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Sample records for vhl disease mutants

  1. Differences in regulation of tight junctions and cell morphology between VHL mutations from disease subtypes

    Directory of Open Access Journals (Sweden)

    Isanova Bella

    2009-07-01

    Full Text Available Abstract Background In von Hippel-Lindau (VHL disease, germline mutations in the VHL tumor suppressor gene cause clear cell renal carcinomas, hemangioblastomas, and pheochromocytomas. The VHL gene product is part of an ubiquitin E3 ligase complex and hypoxia-inducible factor alpha (HIF-α is a key substrate, although additional VHL functions have been described. A genotype-phenotype relationship exists in VHL disease such that specific VHL mutations elicit certain subsets of these tumors. Here, we examine VHL genotype-phenotype correlations at the cellular level, focusing on the regulation of tight junctions and cell morphology. Methods Wild-type and various mutant VHL proteins representing VHL disease subtypes were stably expressed in 3 VHL-negative renal carcinoma cell lines. Using these cell lines, the roles of various VHL-associated cellular functions in regulation of cell morphology were investigated. Results As a whole, type 1 mutants varied greatly from type 2 mutants, demonstrating high levels of HIF-2α, cyclin D1 and α5 integrin, lower p27 levels, and a spindly, fibroblastic cellular appearance. Type 2 mutations demonstrated an epithelial morphology similar to wild-type VHL in the majority of the renal cell lines used. Knockdown of p27 in cells with wild-type VHL led to perturbations of both epithelial morphology and ZO-1 localization to tight junctions. ZO-1 localization correlated well with VHL disease subtypes, with greater mislocalization observed for genotypes associated with a higher risk of renal carcinoma. HIF-2α knockdown in 786-O partially restored ZO-1 localization, but did not restore an epithelial morphology. Conclusion VHL has both HIF-α dependent and HIF-α independent functions in regulating tight junctions and cell morphology that likely impact the clinical phenotypes seen in VHL disease.

  2. VHL Manifestations

    Science.gov (United States)

    ... Us Website References Search Patients / What is VHL? / Manifestations People who have VHL disease may experience tumors ... very important to check regularly for possible VHL manifestations throughout a person’s lifetime. Most of these VHL ...

  3. Von Hippel-Lindau disease (vHL)

    DEFF Research Database (Denmark)

    Binderup, Marie Louise Mølgaard; Bisgaard, Søs Marie Luise; Harbud, Vibeke

    2013-01-01

    These clinical guidelines outline the criteria and recommendations for diagnostic and genetic work-up of families suspected of von Hippel-Lindau disease (vHL), as well as recommendations for prophylactic surveillance for vHL patients. The guideline has been composed by the Danish Coordination Group...... for vHL which is comprised of Danish doctors and specialists interested in vHL. The recommendations are based on longstanding clinical experience, Danish original research, and extensive review of the international literature. vHL is a hereditary multi-tumour disease caused by germline mutations...... cell carcinoma), the adrenal glands (pheochromocytoma), the pancreas, as well as in other organs. As many different organs can be affected, several medical specialities often take part in both diagnosis and treatment of manifestations. vHL should be suspected in individuals with a family history...

  4. Von Hippel-Lindau disease (vHL)

    DEFF Research Database (Denmark)

    Binderup, Marie Louise Mølgaard; Bisgaard, Søs Marie Luise; Harbud, Vibeke

    2013-01-01

    These clinical guidelines outline the criteria and recommendations for diagnostic and genetic work-up of families suspected of von Hippel-Lindau disease (vHL), as well as recommendations for prophylactic surveillance for vHL patients. The guideline has been composed by the Danish Coordination Group...... for vHL which is comprised of Danish doctors and specialists interested in vHL. The recommendations are based on longstanding clinical experience, Danish original research, and extensive review of the international literature. vHL is a hereditary multi-tumour disease caused by germline mutations...... of the disease, and/or in individuals with a vHL-associated manifestation; i.e. a hemangioblastoma in the retina or the central nervous system, familial or bilateral pheochromocytomas, familial, multiple, or early onset renal cell carcinomas, and in individuals with an endolymphatic sac tumour in the inner ear...

  5. Surveillance in von Hippel-Lindau disease (vHL)

    DEFF Research Database (Denmark)

    Poulsen, Marie Louise Mølgaard; Budtz-Jørgensen, E; Bisgaard, M L

    2010-01-01

    54 living vHL-mutation carriers, risks of intercurrent manifestations in-between surveillance examinations were determined and clinical consequences of surveillance findings evaluated. Current recommendations of annual ophthalmic and abdominal examinations corresponded to acceptably low intercurrent...... for the patient. Also, pre-symptomatic surveillance increased cumulative incidence of clinical vHL diagnosis from 46% to 72% and from 89% to 94% by age 30 and 50 years, respectively. The present results promote optimization of surveillance, expectantly improving clinical vHL outcomes....

  6. Von Hippel-Lindau disease (vHL)

    DEFF Research Database (Denmark)

    Binderup, Marie Louise Mølgaard; Bisgaard, Søs Marie Luise; Harbud, Vibeke

    2013-01-01

    cell carcinoma), the adrenal glands (pheochromocytoma), the pancreas, as well as in other organs. As many different organs can be affected, several medical specialities often take part in both diagnosis and treatment of manifestations. vHL should be suspected in individuals with a family history...

  7. Von Hippel-Lindau disease (vHL)

    DEFF Research Database (Denmark)

    Binderup, Marie Louise Mølgaard; Bisgaard, Søs Marie Luise; Harbud, Vibeke

    2013-01-01

    in the VHL gene. vHL is inherited in an autosomal dominant manner. Predisposed individuals are advised to undergo prophylactic examinations, as they are at lifelong risk of developing multiple cysts and tumours, especially in the cerebellum, the spinal cord, the retina (hemangioblastomas), the kidneys (renal...... are recommended to start in infancy with annual paediatric examinations and ophthalmoscopy until the age of five years. From five to 14 years, annual plasma-metanephrine and plasma-normetanephrine tests, as well as annual hearing examinations are added. Also, an MRI (Magnetic Resonance Imaging) examination....../MRI of the abdomen, e) annual plasma-metanephrine, plasma-normetanephrine, and plasma-chromogranin A tests, and f) annual hearing examination at a department of audiology. It is advised that one doctor takes on the responsibility of coordination of and referral to the many examinations, and the communication...

  8. Hypoxia-inducible factor-2α stabilizes the von Hippel-Lindau (VHL) disease suppressor, Myb-related protein 2.

    Science.gov (United States)

    Okumura, Fumihiko; Joo-Okumura, Akiko; Nakatsukasa, Kunio; Kamura, Takumi

    2017-01-01

    Ubiquitin ligase von Hippel-Lindau tumor suppressor (pVHL) negatively regulates protein levels of hypoxia-inducible factor-α (HIF-α). Loss of pVHL causes HIF-α accumulation, which contributes to the pathogenesis of von Hippel-Lindau (VHL) disease. In contrast, v-Myb avian myeloblastosis viral oncogene homolog-like 2 (MYBL2; B-Myb), a transcription factor, prevents VHL pathogenesis by regulating gene expression of HIF-independent pathways. Both HIF-α and B-Myb are targets of pVHL-mediated polyubiquitination and proteasomal degradation. Here, we show that knockdown of HIF-2α induces downregulation of B-Myb in 786-O cells, which are deficient in pVHL, and this downregulation is prevented by proteasome inhibition. In the presence of pVHL and under hypoxia-like conditions, B-Myb and HIF-2α are both upregulated, and the upregulation of B-Myb requires expression of HIF-2α. We also show that HIF-2α and B-Myb interact in the nucleus, and this interaction is mediated by the central region of HIF-2α and the C-terminal region of B-Myb. These data indicate that oncogenic HIF-2α stabilizes B-Myb to suppress VHL pathogenesis.

  9. VHL type 2B mutations retain VBC complex form and function.

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    Kathryn E Hacker

    Full Text Available von Hippel-Lindau disease is characterized by a spectrum of hypervascular tumors, including renal cell carcinoma, hemangioblastoma, and pheochromocytoma, which occur with VHL genotype-specific differences in penetrance. VHL loss causes a failure to regulate the hypoxia inducible factors (HIF-1alpha and HIF-2alpha, resulting in accumulation of both factors to high levels. Although HIF dysregulation is critical to VHL disease-associated renal tumorigenesis, increasing evidence points toward gradations of HIF dysregulation contributing to the degree of predisposition to renal cell carcinoma and other manifestations of the disease.This investigation examined the ability of disease-specific VHL missense mutations to support the assembly of the VBC complex and to promote the ubiquitylation of HIF. Our interaction analysis supported previous observations that VHL Type 2B mutations disrupt the interaction between pVHL and Elongin C but maintain partial regulation of HIF. We additionally demonstrated that Type 2B mutant pVHL forms a remnant VBC complex containing the active members ROC1 and Cullin-2 which retains the ability to ubiquitylate HIF-1alpha.Our results suggest that subtypes of VHL mutations support an intermediate level of HIF regulation via a remnant VBC complex. These findings provide a mechanism for the graded HIF dysregulation and genetic predisposition for cancer development in VHL disease.

  10. Identification of 3 novel VHL germ-line mutations in Danish VHL patients

    DEFF Research Database (Denmark)

    Dandanell, Mette; Friis-Hansen, Lennart Jan; Sunde, Lone

    2012-01-01

    von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome in which the patients develop retinal and central nervous system hemangioblastomas, pheochromocytomas and clear-cell renal tumors. The autosomal dominant disease is caused by mutations in the VHL gene.......von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome in which the patients develop retinal and central nervous system hemangioblastomas, pheochromocytomas and clear-cell renal tumors. The autosomal dominant disease is caused by mutations in the VHL gene....

  11. Prevalence, birth incidence, and penetrance of von Hippel-Lindau disease (vHL) in Denmark

    DEFF Research Database (Denmark)

    Binderup, Marie Louise Mølgaard; Galanakis, Michael Carter Bisgaard; Budtz-Jørgensen, Esben

    2017-01-01

    . We further used national health registers to identify individuals who fulfilled the clinical diagnostic vHL criteria based on their registered diagnostic codes, but had not been diagnosed with vHL. We also assessed the medical histories of first-degree relatives to identify familial cases. This study...

  12. Parallel Regulation of von Hippel-Lindau Disease by pVHL-Mediated Degradation of B-Myb and Hypoxia-Inducible Factor α

    Science.gov (United States)

    Uematsu, Keiji; Byrne, Stuart D.; Hirano, Mie; Joo-Okumura, Akiko; Nishikimi, Akihiko; Shuin, Taro; Fukui, Yoshinori; Nakatsukasa, Kunio

    2016-01-01

    pVHL, the protein product of the von Hippel-Lindau (VHL) tumor suppressor gene, is a ubiquitin ligase that targets hypoxia-inducible factor α (HIF-α) for proteasomal degradation. Although HIF-α activation is necessary for VHL disease pathogenesis, constitutive activation of HIF-α alone did not induce renal clear cell carcinomas and pheochromocytomas in mice, suggesting the involvement of an HIF-α-independent pathway in VHL pathogenesis. Here, we show that the transcription factor B-Myb is a pVHL substrate that is degraded via the ubiquitin-proteasome pathway and that vascular endothelial growth factor (VEGF)- and/or platelet-derived growth factor (PDGF)-dependent tyrosine 15 phosphorylation of B-Myb prevents its degradation. Mice injected with B-Myb knockdown 786-O cells developed dramatically larger tumors than those bearing control cell tumors. Microarray screening of B-Myb-regulated genes showed that the expression of HIF-α-dependent genes was not affected by B-Myb knockdown, indicating that B-Myb prevents HIF-α-dependent tumorigenesis through an HIF-α-independent pathway. These data indicate that the regulation of B-Myb by pVHL plays a critical role in VHL disease. PMID:27090638

  13. Playing Tag with HIF: The VHL Story

    Directory of Open Access Journals (Sweden)

    Sherri K. Leung

    2002-01-01

    Full Text Available Inactivation of the von Hippel-Lindau (VHL tumour suppressor gene product pVHL is the cause of inherited VHL disease and is associated with sporadic kidney cancer. pVHL is found in a multiprotein complex with elongins B/C, Cul2, and Rbx1 forming an E3 ubiquitin ligase complex called VEC. This modular enzyme targets the α subunits of hypoxia-inducible factor (HIF for ubiquitin-mediated destruction. Consequently, tumour cells lacking functional pVHL overproduce the products of HIF-target genes such as vascular endothelial growth factor (VEGF, which promotes angiogenesis. This likely accounts for the hypervascular nature of VHL-associated neoplasms. Although pVHL has been linked to the cell-cycle, differentiation, and the regulation of extracellular matrix assembly, microenvironment pH, and tissue invasiveness, this review will focus on the recent insights into the molecular mechanisms governing the E3 ubiquitin ligase function of VEC.

  14. Homology modeling and molecular dynamics simulation of the HIF2α degradation-related HIF2α-VHL complex.

    Science.gov (United States)

    Dong, Xiaotian; Su, Xiaoru; Yu, Jiong; Liu, Jingqi; Shi, Xiaowei; Pan, Qiaoling; Yang, Jinfeng; Chen, Jiajia; Li, Lanjuan; Cao, Hongcui

    2017-01-01

    R)-pVHL mutants on the basis of RMSD, DSSP, binding free energy, and hydrogen bonding. The experimental data indicate that the stability of the structural model of the HIF2α-pVHL complex is higher than that of the mutants, consistently with clinical observations. The structural model of the HIF2α-pVHL complex presented in this study enhances understanding of how HIF2α is captured by pVHL. Moreover, the important contact amino acids that we identified may be useful in the development of drugs to treat HIF2a-related diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Potential value of EUS in pancreatic surveillance of VHL patients

    NARCIS (Netherlands)

    van Asselt, Sophie Josephien; Brouwers, Adrienne H; van Dullemen, Hendrik M; van der Jagt, Eric J; Bongaerts, Alfons H; Koopmans, Klaas P; Kema, Ido; Zonnenberg, Bernard A; Timmers, Henri Jlm; de Herder, Wouter; Sluiter, Wim; de Vries, Elisabeth G E; Links, T P

    Background: Patients with von Hippel-Lindau (VHL) disease are prone to develop pancreatic neuroendocrine tumors (pNETs). However, the best imaging technique for early detection of pNETs in VHL is currently unknown. In a head-to-head comparison, we evaluated endoscopic ultrasound (EUS) and

  16. LOSS OF JAK2 REGULATION VIA VHL-SOCS1 E3 UBIQUITIN HETEROCOMPLEX UNDERLIES CHUVASH POLYCYTHEMIA

    Science.gov (United States)

    Russell, Ryan C.; Sufan, Roxana I.; Zhou, Bing; Heir, Pardeep; Bunda, Severa; Sybingco, Stephanie S.; Greer, Samantha N.; Roche, Olga; Heathcote, Samuel A.; Chow, Vinca W.K.; Boba, Lukasz M.; Richmond, Terri D.; Hickey, Michele M.; Barber, Dwayne L.; Cheresh, David A.; Simon, M. Celeste; Irwin, Meredith S.; Kim, William Y.; Ohh, Michael

    2011-01-01

    SUMMARY Chuvash polycythemia (CP) is a rare congenital form of polycythemia caused by homozygous R200W and H191D mutations in the von Hippel-Lindau (VHL) gene whose gene product is the principal negative regulator of hypoxia-inducible factor. However, the molecular mechanisms underlying some of the hallmark features of CP such as hypersensitivity to erythropoietin are unclear. Here, we show that VHL directly binds suppressor of cytokine signalling 1 (SOCS1) to form a heterodimeric E3 ligase that targets phosphorylated (p)JAK2 for ubiquitin-mediated destruction. In contrast, CP-associated VHL mutants have altered affinity for SOCS1 and fail to engage and degrade pJAK2. Systemic administration of a highly selective JAK2 inhibitor, TG101209, reverses the disease phenotype in vhlR200W/R200W knock-in mice, a model that faithfully recapitulates human CP. These results reveal VHL as a SOCS1-cooperative negative regulator of JAK2 and provide compelling biochemical and preclinical evidence for JAK2- targeted therapy in CP patients. PMID:21685897

  17. Loss of JAK2 regulation via a heterodimeric VHL-SOCS1 E3 ubiquitin ligase underlies Chuvash polycythemia.

    Science.gov (United States)

    Russell, Ryan C; Sufan, Roxana I; Zhou, Bing; Heir, Pardeep; Bunda, Severa; Sybingco, Stephanie S; Greer, Samantha N; Roche, Olga; Heathcote, Samuel A; Chow, Vinca W K; Boba, Lukasz M; Richmond, Terri D; Hickey, Michele M; Barber, Dwayne L; Cheresh, David A; Simon, M Celeste; Irwin, Meredith S; Kim, William Y; Ohh, Michael

    2011-06-19

    Chuvash polycythemia is a rare congenital form of polycythemia caused by homozygous R200W and H191D mutations in the VHL (von Hippel-Lindau) gene, whose gene product is the principal negative regulator of hypoxia-inducible factor. However, the molecular mechanisms underlying some of the hallmark abnormalities of Chuvash polycythemia, such as hypersensitivity to erythropoietin, are unclear. Here we show that VHL directly binds suppressor of cytokine signaling 1 (SOCS1) to form a heterodimeric E3 ligase that targets phosphorylated JAK2 (pJAK2) for ubiquitin-mediated destruction. In contrast, Chuvash polycythemia-associated VHL mutants have altered affinity for SOCS1 and do not engage with and degrade pJAK2. Systemic administration of a highly selective JAK2 inhibitor, TG101209, reversed the disease phenotype in Vhl(R200W/R200W) knock-in mice, an experimental model that recapitulates human Chuvash polycythemia. These results show that VHL is a SOCS1-cooperative negative regulator of JAK2 and provide biochemical and preclinical support for JAK2-targeted therapy in individuals with Chuvash polycythemia.

  18. Dicty_cDB: VHL444 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available library) VHL444 (Link to dictyBase) - - - Contig-U15033-1 VHL444P (Link to Original site) VHL444F 595 VHL444Z...VHL444Z 674 VHL444P 1249 - - Show VHL444 Library VH (Link to library) Clone ID VHL444 (Link to dictyBase)...tsukuba.ac.jp/CSM/VH/VHL4-B/VHL444Q.Seq.d/ Representative seq. ID VHL444P (Link to Original site) Representative...Representative DNA sequence >VHL444 (VHL444Q) /CSM/VH/VHL4-B/VHL444Q.Seq.d/ CACTGTTGGCCTACTGGTATAGTTACA...significant alignments: (bits) Value VHL444 (VHL444Q) /CSM/VH/VHL4-B/VHL444Q.Seq.d/ 2420 0.0 VHN389 (VHN389Q)

  19. Von Hippel-Lindau (VHL inactivation in sporadic clear cell renal cancer: associations with germline VHL polymorphisms and etiologic risk factors.

    Directory of Open Access Journals (Sweden)

    Lee E Moore

    2011-10-01

    Full Text Available Renal tumor heterogeneity studies have utilized the von Hippel-Lindau VHL gene to classify disease into molecularly defined subtypes to examine associations with etiologic risk factors and prognosis. The aim of this study was to provide a comprehensive analysis of VHL inactivation in clear cell renal tumors (ccRCC and to evaluate relationships between VHL inactivation subgroups with renal cancer risk factors and VHL germline single nucleotide polymorphisms (SNPs. VHL genetic and epigenetic inactivation was examined among 507 sporadic RCC/470 ccRCC cases using endonuclease scanning and using bisulfite treatment and Sanger sequencing across 11 CpG sites within the VHL promoter. Case-only multivariate analyses were conducted to identify associations between alteration subtypes and risk factors. VHL inactivation, either through sequence alterations or promoter methylation in tumor DNA, was observed among 86.6% of ccRCC cases. Germline VHL SNPs and a haplotype were associated with promoter hypermethylation in tumor tissue (OR = 6.10; 95% CI: 2.28-16.35, p = 3.76E-4, p-global = 8E-5. Risk of having genetic VHL inactivation was inversely associated with smoking due to a higher proportion of wild-type ccRCC tumors [former: OR = 0.70 (0.20-1.31 and current: OR = 0.56 (0.32-0.99; P-trend = 0.04]. Alteration prevalence did not differ by histopathologic characteristics or occupational exposure to trichloroethylene. ccRCC cases with particular VHL germline polymorphisms were more likely to have VHL inactivation through promoter hypermethylation than through sequence alterations in tumor DNA, suggesting that the presence of these SNPs may represent an example of facilitated epigenetic variation (an inherited propensity towards epigenetic variation in renal tissue. A proportion of tumors from current smokers lacked VHL alterations and may represent a biologically distinct clinical entity from inactivated cases.

  20. Difference in CXCR4 expression between sporadic and VHL-related hemangioblastoma

    NARCIS (Netherlands)

    Kruizinga, Roeliene C; van Marion, Denise M S; den Dunnen, Wilfred F A; de Groot, Jan C; Hoving, Eelco W; Oosting, Sjoukje F; Timmer-Bosscha, Hetty; Derks, Rosalie P H; Cornelissen, Chantal; van der Luijt, Rob B; Links, Thera P; de Vries, Elisabeth G E; Walenkamp, Annemiek M E

    2016-01-01

    Central nervous system hemangioblastomas occur sporadically and in patients with von Hippel-Lindau (VHL) disease due to a VHL germline mutation. This mutation leads to enhanced transcription of chemokine receptor 4 (CXCR4), its ligand (CXCL12) and vascular endothelial growth factor A (VEGFA). We

  1. [Prenatal exclusion of von Hippel-Lindau syndrome in a Mexican family carrying a novel VHL gene mutation].

    Science.gov (United States)

    Chacón-Camacho, Oscar Francisco; Benitez-Granados, Jesús; Zenteno, Juan Carlos

    2014-03-01

    von Hippel-Lindau (VHL) disease is an autosomal dominant and familial multisystemic syndrome that is caused by the inactivation of the VHL gene and it is characterized by diverse types of high vasculated tumours of benign and malign nature. In this work we describe the clinical characteristics and the prenatal diagnosis of a woman with VHL. Describe the first exclusion prenatal case by DNA analysis of the VHL syndrome in Latinoamerican population. Analysis of a Mexican familial pedigree showed 5 affected subjects with VHL on 3 consecutive generations. The proband was a 7 weeks pregnancy woman who was referred to our service for familiar and personal history of this disease. Maternal DNA was obtained from peripheral blood leukocytes, while fetal DNA was isolated from amniotic liquid cells on the 15th week. The maternal and fetal DNA analysis were done by the Polymerase Chain reaction (PCR) and the direct nucleotide sequence of the VHL gene. A novel mutation (c. 161_168 dup GGAGGCCG) in the VHL gene was identified in maternal DNA. Fetal DNA analysis indicated that the fetus inherited the wild-type allele from the mother. A novel VHL gene mutation was identified in a familial case of the disease, expanding the mutational spectrum in this disorder. The molecular prenatal testing in the affected woman at 15 weeks of gestation, demonstrated that the fetus did nor inherited the mutated allele. To the best of our knowledge, this is the first example of prenatal-molecular exclusion on VHL syndrome in Latinoamerica population.

  2. Dicty_cDB: VHL663 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available VH (Link to library) VHL663 (Link to dictyBase) - - - Contig-U15767-1 VHL663P (Link... to Original site) VHL663F 574 VHL663Z 702 VHL663P 1256 - - Show VHL663 Library VH (Link to library) Clone ID VHL663 (Link to dict...yBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U15767-1 Original site URL http://dict...WPDGFKYFFVDNQAGDSESAKSGKNLPIQRDIELNWNGEAYEYSNSNYFPINGQG FNDVSYPV--- ---SYATGKCEPDSSLCNDNNICTIDICVHEGILDGLPQG...ik rqelvgqmvlsifl*itklviqnlpnlvkifqfkeiss*igmekhmniviqitsqltdkv smm*aiq--- ---SYATGKCEPDSSLCNDNNICTIDICVHEGI

  3. Dicty_cDB: VHL434 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available VH (Link to library) VHL434 (Link to dictyBase) - - - Contig-U16336-1 VHL434P (Link... to Original site) VHL434F 546 VHL434Z 778 VHL434P 1304 - - Show VHL434 Library VH (Link to library) Clone ID VHL434 (Link to dict...yBase) Atlas ID - NBRP ID - dictyBase ID - Link to Contig Contig-U16336-1 Original site URL http://dict...EVMSCNKFSSKRIGYLAASQSFNEGTDVIVLATHQIRKDFLS SNQSEAYLALNCLSNICTTDLARELANDILTLLSTQKTHILKRAITVLYKIFLRYPES-- - --...GFDISWASFKIVEVMSCNKFSSKRIGYLAASQSFNEGTDVIVLATHQIRKDFLS SNQSEAYLALNCLSNICTTDLARELANDILTLLSTQKTHILKRAITVLYKIFL

  4. A patient with bilateral pheochromocytoma as part of a Von Hippel-Lindau (VHL syndrome type 2C

    Directory of Open Access Journals (Sweden)

    Rinkes Inne

    2007-10-01

    Full Text Available Abstract Background Von Hippel-Lindau (VHL disease is an autosomal dominant inherited disease. It is relatively recent that type 2C was identified as a separate group solely presenting with pheochromocytomas. As an illustration, an interesting case is presented of a pregnant woman with refractory hypertension. It proved to be the first manifestation of bilateral pheochromocytomas. The family history may indicate the diagnosis, but only identification of a germ line mutation in the DNA of a patient will confirm carriership. Case presentation A 27 year pregnant patient with intra uterine growth retardation presented with hypertension and pre-eclampsia. Magnetic resonance imaging revealed bilateral adrenal pheochromocytoma. She underwent laparoscopic adrenelectomy and a missense mutation (Gly93Ser in exon 1 of the VHL gene on chromosome 3 (p25 – p26 was shown in the patient, her father and her daughter confirming the diagnosis of VHL. Conclusion In almost all VHL families molecular genetic analysis of DNA will demonstrate an inherited mutation. Because of the involvement in several organs, periodic clinical evaluation should take place in a well coordinated, multidisciplinary setting. VHL disease can be classified into several subtypes. VHL type 2C patients present with pheochromocytomas without evidence of haemangioblastomas in the central nervous system and/or retina and a low risk of renal cell carcinoma. Therefore, in such families, periodic clinical screening can be focussed on pheochromocytomas.

  5. Pregnancy-related hemangioblastoma progression and complications in von Hippel-Lindau disease.

    NARCIS (Netherlands)

    Frantzen, C.; Kruizinga, R.C.; Asselt, S.J. van; Zonnenberg, B.A.; Lenders, J.W.M.; Herder, W.W. de; Walenkamp, A.M.; Giles, R.H.; Hes, F.J.; Sluiter, W.J.; Pampus, M.G. van; Links, T.P.

    2012-01-01

    OBJECTIVE: We studied the reciprocal effect of pregnancy and von Hippel-Lindau (VHL) disease by analyzing the influence of pregnancy on VHL disease-related lesions and VHL disease on pregnancy outcome. METHODS: Medical charts and imaging reports from the VHL disease expertise centers in the

  6. Identification of the Lipodepsipeptide MDN-0066, a Novel Inhibitor of VHL/HIF Pathway Produced by a New Pseudomonas Species.

    Directory of Open Access Journals (Sweden)

    Bastien Cautain

    Full Text Available Throughout recent history, metabolites of microbial origin have had an extraordinary impact on the welfare of humanity. In fact, natural products have largely been--and still are--considered an exceedingly valuable platform for the discovery of new drugs against diverse pathologies. Such value is partly due to their higher complexity and chemical diversity as compared to those of synthetic and combinatorial compounds. Mutations in the Von Hippel-Lindau (vhl gene are responsible for VHL disease, congenital polycythemia, and are found in many sporadic tumor types. The primary cause of morbidity and mortality for these patients arises from progression of Renal Cell Carcinoma (RCC or end-stage renal disease. Inactivation of the Von Hippel-Lindau (vhl tumor suppressor gene arises in the majority of Renal Cell Carcinoma (RCC as well as in other types of cancer and is associated with a high degree of vascularization and poor prognosis. Loss of pVHL function thus represents a pathognomonic molecular defect for therapeutic exploitation. In this study, renal carcinoma cell lines with naturally occurring vhl mutations (RCC4 VA and their genetically matched wild-type vhl (RCC4 VHL counterparts were seeded onto 96-well plates and treated with a collection of 1,040 organic extracts obtained from 130 bacterial strains belonging to at least 25 genera of the phyla Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes. This strategy allowed us to identify several extracts obtained from bacterial strain F-278,770T, the type strain of the recently proposed new species Pseudomonas granadensis, showing biological activities not associated with previously known bioactive metabolites. The fractionation and structural elucidation of one of these extracts led to the discovery of a new lipodepsipeptide (MDN-0066 with specific toxicity in pVHL deficient cells that is not detectable in cells with pVHL expression rescue. This specific toxicity is associated with

  7. Identification of the Lipodepsipeptide MDN-0066, a Novel Inhibitor of VHL/HIF Pathway Produced by a New Pseudomonas Species.

    Science.gov (United States)

    Cautain, Bastien; de Pedro, Nuria; Schulz, Christian; Pascual, Javier; Sousa, Thiciana da S; Martin, Jesús; Pérez-Victoria, Ignacio; Asensio, Francisco; González, Ignacio; Bills, Gerald F; Reyes, Fernando; Genilloud, Olga; Vicente, Francisca

    2015-01-01

    Throughout recent history, metabolites of microbial origin have had an extraordinary impact on the welfare of humanity. In fact, natural products have largely been--and still are--considered an exceedingly valuable platform for the discovery of new drugs against diverse pathologies. Such value is partly due to their higher complexity and chemical diversity as compared to those of synthetic and combinatorial compounds. Mutations in the Von Hippel-Lindau (vhl) gene are responsible for VHL disease, congenital polycythemia, and are found in many sporadic tumor types. The primary cause of morbidity and mortality for these patients arises from progression of Renal Cell Carcinoma (RCC) or end-stage renal disease. Inactivation of the Von Hippel-Lindau (vhl) tumor suppressor gene arises in the majority of Renal Cell Carcinoma (RCC) as well as in other types of cancer and is associated with a high degree of vascularization and poor prognosis. Loss of pVHL function thus represents a pathognomonic molecular defect for therapeutic exploitation. In this study, renal carcinoma cell lines with naturally occurring vhl mutations (RCC4 VA) and their genetically matched wild-type vhl (RCC4 VHL) counterparts were seeded onto 96-well plates and treated with a collection of 1,040 organic extracts obtained from 130 bacterial strains belonging to at least 25 genera of the phyla Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes. This strategy allowed us to identify several extracts obtained from bacterial strain F-278,770T, the type strain of the recently proposed new species Pseudomonas granadensis, showing biological activities not associated with previously known bioactive metabolites. The fractionation and structural elucidation of one of these extracts led to the discovery of a new lipodepsipeptide (MDN-0066) with specific toxicity in pVHL deficient cells that is not detectable in cells with pVHL expression rescue. This specific toxicity is associated with apoptosis

  8. Altered protein dynamics of disease-associated lamin A mutants

    Directory of Open Access Journals (Sweden)

    Worman Howard J

    2004-12-01

    Full Text Available Abstract Background Recent interest in the function of the nuclear lamina has been provoked by the discovery of lamin A/C mutations in the laminopathy diseases. However, it is not understood why mutations in lamin A give such a range of tissue-specific phenotypes. Part of the problem in rationalising genotype-phenotype correlations in the laminopathies is our lack of understanding of the function of normal and mutant lamin A. To investigate this we have used photobleaching in human cells to analyse the dynamics of wild-type and mutant lamin A protein at the nuclear periphery. Results We have found that a large proportion of wild-type lamin A at the nuclear periphery is immobile, but that there is some slow movement of lamin A within the nuclear lamina. The mobility of an R482W mutant lamin A was indistinguishable from wild-type, but increased mobility of L85R and L530P mutant proteins within the nuclear lamina was found. However, the N195K mutant shows the most enhanced protein mobility, both within the nucleoplasm and within the lamina. Conclusion The slow kinetics of lamin A movement is compatible with its incorporation into a stable polymer that only exchanges subunits very slowly. All of the myopathy-associated lamin A mutants that we have studied show increased protein movement compared with wild-type. In contrast, the dynamic behaviour of the lipodystrophy-associated lamin A mutant was indistinguishable from wild-type. This supports the hypothesis that the underlying defect in lamin A function is quite distinct in the laminopathies that affect striated muscle, compared to the diseases that affect adipose tissue. Our data are consistent with an alteration in the stability of the lamin A molecules within the higher-order polymer at the nuclear lamina in myopathies.

  9. Vhl deletion in osteoblasts boosts cellular glycolysis and improves global glucose metabolism.

    Science.gov (United States)

    Dirckx, Naomi; Tower, Robert J; Mercken, Evi M; Vangoitsenhoven, Roman; Moreau-Triby, Caroline; Breugelmans, Tom; Nefyodova, Elena; Cardoen, Ruben; Mathieu, Chantal; Van der Schueren, Bart; Confavreux, Cyrille B; Clemens, Thomas L; Maes, Christa

    2018-02-12

    The skeleton has emerged as an important regulator of systemic glucose homeostasis, with osteocalcin and insulin representing prime mediators of the interplay between bone and energy metabolism. However, genetic evidence indicates that osteoblasts can influence global energy metabolism through additional, as yet unknown, mechanisms. Here, we report that constitutive or postnatally induced deletion of the hypoxia signaling pathway component von Hippel-Lindau (VHL) in skeletal osteolineage cells of mice led to high bone mass as well as hypoglycemia and increased glucose tolerance, not accounted for by osteocalcin or insulin. In vitro and in vivo data indicated that Vhl-deficient osteoblasts displayed massively increased glucose uptake and glycolysis associated with upregulated HIF-target gene expression, resembling the Warburg effect that typifies cancer cells. Overall, the glucose consumption by the skeleton was increased in the mutant mice, as revealed by 18F-FDG radioactive tracer experiments. Moreover, the glycemia levels correlated inversely with the level of skeletal glucose uptake, and pharmacological treatment with the glycolysis inhibitor dichloroacetate (DCA), which restored glucose metabolism in Vhl-deficient osteogenic cells in vitro, prevented the development of the systemic metabolic phenotype in the mutant mice. Altogether, these findings reveal a novel link between cellular glucose metabolism in osteoblasts and whole-body glucose homeostasis, controlled by local hypoxia signaling in the skeleton.

  10. Allele-selective suppression of mutant genes in polyglutamine diseases.

    Science.gov (United States)

    Liu, Chia-Rung; Cheng, Tzu-Hao

    2015-01-01

    Polyglutamine (polyQ) diseases are heritable dominant neurological disorders, caused by abnormal CAG tri-nucleotide expansion in the coding sequence of affected genes. Extension of CAG repeats results in the production of aberrant gene products that are deleterious to neurons, such as transcripts with a CAG stem-loop secondary structure, and proteins containing a long stretch of polyQ residues. Thus, determining methods for the prevention or elimination of these mutant gene products from neuronal cells and translating this knowledge to clinical application are currently important goals in the fields of neurology and neurogenetics. Recently, several studies have revealed intriguing findings related to the allele-selective regulation of CAG-expanded genes, and have proposed novel designs to selectively diminish the mutant polyQ proteins. In this review, we focus on the genes, genetically engineered proteins, and oligonucleotides that show potential to modulate the expression of mutant genes. We also discuss their respective molecular functions at the levels of transcription, translation, and post-translation.

  11. Von Hippel-Lindau Disease (VHL)

    Science.gov (United States)

    ... ALS) Information Page NINDS Anencephaly Information Page NINDS Angelman Syndrome Information Page NINDS Antiphospholipid Syndrome Information Page ... ALS) Information Page NINDS Anencephaly Information Page NINDS Angelman Syndrome Information Page NINDS Antiphospholipid Syndrome Information Page ...

  12. Small activating RNA induced expression of VHL gene in renal cell carcinoma.

    Science.gov (United States)

    Kang, Moo Rim; Park, Ki Hwan; Lee, Chang Woo; Lee, Myeong Youl; Han, Sang-Bae; Li, Long-Cheng; Kang, Jong Soon

    2018-02-06

    Recent studies have reported that chemically synthesized double-stranded RNAs (dsRNAs), also known as small activating RNA (saRNAs), can specifically induce gene expression by targeting promoter sequences by a mechanism termed RNA activation (RNAa). In the present study, we designed 4 candidate saRNAs targeting the Von Hippel-Lindau (VHL) gene promoter. Among these saRNAs, dsVHL-821 significantly inhibited cell growth by up-regulating VHL at both the mRNA and protein levels in renal cell carcinoma 769-P cells. Functional analysis showed that dsVHL-821 induced apoptosis by increasing p53, decreasing Bcl-xL, activating caspase 3/7 and poly-ADP-ribose polymerase in a dose-dependent manner. Chromatin immunoprecipitation analysis revealed that dsVHL-821 increased the enrichment of Ago2 and RNA polymerase II at the dsVHL-821 target site. In addition, Ago2 depletion significantly suppressed dsVHL-821-induced up-regulation of VHL gene expression and related effects. Single transfection of dsVHL-821 caused long-lasting (14 days) VHL up-regulation. Furthermore, the activation of VHL by dsVHL-821 was accompanied by an increase in dimethylation of histone 3 at lysine 4 (H3K4me2) and acetylation of histone 4 (H4ac) and a decrease in dimethylation of histone 3 at lysine 9 (H3K9me2) and lysine 27 (H3K27me2) in the dsVHL-821 target region. Taken together, these results demonstrate that dsVHL-821, a novel saRNA for VHL, induces the expression of the VHL gene by epigenetic changes, leading to inhibition of cell growth and induction of apoptosis, and suggest that targeted activation of VHL by dsVHL-821 may be explored as a novel treatment of renal cell carcinoma. Copyright © 2018 Elsevier Ltd. All rights reserved.

  13. Novel homozygous VHL mutation in exon 2 is associated with congenital polycythemia but not with cancer.

    Science.gov (United States)

    Lanikova, Lucie; Lorenzo, Felipe; Yang, Chunzhang; Vankayalapati, Hari; Drachtman, Richard; Divoky, Vladimir; Prchal, Josef T

    2013-05-09

    Germline von Hippel-Lindau (VHL) gene mutations underlie dominantly inherited familial VHL tumor syndrome comprising a predisposition for renal cell carcinoma, pheochromocytoma/paraganglioma, cerebral hemangioblastoma, and endolymphatic sac tumors. However, recessively inherited congenital polycythemia, exemplified by Chuvash polycythemia, has been associated with 2 separate 3' VHL gene mutations in exon 3. It was proposed that different positions of loss-of-function VHL mutations are associated with VHL syndrome cancer predisposition and only C-terminal domain-encoding VHL mutations would cause polycythemia. However, now we describe a new homozygous VHL exon 2 mutation of the VHL gene:(c.413C>T):P138L, which is associated in the affected homozygote with congenital polycythemia but not in her, or her-heterozygous relatives, with cancer or other VHL syndrome tumors. We show that VHL(P138L) has perturbed interaction with hypoxia-inducible transcription factor (HIF)1α. Further, VHL(P138L) protein has decreased stability in vitro. Similarly to what was reported in Chuvash polycythemia and some other instances of HIFs upregulation, VHL(P138L) erythroid progenitors are hypersensitive to erythropoietin. Interestingly, the level of RUNX1/AML1 and NF-E2 transcripts that are specifically upregulated in acquired polycythemia vera were also upregulated in VHL(P138L) granulocytes.

