    DEFF Research Database (Denmark)

    van der Vegt, J P M; van Nuenen, B F L; Bloem, B R

    2009-01-01

    by the discovery of mutations in single genes that can cause autosomal dominant (alpha-synuclein (SNCA)) and leucine rich repeat kinase 2 (LRRK2) gene) or recessive (Parkin, PTEN-induced putative kinase 1 (PINK1), DJ-1, and ATP13A2 gene) forms of PD. Here, we review how structural and functional neuroimaging...... of individuals carrying a mutation in one of the PD genes has offered a unique avenue of research into the pathogenesis of PD. In symptomatic mutation carriers (i.e. those with overt disease), brain mapping can help to link the molecular pathogenesis of PD more directly with functional and structural changes...... monogenic forms of PD, common polymorphisms in genes that influence mono-aminergic signaling or synaptic plasticity may have modifying effects on distinct aspects of PD. We also discuss how functional and structural neuroimaging can be used to better characterize these genotype-phenotype correlations....

  1. Impaired sense of smell and color discrimination in monogenic and idiopathic Parkinson's disease

    DEFF Research Database (Denmark)

    Kertelge, Lena; Brüggemann, Norbert; Schmidt, Alexander

    2010-01-01

    deficits are linked. We examined 100 patients with IPD, 27 manifesting mutation carriers (MC), 20 nonmanifesting mutation carriers (NMC), and 110 controls. Participants underwent a standardized neurological examination, the University of Pennsylvania Smell Identification Test (UPSIT), the Farnsworth...... groups (MC 13.8 ± 11.9, NMC 19.6 ± 13.0, controls 33.8 ± 22.4). Within MC, carriers of two mutations in Parkin and PINK1 showed higher UPSIT percentiles than LRRK2 and SNCA carriers. Color discrimination was reduced in IPD (FM total error score 134.8 ± 92.7). In MC (122.4 ± 142.4), the reduction was most...

  2. A Tangled Web - Tau and Sporadic Parkinson's Disease

    Directory of Open Access Journals (Sweden)

    Selina Wray

    2010-12-01

    Full Text Available Parkinson's disease (PD represents a major challenge for health care systems around the world: it is the most common degenerative movement disorder of old age, affecting over 100,000 people in the UK alone. A great deal of progress has been made in understanding the molecular basis of PD by taking advantage of advances in genetics, initially by the identification of genes responsible for rare mendellian forms of PD (outlined in table one, and more recently by applying genome wide association studies (GWAS to the sporadic form of the disease. Several such GWAS have now been carried out, with a meta-analysis currently under way. Using over 6000 cases and 10000 controls, two of these studies have identified variation at a number of loci as being associated with an increased risk of disease. Three genes stand out as candidates from these studies – the SNCA gene, coding for α -synuclein, the LRRK2 gene, coding for leucine rich repeat kinase 2, and MAPT, coding for the microtubule associated protein tau. Point mutations in α -synuclein, along with gene multiplication events, result in autosomal dominant PD, often with a significant dementia component. In addition to this, α -synuclein is the principle component of the main pathological hallmark of PD, the Lewy body. Mutations in LRRK2 are the most common genetic cause of PD, and so again were a likely candidate for a susceptibility locus for the sporadic form of disease. More surprising, perhaps, was the identification of tau as a susceptibility factor for Parkinson's. In this review we will outline the role of tau in neurodegeneration and in different forms of parkinsonism, and speculate as to what the functional basis of this association might be.

  3. Prodromal Parkinsonism and Neurodegenerative Risk Stratification in REM Sleep Behavior Disorder.

    Science.gov (United States)

    Barber, Thomas R; Lawton, Michael; Rolinski, Michal; Evetts, Samuel; Baig, Fahd; Ruffmann, Claudio; Gornall, Aimie; Klein, Johannes C; Lo, Christine; Dennis, Gary; Bandmann, Oliver; Quinnell, Timothy; Zaiwalla, Zenobia; Ben-Shlomo, Yoav; Hu, Michele T M

    2017-08-01

    Rapid eye movement (REM) sleep behavior disorder (RBD) is the most specific marker of prodromal alpha-synucleinopathies. We sought to delineate the baseline clinical characteristics of RBD and evaluate risk stratification models. Clinical assessments were performed in 171 RBD, 296 control, and 119 untreated Parkinson's (PD) participants. Putative risk measures were assessed as predictors of prodromal neurodegeneration, and Movement Disorders Society (MDS) criteria for prodromal PD were applied. Participants were screened for common leucine-rich repeat kinase 2 (LRRK2)/glucocerebrosidase gene (GBA) gene mutations. Compared to controls, participants with RBD had higher rates of solvent exposure, head injury, smoking, obesity, and antidepressant use. GBA mutations were more common in RBD, but no LRRK2 mutations were found. RBD participants performed significantly worse than controls on Unified Parkinson's Disease Rating Scale (UPDRS)-III, timed "get-up-and-go", Flamingo test, Sniffin Sticks, and cognitive tests and had worse measures of constipation, quality of life (QOL), and orthostatic hypotension. For all these measures except UPDRS-III, RBD and PD participants were equally impaired. Depression, anxiety, and apathy were worse in RBD compared to PD participants. Stratification of people with RBD according to antidepressant use, obesity, and age altered the odds ratio (OR) of hyposmia compared to controls from 3.4 to 45.5. 74% (95% confidence interval [CI] 66%, 80%) of RBD participants met the MDS criteria for probable prodromal Parkinson's compared to 0.3% (95% CI 0.009%, 2%) of controls. RBD are impaired across a range of clinical measures consistent with prodromal PD and suggestive of a more severe nonmotor subtype. Clinical risk stratification has the potential to select higher risk patients for neuroprotective interventions. © Sleep Research Society 2017. Published by Oxford University Press [on behalf of the Sleep Research Society].

  4. Mutagenesis at the ad-3A and ad-3B loci in haploid UV-sensitive strains of Neurospora crassa. Pt. 6

    International Nuclear Information System (INIS)

    De Serres, F.J.; Inoue, H.; Schuepbach, M.E.

    1983-01-01

    Genetic characterization of ad-3B mutants induced in wild-type and UV-sensitive strains has revealed qualitative differences between the spectra of genetic alterations at the molecular level. Ad-3B mutants induced in the two nucleotide excision-repair-deficient strains upr-1 and uvs-2 had significantly lower frequencies of nonpolarized complementation patterns and higher frequencies of noncomplementing mutants than ad-3B mutants induced in the wild-type strain in samples induced by either UV, #betta#-rays, 4NQO or MNNG. In these same samples ad-3B mutants induced in uvs-4, uvs-5 or uvs-6 did not differ significantly from those induced in the wild-type strain. After ICR-170 treatment, ad-3B mutants induced in the UV-sensitive strains did not differ significantly from those induced in wild-type. The comparisons in the present and previous studies demonstrate that the process of mutation-induction in the ad-3 region is under the control of other loci that not only alter mutant recovery quantitatively but also qualitatively. These data have important implications for comparative chemical mutagenesis, since the spectrum of genetic alterations produced by a given agent can be modified markedly as a result of defects in DNA repair. (orig./AJ)

  5. Mutagenic effect of radionuclides incorporated into DNA of Drosophila melanogaster. Progress report, December 15, 1982-July 15, 1983

    International Nuclear Information System (INIS)

    Lee, W.R.

    1983-01-01

    The molecular changes in DNA of mutations induced at the well-defined locus alcohol dehydrogenase (Adh) in Drosophila melanogaster were compared between null mutants induced by x-rays, the alkylating agent N-ethyl-N-nitrosourea (ENU) and decay of tritium incorporated into specific sites of DNA

  6. Development of Database Software with Plant Mutant Resources

    International Nuclear Information System (INIS)

    Namgoong, Won; Lee, M. J.; Kim, J. D.; Ma, N. K.

    2007-03-01

    In this research, mutants induced by nuclear radiation are developed information computerised system. The status and progress on the collection, identification and utilization of mutants in Korea are introduced. And it was produced home page, manual, test record, construction of system

  7. Isozyme patterns of powdery mildew resistant wheat mutants

    International Nuclear Information System (INIS)

    Xia Wengau; Li Zhengkui; Wang Kefeng

    1989-01-01

    Full Text: Wheat mutants induced by gamma irradiation and showing improved resistance to powdery mildew were analysed for isozymes. The peroxidase band 3A could be related to the disease reaction. The band 3A is absent in resistant mutants, the higher the activity of band 3A the greater the susceptibility. (author)

  8. The research progress on plant mutant germplasm resources in China

    International Nuclear Information System (INIS)

    He Cexi; Ji Linzhen; Zhao Shirong

    1991-07-01

    Mutants induced by nuclear radiation or other mutagens are new artificial germplasm resources. Some mutants have been applied in plant breeding and great achievements have been reached. The status and progress on the collection, identification and utilization of mutants in China are introduced. A proposal for developing mutant germplasm resources with good agronomic characters is suggested

  9. Molecular analysis of mutants of the Neurospora adenylosuccinate ...

    Indian Academy of Sciences (India)

    2012-08-07

    Aug 7, 2012 ... and mutants induced with X-ray, UV or chemical mutagens. ... We have sequenced the ad-8 locus from 13 of these mutants and identified the molecular nature ..... mutants in yeast by selection for constitutive behavior in pig-.

  10. 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

  11. Genetics of amyloglucosidase production in Aspergillus niger and Aspergillus awamori

    International Nuclear Information System (INIS)

    Bonatelli Junior, R.; Umbuzeiro-Valent, G.; Masiero, M.; Vialta, A.; Calil, M.R.

    1984-01-01

    The results obtained with the isolation and characterization of mutants induced by irradiating 5-8 days old spores with ultraviolet light are presented. The modification in the amyloglucosidase accumulation, the allelic interactions of these mutations and the inhibitory effects of glycerol are reported. (M.A.C.) [pt

  12. Molecular markers for granulovacuolar degeneration are present in rimmed vacuoles.

    Directory of Open Access Journals (Sweden)

    Masahiro Nakamori

    Full Text Available BACKGROUND: Rimmed vacuoles (RVs are round-oval cytoplasmic inclusions, detected in muscle cells of patients with myopathies, such as inclusion body myositis (IBM and distal myopathy with RVs (DMRV. Granulovacuolar degeneration (GVD bodies are spherical vacuoles containing argentophilic and hematoxyphilic granules, and are one of the pathological hallmarks commonly found in hippocampal pyramidal neurons of patients with aging-related neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. These diseases are common in the elderly and share some pathological features. Therefore, we hypothesized that mechanisms of vacuolar formation in RVs and GVD bodies are common despite their role in two differing pathologies. We explored the components of RVs by immunohistochemistry, using antibodies for GVD markers. METHODS: Subjects included one AD case, eight cases of sporadic IBM, and three cases of DMRV. We compared immunoreactivity and staining patterns for GVD markers. These markers included: (1 tau-modifying proteins (caspase 3, cyclin-dependent kinase 5 [CDK5], casein kinase 1δ [CK1δ], and c-jun N-terminal kinase [JNK], (2 lipid raft-associated materials (annexin 2, leucine-rich repeat kinase 2 [LRRK2], and flotillin-1, and (3 other markers (charged multi-vesicular body protein 2B [CHMP2B] and phosphorylated transactive response DNA binding protein-43 [pTDP43] in both GVD bodies and RVs. Furthermore, we performed double staining of each GVD marker with pTDP43 to verify the co-localization. RESULTS: GVD markers, including lipid raft-associated proteins and tau kinases, were detected in RVs. CHMP2B, pTDP43, caspase 3, LRRK2, annexin 2 and flotillin-1 were detected on the rim and were diffusely distributed in the cytoplasm of RV-positive fibers. CDK5, CK1δ and JNK were detected only on the rim. In double staining experiments, all GVD markers colocalized with pTDP43 in RVs. CONCLUSIONS: These results suggest that RVs of muscle

  13. Distinct mechanisms of axonal globule formation in mice expressing human wild type α-synuclein or dementia with Lewy bodies-linked P123H ß-synuclein

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    Sekigawa Akio

    2012-09-01

    Full Text Available Abstract Background Axonopathy is critical in the early pathogenesis of neurodegenerative diseases, including Parkinson’s disease (PD and dementia with Lewy bodies (DLB. Axonal swellings such as globules and spheroids are a distinct feature of axonopathy and our recent study showed that transgenic (tg mice expressing DLB-linked P123H β-synuclein (P123H βS were characterized by P123H βS-immunoreactive axonal swellings (P123H βS-globules. Therefore, the objectives of this study were to evaluate α-synuclein (αS-immunoreactive axonal swellings (αS-globules in the brains of tg mice expressing human wild-type αS and to compare them with the globules in P123H βS tg mice. Results In αS tg mice, αS-globules were formed in an age-dependent manner in various brain regions, including the thalamus and basal ganglia. These globules were composed of autophagosome-like membranous structures and were reminiscent of P123H βS-globules in P123H βS tg mice. In the αS-globules, frequent clustering and deformation of mitochondria were observed. These changes were associated with oxidative stress, based on staining of nitrated αS and 4-hydroxy-2-nonenal (4-HNE. In accord with the absence of mitochondria in the P123H βS-globules, staining of nitrated αS and 4-HNE in these globules was weaker than that for αS-globules. Leucine-rich repeat kinase 2 (LRRK2, the PARK8 of familial PD, was detected exclusively in αS-globules, suggesting a specific role of this molecule in these globules. Conclusions Lysosomal pathology was similarly observed for both αS- and P123H βS-globules, while oxidative stress was associated with the αS-globules, and to a lesser extent with the P123H βS-globules. Other pathologies, such as mitochondrial alteration and LRRK2 accumulation, were exclusively detected for αS-globules. Collectively, both αS- and P123H βS-globules were formed through similar but distinct pathogenic mechanisms. Our findings suggest that synuclein

  14. Genetic or pharmacological activation of the Drosophila PGC-1α ortholog spargel rescues the disease phenotypes of genetic models of Parkinson's disease.

    Science.gov (United States)

    Ng, Chee-Hoe; Basil, Adeline H; Hang, Liting; Tan, Royston; Goh, Kian-Leong; O'Neill, Sharon; Zhang, Xiaodong; Yu, Fengwei; Lim, Kah-Leong

    2017-07-01

    Despite intensive research, the etiology of Parkinson's disease (PD) remains poorly understood and the disease remains incurable. However, compelling evidence gathered over decades of research strongly support a role for mitochondrial dysfunction in PD pathogenesis. Related to this, PGC-1α, a key regulator of mitochondrial biogenesis, has recently been proposed to be an attractive target for intervention in PD. Here, we showed that silencing of expression of the Drosophila PGC-1α ortholog spargel results in PD-related phenotypes in flies and also seem to negate the effects of AMPK activation, which we have previously demonstrated to be neuroprotective, that is, AMPK-mediated neuroprotection appears to require PGC-1α. Importantly, we further showed that genetic or pharmacological activation of the Drosophila PGC-1α ortholog spargel is sufficient to rescue the disease phenotypes of Parkin and LRRK2 genetic fly models of PD, thus supporting the proposed use of PGC-1α-related strategies for neuroprotection in PD. Copyright © 2017 National Neuroscience Institute. Published by Elsevier Inc. All rights reserved.

  15. The Monoamine Brainstem Reticular Formation as a Paradigm for Re-Defining Various Phenotypes of Parkinson's Disease Owing Genetic and Anatomical Specificity.

    Science.gov (United States)

    Gambardella, Stefano; Ferese, Rosangela; Biagioni, Francesca; Busceti, Carla L; Campopiano, Rosa; Griguoli, Anna M P; Limanaqi, Fiona; Novelli, Giuseppe; Storto, Marianna; Fornai, Francesco

    2017-01-01

    The functional anatomy of the reticular formation (RF) encompasses a constellation of brain regions which are reciprocally connected to sub-serve a variety of functions. Recent evidence indicates that neuronal degeneration within one of these regions spreads synaptically along brainstem circuitries. This is exemplified by the recruitment of various brainstem reticular nuclei in specific Parkinson's disease (PD) phenotypes, and by retrospective analysis of lethargic post-encephalitic parkinsonism. In fact, the spreading to various monoamine reticular nuclei can be associated with occurrence of specific motor and non-motor symptoms (NMS). This led to re-consider PD as a brainstem monoamine disorder (BMD). This definition surpasses the anatomy of meso-striatal motor control to include a variety of non-motor domains. This concept clearly emerges from the quite specific clinical-anatomical correlation which can be drawn in specific paradigms of PD genotypes. Therefore, this review article focuses on the genetics and neuroanatomy of three PD genotypes/phenotypes which can be selected as prototype paradigms for a differential recruitment of the RF leading to differential occurrence of NMS: (i) Parkin-PD, where NMS are rarely reported; (ii) LRRK2-PD and slight SNC point mutations, where the prevalence of NMS resembles idiopathic PD; (iii) Severe SNCA point mutations and multiplications, where NMS are highly represented.

  16. The Monoamine Brainstem Reticular Formation as a Paradigm for Re-Defining Various Phenotypes of Parkinson’s Disease Owing Genetic and Anatomical Specificity

    Science.gov (United States)

    Gambardella, Stefano; Ferese, Rosangela; Biagioni, Francesca; Busceti, Carla L.; Campopiano, Rosa; Griguoli, Anna M. P.; Limanaqi, Fiona; Novelli, Giuseppe; Storto, Marianna; Fornai, Francesco

    2017-01-01

    The functional anatomy of the reticular formation (RF) encompasses a constellation of brain regions which are reciprocally connected to sub-serve a variety of functions. Recent evidence indicates that neuronal degeneration within one of these regions spreads synaptically along brainstem circuitries. This is exemplified by the recruitment of various brainstem reticular nuclei in specific Parkinson’s disease (PD) phenotypes, and by retrospective analysis of lethargic post-encephalitic parkinsonism. In fact, the spreading to various monoamine reticular nuclei can be associated with occurrence of specific motor and non-motor symptoms (NMS). This led to re-consider PD as a brainstem monoamine disorder (BMD). This definition surpasses the anatomy of meso-striatal motor control to include a variety of non-motor domains. This concept clearly emerges from the quite specific clinical-anatomical correlation which can be drawn in specific paradigms of PD genotypes. Therefore, this review article focuses on the genetics and neuroanatomy of three PD genotypes/phenotypes which can be selected as prototype paradigms for a differential recruitment of the RF leading to differential occurrence of NMS: (i) Parkin-PD, where NMS are rarely reported; (ii) LRRK2-PD and slight SNC point mutations, where the prevalence of NMS resembles idiopathic PD; (iii) Severe SNCA point mutations and multiplications, where NMS are highly represented. PMID:28458632

  17. A strategy for the generation, characterization and distribution of animal models by The Michael J. Fox Foundation for Parkinson’s Research

    Directory of Open Access Journals (Sweden)

    Marco A. S. Baptista

    2013-11-01

    Full Text Available Progress in Parkinson’s disease (PD research and therapeutic development is hindered by many challenges, including a need for robust preclinical animal models. Limited availability of these tools is due to technical hurdles, patent issues, licensing restrictions and the high costs associated with generating and distributing these animal models. Furthermore, the lack of standardization of phenotypic characterization and use of varying methodologies has made it difficult to compare outcome measures across laboratories. In response, The Michael J. Fox Foundation for Parkinson’s Research (MJFF is directly sponsoring the generation, characterization and distribution of preclinical rodent models, enabling increased access to these crucial tools in order to accelerate PD research. To date, MJFF has initiated and funded the generation of 30 different models, which include transgenic or knockout models of PD-relevant genes such as Park1 (also known as Park4 and SNCA, Park8 (LRRK2, Park7 (DJ-1, Park6 (PINK1, Park2 (Parkin, VPS35, EiF4G1 and GBA. The phenotypic characterization of these animals is performed in a uniform and streamlined manner at independent contract research organizations. Finally, MJFF created a central repository at The Jackson Laboratory (JAX that houses both non-MJFF and MJFF-generated preclinical animal models. Funding from MJFF, which subsidizes the costs involved in transfer, rederivation and colony expansion, has directly resulted in over 2500 rodents being distributed to the PD community for research use.

  18. Classic and New Animal Models of Parkinson's Disease

    Directory of Open Access Journals (Sweden)

    Javier Blesa

    2012-01-01

    Full Text Available Neurological disorders can be modeled in animals so as to recreate specific pathogenic events and behavioral outcomes. Parkinson’s Disease (PD is the second most common neurodegenerative disease of an aging population, and although there have been several significant findings about the PD disease process, much of this process still remains a mystery. Breakthroughs in the last two decades using animal models have offered insights into the understanding of the PD disease process, its etiology, pathology, and molecular mechanisms. Furthermore, while cellular models have helped to identify specific events, animal models, both toxic and genetic, have replicated almost all of the hallmarks of PD and are useful for testing new neuroprotective or neurorestorative strategies. Moreover, significant advances in the modeling of additional PD features have come to light in both classic and newer models. In this review, we try to provide an updated summary of the main characteristics of these models as well as the strengths and weaknesses of what we believe to be the most popular PD animal models. These models include those produced by 6-hydroxydopamine (6-OHDA, 1-methyl-1,2,3,6-tetrahydropiridine (MPTP, rotenone, and paraquat, as well as several genetic models like those related to alpha-synuclein, PINK1, Parkin and LRRK2 alterations.

  19. Advances with microRNAs in Parkinson’s disease research

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    Ma L

    2013-10-01

    Full Text Available Liuqing Ma,1,2,* Liangming Wei,3,* Fei Wu,2,* Zhenhua Hu,2 Zhenguo Liu,1 Weien Yuan2 1Department of Neurology, Xinhua Hospital affiliated with Shanghai JiaoTong University School of Medicine, Shanghai, People’s Republic of China; 2School of Pharmacy, Shanghai JiaoTong University, Shanghai, People’s Republic of China; 3Research Institute of Micro/Nano Science and Technology, Shanghai JiaoTong University, Shanghai, People’s Republic of China *These authors contributed equally to this work Abstract: Parkinson’s disease (PD is the second-most common age-dependent neurodegenerative disorder and is caused by severe degeneration of dopaminergic neurons in the substantia nigra pars compacta. Unfortunately, current treatment only targets symptoms and involves dopamine replacement therapy, which does not counteract progressive degeneration. MicroRNAs (miRNAs are a class of small RNA molecules implicated in post-transcriptional regulation of gene expression during development. Recent studies show that miRNAs are playing an important role in the pathophysiology of PD. miRNA-based therapy is a powerful tool with which to study gene function, investigate the mechanism of the disease, and validate drug targets. In this review, we focus on the recent advances of the use of miRNAs in the pathogenesis of PD. Keywords: miRNA, α-synuclein, LRRK2, miRNA-based therapy, pathophysiology

  20. Genetic perspective on the role of the autophagy-lysosome pathway in Parkinson disease.

    Science.gov (United States)

    Gan-Or, Ziv; Dion, Patrick A; Rouleau, Guy A

    2015-01-01

    Parkinson disease (PD), once considered as a prototype of a sporadic disease, is now known to be considerably affected by various genetic factors, which interact with environmental factors and the normal process of aging, leading to PD. Large studies determined that the hereditary component of PD is at least 27%, and in some populations, single genetic factors are responsible for more than 33% of PD patients. Interestingly, many of these genetic factors, such as LRRK2, GBA, SMPD1, SNCA, PARK2, PINK1, PARK7, SCARB2, and others, are involved in the autophagy-lysosome pathway (ALP). Some of these genes encode lysosomal enzymes, whereas others correspond to proteins that are involved in transport to the lysosome, mitophagy, or other autophagic-related functions. Is it possible that all these factors converge into a single pathway that causes PD? In this review, we will discuss these genetic findings and the role of the ALP in the pathogenesis of PD and will try to answer this question. We will suggest a novel hypothesis for the pathogenic mechanism of PD that involves the lysosome and the different autophagy pathways.

  1. Visual dysfunction in Parkinson’s disease

    Science.gov (United States)

    Weil, Rimona S.; Schrag, Anette E.; Warren, Jason D.; Crutch, Sebastian J.; Lees, Andrew J.; Morris, Huw R.