  14. Management Strategies and Outcomes for VHL-related Craniospinal Hemangioblastomas

    Directory of Open Access Journals (Sweden)

    Christ Ordookhanian

    2017-08-01

    Full Text Available Hemangioblastomas are rare and benign tumors accounting for less than 2% of all central nervous system (CNS tumors. The vast majority of hemangioblastomas occur sporadically, whereas a small number of cases, especially in younger patients, are associated with Von Hippel–Lindau (VHL syndrome. It is thought that loss of tumor suppressor function of the VHL gene results in stabilization of hypoxia-inducible factor alpha with downstream activation of cellular proliferative and angiogenic genes that promote tumorigenesis. VHL-related hemangioblastomas predominantly occur in the cerebellum and spine. Lesions are often diagnosed on contrast-enhanced craniospinal MRIs, and the diagnosis of VHL occurs through assessment for germline VHL mutations. Surgical resection remains the primary treatment modality for symptomatic or worrisome lesions, with excellent local control rates and neurological outcomes. Stereotactic radiotherapy can be employed in patients who are deemed high risk for surgery, have multiple lesions, or have non-resectable lesions. Given the tendency for development of either new or multiple lesions, close radiographic surveillance is often recommended for asymptomatic lesions.

  15. Role of VEGFA, CXCR4 and VHL mutation in tumour behaviour

    NARCIS (Netherlands)

    Kruizinga, Roeliene

    2014-01-01

    De ziekte van Von Hippel Lindau (VHL) is een zeldzaam kankersyndroom. Patiënten met deze ziekte krijgen zowel goedaardige als kwaadaardige tumoren in verschillende organen. VHL-patiënten hebben een niet goed werkend VHL-eiwit waardoor er meer CXCR4, een chemokine receptor, en VEGFA, een

  16. Zebrafish mutants in the von Hippel-Lindau tumor suppressor display a hypoxic response and recapitulate key aspects of Chuvash polycythemia

    NARCIS (Netherlands)

    van Rooijen, E.; Voest, E.E.; Logister, I.; Korving, J.; Schwerte, T.; Schulte-Merker, S.; Giles, R.H.; van Eeden, F.J.

    2009-01-01

    We have generated 2 zebrafish lines carrying inactivating germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene ortholog vhl. Mutant embryos display a general systemic hypoxic response, including the up-regulation of hypoxia-induced genes by 1 day after fertilization and a severe

  17. Role of VHL, HIF1A and SDH on the expression of miR-210: Implications for tumoral pseudo-hypoxic fate.

    Science.gov (United States)

    Merlo, Anna; Bernardo-Castiñeira, Cristóbal; Sáenz-de-Santa-María, Inés; Pitiot, Ana S; Balbín, Milagros; Astudillo, Aurora; Valdés, Nuria; Scola, Bartolomé; Del Toro, Raquel; Méndez-Ferrer, Simón; Piruat, José I; Suarez, Carlos; Chiara, María-Dolores

    2017-01-24

    The hypoxia-inducible factor 1α (HIF-1α) and its microRNA target, miR-210, are candidate tumor-drivers of metabolic reprogramming in cancer. Neuroendocrine neoplasms such as paragangliomas (PGLs) are particularly appealing for understanding the cancer metabolic adjustments because of their associations with deregulations of metabolic enzymes, such as succinate dehydrogenase (SDH), and the von Hippel Lindau (VHL) gene involved in HIF-1α stabilization. However, the role of miR-210 in the pathogenesis of SDH-related tumors remains an unmet challenge. Herein is described an in vivo genetic analysis of the role of VHL, HIF1A and SDH on miR-210 by using knockout murine models, siRNA gene silencing, and analyses of human tumors. HIF-1α knockout abolished hypoxia-induced miR-210 expression in vivo but did not alter its constitutive expression in paraganglia. Normoxic miR-210 levels substantially increased by complete, but not partial, VHL silencing in paraganglia of knockout VHL-mice and by over-expression of p76del-mutated pVHL. Similarly, VHL-mutated PGLs, not those with decreased VHL-gene/mRNA dosage, over-expressed miR-210 and accumulate HIF-1α in most tumor cells. Ablation of SDH activity in SDHD-null cell lines or reduction of the SDHD or SDHB protein levels elicited by siRNA-induced gene silencing did not induce miR-210 whereas the presence of SDH mutations in PGLs and tumor-derived cell lines was associated with mild increase of miR-210 and the presence of a heterogeneous, HIF-1α-positive and HIF-1α-negative, tumor cell population. Thus, activation of HIF-1α is likely an early event in VHL-defective PGLs directly linked to VHL mutations, but it is a late event favored but not directly triggered by SDHx mutations. This combined analysis provides insights into the mechanisms of HIF-1α/miR-210 regulation in normal and tumor tissues potentially useful for understanding the pathogenesis of cancer and other diseases sharing similar underpinnings.

  18. Kidney Tumor in a von Hippel-Lindau (VHL) Patient With Intensely Increased Activity on 68Ga-DOTA-TATE PET/CT.

    Science.gov (United States)

    Papadakis, Georgios Z; Millo, Corina; Sadowski, Samira M; Bagci, Ulas; Patronas, Nicholas J

    2016-12-01

    Renal and pancreatic cysts and tumors are the most common visceral manifestations of von Hippel-Lindau (VHL) disease, a heritable multisystem cancer syndrome characterized by development of a variety of malignant and benign tumors. We report a case of a VHL patient with multiple renal cystic and complex cystic/solid lesions. The patient underwent Ga-DOTA-TATE-PET/CT showing intensely increased activity by a solid lesion which demonstrated enhancement on both CT and MRI scans, raising high suspicion for malignancy. The presented case indicates application of SSTR-imaging using Ga-DOTA-conjugated peptides in VHL-patients and emphasizes the need for cautious interpretation of renal parenchyma Ga-DOTATATE activity.

  19. Endemic polycythemia in Russia: mutation in the VHL gene.

    Science.gov (United States)

    Ang, Sonny O; Chen, Hua; Gordeuk, Victor R; Sergueeva, Adelina I; Polyakova, Lydia A; Miasnikova, Galina Y; Kralovics, Robert; Stockton, David W; Prchal, Josef T

    2002-01-01

    Chuvash polycythemia (CP) is an autosomal recessive condition that is endemic in the Russian mid-Volga River region of Chuvashia. We previously found that CP patients may have increased serum erythropoietin (EPO) levels, ruled out linkage to both the EPO and EPO receptor (EPOR) gene loci, and hypothesized that the defect may lie in the oxygen homeostasis pathway. We now report a study of five multiplex Chuvash families which confirms that CP is associated with significant elevations of serum EPO levels and rules out a location for the CP gene on chromosome 11 as had been reported by other investigators or a mutation of the HIF-1 alpha gene. Using a genome-wide screen, we localized a region on chromosome 3 with a LOD score >2. After sequencing three candidate genes, we identified a C to T transition at nucleotide 598 (an R200W mutation) in the von Hippel-Lindau (VHL) gene. The VHL protein (pVHL) downregulates the alpha subunit of hypoxia-inducible factor 1 (HIF-1 alpha), the main regulator of hypoxia adaptation, by targeting the protein for degradation. In the simplest scenario, disruption of pVHL function causes a failure to degrade HIF-1 alpha resulting in accumulation of HIF-1 alpha, upregulation of downstream target genes such as EPO, and the clinical manifestation of polycythemia. These findings strongly suggest that CP is a congenital disorder of oxygen homeostasis.

  20. Normal and mutant HTT interact to affect clinical severity and progression in Huntington disease

    DEFF Research Database (Denmark)

    Aziz, N A; Jurgens, C K; Landwehrmeyer, G B

    2009-01-01

    OBJECTIVE: Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion in the HD gene (HTT). We aimed to assess whether interaction between CAG repeat sizes in the mutant and normal allele could affect disease severity and progression. METHODS: Using...... with less severe symptoms and pathology. CONCLUSIONS: Increasing CAG repeat size in normal HTT diminishes the association between mutant CAG repeat size and disease severity and progression in Huntington disease. The underlying mechanism may involve interaction of the polyglutamine domains of normal...

  1. Phospholamban Is Downregulated by pVHL-Mediated Degradation through Oxidative Stress in Failing Heart

    Directory of Open Access Journals (Sweden)

    Shunichi Yokoe

    2017-10-01

    Full Text Available The E3 ubiquitin ligase, von Hippel–Lindau (VHL, regulates protein expression by polyubiquitination. Although the protein VHL (pVHL was reported to be involved in the heart function, the underlying mechanism is unclear. Here, we show that pVHL was upregulated in hearts from two types of genetically dilated cardiomyopathy (DCM mice models. In comparison with the wild-type mouse, both DCM mice models showed a significant reduction in the expression of phospholamban (PLN, a potent inhibitor of sarco(endoplasmic reticulum Ca2+-ATPase, and enhanced interaction between pVHL and PLN. To clarify whether pVHL is involved in PLN degradation in failing hearts, we used carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial membrane potential (MMP-lowering reagent, to mimic the heart failure condition in PLN-expressing HEK293 cells and found that CCCP treatment resulted in PLN degradation and increased interaction between PLN and pVHL. However, these effects were reversed with the addition of N-acetyl-l-cysteine. Furthermore, the co-transfection of VHL and PLN in HEK293 cells decreased PLN expression under oxidative stress, whereas knockdown of VHL increased PLN expression both under normal and oxidative stress conditions. Together, we propose that oxidative stress upregulates pVHL expression to induce PLN degradation in failing hearts.

  2. Proteomic analysis of a disease-resistance-enhanced lesion mimic mutant spotted leaf 5 in rice

    OpenAIRE

    Chen, Xifeng; Fu, Shufang; Zhang, Pinghua; Gu, Zhimin; Liu, Jianzhong; Qian, Qian; Ma, Bojun

    2013-01-01

    Background A lesion-mimic mutant in rice (Oryza sativa L.), spotted leaf 5 (spl5), displays a disease-resistance-enhanced phenotype, indicating that SPL5 negatively regulates cell death and resistance responses. To understand the molecular mechanisms of SPL5 mutation-induced cell death and resistance responses, a proteomics-based approach was used to identify differentially accumulated proteins between the spl5 mutant and wild type (WT). Results Proteomic data from two-dimensional gel electro...

  3. Selective processing and metabolism of disease-causing mutant prion proteins.

    Directory of Open Access Journals (Sweden)

    Aarthi Ashok

    2009-06-01

    Full Text Available Prion diseases are fatal neurodegenerative disorders caused by aberrant metabolism of the cellular prion protein (PrP(C. In genetic forms of these diseases, mutations in the globular C-terminal domain are hypothesized to favor the spontaneous generation of misfolded PrP conformers (including the transmissible PrP(Sc form that trigger downstream pathways leading to neuronal death. A mechanistic understanding of these diseases therefore requires knowledge of the quality control pathways that recognize and degrade aberrant PrPs. Here, we present comparative analyses of the biosynthesis, trafficking, and metabolism of a panel of genetic disease-causing prion protein mutants in the C-terminal domain. Using quantitative imaging and biochemistry, we identify a misfolded subpopulation of each mutant PrP characterized by relative detergent insolubility, inaccessibility to the cell surface, and incomplete glycan modifications. The misfolded populations of mutant PrPs were neither recognized by ER quality control pathways nor routed to ER-associated degradation despite demonstrable misfolding in the ER. Instead, mutant PrPs trafficked to the Golgi, from where the misfolded subpopulation was selectively trafficked for degradation in acidic compartments. Surprisingly, selective re-routing was dependent not only on a mutant globular domain, but on an additional lysine-based motif in the highly conserved unstructured N-terminus. These results define a specific trafficking and degradation pathway shared by many disease-causing PrP mutants. As the acidic lysosomal environment has been implicated in facilitating the conversion of PrP(C to PrP(Sc, our identification of a mutant-selective trafficking pathway to this compartment may provide a cell biological basis for spontaneous generation of PrP(Sc in familial prion disease.

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

    Science.gov (United States)

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

    2017-06-01

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

  5. Survival and causes of death in patients with von Hippel-Lindau disease

    DEFF Research Database (Denmark)

    Binderup, Marie Louise Mølgaard; Jensen, Annette Møller; Budtz-Jørgensen, Esben

    2017-01-01

    . Surveillance is especially beneficial for truncating mutation carriers, who have the greatest RCC and central nervous system (CNS) hemangioblastoma risk.  Conclusions: vHL survival has improved over time and has become closer to that of siblings without vHL and the general population. Even though the risk of v......Background: Historically, the survival of patients with von Hippel-Lindau disease (vHL) has been poorer than that of the general population. We aimed to determine whether the survival of VHL mutation carriers and their risk of vHL-related death has changed over time and how it has been affected...... by sex, genotype and surveillance attendance.  Methods: In a retrospective cohort study, we included all known Danish vHL families with a VHL mutation. We assessed the survival and causes of death for 143 VHL mutation carriers using Cox regression models and compared vHL survival with that of 137...

  6. ERK5/BMK1 Is a Novel Target of the Tumor Suppressor VHL: Implication in Clear Cell Renal Carcinoma12

    Science.gov (United States)

    Arias-González, Laura; Moreno-Gimeno, Inmaculada; del Campo, Antonio Rubio; Serrano-Oviedo, Leticia; Valero, María Llanos; Esparís-Ogando, Azucena; de la Cruz-Morcillo, Miguel Ángel; Melgar-Rojas, Pedro; García-Cano, Jesús; Cimas, Francisco José; Hidalgo, María José Ruiz; Prado, Alfonso; Callejas-Valera, Juan Luis; Nam-Cha, Syong Hyun; Giménez-Bachs, José Miguel; Salinas-Sánchez, Antonio S; Pandiella, Atanasio; del Peso, Luis; Sánchez-Prieto, Ricardo

    2013-01-01

    Extracellular signal-regulated kinase 5 (ERK5), also known as big mitogen-activated protein kinase (MAPK) 1, is implicated in a wide range of biologic processes, which include proliferation or vascularization. Here, we show that ERK5 is degraded through the ubiquitin-proteasome system, in a process mediated by the tumor suppressor von Hippel-Lindau (VHL) gene, through a prolyl hydroxylation-dependent mechanism. Our conclusions derive from transient transfection assays in Cos7 cells, as well as the study of endogenous ERK5 in different experimental systems such as MCF7, HMEC, or Caki-2 cell lines. In fact, the specific knockdown of ERK5 in pVHL-negative cell lines promotes a decrease in proliferation and migration, supporting the role of this MAPK in cellular transformation. Furthermore, in a short series of fresh samples from human clear cell renal cell carcinoma, high levels of ERK5 correlate with more aggressive and metastatic stages of the disease. Therefore, our results provide new biochemical data suggesting that ERK5 is a novel target of the tumor suppressor VHL, opening a new field of research on the role of ERK5 in renal carcinomas. PMID:23730213

  7. ERK5/BMK1 Is a Novel Target of the Tumor Suppressor VHL: Implication in Clear Cell Renal Carcinoma

    Directory of Open Access Journals (Sweden)

    Laura Arias-González

    2013-06-01

    Full Text Available Extracellular signal-regulated kinase 5 (ERK5, also known as big mitogen-activated protein kinase (MAPK 1, is implicated in a wide range of biologic processes, which include proliferation or vascularization. Here, we show that ERK5 is degraded through the ubiquitin-proteasome system, in a process mediated by the tumor suppressor von Hippel-Lindau (VHL gene, through a prolyl hydroxylation-dependent mechanism. Our conclusions derive from transient transfection assays in Cos7 cells, as well as the study of endogenous ERK5 in different experimental systems such as MCF7, HMEC, or Caki-2 cell lines. In fact, the specific knockdown of ERK5 in pVHL-negative cell lines promotes a decrease in proliferation and migration, supporting the role of this MAPK in cellular transformation. Furthermore, in a short series of fresh samples from human clear cell renal cell carcinoma, high levels of ERK5 correlate with more aggressive and metastatic stages of the disease. Therefore, our results provide new biochemical data suggesting that ERK5 is a novel target of the tumor suppressor VHL, opening a new field of research on the role of ERK5 in renal carcinomas.

  8. Kinase Inhibitor Profiling Reveals Unexpected Opportunities to Inhibit Disease-Associated Mutant Kinases

    Directory of Open Access Journals (Sweden)

    Krisna C. Duong-Ly

    2016-02-01

    Full Text Available Small-molecule kinase inhibitors have typically been designed to inhibit wild-type kinases rather than the mutant forms that frequently arise in diseases such as cancer. Mutations can have serious clinical implications by increasing kinase catalytic activity or conferring therapeutic resistance. To identify opportunities to repurpose inhibitors against disease-associated mutant kinases, we conducted a large-scale functional screen of 183 known kinase inhibitors against 76 recombinant mutant kinases. The results revealed lead compounds with activity against clinically important mutant kinases, including ALK, LRRK2, RET, and EGFR, as well as unexpected opportunities for repurposing FDA-approved kinase inhibitors as leads for additional indications. Furthermore, using T674I PDGFRα as an example, we show how single-dose screening data can provide predictive structure-activity data to guide subsequent inhibitor optimization. This study provides a resource for the development of inhibitors against numerous disease-associated mutant kinases and illustrates the potential of unbiased profiling as an approach to compound-centric inhibitor development.

  9. Generalized Degenerative Joint Disease in Osteoprotegerin (Opg) Null Mutant Mice.

    Science.gov (United States)

    Bolon, B; Grisanti, M; Villasenor, K; Morony, S; Feige, U; Simonet, W S

    2015-09-01

    Bone structure is modulated by the interaction between receptor activator of nuclear factor-κB (RANK) and RANK ligand (RANKL). Osteoprotegerin (OPG), a decoy receptor for RANKL, modifies osteoclast-mediated bone resorption directly and spares articular cartilage indirectly in rodents with immune-mediated arthritis by preventing subchondral bone destruction. The OPG/RANKL balance also seems to be critical in maintaining joint integrity in osteoarthritis, a condition featuring articular bone and cartilage damage in the absence of profound inflammation. The current study explored the role of OPG in sparing articular cartilage by evaluating joint lesions in adult C57BL/6J mice lacking osteoprotegerin (Opg (-) (/-)). At 3, 5, 7, 9, and 12 months of age, both sexes of Opg (-) (/-) mice developed severe degenerative joint disease (DJD) characterized by progressive loss of cartilage matrix and eventually articular cartilage. Lesions developed earlier and more severely in Opg (-) (/-) mice relative to age-matched, wild-type (Opg (+) (/+)), or heterozygous (Opg (+) (/-)) littermates (P ≤ .05). The femorotibial joint was affected bilaterally at 3 months, while other key weight-bearing diarthrodial joints (eg, coxofemoral, scapulohumeral, humeroradioulnar) were affected later and unilaterally. Cortical bone in subchondral plates and long bone diaphyses of Opg (-) (/-) mice but not Opg (+/+) or Opg (+) (/-) animals was osteoporotic by 3 months of age (P ≤ .05); the extent of porosity was less than the degree of DJD. Closure of the physes in long bones (P ≤ .05) and cartilage retention in the femoral primary spongiosa (P ≤ .05) affected chiefly Opg (-) (/-) mice. These data suggest that OPG plays an essential direct role in maintaining cartilage integrity in the articular surfaces and physes. © The Author(s) 2015.

  10. The VHL-dependent regulation of microRNAs in renal cancer

    Directory of Open Access Journals (Sweden)

    Rawlings Lesley H

    2010-10-01

    Full Text Available Abstract Background The commonest histological type of renal cancer, clear cell renal cell carcinoma (cc RCC, is associated with genetic and epigenetic changes in the von Hippel-Lindau (VHL tumour suppressor. VHL inactivation leads to induction of hypoxia-inducible factors (HIFs and a hypoxic pattern of gene expression. Differential levels of specific microRNAs (miRNAs are observed in several tumours when compared to normal tissue. Given the central role of VHL in renal cancer formation, we examined the VHL-dependent regulation of miRNAs in renal cancer. Methods VHL-dependent miRNA expression in cc RCC was determined by microarray analysis of renal cell line RCC4 with mutated VHL (RCC4-VHL and reintroduced wild-type VHL (RCC4 + VHL. Five miRNAs highly upregulated in RCC4 + VHL and five miRNAs highly downregulated in RCC4 + VHL were studied further, in addition to miR-210, which is regulated by the HIF-VHL system. miRNA expression was also measured in 31 cc RCC tumours compared to adjacent normal tissue. Results A significant increase in miR-210, miR-155 and miR-21 expression was observed in the tumour tissue. miR-210 levels also showed a correlation with a HIF-regulated mRNA, carbonic anhydrase IX (CAIX, and with VHL mutation or promoter methylation. An inverse correlation was observed between miR-210 expression and patient survival, and a putative target of miR-210, iron-sulfur cluster assembly protein (ISCU1/2, shows reciprocal levels of mRNA expression in the tumours. Conclusions We have identified VHL-regulated miRNAs and found that for some the regulation is HIF-dependent and for others it is HIF-independent. This pattern of regulation was also seen in renal cancer tissue for several of these miRNAs (miR-210, miR-155, let-7i and members of the miR-17-92 cluster when compared with normal tissue. miR-210 showed marked increases in expression in renal cancer and levels correlated with patient survival. The inverse correlation between miR-210

  11. Mutant Huntingtin Does Not Affect the Intrinsic Phenotype of Human Huntington's Disease T Lymphocytes.

    Science.gov (United States)

    Miller, James R C; Träger, Ulrike; Andre, Ralph; Tabrizi, Sarah J

    2015-01-01

    Huntington's disease is a fatal neurodegenerative condition caused by a CAG repeat expansion in the huntingtin gene. The peripheral innate immune system is dysregulated in Huntington's disease and may contribute to its pathogenesis. However, it is not clear whether or to what extent the adaptive immune system is also involved. Here, we carry out the first comprehensive investigation of human ex vivo T lymphocytes in Huntington's disease, focusing on the frequency of a range of T lymphocyte subsets, as well as analysis of proliferation, cytokine production and gene transcription. In contrast to the innate immune system, the intrinsic phenotype of T lymphocytes does not appear to be affected by the presence of mutant huntingtin, with Huntington's disease T lymphocytes exhibiting no significant functional differences compared to control cells. The transcriptional profile of T lymphocytes also does not appear to be significantly affected, suggesting that peripheral immune dysfunction in Huntington's disease is likely to be mediated primarily by the innate rather than the adaptive immune system. This study increases our understanding of the effects of Huntington's disease on peripheral tissues, while further demonstrating the differential effects of the mutant protein on different but related cell types. Finally, this study suggests that the potential use of novel therapeutics aimed at modulating the Huntington's disease innate immune system should not be extended to include the adaptive immune system.

  12. Glucosylsphingosine Promotes α-Synuclein Pathology in Mutant GBA-Associated Parkinson's Disease.

    Science.gov (United States)

    Taguchi, Yumiko V; Liu, Jun; Ruan, Jiapeng; Pacheco, Joshua; Zhang, Xiaokui; Abbasi, Justin; Keutzer, Joan; Mistry, Pramod K; Chandra, Sreeganga S

    2017-10-04

    Glucocerebrosidase 1 (GBA) mutations responsible for Gaucher disease (GD) are the most common genetic risk factor for Parkinson's disease (PD). Although the genetic link between GD and PD is well established, the underlying molecular mechanism(s) are not well understood. We propose that glucosylsphingosine, a sphingolipid accumulating in GD, mediates PD pathology in GBA-associated PD. We show that, whereas GD-related sphingolipids (glucosylceramide, glucosylsphingosine, sphingosine, sphingosine-1-phosphate) promote α-synuclein aggregation in vitro, glucosylsphingosine triggers the formation of oligomeric α-synuclein species capable of templating in human cells and neurons. Using newly generated GD/PD mouse lines of either sex [Gba mutant (N370S, L444P, KO) crossed to α-synuclein transgenics], we show that Gba mutations predispose to PD through a loss-of-function mechanism. We further demonstrate that glucosylsphingosine specifically accumulates in young GD/PD mouse brain. With age, brains exhibit glucosylceramide accumulations colocalized with α-synuclein pathology. These findings indicate that glucosylsphingosine promotes pathological aggregation of α-synuclein, increasing PD risk in GD patients and carriers.SIGNIFICANCE STATEMENT Parkinson's disease (PD) is a prevalent neurodegenerative disorder in the aging population. Glucocerebrosidase 1 mutations, which cause Gaucher disease, are the most common genetic risk factor for PD, underscoring the importance of delineating the mechanisms underlying mutant GBA-associated PD. We show that lipids accumulating in Gaucher disease, especially glucosylsphingosine, play a key role in PD pathology in the brain. These data indicate that ASAH1 (acid ceramidase 1) and GBA2 (glucocerebrosidase 2) enzymes that mediate glucosylsphingosine production and metabolism are attractive therapeutic targets for treating mutant GBA-associated PD. Copyright © 2017 the authors 0270-6474/17/379617-15$15.00/0.

  13. Differential disease resistance response in the barley necrotic mutant nec1

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    Kunga Laura

    2011-04-01

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

  14. VHL genetic alteration in CCRCC does not determine de-regulation of HIF, CAIX, hnRNP A2/B1 and osteopontin.

    LENUS (Irish Health Repository)

    Nyhan, Michelle J

    2012-01-31

    BACKGROUND: von Hippel-Lindau (VHL) tumour suppressor gene inactivation is associated with clear cell renal cell carcinoma (CCRCC) development. The VHL protein (pVHL) has been proposed to regulate the expression of several proteins including Hypoxia Inducible Factor-alpha (HIF-alpha), carbonic anhydrase (CA)IX, heterogeneous nuclear ribonucleoprotein (hnRNP) A2\\/B1 and osteopontin. pVHL has been characterized in vitro, however, clinical studies are limited. We evaluated the impact of VHL genetic alterations on the expression of several pVHL protein targets in paired normal and tumor tissue. METHODS: The VHL gene was sequenced in 23 CCRCC patients and VHL transcript levels were evaluated by real-time RT-PCR. Expression of pVHL\\'s protein targets were determined by Western blotting in 17 paired patient samples. RESULTS: VHL genetic alterations were identified in 43.5% (10\\/23) of CCRCCs. HIF-1alpha, HIF-2alpha and CAIX were up-regulated in 88.2% (15\\/17), 100% (17\\/17) and 88.2% (15\\/17) of tumors respectively and their expression is independent of VHL status. hnRNP A2\\/B1 and osteopontin expression was variable in CCRCCs and had no association with VHL genetic status. CONCLUSION: As expression of these proposed pVHL targets can be achieved independently of VHL mutation (and possibly by hypoxia alone), these data suggests that other pVHL targets may be more crucial in renal carcinogenesis.

  15. The phenotype of polycythemia due to Croatian homozygous VHL (571C>G:H191D) mutation is different from that of Chuvash polycythemia (VHL 598C>T:R200W).

    Science.gov (United States)

    Tomasic, Nikica Ljubas; Piterkova, Lucie; Huff, Chad; Bilic, Ernest; Yoon, Donghoon; Miasnikova, Galina Y; Sergueeva, Adelina I; Niu, Xiaomei; Nekhai, Sergei; Gordeuk, Victor; Prchal, Josef T

    2013-04-01

    Mutations of VHL (a negative regulator of hypoxia-inducible factors) have position-dependent distinct cancer phenotypes. Only two known inherited homozygous VHL mutations exist and they cause polycythemia: Chuvash R200W and Croatian H191D. We report a second polycythemic Croatian H191D homozygote distantly related to the first propositus. Three generations of both families were genotyped for analysis of shared ancestry. Biochemical and molecular tests were performed to better define their phenotypes, with an emphasis on a comparison with Chuvash polycythemia. The VHL H191D mutation did not segregate in the family defined by the known common ancestors of the two subjects, suggesting a high prevalence in Croatians, but haplotype analysis indicated an undocumented common ancestor ∼six generations ago as the founder of this mutation. We show that erythropoietin levels in homozygous VHL H191D individuals are higher than in VHL R200W patients of similar ages, and their native erythroid progenitors, unlike Chuvash R200W, are not hypersensitive to erythropoietin. This observation contrasts with a report suggesting that polycythemia in VHL R200W and H191D homozygotes is due to the loss of JAK2 regulation from VHL R200W and H191D binding to SOCS1. In conclusion, our studies further define the hematologic phenotype of VHL H191D and provide additional evidence for phenotypic heterogeneity associated with the positional effects of VHL mutations.

  16. Significance of Coronavirus Mutants in Feces and Diseased Tissues of Cats Suffering from Feline Infectious Peritonitis

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    Niels C. Pedersen

    2009-08-01

    Full Text Available The internal FECV→FIPV mutation theory and three of its correlates were tested in four sibs/half-sib kittens, a healthy contact cat, and in four unrelated cats that died of FIP at geographically disparate regions. Coronavirus from feces and extraintestinal FIP lesions from the same cat were always >99% related in accessory and structural gene sequences. SNPs and deletions causing a truncation of the 3c gene product were found in almost all isolates from the diseased tissues of the eight cats suffering from FIP, whereas most, but not all fecal isolates from these same cats had intact 3c genes. Other accessory and structural genes appeared normal in both fecal and lesional viruses. Deliterious mutations in the 3c gene were unique to each cat, indicating that they did not originate in one cat and were subsequently passed horizontally to the others. Compartmentalization of the parental and mutant forms was not absolute; virus of lesional type was sometimes found in feces of affected cats and virus identical to fecal type was occasionally identified in diseased tissues. Although 3c gene mutants in this study were not horizontally transmitted, the parental fecal virus was readily transmitted by contact from a cat that died of FIP to its housemate. There was a high rate of mutability in all structural and accessory genes both within and between cats, leading to minor genetic variants. More than one variant could be identified in both diseased tissues and feces of the same cat. Laboratory cats inoculated with a mixture of two closely related variants from the same FIP cat developed disease from one or the other variant, but not both. Significant genetic drift existed between isolates from geographically distinct regions of the Western US.

  17. Characterization of Drosophila Saposin-related mutants as a model for lysosomal sphingolipid storage diseases

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    Julia Sellin

    2017-06-01

    Full Text Available Sphingolipidoses are inherited diseases belonging to the class of lysosomal storage diseases (LSDs, which are characterized by the accumulation of indigestible material in the lysosome caused by specific defects in the lysosomal degradation machinery. While some LSDs can be efficiently treated by enzyme replacement therapy (ERT, this is not possible if the nervous system is affected due to the presence of the blood-brain barrier. Sphingolipidoses in particular often present as severe, untreatable forms of LSDs with massive sphingolipid and membrane accumulation in lysosomes, neurodegeneration and very short life expectancy. The digestion of intralumenal membranes within lysosomes is facilitated by lysosomal sphingolipid activator proteins (saposins, which are cleaved from a prosaposin precursor. Prosaposin mutations cause some of the severest forms of sphingolipidoses, and are associated with perinatal lethality in mice, hampering studies on disease progression. We identify the Drosophila prosaposin orthologue Saposin-related (Sap-r as a key regulator of lysosomal lipid homeostasis in the fly. Its mutation leads to a typical spingolipidosis phenotype with an enlarged endolysosomal compartment and sphingolipid accumulation as shown by mass spectrometry and thin layer chromatography. Sap-r mutants show reduced viability with ∼50% survival to adulthood, allowing us to study progressive neurodegeneration and analyze their lipid profile in young and aged flies. Additionally, we observe a defect in sterol homeostasis with local sterol depletion at the plasma membrane. Furthermore, we find that autophagy is increased, resulting in the accumulation of mitochondria in lysosomes, concomitant with increased oxidative stress. Together, we establish Drosophila Sap-r mutants as a lysosomal storage disease model suitable for studying the age-dependent progression of lysosomal dysfunction associated with lipid accumulation and the resulting pathological

  18. Uptake of genetic testing and long-term tumor surveillance in von Hippel-Lindau disease

    OpenAIRE

    Chávez Mireya; Rodríguez Yaneth; Sosa Ana-Luisa; Yescas Petra; Familiar Itziar; De Biase Irene; Ochoa Adriana; Alonso Elisa; Rasmussen Astrid; López-López Marisol; Bidichandani Sanjay I

    2010-01-01

    Abstract Background von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations in the VHL gene. Patients have significant morbidity and mortality secondary to vascular tumors. Disease management is centered on tumor surveillance that allows early detection and treatment. Presymptomatic genetic testing is therefore recommended, including in at-risk children. Methods We tested 17 families (n = 109 individuals) for VHL mutations including 43 children under the a...

  19. Multiple Components of the VHL Tumor Suppressor Complex Are Frequently Affected by DNA Copy Number Loss in Pheochromocytoma

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    David A. Rowbotham

    2014-01-01

    Full Text Available Pheochromocytomas (PCC are rare tumors that arise in chromaffin tissue of the adrenal gland. PCC are frequently inherited through predisposing mutations in genes such as the von Hippel-Lindau (VHL tumor suppressor. VHL is part of the VHL elongin BC protein complex that also includes CUL2/5, TCEB1, TCEB2, and RBX1; in normoxic conditions this complex targets hypoxia-inducible factor 1 alpha (HIF1A for degradation, thus preventing a hypoxic response. VHL inactivation by genetic mechanisms, such as mutation and loss of heterozygosity, inhibits HIF1A degradation, even in the presence of oxygen, and induces a pseudohypoxic response. However, the described <10% VHL mutation rate cannot account for the high frequency of hypoxic response observed. Indeed, little is known about genetic mechanisms disrupting other complex component genes. Here, we show that, in a panel of 171 PCC tumors, 59.6% harbored gene copy number loss (CNL of at least one complex component. CNL significantly reduced gene expression and was associated with enrichment of gene targets controlled by HIF1. Interestingly, we show that VHL-related renal clear cell carcinoma harbored disruption of VHL alone. Our results indicate that VHL elongin BC protein complex components other than VHL could be important for PCC tumorigenesis and merit further investigation.

  20. Inhibiting sphingosine kinase 2 mitigates mutant Huntingtin-induced neurodegeneration in neuron models of Huntington disease.

    Science.gov (United States)

    Moruno-Manchon, Jose F; Uzor, Ndidi-Ese; Blasco-Conesa, Maria P; Mannuru, Sishira; Putluri, Nagireddy; Furr-Stimming, Erin E; Tsvetkov, Andrey S

    2017-04-01

    Huntington disease (HD) is the most common inherited neurodegenerative disorder. It has no cure. The protein huntingtin causes HD, and mutations to it confer toxic functions to the protein that lead to neurodegeneration. Thus, identifying modifiers of mutant huntingtin-mediated neurotoxicity might be a therapeutic strategy for HD. Sphingosine kinases 1 (SK1) and 2 (SK2) synthesize sphingosine-1-phosphate (S1P), a bioactive lipid messenger critically involved in many vital cellular processes, such as cell survival. In the nucleus, SK2 binds to and inhibits histone deacetylases 1 and 2 (HDAC1/2). Inhibiting both HDACs has been suggested as a potential therapy in HD. Here, we found that SK2 is nuclear in primary neurons and, unexpectedly, overexpressed SK2 is neurotoxic in a dose-dependent manner. SK2 promotes DNA double-strand breaks in cultured primary neurons. We also found that SK2 is hyperphosphorylated in the brain samples from a model of HD, the BACHD mice. These data suggest that the SK2 pathway may be a part of a pathogenic pathway in HD. ABC294640, an inhibitor of SK2, reduces DNA damage in neurons and increases survival in two neuron models of HD. Our results identify a novel regulator of mutant huntingtin-mediated neurotoxicity and provide a new target for developing therapies for HD. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. Autosomal dominant cutis laxa with severe lung disease: synthesis and matrix deposition of mutant tropoelastin.

    Science.gov (United States)

    Urban, Zsolt; Gao, Jimin; Pope, F Michael; Davis, Elaine C

    2005-06-01

    Cutis laxa (CL) is a heterogeneous group of genetic and acquired disorders with at least two autosomal dominant forms caused by mutations in the elastin and fibulin-5 genes, respectively. To define the molecular basis of CL in patients negative for point mutations in the elastin gene, metabolic labeling and immunoprecipitation experiments were used to study the synthesis of elastin in dermal fibroblasts. In addition to the normal 68 kDa tropoelastin (TE) protein, an abnormal, 120 kDa polypeptide was detected in the proband and her affected daughter in a CL family characterized by hernias and unusually severe and early-onset pulmonary disease including bronchiectasis and pulmonary emphysema. Mutational and gene expression studies established that affected individuals in this family carried a partial tandem duplication in the elastin locus. Immunoprecipitation experiments showed that the mutant TE was partially secreted and partially retained intracellularly. A polyclonal antibody raised against a unique peptide in the mutant TE molecule showed both intracellular and matrix staining. We conclude that elastin mutations can cause CL associated with a severe pulmonary phenotype. Synthesis of abnormal TE may interfere with elastic fiber function through a dominant-negative or a gain of function mechanism.

  2. Mutant huntingtin activates Nrf2-responsive genes and impairs dopamine synthesis in a PC12 model of Huntington's disease

    NARCIS (Netherlands)

    van Roon-Mom, W.M.C.; Pepers, B.A.; 't Hoen, P.A.C.; Verwijmeren, C.A.C.M.; den Dunnen, J.T.; Dorsman, J.C.; van Ommen, G.B.

    2008-01-01

    Background: Huntington's disease is a progressive autosomal dominant neurodegenerative disorder that is caused by a CAG repeat expansion in the HD or Huntington's disease gene. Although micro array studies on patient and animal tissue provide valuable information, the primary effect of mutant

  3. Plasma mutant α-galactosidase A protein and globotriaosylsphingosine level in Fabry disease

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    Takahiro Tsukimura

    2014-01-01

    Full Text Available Fabry disease is an X-linked genetic disorder characterized by deficient activity of α-galactosidase A (GLA and accumulation of glycolipids, and various GLA gene mutations lead to a wide range of clinical phenotypes from the classic form to the later-onset one. To investigate the biochemical heterogeneity and elucidate the basis of the disease using available clinical samples, we measured GLA activity, GLA protein and accumulated globotriaosylsphingosine (Lyso-Gb3, a biomarker of this disease, in plasma samples from Fabry patients. The analysis revealed that both the enzyme activity and the protein level were apparently decreased, and the enzyme activity was well correlated with the protein level in many Fabry patients. In these cases, a defect of biosynthesis or excessive degradation of mutant GLAs should be involved in the pathogenesis, and the residual protein level would determine the accumulation of Lyso-Gb3 and the severity of the disease. However, there are some exceptional cases, i.e., ones harboring p.C142Y, p.R112H and p.M296I, who exhibit a considerable amount of GLA protein. Especially, a subset of Fabry patients with p.R112H or p.M296I has been attracted interest because the patients exhibit almost normal plasma Lyso-Gb3 concentration. Structural analysis revealed that C142Y causes a structural change at the entrance of the active site. It will lead to a complete enzyme activity deficiency, resulting in a high level of plasma Lyso-Gb3 and the classic Fabry disease. On the other hand, it is thought that R112H causes a relatively large structural change on the molecular surface, and M296I a small one in a restricted region from the core to the surface, both the structural changes being far from the active site. These changes will cause not only partial degradation but also degeneration of the mutant GLA proteins, and the degenerated enzymes exhibiting small and residual activity remain and probably facilitate degradation of Lyso-Gb3

  4. Pancreatic cyst development: insights from von Hippel-Lindau disease

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    van Asselt Sophie J

    2013-02-01

    Full Text Available Abstract Pancreatic cysts are a heterogeneous group of lesions, which can be benign or malignant. Due to improved imaging techniques, physicians are more often confronted with pancreatic cysts. Little is known about the origin of pancreatic cysts in general. Von Hippel-Lindau (VHL disease is an atypical ciliopathy and inherited tumor syndrome, caused by a mutation in the VHL tumor suppressor gene encoding the VHL protein (pVHL. VHL patients are prone to develop cysts and neuroendocrine tumors in the pancreas in addition to several other benign and malignant neoplasms. Remarkably, pancreatic cysts occur in approximately 70% of VHL patients, making it the only hereditary tumor syndrome with such a discernible expression of pancreatic cysts. Cellular loss of pVHL due to biallelic mutation can model pancreatic cystogenesis in other organisms, suggesting a causal relationship. Here, we give a comprehensive overview of various pVHL functions, focusing on those that can potentially explain pancreatic cyst development in VHL disease. Based on preclinical studies, cilia loss in ductal cells is probably an important early event in pancreatic cyst development.

  5. Characterization of endolymphatic sac tumors and von Hippel-Lindau disease in the International Endolymphatic Sac Tumor Registry.

    Science.gov (United States)

    Bausch, Birke; Wellner, Ulrich; Peyre, Mathieu; Boedeker, Carsten C; Hes, Frederik J; Anglani, Mariagiulia; de Campos, Jose M; Kanno, Hiroshi; Maher, Eamonn R; Krauss, Tobias; Sansó, Gabriela; Barontini, Marta; Letizia, Claudio; Hader, Claudia; Schiavi, Francesca; Zanoletti, Elisabetta; Suárez, Carlos; Offergeld, Christian; Malinoc, Angelica; Zschiedrich, Stefan; Glasker, Sven; Bobin, Serge; Sterkers, Olivier; Ba Huy, Patrice Tran; Giraud, Sophie; Links, Thera; Eng, Charis; Opocher, Giuseppe; Richard, Stephane; Neumann, Hartmut P H

    2016-04-01

    Endolymphatic sac tumors (ELSTs) are, with a prevalence of up to 16%, a component of von Hippel-Lindau (VHL) disease. Data from international registries regarding heritable fraction and characteristics, germline VHL mutation frequency, and prevalence are lacking. Systematic registration of ELSTs from international centers of otorhinolaryngology and from multidisciplinary VHL centers' registries was performed. Molecular genetic analyses of the VHL gene were offered to all patients. Our population-based registry comprised 93 patients with ELST and 1789 patients with VHL. The prevalence of VHL germline mutations in apparently sporadic ELSTs was 39%. The prevalence of ELSTs in patients with VHL was 3.6%. ELST was the initial manifestation in 32% of patients with VHL-ELST. Prevalence of ELST in VHL disease is much lower compared to the literature. VHL-associated ELSTs can be the first presentation of the syndrome and mimic sporadic tumors, thus emphasizing the need of molecular testing in all presentations of ELST. © 2015 Wiley Periodicals, Inc. Head Neck 38: 673-679, 2016. © 2015 Wiley Periodicals, Inc.

  6. Pathology of the Nervous System in Von Hippel-Lindau Disease

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    Alexander O. Vortmeyer

    2015-06-01

    Full Text Available Von Hippel-Lindau (VHL disease is a tumor syndrome that frequently involves the central nervous system (CNS. It is caused by germline mutation of the VHL gene. Subsequent VHL inactivation in selected cells is followed by numerous well-characterized molecular consequences, in particular, activation and stabilization of hypoxia-inducible factors HIF1 and HIF2. The link between VHL gene inactivation and tumorigenesis remains poorly understood. Hemangioblastomas are the most common manifestation in the CNS; however, CNS invasion by VHL disease-associated endolymphatic sac tumors or metastatic renal cancer also occur, and their differentiation from primary hemangioblastoma may be challenging. Finally, in this review, we present recent morphologic insights on the developmental concept of VHL tumorigenesis which is best explained by pathologic persistence of temporary embryonic progenitor cells. 

  7. Differences in endoplasmic-reticulum quality control determine the cellular response to disease-associated mutants of proteolipid protein.

    Science.gov (United States)

    Roboti, Peristera; Swanton, Eileithyia; High, Stephen

    2009-11-01

    Missense mutations in human PLP1, the gene encoding myelin proteolipid protein (PLP), cause dysmyelinating Pelizaeus-Merzbacher disease of varying severity. Although disease pathology has been linked to retention of misfolded PLP in the endoplasmic reticulum (ER) and induction of the unfolded protein response (UPR), the molecular mechanisms that govern phenotypic heterogeneity remain poorly understood. To address this issue, we examined the cellular response to missense mutants of PLP that are associated with distinct disease phenotypes. We found that the mild-disease-associated mutants, W162L and G245A, were cleared from the ER comparatively quickly via proteasomal degradation and/or ER exit. By contrast, the more ;aggressive' A242V mutant, which causes severe disease, was significantly more stable, accumulated at the ER and resulted in a specific activation of the UPR. On the basis of these findings, we propose that the rate at which mutant PLP proteins are cleared from the ER modulates disease severity by determining the extent to which the UPR is activated.

  8. Mutant α-Synuclein Overexpression Induces Stressless Pacemaking in Vagal Motoneurons at Risk in Parkinson's Disease.

    Science.gov (United States)

    Lasser-Katz, Efrat; Simchovitz, Alon; Chiu, Wei-Hua; Oertel, Wolfgang H; Sharon, Ronit; Soreq, Hermona; Roeper, Jochen; Goldberg, Joshua A

    2017-01-04

    α-Synuclein overexpression (ASOX) drives the formation of toxic aggregates in neurons vulnerable in Parkinson's disease (PD), including dopaminergic neurons of the substantia nigra (SN) and cholinergic neurons of the dorsal motor nucleus of the vagus (DMV). Just as these populations differ in when they exhibit α-synucleinopathies during PD pathogenesis, they could also differ in their physiological responses to ASOX. An ASOX-mediated hyperactivity of SN dopamine neurons, which was caused by oxidative dysfunction of Kv4.3 potassium channels, was recently identified in transgenic (A53T-SNCA) mice overexpressing mutated human α-synuclein. Noting that DMV neurons display extensive α-synucleinopathies earlier than SN dopamine neurons while exhibiting milder cell loss in PD, we aimed to define the electrophysiological properties of DMV neurons in A53T-SNCA mice. We found that DMV neurons maintain normal firing rates in response to ASOX. Moreover, Kv4.3 channels in DMV neurons exhibit no oxidative dysfunction in the A53T-SNCA mice, which could only be recapitulated in wild-type mice by glutathione dialysis. Two-photon imaging of redox-sensitive GFP corroborated the finding that mitochondrial oxidative stress was diminished in DMV neurons in the A53T-SNCA mice. This reduction in oxidative stress resulted from a transcriptional downregulation of voltage-activated (Cav) calcium channels in DMV neurons, which led to a reduction in activity-dependent calcium influx via Cav channels. Thus, ASOX induces a homeostatic remodeling with improved redox signaling in DMV neurons, which could explain the differential vulnerability of SN dopamine and DMV neurons in PD and could promote neuroprotective strategies that emulate endogenous homeostatic responses to ASOX (e.g., stressless pacemaking) in DMV neurons. Overexpression of mutant α-synuclein causes Parkinson's disease, presumably by driving neurodegeneration in vulnerable neuronal target populations. However, the extent of

  9. Dimerization of the transmembrane domain of amyloid precursor proteins and familial Alzheimer's disease mutants

    Directory of Open Access Journals (Sweden)

    Fraser Paul E

    2008-01-01

    Full Text Available Abstract Background Amyloid precursor protein (APP is enzymatically cleaved by γ-secretase to form two peptide products, either Aβ40 or the more neurotoxic Aβ42. The Aβ42/40 ratio is increased in many cases of familial Alzheimer's disease (FAD. The transmembrane domain (TM of APP contains the known dimerization motif GXXXA. We have investigated the dimerization of both wild type and FAD mutant APP transmembrane domains. Results Using synthetic peptides derived from the APP-TM domain, we show that this segment is capable of forming stable transmembrane dimers. A model of a dimeric APP-TM domain reveals a putative dimerization interface, and interestingly, majority of FAD mutations in APP are localized to this interface region. We find that FAD-APP mutations destabilize the APP-TM dimer and increase the population of APP peptide monomers. Conclusion The dissociation constants are correlated to both the Aβ42/Aβ40 ratio and the mean age of disease onset in AD patients. We also show that these TM-peptides reduce Aβ production and Aβ42/Aβ40 ratios when added to HEK293 cells overexpressing the Swedish FAD mutation and γ-secretase components, potentially revealing a new class of γ-secretase inhibitors.

  10. VHL and HIF-1α: gene variations and prognosis in early-stage clear cell renal cell carcinoma.

    Science.gov (United States)

    Lessi, Francesca; Mazzanti, Chiara Maria; Tomei, Sara; Di Cristofano, Claudio; Minervini, Andrea; Menicagli, Michele; Apollo, Alessandro; Masieri, Lorenzo; Collecchi, Paola; Minervini, Riccardo; Carini, Marco; Bevilacqua, Generoso

    2014-03-01

    Von Hipple-Lindau gene (VHL) inactivation represents the most frequent abnormality in clear cell renal cell carcinoma (ccRCC). Hypoxia-inducible factor-1α (HIF-1α) expression is regulated by O2 level. In normal O2 conditions, VHL binds HIF-1α and allows HIF-1α proteasomal degradation. A single-nucleotide polymorphism (SNP) has been found located in the oxygen-dependent degradation domain at codon 582 (C1772T, rs11549465, Pro582Ser). In hypoxia, VHL/HIF-1α interaction is abolished and HIF-1α activates target genes in the nucleus. This study analyzes the impact of genetic alterations and protein expression of VHL and the C1772T SNP of HIF-1α gene (HIF-1α) on prognosis in early-stage ccRCC (pT1a, pT1b, and pT2). Mutational analysis of the entire VHL sequence and the genotyping of HIF-1α C1772T SNP were performed together with VHL promoter methylation analysis and loss of heterozygosis (LOH) analysis at (3p25) locus. Data obtained were correlated with VHL and HIF-1α protein expression and with tumor-specific survival (TSS). VHL mutations, methylation status, and LOH were detected in 51, 11, and 12% of cases, respectively. Our results support the association between biallelic alterations and/or VHL silencing with a worse TSS. Moreover, we found a significant association between the HIF-1α C1772C genotype and a worse TSS. The same association was found when testing the presence of HIF-1α protein in the nucleus. Our results highlight the role of VHL/HIF-1α pathway in RCC and support the molecular heterogeneity of early-stage ccRCC. More important, we show the involvement of HIF-1α C1772T SNP in ccRCC progression.

  11. Identification of key uric acid synthesis pathway in a unique mutant silkworm Bombyx mori model of Parkinson's disease.

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    Hiroko Tabunoki

    Full Text Available Plasma uric acid (UA levels decrease following clinical progression and stage development of Parkinson's disease (PD. However, the molecular mechanisms underlying decreases in plasma UA levels remain unclear, and the potential to apply mutagenesis to a PD model has not previously been discovered. We identified a unique mutant of the silkworm Bombyx mori (B.mori op. Initially, we investigated the causality of the phenotypic "op" by microarray analysis using our constructed KAIKO functional annotation pipeline. Consequently, we found a novel UA synthesis-modulating pathway, from DJ-1 to xanthine oxidase, and established methods for large-scale analysis of gene expression in B. mori. We found that the mRNA levels of genes in this pathway were significantly lower in B. mori op mutants, indicating that downstream events in the signal transduction cascade might be prevented. Additionally, levels of B.mori tyrosine hydroxylase (TH and DJ-1 mRNA were significantly lower in the brain of B. mori op mutants. UA content was significantly lower in the B. mori op mutant tissues and hemolymph. The possibility that the B. mori op mutant might be due to loss of DJ-1 function was supported by the observed vulnerability to oxidative stress. These results suggest that UA synthesis, transport, elimination and accumulation are decreased by environmental oxidative stress in the B. mori op mutant. In the case of B. mori op mutants, the relatively low availability of UA appears to be due both to the oxidation of DJ-1 and to its expenditure to mitigate the effects of environmental oxidative stress. Our findings are expected to provide information needed to elucidate the molecular mechanism of decreased plasma UA levels in the clinical stage progression of PD.

  12. Mutant huntingtin protein expression and blood-spinal cord barrier dysfunction in huntington disease.

    Science.gov (United States)

    Sciacca, Giacomo; Cicchetti, Francesca

    2017-12-01

    The aim of the study was to assess the distribution, frequency, and specific location of mutant huntingtin protein (mHTT) aggregates-the pathological hallmark of Huntington disease (HD)-within the various compartments of the spinal cord and their potential impact on the local vasculature and blood-spinal cord barrier (BSCB). We performed a series of postmortem immunohistochemical and immunofluorescent stainings, as well as Western blot analyses, on cervical and lumbar sections of the spinal cord in patients diagnosed with HD (n = 11 of all grades of disease severity) along with sex- and age-matched healthy controls (n = 9). We observed that mHTT was preferably expressed within the anterior horn of the gray matter, in both cervical and lumbar sections. At the cellular level, mHTT aggregates were more often encountered in the extracellular matrix but could also be observed within cell bodies and neurites as well as within the endothelium of blood vessels with an increase in the density of small blood vessels in cervical sections of HD cases. These vasculature changes were accompanied with features of BSCB leakage, as assessed by the presence of increased levels of fibrinogen in the surrounding parenchyma and enhanced leukocyte infiltration. This alteration in BSCB integrity may be explained, in part, by the dysregulation we found in some of the main proteins associated with it such as junctional adhesion molecule-1 and vascular endothelial cadherin. These observations have important implications for our understanding of HD pathology and may also have significant therapeutic implications. Ann Neurol 2017;82:981-994. © 2017 American Neurological Association.

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

    Science.gov (United States)

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

    2017-02-01

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

  14. Clearance of mutant huntingtin.

    Science.gov (United States)

    Li, Xiao-Jiang; Li, He; Li, Shihua

    2010-07-01

    Mutant huntingtin (htt) carries an expanded polyglutamine (polyQ) repeat (> 36 glutamines) in its N-terminal region, which leads htt to become misfolded and kill neuronal cells in Huntington disease (HD). The cytotoxicity of N-terminal mutant htt fragments is evident by severe neurological phenotypes of transgenic mice that express these htt fragments. Clearance of mutant htt is primarily mediated by the ubiquitin-proteasomal sysmtem (UPS) and autophagy. However, the relative efficiency of these two systems to remove toxic forms of mutant htt has not been rigorously compared. Using cellular and mouse models of HD, we found that inhibiting the UPS leads to a greater accumulation of mutant htt than inhibiting autophagy. Moreover, N-terminal mutant htt fragments, but not full-length mutant htt, accumulate in the HD mouse brains after inhibiting the UPS. These findings suggest that the UPS is more efficient than autophagy to remove N-terminal mutant htt.

  15. Structural differences between wild-type and fish eye disease mutant of lecithin:cholesterol acyltransferase.

    Science.gov (United States)

    Reshetnyak, Yana; Tchedre, Kissaou T; Nair, Maya P; Pritchard, P Haydn; Lacko, Andras G

    2006-08-01

    Fluorescence spectroscopy has been used to investigate the conformational changes that occur upon binding of wild type (WT) and mutant (Thr123Ile) lecithin:cholesterol acyltransferase (LCAT) to the potential substrates (dioleoyl-phosphatidyl choline [DOPC] and high density lipoprotein [HDL]). For a detailed analysis of structural differences between WT and mutant LCAT, we performed decompositional analysis of a set of tryptophan fluorescence spectra, measured at increasing concentrations of external quenchers (acrylamide and KI). The data obtained show that Thr123Ile mutation in LCAT leads to a conformation that is likely to be more rigid (less mobile/flexible) than that of the WT protein with a redistribution of charged residues around exposed tryptophan fluorophores. We propose that the redistribution of charged residues in mutant LCAT may be a major factor responsible for the dramatically reduced activity of the enzyme with HDL and reconstituted high density lipoprotein (rHDL).

  16. Psychosocial impact of Von Hippel-Lindau disease : levels and sources of distress

    NARCIS (Netherlands)

    Lammens, C. R. M.; Bleiker, E. M. A.; Verhoef, S.; Hes, F. J.; Ausems, M. G. E. M.; Majoor-Krakauer, D.; Sijmons, R. H.; van der Luijt, R. B.; van den Ouweland, A. M. W.; Van Os, Tam; Hoogerbrugge, N.; Garcia, E. B. Gomez; Dommering, C. J.; Gundy, C. M.; Aaronson, N. K.

    Von Hippel-Lindau disease (VHL) is a hereditary tumor susceptibility syndrome, characterized by an increased risk of developing multiple benign and malignant tumors at various sites and ages with limited preventive options. This study evaluates the prevalence of distress among VHL family members and

  17. Preimplantation genetic diagnosis of Von Hippel-Lindau disease cancer syndrome by combined mutation and segregation analysis

    Directory of Open Access Journals (Sweden)

    Denilce R. Sumita

    2007-03-01

    Full Text Available Von Hippel-Lindau (VHL disease is an autosomal dominant cancer syndrome, associated with the development of tumors and cysts in multiple organ systems, whose expression and age of onset are highly variable. The VHL disease tumor suppressor gene (VHL maps to 3p25-p26 and mutations ranging from a single base change to large deletions have been detected in patients with VHL disease. We developed a single cell PCR protocol for preimplantation genetic diagnosis (PGD of VHL disease to select unaffected embryos on the basis of the detection of the specific mutation and segregation analysis of polymorphic linked markers. Multiplex-nested PCR using single buccal cells of an affected individual were performed in order to test the accuracy and reliability of this single-cell protocol. For each locus tested, amplification efficiency was 83% to 87% and allelic drop-out rates ranged from 12% to 8%. Three VHL disease PGD cycles were performed on cells from a couple with paternal transmission of a 436delC mutation in exon 2 of the VHL gene, leading to the identification of three unaffected embryos. Independent of the mutation present, this general PGD protocol for the diagnosis of VHL disease can be used in families informative for either the D3S1038 or D3S1317 microsatellite markers.

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

    Directory of Open Access Journals (Sweden)

    Ting-Wei Mu

    2008-02-01

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

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

    Science.gov (United States)

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

    2017-06-01

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

  20. High throughput screening for small molecule therapy for Gaucher disease using patient tissue as the source of mutant glucocerebrosidase.

    Directory of Open Access Journals (Sweden)

    Ehud Goldin

    Full Text Available Gaucher disease (GD, the most common lysosomal storage disorder, results from the inherited deficiency of the lysosomal enzyme glucocerebrosidase (GCase. Previously, wildtype GCase was used for high throughput screening (HTS of large collections of compounds to identify small molecule chaperones that could be developed as new therapies for GD. However, the compounds identified from HTS usually showed reduced potency later in confirmatory cell-based assays. An alternate strategy is to perform HTS on mutant enzyme to identify different lead compounds, including those enhancing mutant enzyme activities. We developed a new screening assay using enzyme extract prepared from the spleen of a patient with Gaucher disease with genotype N370S/N370S. In tissue extracts, GCase is in a more native physiological environment, and is present with the native activator saposin C and other potential cofactors. Using this assay, we screened a library of 250,000 compounds and identified novel modulators of mutant GCase including 14 new lead inhibitors and 30 lead activators. The activities of some of the primary hits were confirmed in subsequent cell-based assays using patient-derived fibroblasts. These results suggest that primary screening assays using enzyme extracted from tissues is an alternative approach to identify high quality, physiologically relevant lead compounds for drug development.

  1. Foot-and-Mouth Disease (FMD) Virus 3C Protease Mutant L127P: Implications for FMD Vaccine Development.

    Science.gov (United States)

    Puckette, Michael; Clark, Benjamin A; Smith, Justin D; Turecek, Traci; Martel, Erica; Gabbert, Lindsay; Pisano, Melia; Hurtle, William; Pacheco, Juan M; Barrera, José; Neilan, John G; Rasmussen, Max

    2017-11-15

    The foot-and-mouth disease virus (FMDV) afflicts livestock in more than 80 countries, limiting food production and global trade. Production of foot-and-mouth disease (FMD) vaccines requires cytosolic expression of the FMDV 3C protease to cleave the P1 polyprotein into mature capsid proteins, but the FMDV 3C protease is toxic to host cells. To identify less-toxic isoforms of the FMDV 3C protease, we screened 3C mutants for increased transgene output in comparison to wild-type 3C using a Gaussia luciferase reporter system. The novel point mutation 3C(L127P) increased yields of recombinant FMDV subunit proteins in mammalian and bacterial cells expressing P1-3C transgenes and retained the ability to process P1 polyproteins from multiple FMDV serotypes. The 3C(L127P) mutant produced crystalline arrays of FMDV-like particles in mammalian and bacterial cells, potentially providing a practical method of rapid, inexpensive FMD vaccine production in bacteria. IMPORTANCE The mutant FMDV 3C protease L127P significantly increased yields of recombinant FMDV subunit antigens and produced virus-like particles in mammalian and bacterial cells. The L127P mutation represents a novel advancement for economical FMD vaccine production. Copyright © 2017 Puckette et al.

  2. Allele-specific suppression of mutant huntingtin using antisense oligonucleotides: providing a therapeutic option for all Huntington disease patients.

    Directory of Open Access Journals (Sweden)

    Niels H Skotte

    Full Text Available Huntington disease (HD is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs. We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder.

  3. Allele-specific suppression of mutant huntingtin using antisense oligonucleotides: providing a therapeutic option for all Huntington disease patients.

    Science.gov (United States)

    Skotte, Niels H; Southwell, Amber L; Østergaard, Michael E; Carroll, Jeffrey B; Warby, Simon C; Doty, Crystal N; Petoukhov, Eugenia; Vaid, Kuljeet; Kordasiewicz, Holly; Watt, Andrew T; Freier, Susan M; Hung, Gene; Seth, Punit P; Bennett, C Frank; Swayze, Eric E; Hayden, Michael R

    2014-01-01

    Huntington disease (HD) is an inherited, fatal neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. The mutant protein causes neuronal dysfunction and degeneration resulting in motor dysfunction, cognitive decline, and psychiatric disturbances. Currently, there is no disease altering treatment, and symptomatic therapy has limited benefit. The pathogenesis of HD is complicated and multiple pathways are compromised. Addressing the problem at its genetic root by suppressing mutant huntingtin expression is a promising therapeutic strategy for HD. We have developed and evaluated antisense oligonucleotides (ASOs) targeting single nucleotide polymorphisms that are significantly enriched on HD alleles (HD-SNPs). We describe our structure-activity relationship studies for ASO design and find that adjusting the SNP position within the gap, chemical modifications of the wings, and shortening the unmodified gap are critical for potent, specific, and well tolerated silencing of mutant huntingtin. Finally, we show that using two distinct ASO drugs targeting the two allelic variants of an HD-SNP could provide a therapeutic option for all persons with HD; allele-specifically for roughly half, and non-specifically for the remainder.

  4. Mutant huntingtin activates Nrf2-responsive genes and impairs dopamine synthesis in a PC12 model of Huntington's disease

    Directory of Open Access Journals (Sweden)

    den Dunnen Johan T

    2008-10-01

    Full Text Available Abstract Background Huntington's disease is a progressive autosomal dominant neurodegenerative disorder that is caused by a CAG repeat expansion in the HD or Huntington's disease gene. Although micro array studies on patient and animal tissue provide valuable information, the primary effect of mutant huntingtin will inevitably be masked by secondary processes in advanced stages of the disease. Thus, cell models are instrumental to study early, direct effects of mutant huntingtin. mRNA changes were studied in an inducible PC12 model of Huntington's disease, before and after aggregates became visible, to identify groups of genes that could play a role in the early pathology of Huntington's disease. Results Before aggregation, up-regulation of gene expression predominated, while after aggregates became visible, down-regulation and up-regulation occurred to the same extent. After aggregates became visible there was a down-regulation of dopamine biosynthesis genes accompanied by down-regulation of dopamine levels in culture, indicating the utility of this model to identify functionally relevant pathways. Furthermore, genes of the anti-oxidant Nrf2-ARE pathway were up-regulated, possibly as a protective mechanism. In parallel, we discovered alterations in genes which may result in increased oxidative stress and damage. Conclusion Up-regulation of gene expression may be more important in HD pathology than previously appreciated. In addition, given the pathogenic impact of oxidative stress and neuroinflammation, the Nrf2-ARE signaling pathway constitutes a new attractive therapeutic target for HD.

  5. Development of synchronous VHL syndrome tumors reveals contingencies and constraints to tumor evolution

    DEFF Research Database (Denmark)

    Fisher, Rosalie; Horswell, Stuart; Rowan, Andrew

    2014-01-01

    with a germline VHL mutation. We report that tumors arising in this context are clonally independent and harbour distinct secondary events exemplified by loss of chromosome 3p, despite an identical genetic background and tissue microenvironment. We propose that divergent mutational and copy number anomalies......Background : Genomic analysis of multi-focal renal cell carcinomas from an individual with a germline VHL mutation offers a unique opportunity to study tumor evolution. Results : We perform whole exome sequencing on four clear cell renal cell carcinomas removed from both kidneys of a patient...... are contingent upon the nature of 3p loss of heterozygosity occurring early in tumorigenesis. However, despite distinct 3p events, genomic, proteomic and immunohistochemical analyses reveal evidence for convergence upon the PI3K-AKT-mTOR signaling pathway. Four germline tumors in this young patient...

  6. Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumour suppressor pVHL

    DEFF Research Database (Denmark)

    Staller, Peter; Sulitkova, Jitka; Lisztwan, Joanna

    2003-01-01

    Organ-specific metastasis is governed, in part, by interactions between chemokine receptors on cancer cells and matching chemokines in target organs. For example, malignant breast cancer cells express the chemokine receptor CXCR4 and commonly metastasize to organs that are an abundant source...... regulates CXCR4 expression owing to its capacity to target hypoxia-inducible factor (HIF) for degradation under normoxic conditions. This process is suppressed under hypoxic conditions, resulting in HIF-dependent CXCR4 activation. An analysis of clear cell renal carcinoma that manifests mutation of the VHL...... gene in most cases revealed an association of strong CXCR4 expression with poor tumour-specific survival. These results suggest a mechanism for CXCR4 activation during tumour cell evolution and imply that VHL inactivation acquired by incipient tumour cells early in tumorigenesis confers not only...

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

    Directory of Open Access Journals (Sweden)

    Jonathan Wills

    2011-03-01

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

  8. Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance.

    OpenAIRE

    Delaney, T P; Friedrich, L.; Ryals, J A

    1995-01-01

    Plants possess multiple resistance mechanisms that guard against pathogen attack. Among these are inducible systems such as systemic acquired resistance (SAR). SAR is activated by pathogen exposure and leads to an increase in salicylic acid (SA), high-level expression of SAR-related genes, and resistance to a spectrum of pathogens. To identify components of the signal transduction pathways regulating SAR, a mutant screen was developed that uses 2,6-dichloroisonicotinic acid as an activator of...

  9. VHL negatively regulates SARS coronavirus replication by modulating nsp16 ubiquitination and stability.

    Science.gov (United States)

    Yu, Xiao; Chen, Shuliang; Hou, Panpan; Wang, Min; Chen, Yu; Guo, Deyin

    2015-04-03

    Eukaryotic cellular and most viral RNAs carry a 5'-terminal cap structure, a 5'-5' triphosphate linkage between the 5' end of the RNA and a guanosine nucleotide (cap-0). SARS coronavirus (SARS-CoV) nonstructural protein nsp16 functions as a methyltransferase, to methylate mRNA cap-0 structure at the ribose 2'-O position of the first nucleotide to form cap-1 structures. However, whether there is interplay between nsp16 and host proteins was not yet clear. In this report, we identified several potential cellular nsp16-interacting proteins from a human thymus cDNA library by yeast two-hybrid screening. VHL, one of these proteins, was proven to interact with nsp16 both in vitro and in vivo. Further studies showed that VHL can inhibit SARS-CoV replication by regulating nsp16 ubiquitination and promoting its degradation. Our results have revealed the role of cellular VHL in the regulation of SARS-CoV replication. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Elevated homocysteine, glutathione and cysteinylglycine concentrations in patients homozygous for the Chuvash polycythemia VHL mutation

    Science.gov (United States)

    Sergueeva, Adelina I.; Miasnikova, Galina Y.; Okhotin, Daniel J.; Levina, Alla A.; Debebe, Zufan; Ammosova, Tatiana; Niu, Xiaomei; Romanova, Elena A.; Nekhai, Sergei; DiBello, Patricia M.; Jacobsen, Donald W.; Prchal, Josef T.; Gordeuk, Victor R.

    2010-01-01

    In Chuvash polycythemia, homozygous von Hippel-Lindau (VHL) 598C>T leads to increased hypoxia inducible factor-1α and 2α, thromboses and lower systemic blood pressures. Circulating homocysteine, glutathione, γ-glutamyltransferase and cysteinylglycine concentrations were higher in 34 VHL598C>T homozygotes than in 37 normal controls and cysteine was lower. Multivariate analysis showed elevated homocysteine independently associated with higher mean systemic blood pressures and elevated glutathione was associated with lower pressures to a similar degree. Among VHL598C>T homozygotes, homocysteine was elevated with low and normal folate concentrations, consistent with a possible defect in the remethylation pathway. The elevated glutathione and γ-glutamyltranserase levels correlated positively with cysteinylglycine, consistent with possible upregulation of a glutathione synthetic enzyme and γ-glutamyltransferase. Cysteinylglycine correlated inversely with cysteine, consistent with possible reduced cysteinyldipeptidase activity. We conclude that up-regulated hypoxia-sensing may influence multiple steps in thiol metabolism. The effects of the resultant elevated levels of homocysteine and glutathione on systemic blood pressure may largely balance each other out. PMID:18223282

  11. Crystal Structure of the Cul2-Rbx1-EloBC-VHL Ubiquitin Ligase Complex.

    Science.gov (United States)

    Cardote, Teresa A F; Gadd, Morgan S; Ciulli, Alessio

    2017-06-06

    Cullin RING E3 ubiquitin ligases (CRLs) function in the ubiquitin proteasome system to catalyze the transfer of ubiquitin from E2 conjugating enzymes to specific substrate proteins. CRLs are large dynamic complexes and attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. The atomic details of whole CRL assembly and interactions that dictate subunit specificity remain elusive. Here we present the crystal structure of a pentameric CRL2VHL complex, composed of Cul2, Rbx1, Elongin B, Elongin C, and pVHL. The structure traps a closed state of full-length Cul2 and a new pose of Rbx1 in a trajectory from closed to open conformation. We characterize hotspots and binding thermodynamics at the interface between Cul2 and pVHL-EloBC and identify mutations that contribute toward a selectivity switch for Cul2 versus Cul5 recognition. Our findings provide structural and biophysical insights into the whole Cul2 complex that could aid future drug targeting. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  12. Characterization of endolymphatic sac tumors and von Hippel-Lindau disease in the International Endolymphatic Sac Tumor Registry

    NARCIS (Netherlands)

    Bausch, Birke; Wellner, Ulrich; Peyre, Mathieu; Boedeker, Carsten C.; Hes, Frederik J.; Anglani, Mariagiulia; de Campos, Jose M.; Kanno, Hiroshi; Maher, Eamonn R.; Krauss, Tobias; Sanso, Gabriela; Barontini, Marta; Letizia, Claudio; Hader, Claudia; Schiavi, Francesca; Zanoletti, Elisabetta; Suarez, Carlos; Offergeld, Christian; Malinoc, Angelica; Zschiedrich, Stefan; Glasker, Sven; Bobin, Serge; Sterkers, Olivier; Huy, Patrice Tran Ba; Giraud, Sophie; Links, Thera; Eng, Charis; Opocher, Giuseppe; Richard, Stephane; Neumann, Hartmut P. H.

    Background. Endolymphatic sac tumors (ELSTs) are, with a prevalence of up to 16%, a component of von Hippel-Lindau (VHL) disease. Data from international registries regarding heritable fraction and characteristics, germline VHL mutation frequency, and prevalence are lacking. Methods. Systematic

  13. Pre-fibrillar α-synuclein mutants cause Parkinson's disease-like non-motor symptoms in Drosophila.

    Directory of Open Access Journals (Sweden)

    Madhu Babu Gajula Balija

    Full Text Available Parkinson's disease (PD is linked to the formation of insoluble fibrillar aggregates of the presynaptic protein α-Synuclein (αS in neurons. The appearance of such aggregates coincides with severe motor deficits in human patients. These deficits are often preceded by non-motor symptoms such as sleep-related problems in the patients. PD-like motor deficits can be recapitulated in model organisms such as Drosophila melanogaster when αS is pan-neurally expressed. Interestingly, both these deficits are more severe when αS mutants with reduced aggregation properties are expressed in flies. This indicates that that αS aggregation is not the primary cause of the PD-like motor symptoms. Here we describe a model for PD in Drosophila which utilizes the targeted expression of αS mutants in a subset of dopadecarboxylase expressing serotonergic and dopaminergic (DA neurons. Our results show that targeted expression of pre-fibrillar αS mutants not only recapitulates PD-like motor symptoms but also the preceding non-motor symptoms such as an abnormal sleep-like behavior, altered locomotor activity and abnormal circadian periodicity. Further, the results suggest that the observed non-motor symptoms in flies are caused by an early impairment of neuronal functions rather than by the loss of neurons due to cell death.

  14. Biochemical Classification of Disease-associated Mutants of RAS-like Protein Expressed in Many Tissues (RIT1).

    Science.gov (United States)

    Fang, Zhenhao; Marshall, Christopher B; Yin, Jiani C; Mazhab-Jafari, Mohammad T; Gasmi-Seabrook, Geneviève M C; Smith, Matthew J; Nishikawa, Tadateru; Xu, Yang; Neel, Benjamin G; Ikura, Mitsuhiko

    2016-07-22

    RAS-like protein expressed in many tissues 1 (RIT1) is a disease-associated RAS subfamily small guanosine triphosphatase (GTPase). Recent studies revealed that germ-line and somatic RIT1 mutations can cause Noonan syndrome (NS), and drive proliferation of lung adenocarcinomas, respectively, akin to RAS mutations in these diseases. However, the locations of these RIT1 mutations differ significantly from those found in RAS, and do not affect the three mutational "hot spots" of RAS. Moreover, few studies have characterized the GTPase cycle of RIT1 and its disease-associated mutants. Here we developed a real-time NMR-based GTPase assay for RIT1 and investigated the effect of disease-associated mutations on GTPase cycle. RIT1 exhibits an intrinsic GTP hydrolysis rate similar to that of H-RAS, but its intrinsic nucleotide exchange rate is ∼4-fold faster, likely as a result of divergent residues near the nucleotide binding site. All of the disease-associated mutations investigated increased the GTP-loaded, activated state of RIT1 in vitro, but they could be classified into two groups with different intrinsic GTPase properties. The S35T, A57G, and Y89H mutants exhibited more rapid nucleotide exchange, whereas F82V and T83P impaired GTP hydrolysis. A RAS-binding domain pulldown assay indicated that RIT1 A57G and Y89H were highly activated in HEK293T cells, whereas T83P and F82V exhibited more modest activation. All five mutations are associated with NS, whereas two (A57G and F82V) have also been identified in urinary tract cancers and myeloid malignancies. Characterization of the effects on the GTPase cycle of RIT1 disease-associated mutations should enable better understanding of their role in disease processes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Characterization of ATP7A missense mutants suggests a correlation between intracellular trafficking and severity of Menkes disease

    DEFF Research Database (Denmark)

    Skjørringe, Tina; Pedersen, Per Amstrup; Thorborg, Sidsel Salling

    2017-01-01

    Menkes disease (MD) is caused by mutations in ATP7A, encoding a copper-transporting P-type ATPase which exhibits copper-dependent trafficking. ATP7A is found in the Trans-Golgi Network (TGN) at low copper concentrations, and in the post-Golgi compartments and the plasma membrane at higher...... led to trapping of the protein in TGN and displayed essentially no activity in a yeast-based functional assay. These were predicted to inhibit the catalytic phosphorylation of the protein. Four mutants showed diffuse post-TGN localization, while two displayed copper dependent trafficking. These six...

  16. Mutant alpha-synuclein-induced degeneration is reduced by parkin in a fly model of Parkinson's disease.

    Science.gov (United States)

    Haywood, Annika F M; Staveley, Brian E

    2006-05-01

    Parkinson's disease (PD) patients show a characteristic loss of motor control caused by the degeneration of dopaminergic neurons. Mutations in the genes that encode alpha-synuclein and parkin have been linked to inherited forms of this disease. The parkin protein functions as a ubiquitin ligase that targets proteins for degradation. Expression of isoforms of human alpha-synuclein in the Drosophila melanogaster nervous system forms the basis of an excellent genetic model that recapitulates phenotypic and behavioural features of PD. Using this model, we analysed the effect of parkin co-expression on the climbing ability of aging flies, their life span, and their retinal degeneration. We have determined that co-expression of parkin can suppress phenotypes caused by expression of mutant alpha-synuclein. In the developing eye, parkin reduces retinal degeneration. When co-expressed in the dopaminergic neurons, the ability to climb is extended over time. If conserved in humans, we suggest that upregulation of parkin may prove a method of suppression for PD induced by mutant forms of alpha-synuclein.

  17. B-RAF mutant alleles associated with Langerhans cell histiocytosis, a granulomatous pediatric disease.

    Directory of Open Access Journals (Sweden)

    Takeshi Satoh

    Full Text Available Langerhans cell histiocytosis (LCH features inflammatory granuloma characterised by the presence of CD1a+ dendritic cells or 'LCH cells'. Badalian-Very et al. recently reported the presence of a canonical (V600EB-RAF mutation in 57% of paraffin-embedded biopsies from LCH granuloma. Here we confirm their findings and report the identification of two novel B-RAF mutations detected in LCH patients.Mutations of B-RAF were observed in granuloma samples from 11 out of 16 patients using 'next generation' pyrosequencing. In 9 cases the mutation identified was (V600EB-RAF. In 2 cases novel polymorphisms were identified. A somatic (600DLATB-RAF insertion mimicked the structural and functional consequences of the (V600EB-RAF mutant. It destabilized the inactive conformation of the B-RAF kinase and resulted in increased ERK activation in 293 T cells. The (600DLATB-RAF and (V600EB-RAF mutations were found enriched in DNA and mRNA from the CD1a+ fraction of granuloma. They were absent from the blood and monocytes of 58 LCH patients, with a lower threshold of sequencing sensitivity of 1%-2% relative mutation abundance. A novel germ line (T599AB-RAF mutant allele was detected in one patient, at a relative mutation abundance close to 50% in the LCH granuloma, blood monocytes and lymphocytes. However, (T599AB-RAF did not destabilize the inactive conformation of the B-RAF kinase, and did not induce increased ERK phosphorylation or C-RAF transactivation.Our data confirmed presence of the (V600EB-RAF mutation in LCH granuloma of some patients, and identify two novel B-RAF mutations. They indicate that (V600EB-RAF and (600DLATB-RAF mutations are somatic mutants enriched in LCH CD1a(+ cells and absent from the patient blood. Further studies are needed to assess the functional consequences of the germ-line (T599AB-RAF allele.