    2016-01-01

    Patients with Parkinson’s disease have a number of specific visual disturbances. These include changes in colour vision and contrast sensitivity and difficulties with complex visual tasks such as mental rotation and emotion recognition. We review changes in visual function at each stage of visual processing from retinal deficits, including contrast sensitivity and colour vision deficits to higher cortical processing impairments such as object and motion processing and neglect. We consider changes in visual function in patients with common Parkinson’s disease-associated genetic mutations including GBA and LRRK2. We discuss the association between visual deficits and clinical features of Parkinson’s disease such as rapid eye movement sleep behavioural disorder and the postural instability and gait disorder phenotype. We review the link between abnormal visual function and visual hallucinations, considering current models for mechanisms of visual hallucinations. Finally, we discuss the role of visuo-perceptual testing as a biomarker of disease and predictor of dementia in Parkinson’s disease. PMID:27412389

  2. Specific gene mutations induced by heavy ions

    International Nuclear Information System (INIS)

    Freeling, M.; Karoly, C.W.; Cheng, D.S.K.

    1980-01-01

    This report summarizes our heavy-ion research rationale, progress, and plans for the near future. The major project involves selecting a group of maize Adh1 mutants induced by heavy ions and correlating their altered behavior with altered DNA nucleotide sequences and sequence arrangements. This research requires merging the techniques of classical genetics and recombinant DNA technology. Our secondary projects involve (1) the use of the Adh gene in the fruit fly, Drosophila melanogaster, as a second system with which to quantify the sort of specific gene mutants induced by heavy ions as compared to x rays, and (2) the development of a maize Adh1 pollen in situ monitor for environmental mutagens

  3. Modification of radiation-induced sex-linked recessive lethal mutation frequency by tocopherol

    International Nuclear Information System (INIS)

    Beckman, C.; Roy, R.M.; Sproule, A.

    1982-01-01

    The present study evaluates the effect of supplementing culture medium with α-tocopherol acetate on the yield of sex-linked recessive lethal mutants induced by X-irradiation in mature sperm of Drosophila. Although tocopherol treatment of males had no impact on the yield of mutations, a drastic reduction in mutation frequency was observed when irradiated males were mated to females raised and subsequently maintained on tocopherol-enriched diet. (orig./MG)

  4. RAPD analysis on male sterility mutant of Lilium asiatic hybrids 'pollyanna' induced by irradiation

    International Nuclear Information System (INIS)

    Jia Yuehui; Zhao Xiangyun; Zhang Kezhong; Huang Shangwu; Lu Changxun

    2005-01-01

    RAPD analysis of 80 random 10-mer primers on Lilium Asiatic hybrids 'pollyanna' and its 20 phenotype male sterility mutants induced by irradiation was carried out. Of the tested primers, 31 primers could produced ideal amplification bands on all materials, 4 primers generated stable different polymorphic bands among 9 mutants and 'pollyanna'. Different polymorphic bands of 7-18 were found among 9 mutants and 'pollyanna'. It was showed that 9 mutants were phenotype male sterility mutant of 'pollyanna'. (authors)

  5. MIBG

    International Nuclear Information System (INIS)

    Orimo, Satoshi

    2010-01-01

    MIBG ([ 123 I]meta-iodobenzylguanidine) exhibits a similar behavior to noradrenaline (NA) in uptake, storage and release at presynaptic nerve ending (sympathetic nerve ending, SNE). This paper explains the outline of MIBG myocardial scintigraphy (MIBG-ms), MIBG-ms in Parkinson disease (PD), Parkinsonism and dementia (D), and patho-morphological base which authors have revealed, of reduced MIBG accumulation in Lewy body disease (DLB). At SNE, MIBG is actively taken up through NA transporter, stored in NA storage vesicle through the vesicle monoamine transporter and released at the nerve excitation by exocytosis, but does not bind to alpha/beta receptors: the mechanism of cardiac MIBG accumulation. Usually, authors inject MIBG at 111 MBq (3 mCi) to patients, and 15-30 min and 3-4 hr later, the early and late heart images are acquisitioned to see the accumulation and retention, respectively, with gamma-/single photon emission computed tomography (SPECT)-cameras. H/M (heart/mediastinum) count ratio and washout rate (difference between early and late counts/early count) for evaluation of sensitivity and specificity are applied for differential diagnosis of the subclasses of above mentioned diseases. For instance, early H/M is significantly lower than control in PD, PD I-II, and D with Lb, but not in multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration and Alzheimer disease. Authors have found through autopsy of 52 patients with DLB, Parkinsonism and/or Alzheimer disease that there are degeneration and denervation of cardiac SN in DLB and PARK 4 (a familial PD caused by LRRK2 alpha-synuclein gene redundancy), which are closely correlated with reduced MIBG accumulation in the heart. Thus the reduction is thought to be a bio-marker of the presence of LB, suggesting MIBG-ms can be a subsidiary diagnostic mean of such diseases as possessing LB. (T.T.)

  6. Web-based genome-wide association study identifies two novel loci and a substantial genetic component for Parkinson's disease.

    Directory of Open Access Journals (Sweden)

    Chuong B Do

    2011-06-01

    Full Text Available Although the causes of Parkinson's disease (PD are thought to be primarily environmental, recent studies suggest that a number of genes influence susceptibility. Using targeted case recruitment and online survey instruments, we conducted the largest case-control genome-wide association study (GWAS of PD based on a single collection of individuals to date (3,426 cases and 29,624 controls. We discovered two novel, genome-wide significant associations with PD-rs6812193 near SCARB2 (p = 7.6 × 10(-10, OR = 0.84 and rs11868035 near SREBF1/RAI1 (p = 5.6 × 10(-8, OR = 0.85-both replicated in an independent cohort. We also replicated 20 previously discovered genetic associations (including LRRK2, GBA, SNCA, MAPT, GAK, and the HLA region, providing support for our novel study design. Relying on a recently proposed method based on genome-wide sharing estimates between distantly related individuals, we estimated the heritability of PD to be at least 0.27. Finally, using sparse regression techniques, we constructed predictive models that account for 6%-7% of the total variance in liability and that suggest the presence of true associations just beyond genome-wide significance, as confirmed through both internal and external cross-validation. These results indicate a substantial, but by no means total, contribution of genetics underlying susceptibility to both early-onset and late-onset PD, suggesting that, despite the novel associations discovered here and elsewhere, the majority of the genetic component for Parkinson's disease remains to be discovered.

  7. Comprehensive research synopsis and systematic meta-analyses in Parkinson's disease genetics: The PDGene database.

    Directory of Open Access Journals (Sweden)

    Christina M Lill

    Full Text Available More than 800 published genetic association studies have implicated dozens of potential risk loci in Parkinson's disease (PD. To facilitate the interpretation of these findings, we have created a dedicated online resource, PDGene, that comprehensively collects and meta-analyzes all published studies in the field. A systematic literature screen of -27,000 articles yielded 828 eligible articles from which relevant data were extracted. In addition, individual-level data from three publicly available genome-wide association studies (GWAS were obtained and subjected to genotype imputation and analysis. Overall, we performed meta-analyses on more than seven million polymorphisms originating either from GWAS datasets and/or from smaller scale PD association studies. Meta-analyses on 147 SNPs were supplemented by unpublished GWAS data from up to 16,452 PD cases and 48,810 controls. Eleven loci showed genome-wide significant (P < 5 × 10(-8 association with disease risk: BST1, CCDC62/HIP1R, DGKQ/GAK, GBA, LRRK2, MAPT, MCCC1/LAMP3, PARK16, SNCA, STK39, and SYT11/RAB25. In addition, we identified novel evidence for genome-wide significant association with a polymorphism in ITGA8 (rs7077361, OR 0.88, P  =  1.3 × 10(-8. All meta-analysis results are freely available on a dedicated online database (www.pdgene.org, which is cross-linked with a customized track on the UCSC Genome Browser. Our study provides an exhaustive and up-to-date summary of the status of PD genetics research that can be readily scaled to include the results of future large-scale genetics projects, including next-generation sequencing studies.

  8. Transcriptomic Profiling of Extracellular RNAs Present in Cerebrospinal Fluid Identifies Differentially Expressed Transcripts in Parkinson’s Disease

    Science.gov (United States)

    Hossein-nezhad, Arash; Fatemi, Roya Pedram; Ahmad, Rili; Peskind, Elaine R.; Zabetian, Cyrus P.; Hu, Shu-Ching; Shi, Min; Wahlestedt, Claes; Zhang, Jing; Faghihi, Mohammad Ali

    2016-01-01

    Background: Parkinson’s disease (PD) is a debilitating neurological disorder for which prognostic and diagnostic biomarkers are lacking. Cerebrospinal fluid (CSF) is an accessible body fluid that comes into direct contact with the central nervous system (CNS) and acts as a nuclease-free repository where RNA transcripts shed by brain tissues can reside for extended periods of time. Objective: We studied the RNA species present in the CSF of PD patients to identify novel diagnostic biomarkers. Methods: Small volumes of CSF from 27 PD patients and 30 healthy age- and sex-matched controls were used for RNA extraction followed by next-generation sequencing (RNA-seq) using the Illumina platform. CSF contains a number of fragmented RNA species that were individually sequenced and analyzed. Comparing PD to control subjects, we observed a pool of dysregulated sequencing tags that were further analyzed and validated by quantitative real-time PCR (qRT-PCR). Results: A total of 201 differentially expressed sequencing tags (DETs), including 92 up-regulated and 109 down-regulated DETs were identified. We validated the following DETs by real time PCR in the patient samples: Dnmt1, Ezh2, CCR3, SSTR5,PTPRC, UBC, NDUFV2, BMP7, SCN9, SCN9 antisense (AC010127.3), and long noncoding RNAs AC079630 and UC001lva.4 (close to the LRRK2 gene locus), as potential PD biomarkers. Conclusions: The CSF is a unique environment that contains many species of RNA. Our work demonstrates that CSF can potentially be used to identify biomarkers for the detection and tracking of disease progression and evaluation of therapeutic outcomes. PMID:26889637

  9. Parkinsonian phenotype in Machado-Joseph disease (MJD/SCA3: a two-case report

    Directory of Open Access Journals (Sweden)

    Vasconcelos João

    2011-10-01

    Full Text Available Abstract Background Machado-Joseph disease (MJD, or spinocerebellar ataxia type 3 (SCA3, is an autosomal dominant neurodegenerative disorder of late onset, which is caused by a CAG repeat expansion in the coding region of the ATXN3 gene. This disease presents clinical heterogeneity, which cannot be completely explained by the size of the repeat tract. MJD presents extrapyramidal motor signs, namely Parkinsonism, more frequently than the other subtypes of autosomal dominant cerebellar ataxias. Although Parkinsonism seems to segregate within MJD families, only a few MJD patients develop parkinsonian features and, therefore, the clinical and genetic aspects of these rare presentations remain poorly investigated. The main goal of this work was to describe two MJD patients displaying the parkinsonian triad (tremor, bradykinesia and rigidity, namely on what concerns genetic variation in Parkinson's disease (PD associated loci (PARK2, LRRK2, PINK1, DJ-1, SNCA, MAPT, APOE, and mtDNA tRNAGln T4336C. Case presentation Patient 1 is a 40 year-old female (onset at 30 years of age, initially with a pure parkinsonian phenotype (similar to the phenotype previously reported for her mother. Patient 2 is a 38 year-old male (onset at 33 years of age, presenting an ataxic phenotype with parkinsonian features (not seen either in other affected siblings or in his father. Both patients presented an expanded ATXN3 allele with 72 CAG repeats. No PD mutations were found in the analyzed loci. However, allelic variants previously associated with PD were observed in DJ-1 and APOE genes, for both patients. Conclusions The present report adds clinical and genetic information on this particular and rare MJD presentation, and raises the hypothesis that DJ-1 and APOE polymorphisms may confer susceptibility to the parkinsonian phenotype in MJD.

  10. [Epidemiology and causes of Parkinson's disease].

    Science.gov (United States)

    Lill, C M; Klein, C

    2017-04-01

    Parkinson's disease (PD) is the second most common neurodegenerative disease and has a growing socioeconomic impact due to demographic changes in the industrial nations. There are several forms of PD, a fraction of which (parkinsonism including three autosomal dominantly (SNCA, LRRK2, VPS35) and three autosomal recessively inherited ones (Parkin, PINK1, DJ-1). In addition, there are a plethora of genes causing atypical forms of parkinsonism. In contrast, idiopathic PD is of a multifactorial nature. Genome-wide association studies have established a total of 26 genetic loci for this form of the disease; however, for most of these loci the underlying functional genetic variants have not yet been identified and the respective disease mechanisms remain unresolved. Furthermore, there are a number of environmental and life style factors that are associated with idiopathic PD. Exposure to pesticides and possibly a history of head trauma represent genuine risk factors. Other PD-associated factors, such as smoking and intake of coffee and alcohol may not represent risk factors per se and the cause-effect relationship has not yet been elucidated for most of these factors. A patient with a positive family history and/or an early age of disease onset should undergo counseling with respect to a possible monogenic form of the disease. Disease prediction based on genetic, environmental and life style factors is not yet possible for idiopathic PD and potential gene-specific therapies are currently in the development or early testing phase.

  11. Lysosomal impairment in Parkinson's disease.

    Science.gov (United States)

    Dehay, Benjamin; Martinez-Vicente, Marta; Caldwell, Guy A; Caldwell, Kim A; Yue, Zhenyue; Cookson, Mark R; Klein, Christine; Vila, Miquel; Bezard, Erwan

    2013-06-01

    Impairment of autophagy-lysosomal pathways (ALPs) is increasingly regarded as a major pathogenic event in neurodegenerative diseases, including Parkinson's disease (PD). ALP alterations are observed in sporadic PD brains and in toxic and genetic rodent models of PD-related neurodegeneration. In addition, PD-linked mutations and post-translational modifications of α-synuclein impair its own lysosomal-mediated degradation, thereby contributing to its accumulation and aggregation. Furthermore, other PD-related genes, such as leucine-rich repeat kinase-2 (LRRK2), parkin, and phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), have been mechanistically linked to alterations in ALPs. Conversely, mutations in lysosomal-related genes, such as glucocerebrosidase (GBA) and lysosomal type 5 P-type ATPase (ATP13A2), have been linked to PD. New data offer mechanistic molecular evidence for such a connection, unraveling a causal link between lysosomal impairment, α-synuclein accumulation, and neurotoxicity. First, PD-related GBA deficiency/mutations initiate a positive feedback loop in which reduced lysosomal function leads to α-synuclein accumulation, which, in turn, further decreases lysosomal GBA activity by impairing the trafficking of GBA from the endoplasmic reticulum-Golgi to lysosomes, leading to neurodegeneration. Second, PD-related mutations/deficiency in the ATP13A2 gene lead to a general lysosomal impairment characterized by lysosomal membrane instability, impaired lysosomal acidification, decreased processing of lysosomal enzymes, reduced degradation of lysosomal substrates, and diminished clearance of autophagosomes, collectively contributing to α-synuclein accumulation and cell death. According to these new findings, primary lysosomal defects could potentially account for Lewy body formation and neurodegeneration in PD, laying the groundwork for the prospective development of new neuroprotective/disease-modifying therapeutic strategies

  12. Comprehensive genetic study of fatty acids helps explain the role of noncoding inflammatory bowel disease associated SNPs and fatty acid metabolism in disease pathogenesis.

    Science.gov (United States)

    Jezernik, Gregor; Potočnik, Uroš

    2018-03-01

    Fatty acids and their derivatives play an important role in inflammation. Diet and genetics influence fatty acid profiles. Abnormalities of fatty acid profiles have been observed in inflammatory bowel diseases (IBD), a group of complex diseases defined by chronic gastrointestinal inflammation. IBD associated fatty acid profile abnormalities were observed independently of nutritional status or disease activity, suggesting a common genetic background. However, no study so far has attempted to look for overlap between IBD loci and fatty acid associated loci or investigate the genetics of fatty acid profiles in IBD. To this end, we conducted a comprehensive genetic study of fatty acid profiles in IBD using iCHIP, a custom microarray platform designed for deep sequencing of immune-mediated disease associated loci. This study identifies 10 loci associated with fatty acid profiles in IBD. The most significant associations were a locus near CBS (p = 7.62 × 10 -8 ) and a locus in LRRK2 (p = 1.4 × 10 -7 ). Of note, this study replicates the FADS gene cluster locus, previously associated with both fatty acid profiles and IBD pathogenesis. Furthermore, we identify 18 carbon chain trans-fatty acids (p = 1.12 × 10 -3 ), total trans-fatty acids (p = 4.49 × 10 -3 ), palmitic acid (p = 5.85 × 10 -3 ) and arachidonic acid (p = 8.58 × 10 -3 ) as significantly associated with IBD pathogenesis. Copyright © 2018 Elsevier Ltd. All rights reserved.

  13. Sec16 in conventional and unconventional exocytosis: Working at the interface of membrane traffic and secretory autophagy?

    Science.gov (United States)

    Tang, Bor Luen

    2017-12-01

    Sec16 is classically perceived to be a scaffolding protein localized to the transitional endoplasmic reticulum (tER) or the ER exit sites (ERES), and has a conserved function in facilitating coat protein II (COPII) complex-mediated ER exit. Recent findings have, however, pointed toward a role for Sec16 in unconventional exocytosis of certain membrane proteins, such as the Cystic fibrosis transmembrane conductance regulator (CFTR) in mammalian cells, and possibly also α-integrin in certain contexts of Drosophila development. In this regard, Sec16 interacts with components of a recently deciphered pathway of stress-induced unconventional exocytosis, which is dependent on the tether protein Golgi reassembly stacking proteins (GRASPs) and the autophagy pathway. Intriguingly, Sec16 also appears to be post-translationally modified by autophagy-related signaling processes. Sec16 is known to be phosphorylated by the atypical extracellular signal regulated kinase 7 (Erk7) upon serum and amino acid starvation, both represent conditions that trigger autophagy. Recent work has also shown that Sec16 is phosphorylated, and thus regulated by the prominent autophagy-initiating Unc-51-like autophagy activating kinase 1 (Ulk1), as well as another autophagy modulator Leucine-rich repeat kinase 2 (Lrrk2). The picture emerging from Sec16's network of physical and functional interactors allows the speculation that Sec16 is situated (and may in yet undefined ways function) at the interface between COPII-mediated exocytosis of conventional vesicular traffic and the GRASP/autophagy-dependent mode of unconventional exocytosis. © 2017 Wiley Periodicals, Inc.

  14. Mutagenic effect of radionuclides incorporated into DNA of Drosophila melanogaster. Progress report, 1981-1982

    International Nuclear Information System (INIS)

    Lee, W.R.

    1982-01-01

    Research during the 1981-1982 year emphasized the development of tests that can distinguish between mutations induced at the alcohol dehydrogenase (Adh) locus in Drosophila melanogaster by ionizing radiation, which induces largely deletions, and mutations that result from single base changes. For development of these tests three alleles at the Adh locus were used which have been shown by sequencing to differ by only a single base change, and for contrast a group of mutants induced by x-rays were used in which at least 71% of the mutants were deletions. Two tests that give complementary information were selected and progress in validation is described

  15. ''2 + 1'' Mechanism as the basis for synergistic action of neutron-photon irradiation of the genome of Drosophila melanogaster spermatozoa

    International Nuclear Information System (INIS)

    Aleksandrov, I.D.; Aleksandrova, M.V.; Lapidus, I.L.

    1996-01-01

    Cytogenetic analysis of polythene chromosomes of Drosophila melanogaster locus-specific mutants induced by consecutive neutron-photon irradiation has shown that their genome contains multiple intra- and inter-chromosome exchange, including triradials, evidencing the synergistic action of such combined exposure. The appearance of the triradials may be only possible on the base of an interaction between a double and a single DNA strand breaks. The important significance of such interaction as the general mechanism for production of chromosome aberrations in irradiated cells of higher eucaryotes had been postulated by N.V. Luchnik as early as 10 years ago, but only nowadays it has been confirmed experimentally

  16. [Genetic improvement of technological characteristics of starters for fermented milk products].

    Science.gov (United States)

    Oganesian, G G; Barsegian, A A; Grigorian, N G; Toptsian, A V

    2010-01-01

    Possibility for improvement of technological characteristics of lactobacilli using mutations of resistance to rifampicin (rif(r)) and streptomycin (str(r)) was studied. Using starter model of Narine Lactobacillus acidophilus INMIA-9602 Armenian diet milk product, it was showed that a possibility for selecting strains with increased rate of milk fermentation and acid production is higher in Rif(r) and Str(r) mutants induced by nitrosoguanidine than in cultures sensitive to antibiotics. The milk products obtained using Rif(r) and Str(r) strains had high viscosity, improved texture, increased amount of alive cells and good organoleptic features.

  17. Development of new iraqi wheat varieties induced by gamma rays

    International Nuclear Information System (INIS)

    Ibrahim, I.F.; Al-Janabi, K.K.; Al-Maaroof, E.M.; Al-Aubaidi, M.O.; Mahmoud, A.H.; Al-Janabi, A.A.

    1991-01-01

    The aim of the present investigation is to study agronomic traits of three wheat mutants induced by gamma rays and compared with their origin 'Saber Beg' during M 8 - M 11 generations. These mutants showed a moderate resistance to leaf rust and lodging, while the origin was susceptible. Also, these mutants surpassed their origin in seed weight of 100 spikes, weight of 1000 kernels and protein yield per unit area. Chemical and physical analyses of mutant flours indicated that it could be used for bread making successfully.2 fig.,4 tab

  18. The experience of induction of mutation on barley in Peru

    International Nuclear Information System (INIS)

    Romero Loli, M.; Pozo Cardenas, M.; Gomez Pando, L.

    1984-01-01

    Work on induced mutation of barley was started in 1978 under the Programme of Cereal Improvement. Barley was irradiated with gamma radiation at doses of 12, 15, 18, 21, and 24 Krad. Radiation doses of 18 and 21 Krad gave the highest frequency of albino and cloroticos mutants. Induced mutation is being carried out in different parts of the country to develop mutants having early germination property. These mutants will play an important role in the late cultivation in the mountain areas of Peru

  19. Screening of respiration deficiency mutants of yeasts (Saccharomyces cerevisiae) induced by ion irradiation and the mtDNA restriction analysis

    International Nuclear Information System (INIS)

    Mao Shuhong; Chinese Academy of Sciences, Beijing; Jin Genming; Wei Zengquan; Xie Hongmei; Ma Qiufeng; Gu Ying

    2005-01-01

    Screening of the respiration deficiency mutants of Saccharomyces cerevisiae induced by 5.19 MeV/u 22 Ne 5+ ion irradiation is reported in this paper. Some respiration deficiency mutants of white colony phenotype, in a condition of selective culture of TTC medium, were obtained. A new and simplified method based on mtDNA restriction analysis is described. The authors found that there are many obvious differences in mtDNAs between wild yeasts and the respiration deficiency mutants. The mechanism of obtaining the respiration deficiency mutants induced by heavy ion irradiation is briefly discussed. (authors)

  20. Molecular characterization of thymidine kinase mutants of human cells induced by densely ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Kronenberg, A; Little, J B

    1989-04-01

    In order to characterize the nature of mutants induced by densely ionizing radiations at an autosomal locus, the authors have isolated a series of 99 thymidine kinase (tk) mutants of human TK6 lymphoblastoid cells iraadiated with either fast neutrons or accelerated argon ions. Individual muant clones were examined for alterations in their restriction fragment pattern after hybridization with a human cDNA probe for tk. A restriction fragment length polymorphism (RFLP) allowed identification of the active tk allele. Among the neutron-induced mutants, 34/52 exhibited loss of the previously active allele while 6/52 exhibited intragenic rearrangements. Among the argon-induced mutants 27/46 exhibited allele loses and 10/46 showed rearrangements within the tk locus. The remaining mutants had restriction patterns indistinguishable from the TK6 parent. Each of the mutant clones was further examined for structural alterations within the c-erbAl locus which has been localized to chromosome 17q11-q22, at some unknown distance from the human tk locus at chromosome 17q21-q22. A substantial proportion (54%) of tk mutants induced by densely ionizing radiation showed loss of the c-erb locus on the homologous chromosome, suggesting that the mutations involve large-scale genetic changes. (author). 51 refs.; 2 figs.; 6 tabs.