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

    Science.gov (United States)

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

    2015-05-01

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

  19. pVHL co-ordinately regulates CXCR4/CXCL12 and MMP2/MMP9 expression in human clear-cell renal cell carcinoma

    DEFF Research Database (Denmark)

    Struckmann, K; Mertz, Kd; Steu, S

    2008-01-01

    Loss of pVHL function, characteristic for clear-cell renal cell carcinoma (ccRCC), causes increased expression of CXCR4 chemokine receptor, which triggers expression of metastasis-associated MMP2/MMP9 in different human cancers. The impact of pVHL on MMP2/MMP9 expression and their relationship...

  20. Understanding mechanisms of GLI-mediated transcription during craniofacial development and disease using the ciliopathic mutant, talpid2

    Directory of Open Access Journals (Sweden)

    Ya-Ting Chang

    2016-10-01

    Full Text Available The primary cilium is a ubiquitous, microtubule-based organelle that cells utilize to transduce molecular signals. Ciliopathies are a group of diseases that are caused by a disruption in the structure or function of the primary cilium. Over 30% of all ciliopathies are primarily defined by their craniofacial phenotypes, which typically include midfacial defects, cleft lip/palate, micrognathia, aglossia and craniosynostosis. The frequency and severity of craniofacial phenotypes in ciliopathies emphasizes the importance of the cilium during development of the craniofacial complex. Molecularly, many ciliopathic mutants, including the avian talpid2 (ta2, report pathologically high levels of full-length GLI3 (GLI3FL, which can go on to function as an activator (GLIA, and reduced production of truncated GLI3 (GLI3T, which can go on to function as a repressor (GLIR. These observations suggest that the craniofacial phenotypes of ciliary mutants like ta2 are caused either by excessive activity of the GLIA or reduced activity of GLIR. To decipher between these two scenarios, we examined GLI3 occupation at the regulatory regions of target genes and subsequent target gene expression. Using in silico strategies we identified consensus GLI binding regions (GBRs in the avian genome and confirmed GLI3 binding to the regulatory regions of its targets by chromatin immunoprecipitation (ChIP. In ta2 mutants, there was a strikingly low number of GLI3 target genes that had significantly increased expression in facial prominences compared to the control embryo and GLI3 occupancy at GBRs associated with target genes was largely reduced. In vitro DNA binding assays, further supported ChIP results, indicated that the excessive GLI3FL generated in ta2 mutants did not bind to GBRs. In light of these results, we explored the possibility of GLI co-regulator proteins playing a role in regulatory mechanism of GLI-mediated transcription. Taken together our studies suggest that

  1. Vhl deletion in renal epithelia causes HIF-1?-dependent, HIF-2?-independent angiogenesis and constitutive diuresis

    OpenAIRE

    Schönenberger, Désirée; Rajski, Michal; Harlander, Sabine; Frew, Ian J

    2016-01-01

    One of the earliest requirements for the formation of a solid tumor is the establishment of an adequate blood supply. Clear cell renal cell carcinomas (ccRCC) are highly vascularized tumors in which the earliest genetic event is most commonly the biallelic inactivation of the VHL tumor suppressor gene, leading to constitutive activation of the HIF-1α and HIF-2α transcription factors, which are known angiogenic factors. However it remains unclear whether either or both HIF-1α or HIF-2α stabili...

  2. The homozygous VHL(D126N) missense mutation is associated with dramatically elevated erythropoietin levels, consequent polycythemia, and early onset severe pulmonary hypertension.

    Science.gov (United States)

    Sarangi, Susmita; Lanikova, Lucie; Kapralova, Katarina; Acharya, Suchitra; Swierczek, Sabina; Lipton, Jeffrey M; Wolfe, Lawrence; Prchal, Josef T

    2014-11-01

    von Hippel-Lindau (VHL) protein is the principal negative regulator of hypoxia sensing mediated by transcription factors. Mutations in exon 3 of the VHL gene lead to Chuvash (VHL(R200W)) and Croatian (VHL(H191D)) polycythemias. Here, we describe an infant of Bangladesh ethnicity with a novel homozygous VHL(D126N) mutation with congenital polycythemia and dramatically elevated erythropoietin (EPO) levels, who developed severe fatal pulmonary hypertension. In contrast to Chuvash polycythemia, erythroid progenitors (BFU-Es) did not reveal a marked EPO hypersensitivity. Further, NF-E2 and RUNX1 transcripts that correlate with BFU-Es EPO hypersensitivity in polycythemic mutations were not elevated. © 2014 Wiley Periodicals, Inc.

  3. Microtubular stability affects pVHL-mediated regulation of HIF-1alpha via the p38/MAPK pathway in hypoxic cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Miao Teng

    Full Text Available BACKGROUND: Our previous research found that structural changes of the microtubule network influence glycolysis in cardiomyocytes by regulating the hypoxia-inducible factor (HIF-1α during the early stages of hypoxia. However, little is known about the underlying regulatory mechanism of the changes of HIF-1α caused by microtubule network alternation. The von Hippel-Lindau tumor suppressor protein (pVHL, as a ubiquitin ligase, is best understood as a negative regulator of HIF-1α. METHODOLOGY/PRINCIPAL FINDINGS: In primary rat cardiomyocytes and H9c2 cardiac cells, microtubule-stabilization was achieved by pretreating with paclitaxel or transfection of microtubule-associated protein 4 (MAP4 overexpression plasmids and microtubule-depolymerization was achieved by pretreating with colchicine or transfection of MAP4 siRNA before hypoxia treatment. Recombinant adenovirus vectors for overexpressing pVHL or silencing of pVHL expression were constructed and transfected in primary rat cardiomyocytes and H9c2 cells. With different microtubule-stabilizing and -depolymerizing treaments, we demonstrated that the protein levels of HIF-1α were down-regulated through overexpression of pVHL and were up-regulated through knockdown of pVHL in hypoxic cardiomyocytes. Importantly, microtubular structure breakdown activated p38/MAPK pathway, accompanied with the upregulation of pVHL. In coincidence, we found that SB203580, a p38/MAPK inhibitor decreased pVHL while MKK6 (Glu overexpression increased pVHL in the microtubule network altered-hypoxic cardiomyocytes and H9c2 cells. CONCLUSIONS/SIGNIFICANCE: This study suggests that pVHL plays an important role in the regulation of HIF-1α caused by the changes of microtubular structure and the p38/MAPK pathway participates in the process of pVHL change following microtubule network alteration in hypoxic cardiomyocytes.

  4. Two childhood pheochromocytoma cases due to von Hippel -Lindau disease, one associated with pancreatic neuroendocrine tumor; a rare manifestation.

    Science.gov (United States)

    Dağdeviren Çakır, Aydilek; Turan, Hande; Aykut, Ayça; Durmaz, Asude; Ercan, Oya; Evliyaoğlu, Olcay

    2017-10-12

    (VHL) disease is an autosomal dominantly inherited disorder characterized by hemangioblastomas of retina and central nervous system (CNS); renal cysts, clear cell carcinoma; PCC; endolymphatic sac tumors; cystadenomas of the epididymis in males, broad ligament of uterus in females; pancreatic cysts, cystadenomas and neuroendocrine tumors. We here report two cases of VHL disease presented with PCC as the first manifestation. Hemangioblastoma of CNS in the first case and PNET in the second case developed during follow- up and led to the diagnosis of VHL disease. Genetic analyses of cases revealed p.Arg161Gln (c.482G>A) and p.Leu129Pro(c.386T>G) heterozygous missense mutation in VHL gene, respectively. In children, PCC may be the only and/or initial manifestation of the disease with delayed manifestations of the syndrome in other organs. PNET is a very rare manifestation of VHL disease. To best of our knowledge, this is the second case in literature, presenting with combination of PNET and bilateral PCC as components of childhood VHL disease. Pediatric patients diagnosed with PCC should be investigated for the genetic causes especially for VHL.

  5. A tailored mouse model of CLN2 disease: A nonsense mutant for testing personalized therapies.

    Science.gov (United States)

    Geraets, Ryan D; Langin, Logan M; Cain, Jacob T; Parker, Camille M; Beraldi, Rosanna; Kovacs, Attila D; Weimer, Jill M; Pearce, David A

    2017-01-01

    The Neuronal Ceroid Lipofuscinoses (NCLs), also known as Batten disease, result from mutations in over a dozen genes. Although, adults are susceptible, the NCLs are frequently classified as pediatric neurodegenerative diseases due to their greater pediatric prevalence. Initial clinical presentation usually consists of either seizures or retinopathy but develops to encompass both in conjunction with declining motor and cognitive function. The NCLs result in premature death due to the absence of curative therapies. Nevertheless, preclinical and clinical trials exist for various therapies. However, the genotypes of NCL animal models determine which therapeutic approaches can be assessed. Mutations of the CLN2 gene encoding a soluble lysosomal enzyme, tripeptidyl peptidase 1 (TPP1), cause late infantile NCL/CLN2 disease. The genotype of the original mouse model of CLN2 disease, Cln2-/-, excludes mutation guided therapies like antisense oligonucleotides and nonsense suppression. Therefore, the purpose of this study was to develop a model of CLN2 disease that allows for the assessment of all therapeutic approaches. Nonsense mutations in CLN2 disease are frequent, the most common being CLN2R208X. Thus, we created a mouse model that carries a mutation equivalent to the human p.R208X mutation. Molecular assessment of Cln2R207X/R207X tissues determined significant reduction in Cln2 transcript abundance and TPP1 enzyme activity. This reduction leads to the development of neurological impairment (e.g. tremors) and neuropathology (e.g. astrocytosis). Collectively, these assessments indicate that the Cln2R207X/R207X mouse is a valid CLN2 disease model which can be used for the preclinical evaluation of all therapeutic approaches including mutation guided therapies.

  6. Von Hippel-Lindau disease: an evaluation of natural history and functional disability

    Science.gov (United States)

    Feletti, Alberto; Anglani, Mariagiulia; Scarpa, Bruno; Schiavi, Francesca; Boaretto, Francesca; Zovato, Stefania; Taschin, Elisa; Gardi, Mario; Zanoletti, Elisabetta; Piermarocchi, Stefano; Murgia, Alessandra; Pavesi, Giacomo; Opocher, Giuseppe

    2016-01-01

    Background Although many studies have been published about specific lesions characterizing von Hippel-Lindau(VHL) disease, none have dealt with the natural history of the whole disease and the consequent disabilities. We aim to define the comprehensive natural history of VHL disease and to describe the functional disabilities and their impact upon patients' quality of life, thereby tailoring the follow-up schedule accordingly. Methods We performed a prospective analysis on 128 VHL-affected patients beginning in 1996. For each affected organ, we defined intervals between the first and subsequent VHL-related manifestations and compared them with current VHL surveillance protocols. We looked for any association of the number of involved organs with age, sex, type of VHL gene mutation, and functional domain mutation. Ultimately, we assessed the organ-specific disabilities caused by VHL disease. Results Hemangioblastomas show different patterns of progression depending on their location, whereas both renal cysts and carcinomas have similar progression rates. Surgery for pheochromocytoma and CNS hemangioblastoma is performed earlier than for pancreatic or renal cancer. The number of involved organs is associated with age but not with sex, type of VHL gene mutation, or functional domain mutation. A thorough analysis of functional disabilities showed that age is related to the first-appearing functional impairment, but it is not predictive of the final number of disabilities. Conclusions Our study defines the disease progression and provides a comprehensive view of the syndrome over time. We analyzed for the first time the functional disability of VHL patients, assessing the progression for each function. PMID:26763786

  7. Defective folding and rapid degradation of mutant proteins is a common disease mechanism in genetic disorders

    DEFF Research Database (Denmark)

    Gregersen, N; Bross, P; Jørgensen, M M

    2000-01-01

    Many disease-causing point mutations do not seriously compromise synthesis of the affected polypeptide but rather exert their effects by impairing subsequent protein folding or stability of the folded protein. This often results in rapid degradation of the affected protein. The concepts...

  8. Defective folding and rapid degradation of mutant proteins is a common disease mechanism in genetic disorders

    DEFF Research Database (Denmark)

    Gregersen, Niels; Bross, Peter; Jørgensen, Malene Munk

    2000-01-01

    Many disease-causing point mutations do not seriously compromise synthesis of the affected polypeptides but rather exert their effects by impairing subsequent protein folding or stability of the folded protein. This often results in rapid degradation of the affected protein. The concepts...

  9. Loss of rad-23 protects against models of motor neuron disease by enhancing mutant protein clearance

    NARCIS (Netherlands)

    A.M. Jablonski (Angela M.); T. Lamitina (Todd); N.F. Liachko (Nicole F.); M. Sabatella (Mariangela); J. Lu (Jiayin); L. Zhang (Lei); L.W. Ostrow (Lyle W.); P. Gupta (Preetika); C.-Y. Wu (Chia-Yen); S. Doshi (Shachee); J. Mojsilovic-Petrovic (Jelena); H. Lans (Hannes); J. Wang (Jiou); B. Kraemer (Brian); R.G. Kalb (Robert G.)

    2015-01-01

    textabstractMisfolded proteins accumulate and aggregate in neurodegenerative disease. The existence of these deposits reflects a derangement in the protein homeostasis machinery. Using a candidate gene screen, we report that loss of RAD-23 protects against the toxicity of proteins known to aggregate

  10. Characteristics of scientific production in Special Education in Virtual Health Library (VHL: a bibliometric study

    Directory of Open Access Journals (Sweden)

    Luciana Pizzani

    2010-12-01

    Full Text Available Objective: To characterize, through bibliometric approach, the scientific literature in this Special Education in the databases of the Virtual Health Library (VHL. The VHL is coordinated by BIREME - Specialized Center of the Pan American Health Organization whose objective is to promote the dissemination and use of scientific information in health. Method: The research methodology was performed by observing the following steps: a literature review on education special and bibliometrics, data collection from the site of BIREME about the presence of special education in the databases, organization, processing and bibliometric analysis of data collected using the software MS Excel and Vantage Point. Results: indicators produced allow signal that the predominant language of scientific production was the Portuguese and the majority of records were written individually, the themes addressed were psychology and developmental psychology. Conclusion: These bibliometric indicators characterizing the state of the art of scientific literature in Special Education at the various bases Data Bireme and also showed a field of interconnections between Health Sciences and Special Education.

  11. ATM mutants

    Indian Academy of Sciences (India)

    First page Back Continue Last page Graphics. ATM mutants. ATM (Ataxia Telangiectasia Mutated). AT2BE and AT5B1 cells – fibroblast cell lines from Ataxia telangiectasia patients. Deletion mutants expressing truncated ATM protein which is inactive. Have been used in studies looking at the role of ATM in DNA damage ...

  12. Differential copper binding to alpha-synuclein and its disease-associated mutants affect the aggregation and amyloid formation.

    Science.gov (United States)

    Ranjan, Priyatosh; Ghosh, Dhiman; Yarramala, Deepthi S; Das, Subhadeep; Maji, Samir K; Kumar, Ashutosh

    2017-02-01

    Copper is an essential trace element required for the proper functioning of various enzymes present in the central nervous system. An imbalance in the copper homeostasis results in the pathology of various neurodegenerative disorders including Parkinson's Disease. Hence, residue specific interaction of Cu2+ to α-Syn along with the familial mutants H50Q and G51D needs to be studied in detail. We investigated the residue specific mapping of Cu2+ binding sites and binding strength using solution-state NMR and ITC respectively. The aggregation kinetics, secondary structural changes, and morphology of the formed fibrils in the presence and absence of Cu2+ were studied using fluorescence, CD, and AFM respectively. Copper binding to α-Syn takes place at three different sites with a higher affinity for the region 48-53. While one of the sites got abolished in the case of H50Q, the mutant G51D showed a binding pattern similar to WT. The aggregation kinetics of these proteins in the presence of Cu2+ showed an enhanced rate of fibril formation with a pronounced effect for G51D. Cu2+ binding results in the destabilization of long-range tertiary interactions in α-Syn leading to the exposure of highly amyloidogenic NAC region which results in the increased rate of fibril formation. Although the residues 48-53 have a stronger affinity for Cu2+ in case of WT and G51D, the binding is not responsible for enhancing the rate of fibril formation in case of H50Q. These findings will help in the better understanding of Cu2+ catalyzed aggregation of synucleins. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Disease causing mutants of TDP-43 nucleic acid binding domains are resistant to aggregation and have increased stability and half-life.

    Science.gov (United States)

    Austin, James A; Wright, Gareth S A; Watanabe, Seiji; Grossmann, J Günter; Antonyuk, Svetlana V; Yamanaka, Koji; Hasnain, S Samar

    2014-03-18

    Over the last two decades many secrets of the age-related human neural proteinopathies have been revealed. A common feature of these diseases is abnormal, and possibly pathogenic, aggregation of specific proteins in the effected tissue often resulting from inherent or decreased structural stability. An archetype example of this is superoxide dismutase-1, the first genetic factor to be linked with amyotrophic lateral sclerosis (ALS). Mutant or posttranslationally modified TAR DNA binding protein-32 (TDP-43) is also strongly associated with ALS and an increasingly large number of other neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD). Cytoplasmic mislocalization and elevated half-life is a characteristic of mutant TDP-43. Furthermore, patient age at the onset of disease symptoms shows a good inverse correlation with mutant TDP-43 half-life. Here we show that ALS and FTLD-associated TDP-43 mutations in the central nucleic acid binding domains lead to elevated half-life and this is commensurate with increased thermal stability and inhibition of aggregation. It is achieved without impact on secondary, tertiary, or quaternary structure. We propose that tighter structural cohesion contributes to reduced protein turnover, increasingly abnormal proteostasis and, ultimately, faster onset of disease symptoms. These results contrast our perception of neurodegenerative diseases as misfolded proteinopathies and delineate a novel path from the molecular characteristics of mutant TDP-43 to aberrant cellular effects and patient phenotype.

  14. A novel mutant allele of SSI2 confers a better balance between disease resistance and plant growth inhibition on Arabidopsis thaliana.

    Science.gov (United States)

    Yang, Wei; Dong, Ran; Liu, Li; Hu, Zhubing; Li, Jing; Wang, Yong; Ding, Xinhua; Chu, Zhaohui

    2016-09-26

    Resistance and growth are opposing characteristics in plants. SA INSENSITIVITY OF npr1-5 (SSI2) encodes a stearoyl-ACP desaturase (S-ACP DES) that has previously been reported to simultaneously enhance resistance and repress growth. Here, we characterize ssi2-2, a novel mutant allele of SSI2 that has two amino acid substitutions. Compared with wild-type and two other mutants of SSI2, ssi2-2 showed intermediate phenotypes in growth size, punctate necrosis, resistance to the bacterial pathogen Pst DC3000, salicylic acid (SA) content, pathogenesis-related (PR) gene levels and 18:1 content. These results indicate that ssi2-2 is a weak mutant of SSI2. Additionally, by using ssi2-2 as an intermediate control, a number of differentially expressed genes were identified in transcriptome profiling analysis. These results suggest that constitutive expression of defense-related genes and repression of IAA signaling-associated genes is present in all SSI2 mutants. Taken together, our results suggest that the weak ssi2-2 mutant maintains a better balance between plant immunity and vegetative growth than other mutants, consequently providing a basis to genetically engineer disease resistance in crop plants.

  15. A novel triple repeat mutant tau transgenic model that mimics aspects of pick's disease and fronto-temporal tauopathies.

    Directory of Open Access Journals (Sweden)

    Edward Rockenstein

    Full Text Available Tauopathies are a group of disorders leading to cognitive and behavioral impairment in the aging population. While four-repeat (4R Tau is more abundant in corticobasal degeneration, progressive supranuclear palsy, and Alzheimer's disease, three-repeat (3R Tau is the most abundant splice, in Pick's disease. A number of transgenic models expressing wild-type and mutant forms of the 4R Tau have been developed. However, few models of three-repeat Tau are available. A transgenic mouse model expressing three-repeat Tau was developed bearing the mutations associated with familial forms of Pick's disease (L266V and G272V mutations. Two lines expressing high (Line 13 and low (Line 2 levels of the three-repeat mutant Tau were analyzed. By Western blot, using antibodies specific to three-repeat Tau, Line 13 expressed 5-times more Tau than Line 2. The Tau expressed by these mice was most abundant in the frontal-temporal cortex and limbic system and was phosphorylated at residues detected by the PHF-1, AT8, CP9 and CP13 antibodies. The higher-expressing mice displayed hyperactivity, memory deficits in the water maze and alterations in the round beam. The behavioral deficits started at 6-8 months of age and were associated with a progressive increase in the accumulation of 3R Tau. By immunocytochemistry, mice from Line 13 displayed extensive accumulation of 3R Tau in neuronal cells bodies in the pyramidal neurons of the neocortex, CA1-3 regions, and dentate gyrus of the hippocampus. Aggregates in the granular cells had a globus appearance and mimic Pick's-like inclusions. There were abundant dystrophic neurites, astrogliosis and synapto-dendritic damage in the neocortex and hippocampus of the higher expresser line. The hippocampal lesions were moderately argyrophilic and Thioflavin-S negative. By electron microscopy, discrete straight filament aggregates were detected in some neurons in the hippocampus. This model holds promise for better understanding the

  16. Characterization of the Xylella fastidiosa PD1311 gene mutant and its suppression of Pierce's disease on grapevines.

    Science.gov (United States)

    Hao, Lingyun; Johnson, Kameka; Cursino, Luciana; Mowery, Patricia; Burr, Thomas J

    2017-06-01

    Xylella fastidiosa causes Pierce's disease (PD) on grapevines, leading to significant economic losses in grape and wine production. To further our understanding of X. fastidiosa virulence on grapevines, we examined the PD1311 gene, which encodes a putative acyl-coenzyme A (acyl-CoA) synthetase, and is highly conserved across Xylella species. It was determined that PD1311 is required for virulence, as the deletion mutant, ΔPD1311, was unable to cause disease on grapevines. The ΔPD1311 strain was impaired in behaviours known to be associated with PD development, including motility, aggregation and biofilm formation. ΔPD1311 also expressed enhanced sensitivity to H 2 O 2 and polymyxin B, and showed reduced survival in grapevine sap, when compared with wild-type X. fastidiosa Temecula 1 (TM1). Following inoculation, ΔPD1311 could not be detected in grape shoots, which may be related to its altered growth and sensitivity phenotypes. Inoculation with ΔPD1311 2 weeks prior to TM1 prevented the development of PD in a significant fraction of vines and eliminated detectable levels of TM1. In contrast, vines inoculated simultaneously with TM1 and ΔPD1311 developed disease at the same level as TM1 alone. In these vines, TM1 populations were distributed similarly to populations in TM1-only inoculated plants. These findings suggest that, through an indirect mechanism, pretreatment of vines with ΔPD1311 suppresses pathogen population and disease. © 2016 BSPP AND JOHN WILEY & SONS LTD.

  17. Potent and selective antisense oligonucleotides targeting single-nucleotide polymorphisms in the Huntington disease gene / allele-specific silencing of mutant huntingtin

    DEFF Research Database (Denmark)

    Carroll, Jeffrey B; Warby, Simon C; Southwell, Amber L

    2011-01-01

    Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by CAG-expansion in the huntingtin gene (HTT) that results in a toxic gain of function in the mutant huntingtin protein (mHTT). Reducing the expression of mHTT is therefore an attractive therapy for HD. However, wild...

  18. Potent and selective antisense oligonucleotides targeting single-nucleotide polymorphisms in the Huntington disease gene / allele-specific silencing of mutant huntingtin

    DEFF Research Database (Denmark)

    Carroll, Jeffrey B; Warby, Simon C; Southwell, Amber L

    2011-01-01

    Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by CAG-expansion in the huntingtin gene (HTT) that results in a toxic gain of function in the mutant huntingtin protein (mHTT). Reducing the expression of mHTT is therefore an attractive therapy for HD. However, wi...

  19. Mutant LRP6 Impairs Endothelial Cell Functions Associated with Familial Normolipidemic Coronary Artery Disease

    Directory of Open Access Journals (Sweden)

    Jian Guo

    2016-07-01

    Full Text Available Mutations in the genes low-density lipoprotein (LDL receptor-related protein-6 (LRP6 and myocyte enhancer factor 2A (MEF2A were reported in families with coronary artery disease (CAD. We intend to determine the mutational spectrum of these genes among hyperlipidemic and normolipidemic CAD families. Forty probands with early-onset CAD were recruited from 19 hyperlipidemic and 21 normolipidemic Chinese families. We sequenced all exons and intron-exon boundaries of LRP6 and MEF2A, and found a novel heterozygous variant in LRP6 from a proband with normolipidemic CAD. This variant led to a substitution of histidine to tyrosine (Y418H in an evolutionarily conserved domain YWTD in exon 6 and was not found in 1025 unrelated healthy individuals. Co-segregated with CAD in the affected family, LRP6Y418H significantly debilitated the Wnt3a-associated signaling pathway, suppressed endothelial cell proliferation and migration, and decreased anti-apoptotic ability. However, it exhibited no influences on low-density lipoprotein cholesterol uptake. Thus, mutation Y418H in LRP6 likely contributes to normolipidemic familial CAD via impairing endothelial cell functions and weakening the Wnt3a signaling pathway.

  20. Inoculation of Swine with Foot-and-Mouth Disease SAP-Mutant Virus Induces Early Protection against Disease

    OpenAIRE

    Díaz-San Segundo, Fayna; Weiss, Marcelo; Pérez-Martín, Eva; Dias, Camila C.; Grubman, Marvin J.; de los Santos, Teresa

    2012-01-01

    Foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) cleaves itself from the viral polyprotein and cleaves the translation initiation factor eIF4G. As a result, host cell translation is inhibited, affecting the host innate immune response. We have demonstrated that Lpro is also associated with degradation of nuclear factor κB (NF-κB), a process that requires Lpro nuclear localization. Additionally, we reported that disruption of a conserved protein domain within the Lpro coding sequen...

  1. Lack of interleukin-1 type 1 receptor enhances the accumulation of mutant huntingtin in the striatum and exacerbates the neurological phenotypes of Huntington's disease mice

    Directory of Open Access Journals (Sweden)

    Wang Chuan-En

    2010-11-01

    Full Text Available Abstract Huntington's disease results from expansion of a glutamine repeat (>36 glutamines in the N-terminal region of huntingtin (htt and is characterized by preferential neurodegeneration in the striatum of the brain. N171-82Q mice that express N-terminal 171 amino acids of htt with an 82-glutamine repeat show severe neurological phenotypes and die early, suggesting that N-terminal mutant htt is pathogenic. In addition, various cellular factors and genetic modifiers are found to modulate the cytotoxicity of mutant htt. Understanding the contribution of these factors to HD pathogenesis will help identify therapeutics for this disease. To investigate the role of interleukin type 1 (IL-1, a cytokine that has been implicated in various neurological diseases, in HD neurological symptoms, we crossed N171-82Q mice to type I IL-1 receptor (IL-1RI knockout mice. Mice lacking IL-1RI and expressing N171-82Q show more severe neurological symptoms than N171-82Q or IL-1RI knockout mice, suggesting that lack of IL-1RI can promote the neuronal toxicity of mutant htt. Lack of IL-1RI also increases the accumulation of transgenic mutant htt in the striatum in N171-82Q mice. Since IL-1RI signaling mediates both toxic and protective effects on neurons, its basal function and protective effects may be important for preventing the neuropathology seen in HD.

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

    Directory of Open Access Journals (Sweden)

    Moritz D. Brandt

    2017-08-01

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

  3. Ga-68 Somatostatin Receptor PET/CT in von Hippel-Lindau Disease

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Jong-Ryool; Min, Jung-Joon [Chonnam National Univ. Hwasun Hospital, Hwasun (Korea, Republic of); Kulkarui, Harshad; Carreras, Cecilia; Schalch, Georg; Baum, Richard P. [Nuclear Medicine and Center for PET/CT, Zentralk Bad Berka, Bad Verka (Germany)

    2012-06-15

    Von Hippel-Lindau (VHL) disease is a dominantly inherited familial cancer syndrome with a variety of benign and malignant tumors such as retinal and central nervous system hemangioblastomas, endolymphatic sac tumors, renalcysts and tumors, pancreatic cysts and tumors, pheochromo-cytomas, and epididymal cystadenomas. Cross-sectional mo-dalities (computed tomography and magnetic resonance imaging) as well as ultrasound play a major role in the initial evaluation and follow-up of the various manifestations of VHL disease. Ga-68-labeled somatostatin receptor analogs already have a significant role in the diagnosis, staging, and therapy management of neuroendocrine neoplasms and neural crest tumors. Herein, we report a case presenting a variety of malignancies in VHL and showing the usefulness of Ga-68 somatostatin receptor PET/CT as a one-stop-shop imaging modality in the management of VHL disease.

  4. A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase

    DEFF Research Database (Denmark)

    Maile, C A; Hingst, Janne Rasmuss; Mahalingan, K K

    2017-01-01

    had significantly higher glycogen content than control horse muscle despite no difference in GS expression. GS activity was significantly higher in muscle from homozygous mutants than from heterozygote and control horses, in the absence and presence of the allosteric regulator, glucose 6 phosphate (G6......P). Muscle from homozygous mutant horses also had significantly increased GS phosphorylation at sites 2+2a and significantly higher AMPKα1 (an upstream kinase) expression than controls, likely reflecting a physiological attempt to reduce GS enzyme activity. Recombinant mutant GS was highly active...

  5. Inactivation of Vhl in Osteochondral Progenitor Cells Causes High Bone Mass Phenotype and Protects Against Age-Related Bone Loss in Adult Mice

    Science.gov (United States)

    Weng, Tujun; Xie, Yangli; Huang, Junlan; Luo, Fengtao; Yi, Lingxian; He, Qifen; Chen, Di; Chen, Lin

    2014-01-01

    Previous studies have shown that disruption of von Hippel–Lindau gene (Vhl) coincides with activation of hypoxia-inducible factor α (HIFα) signaling in bone cells and plays an important role in bone development, homeostasis, and regeneration. It is known that activation of HIF1α signaling in mature osteoblasts is central to the coupling between angiogenesis and bone formation. However, the precise mechanisms responsible for the coupling between skeletal angiogenesis and osteogenesis during bone remodeling are only partially elucidated. To evaluate the role of Vhl in bone homeostasis and the coupling between vascular physiology and bone, we generated mice lacking Vhl in osteochondral progenitor cells (referred to as Vhl cKO mice) at postnatal and adult stages in a tamoxifen-inducible manner and changes in skeletal morphology were assessed by micro–computed tomography (µCT), histology, and bone histomorphometry. We found that mice with inactivation of Vhl in osteochondral progenitor cells at the postnatal stage largely phenocopied that of mice lacking Vhl in mature osteoblasts, developing striking and progressive accumulation of cancellous bone with increased microvascular density and bone formation. These were accompanied with a significant increase in osteoblast proliferation, upregulation of differentiation marker Runx2 and osteocalcin, and elevated expression of vascular endothelial growth factor (VEGF) and phosphorylation of Smad1/5/8. In addition, we found that Vhl deletion in osteochondral progenitor cells in adult bone protects mice from aging-induced bone loss. Our data suggest that the VHL-mediated signaling in osteochondral progenitor cells plays a critical role in bone remodeling at postnatal/adult stages through coupling osteogenesis and angiogenesis. © 2014 American Society for Bone and Mineral Research. PMID:23999831

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

    Directory of Open Access Journals (Sweden)

    Zeng YX

    2016-04-01

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

  7. 68Ga-DOTA-NOC PET/CT detects somatostatin receptors expression in von hippel-lindau cerebellar disease.

    Science.gov (United States)

    Ambrosini, Valentina; Campana, Davide; Allegri, Vincenzo; Opocher, Giuseppe; Fanti, Stefano

    2011-01-01

    A case of Von-Hippel Lindau (VHL) disease has been studied using 68Ga-DOTA-NOC PET/CT. PET/CT demonstrated the presence of somatostatin receptors within 2 focal areas in the cerebellum corresponding to the lesions detected by MRI. Considering the heterogeneous lesions localizations in VHL disease, PET/CT may be a useful imaging modality for diagnosing lesions of central nervous system and neuroendocrine lesions and for direct demonstration of somatostatin receptors for targeted treatment.

  8. Characterization of a rat model of Huntington's disease based on targeted expression of mutant huntingtin in the forebrain using adeno-associated viral vectors.

    Science.gov (United States)

    Gabery, Sanaz; Sajjad, Muhammad U; Hult, Sofia; Soylu, Rana; Kirik, Deniz; Petersén, Åsa

    2012-09-01

    Huntington's disease (HD) is a fatal neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (htt) gene. Neuropathology is most severe in the striatum and cerebral cortex. As mutant htt is ubiquitously expressed, it has not been possible to establish clear structure-to-function relationships for the clinical aspects. In the present study, we have injected recombinant adeno-associated viral vectors of serotype 5 (rAAV5) expressing an 853-amino-acid fragment of htt with either 79 (mutant) or 18 (wild-type) glutamines (Q) in the dorsal striatum of neonatal rats to achieve expression of htt in the forebrain. Rats were followed for 6 months and compared with control rats. Neuropathological assessment showed long-term expression of the green fluorescent protein (GFP) transgene (used as a marker protein) and accumulation of htt inclusions in the cerebral cortex with the rAAV5-htt-79Q vectors. We estimated that around 10% of NeuN-positive cells in the cerebral cortex and 2% of DARPP-32 neurons in the striatum were targeted with the GFP-expressing vector. Formation of intracellular htt inclusions was not associated with neuronal loss, gliosis or microglia activation and did not lead to altered motor activity or changes in body weight. However, the same mutant htt vector caused orexin loss in the hypothalamus - another area known to be affected in HD. In conclusion, our results demonstrate that widespread forebrain expression of mutant htt can be achieved using rAAV5-vectors and suggest that this technique can be further explored to study region-specific effects of mutant htt or other disease-causing genes in the brain. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  9. Rare presentation of familial paraganglioma without evidence of mutation in the SDH, RET and VHL genes: towards further genetic heterogeneity.

    Science.gov (United States)

    Persu, Alexandre; Amyere, Mustapha; Gutierrez-Roelens, Ilse; Rustin, Pierre; Sempoux, Christine; Lecouvet, Frédéric E; Van Beers, Bernard E; Horsmans, Yves; De Plaen, Jean-François; MarcHamoir; Vikkula, Miikka

    2009-01-01

    Mutations in genes encoding succinate dehydrogenase and its anchoring subunits (SDH genes) are at the origin of hereditary head and neck paraganglioma (PGL) and a subset of apparently sporadic pheochromocytoma. We describe a family including three patients harbouring bilateral head and neck PGL diagnosed before 25 years of age. Multiple hypervascular hepatic lesions were subsequently discovered in two of them. In both, liver biopsy confirmed the diagnosis of PGL. In addition, in one patient, MRI disclosed multiple target-like lesions of the spine, highly suggestive of metastatic PGL. Family history was compatible with autosomal dominant inheritance with possible maternal imprinting. Combined single-strand conformation polymorphism and heteroduplex analysis followed by sequencing did not show any mutation of the coding parts of SDHB, SDHC, SDHD, RET or VHL genes. Screening of copy number alterations and loss of heterozygosity in the three affected family members showed no deletion or amplification of the SDH, RET and VHL genes. Furthermore, succinate dehydrogenase activity measured in a liver PGL sample was not significantly decreased in the affected patient as compared with controls, underscoring the exclusion of the SDH genes. To our knowledge, this is the first reported family of hereditary head and neck PGL with metastatic dissemination in the liver and the spine. A large body of evidence supports the absence of mutations in SDH, RET and VHL genes, which suggests the existence of a yet unknown gene at the origin of this particular form of familial PGL.

  10. Identification of Somatic Mutations in the von Hippel–Lindau (VHL Gene in a Patient With Renal Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Wen-Chung Wang

    2009-11-01

    Full Text Available One of the known causal molecular events in renal cell carcinoma is somatic mutation in the von Hippel–Lindau (VHL gene. Our study describes a 51-year-old Taiwanese man who had bilateral renal cell carcinoma. The patient underwent radical nephrectomy without postoperative chemotherapy or radiotherapy, and is still alive after renal transplantation without tumor recurrence after > 5 years. To clarify his predisposition for bilateral tumors, we performed molecular genetic analysis of the VHL gene in this study. Polymerase chain reaction–single-strand conformation polymorphism and direct sequencing were performed on DNA of blood samples and paraffin-embedded tumor specimens from this patient. DNA from peripheral blood lymphocytes tested negative for germline mutations. However, there were two heterozygous alleles in the promoter and 3′ untranslated regions of this gene. Nonetheless, the DNA from his tumors showed loss of heterozygosity (LOH in these two loci. In addition to the LOH, we identified some different somatic mutations in his tumor tissues: C287T and G460A in the right-sided tumor, and G244A and G390A in the left-sided tumor. The possible roles of these genetic polymorphisms and point mutations in his renal tumorigenesis are discussed. This report provides new insights into renal cell carcinoma that result from VHL gene alterations in Taiwan.

  11. The Arabidopsis defence response mutant esa1 as a tool to discover novel resistance traits against Fusarium diseases

    NARCIS (Netherlands)

    Hemelrijck, van W.; Wouters, P.F.J.; Brouwer, M.; Windelinckx, A.; Goderis, I.J.W.M.; Bolle, De M.F.C.; Thomma, B.P.H.J.; Cammue, B.P.A.; Delauré, S.L.

    2006-01-01

    The Arabidopsis thaliana mutant esa1 was previously shown to exhibit enhanced susceptibility to the necrotrophic fungal pathogens Alternaria brassicicola, Botrytis cinerea and Plectosphaerella cucumerina. In this work, we tried to elaborate on this susceptibility by investigating whether the esa1

  12. Polycystic kidney disease in the medaka (Oryzias latipes pc mutant caused by a mutation in the Gli-Similar3 (glis3 gene.