  1. Molecular analysis of waxy mutants in rice

    International Nuclear Information System (INIS)

    Yatou, O.; Amano, E.

    1990-01-01

    Full text: The 'waxy' gene is a structural gene coding a glycosyl transferase which synthesises amylose in the endosperm tissue. 'Non-waxy' rice cultivars have an active gene and their amylose content is 18-25% depending upon gene performance and modifier genes. In 'waxy' rice, no amylose is found because the enzyme is absent. In mutants induced by gamma rays, neutrons, EI or EMS, amylose content ranged from 0 to 20%, i.e. there are intermediate phenotypes as well. Some of them had the same amount of the enzyme as a 'non-waxy' cultivar, even fully 'waxy' mutants showed a certain amount of the enzyme. This suggests that in mutants there may be no structural change in the enzyme gene but the enzyme produced might be less active. By molecular analysis of the mutants' genes it was found that only two mutants induced by thermal neutrons show structural alterations, the changes in other mutants are either too small to be detected by Southern analysis or are outside the structural gene in question. (author)

  2. Mutagenic effect of tritium on DNA of Drosophila melanogaster: Technical progress report, December 15, 1986-July 15, 1987

    International Nuclear Information System (INIS)

    Lee, W.R.

    1987-01-01

    Recombinant DNA techniques were used to analyze mutants induced by either tritium or x-ray. Mutations induced at the alcohol dehydrogenase locus (Adh) in Drosophila melanogaster were first characterized by genetic complementation tests to determine if a multi-locus deletion has occurred. Mutants that are intragenic as defined by the complementation test are then placed opposite a deficiency so that the DNA from the mutant allele may be extracted and analyzed. Part I of the project is to analyze mutants induced by ionizing radiation with molecular techniques, and part II is to determine the molecular effects of these mutant phenotypes on their expression in the polypeptide produced by the mutant gene. Part III of this project consists of inducing mutants with tritiated water at the Adh locus in D. melanogaster. We have reported the development of a feeding method for exposing male D. melanogaster to tritiated water that would give a range in dose from 6.66 Gy to 26.64 Gy. This method of exposing Drosophila was used first to study a dose response curve for tritium using as a genetic endpoint the sex-linked recessive lethal test. 3 figs., 1 tab

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

    International Nuclear Information System (INIS)

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

    1984-01-01

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

  4. Molecular and biochemical analyses of spontaneous and X-ray-induced mutants in human lymphoblastoid cells

    Energy Technology Data Exchange (ETDEWEB)

    Liber, H L; Call, K M; Little, J B

    1987-05-01

    The authors have isolated a series of 14 spontaneously arising and 28 X-ray-induced mutants at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in human lymphoblastoid cells. Among the spontaneous mutants, 5/14 (36%) had detectable alterations in their restriction fragment pattern after hybridization with a human cDNA probe for hgprt. Of the 10 remaining mutants, 4 had partial HGPRT enzyme activity, which suggested that they contained point mutations. Among the 28 mutants induced by 150 rad of X-rays, 15 (54%) had deletions of part or all of the hgprt gene. Of the remaining 13 (18% overall) 5 had partial HGPRT enzyme activity, which suggested that they contained point mutations. These data imply that in this human cell system, X-rays induce both point mutants which have residual enzyme activity as well as mutations involving relatively large deletions of DNA. 48 reference, 1 figure, 4 tables.

  5. An induced early mutant in finger millet Eleusin coranaca Gaertn

    International Nuclear Information System (INIS)

    Shivashankar, G.; Kempanna, C.; Viswanatha, S.R.

    1975-01-01

    Among the several collections of ragi (Eleusine coracana. Gaertn.) from all over the world, the strain 'H.E.S.927' has been found to be the highest yielder compared to other cultivars. Mutation studies have been conducted on this variety at Bangalore in India to make it suitable for the rainfall pattern of the ragi tract of the Karnataka state. A mutant induced by gamma radiation at 30 K rad has been stabilized. This mutant is early by 30-35 days with comparatively better straw quality. Various morphological characters concerning the yield and yield attributes and the possibility of exploiting it as a genotype for future breeding work and as a promising variety is discussed. Another promising mutant with earhead measuring 15 cm as against the earhead length of 5 to 10 cm of the cultivated varieties is illustrated. (K.B.)

  6. Progress in the exploitation of new dwarfing genes in Chinese rice breeding

    International Nuclear Information System (INIS)

    Zhu, L.H.; Xie, Z.Q.

    1990-01-01

    Full text: Use of dwarfing genes in rice breeding in China began in the late 1950's. Since then, the major source of dwarfism has been 'Ai-zi-Zhan' with a gene allelic to sd-1 from 'Dee-Geo-Woo-Gen'. Since the 1970's, Chinese rice breeders paid attention to exploring new dwarfing gene sources and these efforts resulted in at least 7 sources non-allelic to sd-1. The late indica variety 'Gui-Yang-Ai No. 1' possessing gene dg(t) or sdg[t) proved to be most promising. The new sources include mutants induced by irradiation in 'Nanjing 11' and 'Nanjing 15'. The new genes are integrated in breeding to further improve plant type and adaptability. (author)

  7. Induction and assay of pure soyabean mutants obtained from gamma irradiation

    International Nuclear Information System (INIS)

    Nasseri Tafti, M.; Rezazadeh, M.; Yousefi, F.; Sabzi, H.

    2002-01-01

    Gamma ray is an electromagnetic type of radiation and produces ions when passing through biological matter. It can be applied in plant breeding to induce variation. The most important character of this ray is to produce changes in DNA structure existing in cell. Mutants induced by irradiation of soybean seeds were assayed for their agronomic traits. Two locations were used for this purpose, Alishtar and Karaj. There were significant differences between soybean mutant lines and their check cv. Williams at 1% level and cv.Clark at 5% level. Line No. 47 with 4782 kg/hect. Possessed the top of the list and next to it line No.38 with 4722 kg/hect. Some mutant lines reached maturity 10 to 12 days earlier than commercial cv s used as check cultivars

  8. Sensitivity to ultraviolet radiation in a dominantly inherited form of xeroderma pigmentosum

    International Nuclear Information System (INIS)

    Imray, F.P.; Relf, W.; Ramsay, R.G.; Kidson, C.; Hockey, A.

    1986-01-01

    An Australian family is described in which a mild form of xeroderma pigmentosum (XP) is inherited as an autosomal dominant trait. Studies of lymphoblastoid cells and fibroblasts from affected person demonstrated sensitivity to ultraviolet (UV) light as judged by diminished clonogenicity and higher frequencies of UV induced chromosome aberrations compared to normal controls. After UV irradiation of dominant XP cells, replicative DNA synthesis was depressed to a greater extent than normal and the level of UV induced DNA repair synthesis was lower than that in normal cells. The level of sister chromatid exchanges and the numbers of 6-thioguanine resistant mutants induced by UV irradiation were equal to those found in normal controls. Although two subjects in the family had skin cancers, this dominant form of XP is not apparently associated with high risk, or large numbers of skin cancers in affected persons. (author)

  9. AID/APOBEC cytosine deaminase induces genome-wide kataegis

    Directory of Open Access Journals (Sweden)

    Lada Artem G

    2012-12-01

    Full Text Available Abstract Clusters of localized hypermutation in human breast cancer genomes, named “kataegis” (from the Greek for thunderstorm, are hypothesized to result from multiple cytosine deaminations catalyzed by AID/APOBEC proteins. However, a direct link between APOBECs and kataegis is still lacking. We have sequenced the genomes of yeast mutants induced in diploids by expression of the gene for PmCDA1, a hypermutagenic deaminase from sea lamprey. Analysis of the distribution of 5,138 induced mutations revealed localized clusters very similar to those found in tumors. Our data provide evidence that unleashed cytosine deaminase activity is an evolutionary conserved, prominent source of genome-wide kataegis events. Reviewers This article was reviewed by: Professor Sandor Pongor, Professor Shamil R. Sunyaev, and Dr Vladimir Kuznetsov.

  10. Mutation induction by ion beams in plants

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, Atsushi [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

    2001-03-01

    The effect of ion beams such as C, He, and Ne ions was investigated on the mutation induction in plants with the expectation that ion beams of high linear energy transfer (LET) can frequently produce large DNA alternation such as inversion, translocation and large deletion rather than point mutation. Mutation frequency was investigated using Arabidopsis visible phenotype loci and was 8 to 33 fold higher for 220 MeV carbon ions than for electrons. Mutation spectrum was investigated on the flower color of chrysanthemum cv to find that flower mutants induced by ion beams show complex and stripe types rather than single color. Polymerase chain reaction analysis was performed to investigate DNA alteration of mutations. In conclusion, the characteristics of ion beams for the mutation induction are 1) high frequency, 2) broad mutation spectrum, and 3) novel mutants. (S. Ohno)

  11. Mutation induction by ion beams in plants

    International Nuclear Information System (INIS)

    Tanaka, Atsushi

    2001-01-01

    The effect of ion beams such as C, He, and Ne ions was investigated on the mutation induction in plants with the expectation that ion beams of high linear energy transfer (LET) can frequently produce large DNA alternation such as inversion, translocation and large deletion rather than point mutation. Mutation frequency was investigated using Arabidopsis visible phenotype loci and was 8 to 33 fold higher for 220 MeV carbon ions than for electrons. Mutation spectrum was investigated on the flower color of chrysanthemum cv to find that flower mutants induced by ion beams show complex and stripe types rather than single color. Polymerase chain reaction analysis was performed to investigate DNA alteration of mutations. In conclusion, the characteristics of ion beams for the mutation induction are 1) high frequency, 2) broad mutation spectrum, and 3) novel mutants. (S. Ohno)

  12. PI5P Triggers ICAM-1 Degradation in Shigella Infected Cells, Thus Dampening Immune Cell Recruitment

    Directory of Open Access Journals (Sweden)

    Frédéric Boal

    2016-02-01

    Full Text Available Shigella flexneri, the pathogen responsible for bacillary dysentery, has evolved multiple strategies to control the inflammatory response. Here, we show that Shigella subverts the subcellular trafficking of the intercellular adhesion molecule-1 (ICAM-1, a key molecule in immune cell recruitment, in a mechanism dependent on the injected bacterial enzyme IpgD and its product, the lipid mediator PI5P. Overexpression of IpgD, but not a phosphatase dead mutant, induced the internalization and the degradation of ICAM-1 in intestinal epithelial cells. Remarkably, addition of permeant PI5P reproduced IpgD effects and led to the inhibition of neutrophil recruitment. Finally, these results were confirmed in an in vivo model of Shigella infection where IpgD-dependent ICAM-1 internalization reduced neutrophil adhesion. In conclusion, we describe here an immune evasion mechanism used by the pathogen Shigella to divert the host cell trafficking machinery in order to reduce immune cell recruitment.

  13. Mutation effects of kojic acid production strain induced by synchrotron radiation of soft X-rays and kinetics of the fermentation

    International Nuclear Information System (INIS)

    Chen Liang; Jiang Shiping; Wan Libiao; Ma Xiaodong; Li Meifang

    2006-01-01

    The irradiation effect on Aspergillus oryzae spores was studied by 0.54 keV X-rays (about the K shell absorption edge of oxygen) from the synchrotron facility at NSRL. A high production mutant for kojic acid was obtained from the spores irradiated by soft X-rays, which accumulated 27.79 g kojic acid per L in 500 mL shake-flask fermentation for 10 days using glucose as carbon source, and has increased about 56% of production than that of the original (17.81 g/L). Also the fermentation conditions were studied with different carbon sources and nitrogen sources. The results showed that the mutant induced by the X-rays of synchrotron radiation would be of potential for high kojic acid production. (authors)

  14. Clonal analysis of the progeny of UV-irradiated cells of Bacillus subtilis (uvr+ and uvr)

    International Nuclear Information System (INIS)

    Lotareva, O.V.; Filippov, V.D.

    1975-01-01

    The revertants to adenine prototrophy or mutants to auxotrophy can be easily identified on synthetic media which are pathly enriched with caseine hydrolyzate and yeast extract. It is shown with the use of these media that 1.5% colonies formed by Bacillus subtilis cells of the original type (ade6 met5) have mutant clones which are initiated by spontaneous revertants to adenine prototrophy. These revertants arise in the time of division of cells in macrocolonies. After plating diluted suspension of irradiated cells those colonies which contain mutant clones formed by spontaneous revertants can be erroneously taken for mixed colonies formed by induced revertants. About 40% mutants to auxotrophy induced by high dose of UV-light in uvr + cells form pure mutant colonies. The same mutants, induced in uvr cells by a five-times as-low UV-dose, usually form mixed colonies

  15. Fibrinogen binding sites P336 and Y338 of clumping factor A are crucial for Staphylococcus aureus virulence.

    Directory of Open Access Journals (Sweden)

    Elisabet Josefsson

    Full Text Available We have earlier shown that clumping factor A (ClfA, a fibrinogen binding surface protein of Staphylococcus aureus, is an important virulence factor in septic arthritis. When two amino acids in the ClfA molecule, P(336 and Y(338, were changed to serine and alanine, respectively, the fibrinogen binding property was lost. ClfAP(336Y(338 mutants have been constructed in two virulent S. aureus strains Newman and LS-1. The aim of this study was to analyze if these two amino acids which are vital for the fibrinogen binding of ClfA are of importance for the ability of S. aureus to generate disease. Septic arthritis or sepsis were induced in mice by intravenous inoculation of bacteria. The clfAP(336Y(338 mutant induced significantly less arthritis than the wild type strain, both with respect to severity and frequency. The mutant infected mice developed also a much milder systemic inflammation, measured as lower mortality, weight loss, bacterial growth in kidneys and lower IL-6 levels. The data were verified with a second mutant where clfAP(336 and Y(338 were changed to alanine and serine respectively. When sepsis was induced by a larger bacterial inoculum, the clfAP(336Y(338 mutants induced significantly less septic death. Importantly, immunization with the recombinant A domain of ClfAP(336SY(338A mutant but not with recombinant ClfA, protected against septic death. Our data strongly suggest that the fibrinogen binding activity of ClfA is crucial for the ability of S. aureus to provoke disease manifestations, and that the vaccine potential of recombinant ClfA is improved by removing its ability to bind fibrinogen.

  16. Fibrinogen binding sites P336 and Y338 of clumping factor A are crucial for Staphylococcus aureus virulence.

    Science.gov (United States)

    Josefsson, Elisabet; Higgins, Judy; Foster, Timothy J; Tarkowski, Andrej

    2008-05-21

    We have earlier shown that clumping factor A (ClfA), a fibrinogen binding surface protein of Staphylococcus aureus, is an important virulence factor in septic arthritis. When two amino acids in the ClfA molecule, P(336) and Y(338), were changed to serine and alanine, respectively, the fibrinogen binding property was lost. ClfAP(336)Y(338) mutants have been constructed in two virulent S. aureus strains Newman and LS-1. The aim of this study was to analyze if these two amino acids which are vital for the fibrinogen binding of ClfA are of importance for the ability of S. aureus to generate disease. Septic arthritis or sepsis were induced in mice by intravenous inoculation of bacteria. The clfAP(336)Y(338) mutant induced significantly less arthritis than the wild type strain, both with respect to severity and frequency. The mutant infected mice developed also a much milder systemic inflammation, measured as lower mortality, weight loss, bacterial growth in kidneys and lower IL-6 levels. The data were verified with a second mutant where clfAP(336) and Y(338) were changed to alanine and serine respectively. When sepsis was induced by a larger bacterial inoculum, the clfAP(336)Y(338) mutants induced significantly less septic death. Importantly, immunization with the recombinant A domain of ClfAP(336)SY(338)A mutant but not with recombinant ClfA, protected against septic death. Our data strongly suggest that the fibrinogen binding activity of ClfA is crucial for the ability of S. aureus to provoke disease manifestations, and that the vaccine potential of recombinant ClfA is improved by removing its ability to bind fibrinogen.

  17. Cytosine arabinoside enhancement of gamma irradiation induced mutations in human T-lymphocytes

    International Nuclear Information System (INIS)

    O'Neill, J.P.; Sullivan, L.M.; Hunter, T.C.; Nicklas, J.A.

    1991-01-01

    The frequency of 6-thioguanine resistant (TGr) mutants induced in human G0 phase T-lymphocytes by 200 cGy of gamma irradiation is greatly enhanced by incubation with cytosine arabinoside (ara-C) after irradiation. The mutant frequency increased with increasing incubation time in ara-C for up to 2 hr. This mutation induction required a phenotypic expression time of 5-8 days mass culture growth, similar to that found with mutants induced by 300 cGy of irradiation alone. Southern blot analysis of 40 isolated mutant clones revealed 8 independent mutations by T-cell receptor (TCR) gene rearrangement patterns. Four of these eight showed hprt gene structural alterations (0.50). An alternative method to allow phenotypic expression was developed to minimize the isolation of hprt/TCR sibling mutants. The use of in situ expression in the microtiter dish wells resulted in the isolation of 17 independent mutations in 19 mutant clones. Ten of these 17 mutations showed hprt structural alterations (0.59). The high fraction of mutations involving structural alterations detected by Southern blot analysis is consistent with the known induction of chromosome aberrations by irradiation plus ara-C treatment. We propose that both the increase in Mf and the increase in the incidence of hprt gene structural alterations are due to the accumulation of strand breaks in repairing regions of DNA under these conditions of ara-C induced inhibition of repair. We further propose that upon release of the ara-C inhibition, these repairing regions can interact to yield both gene mutations and chromosome aberrations

  18. Immune-related genetic enrichment in frontotemporal dementia: An analysis of genome-wide association studies.

    Directory of Open Access Journals (Sweden)

    Iris Broce

    2018-01-01

    Full Text Available Converging evidence suggests that immune-mediated dysfunction plays an important role in the pathogenesis of frontotemporal dementia (FTD. Although genetic studies have shown that immune-associated loci are associated with increased FTD risk, a systematic investigation of genetic overlap between immune-mediated diseases and the spectrum of FTD-related disorders has not been performed.Using large genome-wide association studies (GWASs (total n = 192,886 cases and controls and recently developed tools to quantify genetic overlap/pleiotropy, we systematically identified single nucleotide polymorphisms (SNPs jointly associated with FTD-related disorders-namely, FTD, corticobasal degeneration (CBD, progressive supranuclear palsy (PSP, and amyotrophic lateral sclerosis (ALS-and 1 or more immune-mediated diseases including Crohn disease, ulcerative colitis (UC, rheumatoid arthritis (RA, type 1 diabetes (T1D, celiac disease (CeD, and psoriasis. We found up to 270-fold genetic enrichment between FTD and RA, up to 160-fold genetic enrichment between FTD and UC, up to 180-fold genetic enrichment between FTD and T1D, and up to 175-fold genetic enrichment between FTD and CeD. In contrast, for CBD and PSP, only 1 of the 6 immune-mediated diseases produced genetic enrichment comparable to that seen for FTD, with up to 150-fold genetic enrichment between CBD and CeD and up to 180-fold enrichment between PSP and RA. Further, we found minimal enrichment between ALS and the immune-mediated diseases tested, with the highest levels of enrichment between ALS and RA (up to 20-fold. For FTD, at a conjunction false discovery rate < 0.05 and after excluding SNPs in linkage disequilibrium, we found that 8 of the 15 identified loci mapped to the human leukocyte antigen (HLA region on Chromosome (Chr 6. We also found novel candidate FTD susceptibility loci within LRRK2 (leucine rich repeat kinase 2, TBKBP1 (TBK1 binding protein 1, and PGBD5 (piggyBac transposable element

  19. The effects of modeled microgravity on growth kinetics, antibiotic susceptibility, cold growth, and the virulence potential of a Yersinia pestis ymoA-deficient mutant and its isogenic parental strain.

    Science.gov (United States)

    Lawal, Abidat; Kirtley, Michelle L; van Lier, Christina J; Erova, Tatiana E; Kozlova, Elena V; Sha, Jian; Chopra, Ashok K; Rosenzweig, Jason A

    2013-09-01

    Previously, we reported that there was no enhancement in the virulence potential (as measured by cell culture infections) of the bacterial pathogen Yersinia pestis (YP) following modeled microgravity/clinorotation growth. We have now further characterized the effects of clinorotation (CR) on YP growth kinetics, antibiotic sensitivity, cold growth, and YP's virulence potential in a murine model of infection. Surprisingly, none of the aforementioned phenotypes were altered. To better understand why CR did not enhance YP's virulence potential as it did for other bacterial pathogens, a YP ΔymoA isogenic mutant in the KIM/D27 background strain that is unable to produce the histone-like YmoA protein and influences DNA topography was used in both cell culture and murine models of infection. YmoA represses type three secretion system (T3SS) virulence gene expression in the yersiniae. Similar to our CR-grown parental YP strain data, the CR-grown ΔymoA mutant induced reduced HeLa cell cytotoxicity with concomitantly decreased Yersinia outer protein E (YopE) and low calcium response V (LcrV) antigen production and secretion. Important, however, were our findings that, although no significant differences were observed in survival of mice infected intraperitoneally with either normal gravity (NG)- or CR-grown parental YP, the ΔymoA mutant induced significantly more mortality in infected mice than did the parental strain following CR growth. Taken together, our data demonstrate that CR did enhance the virulence potential of the YP ΔymoA mutant in a murine infection model (relative to the CR-grown parental strain), despite inducing less HeLa cell rounding in our cell culture infection assay due to reduced T3SS activity. Therefore, CR, which induces a unique type of bacterial stress, might be enhancing YP's virulence potential in vivo through a T3SS-independent mechanism when the histone-like YmoA protein is absent.

  20. hREV3 is essential for error-prone translesion synthesis past UV or benzo[a]pyrene diol epoxide-induced DNA lesions in human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Li Ziqiang; Zhang Hong; McManus, Terrence P.; McCormick, J. Justin; Lawrence, Christopher W.; Maher, Veronica M

    2002-12-29

    In S. cerevisiae, the REV3 gene, encoding the catalytic subunit of polymerase zeta, is involved in translesion synthesis and required for the production of mutations induced by ultraviolet radiation (UV) photoproducts and other DNA fork-blocking lesions, and for the majority of spontaneous mutations. To determine whether hREV3, the human homolog of yeast REV3, is similarly involved in error-prone translesion synthesis past UV photoproducts and other lesions that block DNA replication, an hREV3 antisense construct under the control of the TetP promoter was transfected into an infinite life span human fibroblast cell strain that expresses a high level of tTAk, the activator of that promoter. Three transfectant strains expressing high levels of hREV3 antisense RNA were identified and compared with their parental cell strain for sensitivity to the cytotoxic and mutagenic effects of UV. The three hREV3 antisense-expressing cell strains were not more sensitive than the parental strain to the cytotoxic effect of UV, but the frequency of mutants induced by UV in their HPRT gene was significantly reduced, i.e. to 14% that of the parent. Two of these hREV3 antisense-expressing cell strains were compared with the parental strain for sensitivity to ({+-})-7{beta},8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydro= benzo[a]pyrene (BPDE). They were not more sensitive than the parent strain to the cytotoxic effect of BPDE, but the frequency of mutants induced was significantly reduced, i.e. in one strain, to 17% that of the parent, and in the other, to 24%. DNA sequencing showed that the kinds of mutations induced by BPDE in the parental and the derivative strains did not differ and were similar to those found previously with finite life span human fibroblasts. The data strongly support the hypothesis that hRev3 plays a critical role in the induction of mutations by UV or BPDE. Because the level of hRev3 protein in human fibroblasts is below the level of antibody

  1. hREV3 is essential for error-prone translesion synthesis past UV or benzo[a]pyrene diol epoxide-induced DNA lesions in human fibroblasts

    International Nuclear Information System (INIS)

    Li Ziqiang; Zhang Hong; McManus, Terrence P.; McCormick, J. Justin; Lawrence, Christopher W.; Maher, Veronica M.