    Directory of Open Access Journals (Sweden)

    Hisashi Hashimoto

    Full Text Available Polycystic kidney disease (PKD is a common hereditary disease in humans. Recent studies have shown an increasing number of ciliary genes that are involved in the pathogenesis of PKD. In this study, the Gli-similar3 (glis3 gene was identified as the causal gene of the medaka pc mutant, a model of PKD. In the pc mutant, a transposon was found to be inserted into the fourth intron of the pc/glis3 gene, causing aberrant splicing of the pc/glis3 mRNA and thus a putatively truncated protein with a defective zinc finger domain. pc/glis3 mRNA is expressed in the epithelial cells of the renal tubules and ducts of the pronephros and mesonephros, and also in the pancreas. Antisense oligonucleotide-mediated knockdown of pc/glis3 resulted in cyst formation in the pronephric tubules of medaka fry. Although three other glis family members, glis1a, glis1b and glis2, were found in the medaka genome, none were expressed in the embryonic or larval kidney. In the pc mutant, the urine flow rate in the pronephros was significantly reduced, which was considered to be a direct cause of renal cyst formation. The cilia on the surface of the renal tubular epithelium were significantly shorter in the pc mutant than in wild-type, suggesting that shortened cilia resulted in a decrease in driving force and, in turn, a reduction in urine flow rate. Most importantly, EGFP-tagged pc/glis3 protein localized in primary cilia as well as in the nucleus when expressed in mouse renal epithelial cells, indicating a strong connection between pc/glis3 and ciliary function. Unlike human patients with GLIS3 mutations, the medaka pc mutant shows none of the symptoms of a pancreatic phenotype, such as impaired insulin expression and/or diabetes, suggesting that the pc mutant may be suitable for use as a kidney-specific model for human GLIS3 patients.

  13. Breeding for blast-disease-resistant and high-yield Thai jasmine rice (Oryza sativa L. cv. KDML 105) mutants using low-energy ion beams

    Energy Technology Data Exchange (ETDEWEB)

    Mahadtanapuk, S. [School of Agriculture and Natural Resources, University of Phayao, Phayao 56000 (Thailand); Teraarusiri, W. [Central Laboratory, University of Phayao, Phayao 56000 (Thailand); Phanchaisri, B. [Science and Technology Research Institute, Chiang Mai University, Chiang Mai 50200 (Thailand); Yu, L.D., E-mail: yuld@frnf.science.cmu.ac.th [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand); Anuntalabhochai, S., E-mail: burinka@hotmail.com [Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand)

    2013-07-15

    Highlights: •N-ion beam bombarded Thai jasmine rice seeds to induce mutation. •Mutants with blast-disease resistance and high yield were screened. •Gene involved in the blast-disease resistance was analyzed. •The gene responsible for the resistance was linked to Spotted leaf protein 11. -- Abstract: Low-energy ion beam was applied on mutation induction for plant breeding of blast-disease-resistant Thai jasmine rice (Oryza sativa L. cv. KDML 105). Seeds of the wild-type rice were bombarded in vacuum by nitrogen ion beam at energy of 60–80 keV to a beam fluence range of 2 × 10{sup 16}–2 × 10{sup 17} ions/cm{sup 2}. The ion-bombarded rice seeds were grown in soil for 2 weeks as transplanted rice in plastic pots at 1 seedling/pot. The seedlings were then screened for blast resistance by Pyricularia grisea inoculation with 10{sup 6} spores/ml concentrations. The blast-resistant rice mutant was planted up to F6 generation with the consistent phenotypic variation. The high percentage of the blast-disease-resistant rice was analyzed with DNA fingerprint. The HAT-RAPD (high annealing temperature-random amplified polymorphic DNA) marker revealed the modified polymorphism fragment presenting in the mutant compared with wild type (KDML 105). The cDNA fingerprints were investigated and the polymorphism fragment was subcloned into pGEM-T easy vector and then sequenced. The sequence of this fragment was compared with those already contained in the database, and the fragment was found to be related to the Spotted leaf protein 11 (Spl11)

  14. In vitro hybridization and separation of hybrids of human adenylosuccinate lyase from wild-type and disease-associated mutant enzymes.

    Science.gov (United States)

    De Zoysa Ariyananda, Lushanti; Antonopoulos, Christina; Currier, Jenna; Colman, Roberta F

    2011-03-01

    Human adenylosuccinate lyase (ASL) deficiency is an inherited metabolic disease in which the majority of the patients are compound heterozygotes for the mutations that occur in the ASL gene. Starting with purified wild-type (WT) and single-mutant human ASL, we generated in vitro hybrids that mimic compound heterozygote ASL. For this study, we used His-tagged WT/non-His-tagged WT, His-tagged WT/non-His-tagged R396C, His-tagged WT/non-His-tagged R396H, His-tagged R194C/non-His-tagged R396C, and His-tagged L311V/non-His-tagged R396H enzyme pairs. We generated various hybrids by denaturing pairs of enzymes in 1 M guanidinium chloride and renaturing them by removing the denaturant. The hybrids were separated on a nickel-nitrilotriacetic acid-agarose column based on the number of His tags present in the enzyme tetramer. Analytical ultracentrifuge data indicate that the hybrids have predominant amounts of heterotetramers. Analysis of the V(max) values of the hybrids indicates that most of the subunits behave independently; however, the hybrid tetramers retain weak positive cooperativity, indicating that there is some interaction between the different subunit types. The interactions between WT and mutant subunits may be advantageous to the parents of ASL deficient patients, while the interactions between some mutant subunits may assist heterozygote ASL deficient patients.

  15. Alzheimer’s Disease Mutant Mice Exhibit Reduced Brain Tissue Stiffness Compared to Wild-type Mice in both Normoxia and following Intermittent Hypoxia Mimicking Sleep Apnea

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    Maria José Menal

    2018-01-01

    Full Text Available BackgroundEvidence from patients and animal models suggests that obstructive sleep apnea (OSA may increase the risk of Alzheimer’s disease (AD and that AD is associated with reduced brain tissue stiffness.AimTo investigate whether intermittent hypoxia (IH alters brain cortex tissue stiffness in AD mutant mice exposed to IH mimicking OSA.MethodsSix-eight month old (B6C3-Tg(APPswe,PSEN1dE985Dbo/J AD mutant mice and wild-type (WT littermates were subjected to IH (21% O2 40 s to 5% O2 20 s; 6 h/day or normoxia for 8 weeks. After euthanasia, the stiffness (E of 200-μm brain cortex slices was measured by atomic force microscopy.ResultsTwo-way ANOVA indicated significant cortical softening and weight increase in AD mice compared to WT littermates, but no significant effects of IH on cortical stiffness and weight were detected. In addition, reduced myelin was apparent in AD (vs. WT, but no significant differences emerged in the cortex extracellular matrix components laminin and glycosaminoglycans when comparing baseline AD and WT mice.ConclusionAD mutant mice exhibit reduced brain tissue stiffness following both normoxia and IH mimicking sleep apnea, and such differences are commensurate with increased edema and demyelination in AD.

  16. Glial innate immunity generated by non-aggregated alpha-synuclein in mouse: differences between wild-type and Parkinson's disease-linked mutants.

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    Cintia Roodveldt

    Full Text Available BACKGROUND: Parkinson's disease (PD is a progressive neurodegenerative disorder characterized pathologically by the presence in the brain of intracellular protein inclusions highly enriched in aggregated alpha-synuclein (α-Syn. Although it has been established that progression of the disease is accompanied by sustained activation of microglia, the underlying molecules and factors involved in these immune-triggered mechanisms remain largely unexplored. Lately, accumulating evidence has shown the presence of extracellular α-Syn both in its aggregated and monomeric forms in cerebrospinal fluid and blood plasma. However, the effect of extracellular α-Syn on cellular activation and immune mediators, as well as the impact of familial PD-linked α-Syn mutants on this stimulation, are still largely unknown. METHODS AND FINDINGS: In this work, we have compared the activation profiles of non-aggregated, extracellular wild-type and PD-linked mutant α-Syn variants on primary glial and microglial cell cultures. After stimulation of cells with α-Syn, we measured the release of Th1- and Th2- type cytokines as well as IP-10/CXCL10, RANTES/CCL5, MCP-1/CCL2 and MIP-1α/CCL3 chemokines. Contrary to what had been observed using cell lines or for the case of aggregated α-Syn, we found strong differences in the immune response generated by wild-type α-Syn and the familial PD mutants (A30P, E46K and A53T. CONCLUSIONS: These findings might contribute to explain the differences in the onset and progression of this highly debilitating disease, which could be of value in the development of rational approaches towards effective control of immune responses that are associated with PD.

  17. Ambroxol-induced rescue of defective glucocerebrosidase is associated with increased LIMP-2 and saposin C levels in GBA1 mutant Parkinson's disease cells.

    Science.gov (United States)

    Ambrosi, Giulia; Ghezzi, Cristina; Zangaglia, Roberta; Levandis, Giovanna; Pacchetti, Claudio; Blandini, Fabio

    2015-10-01

    Heterozygous mutations in GBA1 gene, encoding for lysosomal enzyme glucocerebrosidase (GCase), are a major risk factor for sporadic Parkinson's disease (PD). Defective GCase has been reported in fibroblasts of GBA1-mutant PD patients and pharmacological chaperone ambroxol has been shown to correct such defect. To further explore this issue, we investigated GCase and elements supporting GCase function and trafficking in fibroblasts from sporadic PD patients--with or without heterozygous GBA1 mutations--and healthy subjects, in basal conditions and following in vitro exposure to ambroxol. We assessed protein levels of GCase, lysosomal integral membrane protein-2 (LIMP-2), which mediates GCase trafficking to lysosomes, GCase endogenous activator saposin (Sap) C and parkin, which is involved in degradation of defective GCase. We also measured activities of GCase and cathepsin D, which cleaves Sap C from precursor prosaposin. GCase activity was reduced in fibroblasts from GBA1-mutant patients and ambroxol corrected this defect. Ambroxol increased cathepsin D activity, GCase and Sap C protein levels in all groups, while LIMP-2 levels were increased only in GBA1-mutant PD fibroblasts. Parkin levels were slightly increased only in the PD group without GBA1 mutations and were not significantly modified by ambroxol. Our study confirms that GCase activity is deficient in fibroblasts of GBA1-mutant PD patients and that ambroxol corrects this defect. The drug increased Sap C and LIMP-2 protein levels, without interfering with parkin. These results confirm that chemical chaperone ambroxol modulates lysosomal markers, further highlighting targets that may be exploited for innovative PD therapeutic strategies. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Von Hippel-Lindau disease associated with myasthenia gravis not related to thymoma

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    Paolo Pozzato

    2013-04-01

    Full Text Available BACKGROUND Von Hippel-Lindau disease (VHL is a rare autosomal dominant inherited disorder characterized by an increased risk of tumours in a number of locations (eyes, brain, adrenal gland, pancreas, liver, kidneys, or other areas of the body. It is caused by germline mutation in the VHL gene. The VHL gene is a tumour suppressor gene that has been identified on the short arm of chromosome 3. CASE REPORT We report a case of a 60 year-old female with the clinical diagnosis of VHL type 1 (cerebellar haemangioblastoma, pancreatic cysts with subsequent steatorrhoea, and bilateral renal carcinoma who developed weakness and fatigability of skeletal muscles, left lid ptosis, snarling expression and nasal timbre speech. Acetylcholine receptor antibodies were negative in serum, while the electrodiagnostic test demonstrated an alteration of neuromuscolar junction which was consistent with the diagnosis of myasthenia gravis. Contrast-enhanced TC scan of the anterior mediastinum was performed, which excluded thymus enlargement. VHL gene evaluation in this patient identified a new mutation (c279delC9 and polymorphism c291C>G. At present the patient still suffers from ataxia and dysmetria due to cerebellar involvement in VHL, while fatigue and lid ptosis improved after the treatment with oral pyridostigmine 60 mg tid. DISCUSSION AND CONCLUSIONS To our knowledge this is the first report of a case of VHL associated with myasthenia gravis without thymoma. A case of VHL associated with a form of myasthenia gravis related to thymoma has been recently reported. In our case the absence of acetylcholine receptor antibodies may suggest a genetic origin also for the myasthenia gravis.

  19. Anatomical distributional defects in mutant genes associated with dominant intermediate Charcot-Marie-Tooth disease type C in an adenovirus-mediated mouse model

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    SeoJin Lee

    2017-01-01

    Full Text Available Dominant intermediate Charcot-Marie-Tooth disease type C (DI-CMTC is a dominantly inherited neuropathy that has been classified primarily based on motor conduction velocity tests but is now known to involve axonal and demyelination features. DI-CMTC is linked to tyrosyl-tRNA synthetase (YARS-associated neuropathies, which are caused by E196K and G41R missense mutations and a single de novo deletion (153-156delVKQV. It is well-established that these YARS mutations induce neuronal dysfunction, morphological symptoms involving axonal degeneration, and impaired motor performance. The present study is the first to describe a novel mouse model of YARS-mutation-induced neuropathy involving a neuron-specific promoter with a deleted mitochondrial targeting sequence that inhibits the expression of YARS protein in the mitochondria. An adenovirus vector system and in vivo techniques were utilized to express YARS fusion proteins with a Flag-tag in the spinal cord, peripheral axons, and dorsal root ganglia. Following transfection of YARS-expressing viruses, the distributions of wild-type (WT YARS and E196K mutant proteins were compared in all expressed regions; G41R was not expressed. The proportion of Flag/green fluorescent protein (GFP double-positive signaling in the E196K mutant-type mice did not significantly differ from that of WT mice in dorsal root ganglion neurons. All adenovirus genes, and even the empty vector without the YARS gene, exhibited GFP-positive signaling in the ventral horn of the spinal cord because GFP in an adenovirus vector is driven by a cytomegalovirus promoter. The present study demonstrated that anatomical differences in tissue can lead to dissimilar expressions of YARS genes. Thus, use of this novel animal model will provide data regarding distributional defects between mutant and WT genes in neurons, the DI-CMTC phenotype, and potential treatment approaches for this disease.

  20. Haemolysis and perturbations in the systemic iron metabolism of suckling, copper-deficient mosaic mutant mice - an animal model of Menkes disease.

    Science.gov (United States)

    Lenartowicz, Małgorzata; Starzyński, Rafał R; Krzeptowski, Wojciech; Grzmil, Paweł; Bednarz, Aleksandra; Ogórek, Mateusz; Pierzchała, Olga; Staroń, Robert; Gajowiak, Anna; Lipiński, Paweł

    2014-01-01

    The biological interaction between copper and iron is best exemplified by the decreased activity of multicopper ferroxidases under conditions of copper deficiency that limits the availability of iron for erythropoiesis. However, little is known about how copper deficiency affects iron homeostasis through alteration of the activity of other copper-containing proteins, not directly connected with iron metabolism, such as superoxide dismutase 1 (SOD1). This antioxidant enzyme scavenges the superoxide anion, a reactive oxygen species contributing to the toxicity of iron via the Fenton reaction. Here, we analyzed changes in the systemic iron metabolism using an animal model of Menkes disease: copper-deficient mosaic mutant mice with dysfunction of the ATP7A copper transporter. We found that the erythrocytes of these mutants are copper-deficient, display decreased SOD1 activity/expression and have cell membrane abnormalities. In consequence, the mosaic mice show evidence of haemolysis accompanied by haptoglobin-dependent elimination of haemoglobin (Hb) from the circulation, as well as the induction of haem oxygenase 1 (HO1) in the liver and kidney. Moreover, the hepcidin-ferroportin regulatory axis is strongly affected in mosaic mice. These findings indicate that haemolysis is an additional pathogenic factor in a mouse model of Menkes diseases and provides evidence of a new indirect connection between copper deficiency and iron metabolism.

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

    Directory of Open Access Journals (Sweden)

    Melissa K Boles

    2009-12-01

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

  2. Breeding for blast-disease-resistant and high-yield Thai jasmine rice (Oryza sativa L. cv. KDML 105) mutants using low-energy ion beams

    Science.gov (United States)

    Mahadtanapuk, S.; Teraarusiri, W.; Phanchaisri, B.; Yu, L. D.; Anuntalabhochai, S.

    2013-07-01

    Low-energy ion beam was applied on mutation induction for plant breeding of blast-disease-resistant Thai jasmine rice (Oryza sativa L. cv. KDML 105). Seeds of the wild-type rice were bombarded in vacuum by nitrogen ion beam at energy of 60-80 keV to a beam fluence range of 2 × 1016-2 × 1017 ions/cm2. The ion-bombarded rice seeds were grown in soil for 2 weeks as transplanted rice in plastic pots at 1 seedling/pot. The seedlings were then screened for blast resistance by Pyricularia grisea inoculation with 106 spores/ml concentrations. The blast-resistant rice mutant was planted up to F6 generation with the consistent phenotypic variation. The high percentage of the blast-disease-resistant rice was analyzed with DNA fingerprint. The HAT-RAPD (high annealing temperature-random amplified polymorphic DNA) marker revealed the modified polymorphism fragment presenting in the mutant compared with wild type (KDML 105). The cDNA fingerprints were investigated and the polymorphism fragment was subcloned into pGEM-T easy vector and then sequenced. The sequence of this fragment was compared with those already contained in the database, and the fragment was found to be related to the Spotted leaf protein 11 (Spl11).

  3. Haemolysis and perturbations in the systemic iron metabolism of suckling, copper-deficient mosaic mutant mice - an animal model of Menkes disease.

    Directory of Open Access Journals (Sweden)

    Małgorzata Lenartowicz

    Full Text Available The biological interaction between copper and iron is best exemplified by the decreased activity of multicopper ferroxidases under conditions of copper deficiency that limits the availability of iron for erythropoiesis. However, little is known about how copper deficiency affects iron homeostasis through alteration of the activity of other copper-containing proteins, not directly connected with iron metabolism, such as superoxide dismutase 1 (SOD1. This antioxidant enzyme scavenges the superoxide anion, a reactive oxygen species contributing to the toxicity of iron via the Fenton reaction. Here, we analyzed changes in the systemic iron metabolism using an animal model of Menkes disease: copper-deficient mosaic mutant mice with dysfunction of the ATP7A copper transporter. We found that the erythrocytes of these mutants are copper-deficient, display decreased SOD1 activity/expression and have cell membrane abnormalities. In consequence, the mosaic mice show evidence of haemolysis accompanied by haptoglobin-dependent elimination of haemoglobin (Hb from the circulation, as well as the induction of haem oxygenase 1 (HO1 in the liver and kidney. Moreover, the hepcidin-ferroportin regulatory axis is strongly affected in mosaic mice. These findings indicate that haemolysis is an additional pathogenic factor in a mouse model of Menkes diseases and provides evidence of a new indirect connection between copper deficiency and iron metabolism.

  4. Connexin mutants and cataracts

    Directory of Open Access Journals (Sweden)

    Eric C Beyer

    2013-04-01

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

  5. Pathological and Clinical Features and Management of Central Nervous System Hemangioblastomas in von Hippel-Lindau Disease

    Directory of Open Access Journals (Sweden)

    Hiroshi Kanno

    2014-08-01

    Full Text Available Central nervous system (CNS hemangioblastoma is the most common manifestation of von Hippel-Lindau (VHL disease. It is found in 70-80% of VHL patients. Hemangioblastoma is a rare form of benign vascular tumor of the CNS, accounting for 2.0% of CNS tumors. It can occur sporadically or as a familial syndrome. CNS hemangioblastomas are typically located in the posterior fossa and the spinal cord. VHL patients usually develop a CNS hemangioblastoma at an early age. Therefore, they require a special routine for diagnosis, treatment and follow-up. The surgical management of symptomatic tumors depends on many factors such as symptom, location, multiplicity, and progression of the tumor. The management of asymptomatic tumors in VHL patients is controversial since CNS hemangioblastomas grow with intermittent quiescent and rapid-growth phases. Preoperative embolization of large solid hemangioblastomas prevents perioperative hemorrhage but is not necessary in every case. Radiotherapy should be reserved for inoperable tumors. Because of complexities of VHL, a better understanding of the pathological and clinical features of hemangioblastoma in VHL is essential for its proper management.

  6. Mutant copper-zinc superoxide dismutase (SOD1) induces protein secretion pathway alterations and exosome release in astrocytes: implications for disease spreading and motor neuron pathology in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Basso, Manuela; Pozzi, Silvia; Tortarolo, Massimo; Fiordaliso, Fabio; Bisighini, Cinzia; Pasetto, Laura; Spaltro, Gabriella; Lidonnici, Dario; Gensano, Francesco; Battaglia, Elisa; Bendotti, Caterina; Bonetto, Valentina

    2013-05-31

    Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread.

  7. A novel humanized mouse model of Huntington disease for preclinical development of therapeutics targeting mutant huntingtin alleles

    DEFF Research Database (Denmark)

    Southwell, Amber L; Skotte, Niels H; Villanueva, Erika B

    2017-01-01

    Huntington disease (HD) is a neurodegenerative disease caused by a mutation in the huntingtin (HTT) gene. HTT is a large protein, interacts with many partners and is involved in many cellular pathways, which are perturbed in HD. Therapies targeting HTT directly are likely to provide the most global...

  8. Chaperone therapy for neuronopathic lysosomal diseases: competitive inhibitors as chemical chaperones for enhancement of mutant enzyme activities.

    Science.gov (United States)

    Suzuki, Yoshiyuki; Ogawa, Seiichiro; Sakakibara, Yasubumi

    2009-05-26

    Chaperone therapy is a newly developed molecular approach to lysosomal diseases, a group of human genetic diseases causing severe brain damage. We found two valienamine derivatives, N-octyl-4-epi-beta-valienamine (NOEV) and N-octyl-beta-valienamine (NOV), as promising therapeutic agents for human beta-galactosidase deficiency disorders (mainly G(M1)-gangliosidosis) and beta-glucosidase deficiency disorders (Gaucher disease), respectively. We briefly reviewed the historical background of research in carbasugar glycosidase inhibitors. Originally NOEV and NOV had been discovered as competitive inhibitors, and then their paradoxical bioactivities as chaperones were confirmed in cultured fibroblasts from patients with these disorders. Subsequently G(M1)-gangliosidosis model mice were developed and useful for experimental studies. Orally administered NOEV entered the brain through the blood-brain barrier, enhanced beta-galactosidase activity, reduced substrate storage, and improved neurological deterioration clinically. Furthermore, we executed computational analysis for prediction of molecular interactions between beta-galactosidase and NOEV. Some preliminary results of computational analysis of molecular interaction mechanism are presented in this article. NOV also showed the chaperone effect toward several beta-glucosidase gene mutations in Gaucher disease. We hope chaperone therapy will become available for some patients with G(M1)-gangliosidosis, Gaucher disease, and potentially other lysosomal storage diseases with central nervous system involvement.

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

    Science.gov (United States)

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

    2002-12-10

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

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

    Science.gov (United States)

    Dasgupta, Nupur; Xu, You-Hai; Oh, Sunghee; Sun, Ying; Jia, Li; Keddache, Mehdi; Grabowski, Gregory A

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Nupur Dasgupta

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

  12. Non-Mendelian transmission in dentatorubral-pallidolysian atrophy and Machado-Joseph disease: The mutant allele is preferentially transmitted in male meiosis

    Energy Technology Data Exchange (ETDEWEB)

    Ikeuchi, Takeshi; Igarashi, Shuichi; Takiyama, Yoshihisa; Onodera, Osamu [Niigata Univ. (Japan)] [and others

    1996-04-01

    Autosomal dominant dentatorubral-pallidoluysian atrophy (DRPLA) and Machado-Joseph disease (MJD) are neurodegenerative disorders caused by CAG trinucleotide repeat expansions. An inverse correlation of age at onset with the length of the expanded CAG trinucleotide repeats has been demonstrated, and the intergenerational instability of the length of the CAG trinucleotide repeats, which is more prominent in paternal than in maternal transmissions, has been shown to underlie the basic mechanisms of anticipation in DRPLA and MJD. Our previous observations on DRPLA and MJD pedigrees, as well as a review of the literature, have suggested that the numbers of affected offspring exceed those of unaffected offspring, which is difficult to explain by the Mendelian principle of random segregation of alleles. In the present study, we analyzed the segregation patterns in 211 transmissions in 24 DRPLA pedigrees and 80 transmissions in 7 MJD pedigrees, with the diagnoses confirmed by molecular testing. Significant distortions in favor of transmission of the mutant alleles were found in male meiosis, where the mutant alleles were transmitted to 62% of all offspring in DRPLA ({chi}{sup 2} = 7.69; P < .01) and 73% in MJD ({chi}{sup 2} = 6.82; P < .01). The results were consistent with meiotic drive in DRPLA and MJD. Since more prominent meiotic instability of the length of the CAG trinucleotide repeats is observed in male meiosis than in female meiosis and meiotic drive is observed only in male meiosis, these results raise the possibility that a common molecular mechanism underlies the meiotic drive and the meiotic instability in male meiosis. 23 refs., 1 tab.

  13. The 'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring '2A-like' sequences.

    Science.gov (United States)

    Donnelly, M L; Hughes, L E; Luke, G; Mendoza, H; ten Dam, E; Gani, D; Ryan, M D

    2001-05-01

    The 2A/2B cleavage of aphtho- and cardiovirus 2A polyproteins is mediated by their 2A proteins 'cleaving' at their own C termini. We have analysed this activity using artificial reporter polyprotein systems comprising green fluorescent protein (GFP) linked via foot-and-mouth disease virus (FMDV) 2A to beta-glucuronidase (GUS) -- forming a single, long, open reading frame. Analysis of the distribution of radiolabel showed a high proportion of the in vitro translation products (approximately 90%) were in the form of the 'cleavage' products GUS and [GFP2A]. Alternative models have been proposed to account for the 'cleavage' activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect. To investigate the mechanism of this cleavage event constructs encoding site-directed mutant and naturally occurring '2A-like' sequences were used to program in vitro translation systems and the gel profiles analysed. Analysis of site-directed mutant 2A sequences showed that 'cleavage' occurred in constructs in which all the candidate nucleophilic residues were substituted -- with the exception of aspartate-12. This residue is not, however, conserved amongst all functional '2A-like' sequences. '2A-like' sequences were identified within insect virus polyproteins, the NS34 protein of type C rotaviruses, repeated sequences in Trypanosoma spp. and a eubacterial alpha-glucosiduronasesequence(Thermatoga maritima aguA). All of the 2A-like sequences analysed were active (to various extents), other than the eubacterial alpha-glucosiduronase 2A-like sequence. This method of control of protein biogenesis may well not, therefore, be confined to members of the PICORNAVIRIDAE: Taken together, these data provide additional evidence that neither FMDV 2A nor '2A-like' sequences are autoproteolytic elements.

  14. Resistance to cutaneous graft-vs.-host disease is not induced in T cell receptor delta gene-mutant mice

    OpenAIRE

    1996-01-01

    The function of murine dendritic epidermal cells (dEC) remains largely speculative, probably because of the lack of a suitable in vivo model, although previous studies suggest that gamma/delta+ dEC may have originally evolved to serve as a self-protection mechanism(s). Our previous study demonstrated that the epidermis of mice that had spontaneously recovered from cutaneous graft-vs-host disease (GVHD) induced by local injection of CD4+ autoreactive T cells contained unexpectedly large number...

  15. Characterization of Marek's disease virus insertion and deletion mutants that lack US1 (ICP22 homolog), US10, and/or US2 and neighboring short-component open reading frames.

    Science.gov (United States)

    Parcells, M S; Anderson, A S; Cantello, J L; Morgan, R W

    1994-01-01

    We report the characterization of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome. A deletion mutant (GA delta 4.8lac) lacks 4.8 kbp of US region DNA, the deleted segment having been replaced by the lacZ gene of Escherichia coli. This deletion results in the loss of the MDV-encoded US1, US10, and US2 homologs of herpes simplex virus type 1, as well as three putative MDV-specific genes, Sorf1, Sorf2, and Sorf3. Two mutants containing lacZ insertions in the US1 and US10 genes have been constructed, and we have previously reported a US2lac insertion mutant (J. L. Cantello, A. S. Anderson, A. Francesconi, and R. W. Morgan, J. Virol. 65:1584-1588, 1991). The isolation of these mutants indicates that the relevant genes are not required for growth of MDV in chicken embryo fibroblasts. The mutants had early growth kinetics indistinguishable from those of their parent viruses; however, 5 to 7 days after being plated, the US1 insertion mutant (US1lac) and the GA delta 4.8lac deletion mutant showed a 5- to 10-fold decrease in virus growth. This decrease in virus accumulation correlated with a 30 to 50% decrease in plaquing efficiency when these viruses were plated onto established versus fresh chicken embryo fibroblast monolayers compared with a 10 to 15% decrease seen for the parent viruses and for the US10lac or US2lac insertion mutants. Finally, GA delta 4.8lac could be reisolated from chickens, indicating that the deleted genes are not required for the infection of chickens following intra-abdominal inoculation of an attenuated serotype 1 MDV. Images PMID:7966617

  16. Potent and selective antisense oligonucleotides targeting single-nucleotide polymorphisms in the Huntington disease gene / allele-specific silencing of mutant huntingtin.

    Science.gov (United States)

    Carroll, Jeffrey B; Warby, Simon C; Southwell, Amber L; Doty, Crystal N; Greenlee, Sarah; Skotte, Niels; Hung, Gene; Bennett, C Frank; Freier, Susan M; Hayden, Michael R

    2011-12-01

    Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by CAG-expansion in the huntingtin gene (HTT) that results in a toxic gain of function in the mutant huntingtin protein (mHTT). Reducing the expression of mHTT is therefore an attractive therapy for HD. However, wild-type HTT protein is essential for development and has critical roles in maintaining neuronal health. Therapies for HD that reduce wild-type HTT may therefore generate unintended negative consequences. We have identified single-nucleotide polymorphism (SNP) targets in the human HD population for the disease-specific targeting of the HTT gene. Using primary cells from patients with HD and the transgenic YAC18 and BACHD mouse lines, we developed antisense oligonucleotide (ASO) molecules that potently and selectively silence mHTT at both exonic and intronic SNP sites. Modification of these ASOs with S-constrained-ethyl (cET) motifs significantly improves potency while maintaining allele selectively in vitro. The developed ASO is potent and selective for mHTT in vivo after delivery to the mouse brain. We demonstrate that potent and selective allele-specific knockdown of the mHTT protein can be achieved at therapeutically relevant SNP sites using ASOs in vitro and in vivo.

  17. Marker-Assisted Molecular Profiling, Deletion Mutant Analysis, and RNA-Seq Reveal a Disease Resistance Cluster Associated with Uromyces appendiculatus Infection in Common Bean Phaseolus vulgaris L.

    Science.gov (United States)

    Todd, Antonette R; Donofrio, Nicole; Sripathi, Venkateswara R; McClean, Phillip E; Lee, Rian K; Pastor-Corrales, Marcial; Kalavacharla, Venu Kal

    2017-05-23

    Common bean (Phaseolus vulgaris L.) is an important legume, useful for its high protein and dietary fiber. The fungal pathogen Uromyces appendiculatus (Pers.) Unger can cause major loss in susceptible varieties of the common bean. The Ur-3 locus provides race specific resistance to virulent strains or races of the bean rust pathogen along with Crg, (Complements resistance gene), which is required for Ur-3-mediated rust resistance. In this study, we inoculated two common bean genotypes (resistant "Sierra" and susceptible crg) with rust race 53 of U. appendiculatus, isolated leaf RNA at specific time points, and sequenced their transcriptomes. First, molecular markers were used to locate and identify a 250 kb deletion on chromosome 10 in mutant crg (which carries a deletion at the Crg locus). Next, we identified differential expression of several disease resistance genes between Mock Inoculated (MI) and Inoculated (I) samples of "Sierra" leaf RNA within the 250 kb delineated region. Both marker assisted molecular profiling and RNA-seq were used to identify possible transcriptomic locations of interest regarding the resistance in the common bean to race 53. Identification of differential expression among samples in disease resistance clusters in the bean genome may elucidate significant genes underlying rust resistance. Along with preserving favorable traits in the crop, the current research may also aid in global sustainability of food stocks necessary for many populations.

  18. Pulmonary artery pressure and iron deficiency in patients with upregulation of hypoxia sensing due to homozygous VHL(R200W) mutation (Chuvash polycythemia).

    Science.gov (United States)

    Sable, Craig A; Aliyu, Zakari Y; Dham, Niti; Nouraie, Mehdi; Sachdev, Vandana; Sidenko, Stanislav; Miasnikova, Galina Y; Polyakova, Lydia A; Sergueeva, Adelina I; Okhotin, Daniel J; Bushuev, Vladimir; Remaley, Alan T; Niu, Xiaomei; Castro, Oswaldo L; Gladwin, Mark T; Kato, Gregory J; Prchal, Josef T; Gordeuk, Victor R

    2012-02-01

    Patients with Chuvash polycythemia, (homozygosity for the R200W mutation in the von Hippel Lindau gene (VHL)), have elevated levels of hypoxia inducible factors HIF-1 and HIF-2, often become iron-deficient secondary to phlebotomy, and have elevated estimated pulmonary artery pressure by echocardiography. The objectives of this study were to provide a comprehensive echocardiographic assessment of cardiovascular physiology and to identify clinical, hematologic and cardiovascular risk factors for elevation of tricuspid regurgitation velocity in children and adults with Chuvash polycythemia. This cross-sectional observational study of 120 adult and pediatric VHL(R200W) homozygotes and 31 controls at outpatient facilities in Chuvashia, Russian Federation included echocardiography assessment of pulmonary artery pressure (tricuspid regurgitation velocity), cardiac volume, and systolic and diastolic function, as well as hematologic and clinical parameters. We determined the prevalence and risk factors for elevation of tricuspid regurgitation velocity in this population and its relationship to phlebotomy. The age-adjusted mean ± SE tricuspid regurgitation velocity was higher in VHL(R200W) homozygotes than controls with normal VHL alleles (2.5±0.03 vs. 2.3±0.05 m/sec, P=0.005). The age-adjusted left ventricular diastolic diameter (4.8±0.05 vs. 4.5±0.09 cm, P=0.005) and left atrial diameter (3.4±0.04 vs. 3.2±0.08 cm, P=0.011) were also greater in the VHL(R200W) homozygotes, consistent with increased blood volume, but the elevation in tricuspid regurgitation velocity persisted after adjustment for these variables. Among VHL(R200W) homozygotes, phlebotomy therapy was associated with lower serum ferritin concentration, and low ferritin independently predicted higher tricuspid regurgitation velocity (standardized beta=0.29; P=0.009). Children and adults with Chuvash polycythemia have higher estimated right ventricular systolic pressure, even after adjustment for

  19. Establishment of PSEN1 mutant induced pluripotent stem cell (iPSC line from an Alzheimer's disease (AD female patient

    Directory of Open Access Journals (Sweden)

    Csilla Nemes

    2016-07-01

    Full Text Available Peripheral blood mononuclear cells (PBMCs were collected from a clinically characterised, early onset Alzheimer's disease patient, carries a heterozygous, pathogenic single nucleotide variation (SNV in Presenilin1 (PSEN1 gene (NM_000021.3(PSEN1:c.265G>C; ClinVar ID: 21r027. The PMBCs were reprogrammed with the human OSKM transcription factors using the Sendai-virus delivery system. The transgene-free iPSC showed pluripotency verified by immunocytochemistry for pluripotency markers and differentiated spontaneously towards the 3 germ layers in vitro. Furthermore, the iPSC line showed normal karyotype. Our model might offer a good platform to study the pathomechanism of familial AD, to identify early biomarkers and also for drug testing.

  20. A novel humanized mouse model of Huntington disease for preclinical development of therapeutics targeting mutant huntingtin alleles.

    Science.gov (United States)

    Southwell, Amber L; Skotte, Niels H; Villanueva, Erika B; Østergaard, Michael E; Gu, Xiaofeng; Kordasiewicz, Holly B; Kay, Chris; Cheung, Daphne; Xie, Yuanyun; Waltl, Sabine; Dal Cengio, Louisa; Findlay-Black, Hailey; Doty, Crystal N; Petoukhov, Eugenia; Iworima, Diepiriye; Slama, Ramy; Ooi, Jolene; Pouladi, Mahmoud A; Yang, X William; Swayze, Eric E; Seth, Punit P; Hayden, Michael R

    2017-03-15

    Huntington disease (HD) is a neurodegenerative disease caused by a mutation in the huntingtin (HTT) gene. HTT is a large protein, interacts with many partners and is involved in many cellular pathways, which are perturbed in HD. Therapies targeting HTT directly are likely to provide the most global benefit. Thus there is a need for preclinical models of HD recapitulating human HTT genetics. We previously generated a humanized mouse model of HD, Hu97/18, by intercrossing BACHD and YAC18 mice with knockout of the endogenous mouse HD homolog (Hdh). Hu97/18 mice recapitulate the genetics of HD, having two full-length, genomic human HTT transgenes heterozygous for the HD mutation and polymorphisms associated with HD in populations of Caucasian descent. We have now generated a companion model, Hu128/21, by intercrossing YAC128 and BAC21 mice on the Hdh-/- background. Hu128/21 mice have two full-length, genomic human HTT transgenes heterozygous for the HD mutation and polymorphisms associated with HD in populations of East Asian descent and in a minority of patients from other ethnic groups. Hu128/21 mice display a wide variety of HD-like phenotypes that are similar to YAC128 mice. Additionally, both transgenes in Hu128/21 mice match the human HTT exon 1 reference sequence. Conversely, the BACHD transgene carries a floxed, synthetic exon 1 sequence. Hu128/21 mice will be useful for investigations of human HTT that cannot be addressed in Hu97/18 mice, for developing therapies targeted to exon 1, and for preclinical screening of personalized HTT lowering therapies in HD patients of East Asian descent. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. Clinical and molecular features of familial and sporadic cases of von Hippel-Lindau disease from Mexico.

    Science.gov (United States)

    Chacon-Camacho, Oscar F; Rodriguez-Dennen, Fernando; Camacho-Molina, Alejandra; Rasmussen, Astrid; Alonso-Vilatela, Elisa; Zenteno, Juan C

    2010-04-01

    von Hippel-Lindau disease (VHL) is an uncommon autosomal dominant condition predisposing to the development of tumours in a variety of body organs and caused by germline mutations in VHL, a tumour suppressor gene located on 3p. Up to 60% of VHL patients show ocular involvement with retinal hemangioblastoma being the most common observed lesion. In this study, we describe the clinical and genetic characteristics of two familial and one apparently non-familial case of VHL ascertained at our institution. Clinical evaluation included ophthalmologic examination and imaging exams for tumours identification; molecular analysis consisted of PCR amplification of the complete VHL gene coding sequence (three exons) and automated nucleotide sequencing. A total of eight affected subjects were demonstrated to carry a causative mutation in VHL. Affected subjects from family #1 had a c.245G > C change, predicting a p.R82P substitution, affected individuals from family #2 were shown to have a c.266T > C change, leading to a p.L89P missense substitution, whereas the apparently non-familial case had a c.298-299insA mutation. One subject from family #2 was a non-penetrant carrier. No ocular anomalies were found in two adult affected subjects carrying the p.L89P mutation. Considerable interfamilial and intrafamilial clinical variability as well as one instance of non penetrance were recorded in these VHL disease cases. Three different mutations were demonstrated, including the c.298-299insA one base insertion, which has been previously described in two unrelated families from our country. Although additional studies are needed, our data suggest that this insertion could be a 'founder' mutation.

  2. A case of von Hippel-Lindau disease with exudative maculopathy

    Directory of Open Access Journals (Sweden)

    Basel T Ba′arah

    2009-01-01

    Full Text Available Von Hippel-Lindau (VHL disease is a rare multisystem familial tumor syndrome of autosomal dominant inheritance. Hallmark lesions include retinal, cerebellum and spinal cord hemangioblastomas, renal cell carcinomas, adrenal pheochromocytomas, angiomatous or cystic lesions of the kidneys, pancreas, and epididymis. We report a case of VHL disease in a 26-year-old patient who presented with exudative macular edema. Ocular and systemic studies revealed the presence of retinal and central nervous system hemangioblastomas, adrenal pheochromocytoma, multiple pancreatic, and kidney cysts. The retinal angiomas were successfully treated with argon laser photocoagulation and cryotherapy.