    2002-01-01

    In S. cerevisiae, the REV3 gene, encoding the catalytic subunit of polymerase zeta, is involved in translesion synthesis and required for the production of mutations induced by ultraviolet radiation (UV) photoproducts and other DNA fork-blocking lesions, and for the majority of spontaneous mutations. To determine whether hREV3, the human homolog of yeast REV3, is similarly involved in error-prone translesion synthesis past UV photoproducts and other lesions that block DNA replication, an hREV3 antisense construct under the control of the TetP promoter was transfected into an infinite life span human fibroblast cell strain that expresses a high level of tTAk, the activator of that promoter. Three transfectant strains expressing high levels of hREV3 antisense RNA were identified and compared with their parental cell strain for sensitivity to the cytotoxic and mutagenic effects of UV. The three hREV3 antisense-expressing cell strains were not more sensitive than the parental strain to the cytotoxic effect of UV, but the frequency of mutants induced by UV in their HPRT gene was significantly reduced, i.e. to 14% that of the parent. Two of these hREV3 antisense-expressing cell strains were compared with the parental strain for sensitivity to (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). They were not more sensitive than the parent strain to the cytotoxic effect of BPDE, but the frequency of mutants induced was significantly reduced, i.e. in one strain, to 17% that of the parent, and in the other, to 24%. DNA sequencing showed that the kinds of mutations induced by BPDE in the parental and the derivative strains did not differ and were similar to those found previously with finite life span human fibroblasts. The data strongly support the hypothesis that hRev3 plays a critical role in the induction of mutations by UV or BPDE. Because the level of hRev3 protein in human fibroblasts is below the level of antibody detection, it was not

  2. Deletions induced by gamma rays in the genome of Escherichia coli

    International Nuclear Information System (INIS)

    Raha, Manidipa; Hutchinson, Franklin

    1991-01-01

    An Escherichia coli lysogen was constructed with a lambda phage bearing a lacZ gene surrounded by about 100 x 10 3 base-pairs of dispensable DNA. The lacZ mutants induced by gamma rays in this lysogen were more than 10% large deletions, ranging in size from 0.6 x 10 -3 to 70 x 10 3 base-pairs. These deletions were centered, not on lacZ, but on a ColE1 origin of DNA replication located 1.2 x 10 3 bases downstream from lacZ, suggesting that this origin of replication was involved in the process by which deletions were formed. In agreement with this hypothesis, a lysogen of the same phage without the ColE1 origin showed a very much lower percentage of radiation-induced deletions, as did a second lysogen of a lambda phage without any known plasmid origin of replication. Indirect evidence is presented for radiation-induced deletions centered on the lambda origin of DNA replication in a lysogen. (author)

  3. Molecular mechanisms of mutagenesis determined by the recombinant DNA technology

    International Nuclear Information System (INIS)

    Lee, W.R.

    1985-01-01

    A study of the alteration of the DNA in the mutant gene can determine mechanisms of mutation by distinguishing between mutations induced by transition, transversion, frameshifts of a single base and deletions involving many base pairs. The association of a specific pattern of response with a mutagen will permit detecting mutants induced by the mutagen with a reduced background by removing mutations induced by other mechanisms from the pool of potential mutants. From analyses of studies that have been conducted, it is quite apparent that there are substantial differences among mutagens in their modes of action. Of 31 x-ray induced mutants, 20 were large deletions while only 3 showed normal Southern blots. Only one mutant produced a sub-unit polypeptide of normal molecular weight and charge in the in vivo test whereas in vitro synthesis produced a second one. In contrast, nine of thirteen EMS induced mutants produced cross-reacting proteins with sub-unit polypeptide molecular weights equivalent to wild type. Two of three ENU induced mutants recently analyzed in our laboratory produced protein with sub-unit polypeptide molecular weight and electrical charge similar to the wild type stock in which the mutants were induced. One ENU induced mutation is a large deletion. 21 refs., 1 fig

  4. Reduction of Aspergillus niger Virulence in Apple Fruits by Deletion of the Catalase Gene cpeB.

    Science.gov (United States)

    Zhang, Meng-Ke; Tang, Jun; Huang, Zhong-Qin; Hu, Kang-Di; Li, Yan-Hong; Han, Zhuo; Chen, Xiao-Yan; Hu, Lan-Ying; Yao, Gai-Fang; Zhang, Hua

    2018-05-30

    Aspergillus niger, a common saprophytic fungus, causes rot in many fruits. We studied the role of a putative catalase-peroxidase-encoding gene, cpeB, in oxidative stress and virulence in fruit. The cpeB gene was deleted in A. niger by homologous recombination, and the Δ cpeB mutant showed decreased CAT activity compared with that of the wild type. The cpeB gene deletion caused increased sensitivity to H 2 O 2 stress, and spore germination was significantly reduced; in addition, the reactive-oxygen-species (ROS) metabolites superoxide anions (·O 2 - ), hydrogen peroxide (H 2 O 2 ), and malondialdehyde (MDA) accumulated in the Δ cpeB mutant during H 2 O 2 stress. Furthermore, ROS metabolism in A. niger infected apples was determined, and our results showed that the Δ cpeB mutant induced an attenuated response in apple fruit during the fruit-pathogen interaction; the cpeB gene deletion significantly reduced the development of lesions, suggesting that the cpeB gene in A. niger is essential for full virulence in apples.

  5. Molecular analysis of mutant and wild type alcohol dehydrogenase alleles from Drosophila

    International Nuclear Information System (INIS)

    Batzer, M.A.

    1988-01-01

    Wild type alcohol dehydrogenase polypeptides (ADH) from Drosophila melanogaster transformants were examined using western blots and polyclonal antiserum specific for Drosophila melanogaster ADH. Mutants induced in Drosophila spermatozoa at the alcohol dehydrogenase (Adh) locus using X-rays, 1-ethyl-1-nitrosourea (ENU) or ethyl methanesulfonate (EMS) were characterized using genetic complementation tests, western blots, Southern blots, northern blots and enzymatic amplification of the Adh locus. Genetic complementation tests showed that 22/30 X-ray-induced mutants, and 3/13 ENU and EMS induced mutants were multi-locus deficiencies. Western blot analysis of the intragenic mutations showed that 4/7 X-ray-induced mutants produced detectable polypeptides, one of which was normal in molecular weight and charge. In contrast 8/10 intragenic ENU and EMS induced mutants produced normal polypeptides. Southern blot analysis showed that 5/7 intragenic X-ray induced mutants and all 10 of the intragenic ENU and EMS induced mutants were normal with respect to the alleles they were derived from

  6. The Ustilago maydis effector Pep1 suppresses plant immunity by inhibition of host peroxidase activity.

    Directory of Open Access Journals (Sweden)

    Christoph Hemetsberger

    Full Text Available The corn smut Ustilago maydis establishes a biotrophic interaction with its host plant maize. This interaction requires efficient suppression of plant immune responses, which is attributed to secreted effector proteins. Previously we identified Pep1 (Protein essential during penetration-1 as a secreted effector with an essential role for U. maydis virulence. pep1 deletion mutants induce strong defense responses leading to an early block in pathogenic development of the fungus. Using cytological and functional assays we show that Pep1 functions as an inhibitor of plant peroxidases. At sites of Δpep1 mutant penetrations, H₂O₂ strongly accumulated in the cell walls, coinciding with a transcriptional induction of the secreted maize peroxidase POX12. Pep1 protein effectively inhibited the peroxidase driven oxidative burst and thereby suppresses the early immune responses of maize. Moreover, Pep1 directly inhibits peroxidases in vitro in a concentration-dependent manner. Using fluorescence complementation assays, we observed a direct interaction of Pep1 and the maize peroxidase POX12 in vivo. Functional relevance of this interaction was demonstrated by partial complementation of the Δpep1 mutant defect by virus induced gene silencing of maize POX12. We conclude that Pep1 acts as a potent suppressor of early plant defenses by inhibition of peroxidase activity. Thus, it represents a novel strategy for establishing a biotrophic interaction.

  7. Immunocytochemistry and fluorescence imaging efficiently identify individual neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures.

    Science.gov (United States)

    Tsunematsu, Hiroto; Uyeda, Akiko; Yamamoto, Nobuhiko; Sugo, Noriyuki

    2017-08-01

    CRISPR/Cas9 system is a powerful method to investigate the role of genes by introducing a mutation selectively and efficiently to specific genome positions in cell and animal lines. However, in primary neuron cultures, this method is affected by the issue that the effectiveness of CRISPR/Cas9 is different in each neuron. Here, we report an easy, quick and reliable method to identify mutants induced by the CRISPR/Cas9 system at a single neuron level, using immunocytochemistry (ICC) and fluorescence imaging. Dissociated cortical cells were transfected with CRISPR/Cas9 plasmids targeting the transcription factor cAMP-response element binding protein (CREB). Fluorescence ICC with CREB antibody and quantitative analysis of fluorescence intensity demonstrated that CREB expression disappeared in a fraction of the transfected neurons. The downstream FOS expression was also decreased in accordance with suppressed CREB expression. Moreover, dendritic arborization was decreased in the transfected neurons which lacked CREB immunoreactivity. Detection of protein expression is efficient to identify individual postmitotic neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures. The present method composed of CRISPR/Cas9 system, ICC and fluorescence imaging is applicable to study the function of various genes at a single-neuron level.

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

    International Nuclear Information System (INIS)

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

    1995-01-01

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

  9. Identification of early flowering mutants from the cut chrysanthemum variety 'Jinba' and research on the physiological characteristics

    International Nuclear Information System (INIS)

    Cai Haiyan; Wen Lizhu; Zheng Chengshu; Sun Xia; Li Yingying

    2013-01-01

    In this study, the morphological characteristics the early flower mutants induced by EMS from the cut chrysanthemum variety 'Jinba' were characterized, and ISSR-PCR was used for molecular identification of mutants. The photosynthetic characteristics, stomatal and vascular bundle cross-section for the mutants were also measured. The florescence of mutants appeared about 59 days earlier than that of controls. The plant height, leaves and flower diameter were all smaller than those of controls, but they grew well. From the PCR amplification, the differences between the mutants and the control were in the present or absent of the amplified bands. The photosynthetic characteristics, such as the net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and intercellular CO_2 concentration were all significantly lower than the controls. The number of mutant's stomata significantly reduced, and some stomatas appeared variation. The vascular bundles including primary phloem and primary xylem displayed difference in the size and shape. All these work lays a foundation for breeding the early flowering of excellent chrysanthemum and further characterization of the early flowering genes. (authors)

  10. Temperature-sensitive host range mutants of herpes simplex virus type 2

    International Nuclear Information System (INIS)

    Koment, R.W.; Rapp, F.

    1975-01-01

    Herpesviruses are capable of several types of infection of a host cell. To investigate the early events which ultimately determine the nature of the virus-host cell interaction, a system was established utilizing temperature-sensitive mutants of herpes simplex virus type 2. Four mutants have been isolated which fail to induce cytopathic effects and do not replicate at 39 C in hamster embryo fibroblast cells. At least one mutant is virus DNA negative. Since intracellular complementation is detectable between pairs of mutants, a virus function is known to be temperature sensitive. However, all four mutants induce cytopathic effects and replicate to parental virus levels in rabbit kidney cells at 39 C. This suggests that a host cell function, lacking or nonfunctional in HEF cells but present in rabbit kidney cells at 39 C, is required for the replication of these mutants in hamster embryo fibroblast cells at 39 C. Therefore, we conclude that these mutants are both temperature sensitive and exhibit host range properties

  11. Human hepatocytes apoptosis induced by replication of hepatitis B virus subgenotypes F1b and F4: Role of basal core promoter and preCore mutations.

    Science.gov (United States)

    Elizalde, María Mercedes; Sevic, Ina; González López Ledesma, María Mora; Campos, Rodolfo Héctor; Barbini, Luciana; Flichman, Diego Martin

    2018-01-01

    In the context of pathogenesis of HBV infection, HBV genotypes and mutants have been shown to affect the natural course of chronic infection and treatment outcomes. In this work, we studied the induction of apoptosis by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. Both subgenotypes F1b and F4 HBV genome transfections induced cell death by apoptosis in human hepatocytes. The BCPdm (A1762T/G1764A) and preCore (G1896A) mutants induced higher levels of apoptosis than the wt virus. This increase in apoptosis was not associated with the enhanced viral replication of the variants. HBV-mediated apoptosis was independent of viral subgenotypes, and associated with the modulation of members of the regulatory Bcl-2 family proteins expression in the mitochondrial apoptotic pathway. Finally, the apoptosis induction increase observed for the preCore mutants suggests that HBeAg might have an anti-apoptotic effect in human hepatocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. 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

  13. Molecular cloning and functional characterization of porcine cyclic GMP-AMP synthase.

    Science.gov (United States)

    Wang, Jiang; Chu, Beibei; Du, Lili; Han, Yingqian; Zhang, Xuemei; Fan, Shuangshuang; Wang, Yueying; Yang, Guoyu

    2015-06-01

    Cyclic GMP-AMP synthase (cGAS), which belongs to the nucleotidyltransferase family, recognizes cytosolic DNA and induces the type I interferon (IFN) pathway through the synthesis of the second messenger cGAMP. In this study, porcine cGAS (p-cGAS) was identified and its tissue distribution, subcellular localization, and functions in innate immunity were characterized. The coding sequence of p-cGAS is 1494 bp long, encodes 497 amino acids, and is most similar (74%) to Bos taurus cGAS. p-cGAS mRNA is abundant in the spleen, duodenum, jejunum, and ileum. The subcellular distribution of p-cGAS is not only in the cytosol, but also on the endoplasmic reticulum (ER) membrane. The overexpression of wild-type p-cGAS in porcine kidney epithelial cells, but not its catalytically inactive mutants, induced IFN-β expression, which was dependent on STING and IRF3. However, the downregulation of p-cGAS by RNA interference markedly reduced IFN-β expression after pseudorabies virus (PRV) infection or poly(dA:dT) transfection. These results demonstrate that p-cGAS is an important DNA sensor, required for IFN-β activation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Brucella abortus 2308ΔNodVΔNodW double-mutant is highly attenuated and confers protection against wild-type challenge in BALB/c mice.

    Science.gov (United States)

    Li, Zhiqiang; Wang, Shuli; Zhang, Jinliang; Yang, Guangli; Yuan, Baodong; Huang, Jie; Han, Jincheng; Xi, Li; Xiao, Yanren; Chen, Chuangfu; Zhang, Hui

    2017-05-01

    Brucellosis is an important zoonotic disease of worldwide distribution, which causes animal and human disease. However, the current Brucella abortus (B. abortus) vaccines (S19 and RB51) have several drawbacks, including residual virulence for animals and humans. Moreover, S19 cannot allow serological differentiation between infected and vaccinated animals. We constructed double deletion (ΔNodVΔNodW) mutant from virulent B. abortus 2308 (S2308) by deleting the genes encoding two-component regulatory system (TCS) in chromosome II in S2308.2308ΔNodVΔNodW was significantly reduced survival in murine macrophages (RAW 264.7) and BALB/c mice. Moreover, the inoculated mice showed no splenomegaly. The mutant induced high protective immunity in BALB/c mice against challenge with S2308, and elicited an anti-Brucella-specific immunoglobulin G (IgG) response and induced the secretion of gamma interferon (IFN-γ) and interleukin-4 (IL-4). Moreover, NODV and NODW antigens would allow the serological differentiation between infected and vaccinated animals. These results suggest that 2308ΔNodVΔNodW mutant is a potential live attenuated vaccine candidate and can be used effectively against bovine brucellosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Brucella abortusΔcydCΔcydD and ΔcydCΔpurD double-mutants are highly attenuated and confer long-term protective immunity against virulent Brucella abortus.

    Science.gov (United States)

    Truong, Quang Lam; Cho, Youngjae; Park, Soyeon; Kim, Kiju; Hahn, Tae-Wook

    2016-01-04

    We constructed double deletion (ΔcydCΔcydD and ΔcydCΔpurD) mutants from virulent Brucella abortus biovar 1 field isolate (BA15) by deleting the genes encoding an ATP-binding cassette-type transporter (cydC and cydD genes) and a phosphoribosylamine-glycine ligase (purD). Both BA15ΔcydCΔcydD and BA15ΔcydCΔpurD double-mutants exhibited significant attenuation of virulence when assayed in murine macrophages or in BALB/c mice. Both double-mutants were readily cleared from spleens by 4 weeks post-inoculation even when inoculated at the dose of 10(8) CFU per mouse. Moreover, the inoculated mice showed no splenomegaly, which indicates that the mutants are highly attenuated. Importantly, the attenuation of in vitro and in vivo growth did not impair the ability of these mutants to confer long-term protective immunity in mice against challenge with B. abortus strain 2308. Vaccination of mice with either mutant induced humoral and cell-mediated immune responses, and provided significantly better protection than commercial B. abortus strain RB51 vaccine. These results suggest that highly attenuated BA15ΔcydCΔcydD and BA15ΔcydCΔpurD mutants can be used effectively as potential live vaccine candidates against bovine brucellosis. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  16. Adaptive response to mutagenesis and its molecular basis in a human T-cell leukemia line primed with a low dose of γ-rays

    International Nuclear Information System (INIS)

    Zhou, P.K.; Xiang, X.Q.; Sun, W.Z.; Liu, X.Y.; Zhang, Y.P.; Wei, K.

    1994-01-01

    The effect was studied of a low dose of γ-ray preexposure on the frequency and molecular spectrum of radiation-induced mutations at the hprt locus in a human T-cell leukemia line. When the cells were preexposed to 0.01 Gy of γ-rays, the yield of mutations induced by a subsequent 2-Gy challenge dose was reduced by 60%, compared with the 2 Gy of irradiation alone. The data of Southern blot analysis showed that 47% of the mutants induced by 2 Gy in the cells without low-dose preexposure were of the deletion or rearranged mutations type. In contrast, in the low-dose radioadapted cells the proportion of this type of 2-Gy-induced mutations decreased to 28%. This is close to the control level (22%) of spontaneous mutations. Our results confirm that a low dose of γ-ray preexposure leads to a decreased susceptibility to gene deletions and rearrangements after high-dose irradiation. (orig.)

  17. Inheritance of the lysozyme inhibitor Ivy was an important evolutionary step by Yersinia pestis to avoid the host innate immune response.

    Science.gov (United States)

    Derbise, Anne; Pierre, François; Merchez, Maud; Pradel, Elizabeth; Laouami, Sabrina; Ricard, Isabelle; Sirard, Jean-Claude; Fritz, Jill; Lemaître, Nadine; Akinbi, Henry; Boneca, Ivo G; Sebbane, Florent

    2013-05-15

    Yersinia pestis (the plague bacillus) and its ancestor, Yersinia pseudotuberculosis (which causes self-limited bowel disease), encode putative homologues of the periplasmic lysozyme inhibitor Ivy and the membrane-bound lysozyme inhibitor MliC. The involvement of both inhibitors in virulence remains subject to debate. Mutants lacking ivy and/or mliC were generated. We evaluated the mutants' ability to counter lysozyme, grow in serum, and/or counter leukocytes; to produce disease in wild-type, neutropenic, or lysozyme-deficient rodents; and to induce host inflammation. MliC was not required for lysozyme resistance and the development of plague. Deletion of ivy decreased Y. pestis' ability to counter lysozyme and polymorphonuclear neutrophils, but it did not affect the bacterium's ability to grow in serum or resist macrophages. Y. pestis lacking Ivy had attenuated virulence, unless animals were neutropenic or lysozyme deficient. The Ivy mutant induced inflammation to a degree similar to that of the parental strain. Last, Y. pseudotuberculosis did not require Ivy to counter lysozyme and for virulence. Ivy is required to counter lysozyme during infection, but its role as a virulence factor is species dependent. Our study also shows that a gene that is not necessary for the virulence of an ancestral bacterium may become essential in the emergence of a new pathogen.

  18. Triple basepair changes within and adjacent to the conserved YY1 motif upstream of the U3 enhancer repeats of SL3-3 murine leukemia virus cause a small but significant shortening of latency of T-lymphoma induction

    International Nuclear Information System (INIS)

    Ma Shiliang; Lovmand, Jette; Soerensen, Annette Balle; Luz, Arne; Schmidt, Joerg; Pedersen, Finn Skou

    2003-01-01

    A highly conserved sequence upstream of the transcriptional enhancer in the U3 of murine leukemia viruses (MLVs) was reported to mediate negative regulation of their expression. In transient expression studies, negative regulation was reported to be conferred by coexpression of the transcription factor YY1, which binds to a motif in the upstream conserved region (UCR). To address the function of the UCR and its YY1-motif in an in vivo model of MLV-host interactions we introduced six consecutive triple basepair mutations into this region of the potent T-lymphomagenic SL3-3 MLV. We report that all mutants have retained their replication competence and that they all, like the SL3-3 wild type (wt), induce T-cell lymphomas when injected into newborn mice of the SWR strain. However, all mutants induced disease with slightly shorter latency periods than the wt SL3-3, suggesting that the YY1 motif as well as its immediate context in the UCR have a negative effect on the pathogenicity of the virus. This result may have implications for the design of retroviral vectors

  19. 3D modeling of genome macroorganization on the basis of its structural changes after action of radiation

    International Nuclear Information System (INIS)

    Aleksandrov, I.D.; Aleksandrova, M.V.; Zaikin, N.S.; Koren'kov, V.V.; Pervushova, O.V.; Stepanenko, V.A.

    2006-01-01

    At present, after 120 years of the theoretical and experimental works, the issue of the genome macroarchitecture as the highest level of interphase chromosome organization in somatic cell nuclei remains still unresolved. The problem of the spatial arrangement of interphase chromosomes in haploid germ cells has never even been studied. A 3D simulation of packaging of the entire second chromosome in Drosophila mature sperms has been performed by using mathematical approaches and visualization methods to present macromolecular structure data. As genetic markers for simulation, frequency and location of the second inversion breakpoints for 72 structural υg mutants induced by ionizing radiation were used supposing that both ends of each inversion are topologically brought together forming loop of appropriate size. For the account of a degree of spatial affinity and visualization of chromosomal loops modern 3D-modeling methods with application of splines, libraries OpenGL, language Delphi, program Gmax were used. According to the model proposed, the entire second chromosome within mature sperm nuclei seems to be packaged in the form of a megarosette-loop structure which may be a basic principle of organization of the genome macro-architecture in animal haploid germ cells

  20. Collection of rice mutants and application studies of their agronomic characters

    International Nuclear Information System (INIS)

    Sun Shuxiang; Jin Wei; Luo Qian; Sheng Ping; Huang Rongmin

    1993-01-01

    More than 1600 accessions of rice mutant germplasm have been collected since 1980, and 1142 accessions of mutants have been identified according to their agronomy and pattern characters. A part of mutants were compared with their original cultivars in eight main agronomic characters. The results showed that the agronomic characters of mutants induced by ionizing radiations changed to both positive and negative directions compared with their original cultivars. Only 6.3% mutants varied in single agronomic character, and 91.1% mutants varied in two to six agronomic characters. Tenetic analysis and Cellular observations were carried out for two kinds of early mutants. It showed that early mutants 'Yuan Feng Zao' are controlled by two independent and incomplete dominant genes. For the dwarf, the reduction of the number of longitudinal cell layers causes the stem shorter and the increase of the number of horizontal cell layers causes the stem wall thicker. More than 100 preserved accessions of mutants were supplied to breeding units as parents or for genetic studies. Sixteen cultivars (lines) were bred from the parents which played an important role in raising the output of rice production

  1. The Bacterial Second Messenger Cyclic di-GMP Regulates Brucella Pathogenesis and Leads to Altered Host Immune Response.