  3. Human Papillomavirus 16 E6 Contributes HIF-1α Induced Warburg Effect by Attenuating the VHL-HIF-1α Interaction

    Directory of Open Access Journals (Sweden)

    Yi Guo

    2014-05-01

    Full Text Available Cervical cancer is still one of the leading causes of cancer deaths in women worldwide, especially in the developing countries. It is a major metabolic character of cancer cells to consume large quantities of glucose and derive more energy by glycolysis even in the presence of adequate oxygen, which is called Warburg effect that can be exaggerated by hypoxia. The high risk subtype HPV16 early oncoprotein E6 contributes host cell immortalization and transformation through interacting with a number of cellular factors. Hypoxia-inducible factor 1α (HIF-1α, a ubiquitously expressed transcriptional regulator involved in induction of numerous genes associated with angiogenesis and tumor growth, is highly increased by HPV E6. HIF-1α is a best-known target of the von Hippel-Lindau tumor suppressor (VHL as an E3 ligase for degradation. In the present work, we found that HPV16 E6 promotes hypoxia induced Warburg effect through hindering the association of HIF-1α and VHL. This disassociation attenuates VHL-mediated HIF-1α ubiquitination and causes HIF-1α accumulation. These results suggest that oncoprotein E6 plays a major role in the regulation of Warburg effect and can be a valuable therapeutic target for HPV-related cancer.

  4. Duplication of the VHL and IRAK2 genes in a patient with mental retardation/multiple congenital anomalies, epilepsy and ectomorphic habitus.

    Science.gov (United States)

    Chabchoub, E; Michils, G; Vermeesch, J R; De Cock, P; Lagae, L; Fryns, J P

    2010-01-01

    Partial 3p duplications are very rare. Often they are reported in translocations involving other chromosomes, whereas deletions encompassing the VHL gene in 3p25.3 predispose to Van-Hippel Lindau syndrome. We report here a paternally-inherited microduplication of 3p25.3 detected by array comparative genomic hybridisation (aCGH) in a 17 year-old male patient presenting with mental retardation and multiple congenital anomalies (MR/MCA), epilepsy and ectomorphic habitus. He has no tumour and there is no history of familial cancer. We refined the duplication by Multiplex Ligation-dependent Probe Amplification (MLPA) to a 251 kb region encompassing the VHL and IRAK2 genes. The duplication is likely to be causal. Interestingly, duplication of IRAK2 can cause epilepsy. Disruption of the GHRL gene can explain the ectomorphic habitus. To our knowledge, this is the smallest 3p duplication encompassing the VHL region. Its prognosis is unknown and a long-term follow-up is essential for an early diagnosis of malignancy.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zuleta, Amparo [Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago (Chile); Center for Molecular Studies of the Cell, Institute of Biomedical Sciences, University of Chile, Santiago (Chile); Vidal, Rene L. [Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago (Chile); Neurounion Biomedical Foundation, Santiago (Chile); Armentano, Donna; Parsons, Geoffrey [Department of Molecular Biology, Genzyme Corporation, 49 New York Avenue, Framingham, MA 01701 (United States); Hetz, Claudio, E-mail: chetz@med.uchile.cl [Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago (Chile); Center for Molecular Studies of the Cell, Institute of Biomedical Sciences, University of Chile, Santiago (Chile); Department of Immunology and Infectious Diseases, Harvard School of Public Health, 651 Huntington Av., Boston, MA 02446 (United States); Neurounion Biomedical Foundation, Santiago (Chile)

    2012-04-13

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

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

    Directory of Open Access Journals (Sweden)

    Frentzel Stefan

    2010-06-01

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

  7. Differential rescue of the renal and hepatic disease in an autosomal recessive polycystic kidney disease mouse mutant. A new model to study the liver lesion.

    OpenAIRE

    Yoder, B. K.; Richards, W. G.; Sommardahl, C.; Sweeney, W. E.; Michaud, E. J.; Wilkinson, J. E.; Avner, E. D.; Woychik, R. P.

    1997-01-01

    Autosomal recessive polycystic kidney disease (ARPKD) is characterized by biliary and renal lesions that produce significant morbidity and mortality. The biliary ductual ectasia and hepatic portal fibrosis associated with ARPKD have not been well studied even though such lesions markedly affect the clinical course of patients after renal replacement therapy such as dialysis or transplantation. Here we describe the generation of a new mouse model to study the hepatic lesions associated with po...

  8. von Hippel-Lindau disease

    DEFF Research Database (Denmark)

    Binderup, Marie Louise Mølgaard; Gimsing, Steen; Kosteljanetz, Michael

    2013-01-01

    Endolymphatic sac tumours (ELSTs) of the inner ear occur in 16% of patients with the hereditary tumor syndrome von Hippel-Lindau disease (vHL). ELSTs of all sizes can cause irreversible hearing loss which can, however, be prevented through early diagnosis and treatment. We aim to emphasize the ch...... the challenges of prophylactic ELST screening and to explore the role of audiometry in pre-symptomatic ELST screening....

  9. Saccharomyces cerevisiae aldolase mutants.

    OpenAIRE

    Lobo, Z

    1984-01-01

    Six mutants lacking the glycolytic enzyme fructose 1,6-bisphosphate aldolase have been isolated in the yeast Saccharomyces cerevisiae by inositol starvation. The mutants grown on gluconeogenic substrates, such as glycerol or alcohol, and show growth inhibition by glucose and related sugars. The mutations are recessive, segregate as one gene in crosses, and fall in a single complementation group. All of the mutants synthesize an antigen cross-reacting to the antibody raised against yeast aldol...

  10. A novel mutant allele of SSI2 confers a better balance between disease resistance and plant growth inhibition on Arabidopsis thaliana

    OpenAIRE

    Yang, Wei; Dong, Ran; Liu, Li; Hu, Zhubing; Li, Jing; Wang, Yong; Ding, Xinhua; Chu, Zhaohui

    2016-01-01

    Background Resistance and growth are opposing characteristics in plants. SA INSENSITIVITY OF npr1-5 (SSI2) encodes a stearoyl-ACP desaturase (S-ACP DES) that has previously been reported to simultaneously enhance resistance and repress growth. Results Here, we characterize ssi2-2, a novel mutant allele of SSI2 that has two amino acid substitutions. Compared with wild-type and two other mutants of SSI2, ssi2-2 showed intermediate phenotypes in growth size, punctate necrosis, resistance to the ...

  11. Cytogenetic abnormalities in tumors of patients with von Hippel-Lindau disease.

    Science.gov (United States)

    Jordan, D K; Patil, S R; Divelbiss, J E; Vemuganti, S; Headley, C; Waziri, M H; Gurll, N J

    1989-10-15

    Von Hippel-Lindau (VHL) disease is an autosomal dominant disorder that causes the development of benign and malignant tumors in several organ systems. Tumors causing significant morbidity include retinal angioma, cerebellar hemangioblastoma (CH), renal cell carcinoma (RCC), and pheochromocytoma (Pheo). Cytogenetic studies of tumors in VHL patients are rare. Cytogenetic findings in tumors from 12 patients with VHL disease, including four RCCs, three CHs, and five Pheos are presented. Three of the four RCC cases were abnormal. Monosomy 3 or a deletion of 3p was present in all three abnormal cases. Complete or partial trisomy of chromosome 5 was present in two cases. A deletion of 14q, trisomy 7, and a missing Y were each observed in one case. These findings indicate that a deletion of 3p may be a primary cytogenetic change in RCCs associated with VHL disease in addition to playing a role in sporadic RCC. Duplications of 5q and deletions of 14q may be important secondary changes in the progression of the malignant phenotype. No visible cytogenetic abnormalities were observed in the three CHs, or in four of the Pheos. One of the five Pheos was found to exhibit mosaic trisomy 7; its significance is unclear at the present time.

  12. VHL loss in renal cell carcinoma leads to up-regulation of CUB domain-containing protein 1 to stimulate PKC{delta}-driven migration.

    Science.gov (United States)

    Razorenova, Olga V; Finger, Elizabeth C; Colavitti, Renata; Chernikova, Sophia B; Boiko, Alexander D; Chan, Charles K F; Krieg, Adam; Bedogni, Barbara; LaGory, Edward; Weissman, Irving L; Broome-Powell, Marianne; Giaccia, Amato J

    2011-02-01

    A common genetic mutation found in clear cell renal cell carcinoma (CC-RCC) is the loss of the von Hippel-Lindau (VHL) gene, which results in stabilization of hypoxia-inducible factors (HIFs), and contributes to cancer progression and metastasis. CUB-domain-containing protein 1 (CDCP1) was shown to promote metastasis in scirrhous and lung adenocarcinomas as well as in prostate cancer. In this study, we established a molecular mechanism linking VHL loss to induction of the CDCP1 gene through the HIF-1/2 pathway in renal cancer. Also, we report that Fyn, which forms a complex with CDCP1 and mediates its signaling to PKCδ, is a HIF-1 target gene. Mechanistically, we found that CDCP1 specifically regulates phosphorylation of PKCδ, but not of focal adhesion kinase or Crk-associated substrate. Signal transduction from CDCP1 to PKCδ leads to its activation, increasing migration of CC-RCC. Furthermore, patient survival can be stratified by CDCP1 expression at the cell surface of the tumor. Taken together, our data indicates that CDCP1 protein might serve as a therapeutic target for CC-RCC.

  13. Uptake of genetic testing and long-term tumor surveillance in von Hippel-Lindau disease

    Directory of Open Access Journals (Sweden)

    Chávez Mireya

    2010-01-01

    Full Text Available Abstract Background von Hippel-Lindau (VHL disease is a hereditary cancer syndrome caused by germline mutations in the VHL gene. Patients have significant morbidity and mortality secondary to vascular tumors. Disease management is centered on tumor surveillance that allows early detection and treatment. Presymptomatic genetic testing is therefore recommended, including in at-risk children. Methods We tested 17 families (n = 109 individuals for VHL mutations including 43 children under the age of 18. Personalized genetic counseling was provided pre and post-test and the individuals undergoing presymptomatic testing filled out questionnaires gathering socio-demographic, psychological and psychiatric data. Mutation analysis was performed by direct sequencing of the VHL gene. Mutation-carriers were screened for VHL disease-related tumors and were offered follow-up annual examinations. Results Mutations were identified in 36 patients, 17 of whom were asymptomatic. In the initial screening, we identified at least one tumor in five of 17 previously asymptomatic individuals. At the end of five years, only 38.9% of the mutation-carriers continued participating in our tumor surveillance program. During this time, 14 mutation carriers developed a total of 32 new tumors, three of whom died of complications. Gender, education, income, marital status and religiosity were not found to be associated with adherence to the surveillance protocol. Follow-up adherence was also independent of pre-test depression, severity of disease, or number of affected family members. The only statistically significant predictor of adherence was being symptomatic at the time of testing (OR = 5; 95% CI 1.2 - 20.3; p = 0.02. Pre-test anxiety was more commonly observed in patients that discontinued follow-up (64.7% vs. 35.3%; p = 0.01. Conclusions The high initial uptake rate of genetic testing for VHL disease, including in minors, allowed the discontinuation of unnecessary screening

  14. Viral vector mediated expression of mutant huntingtin in the dorsal raphe produces disease-related neuropathology but not depressive-like behaviors in wildtype mice.

    Science.gov (United States)

    Pitzer, Mark; Lueras, Jordan; Warden, Anna; Weber, Sydney; McBride, Jodi

    2015-05-22

    Huntington׳s disease (HD) is a neurodegenerative disorder caused by a mutation in the HTT gene (mHTT) encoding the protein huntingtin. An expansion in the gene׳s CAG repeat length renders a misfolded, dysfunctional protein with an abnormally long glutamine (Q) stretch at the N terminus that often incorporates into inclusion bodies and leads to neurodegeneration in many regions of the brain. HD is characterized by motor and cognitive decline as well as mood disorders, with depression being particularly common. Approximately 40% of the HD population suffers from depressive symptoms. Because these symptoms often manifest a decade or more prior to the knowledge that the person is at risk for the disease, a portion of the early depression in HD appears to be a consequence of the pathology arising from expression of the mutant gene. While the depression in HD patients is often treated with serotonin agonists, there is scant experimental evidence that the depression in HD responds well to these serotonin treatments or in a similar manner to how non-HD depression tends to respond. Additionally, at very early sub-threshold depression levels, abnormal changes in several neuronal populations are already detectable in HD patients, suggesting that a variety of brain structures may be involved. Taken together, the serotonin system is a viable candidate. However, at present there is limited evidence of the precise nuclei or circuits that play a role in HD depression. With this in mind, the current study was designed to control for the widespread brain neuropathology that occurs in HD and in transgenic mouse models of HD and focuses specifically on the influence of the midbrain dorsal raphe nucleus (DRN). The DRN provides the majority of the serotonin to the forebrain and exhibits cell loss in non-HD depression. Therefore, we employed a viral vector delivery system to investigate whether the over-expression of mHTT in the DRN׳s ventral sub-nuclei alone is sufficient to produce

  15. Mutant HSPB1 causes loss of translational repression by binding to PCBP1, an RNA binding protein with a possible role in neurodegenerative disease

    National Research Council Canada - National Science Library

    Geuens, Thomas; De Winter, Vicky; Rajan, Nicholas; Achsel, Tilmann; Mateiu, Ligia; Almeida-Souza, Leonardo; Asselbergh, Bob; Bouhy, Delphine; Auer-Grumbach, Michaela; Bagni, Claudia; Timmerman, Vincent

    2017-01-01

    .... So far disease causing mutations in HSPB1 have been associated with neurodegenerative diseases such as distal hereditary motor neuropathy, Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis...

  16. Management of endolymphatic sac tumors: sporadic cases and von Hippel-Lindau disease.

    Science.gov (United States)

    Nevoux, Jérôme; Nowak, Catherine; Vellin, Jean-François; Lepajolec, Christine; Sterkers, Olivier; Richard, Stéphane; Bobin, Serge

    2014-06-01

    To analyze the difference between the endolymphatic sac tumors (ELSTs) in sporadic cases and in von Hippel-Lindau (VHL) disease. Retrospective case review in a tertiary referral center. Fourteen cases of ELST, occurring since 1998, were reviewed. We analyzed the initial symptoms, characteristics of the tumor, treatment, sequelae, and follow-up for each group. The ELSTs were sporadic in 6 cases and associated with VHL disease in 8 cases. The mean age at the time of the first surgery was 26 years (range, 12-41). All except two of the patients presented with a unilateral tumor. The initial symptoms were hearing loss (n = 9), tinnitus (n = 7), and/or vertigo (n = 5). Hearing loss was more prevalent in the sporadic cases. Preoperative arteriography was performed for 4 patients, with embolization performed for 1 patient. The size of the tumor was significantly larger in the sporadic cases (31.7 mm) than in the cases of VHL disease (19.3 mm). The surgical approach was more extensive in the sporadic cases. The surgeons found 2 types of tumors. Cystic tumors with massive bleeding invading the surrounding structures (the dura mater or jugular bulb) were more common in the sporadic cases. Fibrous tumors that infiltrate the bone and have moderate bleeding were more common in the cases associated with VHL disease.Two patients with small lesions were not operated on but were followed for 6 years without tumor growth. They died of metastasis from gastric and kidney cancer. Four recurrences occurred during the 14 years of follow-up. Four facial palsies and 8 cases of profound deafness were encountered postoperatively. Sporadic tumors are more aggressive than those associated with VHL disease. Complete surgical resection should be the goal of treatment. Preoperative angiography with embolization is recommended. In some cases, embolization may be impossible, and preoperative or postoperative radiotherapy should be discussed.

  17. Using the avian mutant talpid2 as a disease model for understanding the oral-facial phenotypes of oral-facial-digital syndrome

    Directory of Open Access Journals (Sweden)

    Elizabeth N. Schock

    2015-08-01

    Full Text Available Oral-facial-digital syndrome (OFD is a ciliopathy that is characterized by oral-facial abnormalities, including cleft lip and/or palate, broad nasal root, dental anomalies, micrognathia and glossal defects. In addition, these individuals have several other characteristic abnormalities that are typical of a ciliopathy, including polysyndactyly, polycystic kidneys and hypoplasia of the cerebellum. Recently, a subset of OFD cases in humans has been linked to mutations in the centriolar protein C2 Ca2+-dependent domain-containing 3 (C2CD3. Our previous work identified mutations in C2CD3 as the causal genetic lesion for the avian talpid2 mutant. Based on this common genetic etiology, we re-examined the talpid2 mutant biochemically and phenotypically for characteristics of OFD. We found that, as in OFD-affected individuals, protein-protein interactions between C2CD3 and oral-facial-digital syndrome 1 protein (OFD1 are reduced in talpid2 cells. Furthermore, we found that all common phenotypes were conserved between OFD-affected individuals and avian talpid2 mutants. In light of these findings, we utilized the talpid2 model to examine the cellular basis for the oral-facial phenotypes present in OFD. Specifically, we examined the development and differentiation of cranial neural crest cells (CNCCs when C2CD3-dependent ciliogenesis was impaired. Our studies suggest that although disruptions of C2CD3-dependent ciliogenesis do not affect CNCC specification or proliferation, CNCC migration and differentiation are disrupted. Loss of C2CD3-dependent ciliogenesis affects the dispersion and directional persistence of migratory CNCCs. Furthermore, loss of C2CD3-dependent ciliogenesis results in dysmorphic and enlarged CNCC-derived facial cartilages. Thus, these findings suggest that aberrant CNCC migration and differentiation could contribute to the pathology of oral-facial defects in OFD.

  18. The pharmacological chaperone AT2220 increases the specific activity and lysosomal delivery of mutant acid alpha-glucosidase, and promotes glycogen reduction in a transgenic mouse model of Pompe disease.

    Directory of Open Access Journals (Sweden)

    Richie Khanna

    Full Text Available Pompe disease is an inherited lysosomal storage disorder that results from a deficiency in acid α-glucosidase (GAA activity due to mutations in the GAA gene. Pompe disease is characterized by accumulation of lysosomal glycogen primarily in heart and skeletal muscles, which leads to progressive muscle weakness. We have shown previously that the small molecule pharmacological chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride binds and stabilizes wild-type as well as multiple mutant forms of GAA, and can lead to higher cellular levels of GAA. In this study, we examined the effect of AT2220 on mutant GAA, in vitro and in vivo, with a primary focus on the endoplasmic reticulum (ER-retained P545L mutant form of human GAA (P545L GAA. AT2220 increased the specific activity of P545L GAA toward both natural (glycogen and artificial substrates in vitro. Incubation with AT2220 also increased the ER export, lysosomal delivery, proteolytic processing, and stability of P545L GAA. In a new transgenic mouse model of Pompe disease that expresses human P545L on a Gaa knockout background (Tg/KO and is characterized by reduced GAA activity and elevated glycogen levels in disease-relevant tissues, daily oral administration of AT2220 for 4 weeks resulted in significant and dose-dependent increases in mature lysosomal GAA isoforms and GAA activity in heart and skeletal muscles. Importantly, oral administration of AT2220 also resulted in significant glycogen reduction in disease-relevant tissues. Compared to daily administration, less-frequent AT2220 administration, including repeated cycles of 4 or 5 days with AT2220 followed by 3 or 2 days without drug, respectively, resulted in even greater glycogen reductions. Collectively, these data indicate that AT2220 increases the specific activity, trafficking, and lysosomal stability of P545L GAA, leads to increased levels of mature GAA in lysosomes, and promotes glycogen reduction in situ. As

  19. The Pharmacological Chaperone AT2220 Increases the Specific Activity and Lysosomal Delivery of Mutant Acid Alpha-Glucosidase, and Promotes Glycogen Reduction in a Transgenic Mouse Model of Pompe Disease

    Science.gov (United States)

    Lun, Yi; Soska, Rebecca; Feng, Jessie; Dhulipala, Rohini; Frascella, Michelle; Garcia, Anadina; Pellegrino, Lee J.; Xu, Su; Brignol, Nastry; Toth, Matthew J.; Do, Hung V.; Lockhart, David J.; Wustman, Brandon A.; Valenzano, Kenneth J.

    2014-01-01

    Pompe disease is an inherited lysosomal storage disorder that results from a deficiency in acid α-glucosidase (GAA) activity due to mutations in the GAA gene. Pompe disease is characterized by accumulation of lysosomal glycogen primarily in heart and skeletal muscles, which leads to progressive muscle weakness. We have shown previously that the small molecule pharmacological chaperone AT2220 (1-deoxynojirimycin hydrochloride, duvoglustat hydrochloride) binds and stabilizes wild-type as well as multiple mutant forms of GAA, and can lead to higher cellular levels of GAA. In this study, we examined the effect of AT2220 on mutant GAA, in vitro and in vivo, with a primary focus on the endoplasmic reticulum (ER)-retained P545L mutant form of human GAA (P545L GAA). AT2220 increased the specific activity of P545L GAA toward both natural (glycogen) and artificial substrates in vitro. Incubation with AT2220 also increased the ER export, lysosomal delivery, proteolytic processing, and stability of P545L GAA. In a new transgenic mouse model of Pompe disease that expresses human P545L on a Gaa knockout background (Tg/KO) and is characterized by reduced GAA activity and elevated glycogen levels in disease-relevant tissues, daily oral administration of AT2220 for 4 weeks resulted in significant and dose-dependent increases in mature lysosomal GAA isoforms and GAA activity in heart and skeletal muscles. Importantly, oral administration of AT2220 also resulted in significant glycogen reduction in disease-relevant tissues. Compared to daily administration, less-frequent AT2220 administration, including repeated cycles of 4 or 5 days with AT2220 followed by 3 or 2 days without drug, respectively, resulted in even greater glycogen reductions. Collectively, these data indicate that AT2220 increases the specific activity, trafficking, and lysosomal stability of P545L GAA, leads to increased levels of mature GAA in lysosomes, and promotes glycogen reduction in situ. As such, AT2220 may

  20. Hirschsprung disease is associated with an L286P mutation in the fifth transmembrane domain of the endothelin-B receptor in the N-ethyl-N-nitrosourea-induced mutant line.

    Science.gov (United States)

    Chen, Bing; Ouyang, Hui-Ling; Wang, Wen-Hua; Yin, Yi-Heng; Yan, Lin-Na; Yang, Bin; Xue, Zheng-Feng

    2016-07-29

    Hirschsprung disease (HSCR), or colonic aganglionosis, is a congenital disorder characterized by the absence of intramural ganglia along variable lengths of the colon, resulting in intestinal obstruction. It is the most common cause of congenital intestinal obstruction, with an incidence of 1 in 5,000 live births. N-ethyl-N-nitrosourea (ENU)-induced mutagenesis is a powerful tool for the study of gene function and the generation of human disease models. In the current study, a novel mutant mouse with aganglionic megacolon and coat color spotting was generated by ENU-induced mutagenesis. Histological and acetylcholinesterase (AChE) whole-mount staining analysis showed a lack of ganglion cells in the colon in mutant mice. The mutation was mapped to chromosome 14 between markers rs30928624 and D14Mit205 (Chr 14 positions 103723921 bp and 105054651 bp). The Ednrb (Chr 14 position 103814625-103844173 bp) was identified as a potential candidate gene in this location. Mutation analysis revealed a T>C missense mutation at nucleotide 857 of the cDNA encoding endothelin receptor B (EDNRB) in which a proline was substituted for the highly conserved Lys-286 residue (L286P) in the fifth transmembrane (TM V) domain of this G protein-coupled receptor. The mutant mouse was named Ednrb(m1yzcm) (Ednrb; mutation 1, Yangzhou University Comparative Medicine Center). The results of the present study implicate the structural importance of the TM V domain in Ednrb function, and the Ednrb(m1yzcm) mouse represents a valuable model for the study of HSCR in humans.

  1. The Heterologous Expression of the p22 RNA Silencing Suppressor of the Crinivirus Tomato Chlorosis Virus from Tobacco Rattle Virus and Potato Virus X Enhances Disease Severity but Does Not Complement Suppressor-Defective Mutant Viruses.

    Science.gov (United States)

    Landeo-Ríos, Yazmín; Navas-Castillo, Jesús; Moriones, Enrique; Cañizares, M. Carmen

    2017-11-24

    To counteract host antiviral RNA silencing, plant viruses express suppressor proteins that function as pathogenicity enhancers. The genome of the Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae) encodes an RNA silencing suppressor, the protein p22, that has been described as having one of the longest lasting local suppressor activities when assayed in Nicotiana benthamiana. Since suppression of RNA silencing and the ability to enhance disease severity are closely associated, we analyzed the effect of expressing p22 in heterologous viral contexts. Thus, we studied the effect of the expression of ToCV p22 from viral vectors Tobacco rattle virus (TRV) and Potato virus X (PVX), and from attenuated suppressor mutants in N. benthamiana plants. Our results show that although an exacerbation of disease symptoms leading to plant death was observed in the heterologous expression of ToCV p22 from both viruses, only in the case of TRV did increased viral accumulation occur. The heterologous expression of ToCV p22 could not complement suppressor-defective mutant viruses.

  2. The Heterologous Expression of the p22 RNA Silencing Suppressor of the Crinivirus Tomato Chlorosis Virus from Tobacco Rattle Virus and Potato Virus X Enhances Disease Severity but Does Not Complement Suppressor-Defective Mutant Viruses

    Directory of Open Access Journals (Sweden)

    Yazmín Landeo-Ríos

    2017-11-01

    Full Text Available To counteract host antiviral RNA silencing, plant viruses express suppressor proteins that function as pathogenicity enhancers. The genome of the Tomato chlorosis virus (ToCV (genus Crinivirus, family Closteroviridae encodes an RNA silencing suppressor, the protein p22, that has been described as having one of the longest lasting local suppressor activities when assayed in Nicotiana benthamiana. Since suppression of RNA silencing and the ability to enhance disease severity are closely associated, we analyzed the effect of expressing p22 in heterologous viral contexts. Thus, we studied the effect of the expression of ToCV p22 from viral vectors Tobacco rattle virus (TRV and Potato virus X (PVX, and from attenuated suppressor mutants in N. benthamiana plants. Our results show that although an exacerbation of disease symptoms leading to plant death was observed in the heterologous expression of ToCV p22 from both viruses, only in the case of TRV did increased viral accumulation occur. The heterologous expression of ToCV p22 could not complement suppressor-defective mutant viruses.

  3. SOMATIC MUTATIONS OF THE VON HIPPEL-LINDAU DISEASE TUMOR-SUPPRESSOR GENE IN NONFAMILIAL CLEAR-CELL RENAL-CARCINOMA

    NARCIS (Netherlands)

    FOSTER, K; PROWSE, A; van den Berg, Anke; FLEMING, S; HULSBEEK, MMF; CROSSEY, PA; RICHARDS, FM; CAIRNS, P; FERGUSONSMITH, MA; BUYS, CHCM; MAHER, ER

    1994-01-01

    Loss of heterozygosity (LOH) studies have suggested that somatic mutations of a tumour suppressor gene or genes on chromosome 3p are a critical event in the pathogenesis of non-familial renal cell carcinoma (RCC). Germline mutations of the von Hippel - Lindau (VHL) disease gene predispose to early

  4. Nicotinamide ribosyl uptake mutants in Haemophilus influenzae.

    Science.gov (United States)

    Herbert, Mark; Sauer, Elizabeta; Smethurst, Graeme; Kraiss, Anita; Hilpert, Anna-Karina; Reidl, Joachim

    2003-09-01

    The gene for the nicotinamide riboside (NR) transporter (pnuC) was identified in Haemophilus influenzae. A pnuC mutant had only residual NR uptake and could survive in vitro with high concentrations of NR, but could not survive in vivo. PnuC may represent a target for the development of inhibitors for preventing H. influenzae disease.

  5. Inhibitors of cathepsin B improve memory and reduce beta-amyloid in transgenic Alzheimer disease mice expressing the wild-type, but not the Swedish mutant, beta-secretase site of the amyloid precursor protein.

    Science.gov (United States)

    Hook, Vivian Y H; Kindy, Mark; Hook, Gregory

    2008-03-21

    Elucidation of Abeta-lowering agents that inhibit processing of the wild-type (WT) beta-secretase amyloid precursor protein (APP) site, present in most Alzheimer disease (AD) patients, is a logical approach for improving memory deficit in AD. The cysteine protease inhibitors CA074Me and E64d were selected by inhibition of beta-secretase activity in regulated secretory vesicles that produce beta-amyloid (Abeta). The regulated secretory vesicle activity, represented by cathepsin B, selectively cleaves the WT beta-secretase site but not the rare Swedish mutant beta-secretase site. In vivo treatment of London APP mice, expressing the WT beta-secretase site, with these inhibitors resulted in substantial improvement in memory deficit assessed by the Morris water maze test. After inhibitor treatment, the improved memory function was accompanied by reduced amyloid plaque load, decreased Abeta40 and Abeta42, and reduced C-terminal beta-secretase fragment derived from APP by beta-secretase. However, the inhibitors had no effects on any of these parameters in mice expressing the Swedish mutant beta-secretase site of APP. The notable efficacy of these inhibitors to improve memory and reduce Abeta in an AD animal model expressing the WT beta-secretase APP site present in the majority of AD patients provides support for CA074Me and E64d inhibitors as potential AD therapeutic agents.

  6. Morphological mutants of garlic

    Energy Technology Data Exchange (ETDEWEB)

    Choudhary, A.D.; Dnyansagar, V.R. (Nagpur Univ. (India). Dept. of Botany)

    1982-01-01

    Cloves of garlic (Allium sativuum Linn.) were exposed to gamma rays with various doses and different concentrations of ethylmethane sulphonate (EMS), diethyl sulphate (dES) and ethylene imine (EI). In the second and third generations, 16 types of morphological mutants were recorded with varied frequencies. Of all the mutagens used, gamma rays were found to be the most effective in inducing the maximum number of mutations followed EI, EMS and dES in that order.

  7. Unfolding intermediates of the mutant His-107-Tyr of human ...

    Indian Academy of Sciences (India)

    The mutant His-107-Tyr of human carbonic anhydrase II (HCA II) is highly unstable and has long been linked to a misfolding disease known as carbonic anhydrase deficiency syndrome (CADS). High temperature unfolding trajectories of the mutant are obtained from classical molecular dynamics simulationsand analyzed in ...

  8. Unfolding intermediates of the mutant His-107-Tyr of human ...

    Indian Academy of Sciences (India)

    Srabani Taraphder

    Abstract. The mutant His-107-Tyr of human carbonic anhydrase II (HCA II) is highly unstable and has long been linked to a misfolding disease known as carbonic anhydrase deficiency syndrome (CADS). High temperature unfolding trajectories of the mutant are obtained from classical molecular dynamics simulations.

  9. Identification of eight new mutations in familial neurogenic diabetes insipidus supports the concept that defective folding of the mutant provasopressin-neurophysin causes the disease

    Energy Technology Data Exchange (ETDEWEB)

    Rittig, S.; Siggaard, C.; Pedersen, E.B. [University Hospital in Aarhus (Denmark)] [and others

    1994-09-01

    Familial neurogenic diabetes insipidus (FNDI) is an autosomal dominant disorder with a uniform phenotype characterized by polyuria, polydipsia and a severe deficiency of arginine vasopressin (AVP). These abnormalities develop postnatally and appear to be due to progressive degeneration of AVP producing neurons. Previous studies in 8 FNDI kindreds have identified 5 different mutations in the gene that codes for the AVP-neurophysin (NP) precursor, AVP-NP. Four kindreds had the same missense mutation in the part of exon 1 that codes for the C-terminal amino acid of the signal peptide (SP). The other 4 had different missense mutations or a codon deletion in exon 2 which codes for the highly conserved part of NP. In the present study, the AVP-NP genes from 8 other kindreds with FNDI were sequenced bidirectionally using sequence and single-stranded DNA amplified by PCR with biotinylated primers flanking each of the 3 exons. We find that each of the 8 kindreds has a different, previously unreported mutation in either the SP coding part of exon 1, in exon 2 or in the variable, NP-coding part of exon 3. Combining these 8 new mutations with the 5 described previously reveals a distribution pattern that corresponds closely to the domains involved in the mutually interactive processes of AVP binding, folding and dimerization of NP. Based on these findings and the clinical features of FNDI, we postulate that the precursors produced by the mutant alleles are cytotoxic because they do not fold or dimerize properly for subsequent packaging and processing.

  10. Molecular analysis of SUMF1 mutations: stability and residual activity of mutant formylglycine-generating enzyme determine disease severity in multiple sulfatase deficiency.

    Science.gov (United States)

    Schlotawa, Lars; Steinfeld, Robert; von Figura, Kurt; Dierks, Thomas; Gärtner, Jutta

    2008-01-01

    Multiple Sulfatase Deficiency (MSD) is a rare inborn autosomal-recessive disorder, which mainly combines clinical features of metachromatic leukodystrophy, mucopolysaccharidosis and X-linked ichthyosis. The clinical course ranges from neonatal severe to mild juvenile cases. MSD is caused by mutations in the SUMF1 gene encoding the formylglycine-generating enzyme (FGE). FGE posttranslationally activates sulfatases by generating formylglycine in their catalytic sites. We analyzed the functional consequences of missense mutations p.A177P, p.W179S, p.A279V and p.R349W with regard to FGE's subcellular localization, enzymatic activity, protein stability, intracellular retention and resulting sulfatase activities. All four mutations did not affect localization of FGE in the endoplasmic reticulum of MSD fibroblasts. However, they decreased its specific enzymatic activity to less than 1% (p.A177P and p.R349W), 3% (p.W179S) or 23% (p.A279V). Protein stability was severely decreased for p.A279V and p.R349W, and almost comparable to wild type for p.A177P and p.W179S. The patient with the mildest clinical phenotype carries the mutation p.A279V leading to decreased FGE protein stability, but high residual enzymatic activity and only slightly reduced sulfatase activities. In contrast, the most severely affected patient carries the mutation p.R349W leading to drastically decreased protein stability, very low residual enzymatic activity and considerably reduced sulfatase activities. Our functional studies provide novel insight into the molecular defect underlying MSD and reveal that both residual enzyme activity and protein stability of FGE contribute to the clinical phenotype. The application of improved functional assays to determine these two molecular parameters of FGE mutants may enable the prediction of the clinical outcome in the future. (c) 2007 Wiley-Liss, Inc.

  11. A mutation at IVS1 + 5 of the von Hippel-Lindau gene resulting in intron retention in transcripts is not pathogenic in a patient with a tongue cancer?: case report

    Directory of Open Access Journals (Sweden)

    Asakawa Takeshi

    2012-03-01

    Full Text Available Abstract Background Von Hippel-Lindau disease (VHL is a dominantly inherited familial cancer syndrome predisposing the patient to a variety of malignant and benign neoplasms, most frequently hemangioblastoma, renal cell carcinoma, pheochromocytoma, and pancreatic tumors. VHL is caused by mutations of the VHL tumor suppressor gene on the short arm of chromosome 3, and clinical manifestations develop if both alleles are inactivated according to the two-hit hypothesis. VHL mutations are more frequent in the coding region and occur occasionally in the splicing region of the gene. Previously, we reported that the loss of heterozygosity (LOH of the VHL gene is common in squamous cell carcinoma tissues of the tongue. Case Presentation We describe a case of squamous cell carcinoma in the tongue caused by a point mutation in the splicing region of the VHL gene and discuss its association with VHL disease. Sequence analysis of DNA extracted from the tumor and peripheral blood of the patient with squamous cell carcinoma revealed a heterozygous germline mutation (c. 340 + 5 G > C in the splice donor sequence in intron 1 of the VHL gene. RT-PCR analysis of the exon1/intron1 junction in RNA from tumor tissue detected an unspliced transcript. Analysis of LOH using a marker with a heterozygous mutation of nucleotides (G or C revealed a deletion of the mutant C allele in the carcinoma tissues. Conclusions The fifth nucleotide G of the splice donor site of the VHL gene is important for the efficiency of splicing at that site. The development of tongue cancer in this patient was not associated with VHL disease because the mutation occurred in only a single allele of the VHL gene and that allele was deleted in tumor cells.

  12. In vitro assessment of recombinant, mutant immunoglobulin G anti-D devoid of hemolytic activity for treatment of ongoing hemolytic disease of the fetus and newborn

    DEFF Research Database (Denmark)

    Nielsen, Leif K; Green, Trine H; Sandlie, Inger

    2008-01-01

    A specific treatment for ongoing hemolytic disease of the fetus and newborn (HDFN) due to anti-D would be very attractive. One approach could be administration to the mother of nonhemolytic anti-D, which by crossing the placenta can block the binding of hemolytic maternal anti-D....

  13. A dominant negative mutant of rab5 inhibits infection of cells by foot-and-mouth disease virus; implications for virus entry

    DEFF Research Database (Denmark)

    Johns, Helen; Berryman, Stephen; Monaghan, Paul

    2009-01-01

    Foot-and-mouth disease virus (FMDV) can use a number of different integrins (alphavβ1, alphavβ3, alphavβ6, and alphavβ8) as receptors to initiate infection. Infection mediated by alphavβ6 is known to occur by clathrin-mediated endocytosis and is dependent on the acidic pH within endosomes...

  14. Preserved Fronto-Striatal Plasticity and Enhanced Procedural Learning in a Transgenic Mouse Model of Alzheimer's Disease Overexpressing Mutant "hAPPswe"

    Science.gov (United States)

    Middei, Silvia; Geracitano, Raffaella; Caprioli, Antonio; Mercuri, Nicola; Ammassari-Teule, Martine

    2004-01-01

    Mutations in the amyloid precursor protein (APP) gene inducing abnormal processing and deposition of [beta]-amyloid protein in the brain have been implicated in the pathogenesis of Alzheimer's disease (AD). Although Tg2576 mice with the Swedish mutation ("hAPPswe") exhibit age-related [Alpha][beta]-plaque formation in brain regions like the…

  15. Native Mutant Huntingtin in Human Brain

    Science.gov (United States)

    Sapp, Ellen; Valencia, Antonio; Li, Xueyi; Aronin, Neil; Kegel, Kimberly B.; Vonsattel, Jean-Paul; Young, Anne B.; Wexler, Nancy; DiFiglia, Marian

    2012-01-01

    Huntington disease (HD) is caused by polyglutamine expansion in the N terminus of huntingtin (htt). Analysis of human postmortem brain lysates by SDS-PAGE and Western blot reveals htt as full-length and fragmented. Here we used Blue Native PAGE (BNP) and Western blots to study native htt in human postmortem brain. Antisera against htt detected a single band broadly migrating at 575–850 kDa in control brain and at 650–885 kDa in heterozygous and Venezuelan homozygous HD brains. Anti-polyglutamine antisera detected full-length mutant htt in HD brain. There was little htt cleavage even if lysates were pretreated with trypsin, indicating a property of native htt to resist protease cleavage. A soluble mutant htt fragment of about 180 kDa was detected with anti-htt antibody Ab1 (htt-(1–17)) and increased when lysates were treated with denaturants (SDS, 8 m urea, DTT, or trypsin) before BNP. Wild-type htt was more resistant to denaturants. Based on migration of in vitro translated htt fragments, the 180-kDa segment terminated ≈htt 670–880 amino acids. If second dimension SDS-PAGE followed BNP, the 180-kDa mutant htt was absent, and 43–50 kDa htt fragments appeared. Brain lysates from two HD mouse models expressed native full-length htt; a mutant fragment formed if lysates were pretreated with 8 m urea + DTT. Native full-length mutant htt in embryonic HD140Q/140Q mouse primary neurons was intact during cell death and when cell lysates were exposed to denaturants before BNP. Thus, native mutant htt occurs in brain and primary neurons as a soluble full-length monomer. PMID:22375012

  16. Neuronopathic Gaucher disease in the mouse: viable combined selective saposin C deficiency and mutant glucocerebrosidase (V394L) mice with glucosylsphingosine and glucosylceramide accumulation and progressive neurological deficits.