    Science.gov (United States)

    Khan, Mike; Harms, Jerome S; Marim, Fernanda M; Armon, Leah; Hall, Cherisse L; Liu, Yi-Ping; Banai, Menachem; Oliveira, Sergio C; Splitter, Gary A; Smith, Judith A

    2016-12-01

    Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host-Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a ΔbpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the ΔbpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase ΔcgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, ΔbpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  2. Quantitative and molecular analyses of mutation in a pSV2gpt transformed CHO cell line

    International Nuclear Information System (INIS)

    Stankowski, L.F. Jr.; Tindall, K.R.; Hsie, A.W.

    1983-01-01

    Following NDA-mediated gene transfer we have isolated a cell line useful for studying gene mutation at the molecular level. This line, AS52, derived from a hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficient Chinese hamster ovary (CHO) cell line, carries a single copy of the E. coli xanthine-guanine phosphoribosyl transferase (XGPRT) gene (gpt) and exhibits a spontaneous mutant frequency of 20 TG/sup r/ mutants/10 6 clonable cells. As with HGPRT - mutants, XGPRT - mutants can be selected in 6-thioguanine. AS52 (XGPRT + ) and wild type CHO (HGPRT + ) cell exhibit almost identical cytotoxic responses to various agents. We observed significant differences in mutation induction by UV light and ethyl methanesulfonate (EMS). Ratios of XGPRT - to HGPRT - mutants induced per unit dose (J/m 2 for UV light and μg/ml for EMS) are 1.4 and 0.70, respectively. Preliminary Southern blot hybridization analyses has been performed on 30 XGPRT - AS52 mutants. A majority of spontaneous mutants have deletions ranging in size from 1 to 4 kilobases (9/19) to complete loss of gpt sequences (4/19); the remainder have no detectable (5/19) or only minor (1/19) alterations. 5/5 UV-induced and 5/6 EMS-induced mutants do not show a detectable change. Similar analyses are underway for mutations induced by x-irradiation and ICR 191 treatment

  3. Functional Analysis of Lactobacillus rhamnosus GG Pili in Relation to Adhesion and Immunomodulatory Interactions with Intestinal Epithelial Cells

    Science.gov (United States)

    Claes, Ingmar; Tytgat, Hanne L. P.; Verhoeven, Tine L. A.; Marien, Eyra; von Ossowski, Ingemar; Reunanen, Justus; Palva, Airi; de Vos, Willem M.; De Keersmaecker, Sigrid C. J.; Vanderleyden, Jos

    2012-01-01

    Lactobacillus rhamnosus GG, a probiotic with good survival capacity in the human gut, has well-documented adhesion properties and health effects. Recently, spaCBA-encoded pili that bind to human intestinal mucus were identified on its cell surface. Here, we report on the phenotypic analysis of a spaCBA pilus knockout mutant in comparison with the wild type and other adhesin mutants. The SpaCBA pilus of L. rhamnosus GG showed to be key for efficient adherence to the Caco-2 intestinal epithelial cell (IEC) line and biofilm formation. Moreover, the spaCBA mutant induces an elevated level of interleukin-8 (IL-8) mRNA in Caco-2 cells compared to the wild type, possibly involving an interaction of lipoteichoic acid with Toll-like receptor 2. In contrast, an L. rhamnosus GG mutant without exopolysaccharides but with an increased exposure of pili leads to the reduced expression of IL-8. Using Transwells to partition bacteria from Caco-2 cells, IL-8 induction is blocked completely regardless of whether wild-type or mutant L. rhamnosus GG cells are used. Taken together, our data suggest that L. rhamnosus GG SpaCBA pili, while promoting strong adhesive interactions with IECs, have a functional role in balancing IL-8 mRNA expression induced by surface molecules such as lipoteichoic acid. PMID:22020518

  4. The different phenotypes of phot- photosynthetic deficient mutants in Euglena gracilis: the frequency of production by ultraviolet irradiation

    International Nuclear Information System (INIS)

    Nicolas, Paul; Heizmann, Philippe; Nigon, Victor

    1982-01-01

    In Euglena gracilis, pigment-less mutants appear spontaneously with a frequency of about 2-5x10 -3 . Ultraviolet-irradiation increases the proportion of chlorophyll-less colonies to an upper limit where green colonies represent 4x10 -4 of the surviving ones. This limit might indicate the occurrence of processes involving repair of the chloroplastic DNA. Most of the photosynthetic-deficient (phot - ) mutants induced by ultraviolet irradiation are characterized by the presence of a reduced number of chloroplast DNA molecules showing deletions (phi - class). Most of the phi - mutants present the phenotype phi - chlo - car - , where neither chlorophyll nor carotenoids are obvious: the phi - chlo - car + mutants, devoid of chlorophyll but containing carotenoids, are obtained among the phi - strains with a frequency lower than 10 -3 . The phot - mutants which belong to the cp - class are characterized by the maintenance of a great number of chloroplastic DNA molecules, where large deletions are absent: their occurrence after ultraviolet irradiation is low [fr

  5. Persistent genetic instability induced by synergistic interaction between x-irradiation and 6-thioguanine

    International Nuclear Information System (INIS)

    Grosovsky, A.J.; Nelson, S.L.; Smith, L.E.

    1995-01-01

    Clonal karyotypic analysis was performed using G-banding on four groups of clones derived from TK6 human lymphoblasts: 25 HPRT - total gene deletion mutants induced by exposure to 2 Gy of x-rays; 8 spontaneous HPRT - total gene deletion mutants; 25 clones irradiated with 2 Gy, not selected with 6-thioguanine. Ten to twenty metaphases were examined for each clone. Extensive karyotypic heterogeneity was observed among x-ray induced HPRT - mutants involving translocations, deletions, duplications and aneuploidy; recovery of chromosomal aberrations and karyotypic heterogeneity was greater than the additive effects of clones treated with x-irradiation or 6-thioguanine alone. This synergistic interaction between x-irradiation and 6-thioguanine was observed despite a 7 day phenotypic expression interval between exposure to the two agents. Thus, x-irradiated TK6 cells appear to be persistently hypersensitive to the induction of genetic instability. Several mutants appeared to exhibit evidence of clonal evolution since aberrant chromosomes observed in one metaphase, were found to be further modified in other metaphases. In order to determine if genetic instability, identified by clonal karyotypic heterogeneity, affected specific locus mutation rates, we utilized the heterozygous thymidine kinase (tk) locus as a genetic marker. Four x-ray induced HPRT - mutants with extensive karyotypic heterogeneity, exhibited mutation rates at tk ranging from 5 to 8 fold higher than the parental TK6 cells. Further analysis, using fractionated low dose radiation exposure, is currently in progress

  6. Induced mutations in sesame breeding

    International Nuclear Information System (INIS)

    Ashri, A.

    2001-01-01

    The scope of induced mutations in sesame (Sesamum indicum L.) breeding is reviewed. So far in Egypt, India, Iraq, Rep. of Korea, and Sri Lanka, 14 officially released varieties have been developed through induced mutations: 12 directly and 2 through cross breeding (one using the 'dt45' induced mutant from Israel). For another variety released in China there are no details. The induced mutations approach was adopted primarily in order to obtain genetic variability that was not available in the germplasm collection. The mutagens commonly applied have been gamma rays, EMS and sodium azide. Sesame seeds can withstand high mutagen doses, and there are genotypic differences in sensitivity between varieties. The mutants induced in the above named countries and others include better yield, improved seed retention, determinate habit, modified plant architecture and size, more uniform and shorter maturation period, earliness, resistance to diseases, genic male sterility, seed coat color, higher oil content and modified fatty acids composition. Some of the induced mutants have already given rise to improved varieties, the breeding value of other mutants is now being assessed and still others can serve as useful markers in genetic studies and breeding programmes. (author)

  7. Analysis of esterase isozyme and SSR for mutagenic progenies induced by space mutation in mustard

    International Nuclear Information System (INIS)

    Shen Jinjuan; Liu Yihua; Zhang Zhaorong; Ran Guangkui; Zhao Shouzhong; Xiao Li

    2012-01-01

    Seeds of five mustard (Brassica juncea Coss) varieties were carried into outer space by 'Shijian No.8' satellite. After five years' consecutive planting and selection, ten relatively stable mutant lines were obtained, which had significant variation in agronomic and economic characters. The mutant lines and their original varieties without space mutation treatment as control were studied by esterase isozyme and SSR analyses. Electrophoresis analysis of esterase isozymes indicated that there were differences between mutant lines and their controls in enzyme types and enzyme activity. Different mustard varieties had different enzymographs, and so did the mutants induced by space mutation, which shows different sensitivity among different mustard varieties. The SSR analysis showed that large differences were found in the SSR loci between mutant lines and their original variety, the variation frequency was between 9.52% and 57.14% with an average frequency of 26.19% for all the mutant lines. Among the mutant SSR loci, about 56.36% showed changes in band number and 43.64% in molecular weight. These results indicated that the ten mutant lines had large genetic difference in phenotype, genomic sequence and gene expression, and the outer space mutation would be an effective method to develop new mustard germplasm and variety. (authors)

  8. The Use of TILLING Technique to Detect Mutations and genetic diversity in Potato

    International Nuclear Information System (INIS)

    Elias, R; Al-Safadi, B; Till, B

    2008-01-01

    TILLING technique has been used, for the first time, to detect genetic variation, at the molecular level, among potato mutants (induced by gamma irradiation) and genetic diversity among 3 potato cultivars. Three potato mutant lines (every mutant represents a cultivar) tolerant to salinity have been used along with their controls. Three primer pairs were designed with the help of Potato Genome Sequencing Consortium on the web and were evaluated using agarose gel then sequencing. Primer pairs passing these tests were fluorescently labeled. Li-Cor based TILLING was applied using 2 forward primers one of them is labeled and 2 reverse primers one of them is labeled. The results have shown the success of using this technique on potato (tetraploid species) where the average density of nucleotide polymorphisms per sample was 16 polymorphisms per 1 kb. The optimal concentration was also determined between 0.1 and 1 ng/ul for potato genomic DNAs to be used in Li-Cor based TILLING assays. (author)

  9. X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa

    International Nuclear Information System (INIS)

    Serres, F.J. de

    1989-01-01

    More extensive genetic tests have been preformed on a series of 832 X-ray-induced specific-locus mutations in the ad-3 region of a 2-component heterokaryon of Neurospora crassa, reported earlier. Using new tester strains and techniques for performing large-scale genetic tests to characterize ad-3 mutants induced in 2-component heterokaryons, new data have been obtained on this sample of X-ray-induced mutants. These new data show that unexpectedly high frequencies of both single-locus mutations and multilocus deletions in the ad-3 region have addition, but separate, sites of recessive lethal damage in the imeediately adjacent genetic regions. The frequencies of these X-ray-induced multiple-locus mutants in the ad-3 region are orders of magnitude higher than expected on the basis of target theory and classical models of chromosome structure during interphase. Current models of interphase chromosome structure in higher eukaryotes as revealed by chromosome 'painting' offer a possible explanation of the Neurospora data. (author). 25 refs.; 5 figs

  10. Pseudomonas aeruginosa mutations in lasI and rhlI quorum sensing systems result in milder chronic lung infection

    DEFF Research Database (Denmark)

    Wu, H; Song, Z; Givskov, Michael

    2001-01-01

    To understand the importance of quorum sensing in chronic Pseudomonas aeruginosa lung infection, the in vivo pathogenic effects of the wild-type P. aeruginosa PAO1 and its double mutant, PAO1 lasI rhlI, in which the signal-generating parts of the quorum sensing systems are defective were compared....... The rat model of P. aeruginosa lung infection was used in the present study. The rats were killed on days 3, 7, 14 and 28 after infection with the P. aeruginosa strains. The results showed that during the early stages of infection, the PAO1 double mutant induced a stronger serum antibody response, higher...... number of lung bacteria, and minor serum IgG and IgG1 responses but increased lung interferon gamma production were detected in the group infected with the PAO1 double mutant when compared with the PAO1-infected group. Delayed immune responses were observed in the PAO1-infected group and they might...

  11. Pseudomonas aeruginosa mutations in lasl and rhll quorum sensing systems result in milder chronic lung infection

    DEFF Research Database (Denmark)

    Wu, H.; Song, Z.J.; Givskov, Michael Christian

    2001-01-01

    To understand the importance of quorum sensing in chronic Pseudomonas aeruginosa lung infection, the in vivo pathogenic effects of the wild-type P aeruginosa PAO1 and its double mutant, PAO1 lasI rhlI, in which the signal-generating parts of the quorum sensing systems are defective were compared....... The rat model of P. aeruginosa lung infection was used in the present study. The rats were killed on days 3, 7, 14 and 28 after infection with the P. aeruginosa strains. The results showed that during the early stages of infection, the PAO1 double mutant induced a stronger serum antibody response, higher...... number of lung bacteria, and minor serum IgG and IgG1 responses but increased lung interferon gamma production were detected in the group infected with the PAO1 double mutant when compared with the PAO1-infected group. Delayed immune responses were observed in the PAO1-infected group and they might...

  12. RFLP analysis of rice semi-dwarf mutation induced by high energy argon ion radiation

    International Nuclear Information System (INIS)

    Zhuang Chuxiong; Hu Weimin; Mei Mantong

    1997-01-01

    Two Indica rice varieties, Bianpizhan and Xiangzhan, and their semi-dwarf mutants induced by high energy argon ion radiation, Ar-10, and Xiang-Ar-1, were examined with restriction fragment length polymorphism (RFLP) analysis by using 97 rice single copy genomic clones mapped on 12 chromosomes of molecular genetic map, combined with 5 restriction enzymes. Among the markers screened, 9 detected polymorphism were between Bianpizhen and Ar-10, and 11 detected polymorphism were between Xiangzhan and Xiang-Ar-1. Moreover, two or more restriction enzymes could generate RFLP patterns when screened with a given marker for several polymorphic markers. Based on the polymorphic allelic loci, the mutation frequencies were estimated as 5.15% and 6.39% for Ar-10 and Xiang-Ar-1 respectively. These results suggested that the nature of mutation on the DNA level was probably large genetic changes rather than point mutation. Genetic analysis and gene tagging of semi-dwarf mutation in one of the mutant line, Ar-10, indicated that this mutation was controlled by a major recessive gene, which was preliminary located on chromosome 4

  13. RFLP Analysis of rice semi dwarf mutation induced by high energy argon ion radiation

    International Nuclear Information System (INIS)

    Zhuang Chuxiong; Hu Weimin; Mei Mantong

    1997-01-01

    Two Indica rice varieties, Bianpizhan and Xiangzhan, and their semi dwarf mutants induced by high energy argon ion radiation, Ar 10, and Xiang Ar 1, were examined with restriction fragment length polymorphism(RFLP)analysis by using 97 rice single copy genomic clones mapped on 12 chromosomes of molecular genetic map, combined with 5 restriction enzymes.Among the markers screened, 9 detected polymorphism were between Bianpizhan and Ar 10, and 11 detected polymorphism were between Xiangzhan and Xiang Ar 1.Moreover, two or more restriction enzymes could generate RFLP patterns when screened with a given marker for several polymorphic markers. Based on the polymorphic allelic loci, the mutation frequencies were estimated as 5 15% and 6 39% for Ar 10 and Xiang Ar 1 respectively.These results suggested that the nature of mutation on the DNA level was probably large genetic changes rather than point mutation.Genetic analysis and gene tagging of semi dwarf mutation in one of the mutant line, Ar 10, indicated that this mutation was controlled by a major recessive gene, which was preliminary located on chromosome 4. (author)

  14. Mutation breeding in vivo and in vitro in vegetatively propagated crops

    International Nuclear Information System (INIS)

    Tulmann Neto, A.; Latado, R.R.; Tsai, S.M.; Derbyshire, M.T.; Yemma, A.F.; Scarpare Filho, J.A.; Ceravolo, L.; Rossi, A.C.; Namekata, T.; Pompeu, J. Jr.; Figueiredo, J.O.; Pio, R.; Tobias Domingues, E.; Santos, P.C.; Boliani, A.

    2001-01-01

    Mutation breeding in vivo and/or in vitro in vegetatively propagated crops as well as somaclonal variation can be used in Brazil in several crops to increase the genetic variability in characteristics of high importance. This was the objective of this research using ornamentals, citrus and bananas. Somaclonal variants can also be useful in these crops, based on the preliminary results observed in banana (Mycosphaerella musicola); where a short plant variant was selected in Brazil and the mutant resistant to yellow sigatoka, selected in Venezuela, showed resistance also in Brazil. Despite the increase in genetic variability in M 1 V 4 generation obtained after in vitro irradiation of meristems in banana, mutants resistant or tolerant to Fusarium were not selected, perhaps due to the limited number of plants evaluated. In citrus the first results from yield trials showed that following bud irradiation, it was possible to select plants of interest, e.g. mutants with a reduced number of seeds in the fruits. In ornamentals mutants induced by gamma rays in this project were released to the farmers. The results obtained in this research showed that biotechnology is a powerful tool that can be used in several ways in association with mutation breeding. (author)

  15. An early function of the adenoviral E1B 55 kDa protein is required for the nuclear relocalization of the cellular p53 protein in adenovirus-infected normal human cells

    International Nuclear Information System (INIS)

    Cardoso, F.M.; Kato, Sayuri E.M.; Huang Wenying; Flint, S. Jane; Gonzalez, Ramon A.

    2008-01-01

    It is well established that the human subgroup C adenovirus type 5 (Ad5) E1B 55 kDa protein can regulate the activity and concentration of the cellular tumor suppressor, p53. However, the contribution(s) of these functions of the E1B protein to viral reproduction remains unclear. To investigate this issue, we examined properties of p53 in normal human cells infected by E1B mutant viruses that display defective entry into the late phase or viral late mRNA export. The steady-state concentrations of p53 were significantly higher in cells infected by the E1B 55 kDa null mutant Hr6 or three mutants carrying small insertions in the E1B 55 kDa protein coding sequence than in Ad5-infected cells. Nevertheless, none of the mutants induced apoptosis in infected cells. Rather, the localization of p53 to E1B containing nuclear sites observed during infection by Ad5 was prevented by mutations that impair interaction of the E1B protein with p53 and/or with the E4 Orf6 protein. These results indicate that the E1B protein fulfills an early function that correlates efficient entry into the late phase with the localization of E1B and p53 in the nucleus of Ad5-infected normal human cells

  16. The nature of radiation-induced mutations at the white locus of Drosophila melanogaster

    International Nuclear Information System (INIS)

    Pastink, A.; Schalet, A.P.; Vreeken, C.; Eeken, J.C.J.; Paradi, E.

    1987-01-01

    X-ray- and neutron-induced mutations at the white locus of Drosophila melanogaster were used to study the nature of radiation-induced genetic damage. Genetic analysis showed the presence of multi-locus deficiencies in 15 out of 31 X-ray mutants and in 26 out of 35 mutants induced by neutrons. The DNA from 11 X-ray and 4 neutron mutants, which were not multi-locus deficiencies, was analyzed by Southern blot-hybridization. Deletions were observed in 2 X-ray and 1 neutron mutant. In combination with cytogenetic techniques, chromosomal rearrangements affecting the white locus (translocations, inversions, etc.) were identified in 3 X-ray and in 2 neutron mutants. A hot-spot for translocation breakpoints was identified in the left arm of the third chromosome. 5 X-ray mutants, which apparently did not contain large deletions, were subjected to further analysis by the nuclease S1 protection method, after cloning of the white gene. In 4 mutants a small deletion could indeed be detected in this way. Thus it seems that by far the main part of X-ray- and neutron-induced white mutants have arisen through large changes in the white gene, especially deletions. (Auth.)

  17. NPR1 protein regulates pathogenic and symbiotic interactions between Rhizobium and legumes and non-legumes.

    Directory of Open Access Journals (Sweden)

    Smadar Peleg-Grossman

    Full Text Available BACKGROUND: Legumes are unique in their ability to establish symbiotic interaction with rhizobacteria from Rhizobium genus, which provide them with available nitrogen. Nodulation factors (NFs produced by Rhizobium initiate legume root hair deformation and curling that entrap the bacteria, and allow it to grow inside the plant. In contrast, legumes and non-legumes activate defense responses when inoculated with pathogenic bacteria. One major defense pathway is mediated by salicylic acid (SA. SA is sensed and transduced to downstream defense components by a redox-regulated protein called NPR1. METHODOLOGY/PRINCIPAL FINDINGS: We used Arabidopsis mutants in SA defense pathway to test the role of NPR1 in symbiotic interactions. Inoculation of Sinorhizobium meliloti or purified NF on Medicago truncatula or nim1/npr1 A. thaliana mutants induced root hair deformation and transcription of early and late nodulins. Application of S. meliloti or NF on M. truncatula or A. thaliana roots also induced a strong oxidative burst that lasted much longer than in plants inoculated with pathogenic or mutualistic bacteria. Transient overexpression of NPR1 in M. truncatula suppressed root hair curling, while inhibition of NPR1 expression by RNAi accelerated curling. CONCLUSIONS/SIGNIFICANCE: We show that, while NPR1 has a positive effect on pathogen resistance, it has a negative effect on symbiotic interactions, by inhibiting root hair deformation and nodulin expression. Our results also show that basic plant responses to Rhizobium inoculation are conserved in legumes and non-legumes.

  18. Mutation induction by ion beams in arabidopsis

    International Nuclear Information System (INIS)

    Tanaka, Atsushi

    1999-01-01

    An investigation was made on characteristics of ion beams for the biological effects and the induction of mutation using Arabidopsis plant as a model plant for the molecular genetics. Here, the characteristics of mutation at the molecular level as well as new mutants induced by ion beams were described. The ast and sep1 were obtained from the offspring of 1488 carbon ion-irradiated seeds respectively. The uvi1-uvi4 mutants were also induced from 1280 M 1 lines. Thus, ion beams can induce not only known mutants such as tt, gl and hy but also novel mutants with high frequency. Even in the tt phenotype, two new mutant loci other than known loci were found. In chrysanthemum, several kinds of single, complex or stripped flower-color mutants that have been never induced by γirradiation, indicating that ion beams could produce a variety of mutants with the same phenotype. In conclusion, ion beams for the mutation induction are characterized by 1) to induce mutants with high frequency, 2) to show broad mutation spectrum and 3) to produce novel mutants. For these reasons, chemical mutagens such as EMS and low LET ionizing radiation such as X-rays and γ-rays will predominantly induce many but small modifications or DNA damages on the DNA strands. As the result, several point mutations will be produced on the genome. On the contrary, ion beams as a high LET ionizing radiation will not cause so many but large and irreparable DNA damage locally, resulting in production of limited number of null mutation. (M.N.)

  19. Pseudomonas Evades Immune Recognition of Flagellin in Both Mammals and Plants

    Science.gov (United States)

    Bardoel, Bart W.; van der Ent, Sjoerd; Pel, Michiel J. C.; Tommassen, Jan; Pieterse, Corné M. J.; van Kessel, Kok P. M.; van Strijp, Jos A. G.

    2011-01-01

    The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host innate immune systems. Recognition of flagellin monomers occurs by flagellin-specific pattern-recognition receptors, such as Toll-like receptor 5 (TLR5) in mammals and flagellin-sensitive 2 (FLS2) in plants. Activation of these immune systems via flagellin leads eventually to elimination of the bacterium from the host. In order to prevent immune activation and thus favor survival in the host, bacteria secrete many proteins that hamper such recognition. In our search for Toll like receptor (TLR) antagonists, we screened bacterial supernatants and identified alkaline protease (AprA) of Pseudomonas aeruginosa as a TLR5 signaling inhibitor as evidenced by a marked reduction in IL-8 production and NF-κB activation. AprA effectively degrades the TLR5 ligand monomeric flagellin, while polymeric flagellin (involved in bacterial motility) and TLR5 itself resist degradation. The natural occurring alkaline protease inhibitor AprI of P. aeruginosa blocked flagellin degradation by AprA. P. aeruginosa aprA mutants induced an over 100-fold enhanced activation of TLR5 signaling, because they fail to degrade excess monomeric flagellin in their environment. Interestingly, AprA also prevents flagellin-mediated immune responses (such as growth inhibition and callose deposition) in Arabidopsis thaliana plants. This was due to decreased activation of the receptor FLS2 and clearly demonstrated by delayed stomatal closure with live bacteria in plants. Thus, by degrading the ligand for TLR5 and FLS2, P. aeruginosa escapes recognition by the innate immune systems of both mammals and plants. PMID:21901099

  20. Pseudomonas evades immune recognition of flagellin in both mammals and plants.

    Directory of Open Access Journals (Sweden)

    Bart W Bardoel

    2011-08-01

    Full Text Available The building blocks of bacterial flagella, flagellin monomers, are potent stimulators of host innate immune systems. Recognition of flagellin monomers occurs by flagellin-specific pattern-recognition receptors, such as Toll-like receptor 5 (TLR5 in mammals and flagellin-sensitive 2 (FLS2 in plants. Activation of these immune systems via flagellin leads eventually to elimination of the bacterium from the host. In order to prevent immune activation and thus favor survival in the host, bacteria secrete many proteins that hamper such recognition. In our search for Toll like receptor (TLR antagonists, we screened bacterial supernatants and identified alkaline protease (AprA of Pseudomonas aeruginosa as a TLR5 signaling inhibitor as evidenced by a marked reduction in IL-8 production and NF-κB activation. AprA effectively degrades the TLR5 ligand monomeric flagellin, while polymeric flagellin (involved in bacterial motility and TLR5 itself resist degradation. The natural occurring alkaline protease inhibitor AprI of P. aeruginosa blocked flagellin degradation by AprA. P. aeruginosa aprA mutants induced an over 100-fold enhanced activation of TLR5 signaling, because they fail to degrade excess monomeric flagellin in their environment. Interestingly, AprA also prevents flagellin-mediated immune responses (such as growth inhibition and callose deposition in Arabidopsis thaliana plants. This was due to decreased activation of the receptor FLS2 and clearly demonstrated by delayed stomatal closure with live bacteria in plants. Thus, by degrading the ligand for TLR5 and FLS2, P. aeruginosa escapes recognition by the innate immune systems of both mammals and plants.

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

    International Nuclear Information System (INIS)

    Suzuki, Keiji; Hei, Tom K.

    1996-01-01

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

  2. Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage*

    Science.gov (United States)

    Comeaux, Evan Q.; Cuya, Selma M.; Kojima, Kyoko; Jafari, Nauzanene; Wanzeck, Keith C.; Mobley, James A.; Bjornsti, Mary-Ann; van Waardenburg, Robert C. A. M.

    2015-01-01

    Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3′-DNA adducts, such as the 3′-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (Hisnuc) that attacks DNA adducts to form a covalent 3′-phosphohistidyl intermediate and a general acid/base His (Hisgab), which resolves the Tdp1-DNA linkage. A Hisnuc to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of Hisgab to Arg. However, here we report that expression of the yeast HisnucAla (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 Hisgab mutants, including H432N and the SCAN1-related H432R. Moreover, the HisnucAla mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the HisnucPhe mutant was catalytically inactive and suppressed Hisgab mutant-induced toxicity. These data suggest that the activity of another nucleophile when Hisnuc is replaced with residues containing a small side chain (Ala, Asn, and Gln), but not with a bulky side chain. Indeed, genetic, biochemical, and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to Hisnuc, can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate. PMID:25609251

  3. Gain-of-function mutations in the gene encoding the tyrosine phosphatase SHP2 induce hydrocephalus in a catalytically dependent manner.

    Science.gov (United States)

    Zheng, Hong; Yu, Wen-Mei; Waclaw, Ronald R; Kontaridis, Maria I; Neel, Benjamin G; Qu, Cheng-Kui

    2018-03-20

    Catalytically activating mutations in Ptpn11 , which encodes the protein tyrosine phosphatase SHP2, cause 50% of Noonan syndrome (NS) cases, whereas inactivating mutations in Ptpn11 are responsible for nearly all cases of the similar, but distinct, developmental disorder Noonan syndrome with multiple lentigines (NSML; formerly called LEOPARD syndrome). However, both types of disease mutations are gain-of-function mutations because they cause SHP2 to constitutively adopt an open conformation. We found that the catalytic activity of SHP2 was required for the pathogenic effects of gain-of-function, disease-associated mutations on the development of hydrocephalus in the mouse. Targeted pan-neuronal knockin of a Ptpn11 allele encoding the active SHP2 E76K mutant resulted in hydrocephalus due to aberrant development of ependymal cells and their cilia. These pathogenic effects of the E76K mutation were suppressed by the additional mutation C459S, which abolished the catalytic activity of SHP2. Moreover, ependymal cells in NSML mice bearing the inactive SHP2 mutant Y279C were also unaffected. Mechanistically, the SHP2 E76K mutant induced developmental defects in ependymal cells by enhancing dephosphorylation and inhibition of the transcription activator STAT3. Whereas STAT3 activity was reduced in Ptpn11 E76K/+ cells, the activities of the kinases ERK and AKT were enhanced, and neural cell-specific Stat3 knockout mice also manifested developmental defects in ependymal cells and cilia. These genetic and biochemical data demonstrate a catalytic-dependent role of SHP2 gain-of-function disease mutants in the pathogenesis of hydrocephalus. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  4. BvrR/BvrS-Controlled Outer Membrane Proteins Omp3a and Omp3b Are Not Essential for Brucella abortus Virulence▿

    Science.gov (United States)

    Manterola, Lorea; Guzmán-Verri, Caterina; Chaves-Olarte, Esteban; Barquero-Calvo, Elías; de Miguel, María-Jesús; Moriyón, Ignacio; Grilló, María-Jesús; López-Goñi, Ignacio; Moreno, Edgardo

    2007-01-01

    The Brucella abortus two-component regulatory system BvrR/BvrS controls the expression of outer membrane proteins (Omp) Omp3a (Omp25) and Omp3b (Omp22). Disruption of bvrS or bvrR generates avirulent mutants with altered cell permeability, higher sensitivity to microbicidal peptides, and complement. Consequently, the role of Omp3a and Omp3b in virulence was examined. Similar to bvrS or bvrR mutants, omp3a and omp3b mutants displayed increased attachment to cells, indicating surface alterations. However, they showed unaltered permeability; normal expression of Omp10, Omp16, Omp19, Omp2b, and Omp1; native hapten polysaccharide; and lipopolysaccharide and were resistant to complement and polymyxin B at ranges similar to those of the wild-type (WT) counterpart. Likewise, omp3a and omp3b mutants were able to replicate in murine macrophages and in HeLa cells, were resistant to the killing action of human neutrophils, and persisted in mice, like the WT strain. Murine macrophages infected with the omp3a mutant generated slightly higher levels of tumor necrosis factor alpha than the WT, whereas the bvrS mutant induced lower levels of this cytokine. Since the absence of Omp3a or Omp3b does not result in attenuation, it can be concluded that BvrR/BvrS influences additional Brucella properties involved in virulence. Our results are discussed in the light of previous works suggesting that disruption of omp3a generates attenuated Brucella strains, and we speculate on the role of group 3 Omps. PMID:17664262

  5. Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana.

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    Juan Du

    Full Text Available Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin or polymeric form (F-actin. Members of the actin-depolymerizing factor (ADF/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1 in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1-actin complex, we constructed a homology model of the AtADF1-actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson-Boltzmann Surface Area (MM-GB/PBSA methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin.

  6. Mutagenesis in human cells with accelerated H and Fe ions

    Science.gov (United States)

    Kronenberg, Amy

    1994-01-01

    The overall goals of this research were to determine the risks of mutation induction and the spectra of mutations induced by energetic protons and iron ions at two loci in human lymphoid cells. During the three year grant period the research has focused in three major areas: (1) the acquisition of sufficient statistics for human TK6 cell mutation experiments using Fe ions (400 MeV/amu), Fe ions (600 MeV/amu) and protons (250 MeV/amu); (2) collection of thymidine kinase- deficient (tk) mutants or hypoxanthine phosphoribosyltransferase deficient (hprt) mutants induced by either Fe 400 MeV/amu, Fe 600 MeV/amu, or H 250 MeV/amu for subsequent molecular analysis; and (3) molecular characterization of mutants isolated after exposure to Fe ions (600 MeV/amu). As a result of the shutdown of the BEVALAC heavy ion accelerator in December 1992, efforts were rearranged somewhat in time to complete our dose-response studies and to complete mutant collections in particular for the Fe ion beams prior to the shutdown. These goals have been achieved. A major effort was placed on collection, re-screening, and archiving of 3 different series of mutants for the various particle beam exposures: tk-ng mutants, tk-sg mutants, and hprt-deficient mutants. Where possible, groups of mutants were isolated for several particle fluences. Comparative analysis of mutation spectra has occured with characterization of the mutation spectrum for hprt-deficient mutants obtained after exposure of TK6 cells to Fe ions (600 MeV/amu) and a series of spontaneous mutants.

  7. Xeroderma Pigmentosum Group A Suppresses Mutagenesis Caused by Clustered Oxidative DNA Adducts in the Human Genome

    Science.gov (United States)

    Sassa, Akira; Kamoshita, Nagisa; Kanemaru, Yuki; Honma, Masamitsu; Yasui, Manabu

    2015-01-01

    Clustered DNA damage is defined as multiple sites of DNA damage within one or two helical turns of the duplex DNA. This complex damage is often formed by exposure of the genome to ionizing radiation and is difficult to repair. The mutagenic potential and repair mechanisms of clustered DNA damage in human cells remain to be elucidated. In this study, we investigated the involvement of nucleotide excision repair (NER) in clustered oxidative DNA adducts. To identify the in vivo protective roles of NER, we established a human cell line lacking the NER gene xeroderma pigmentosum group A (XPA). XPA knockout (KO) cells were generated from TSCER122 cells derived from the human lymphoblastoid TK6 cell line. To analyze the mutagenic events in DNA adducts in vivo, we previously employed a system of tracing DNA adducts in the targeted mutagenesis (TATAM), in which DNA adducts were site-specifically introduced into intron 4 of thymidine kinase genes. Using the TATAM system, one or two tandem 7,8-dihydro-8-oxoguanine (8-oxoG) adducts were introduced into the genomes of TSCER122 or XPA KO cells. In XPA KO cells, the proportion of mutants induced by a single 8-oxoG (7.6%) was comparable with that in TSCER122 cells (8.1%). In contrast, the lack of XPA significantly enhanced the mutant proportion of tandem 8-oxoG in the transcribed strand (12%) compared with that in TSCER122 cells (7.4%) but not in the non-transcribed strand (12% and 11% in XPA KO and TSCER122 cells, respectively). By sequencing the tandem 8-oxoG-integrated loci in the transcribed strand, we found that the proportion of tandem mutations was markedly increased in XPA KO cells. These results indicate that NER is involved in repairing clustered DNA adducts in the transcribed strand in vivo. PMID:26559182

  8. Charged-particle mutagenesis 2. Mutagenic effects of high energy charged particles in normal human fibroblasts

    Science.gov (United States)

    Chen, D. J.; Tsuboi, K.; Nguyen, T.; Yang, T. C.

    1994-01-01

    The biological effects of high Linear Energy Transfer (LET) charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 sq micrometer and 0.09 to 5.56 x 10(exp -3) sq micrometer respectively. The maximum values were obtained by Fe-56 with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(exp -5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.

  9. Charged-particle mutagenesis II. Mutagenic effects of high energy charged particles in normal human fibroblasts

    Science.gov (United States)

    Chen, D. J.; Tsuboi, K.; Nguyen, T.; Yang, T. C.

    1994-01-01

    The biological effects of high LET charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 micrometer2 and 0.09 to 5.56 x 10(-3) micrometer2, respectively. The maximum values were obtained by 56Fe with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(-5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.

  10. ELANE mutant-specific activation of different UPR pathways in congenital neutropenia.

    Science.gov (United States)

    Nustede, Rainer; Klimiankou, Maksim; Klimenkova, Olga; Kuznetsova, Inna; Zeidler, Cornelia; Welte, Karl; Skokowa, Julia

    2016-01-01

    A number of studies have demonstrated induction of the unfolded protein response (UPR) in patients with severe congenital neutropenia (CN) harbouring mutations of ELANE, encoding neutrophil elastase. Why UPR is not activated in patients with cyclic neutropenia (CyN) carrying the same ELANE mutations is unclear. We evaluated the effects of ELANE mutants on UPR induction in myeloid cells from CN and CyN patients, and analysed whether additional CN-specific defects contribute to the differences in UPR induction between CN and CyN patients harbouring identical ELANE mutations. We investigated CN-specific p.C71R and p.V174_C181del (NP_001963.1) and CN/CyN-shared p.S126L (NP_001963.1) ELANE mutants. We found that transduction of haematopoietic cells with p.C71R, but not with p.V174_C181del or p.S126L ELANE mutants induced expression of ATF6, and the ATF6 target genes PPP1R15A, DDIT3 and HSPA5. Recently, we found that levels of secretory leucocyte protease inhibitor (SLPI), a natural ELANE inhibitor, are diminished in myeloid cells from CN patients, but not CyN patients. Combined knockdown of SLPI by shRNA and transduction of ELANE p.S126L in myeloid cells led to elevated levels of ATF6, PPP1R15A and HSPA5 RNA, suggesting that normal levels of SLPI in CyN patients might protect them from the UPR induced by mutant ELANE. In summary, different ELANE mutants have different effects on UPR activation, and SLPI regulates the extent of ELANE-triggered UPR. © 2015 John Wiley & Sons Ltd.

  11. Analysis of human HPRT- deletion mutants by the microarray-CGH (comparative genomic hybridization)

    International Nuclear Information System (INIS)

    Kodaira, M.; Sasaki, K.; Tagawa, H.; Omine, H.; Kushiro, J.; Takahashi, N.; Katayama, H.

    2003-01-01

    We are trying to evaluate genetic effects of radiation on human using mutation frequency as an indicator. For the efficient detection of mutations, it is important to understand the mechanism and the characteristics of radiation-induced mutations. We have started the analysis of hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutants induced by X-ray in order to clarify the deletion size and the mutation-distribution. We analyzed 39 human X-ray induced HPRT-deletion mutants by using the microarray-CGH. The array for this analysis contains 57 BAC clones covering as much as possible of the 4Mb of the 5' side and 10Mb of the 3' side of the HPRT gene based on the NCBI genome database. DNA from parent strain and each HPRT-mutant strain are labeled with Cy5 and Cy3 respectively, and were mixed and hybridized on the array. Fluorescent intensity ratio of the obtained spots was analyzed using software we developed to identify clones corresponding to the deletion region. The deletion in these strains ranged up to 3.5 Mb on the 5' side and 6 Mb on the 3' side of the HPRT gene. Deletions in 13 strains ended around BAC clones located at about 3 Mb on the 5' side. On the 3' side, deletions extended up to the specific clones located at 1.5 Mb in 11 strains. The mutations seem to be complex on the 3' end of deletion; some accompanied duplications with deletions and others could not be explained by one mutation event. We need to confirm these results, taking into account the experimental reproducibility and the accuracy of the published genetic map. The results of the research using the microarray-CGH help us to search the regions where deletions are easily induced and to identify the factors affecting the range of deletions

  12. Mutant genes in pea breeding

    International Nuclear Information System (INIS)

    Swiecicki, W.K.

    1990-01-01

    Full text: Mutations of genes Dpo (dehiscing pods) and A (anthocyanin synthesis) played a role in pea domestication. A number of other genes were important in cultivar development for 3 types of usage (dry seeds, green vegetable types, fodder), e.g. fn, fna, le, p, v, fas and af. New genes (induced and spontaneous), are important for present ideotypes and are registered by the Pisum Genetics Association (PGA). Comparison of a pea variety ideotype with the variation available in gene banks shows that breeders need 'new' features. In mutation induction experiments, genotype, mutagen and method of treatment (e.g. combined or fractionated doses) are varied for broadening the mutation spectrum and selecting more genes of agronomic value. New genes are genetically analysed. In Poland, some mutant varieties with the gene afila were registered, controlling lodging by a shorter stem and a higher number of internodes. Really non-lodging pea varieties could strongly increase seed yield. But the probability of detecting a major gene for lodging resistance is low. Therefore, mutant genes with smaller influence on plant architecture are sought, to combine their effect by crossing. Promising seem to be the genes rogue, reductus and arthritic as well as a number of mutant genes not yet genetically identified. The gene det for terminal inflorescence - similarly to Vicia faba - changes plant development. Utilisation of assimilates and ripening should be better. Improvement of harvest index should give higher seed yield. A number of genes controlling disease resistance are well known (eg. Fw, Fnw, En, mo and sbm). Important in mass screening of resistance are closely linked gene markers. Pea gene banks collect respective lines, but mutants induced in highly productive cultivars would be better. Inducing gene markers sometimes seems to be easier than transfer by crossing. Mutation induction in pea breeding is probably more important because a high number of monogenic features are

  13. Cytotoxic and mutagenic effects of high let charged particles on human skin fibroblasts

    International Nuclear Information System (INIS)

    Tsuboi, Koji.; Park, M.S.; Chen, D.J.; Yang, T.C.

    1992-01-01

    Cytotoxic and mutagenic effects of high LET charged particles were quantitatively measured using primary cultures of human skin fibroblasts. The span of LETs selected were from 25 keV/μm(330 MeV/u) to 920 keV/μm (600 MeV/u). Mutations were scored at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus using 6-thioguanine (6-TG) for selection. Exposure to these high LET charged particles resulted in exponential survival curves whereas mutant induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/μm. The inactivation cross-section (σ i ) and the action cross-section for mutant induction (σ m ) ranged from 2.2 to 92.0 μm 2 and 0.09 to 5.56 x 10 -3 μm 2 respectively, the maximum values were obtained by 56 Fe with an LET of 200 keV/μm. The mutagenicity (σ m /σ i ) ranged from 2.05 to 7.99 x 10 -5 with the maximum value at 150 keV/μm. Furthermore, the results of multiplex polymerase chain reaction (PCR) of some of the mutants induced by charged particles indicate that higher LET beams are more likely to cause larger deletions in the hprt locus. (author)

  14. Single Amino Acid Modification of Adeno-Associated Virus Capsid Changes Transduction and Humoral Immune Profiles

    Science.gov (United States)

    Diprimio, Nina; Bowles, Dawn E.; Hirsch, Matthew L.; Monahan, Paul E.; Asokan, Aravind; Rabinowitz, Joseph; Agbandje-McKenna, Mavis

    2012-01-01

    Adeno-associated virus (AAV) vectors have the potential to promote long-term gene expression. Unfortunately, humoral immunity restricts patient treatment and in addition provides an obstacle to the potential option of vector readministration. In this study, we describe a comprehensive characterization of the neutralizing antibody (NAb) response to AAV type 1 (AAV1) through AAV5 both in vitro and in vivo. These results demonstrated that NAbs generated from one AAV type are unable to neutralize the transduction of other types. We extended this observation by demonstrating that a rationally engineered, muscle-tropic AAV2 mutant containing 5 amino acid substitutions from AAV1 displayed a NAb profile different from those of parental AAV2 and AAV1. Here we found that a single insertion of Thr from AAV1 into AAV2 capsid at residue 265 preserved high muscle transduction, while also changing the immune profile. To better understand the role of Thr insertion at position 265, we replaced all 20 amino acids and evaluated both muscle transduction and the NAb response. Of these variants, 8 mutants induced higher muscle transduction than AAV2. Additionally, three classes of capsid NAb immune profile were defined based on the ability to inhibit transduction from AAV2 or mutants. While no relationship was found between transduction, amino acid properties, and NAb titer or its cross-reactivity, these studies map a critical capsid motif involved in all steps of AAV infectivity. Our results suggest that AAV types can be utilized not only as templates to generate mutants with enhanced transduction efficiency but also as substrates for repeat administration. PMID:22593151

  15. Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

    Science.gov (United States)

    Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

    2014-01-17

    The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

  16. Finding the Needle in the Haystack—the Use of Microfluidic Droplet Technology to Identify Vitamin-Secreting Lactic Acid Bacteria

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    Jun Chen

    2017-05-01

    Full Text Available Efficient screening technologies aim to reduce both the time and the cost required for identifying rare mutants possessing a phenotype of interest in a mutagenized population. In this study, we combined a mild mutagenesis strategy with high-throughput screening based on microfluidic droplet technology to identify Lactococcus lactis variants secreting vitamin B2 (riboflavin. Initially, we used a roseoflavin-resistant mutant of L. lactis strain MG1363, JC017, which secreted low levels of riboflavin. By using fluorescence-activated droplet sorting, several mutants that secreted riboflavin more efficiently than JC017 were readily isolated from the mutagenesis library. The screening was highly efficient, and candidates with as few as 1.6 mutations per million base pairs (Mbp were isolated. The genetic characterization revealed that riboflavin production was triggered by mutations inhibiting purine biosynthesis, which is surprising since the purine nucleotide GTP is a riboflavin precursor. Purine starvation in the mutants induced overexpression of the riboflavin biosynthesis cluster ribABGH. When the purine starvation was relieved by purine supplementation in the growth medium, the outcome was an immediate downregulation of the riboflavin biosynthesis cluster and a reduction in riboflavin production. Finally, by applying the new isolates in milk fermentation, the riboflavin content of milk (0.99 mg/liter was improved to 2.81 mg/liter, compared with 0.66 mg/liter and 1.51 mg/liter by using the wild-type strain and the original roseoflavin-resistant mutant JC017, respectively. The results obtained demonstrate how powerful classical mutagenesis can be when combined with droplet-based microfluidic screening technology for obtaining microorganisms with useful attributes.

  17. A Major Facilitator Superfamily Transporter-Mediated Resistance to Oxidative Stress and Fungicides Requires Yap1, Skn7, and MAP Kinases in the Citrus Fungal Pathogen Alternaria alternata.

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    Li-Hung Chen

    Full Text Available Major Facilitator Superfamily (MFS transporters play an important role in multidrug resistance in fungi. We report an AaMFS19 gene encoding a MFS transporter required for cellular resistance to oxidative stress and fungicides in the phytopathogenic fungus Alternaria alternata. AaMFS19, containing 12 transmembrane domains, displays activity toward a broad range of substrates. Fungal mutants lacking AaMFS19 display profound hypersensitivities to cumyl hydroperoxide, potassium superoxide, many singlet oxygen-generating compounds (eosin Y, rose Bengal, hematoporphyrin, methylene blue, and cercosporin, and the cell wall biosynthesis inhibitor, Congo red. AaMFS19 mutants also increase sensitivity to copper ions, clotrimazole, fludioxonil, and kocide fungicides, 2-chloro-5-hydroxypyridine (CHP, and 2,3,5-triiodobenzoic acid (TIBA. AaMFS19 mutants induce smaller necrotic lesions on leaves of a susceptible citrus cultivar. All observed phenotypes in the mutant are restored by introducing and expressing a wild-type copy of AaMFS19. The wild-type strain of A. alternata treated with either CHP or TIBA reduces radial growth and formation and germination of conidia, increases hyphal branching, and results in decreased expression of the AaMFS19 gene. The expression of AaMFS19 is regulated by the Yap1 transcription activator, the Hog1 and Fus3 mitogen-activated protein (MAP kinases, the 'two component' histidine kinase, and the Skn7 response regulator. Our results demonstrate that A. alternata confers resistance to different chemicals via a membrane-bound MFS transporter.

  18. Radiation mutagenesis from molecular and genetic points of view

    International Nuclear Information System (INIS)

    Chen, D.J.C.; Park, M.S.; Okinaka, R.T.; Jaberaboansari, A.