    Science.gov (United States)

    Sun, Ying; Liou, Benjamin; Ran, Huimin; Skelton, Matthew R; Williams, Michael T; Vorhees, Charles V; Kitatani, Kazuyuki; Hannun, Yusuf A; Witte, David P; Xu, You-Hai; Grabowski, Gregory A

    2010-03-15

    Gaucher disease is caused by defective acid beta-glucosidase (GCase) function. Saposin C is a lysosomal protein needed for optimal GCase activity. To test the in vivo effects of saposin C on GCase, saposin C deficient mice (C-/-) were backcrossed to point mutated GCase (V394L/V394L) mice. The resultant mice (4L;C*) began to exhibit CNS abnormalities approximately 30 days: first as hindlimb paresis, then progressive tremor and ataxia. Death occurred approximately 48 days due to neurological deficits. Axonal degeneration was evident in brain stem, spinal cord and white matter of cerebellum accompanied by increasing infiltration of the brain stem, cortex and thalamus by CD68 positive microglial cells and activation of astrocytes. Electron microscopy showed inclusion bodies in neuronal processes and degenerating cells. Accumulation of p62 and Lamp2 were prominent in the brain suggesting the impairment of autophagosome/lysosome function. This phenotype was different from either V394L/V394L or C-/- alone. Relative to V394L/V394L mice, 4L;C* mice had diminished GCase protein and activity. Marked increases (20- to 30-fold) of glucosylsphingosine (GS) and moderate elevation (1.5- to 3-fold) of glucosylceramide (GC) were in 4L;C* brains. Visceral tissues had increases of GS and GC, but no storage cells were found. Neuronal cells in thick hippocampal slices from 4L;C* mice had significantly attenuated long-term potentiation, presumably resulting from substrate accumulation. The 4L;C* mouse mimics the CNS phenotype and biochemistry of some type 3 (neuronopathic) variants of Gaucher disease and is a unique model suitable for testing pharmacological chaperone and substrate reduction therapies, and investigating the mechanisms of neuronopathic Gaucher disease.

  17. Structures of TorsinA and its disease-mutant complexed with an activator reveal the molecular basis for primary dystonia

    Energy Technology Data Exchange (ETDEWEB)

    Demircioglu, F. Esra; Sosa, Brian A.; Ingram, Jessica; Ploegh, Hidde L.; Schwartz, Thomas U.

    2016-08-04

    The most common cause of early onset primary dystonia, a neuromuscular disease, is a glutamate deletion (ΔE) at position 302/303 of TorsinA, a AAA+ ATPase that resides in the endoplasmic reticulum. While the function of TorsinA remains elusive, the ΔE mutation is known to diminish binding of two TorsinA ATPase activators: lamina-associated protein 1 (LAP1) and its paralog, luminal domain like LAP1 (LULL1). Using a nanobody as a crystallization chaperone, we obtained a 1.4 Å crystal structure of human TorsinA in complex with LULL1. This nanobody likewise stabilized the weakened TorsinAΔE-LULL1 interaction, which enabled us to solve its structure at 1.4 Å also. A comparison of these structures shows, in atomic detail, the subtle differences in activator interactions that separate the healthy from the diseased state. This information may provide a structural platform for drug development, as a small molecule that rescues TorsinAΔE could serve as a cure for primary dystonia.

  18. Differential gene expression in porcine SK6 cells infected with wild-type and SAP domain-mutant foot-and-mouth disease virus.

    Science.gov (United States)

    Ni, Zixin; Yang, Fan; Cao, Weijun; Zhang, Xiangle; Jin, Ye; Mao, Ruoqing; Du, Xiaoli; Li, Weiwei; Guo, Jianhong; Liu, Xiangtao; Zhu, Zixiang; Zheng, Haixue

    2016-06-01

    Foot-and-mouth disease virus (FMDV) is the causative agent of a highly contagious disease in livestock. The viral proteinase L(pro) of FMDV is involved in pathogenicity, and mutation of the L(pro) SAP domain reduces FMDV pathogenicity in pigs. To determine the gene expression profiles associated with decreased pathogenicity in porcine cells, we performed transcriptome analysis using next-generation sequencing technology and compared differentially expressed genes in SK6 cells infected with FMDV containing L(pro) with either a wild-type or mutated version of the SAP domain. This analysis yielded 1,853 genes that exhibited a ≥ 2-fold change in expression and was validated by real-time quantitative PCR detection of several differentially expressed genes. Many of the differentially expressed genes correlated with antiviral responses corresponded to genes associated with transcription factors, immune regulation, cytokine production, inflammatory response, and apoptosis. Alterations in gene expression profiles may be responsible for the variations in pathogenicity observed between the two FMDV variants. Our results provided genes of interest for the further study of antiviral pathways and pathogenic mechanisms related to FMDV L(pro).

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

    Directory of Open Access Journals (Sweden)

    Peter Novodvorsky

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

  20. Misfolded opsin mutants display elevated β-sheet structure.

    Science.gov (United States)

    Miller, Lisa M; Gragg, Megan; Kim, Tae Gyun; Park, Paul S-H

    2015-10-07

    Mutations in rhodopsin can cause misfolding and aggregation of the receptor, which leads to retinitis pigmentosa, a progressive retinal degenerative disease. The structure adopted by misfolded opsin mutants and the associated cell toxicity is poorly understood. Förster resonance energy transfer (FRET) and Fourier transform infrared (FTIR) microspectroscopy were utilized to probe within cells the structures formed by G188R and P23H opsins, which are misfolding mutants that cause autosomal dominant retinitis pigmentosa. Both mutants formed aggregates in the endoplasmic reticulum and exhibited altered secondary structure with elevated β-sheet and reduced α-helical content. The newly formed β-sheet structure may facilitate the aggregation of misfolded opsin mutants. The effects observed for the mutants were unrelated to retention of opsin molecules in the endoplasmic reticulum itself. Copyright © 2015 Federation of European Biochemical Societies. All rights reserved.

  1. Through the looking glass: an exploratory study of the lived experiences and unmet needs of families affected by Von Hippel-Lindau disease.

    Science.gov (United States)

    Kasparian, Nadine A; Rutstein, Alison; Sansom-Daly, Ursula M; Mireskandari, Shab; Tyler, Janet; Duffy, Jessica; Tucker, Katherine M

    2015-01-01

    Despite well-established protocols for the medical management of Von Hippel-Lindau disease (VHL), families affected by this rare tumour syndrome continue to face numerous psychological, social, and practical challenges. To our knowledge, this is one of the first qualitative studies to explore the psychosocial difficulties experienced by families affected by VHL. A semi-structured interview was developed to explore patients' and carers' experiences of VHL along several life domains, including: self-identity and self-esteem, interpersonal relationships, education and career opportunities, family communication, physical health and emotional well-being, and supportive care needs. Quantitative measures were also used to examine the prevalence of anxiety, depression, and disease-specific distress in this sample. Participants were recruited via the Hereditary Cancer Clinic at the Prince of Wales Hospital in Sydney, Australia. A total of 23 individual telephone interviews were conducted (15 patients, 8 carers), yielding a response rate of 75%. A diverse range of experiences were reported, including: sustained uncertainty about future tumour development, frustration regarding the need for lifelong medical screening, strained family relationships, difficulties communicating with others about VHL, perceived social isolation and limited career opportunities, financial and care-giving burdens, complex decisions in relation to childbearing, and difficulties accessing expert medical and psychosocial care. Participants also provided examples of psychological growth and resilience, and voiced support for continued efforts to improve supportive care services. More sophisticated systems for connecting VHL patients and their families with holistic, empathic, and person-centred medical and psychosocial care are urgently needed.

  2. Mutant IDH1 Downregulates ATM and Alters DNA Repair and Sensitivity to DNA Damage Independent of TET2

    National Research Council Canada - National Science Library

    Inoue, Satoshi; Li, Wanda Y; Tseng, Alan; Beerman, Isabel; Elia, Andrew J; Bendall, Sean C; Lemonnier, François; Kron, Ken J; Cescon, David W; Hao, Zhenyue; Lind, Evan F; Takayama, Naoya; Planello, Aline C; Shen, Shu Yi; Shih, Alan H; Larsen, Dana M; Li, Qinxi; Snow, Bryan E; Wakeham, Andrew; Haight, Jillian; Gorrini, Chiara; Bassi, Christian; Thu, Kelsie L; Murakami, Kiichi; Elford, Alisha R; Ueda, Takeshi; Straley, Kimberly; Yen, Katharine E; Melino, Gerry; Cimmino, Luisa; Aifantis, Iannis; Levine, Ross L; De Carvalho, Daniel D; Lupien, Mathieu; Rossi, Derrick J; Nolan, Garry P; Cairns, Rob A; Mak, Tak W

    2016-01-01

    ...) but their mechanism is not fully understood. It is thought that IDH1 mutants act by inhibiting TET2 to alter DNA methylation, but there are significant unexplained clinical differences between IDH1- and TET2-mutant diseases...

  3. Endolymphatic sac tumour in von Hippel-Lindau disease: management strategies.

    Science.gov (United States)

    Zanoletti, E; Girasoli, L; Borsetto, D; Opocher, G; Mazzoni, A; Martini, A

    2017-10-01

    Endolymphatic sac tumour (ELST) is infrequent, as emerges from small series reported in the literature. It is a slow-growing malignancy with local aggressiveness and a low risk of distant metastases. It is often misdiagnosed because of the late onset of symptoms and difficulty in obtaining a biopsy. Its frequency is higher in von Hippel-Lindau (VHL) disease (a genetic systemic syndrome involving multiple tumours), with a prevalence of around 25%. The diagnosis is based on radiology, with specific patterns on contrast-enhanced MRI and typical petrous bone erosion on bone CT scan. Our experience of ELST in the years between 2012-2015 concerns 7 cases, one of which was bilateral, in patients with VHL disease. Four of the 7 patients underwent 5 surgical procedures at our institution. Each case is described in detail, including clinical symptoms, and the intervals between symptom onset, diagnosis and therapy. Postoperative morbidity was low after early surgery on small tumours, whereas extensive surgery for large tumours was associated with loss of cranial nerve function (especially VII, IX, X). The critical sites coinciding with loss of neurological function were the fallopian canal, jugular foramen, petrous apex and intradural extension into the posterior cranial fossa. Early surgery on small ELST is advocated for patients with VHL disease, in whom screening enables a prompt diagnosis and consequently good prognosis. © Copyright by Società Italiana di Otorinolaringologia e Chirurgia Cervico-Facciale, Rome, Italy.

  4. Aequorin mutants with increased thermostability.

    Science.gov (United States)

    Qu, Xiaoge; Rowe, Laura; Dikici, Emre; Ensor, Mark; Daunert, Sylvia

    2014-09-01

    Bioluminescent labels can be especially useful for in vivo and live animal studies due to the negligible bioluminescence background in cells and most animals, and the non-toxicity of bioluminescent reporter systems. Significant thermal stability of bioluminescent labels is essential, however, due to the longitudinal nature and physiological temperature conditions of many bioluminescent-based studies. To improve the thermostability of the bioluminescent protein aequorin, we employed random and rational mutagenesis strategies to create two thermostable double mutants, S32T/E156V and M36I/E146K, and a particularly thermostable quadruple mutant, S32T/E156V/Q168R/L170I. The double aequorin mutants, S32T/E156V and M36I/E146K, retained 4 and 2.75 times more of their initial bioluminescence activity than wild-type aequorin during thermostability studies at 37 °C. Moreover, the quadruple aequorin mutant, S32T/E156V/Q168R/L170I, exhibited more thermostability at a variety of temperatures than either double mutant alone, producing the most thermostable aequorin mutant identified thus far.

  5. Analysis of Mycobacterium avium subsp. paratuberculosis mutant libraries reveals loci-dependent transcription biases and strategies to novel mutant discovery

    Science.gov (United States)

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in ruminants and it has been implicated as a cause of Crohn’s disease in humans. The generation of comprehensive random mutant banks by transposon mutagenesis is a fundamental wide genomic technology utilized...

  6. Proteomic analysis of signaling network regulation in renal cell carcinomas with differential hypoxia-inducible factor-2α expression.

    Directory of Open Access Journals (Sweden)

    Lokesh Dalasanur Nagaprashantha

    Full Text Available BACKGROUND: The loss of von Hippel-Lindau (VHL protein function leads to highly vascular renal tumors characterized by an aggressive course of disease and refractoriness to chemotherapy and radiotherapy. Loss of VHL in renal tumors also differs from tumors of other organs in that the oncogenic cascade is mediated by an increase in the levels of hypoxia-inducible factor-2α (HIF2α instead of hypoxia-inducible factor-1α (HIF1α. METHODS AND PRINCIPAL FINDINGS: We used renal carcinoma cell lines that recapitulate the differences between mutant VHL and wild-type VHL genotypes. Utilizing a method relying on extracted peptide intensities as a label-free approach for quantitation by liquid chromatography-mass spectrometry, our proteomics study revealed regulation of key proteins important for cancer cell survival, proliferation and stress-resistance, and implicated differential regulation of signaling networks in VHL-mutant renal cell carcinoma. We also observed upregulation of cellular energy pathway enzymes and the stress-responsive mitochondrial 60-kDa heat shock protein. Finding reliance on glutaminolysis in VHL-mutant renal cell carcinoma was of particular significance, given the generally predominant dependence of tumors on glycolysis. The data have been deposited to the ProteomeXchange with identifier PXD000335. CONCLUSIONS AND SIGNIFICANCE: Pathway analyses provided corroborative evidence for differential regulation of molecular and cellular functions influencing cancer energetics, metabolism and cell proliferation in renal cell carcinoma with distinct VHL genotype. Collectively, the differentially regulated proteome characterized by this study can potentially guide translational research specifically aimed at effective clinical interventions for advanced VHL-mutant, HIF2α-over-expressing tumors.

  7. Occurrence of a 2-bp (AT) deletion allele and a nonsense (G-to-T) mutant allele at the E2 (DBT) locus of six patients with maple syrup urine disease: Multiple-exon skipping as a secondary effect of the mutations

    Energy Technology Data Exchange (ETDEWEB)

    Fisher, C.W.; Fisher, C.R.; Chuang, J.L.; Lau, K.S.; Chuang, D.T.; Cox, R.P. (Univ. of Texas, Dallas (United States))

    1993-02-01

    The authors have identified two novel mutant alleles in the transacylase (E2) gene of the human branched-chain [alpha]-keto acid dehydrogenase (BCKAD) complex in 6 of 38 patients with maple syrup urine disease (MSUD). One mutation, a 2-bp (AT) deletion in exon 2 of the E2 gene, causes a frameshift downstream of residue ([minus]26) in the mitochondrial targeting presequence. The second mutation, a G-to-T transversion in exon 6 of the E2 gene, produces a premature stop codon at Glu-163 (E163*). Transfection of constructs harboring the E163* mutation into an E2-deficient MSUD cell line produced a truncated E2 subunit. However, this mutant E2 chain is unable to assemble into a 24-mer cubic structure and is degraded in the cell. The 2-bp (AT) deletion and the E163* mutant alleles occur in either the homozygous or compound-heterozygous state in the 6 of 38 unrelated MSUD patients studied. Moreover, an array of precise single- and multiple-exon deletions were observed in many amplified E2 mutant cDNAs. The latter results appear to represent secondary effects on RNA processing that are associated with the MSUD mutations at the E2 locus. 30 refs., 8 figs.

  8. Wild Accessions and Mutant Resources

    DEFF Research Database (Denmark)

    Kawaguchi, Masayoshi; Sandal, Niels Nørgaard

    2014-01-01

    Lotus japonicus, Lotus burttii, and Lotus filicaulis are species of Lotus genus that are utilized for molecular genetic analysis such as the construction of a linkage map and QTL analysis. Among them, a number of mutants have been isolated from two wild accessions: L. japonicus Gifu B-129 and Miy...

  9. Normal aging modulates the neurotoxicity of mutant huntingtin.

    Directory of Open Access Journals (Sweden)

    Elsa Diguet

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

  10. Characterization of resistant tomato mutants to bacterial canker ...

    African Journals Online (AJOL)

    Yomi

    2012-04-19

    Apr 19, 2012 ... A small scale ethylmethanesulfonate (EMS) mutation was used to obtain resistant mutant plants to bacterial canker disease caused by Clavibacter michiganensis subsp. michiganensis isolate 2 (Cmm2). Susceptible EBR3 tomato line (200) seeds were mutagenised with the chemical EMS. Of the ...

  11. Characterization of resistant tomato mutants to bacterial canker ...

    African Journals Online (AJOL)

    A small scale ethylmethanesulfonate (EMS) mutation was used to obtain resistant mutant plants to bacterial canker disease caused by Clavibacter michiganensis subsp. michiganensis isolate 2 (Cmm2). Susceptible EBR3 tomato line (200) seeds were mutagenised with the chemical EMS. Of the constructed M2 population, ...

  12. Defective glycinergic synaptic transmission in zebrafish motility mutants

    Directory of Open Access Journals (Sweden)

    Hiromi Hirata

    2010-01-01

    Full Text Available Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Recently, in vivo analysis of glycinergic synaptic transmission has been pursued in zebrafish using molecular genetics. An ENU mutagenesis screen identified two behavioral mutants that are defective in glycinergic synaptic transmission. Zebrafish bandoneon (beo mutants have a defect in glrbb, one of the duplicated glycine receptor (GlyR β subunit genes. These mutants exhibit a loss of glycinergic synaptic transmission due to a lack of synaptic aggregation of GlyRs. Due to the consequent loss of reciprocal inhibition of motor circuits between the two sides of the spinal cord, motor neurons activate simultaneously on both sides resulting in bilateral contraction of axial muscles of beo mutants, eliciting the so-called ‘accordion’ phenotype. Similar defects in GlyR subunit genes have been observed in several mammals and are the basis for human hyperekplexia/startle disease. By contrast, zebrafish shocked (sho mutants have a defect in slc6a9, encoding GlyT1, a glycine transporter that is expressed by astroglial cells surrounding the glycinergic synapse in the hindbrain and spinal cord. GlyT1 mediates rapid uptake of glycine from the synaptic cleft, terminating synaptic transmission. In zebrafish sho mutants, there appears to be elevated extracellular glycine resulting in persistent inhibition of postsynaptic neurons and subsequent reduced motility, causing the ‘twitch once’ phenotype. We review current knowledge regarding zebrafish ‘accordion’ and ‘twitch once’ mutants, including beo and sho, and report the identification of a new α2 subunit that revises the phylogeny of zebrafish GlyRs.

  13. Problem-Solving Test: Tryptophan Operon Mutants

    Science.gov (United States)

    Szeberenyi, Jozsef

    2010-01-01

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

  14. Endolymphatic sac tumor with von Hippel-Lindau disease: report of a case with atypical pathology of endolymphatic sac tumor.

    Science.gov (United States)

    Yang, Xiang; Liu, Xue-Song; Fang, Yuan; Zhang, Xiu-Hui; Zhang, Yue-Kang

    2014-01-01

    The authors described a case of a patient with co-existing endolymphatic sac tumor (ELST) and hemangioblastoma in the posterior cranial fossa, which belonged to a subtype of Von Hippel-Lindau (VHL) disease confirmed by the test of VHL-gene. The signs in this 42-year-old female included intermittent headache and dizziness. Imaging revealed a giant mass in the right cerebellopontine angle (CPA) region and another lesion in the left cerebellar hemisphere. The results of biopsy after two operations confirmed the diagnosis respectively. Both of the tumors were resected totally. Nevertheless, we had to confess the misdiagnosis as vascular tumor instead of ELST at the initial diagnosis because of the rarity of ELST associated with atypical histological characteristics. The purposes we reported this case were to describe the atypical pathological feature of ELST and the mutation of germline VHL not mentioned in previously literature, furthermore, to foster understanding of ELSTs with the avoidance of the similar misdiagnosis as far as possible in future.

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

    Science.gov (United States)

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

    2016-05-01

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

  16. Reversal of mutant myocilin non-secretion and cell killing: implications for glaucoma

    National Research Council Canada - National Science Library

    Liu, Yuhui; Vollrath, Douglas

    2004-01-01

    .... Mutations of the myocilin gene are one cause of autosomal dominant juvenile- and adult-onset primary open angle glaucoma, but the mechanism by which mutant myocilins cause disease is poorly understood...

  17. Ambroxol as a pharmacological chaperone for mutant glucocerebrosidase.

    Science.gov (United States)

    Bendikov-Bar, Inna; Maor, Gali; Filocamo, Mirella; Horowitz, Mia

    2013-02-01

    Gaucher disease (GD) is characterized by accumulation of glucosylceramide in lysosomes due to mutations in the GBA1 gene encoding the lysosomal hydrolase β-glucocerebrosidase (GCase). The disease has a broad spectrum of phenotypes, which were divided into three different Types; Type 1 GD is not associated with primary neurological disease while Types 2 and 3 are associated with central nervous system disease. GCase molecules are synthesized on endoplasmic reticulum (ER)-bound polyribosomes, translocated into the ER and following modifications and correct folding, shuttle to the lysosomes. Mutant GCase molecules, which fail to fold correctly, undergo ER associated degradation (ERAD) in the proteasomes, the degree of which is one of the factors that determine GD severity. Several pharmacological chaperones have already been shown to assist correct folding of mutant GCase molecules in the ER, thus facilitating their trafficking to the lysosomes. Ambroxol, a known expectorant, is one such chaperone. Here we show that ambroxol increases both the lysosomal fraction and the enzymatic activity of several mutant GCase variants in skin fibroblasts derived from Type 1 and Type 2 GD patients. Copyright © 2012 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2018-02-02

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

  19. The genetic basis of kidney cancer: a metabolic disease

    Science.gov (United States)

    Linehan, W. Marston; Srinivasan, Ramaprasad; Schmidt, Laura S.

    2010-01-01

    Kidney cancer is not a single disease but encompasses a number of different types of cancer that occur in the kidney, each caused by a different gene with a different histology and clinical course that responds differently to therapy. Each of the seven known kidney cancer genes, VHL, MET, FLCN, TSC1, TSC2, FH and SDH, is involved in pathways that respond to metabolic stress and/or nutrient stimulation. The VHL protein is a component of the oxygen and iron sensing pathway that regulates HIF levels in the cell. HGF/MET signaling affects the LKB1/AMPK energy sensing cascade. The FLCN/FNIP1/FNIP2 complex binds AMPK and therefore may interact with the cellular energy and nutrient sensing pathways, AMPK-TSC1/2-mTOR and PI3K-Akt-mTOR. TSC1/TSC2 are downstream of AMPK and negatively regulate mTOR in response to cellular energy deficit. FH and SDH play a central role in the mitochondrial tricarboxylic acid (TCA) cycle whose activities are coupled to energy production through oxidative phosphorylation. Mutations in each of these kidney cancer genes result in dysregulation of metabolic pathways involved in oxygen, iron, energy and/or nutrient sensing suggesting that kidney cancer is a disease of cell metabolism. Targeting the fundamental metabolic abnormalities in kidney cancer provides a unique opportunity for the development of more effective forms of therapy for this disease. PMID:20448661

  20. Incomplete flagellar structures in Escherichia coli mutants.

    OpenAIRE

    Suzuki, T; Komeda, Y

    1981-01-01

    Escherichia coli mutants with defects in 29 flagellar genes identified so far were examined by electron microscopy for possession of incomplete flagellar structures in membrane-associated fractions. The results are discussed in consideration of the known transcriptional interaction of flagellar genes. Hook-basal body structures were detected in flaD, flaS, flaT, flbC, and hag mutants. The flaE mutant had a polyhook-basal body structure. An intact basal body appeared in flaK mutants. Putative ...

  1. IGFBP2 expression predicts IDH-mutant glioma patient survival.

    Science.gov (United States)

    Huang, Lin Eric; Cohen, Adam L; Colman, Howard; Jensen, Randy L; Fults, Daniel W; Couldwell, William T

    2017-01-03

    Mutations of the isocitrate dehydrogenase (IDH) 1 and 2 genes occur in ~80% of lower-grade (WHO grade II and grade III) gliomas. Mutant IDH produces (R)-2-hydroxyglutarate, which induces DNA hypermethylation and presumably drives tumorigenesis. Interestingly, IDH mutations are associated with improved survival in glioma patients, but the underlying mechanism for the difference in survival remains unclear. Through comparative analyses of 286 cases of IDH-wildtype and IDH-mutant lower-grade glioma from a TCGA data set, we report that IDH-mutant gliomas have increased expression of tumor-suppressor genes (NF1, PTEN, and PIK3R1) and decreased expression of oncogenes(AKT2, ARAF, ERBB2, FGFR3, and PDGFRB) and glioma progression genes (FOXM1, IGFBP2, and WWTR1) compared with IDH-wildtype gliomas. Furthermore, each of these genes is prognostic in overall gliomas; however, within the IDH-mutant group, none remains prognostic except IGFBP2 (encodinginsulin-like growth factor binding protein 2). Through validation in an independent cohort, we show that patients with low IGFBP2 expressiondisplay a clear advantage in overall and disease-free survival, whereas those with high IGFBP2 expressionhave worse median survival than IDH-wildtype patients. These observations hold true across different histological and molecular subtypes of lower-grade glioma. We propose therefore that an unexpected biological consequence of IDH mutations in glioma is to ameliorate patient survival by promoting tumor-suppressor signaling while inhibiting that of oncogenes, particularly IGFBP2.

  2. Modelling the evolution and spread of HIV immune escape mutants.

    Science.gov (United States)

    Fryer, Helen R; Frater, John; Duda, Anna; Roberts, Mick G; Phillips, Rodney E; McLean, Angela R

    2010-11-18

    During infection with human immunodeficiency virus (HIV), immune pressure from cytotoxic T-lymphocytes (CTLs) selects for viral mutants that confer escape from CTL recognition. These escape variants can be transmitted between individuals where, depending upon their cost to viral fitness and the CTL responses made by the recipient, they may revert. The rates of within-host evolution and their concordant impact upon the rate of spread of escape mutants at the population level are uncertain. Here we present a mathematical model of within-host evolution of escape mutants, transmission of these variants between hosts and subsequent reversion in new hosts. The model is an extension of the well-known SI model of disease transmission and includes three further parameters that describe host immunogenetic heterogeneity and rates of within host viral evolution. We use the model to explain why some escape mutants appear to have stable prevalence whilst others are spreading through the population. Further, we use it to compare diverse datasets on CTL escape, highlighting where different sources agree or disagree on within-host evolutionary rates. The several dozen CTL epitopes we survey from HIV-1 gag, RT and nef reveal a relatively sedate rate of evolution with average rates of escape measured in years and reversion in decades. For many epitopes in HIV, occasional rapid within-host evolution is not reflected in fast evolution at the population level.

  3. Signature-Tagged Mutagenesis of Pasteurella multocida Identifies Mutants Displaying Differential Virulence Characteristics in Mice and Chickens

    OpenAIRE

    Harper, Marina; Boyce, John D.; Wilkie, Ian W.; Adler, Ben

    2003-01-01

    Pasteurella multocida is the causative agent of fowl cholera in birds. Signature-tagged mutagenesis (STM) was used to identify potential virulence factors in a mouse septicemia disease model and a chicken fowl cholera model. A library of P. multocida mutants was constructed with a modified Tn916 and screened for attenuation in both animal models. Mutants identified by the STM screening were confirmed as attenuated by competitive growth assays in both chickens and mice. Of the 15 mutants ident...

  4. Generation and characterization of pigment mutants of ...

    African Journals Online (AJOL)

    The induced mutagenesis method for deriving pigment mutants of a green microalga, Chlamydomonas reinhardtii CC-124 and their pigment composition as well as ability to assess mutability of contaminated aquatic ecosystems were studied. In the present study, 14086 mutants (colonies) were obtained by exposure of the ...

  5. Cadmium-Sensitive Mutants of Arabidopsis thaliana.

    Science.gov (United States)

    Howden, R; Cobbett, C S

    1992-09-01

    A screening procedure for identifying Cd-sensitive mutants of Arabidopsis thaliana is described. With this procedure, two Cd-sensitive mutants were isolated. These represent independent mutations in the same locus, referred to as CAD1. Genetic analysis has shown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus. Crosses of the mutant to marker strains showed that the mutation is closely linked to the tt3 locus on chromosome 5. In addition to Cd, the mutants are also significantly more sensitive to mercuric ions and only slightly more sensitive to Cu and Zn, while being no more sensitive than the wild type to Mn, thus indicating a degree of specificity in the mechanism affected by the mutation. Undifferentiated callus tissue is also Cd sensitive, suggesting that the mutant phenotype is expressed at the cellular level. Both wild-type and mutant plants showed increased sensitivity to Cd in the presence of buthionine sulfoximine, an inhibitor of the biosynthesis of the cadmium-binding (gamma-glutamylcysteine)(n)-glycine peptides, suggesting that the mutant is still able to synthesize these peptides. However, the effects of a cad1 mutation and buthionine sulfoximine together on cadmium sensitivity are essentially nonadditive, indicating that they may affect different aspects of the same detoxification mechanism. Assays of Cd uptake by intact plants indicate that the mutant is deficient in its ability to sequester Cd.

  6. Induced High Lysine Mutants in Barley

    DEFF Research Database (Denmark)

    Doll, Hans; Køie, B.; Eggum, B. O.

    1974-01-01

    Screening of mutagenically treated materials by combined Kjeldahl nitrogen and dye-binding capacity determinations disclosed fourteen barley mutants, which have from a few to about 40 per cent more lysine in the protein and one mutant with 10 per cent less lysine in the protein than the parent...

  7. Los mutantes de la escuela

    Directory of Open Access Journals (Sweden)

    Diego Armando Jaramillo-Ocampo

    2013-01-01

    Full Text Available El presente artículo muestra los resultados parciales del estudio “Juegos en el recreo escolar: un escenario para la formación ciudadana”, cuya pretensión fue comprender los imaginarios sociales de juego en el recreo escolar y su relación con la convivencia social desde la proximidad del enfoque de complementariedad y el diseño de investigación emergente, planteado por Murcia y Jaramillo (2008. Se presentan los desarrollos logrados en dos categorías centrales del estudio: el patio y el cuerpo; dos categorías que mutan constantemente como entidades vivas en la escuela, hacia la configuración de sujetos que reconocen en el otro y lo otro su posibilidad. La escuela viva, donde es posible “ser en relación con”… se reduce a un espacio temporal y físico, limitado por la campana, “el recreo”. El texto muestra, desde la voz de los actores, esa vida que se da y se quita en la escuela y que se posiciona como una más de las imposiciones normalizadas para controlar. Reconoce, finalmente, una propuesta desde la posibilidad que estos dos mutantes propician para una escuela libre y dinámica.

  8. Biocontrol potential of salinity tolerant mutants of Trichoderma harzianum against Fusarium oxysporum Potencial de biocontrole de mutantes sal-tolerantes de Trichoderma harzianum contra Fusarium oxysporum

    Directory of Open Access Journals (Sweden)

    Hassan Abdel-Latif A. Mohamed

    2006-06-01

    Full Text Available Exposing a wild-type culture of Trichoderma harzianum to gamma irradiation induced two stable salt-tolerant mutants (Th50M6 and Th50M11. Under saline conditions, both mutants greatly surpassed their wild type strain in growth rate, sporulation and biological proficiency against Fusarium oxysporum, the causal agent of tomato wilt disease. Tolerant T. harzianum mutants detained a capability to grow and convinced sporulation in growth media containing up to 69 mM NaCl. In comparison with their parent strain, characterization of both mutants confirmed that they have reinforced contents of proline and hydroxyproline, relatively higher sodium content compared to potassium, calcium or magnesium contents, higher level of total phenols. Electrophoretic analysis of total soluble proteins in the salt tolerance mutant Th50M6 showed different bands accumulated in response to 69 mM NaCl. Data also showed that mutants produce certain active metabolites, such as chitinases, cellulases, beta-galactosidases, as well as, some antibiotics i.e., trichodermin, gliotoxin and gliovirin. Trichoderma mutants significantly reduced wilt disease incidence and improved yield and mineral contents of tomato plants under both saline and non-saline soil conditions, as well as, under infested and natural conditions. T. harzianum mutants were also more efficient in dropping the F. oxysporum growth in rhizosphere compared to the wild type strain. Population density of both mutants in rhizosphere far exceeded that of T. harzianum wild type strain.A exposição de uma cepa selvagem de Trichoderma harzianum à irradiação gama induziu dois mutantes tolerantes a sal (Th50M6 e Th50M11. Em condições salinas, os dois mutantes foram muito superiores à cepa selvagem em relação à velocidade de multiplicação, esporulação e eficiência contra Fusarium oxysporum, o agente causador da doença wilt do tomate. Os mutantes tolerantes foram capazes de multiplicação e esporulação em

  9. The Truncated C-terminal Fragment of Mutant ATXN3 Disrupts Mitochondria Dynamics in Spinocerebellar Ataxia Type 3 Models

    Directory of Open Access Journals (Sweden)

    Jung-Yu Hsu

    2017-06-01

    Full Text Available Spinocerebellar ataxia type 3 (SCA3, known as Machado-Joseph disease, is an autosomal dominant disease caused by an abnormal expansion of polyglutamine in ATXN3 gene, leading to neurodegeneration in SCA3 patients. Similar to other neurodegenerative diseases, the dysfunction of mitochondria is observed to cause neuronal death in SCA3 patients. Based on previous studies, proteolytic cleavage of mutant ATXN3 is found to produce truncated C-terminal fragments in SCA3 models. However, whether these truncated mutant fragments disturb mitochondrial functions and result in pathological death is still unclear. Here, we used neuroblastoma cell and transgenic mouse models to examine the effects of truncated mutant ATXN3 on mitochondria functions. In different models, we observed truncated mutant ATXN3 accelerated the formation of aggregates, which translocated into the nucleus to form intranuclear aggregates. In addition, truncated mutant ATXN3 caused more mitochondrial fission, and decreased the expression of mitochondrial fusion markers, including Mfn-1 and Mfn-2. Furthermore, truncated mutant ATXN3 decreased the mitochondrial membrane potential, increased reactive oxygen species and finally increased cell death rate. In transgenic mouse models, truncated mutant ATXN3 also led to more mitochondrial dysfunction, neurodegeneration and cell death in the cerebellums. This study supports the toxic fragment hypothesis in SCA3, and also provides evidence that truncated mutant ATXN3 is severer than full-length mutant one in vitro and in vivo.

  10. Arabidopsis mutant bik1 exhibits strong resistance to Plasmodiophora brassicae

    Directory of Open Access Journals (Sweden)

    Tao Chen

    2016-09-01

    Full Text Available Botrytis-induced kinase1 (BIK1, a receptor-like cytoplasmic kinase, plays an important role in resistance against pathogens and insects in Arabidopsis thaliana. However, it remains unknown whether BIK1 functions against Plasmodiophora brassicae, an obligate biotrophic protist that attacks cruciferous plants and induces gall formation on roots. Here, we investigated the potential roles of receptors FLS2, BAK1 and BIK1 in the infection of P. brassicae cruciferous plants. Wild-type plants, fls2 and bak1 mutants showed typical symptom on roots, and the galls were filled with large quantities of resting spores, while bik1 mutant plants exhibited strong resistance to P. brassicae. Compared with that of the wild-type plants, the root hair and cortical infection rate of bik1 mutant were significantly reduced by about 40-50%. A considerable portion of bik1 roots failed to form typical galls. Even if some small galls were formed, they were filled with multinucleate secondary plasmodia. The bik1 plants accumulated less reactive oxygen species (ROS at infected roots than other mutants and wild-type plants. Exogenous salicylic acid (SA treatment alleviated the clubroot symptoms in wild-type plants, and the expression of the SA signaling marker gene PR1 was significantly increased in bik1. Both sid2 (salicylic acid induction-deficient 2 and npr1-1 (non-expresser of PR genes that regulate systemic acquired resistance (SAR mutants showed increased susceptibility to P. brassicae compared with wild-type plants. These results suggest that the resistance of bik1 to P. brassicae is possibly mediated by SA inducible mechanisms enhance the resistance to clubroot disease.

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

    Directory of Open Access Journals (Sweden)

    Chao Liu

    2015-08-01

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

  12. Mitochondrial quality control: Cell-type-dependent responses to pathological mutant mitochondrial DNA.

    Science.gov (United States)

    Malena, Adriana; Pantic, Boris; Borgia, Doriana; Sgarbi, Gianluca; Solaini, Giancarlo; Holt, Ian J; Spinazzola, Antonella; Perissinotto, Egle; Sandri, Marco; Baracca, Alessandra; Vergani, Lodovica

    2016-11-01

    Pathological mutations in the mitochondrial DNA (mtDNA) produce a diverse range of tissue-specific diseases and the proportion of mutant mitochondrial DNA can increase or decrease with time via segregation, dependent on the cell or tissue type. Previously we found that adenocarcinoma (A549.B2) cells favored wild-type (WT) mtDNA, whereas rhabdomyosarcoma (RD.Myo) cells favored mutant (m3243G) mtDNA. Mitochondrial quality control (mtQC) can purge the cells of dysfunctional mitochondria via mitochondrial dynamics and mitophagy and appears to offer the perfect solution to the human diseases caused by mutant mtDNA. In A549.B2 and RD.Myo cybrids, with various mutant mtDNA levels, mtQC was explored together with macroautophagy/autophagy and bioenergetic profile. The 2 types of tumor-derived cell lines differed in bioenergetic profile and mitophagy, but not in autophagy. A549.B2 cybrids displayed upregulation of mitophagy, increased mtDNA removal, mitochondrial fragmentation and mitochondrial depolarization on incubation with oligomycin, parameters that correlated with mutant load. Conversely, heteroplasmic RD.Myo lines had lower mitophagic markers that negatively correlated with mutant load, combined with a fully polarized and highly fused mitochondrial network. These findings indicate that pathological mutant mitochondrial DNA can modulate mitochondrial dynamics and mitophagy in a cell-type dependent manner and thereby offer an explanation for the persistence and accumulation of deleterious variants.

  13. Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes

    Science.gov (United States)

    Pratt, Ashley J.; Shin, David S.; Merz, Gregory E.; Rambo, Robert P.; Lancaster, W. Andrew; Dyer, Kevin N.; Borbat, Peter P.; Poole, Farris L.; Adams, Michael W. W.; Freed, Jack H.; Crane, Brian R.; Tainer, John A.; Getzoff, Elizabeth D.