    1993-01-01

    An important biological effect of ionizing radiation on living organisms is mutation induction. Mutation is also a primary event in the etiology of cancer. The chain events, from induction of DNA damage by ionizing radiation to processing of these damages by the cellular repair/replication machinery, that lead to mutation are not well understood. The development of quantitative methods for measuring mutation-induction, such as the HPRT system, in cultured mammalian cells has provided an estimate of the mutagenic effects of x- and γ-rays as wen as of high LET radiation in both rodent and human cells. A major conclusion from these mutagenesis data is that high LET radiation induces mutations more efficiently than g-rays. Molecular analysis of mutations induced by sparsely ionizing radiation have detected major structural alterations at the gene level. Our molecular results based on analysis of human HPRT deficient mutants induced by γ-rays, α-particles and high energy charged particles indicate that higher LET radiation induce more total and large deletion mutations than γ-rays. Utilizing molecular techniques including polymerase chain reaction (PCR), Single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and Direct DNA sequencing, mutational spectra induced by ionizing radiation have been compared in different cell systems. Attempts have also been made to determine the mutagenic potential and the nature of mutation induced by low dose rate γ-rays. Defective repair, in the form of either a diminished capability for repair or inaccurate repair, can lead to increased risk of heritable mutations from radiation exposure. Therefore, the effects of DNA repair deficiency on the mutation induction in mammalian cells is reviewed

  19. Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

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    Anna Lundin

    2014-05-01

    Full Text Available Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs, a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6, a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV, and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

  20. Mnn10 Maintains Pathogenicity in Candida albicans by Extending α-1,6-Mannose Backbone to Evade Host Dectin-1 Mediated Antifungal Immunity.

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    Shi Qun Zhang

    2016-05-01

    Full Text Available The cell wall is a dynamic structure that is important for the pathogenicity of Candida albicans. Mannan, which is located in the outermost layer of the cell wall, has been shown to contribute to the pathogenesis of C. albicans, however, the molecular mechanism by which this occurs remains unclear. Here we identified a novel α-1,6-mannosyltransferase encoded by MNN10 in C. albicans. We found that Mnn10 is required for cell wall α-1,6-mannose backbone biosynthesis and polysaccharides organization. Deletion of MNN10 resulted in significant attenuation of the pathogenesis of C. albicans in a murine systemic candidiasis model. Inhibition of α-1,6-mannose backbone extension did not, however, impact the invasive ability of C. albicans in vitro. Notably, mnn10 mutant restored the invasive capacity in athymic nude mice, which further supports the notion of an enhanced host antifungal defense related to this backbone change. Mnn10 mutant induced enhanced Th1 and Th17 cell mediated antifungal immunity, and resulted in enhanced recruitment of neutrophils and monocytes for pathogen clearance in vivo. We also demonstrated that MNN10 could unmask the surface β-(1,3-glucan, a crucial pathogen-associated molecular pattern (PAMP of C. albicans recognized by host Dectin-1. Our results demonstrate that mnn10 mutant could stimulate an enhanced Dectin-1 dependent immune response of macrophages in vitro, including the activation of nuclear factor-κB, mitogen-activated protein kinase pathways, and secretion of specific cytokines such as TNF-α, IL-6, IL-1β and IL-12p40. In summary, our study indicated that α-1,6-mannose backbone is critical for the pathogenesis of C. albicans via shielding β-glucan from recognition by host Dectin-1 mediated immune recognition. Moreover, our work suggests that inhibition of α-1,6-mannose extension by Mnn10 may represent a novel modality to reduce the pathogenicity of C. albicans.

  1. Mutant INS-gene induced diabetes of youth: proinsulin cysteine residues impose dominant-negative inhibition on wild-type proinsulin transport.

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    Ming Liu

    2010-10-01

    Full Text Available Recently, a syndrome of Mutant INS-gene-induced Diabetes of Youth (MIDY, derived from one of 26 distinct mutations has been identified as a cause of insulin-deficient diabetes, resulting from expression of a misfolded mutant proinsulin protein in the endoplasmic reticulum (ER of insulin-producing pancreatic beta cells. Genetic deletion of one, two, or even three alleles encoding insulin in mice does not necessarily lead to diabetes. Yet MIDY patients are INS-gene heterozygotes; inheritance of even one MIDY allele, causes diabetes. Although a favored explanation for the onset of diabetes is that insurmountable ER stress and ER stress response from the mutant proinsulin causes a net loss of beta cells, in this report we present three surprising and interlinked discoveries. First, in the presence of MIDY mutants, an increased fraction of wild-type proinsulin becomes recruited into nonnative disulfide-linked protein complexes. Second, regardless of whether MIDY mutations result in the loss, or creation, of an extra unpaired cysteine within proinsulin, Cys residues in the mutant protein are nevertheless essential in causing intracellular entrapment of co-expressed wild-type proinsulin, blocking insulin production. Third, while each of the MIDY mutants induces ER stress and ER stress response; ER stress and ER stress response alone appear insufficient to account for blockade of wild-type proinsulin. While there is general agreement that ultimately, as diabetes progresses, a significant loss of beta cell mass occurs, the early events described herein precede cell death and loss of beta cell mass. We conclude that the molecular pathogenesis of MIDY is initiated by perturbation of the disulfide-coupled folding pathway of wild-type proinsulin.

  2. Induction and use of artificial mutants in sweet potato

    International Nuclear Information System (INIS)

    Marumine, Shokichi

    1984-01-01

    X-ray, ethylene imine, 32 P and 60 Co were used as mutagen for sweet potato mutation breeding and visible variations were observed for all mutagen. In the case of 60 Co irradiation, mutation rate of skin color is 0.5-1.3% based on cutting. Direction and variation of dry matter and tuber yield of mutants which were induced by 32 P and/or 60 Co irradiation showed more deteriorative variation than progressive variation but some induced mutant lines show same or superior characters than original line. In the case of 32 P irradiation to tuber, obstruction is not so much up to dese of 10,000 μci per tuber but treatment of 330 μci per cutting approximate to LD 50 . By tuber treatment with 60 Co gamma rays, suppression of sprouting occurred in dose of 30kR. Tendency to increase a variation was not observed at higher doses. 50-200 μci per cutting or 300-500 μci per tuber in 32 P treatment and 15 kR in 60 Co gamma-irradiation for tuber seemed to be optimum dosages. Hybrid seed of mutant selected for dry matter content was compared with that of original line and it was concluded that the variation of selected line was genetic. Mutant induced by 32 P and 60 Co treatment was used as a parental material and progeny of the cross was selected for practical characters. As a result, a line of higher starch yield with high resistance to pest and disease was selected and this line was used as parental material of further breeding. (author)

  3. Modes of overinitiation, dnaA gene expression, and inhibition of cell division in a novel cold-sensitive hda mutant of Escherichia coli.

    Science.gov (United States)

    Fujimitsu, Kazuyuki; Su'etsugu, Masayuki; Yamaguchi, Yoko; Mazda, Kensaku; Fu, Nisi; Kawakami, Hironori; Katayama, Tsutomu

    2008-08-01

    The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the beta clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25 degrees C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25 degrees C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42 degrees C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25 degrees C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25 degrees C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway.

  4. Modes of Overinitiation, dnaA Gene Expression, and Inhibition of Cell Division in a Novel Cold-Sensitive hda Mutant of Escherichia coli▿

    Science.gov (United States)

    Fujimitsu, Kazuyuki; Su'etsugu, Masayuki; Yamaguchi, Yoko; Mazda, Kensaku; Fu, Nisi; Kawakami, Hironori; Katayama, Tsutomu

    2008-01-01

    The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the β clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25°C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25°C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42°C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25°C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25°C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway. PMID:18502852

  5. Targeting NF-κB RelA/p65 phosphorylation overcomes RITA resistance.

    Science.gov (United States)

    Bu, Yiwen; Cai, Guoshuai; Shen, Yi; Huang, Chenfei; Zeng, Xi; Cao, Yu; Cai, Chuan; Wang, Yuhong; Huang, Dan; Liao, Duan-Fang; Cao, Deliang

    2016-12-28

    Inactivation of p53 occurs frequently in various cancers. RITA is a promising anticancer small molecule that dissociates p53-MDM2 interaction, reactivates p53 and induces exclusive apoptosis in cancer cells, but acquired RITA resistance remains a major drawback. This study found that the site-differential phosphorylation of nuclear factor-κB (NF-κB) RelA/p65 creates a barcode for RITA chemosensitivity in cancer cells. In naïve MCF7 and HCT116 cells where RITA triggered vast apoptosis, phosphorylation of RelA/p65 increased at Ser536, but decreased at Ser276 and Ser468; oppositely, in RITA-resistant cells, RelA/p65 phosphorylation decreased at Ser536, but increased at Ser276 and Ser468. A phosphomimetic mutation at Ser536 (p65/S536D) or silencing of endogenous RelA/p65 resensitized the RITA-resistant cells to RITA while the phosphomimetic mutant at Ser276 (p65/S276D) led to RITA resistance of naïve cells. In mouse xenografts, intratumoral delivery of the phosphomimetic p65/S536D mutant increased the antitumor activity of RITA. Furthermore, in the RITA-resistant cells ATP-binding cassette transporter ABCC6 was upregulated, and silencing of ABCC6 expression in these cells restored RITA sensitivity. In the naïve cells, ABCC6 delivery led to RITA resistance and blockage of p65/S536D mutant-induced RITA sensitivity. Taken together, these data suggest that the site-differential phosphorylation of RelA/p65 modulates RITA sensitivity in cancer cells, which may provide an avenue to manipulate RITA resistance. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. The effect of dithiothreitol on radiation-induced genetic damage in Arabidopsis thaliana (L) Heynh

    International Nuclear Information System (INIS)

    Dellaert, L.M.W.

    1980-01-01

    A study was made on the effect of dithiothreitol (DTT; present during irradiation) on M 1 ovule sterility, M 2 embryonic lethals, M 2 chlorophyll mutants and M 2 viable mutants induced with fast neutrons or X-rays in Arabidopsis thaliana. DTT provides considerable protection against both fast-neutron and X-ray induced genetic damage. However, a higher protection was observed against M 1 ovule sterility, than against embryonic lethals, chlorophylls and viable mutants. This implies a significant DTT-induced spectral shift (0.01 < p < 0.05), i.e. a shift in the relative frequencies of the different genetic parameters. This spectral shift is explained on the basis of a specific DTT protection against radiation-induced strand breaks, and by differences in the ratio strand breaks/base damage for the genetic parameters concerned, i.e. a higher ratio for ovule sterility than for the other parameters. The induction of the genetic damage by ionizing radiation, either with or without DTT, is described by a mathematical model, which includes both strand breaks and base damage. The model shows that the resolving power of a test for a 'mutation'spectral shift depends on the relative values of the strandbreak reduction factor of -SH compounds and on the ratio strand breaks/base damage of the genetic parameters. For each genetic parameter the DTT damage reduction factor (DRF) is calculated per irradiation dose, and in addition the average (over-all doses) ratio strand breaks/base damage. (orig.)

  7. Heterozygous effects of irradiated chromosomes on viability in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Simmons, M.J.

    1976-01-01

    Two large experiments were conducted in order to evaluate the heterozygous effects of irradiated chromosomes on viability. Mutations were accumulated on several hundred second chromosomes by delivering doses of 2,500R over either two or four generations for total x-ray exposures of 5,000R or 10,000R. Chromosomes treated with 5,000R were screened for lethals after the first treatment, and surviving nonlethals were used to generate families of fully treated chromosomes. The members of these families shared the effects of the first irradiation, but differed with respect to those of the second. The chromosomes treated with 10,000R were not grouped into families since mutations were accumulated independently on each chromosome in that experiment. Heterozygous effects on viability of the irradiated chromosomes were tested in both isogenic (homozygous) and nonisogenic (heterozygous) genetic backgrounds. In conjunction with these tests, homozygous viabilities were determined by the marked-inversion technique. This permitted a separation of the irradiated chromosomes into those which were drastic when made homozygous and those which were not. The results indicate that drastic chromosomes have deleterious effects in heterozygous condition, since viability was reduced by 2 to 4 percent in tests performed with the 10,000R chromosomes, and by 1 percent in those involving the 5,000R material. Within a series of tests, the effects were more pronounced when the genetic background was homozygous. These results suggest that the mutants induced by high doses of x-rays are principally drastic ones which show deleterious effects on viability in heterozygous condition

  8. Attenuated bioluminescent Brucella melitensis mutants GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091) confer protection in mice.

    Science.gov (United States)

    Rajashekara, Gireesh; Glover, David A; Banai, Menachem; O'Callaghan, David; Splitter, Gary A

    2006-05-01

    In vivo bioluminescence imaging is a persuasive approach to investigate a number of issues in microbial pathogenesis. Previously, we have applied bioluminescence imaging to gain greater insight into Brucella melitensis pathogenesis. Endowing Brucella with bioluminescence allowed direct visualization of bacterial dissemination, pattern of tissue localization, and the contribution of Brucella genes to virulence. In this report, we describe the pathogenicity of three attenuated bioluminescent B. melitensis mutants, GR019 (virB4), GR024 (galE), and GR026 (BMEI1090-BMEI1091), and the dynamics of bioluminescent virulent bacterial infection following vaccination with these mutants. The virB4, galE, and BMEI1090-BMEI1091 mutants were attenuated in interferon regulatory factor 1-deficient (IRF-1(-/-)) mice; however, only the GR019 (virB4) mutant was attenuated in cultured macrophages. Therefore, in vivo imaging provides a comprehensive approach to identify virulence genes that are relevant to in vivo pathogenesis. Our results provide greater insights into the role of galE in virulence and also suggest that BMEI1090 and downstream genes constitute a novel set of genes involved in Brucella virulence. Survival of the vaccine strain in the host for a critical period is important for effective Brucella vaccines. The galE mutant induced no changes in liver and spleen but localized chronically in the tail and protected IRF-1(-/-) and wild-type mice from virulent challenge, implying that this mutant may serve as a potential vaccine candidate in future studies and that the direct visualization of Brucella may provide insight into selection of improved vaccine candidates.

  9. Delayed chromosomal instability caused by large deletion

    International Nuclear Information System (INIS)

    Ojima, M.; Suzuki, K.; Kodama, S.; Watanabe, M.

    2003-01-01

    Full text: There is accumulating evidence that genomic instability, manifested by the expression of delayed phenotypes, is induced by X-irradiation but not by ultraviolet (UV) light. It is well known that ionizing radiation, such as X-rays, induces DNA double strand breaks, but UV-light mainly causes base damage like pyrimidine dimers and (6-4) photoproducts. Although the mechanism of radiation-induced genomic instability has not been thoroughly explained, it is suggested that DNA double strand breaks contribute the induction of genomic instability. We examined here whether X-ray induced gene deletion at the hprt locus induces delayed instability in chromosome X. SV40-immortalized normal human fibroblasts, GM638, were irradiated with X-rays (3, 6 Gy), and the hprt mutants were isolated in the presence of 6-thioguanine (6-TG). A 2-fold and a 60-fold increase in mutation frequency were found by 3 Gy and 6 Gy irradiation, respectively. The molecular structure of the hprt mutations was determined by multiplex polymerase chain reaction of nine exons. Approximately 60% of 3 Gy mutants lost a part or the entire hprt gene, and the other mutants showed point mutations like spontaneous mutants. All 6 Gy mutants show total gene deletion. The chromosomes of the hprt mutants were analyzed by Whole Human Chromosome X Paint FISH or Xq telomere FISH. None of the point or partial gene deletion mutants showed aberrations of X-chromosome, however total gene deletion mutants induced translocations and dicentrics involving chromosome X. These results suggest that large deletion caused by DNA double strand breaks destabilizes chromosome structure, which may be involved in an induction of radiation-induced genomic instability

  10. Reducing cytoplasmic polyamine oxidase activity in Arabidopsis increases salt and drought tolerance by reducing reactive oxygen species production and increasing defense gene expression

    Directory of Open Access Journals (Sweden)

    G.H.M. eSagor

    2016-02-01

    Full Text Available The link between polyamine oxidases (PAOs, which function in polyamine catabolism, and stress responses remains elusive. Here, we address this issue using Arabidopsis pao mutants in which the expression of the five PAO genes is knocked-out or knocked-down. As the five single pao mutants and wild type (WT showed similar response to salt stress, we tried to generate the mutants that have either the cytoplasmic PAO pathway (pao1 pao5 or the peroxisomal PAO pathway (pao2 pao3 pao4 silenced. However, the latter triple mutant was not obtained. Thus, in this study, we used two double mutants, pao1 pao5 and pao2 pao4. Of interest, pao1 pao5 mutant was NaCl- and drought-tolerant, whereas pao2 pao4 showed similar sensitivity to those stresses as WT. To reveal the underlying mechanism of salt tolerance, further analyses were performed. Na uptake of the mutant (pao1 pao5 decreased to 75% of WT. PAO activity of the mutant was reduced to 62% of WT. The content of reactive oxygen species (ROS such as hydrogen peroxide, a reaction product of PAO action, and superoxide anion in the mutant became 81% and 72% of the levels in WT upon salt treatment. The mutant contained 2.8-fold higher thermospermine compared to WT. Moreover, the mutant induced the genes of salt overly sensitive-, abscisic acid (ABA-dependent- and ABA-independent- pathways more strongly than WT upon salt treatment. The results suggest that the Arabidopsis plant silencing cytoplasmic PAOs shows salinity tolerance by reducing ROS production and strongly inducing subsets of stress-responsive genes under stress conditions.

  11. The RNA Chaperone Hfq Is Involved in Stress Tolerance and Virulence in Uropathogenic Proteus mirabilis

    Science.gov (United States)

    Wang, Min-Cheng; Liaw, Shwu-Jen

    2014-01-01

    Hfq is a bacterial RNA chaperone involved in the riboregulation of diverse genes via small noncoding RNAs. Here, we show that Hfq is critical for the uropathogenic Proteus mirabilis to effectively colonize the bladder and kidneys in a murine urinary tract infection (UTI) model and to establish burned wound infection of the rats. In this regard, we found the hfq mutant induced higher IL-8 and MIF levels of uroepithelial cells and displayed reduced intra-macrophage survival. The loss of hfq affected bacterial abilities to handle H2O2 and osmotic pressures and to grow at 50°C. Relative to wild-type, the hfq mutant had reduced motility, fewer flagella and less hemolysin expression and was less prone to form biofilm and to adhere to and invade uroepithelial cells. The MR/P fimbrial operon was almost switched to the off phase in the hfq mutant. In addition, we found the hfq mutant exhibited an altered outer membrane profile and had higher RpoE expression, which indicates the hfq mutant may encounter increased envelope stress. With the notion of envelope disturbance in the hfq mutant, we found increased membrane permeability and antibiotic susceptibilities in the hfq mutant. Finally, we showed that Hfq positively regulated the RpoS level and tolerance to H2O2 in the stationary phase seemed largely mediated through the Hfq-dependent RpoS expression. Together, our data indicate that Hfq plays a critical role in P. mirabilis to establish UTIs by modulating stress responses, surface structures and virulence factors. This study suggests Hfq may serve as a scaffold molecule for development of novel anti-P. mirabilis drugs and P. mirabilis hfq mutant is a vaccine candidate for preventing UTIs. PMID:24454905

  12. Xeroderma Pigmentosum Group A Suppresses Mutagenesis Caused by Clustered Oxidative DNA Adducts in the Human Genome.

    Science.gov (United States)

    Sassa, Akira; Kamoshita, Nagisa; Kanemaru, Yuki; Honma, Masamitsu; Yasui, Manabu

    2015-01-01

    Clustered DNA damage is defined as multiple sites of DNA damage within one or two helical turns of the duplex DNA. This complex damage is often formed by exposure of the genome to ionizing radiation and is difficult to repair. The mutagenic potential and repair mechanisms of clustered DNA damage in human cells remain to be elucidated. In this study, we investigated the involvement of nucleotide excision repair (NER) in clustered oxidative DNA adducts. To identify the in vivo protective roles of NER, we established a human cell line lacking the NER gene xeroderma pigmentosum group A (XPA). XPA knockout (KO) cells were generated from TSCER122 cells derived from the human lymphoblastoid TK6 cell line. To analyze the mutagenic events in DNA adducts in vivo, we previously employed a system of tracing DNA adducts in the targeted mutagenesis (TATAM), in which DNA adducts were site-specifically introduced into intron 4 of thymidine kinase genes. Using the TATAM system, one or two tandem 7,8-dihydro-8-oxoguanine (8-oxoG) adducts were introduced into the genomes of TSCER122 or XPA KO cells. In XPA KO cells, the proportion of mutants induced by a single 8-oxoG (7.6%) was comparable with that in TSCER122 cells (8.1%). In contrast, the lack of XPA significantly enhanced the mutant proportion of tandem 8-oxoG in the transcribed strand (12%) compared with that in TSCER122 cells (7.4%) but not in the non-transcribed strand (12% and 11% in XPA KO and TSCER122 cells, respectively). By sequencing the tandem 8-oxoG-integrated loci in the transcribed strand, we found that the proportion of tandem mutations was markedly increased in XPA KO cells. These results indicate that NER is involved in repairing clustered DNA adducts in the transcribed strand in vivo.

  13. Mutation induction by ion beams in arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, Atsushi [Japan Atomic Energy Research Inst., Takasaki, Gunma (Japan). Takasaki Radiation Chemistry Research Establishment

    1999-07-01

    An investigation was made on characteristics of ion beams for the biological effects and the induction of mutation using Arabidopsis plant as a model plant for the molecular genetics. Here, the characteristics of mutation at the molecular level as well as new mutants induced by ion beams were described. The ast and sep1 were obtained from the offspring of 1488 carbon ion-irradiated seeds respectively. The uvi1-uvi4 mutants were also induced from 1280 M{sub 1} lines. Thus, ion beams can induce not only known mutants such as tt, gl and hy but also novel mutants with high frequency. Even in the tt phenotype, two new mutant loci other than known loci were found. In chrysanthemum, several kinds of single, complex or stripped flower-color mutants that have been never induced by {gamma}irradiation, indicating that ion beams could produce a variety of mutants with the same phenotype. In conclusion, ion beams for the mutation induction are characterized by 1) to induce mutants with high frequency, 2) to show broad mutation spectrum and 3) to produce novel mutants. For these reasons, chemical mutagens such as EMS and low LET ionizing radiation such as X-rays and {gamma}-rays will predominantly induce many but small modifications or DNA damages on the DNA strands. As the result, several point mutations will be produced on the genome. On the contrary, ion beams as a high LET ionizing radiation will not cause so many but large and irreparable DNA damage locally, resulting in production of limited number of null mutation. (M.N.)

  14. Gamma-ray mutagenesis studies in a new human-hamster hybrid, A(L)CD59(+/-), which has two human chromosomes 11 but is hemizygous for the CD59 gene

    Science.gov (United States)

    Kraemer, S. M.; Vannais, D. B.; Kronenberg, A.; Ueno, A.; Waldren, C. A.; Chatterjee, A. (Principal Investigator)

    2001-01-01

    Kraemer, S. M., Vannais, D. B., Kronenberg, A., Ueno, A. and Waldren, C. A. Gamma-Ray Mutagenesis Studies in a New Human-Hamster Hybrid, A(L)CD59(+/-), which has Two Human Chromosomes 11 but is Hemizygous for the CD59 Gene. Radiat. Res. 156, 10-19 (2001).We have developed a human-CHO hybrid cell line, named A(L)CD59(+/-), which has two copies of human chromosome 11 but is hemizygous for the CD59 gene and the CD59 cell surface antigen that it encodes. Our previous studies used the A(L) and A(L)C hybrids that respectively contain one or two sets of CHO chromosomes plus a single copy of human chromosome 11. The CD59 gene at 11p13.5 and the CD59 antigen encoded by it are the principal markers used in our mutagenesis studies. The hybrid A(L)CD59(+/-) contains two copies of human chromosome 11, only one of which carries the CD59 gene. The incidence of CD59 (-) mutants (formerly called S1(-)) induced by (137)Cs gamma rays is about fivefold greater in A(L)CD59(+/-) cells than in A(L) cells. Evidence is presented that this increase in mutant yield is due to the increased induction of certain classes of large chromosomal mutations that are lethal to A(L) cells but are tolerated in the A(L)CD59(+/-) hybrid. In addition, significantly more of the CD59 (-) mutants induced by (137)Cs gamma rays in A(L)CD59(+/-) cells display chromosomal instability than in A(L) cells. On the other hand, the yield of gamma-ray-induced CD59 (-) mutants in A(L)CD59(+/-) cells is half that of the A(L)C hybrid, which also tolerates very large mutations but has only one copy of human chromosome 11. We interpret the difference in mutability as evidence that repair processes involving the homologous chromosomes 11 play a role in determining mutant yields. The A(L)CD59(+/-) hybrid provides a useful new tool for quantifying mutagenesis and shedding light on mechanisms of genetic instability and mutagenesis.

  15. Booster vaccination with safe, modified, live-attenuated mutants of Brucella abortus strain RB51 vaccine confers protective immunity against virulent strains of B. abortus and Brucella canis in BALB/c mice.

    Science.gov (United States)

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

    2015-11-01

    Brucella abortus attenuated strain RB51 vaccine (RB51) is widely used in prevention of bovine brucellosis. Although vaccination with this strain has been shown to be effective in conferring protection against bovine brucellosis, RB51 has several drawbacks, including residual virulence for animals and humans. Therefore, a safe and efficacious vaccine is needed to overcome these disadvantages. In this study, we constructed several gene deletion mutants (ΔcydC, ΔcydD and ΔpurD single mutants, and ΔcydCΔcydD and ΔcydCΔpurD double mutants) of RB51 with the aim of increasing the safety of the possible use of these mutants as vaccine candidates. The RB51ΔcydC, RB51ΔcydD, RB51ΔpurD, RB51ΔcydCΔcydD and RB51ΔcydCΔpurD mutants exhibited significant attenuation of virulence when assayed in murine macrophages in vitro or in BALB/c mice. A single intraperitoneal immunization with RB51ΔcydC, RB51ΔcydD, RB51ΔcydCΔcydD or RB51ΔcydCΔpurD mutants was rapidly cleared from mice within 3 weeks, whereas the RB51ΔpurD mutant and RB51 were detectable in spleens until 4 and 7 weeks, respectively. Vaccination with a single dose of RB51 mutants induced lower protective immunity in mice than did parental RB51. However, a booster dose of these mutants provided significant levels of protection in mice against challenge with either the virulent homologous B. abortus strain 2308 or the heterologous Brucella canis strain 26. In addition, these mutants were found to induce a mixed but T-helper-1-biased humoral and cellular immune response in immunized mice. These data suggest that immunization with a booster dose of attenuated RB51 mutants provides an attractive strategy to protect against either bovine or canine brucellosis.

  16. Impaired CK1 delta activity attenuates SV40-induced cellular transformation in vitro and mouse mammary carcinogenesis in vivo.

    Directory of Open Access Journals (Sweden)

    Heidrun Hirner

    Full Text Available Simian virus 40 (SV40 is a powerful tool to study cellular transformation in vitro, as well as tumor development and progression in vivo. Various cellular kinases, among them members of the CK1 family, play an important role in modulating the transforming activity of SV40, including the transforming activity of T-Ag, the major transforming protein of SV40, itself. Here we characterized the effects of mutant CK1δ variants with impaired kinase activity on SV40-induced cell transformation in vitro, and on SV40-induced mammary carcinogenesis in vivo in a transgenic/bi-transgenic mouse model. CK1δ mutants exhibited a reduced kinase activity compared to wtCK1δ in in vitro kinase assays. Molecular modeling studies suggested that mutation N172D, located within the substrate binding region, is mainly responsible for impaired mutCK1δ activity. When stably over-expressed in maximal transformed SV-52 cells, CK1δ mutants induced reversion to a minimal transformed phenotype by dominant-negative interference with endogenous wtCK1δ. To characterize the effects of CK1δ on SV40-induced mammary carcinogenesis, we generated transgenic mice expressing mutant CK1δ under the control of the whey acidic protein (WAP gene promoter, and crossed them with SV40 transgenic WAP-T-antigen (WAP-T mice. Both WAP-T mice as well as WAP-mutCK1δ/WAP-T bi-transgenic mice developed breast cancer. However, tumor incidence was lower and life span was significantly longer in WAP-mutCK1δ/WAP-T bi-transgenic animals. The reduced CK1δ activity did not affect early lesion formation during tumorigenesis, suggesting that impaired CK1δ activity reduces the probability for outgrowth of in situ carcinomas to invasive carcinomas. The different tumorigenic potential of SV40 in WAP-T and WAP-mutCK1δ/WAP-T tumors was also reflected by a significantly different expression of various genes known to be involved in tumor progression, specifically of those involved in wnt-signaling and DNA

  17. Using reverse genetics to manipulate the NSs gene of the Rift Valley fever virus MP-12 strain to improve vaccine safety and efficacy.

    Science.gov (United States)

    Kalveram, Birte; Lihoradova, Olga; Indran, Sabarish V; Ikegami, Tetsuro

    2011-11-01

    NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.

  18. Transcriptomic analysis of vulvovaginal candidiasis identifies a role for the NLRP3 inflammasome.

    Science.gov (United States)

    Bruno, Vincent M; Shetty, Amol C; Yano, Junko; Fidel, Paul L; Noverr, Mairi C; Peters, Brian M

    2015-04-21

    Treatment of vulvovaginal candidiasis (VVC), caused most frequently by Candida albicans, represents a significant unmet clinical need. C. albicans, as both a commensal and a pathogenic organism, has a complex and poorly understood interaction with the vaginal environment. Understanding the complex nature of this relationship is necessary for the development of desperately needed therapies to treat symptomatic infection. Using transcriptome sequencing (RNA-seq), we characterized the early murine vaginal and fungal transcriptomes of the organism during VVC. Network analysis of host genes that were differentially expressed between infected and naive mice predicted the activation or repression of several signaling pathways that have not been previously associated with VVC, including NLRP3 inflammasome activation. Intravaginal challenge of Nlrp3(-/-) mice with C. albicans demonstrated severely reduced levels of polymorphonuclear leukocytes (PMNs), alarmins, and inflammatory cytokines, including interleukin-1β (IL-1β) (the hallmarks of VVC immunopathogenesis) in vaginal lavage fluid. Intravaginal administration of wild-type (WT) mice with glyburide, a potent inhibitor of the NLRP3 inflammasome, reduced PMN infiltration and IL-1β to levels comparable to those observed in Nlrp3(-/-) mice. Furthermore, RNA-seq analysis of C. albicans genes indicated robust expression of hypha-associated secreted aspartyl proteinases 4, 5, and 6 (SAP4-6), which are known inflammasome activators. Despite colonization similar to that of the WT strain, ΔSAP4-6 triple and ΔSAP5 single mutants induced significantly less PMN influx and IL-1β during intravaginal challenge. Our findings demonstrate a novel role for the inflammasome in the immunopathogenesis of VVC and implicate the hypha-associated SAPs as major C. albicans virulence determinants during vulvovaginal candidiasis. Vaginitis, most commonly caused by the fungus Candida albicans, results in significant quality-of-life issues for

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

    Directory of Open Access Journals (Sweden)

    Nasie eConstantino

    2013-12-01

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

  20. Relationship between phytohormones and genic male sterility induced by space flight in maize

    International Nuclear Information System (INIS)

    Cao Moju; Cheng Jiang; Wang Jing; Zhang Caibo; Pan Guangtang; Rong Tingzhao

    2010-01-01

    High performance liquid chromatography (HPLC) was adopted to analyze the content of endogenous hormones such as zeatin (ZT), gibberellin (GA s ), auxin (IAA) and abscisic acid (ABA) in male fertile plants and male sterile plants of maize derived from sister-cross population, which was produced with male sterile mutant induced by space flight. Exogenous GA s treatment was applied to plants of sister-cross population of maize, and the fertility of the maize plant both in the exogenous GA s treatment plots and the control plot was investigated. The results showed that the fertility segregation ratios of sister-cross populations did not change with exogenous GA s treatment. At the seedling stage, only the differences of IAA and ABA contents in leaf between fertile and sterile plants were significant at the 0. 05 level, and the differences of ZT, GA s , IAA and ABA contents between sterile and fertile plants all were at 0. 05 or 0. 01 level significant, at the jointing stage. At the uninucleate microspore stage the differences of IAA and ABA contents in anther were significant at 0. 01 level, but at the binucleate pollen stage only the difference of ABA content was significant at 0. 01 level. During the four different developmental stages, the contents of ZT, GA s and IAA were higher in fertile plants than in sterile plants, but the content of ABA was lower in fertile plants than in sterile plants in the two different tissues. It was concluded that exogenous GA s treatment could not alter the fertility of maize plants, so this mutant did not belong to the GA s sensitive type. The changes of the hormone level or the hormones ratio might be related to the pollen abortion of the male sterile material used in this experiment, and it seems that ABA and IAA may have a closer relationship with the fertility expression of the material used in this experiment than ZT and GA s according to the significance tests. (authors)

  1. Nitrate assimilation pathway (NAP): role of structural (nit) and transporter (ntr1) genes in Fusarium oxysporum f.sp. lycopersici growth and pathogenicity.

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    Gomez-Gil, Lucia; Camara Almiron, Jesus; Rodriguez Carrillo, Patricia Lizett; Olivares Medina, Cindy Nayely; Bravo Ruiz, Gustavo; Romo Rodriguez, Pamela; Corrales Escobosa, Alma Rosa; Gutierrez Corona, Felix; Roncero, M Isabel

    2018-04-01

    The tomato pathogen Fusarium oxysporum f.sp. lycopersici possesses the capability to use nitrate as the only nitrogen source under aerobic and anaerobic conditions and to activate virulence-related functions when cultivated in the presence of nitrate, but not in ammonium. The genome of F. oxysporum f.sp. lycopersici encodes three paralogs nitrate reductase (NR) genes (nit1, nit2 and nit3) and one predicted ortholog of the Aspergillus nidulans high-affinity nitrate/nitrite transporters NtrA and NtrB, named ntr1. We set out to clarify the role of nit1, nit2, nit3 and ntr1 genes in nitrate assimilation and in the virulence of F. oxysporum f.sp. lycopersici. Quantitative RT-PCR analysis revealed that only nit1, nit2 and ntr1 are expressed at significant levels during growth in nitrate as the only nitrogen source. Targeted deletion of nit1 and ntr1, but not of nit2 or nit3, severely impaired growth of F. oxysporum on nitrate as nitrogen source, indicating that Nit1 and Ntr1 proteins are involved in nitrate assimilation by the fungus; biochemical analysis of nit mutants indicated that Nit1 and Nit2 enzymes contribute to about 50 and 30% of the total NR activity, respectively. In addition, a spontaneous chlorate-resistant mutant derived from F. oxysporum 4287, denoted NitFG, was characterized, showing inability to grow in nitrate under aerobic and anaerobic conditions and low levels of NR activity, in spite of its increased transcription levels of nit1 and nit2 genes. Tomato plant infection assays showed that NitFG and ∆ntr1 mutants induced an earlier death in tomato plants, whereas the single mutants ∆nit1, ∆nit2 and ∆nit1∆nit2 double mutant showed a mortality rate similar to the wild-type strain. Taken together, these results indicate that the Nit1 and Ntr1 proteins are important for nitrate assimilation of F. oxysporum f.sp. lycopersici incubated under aerobic and anaerobic conditions and that this metabolic process is not essential for the virulence of

  2. Mutations of amino acids in the DNA-recognition domain of Epstein-Barr virus ZEBRA protein alter its sub-nuclear localization and affect formation of replication compartments

    International Nuclear Information System (INIS)

    Park, Richard; Heston, Lee; Shedd, Duane; Delecluse, Henri-Jacques; Miller, George

    2008-01-01

    foci. The speckled appearance of R179A and Y180E was more regular and clearly defined in EBV-positive than in EBV-negative 293 cells. The Y180E late-mutant induced EA-D, but prevented EA-D from localizing to globular replication compartments. These results show that individual amino acids within the basic domain influence localization of the ZEBRA protein and its capacity to induce EA-D to become located in mature viral replication compartments. Furthermore, these mutant ZEBRA proteins delineate several stages in the processes of nuclear re-organization which accompany lytic EBV replication

  3. Interactions of Francisella tularensis with Alveolar Type II Epithelial Cells and the Murine Respiratory Epithelium.

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    Matthew Faron

    Full Text Available Francisella tularensis is classified as a Tier 1 select agent by the CDC due to its low infectious dose and the possibility that the organism can be used as a bioweapon. The low dose of infection suggests that Francisella is unusually efficient at evading host defenses. Although ~50 cfu are necessary to cause human respiratory infection, the early interactions of virulent Francisella with the lung environment are not well understood. To provide additional insights into these interactions during early Francisella infection of mice, we performed TEM analysis on mouse lungs infected with F. tularensis strains Schu S4, LVS and the O-antigen mutant Schu S4 waaY::TrgTn. For all three strains, the majority of the bacteria that we could detect were observed within alveolar type II epithelial cells at 16 hours post infection. Although there were no detectable differences in the amount of bacteria within an infected cell between the three strains, there was a significant increase in the amount of cellular debris observed in the air spaces of the lungs in the Schu S4 waaY::TrgTn mutant compared to either the Schu S4 or LVS strain. We also studied the interactions of Francisella strains with human AT-II cells in vitro by characterizing the ability of these three strains to invade and replicate within these cells. Gentamicin assay and confocal microscopy both confirmed that F. tularensis Schu S4 replicated robustly within these cells while F. tularensis LVS displayed significantly lower levels of growth over 24 hours, although the strain was able to enter these cells at about the same level as Schu S4 (1 organism per cell, as determined by confocal imaging. The Schu S4 waaY::TrgTn mutant that we have previously described as attenuated for growth in macrophages and mouse virulence displayed interesting properties as well. This mutant induced significant airway inflammation (cell debris and had an attenuated growth phenotype in the human AT-II cells. These

  4. Pseudomonas-derived ceramidase induces production of inflammatory mediators from human keratinocytes via sphingosine-1-phosphate.

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    Ami Oizumi

    Full Text Available Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD. A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P stimulated the production of inflammatory mediators such as TNF-α and IL-8 from three-dimensionally cultured human primary keratinocytes (termed "3D keratinocytes", which form a stratum corneum. PaCDase alone did not affect TNF-α gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-α, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-α, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-α-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-α, mRNA. PaCDase induced NF-κB p65 phosphorylation. The NF-κB inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-κB p65 phosphorylation and reduction in the protein level of the NF-κB inhibitor IκBα. Collectively, these findings suggest that (i 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii S1P induces the production

  5. Streptococcus gordonii lipoproteins induce IL-8 in human periodontal ligament cells.

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    Kim, A Reum; Ahn, Ki Bum; Kim, Hyun Young; Seo, Ho Seong; Kum, Kee-Yeon; Yun, Cheol-Heui; Han, Seung Hyun

    2017-11-01

    Streptococcus gordonii, a Gram-positive oral bacterium, is a life-threatening pathogen that causes infective endocarditis. It is frequently isolated from the periapical lesions of patients with apical periodontitis and has thus been implicated in inflammatory responses. However, little is known about the virulence factors of S. gordonii responsible for the induction of inflammatory responses in the periapical areas. Here, we investigated the role of S. gordonii cell wall-associated virulence factors on interleukin (IL)-8 induction in human periodontal ligament (PDL) cells using ethanol-inactivated wild-type S. gordonii, a lipoteichoic acid (LTA)-deficient mutant (ΔltaS), and a lipoprotein-deficient mutant (Δlgt). Wild-type S. gordonii induced IL-8 expression at both the protein and mRNA levels in human PDL cells in a dose- and time-dependent manner. A transient transfection and reporter gene assay demonstrated that wild-type S. gordonii activated Toll-like receptor 2 (TLR2). Additionally, IL-8 production induced by wild-type S. gordonii was substantially inhibited by anti-TLR2-neutralizing antibodies. Both wild-type S. gordonii and the ΔltaS mutant induced IL-8 production; however, this response was not observed when cells were stimulated with the Δlgt mutant. Interestingly, lipoproteins purified from S. gordonii induced IL-8 production, whereas purified LTA did not. In addition, purified lipoproteins stimulated TLR2 more potently than LTA. Furthermore, S. gordonii-induced IL-8 expression was specifically inhibited by blocking p38 kinase, while lipoprotein-induced IL-8 expression was inhibited by blocking p38 kinase, ERK, or JNK. Of particular note, exogenous addition of purified S. gordonii lipoproteins enhanced Δlgt-induced IL-8 production in human PDL cells to an extent similar to that induced by the wild-type strain. Collectively, these results suggest that lipoproteins are an important component of S. gordonii for the induction of IL-8 production in human

  6. Mast cell deficiency attenuates acupuncture analgesia for mechanical pain using c-kit gene mutant rats.

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    Cui, Xiang; Liu, Kun; Xu, Dandan; Zhang, Youyou; He, Xun; Liu, Hao; Gao, Xinyan; Zhu, Bing

    2018-01-01

    Acupuncture therapy plays a pivotal role in pain relief, and increasing evidence demonstrates that mast cells (MCs) may mediate acupuncture analgesia. The present study aims to investigate the role of MCs in acupuncture analgesia using c-kit gene mutant-induced MC-deficient rats. WsRC-Ws/Ws rats and their wild-type (WT) littermates (WsRC-+/+) were used. The number of MCs in skin of ST36 area was compared in two rats after immunofluorescence labeling. Mechanical withdrawal latency (MWL), mechanical withdrawal threshold (MWT), and thermal withdrawal latency (TWL) were measured on bilateral plantar for pain threshold evaluation before and after each stimulus. Acupuncture- and moxibustion-like stimuli (43°C, 46°C heat, 1 mA electroacupuncture [EA], 3 mA EA, and manual acupuncture [MA]) were applied randomly on different days. Fewer MCs were observed in the skin of ST36 in mutant rats compared to WT rats ( P 0.05). Bilateral MWL and MWT in WsRC-+/+ rats increased significantly after each stimulus compared to baseline ( P <0.01, P <0.001). In WsRC-Ws/Ws rats, only noxious stimuli could produce anti-nociceptive effects for mechanical pain (46°C, 3 mA EA, MA) ( P <0.01, P <0.001). Additionally, the net increases in MWL and MWT induced by most stimuli were greater in WT than in mutant rats ( P <0.05). For thermal nociception, either high- or low-intensity stimuli could significantly augment TWL in two rats ( P <0.001), and the net increases of TWL evoked by most stimuli were to the same extent in two genetic variants. MCs influence the basic mechanical but not thermal pain threshold. MCs participate in acupuncture analgesia in mechanical but not in thermal nociception, in that MC deficiency may attenuate the mechanical analgesia evoked by high-intensity stimuli and eliminate analgesia provoked by low-intensity stimuli.

  7. Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels.

    Science.gov (United States)

    Fu, Ssu-Ju; Jeng, Chung-Jiuan; Ma, Chia-Hao; Peng, Yi-Jheng; Lee, Chi-Ming; Fang, Ya-Ching; Lee, Yi-Ching; Tang, Sung-Chun; Hu, Meng-Chun; Tang, Chih-Yung

    2017-03-01

    Voltage-gated Ca V 2.1 channels comprise a pore-forming α 1A subunit with auxiliary α 2 δ and β subunits. Ca V 2.1 channels play an essential role in regulating synaptic signaling. Mutations in the human gene encoding the Ca V 2.1 subunit are associated with the cerebellar disease episodic ataxia type 2 (EA2). Several EA2-causing mutants exhibit impaired protein stability and exert dominant-negative suppression of Ca V 2.1 wild-type (WT) protein expression via aberrant proteasomal degradation. Here, we set out to delineate the protein degradation mechanism of human Ca V 2.1 subunit by identifying RNF138, an E3 ubiquitin ligase, as a novel Ca V 2.1-binding partner. In neurons, RNF138 and Ca V 2.1 coexist in the same protein complex and display notable subcellular colocalization at presynaptic and postsynaptic regions. Overexpression of RNF138 promotes polyubiquitination and accelerates protein turnover of Ca V 2.1. Disrupting endogenous RNF138 function with a mutant (RNF138-H36E) or shRNA infection significantly upregulates the Ca V 2.1 protein level and enhances Ca V 2.1 protein stability. Disrupting endogenous RNF138 function also effectively rescues the defective protein expression of EA2 mutants, as well as fully reversing EA2 mutant-induced excessive proteasomal degradation of Ca V 2.1 WT subunits. RNF138-H36E coexpression only partially restores the dominant-negative effect of EA2 mutants on Ca V 2.1 WT functional expression, which can be attributed to defective membrane trafficking of Ca V 2.1 WT in the presence of EA2 mutants. We propose that RNF138 plays a critical role in the homeostatic regulation of Ca V 2.1 protein level and functional expression and that RNF138 serves as the primary E3 ubiquitin ligase promoting EA2-associated aberrant degradation of human Ca V 2.1 subunits. SIGNIFICANCE STATEMENT Loss-of-function mutations in the human Ca V 2.1 subunit are linked to episodic ataxia type 2 (EA2), a dominantly inherited disease characterized by

  8. Proteomic analysis of oil bodies in mature Jatropha curcas seeds with different lipid content.

    Science.gov (United States)

    Liu, Hui; Wang, Cuiping; Chen, Fan; Shen, Shihua

    2015-01-15

    To reveal the difference among three mature Jatropha curcas seeds (JcVH, variant with high lipid content; JcW, wild type and JcVL, variant with low lipid content) with different lipid content, comparative proteomics was employed to profile the changes of oil body (OB) associated protein species by using gels-based proteomic technique. Eighty-three protein species were successfully identified through LTQ-ES-MS/MS from mature JcW seeds purified OBs. Two-dimensional electrophoresis analysis of J. curcas OB associated protein species revealed they had essential interactions with other organelles and demonstrated that oleosin and caleosin were the most abundant OB structural protein species. Twenty-eight OB associated protein species showed significant difference among JcVH, JcW and JcVL according to statistical analysis. Complementary transient expression analysis revealed that calcium ion binding protein (CalBP) and glycine-rich RNA binding protein (GRP) were well targeted in OBs apart from the oleosins. This study demonstrated that ratio of lipid content to caleosins abundance was involved in the regulation of OB size, and the mutant induced by ethylmethylsulfone treatment might be related to the caleosin like protein species. These findings are important for biotechnological improvement with the aim to alter the lipid content in J. curcas seeds. The economic value of Jatropha curcas largely depends on the lipid content in seeds which are mainly stored in the special organelle called oil bodies (OBs). In consideration of the biological importance and applications of J. curcas OB in seeds, it is necessary to further explore the components and functions of J. curcas OBs. Although a previous study concerning the J. curcas OB proteome revealed oleosins were the major OB protein component and additional protein species were similar to those in other oil seed plants, these identified OB associated protein species were corresponding to the protein bands instead of protein

  9. El citoesqueleto en la infección con virus dengue

    Directory of Open Access Journals (Sweden)

    Francisco Javier Díaz Castrillón

    2004-03-01

    de vimentina cambian su patrón reticular, formando prolongaciones celulares en donde se detecta aglomeración de haces con un aumento en inmunorreactividad.

    Estos hallazgos son compatibles con el efecto citopático del DV, pero se requieren anticuerpos que marquen específicamente los viriones, para poder vislumbrar la posible interacción entre el citoesqueleto y los virus. Estamos probando otros anticuerpos, y esperamos lograr detectar con más detalle este fenómeno. Las perspectivas son promisorias y nos darán aportes originales, pues hasta ahora no hay reportes al respecto. Además, estos datos pueden ser de utilidad para comprender la patogénesis del dengue desde la perspectiva de la biología celular.

    REFERENCIAS

    1. ARCANGELETTI MC, PINARDI F, MISSORINI S, DE CONTO F, CONTI G, PORTINCASA P, SCHERRER K, CHEZZI C. 1997. Modification of cytoskeleton and prosome networks in relation to protein synthesis in influenza A virus-infected LLC-MK2 cells. Virus Res. 51: 19-34.

    2. GALLEGO-GÓMEZ, J.C. 2003. El Dengue Hemorrágico: Emergencia, Re-emergencia y Globalización de la Pobreza. Simposio Anual “Tópicos en Enfermedades Infecciosas” del Depto. Microb. Parasit., Fac. Medicina, Universidad de Antioquia. Septiembre 24.

    3. GALLEGO-GÓMEZ, J.C., et al. “Vaccinia Virus and their Attenuated Mutants induce Epithelial to Mesenchymal Transition” XIV th International Poxvirus and Iridovirus Workshop Lake