    2014-01-01

    Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu2+ binding site and defined cross-dimer copper–copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies. PMID:25316790

  14. Rescue of a nephrogenic diabetes insipidus-causing vasopressin V-2 receptor mutant by cell-penetrating peptides

    NARCIS (Netherlands)

    Oueslati, Morad; Hermosilla, Ricardo; Schoenenberger, Eva; Oorschot, Viola; Beyermann, Michael; Wiesner, Burkhard; Schmidt, Antje; Klumperman, Judith; Rosenthal, Walter; Schuelein, Ralf

    2007-01-01

    Mutant membrane proteins are frequently retained in the early secretory pathway by a quality control system, thereby causing disease. An example are mutants of the vasopressin V-2 receptor (V2R) leading to nephrogenic diabetes insipidus. Transport-defective V(2)Rs fall into two classes: those

  15. Cell culture attenuation eliminates rMd5deltaMeq-induced bursal and thymic atrophy and renders the mutant virus as an effective and safe vaccine against Marek's disease

    Science.gov (United States)

    Marek’s disease virus (MDV) encodes a basic leucine zipper oncoprotein, meq, which structurally resembles jun/fos family of transcriptional activators. It has been clearly demonstrated that deletion of meq results in loss of transformation and oncogenic capacity of MDV. The rMd5'meq virus provided s...

  16. Separation of foot-and-mouth disease virus leader protein activities; identification of mutants that retain efficient self-processing activity but poorly induce eIF4G cleavage

    DEFF Research Database (Denmark)

    Belsham, Graham; Hua Guan, Su

    2017-01-01

    Foot-and-mouth disease virus (FMDV) is a picornavirus and its RNA genome encodes a large polyprotein. The N-terminal part of this polyprotein is the Leader protein, a cysteine protease, termed Lpro. The virus causes the rapid inhibition of host cell capdependent protein synthesis within infected...

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

    Energy Technology Data Exchange (ETDEWEB)

    Busch, Albert; Kiel, Tilman; Heupel, Wolfgang-M. [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany); Wehnert, Manfred [Institute of Human Genetics, University of Greifswald, Greifswald (Germany); Huebner, Stefan, E-mail: stefan.huebner@mail.uni-wuerzburg.de [University of Wuerzburg, Institute of Anatomy and Cell Biology, Koellikerstrasse 6, 97070 Wuerzburg (Germany)

    2009-08-15

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

  18. Responses to novelty in staggerer mutant mice.

    Science.gov (United States)

    Misslin, R; Cigrang, M; Guastavino, J M

    1986-01-01

    Responses to novelty in normal C57BL/6 and staggerer mutant mice were recorded. The normal mice confronted a novel object in their familiar environment showed avoidance and burying responses while the staggerer mutant mice contacted it. When given the opportunity to move around freely in simultaneously presented novel and familiar environments, the mutant mice more quickly entered the novel areas than normal animals. these data reveal a significant decrease in the neophobic components of the neotic behaviour in the staggerer mice. However, since the mutant mice did not show a locomotor deficit, the impairment of neophobia seems not to be due to the gait abnormalities of these animals. The results support the view that the cerebellum may contribute to the organization of complex behaviours. Copyright © 1986. Published by Elsevier B.V.

  19. Robust mutant strain design by pessimistic optimization.

    Science.gov (United States)

    Apaydin, Meltem; Xu, Liang; Zeng, Bo; Qian, Xiaoning

    2017-10-03

    Flux Balance Analysis (FBA) based mathematical modeling enables in silico prediction of systems behavior for genome-scale metabolic networks. Computational methods have been derived in the FBA framework to solve bi-level optimization for deriving "optimal" mutant microbial strains with targeted biochemical overproduction. The common inherent assumption of these methods is that the surviving mutants will always cooperate with the engineering objective by overproducing the maximum desired biochemicals. However, it has been shown that this optimistic assumption may not be valid in practice. We study the validity and robustness of existing bi-level methods for strain optimization under uncertainty and non-cooperative environment. More importantly, we propose new pessimistic optimization formulations: P-ROOM and P-OptKnock, aiming to derive robust mutants with the desired overproduction under two different mutant cell survival models: (1) ROOM assuming mutants have the minimum changes in reaction fluxes from wild-type flux values, and (2) the one considered by OptKnock maximizing the biomass production yield. When optimizing for desired overproduction, our pessimistic formulations derive more robust mutant strains by considering the uncertainty of the cell survival models at the inner level and the cooperation between the outer- and inner-level decision makers. For both P-ROOM and P-OptKnock, by converting multi-level formulations into single-level Mixed Integer Programming (MIP) problems based on the strong duality theorem, we can derive exact optimal solutions that are highly scalable with large networks. Our robust formulations P-ROOM and P-OptKnock are tested with a small E. coli core metabolic network and a large-scale E. coli iAF1260 network. We demonstrate that the original bi-level formulations (ROOM and OptKnock) derive mutants that may not achieve the predicted overproduction under uncertainty and non-cooperative environment. The knockouts obtained by the

  20. Characterization of MarR Superrepressor Mutants

    OpenAIRE

    Alekshun, Michael N.; Levy, Stuart B.

    1999-01-01

    MarR negatively regulates expression of the multiple antibiotic resistance (mar) locus in Escherichia coli. Superrepressor mutants, generated in order to study regions of MarR required for function, exhibited altered inducer recognition properties in whole cells and increased DNA binding to marO in vitro. Mutations occurred in three areas of the relatively small MarR protein (144 amino acids). It is surmised that superrepression results from increased DNA binding activities of these mutant pr...

  1. Patulin degradation in saccharomyces cerevisiae: Sensitive mutants.

    Science.gov (United States)

    Thonart, P; Sumbu, Z L; Bechet, J

    1985-03-01

    The present experiments (sensitive mutants and transient inhibition of growth) are compatible with the synthesis of an inductible detoxifying substance in the wild type strain. This substance could be glutathione because glutathione detoxification scheme essentially involves properties of the SH group and it is well known that patulin reacts with sulfhy dril groups.Studies are presently being carried out with sensitive mutants to establish definitively the relation between intracellular pool of glutathone and the resistance mechanism of a yeast to patulin.

  2. Protection from genital herpes disease, seroconversion and latent infection in a non-lethal murine genital infection model by immunization with an HSV-2 replication-defective mutant virus.

    Science.gov (United States)

    Diaz, Fernando M; Knipe, David M

    2016-01-15

    Viral vaccines have traditionally protected against disease, but for viruses that establish latent infection, it is desirable for the vaccine to reduce infection to reduce latent infection and reactivation. While seroconversion has been used in clinical trials of herpes simplex virus (HSV) vaccines to measure protection from infection, this has not been modeled in animal infection systems. To measure the ability of a genital herpes vaccine candidate to protect against various aspects of infection, we established a non-lethal murine model of genital HSV-2 infection, an ELISA assay to measure antibodies specific for infected cell protein 8 (ICP8), and a very sensitive qPCR assay. Using these assays, we observed that immunization with HSV-2 dl5-29 virus reduced disease, viral shedding, seroconversion, and latent infection by the HSV-2 challenge virus. Therefore, it may be feasible to obtain protection against genital disease, seroconversion and latent infection by immunization, even if sterilizing immunity is not achieved. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Modulation of mutant huntingtin N-terminal cleavage and its effect on aggregation and cell death

    NARCIS (Netherlands)

    Juenemann, Katrin; Weisse, Christina; Reichmann, Denise; Kaether, Christoph; Calkhoven, Cornelis F.; Schilling, Gabriele

    2011-01-01

    Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion near the N-terminus of huntingtin. A neuropathological hallmark of Huntington's disease is the presence of intracellular aggregates composed of mutant huntingtin N-terminal fragments in human postmortem

  4. Copper-sensitive mutant of Arabidopsis thaliana.

    Science.gov (United States)

    van Vliet, C; Anderson, C R; Cobbett, C S

    1995-11-01

    A Cu-sensitive mutant, cup1-1, of Arabidopsis thaliana has a pattern of heavy-metal sensitivity different from that of the cad1 and cad2 mutants, which are deficient in phytochelatin biosynthesis. The latter are significantly sensitive to Cd and Hg and only slightly sensitive to Cu, whereas the cup1-1 mutant is significantly sensitive to Cu, slightly sensitive to Cd, and not more sensitive to Hg, compared to the wild type. Genetic analysis has shown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus, which has been mapped to chromosome 1. Genetic and biochemical studies demonstrate that the cup1-1 mutant is not affected in phytochelatin biosynthesis or function. The sensitive phenotype of the cup1-1 mutant is associated with, and probably due to, increased accumulation of higher levels of Cd and Cu compared with the wild type. Consistent with this, a Cu-inducible, root-specific metallothionein gene, MT2a, is expressed in cup1-1 roots under conditions in which it is not expressed in the wild type. Undifferentiated cup1-1 callus tissue did not show the Cu-sensitive phenotype, suggesting that the mutant phenotype, in contrast to cad1 and cad2, is not expressed at the cellular level.

  5. Plasmodium berghei: in vivo generation and selection of karyotype mutants and non-gametocyte producer mutants

    NARCIS (Netherlands)

    Janse, C. J.; Ramesar, J.; van den Berg, F. M.; Mons, B.

    1992-01-01

    We previously reported that karyotype and gametocyte-producer mutants spontaneously arose during in vivo asexual multiplication of Plasmodium berghei. Here we studied the rate of selection of these mutants in vivo. Gametocyte production and karyotype pattern were established at regular intervals

  6. Comparative Proteomic Analysis of Wild-Type and SAP Domain Mutant Foot-and-Mouth Disease Virus-Infected Porcine Cells Identifies the Ubiquitin-Activating Enzyme UBE1 Required for Virus Replication.

    Science.gov (United States)

    Zhu, Zixiang; Yang, Fan; Zhang, Keshan; Cao, Weijun; Jin, Ye; Wang, Guoqing; Mao, Ruoqing; Li, Dan; Guo, Jianhong; Liu, Xiangtao; Zheng, Haixue

    2015-10-02

    Leader protein (L(pro)) of foot-and-mouth disease virus (FMDV) manipulates the activities of several host proteins to promote viral replication and pathogenicity. L(pro) has a conserved protein domain SAP that is suggested to subvert interferon (IFN) production to block antiviral responses. However, apart from blocking IFN production, the roles of the SAP domain during FMDV infection in host cells remain unknown. Therefore, we identified host proteins associated with the SAP domain of L(pro) by a high-throughput quantitative proteomic approach [isobaric tags for relative and absolute quantitation (iTRAQ) in conjunction with liquid chromatography/electrospray ionization tandem mass spectrometry]. Comparison of the differentially regulated proteins in rA/FMDVΔmSAP- versus rA/FMDV-infected SK6 cells revealed 45 down-regulated and 32 up-regulated proteins that were mostly associated with metabolic, ribosome, spliceosome, and ubiquitin-proteasome pathways. The results also imply that the SAP domain has a function similar to SAF-A/B besides its potential protein inhibitor of activated signal transducer and activator of transcription (PIAS) function. One of the identified proteins UBE1 was further analyzed and displayed a novel role for the SAP domain of L(pro). Overexpression of UBE1 enhanced the replication of FMDV, and knockdown of UBE1 decreased FMDV replication. This shows that FMDV manipulates UBE1 for increased viral replication, and the SAP domain was involved in this process.

  7. Escherichia coli mutants with a temperature-sensitive alcohol dehydrogenase.

    OpenAIRE

    Lorowitz, W; CLARK, D.

    1982-01-01

    Mutants of Escherichia coli resistant to allyl alcohol were selected. Such mutants were found to lack alcohol dehydrogenase. In addition, mutants with temperature-sensitive alcohol dehydrogenase activity were obtained. These mutations, designated adhE, are all located at the previously described adh regulatory locus. Most adhE mutants were also defective in acetaldehyde dehydrogenase activity.

  8. Targeting mutant NRAS signaling pathways in melanoma.

    Science.gov (United States)

    Vu, Ha Linh; Aplin, Andrew E

    2016-05-01

    Cutaneous melanoma is a devastating form of skin cancer and its incidence is increasing faster than any other preventable cancer in the United States. The mutant NRAS subset of melanoma is more aggressive and associated with poorer outcomes compared to non-NRAS mutant melanoma. The aggressive nature and complex molecular signaling conferred by this transformation has evaded clinically effective treatment options. This review examines the major downstream effectors of NRAS relevant in melanoma and the associated advances made in targeted therapies that focus on these effector pathways. We outline the history of MEK inhibition in mutant NRAS melanoma and recent advances with newer MEK inhibitors. Since MEK inhibitors will likely be optimized when combined with other targeted therapies, we focus on recently identified targets that can be used in combination with MEK inhibitors. Published by Elsevier Ltd.

  9. High Persister Mutants in Mycobacterium tuberculosis.

    Directory of Open Access Journals (Sweden)

    Heather L Torrey

    Full Text Available Mycobacterium tuberculosis forms drug-tolerant persister cells that are the probable cause of its recalcitrance to antibiotic therapy. While genetically identical to the rest of the population, persisters are dormant, which protects them from killing by bactericidal antibiotics. The mechanism of persister formation in M. tuberculosis is not well understood. In this study, we selected for high persister (hip mutants and characterized them by whole genome sequencing and transcriptome analysis. In parallel, we identified and characterized clinical isolates that naturally produce high levels of persisters. We compared the hip mutants obtained in vitro with clinical isolates to identify candidate persister genes. Genes involved in lipid biosynthesis, carbon metabolism, toxin-antitoxin systems, and transcriptional regulators were among those identified. We also found that clinical hip isolates exhibited greater ex vivo survival than the low persister isolates. Our data suggest that M. tuberculosis persister formation involves multiple pathways, and hip mutants may contribute to the recalcitrance of the infection.

  10. MicroNeurotrophins Improve Survival in Motor Neuron-Astrocyte Co-Cultures but Do Not Improve Disease Phenotypes in a Mutant SOD1 Mouse Model of Amyotrophic Lateral Sclerosis.

    Directory of Open Access Journals (Sweden)

    Kelly E Glajch

    Full Text Available Amyotrophic Lateral Sclerosis (ALS is a neurodegenerative disease caused by loss of motor neurons. ALS patients experience rapid deterioration in muscle function with an average lifespan of 3-5 years after diagnosis. Currently, the most effective therapeutic only extends lifespan by a few months, thus highlighting the need for new and improved therapies. Neurotrophic factors (NTFs are important for neuronal development, maintenance, and survival. NTF treatment has previously shown efficacy in pre-clinical ALS models. However, clinical trials using NTFs produced no major improvements in ALS patients, due in part to the limited blood brain barrier (BBB penetration. In this study we assessed the potential neuroprotective effects of a novel class of compounds known as MicroNeurotrophins (MNTs. MNTs are derivatives of Dehydroepiandrosterone (DHEA, an endogenous neurosteroid that can cross the BBB and bind to tyrosine kinase receptors mimicking the pro-survival effects of NTFs. Here we sought to determine whether MNTs were neuroprotective in two different models of ALS. Our results demonstrate that BNN27 (10 μM attenuated loss of motor neurons co-cultured with astrocytes derived from human ALS patients with SOD1 mutations via the reduction of oxidative stress. Additionally, in the G93A SOD1 mouse, BNN27 (10 mg/kg treatment attenuated motor behavioral impairment in the paw grip endurance and rotarod tasks at postnatal day 95 in female but not male mice. In contrast, BNN27 (10 mg/kg and 50 mg/kg treatment did not alter any other behavioral outcome or neuropathological marker in male or female mice. Lastly, BNN27 was not detected in post-mortem brain or spinal cord tissue of treated mice due to the rapid metabolism of BNN27 by mouse hepatocytes relative to human hepatocytes. Together, these findings demonstrate that BNN27 treatment failed to yield significant neuroprotective effects in the G93A SOD1 model likely due to its rapid rate of metabolism in

  11. Aging Kit mutant mice develop cardiomyopathy.

    Directory of Open Access Journals (Sweden)

    Lei Ye

    Full Text Available Both bone marrow (BM and myocardium contain progenitor cells expressing the c-Kit tyrosine kinase. The aims of this study were to determine the effects of c-Kit mutations on: i. myocardial c-Kit(+ cells counts and ii. the stability of left ventricular (LV contractile function and structure during aging. LV structure and contractile function were evaluated (echocardiography in two groups of Kit mutant (W/Wv and W41/W42 and in wild type (WT mice at 4 and 12 months of age and the effects of the mutations on LV mass, vascular density and the numbers of proliferating cells were also determined. In 4 month old Kit mutant and WT mice, LV ejection fractions (EF and LV fractional shortening rates (FS were comparable. At 12 months of age EF and FS were significantly decreased and LV mass was significantly increased only in W41/W42 mice. Myocardial vascular densities and c-Kit(+ cell numbers were significantly reduced in both mutant groups when compared to WT hearts. Replacement of mutant BM with WT BM at 4 months of age did not prevent these abnormalities in either mutant group although they were somewhat attenuated in the W/Wv group. Notably BM transplantation did not prevent the development of cardiomyopathy in 12 month W41/W42 mice. The data suggest that decreased numbers and functional capacities of c-Kit(+ cardiac resident progenitor cells may be the basis of the cardiomyopathy in W41/W42 mice and although defects in mutant BM progenitor cells may prove to be contributory, they are not causal.

  12. Generation and screening of a comprehensive Mycobacterium avium subsp. paratuberculosis transposon mutant bank.

    Science.gov (United States)

    Rathnaiah, Govardhan; Lamont, Elise A; Harris, N Beth; Fenton, Robert J; Zinniel, Denise K; Liu, Xiaofei; Sotos, Josh; Feng, Zhengyu; Livneh-Kol, Ayala; Shpigel, Nahum Y; Czuprynski, Charles J; Sreevatsan, Srinand; Barletta, Raúl G

    2014-01-01

    Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95%) was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated.

  13. Ovarian abnormalities in the staggerer mutant mouse.

    Science.gov (United States)

    Guastavino, Jean-Marie; Boufares, Salima; Crusio, Wim E

    2005-08-24

    Disturbances in several reproductive functions of the staggerer cerebellar mutant mouse have been observed. In this study, reproductive efficiency of staggerer mice was compared to normal mice by recording the number of pups produced and the number of oocytes occurring. It was found that staggerer mothers produced smaller litters than controls and the number of oocytes produced in their ovaries was reduced by the staggerer mutation. These results indicate a pleiotropic effect on fertility of the Rora(sg) gene underlying the cerebellar abnormalities of the staggerer mutant.

  14. Ovarian Abnormalities in the Staggerer Mutant Mouse

    Directory of Open Access Journals (Sweden)

    Jean-Marie Guastavino

    2005-01-01

    Full Text Available Disturbances in several reproductive functions of the staggerer cerebellar mutant mouse have been observed. In this study, reproductive efficiency of staggerer mice was compared to normal mice by recording the number of pups produced and the number of oocytes occurring. It was found that staggerer mothers produced smaller litters than controls and the number of oocytes produced in their ovaries was reduced by the staggerer mutation. These results indicate a pleiotropic effect on fertility of the Rorasg gene underlying the cerebellar abnormalities of the staggerer mutant.

  15. Genetic basis of kidney cancer: Role of genomics for the development of disease-based therapeutics

    Science.gov (United States)

    Linehan, W. Marston

    2012-01-01

    Kidney cancer is not a single disease; it is made up of a number of different types of cancer, including clear cell, type 1 papillary, type 2 papillary, chromophobe, TFE3, TFEB, and oncocytoma. Sporadic, nonfamilial kidney cancer includes clear cell kidney cancer (75%), type 1 papillary kidney cancer (10%), papillary type 2 kidney cancer (including collecting duct and medullary RCC) (5%), the microphalmia-associated transcription (MiT) family translocation kidney cancers (TFE3, TFEB, and MITF), chromophobe kidney cancer (5%), and oncocytoma (5%). Each has a distinct histology, a different clinical course, responds differently to therapy, and is caused by mutation in a different gene. Genomic studies identifying the genes for kidney cancer, including the VHL, MET, FLCN, fumarate hydratase, succinate dehydrogenase, TSC1, TSC2, and TFE3 genes, have significantly altered the ways in which patients with kidney cancer are managed. While seven FDA-approved agents that target the VHL pathway have been approved for the treatment of patients with advanced kidney cancer, further genomic studies, such as whole genome sequencing, gene expression patterns, and gene copy number, will be required to gain a complete understanding of the genetic basis of kidney cancer and of the kidney cancer gene pathways and, most importantly, to provide the foundation for the development of effective forms of therapy for patients with this disease. PMID:23038766

  16. Interleukin-1 hyperproduction by in vitro activated peripheral macrophages from cerebellar mutant mice.

    Science.gov (United States)

    Kopmels, B; Wollman, E E; Guastavino, J M; Delhaye-Bouchaud, N; Fradelizi, D; Mariani, J

    1990-12-01

    Several mutations in mice produce complex patterns of neuronal degeneration of the cerebellum and of its afferent pathways. In the staggerer (sg/sg) mutant, atrophy of the lymphoid organs and immunological abnormalities have been described. To search for a possible link between the neurological and the immune disorders in this mutant, we studied the production by its peripheral macrophages of interleukin-1 (IL-1), which roles in both immune and nervous systems are well established. Suspensions of peritoneal and/or spleen macrophages from mutants and their appropriate controls were stimulated in vitro by lipopolysaccharide. Northern and dot blots, performed with murine IL-1 cDNA probes, revealed a clear-cut hyperexpression of IL-1 mRNA in staggerer macrophages. An IL-1 bioassay using the IL-1-responsive D10.G4 cell line also revealed a sixfold increase of IL-1 activity in the macrophage supernatants of staggerer mutant mice. The hyperproduction was found in 3-week to 1-year-old staggerer and also in heterozygous (+/sg) mice. A similar phenomenon existed in cerebellar mutants lurcher, Purkinje cell degeneration (pcd), and to a lesser extent reeler and wobbler, but was absent in the neurological mutants weaver, jimpy, and motor end plate disease (medH). These observations establish that in several point mutations in mice, central nervous degeneration is associated with dysregulation of IL-1 production by peripheral macrophages.

  17. Phenotypic Characterization of a Comprehensive Set of MAPK1/ERK2 Missense Mutants

    Directory of Open Access Journals (Sweden)

    Lisa Brenan

    2016-10-01

    Full Text Available Tumor-specific genomic information has the potential to guide therapeutic strategies and revolutionize patient treatment. Currently, this approach is limited by an abundance of disease-associated mutants whose biological functions and impacts on therapeutic response are uncharacterized. To begin to address this limitation, we functionally characterized nearly all (99.84% missense mutants of MAPK1/ERK2, an essential effector of oncogenic RAS and RAF. Using this approach, we discovered rare gain- and loss-of-function ERK2 mutants found in human tumors, revealing that, in the context of this assay, mutational frequency alone cannot identify all functionally impactful mutants. Gain-of-function ERK2 mutants induced variable responses to RAF-, MEK-, and ERK-directed therapies, providing a reference for future treatment decisions. Tumor-associated mutations spatially clustered in two ERK2 effector-recruitment domains yet produced mutants with opposite phenotypes. This approach articulates an allele-characterization framework that can be scaled to meet the goals of genome-guided oncology.

  18. Phenotypic Characterization of a Comprehensive Set of MAPK1/ERK2 Missense Mutants.

    Science.gov (United States)

    Brenan, Lisa; Andreev, Aleksandr; Cohen, Ofir; Pantel, Sasha; Kamburov, Atanas; Cacchiarelli, Davide; Persky, Nicole S; Zhu, Cong; Bagul, Mukta; Goetz, Eva M; Burgin, Alex B; Garraway, Levi A; Getz, Gad; Mikkelsen, Tarjei S; Piccioni, Federica; Root, David E; Johannessen, Cory M

    2016-10-18

    Tumor-specific genomic information has the potential to guide therapeutic strategies and revolutionize patient treatment. Currently, this approach is limited by an abundance of disease-associated mutants whose biological functions and impacts on therapeutic response are uncharacterized. To begin to address this limitation, we functionally characterized nearly all (99.84%) missense mutants of MAPK1/ERK2, an essential effector of oncogenic RAS and RAF. Using this approach, we discovered rare gain- and loss-of-function ERK2 mutants found in human tumors, revealing that, in the context of this assay, mutational frequency alone cannot identify all functionally impactful mutants. Gain-of-function ERK2 mutants induced variable responses to RAF-, MEK-, and ERK-directed therapies, providing a reference for future treatment decisions. Tumor-associated mutations spatially clustered in two ERK2 effector-recruitment domains yet produced mutants with opposite phenotypes. This approach articulates an allele-characterization framework that can be scaled to meet the goals of genome-guided oncology. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  19. Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks

    Science.gov (United States)

    Yang, Hee-Jeong; Bogomolnaya, Lydia M.; Elfenbein, Johanna R.; Endicott-Yazdani, Tiana; Reynolds, M. Megan; Porwollik, Steffen; Cheng, Pui; Xia, Xiao-Qin

    2016-01-01

    Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection. PMID:26857572

  20. Generation and characterization of pigment mutants of ...

    African Journals Online (AJOL)

    acer

    The result of bio-test, using the resulting pigment mutant of C. reinhardtii 124y-1 showed that mutagenic activity was observed significantly in both Tekeli River and Pavlodar Oil Refinery in Kazakhstan; the waste water of the. Pavlodar Oil Refinery had high-toxicity while the water of the Tekeli River had medium-toxicity.

  1. Avirulent mutants of Macrophomina phaseolina and Aspergillus ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Biosciences; Volume 25; Issue 1. Avirulent mutants of Macrophomina phaseolina and Aspergillus fumigatus initiate infection in Phaseolus mungo in the presence of phaseo-linone; levamisole gives protection. Suchandra Sett Santosh K Mishra Kazia I Siddiqui. Articles Volume 25 Issue 1 March ...

  2. Ethanol production using engineered mutant E. coli

    Science.gov (United States)

    Ingram, Lonnie O.; Clark, David P.

    1991-01-01

    The subject invention concerns novel means and materials for producing ethanol as a fermentation product. Mutant E. coli are transformed with a gene coding for pyruvate decarboxylase activity. The resulting system is capable of producing relatively large amounts of ethanol from a variety of biomass sources.

  3. Flocculation phenomenon of a mutant flocculent Saccharomyces ...

    African Journals Online (AJOL)

    Flocculation phenomenon of a mutant flocculent Saccharomyces cerevisiae strain: Effects of metal ions, sugars, temperature, pH, protein-denaturants and ... was in the early stationary growth phase, which coincided with glucose depletion in the batch fermentation for the production of ethanol from kitchen refuse medium.

  4. Mutant PTEN in Cancer : Worse Than Nothing

    NARCIS (Netherlands)

    Leslie, Nick R; den Hertog, Jeroen

    2014-01-01

    Tumor suppressors block the development of cancer and are often lost during tumor development. Papa et al. show that partial loss of normal PTEN tumor suppressor function can be compounded by additional disruption caused by the expression of inactive mutant PTEN protein. This has significant

  5. Mutant IDH1 Disrupts the Mouse Subventricular Zone and Alters Brain Tumor Progression.

    Science.gov (United States)

    Pirozzi, Christopher J; Carpenter, Austin B; Waitkus, Matthew S; Wang, Catherine Y; Zhu, Huishan; Hansen, Landon J; Chen, Lee H; Greer, Paula K; Feng, Jie; Wang, Yu; Bock, Cheryl B; Fan, Ping; Spasojevic, Ivan; McLendon, Roger E; Bigner, Darell D; He, Yiping; Yan, Hai

    2017-05-01

    IDH1 mutations occur in the majority of low-grade gliomas and lead to the production of the oncometabolite, D-2-hydroxyglutarate (D-2HG). To understand the effects of tumor-associated mutant IDH1 (IDH1-R132H) on both the neural stem cell (NSC) population and brain tumorigenesis, genetically faithful cell lines and mouse model systems were generated. Here, it is reported that mouse NSCs expressing Idh1-R132H displayed reduced proliferation due to p53-mediated cell-cycle arrest as well as a decreased ability to undergo neuronal differentiation. In vivo , Idh1-R132H expression reduced proliferation of cells within the germinal zone of the subventricular zone (SVZ). The NSCs within this area were dispersed and disorganized in mutant animals, suggesting that Idh1-R132H perturbed the NSCs and the microenvironment from which gliomas arise. In addition, tumor-bearing animals expressing mutant Idh1 displayed a prolonged survival and also overexpressed Olig2, features consistent with IDH1-mutated human gliomas. These data indicate that mutant Idh1 disrupts the NSC microenvironment and the candidate cell-of-origin for glioma; thus, altering the progression of tumorigenesis. In addition, this study provides a mutant Idh1 brain tumor model that genetically recapitulates human disease, laying the foundation for future investigations on mutant IDH1 -mediated brain tumorigenesis and targeted therapy. Implications: Through the use of a conditional mutant mouse model that confers a less aggressive tumor phenotype, this study reveals that mutant Idh1 impacts the candidate cell-of-origin for gliomas. Mol Cancer Res; 15(5); 507-20. ©2017 AACR . ©2017 American Association for Cancer Research.

  6. Mutant p53 Amplifies Epidermal Growth Factor Receptor Family Signaling to Promote Mammary Tumorigenesis.

    Science.gov (United States)

    Yallowitz, Alisha R; Li, Dun; Lobko, Anthony; Mott, Daniel; Nemajerova, Alice; Marchenko, Natalia

    2015-04-01

    The EGFR family (ErbB2/Her2 and EGFR/ErbB1/Her1) often modulates the transcriptional program involved in promoting mammary tumorigenesis. In humans, the majority of ErbB2-positive sporadic breast cancers harbor p53 mutations, which correlate with poor prognosis. Also, the extremely high incidence of ErbB2-positive breast cancer in women with p53 germline mutations (Li-Fraumeni syndrome) suggests a key role of mutant p53 specifically in ErbB2-mediated mammary tumorigenesis. To examine the role of mutant p53 during ErbB2-mediated mammary tumorigenesis, a mutant p53 allele (R172H) was introduced into the (MMTV)-ErbB2/Neu mouse model system. Interestingly, we show in heterozygous p53 mice that mutant p53 R172H is a more potent activator of ErbB2-mediated mammary tumorigenesis than simple loss of p53. The more aggressive disease in mutant p53 animals was reflected by earlier tumor onset, increased mammary tumor multiplicity, and shorter survival. These in vivo and in vitro data provide mechanistic evidence that mutant p53 amplifies ErbB2 and EGFR signaling to promote the expansion of mammary stem cells and induce cell proliferation. This study identifies mutant p53 as an essential player in ErbB2 and EGFR-mediated mammary tumorigenesis and indicates the potential translational importance of targeting mutant p53 in this subset of patients with breast cancer. ©2015 American Association for Cancer Research.

  7. Erwinia amylovora pyrC mutant causes fire blight despite pyrimidine auxotrophy.

    Science.gov (United States)

    Ramos, L S; Sinn, J P; Lehman, B L; Pfeufer, E E; Peter, K A; McNellis, T W

    2015-06-01

    Erwinia amylovora bacteria cause fire blight disease, which affects apple and pear production worldwide. The Erw. amylovora pyrC gene encodes a predicted dihydroorotase enzyme involved in pyrimidine biosynthesis. Here, we discovered that the Erw. amylovora pyrC244::Tn5 mutant was a uracil auxotroph. Unexpectedly, the Erw. amylovora pyrC244::Tn5 mutant grew as well as the wild-type in detached immature apple and pear fruits. Fire blight symptoms caused by the pyrC244::Tn5 mutant in immature apple and pear fruits were attenuated compared to those caused by the wild-type. The pyrC244::Tn5 mutant also caused severe fire blight symptoms in apple tree shoots. A plasmid-borne copy of the wild-type pyrC gene restored prototrophy and symptom induction in apple and pear fruit to the pyrC244::Tn5 mutant. These results suggest that Erw. amylovora can obtain sufficient pyrimidine from the host to support bacterial growth and fire blight disease development, although de novo pyrimidine synthesis by Erw. amylovora is required for full symptom development in fruits. Significance and impact of the study: This study provides information about the fire blight host-pathogen interaction. Although the Erwinia amylovora pyrC mutant was strictly auxotrophic for pyrimidine, it grew as well as the wild-type in immature pear and apple fruits and caused severe fire blight disease in apple trees. This suggests that Erw. amylovora can obtain sufficient pyrimidines from host tissue to support growth and fire blight disease development. This situation contrasts with findings in some human bacterial pathogens, which require de novo pyrimidine synthesis for growth in host blood, for example. © 2015 The Society for Applied Microbiology.

  8. Physiology and pathogenicity of cpdB deleted mutant of avian pathogenic Escherichia coli.

    Science.gov (United States)

    Liu, Huifang; Chen, Liping; Si, Wei; Wang, Chunlai; Zhu, Fangna; Li, Guangxing; Liu, Siguo

    2017-04-01

    Avian colibacillosis is one of the most common infectious diseases caused partially or entirely by avian pathogenic Escherichia coli (APEC) in birds. In addition to spontaneous infection, APEC can also cause secondary infections that result in greater severity of illness and greater losses to the poultry industry. In order to assess the role of 2', 3'-cyclic phosphodiesterase (cpdB) in APEC on disease physiology and pathogenicity, an avian pathogenic Escherichia coli-34 (APEC-34) cpdB mutant was obtained using the Red system. The cpdB mutant grew at a slower rate than the natural strain APEC-34. Scanning electron microscopy (SEM) indicated that the bacteria of the cpdB mutant were significantly longer than the bacteria observed in the natural strain (P<0.01), and that the width of the cpdB mutant was significantly smaller than its natural counterpart (P<0.01). In order to evaluate the role of cpdB in APEC in the colonization of internal organs (lung, liver and spleen) in poultry, seven-day-old SPF chicks were infected with 10 9 CFU/chick of the cpdB mutant or the natural strain. No colonizations of cpdB mutants were observed in the internal organs 10days following the infection, though numerous natural strains were observed at 20days following infection. Additionally, the relative expression of division protein ftsZ, outer membrane protein A ompA, ferric uptake regulator fur and tryptophanase tnaA genes in the mutant strain were all significantly lower than in the natural strain (P<0.05 or P<0.01). These results suggested that cpdB is involved in the long-term colonization of APEC in the internal organs of the test subjects. The deletion of the cpdB gene also significantly affected the APEC growth and morphology. Copyright © 2016. Published by Elsevier Ltd.

  9. Elevated oxidative membrane damage associated with genetic modifiers of Lyst-mutant phenotypes.

    Directory of Open Access Journals (Sweden)

    Colleen M Trantow

    2010-07-01

    Full Text Available LYST is a large cytosolic protein that influences the biogenesis of lysosome-related organelles, and mutation of the encoding gene, LYST, can cause Chediak-Higashi syndrome. Recently, Lyst-mutant mice were recognized to also exhibit an iris disease resembling exfoliation syndrome, a common cause of glaucoma in humans. Here, Lyst-mutant iris phenotypes were used in a search for genes that influence Lyst pathways. In a candidate gene-driven approach, albino Lyst-mutant mice homozygous for a mutation in Tyr, whose product is key to melanin synthesis within melanosomes, exhibited complete rescue of Lyst-mutant iris phenotypes. In a genetic background-driven approach using a DBA/2J strain of congenic mice, an interval containing Tyrp1 enhanced Lyst-dependent iris phenotypes. Thus, both experimental approaches implicated the melanosome, an organelle that is a potential source of oxidative stress, as contributing to the disease phenotype. Confirming an association with oxidative damage, Lyst mutation resulted in genetic context-sensitive changes in iris lipid hydroperoxide levels, being lowest in albino and highest in DBA/2J mice. Surprisingly, the DBA/2J genetic background also exposed a late-onset neurodegenerative phenotype involving cerebellar Purkinje-cell degeneration. These results identify an association between oxidative damage to lipid membranes and the severity of Lyst-mutant phenotypes, revealing a new mechanism that contributes to pathophysiology involving LYST.

  10. A sialidase mutant displaying trans-sialidase activity.

    Science.gov (United States)

    Paris, Gastón; Ratier, Laura; Amaya, María Fernanda; Nguyen, Tong; Alzari, Pedro M; Frasch, Alberto Carlos C

    2005-01-28

    Trypanosoma cruzi, the agent of Chagas disease, expresses a modified sialidase, the trans-sialidase, which transfers sialic acid from host glycoconjugates to beta-galactose present in parasite mucins. Another American trypanosome, Trypanosoma rangeli, expresses a homologous protein that has sialidase activity but is devoid of transglycosidase activity. Based on the recently determined structures of T.rangeli sialidase (TrSA) and T.cruzi trans-sialidase (TcTS), we have now constructed mutants of TrSA with the aim of studying the relevant residues in transfer activity. Five mutations, Met96-Val, Ala98-Pro, Ser120-Tyr, Gly249-Tyr and Gln284-Pro, were enough to obtain a sialidase mutant (TrSA(5mut)) with trans-sialidase activity; and a sixth mutation increased the activity to about 10% that of wild-type TcTS. The crystal structure of TrSA(5mut) revealed the formation of a trans-sialidase-like binding site for the acceptor galactose, primarily defined by the phenol group of Tyr120 and the indole ring of Trp313, which adopts a new conformation, similar to that in TcTS, induced by the Gln284-Pro mutation. The transition state analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA), which inhibits sialidases but is a poor inhibitor of trans-sialidase, was used to probe the active site conformation of mutant enzymes. The results show that the presence of a sugar acceptor binding-site, the fine-tuning of protein-substrate interactions and the flexibility of crucial active site residues are all important to achieve transglycosidase activity from the TrSA sialidase scaffold.

  11. Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening

    NARCIS (Netherlands)

    Verissimo, Carla S; Overmeer, René M; Ponsioen, Bas; Drost, Jarno; Mertens, Sander; Verlaan-Klink, Ingrid; Gerwen, Bastiaan van; van der Ven, Marieke; Wetering, Marc van de; Egan, David A; Bernards, René; Clevers, Hans; Bos, Johannes L; Snippert, Hugo J

    2016-01-01

    Colorectal cancer (CRC) organoids can be derived from almost all CRC patients and therefore capture the genetic diversity of this disease. We assembled a panel of CRC organoids carrying either wild-type or mutant RAS, as well as normal organoids and tumor organoids with a CRISPR-introduced oncogenic

  12. Management of renal cell carcinoma in von Hippel-Lindau disease.

    Science.gov (United States)

    Hes, F J; Slootweg, P J; van Vroonhoven, T J; Hené, R J; Feldberg, M A; Zewald, R A; Ploos van Amstel, J K; Höppener, J W; Pearson, P L; Lips, C J

    1999-01-01

    An evaluation of nephron-sparing surgery (NSS) or radical nephrectomy (RN) for treating renal cell carcinoma (RCC) in patients with von Hippel-Lindau disease (VHL) was carried out. Between 1976 and 1997, 10 patients with RCC from four VHL families, of whom seven were from one family, were studied by clinical and histopathological examination. Before 1991, three patients were treated using RN, and thereafter five patients were treated using NSS. Two patients were not operated on. RCCs in our patients showed a slow growth rate (on average 0.3 cm year-1), and asymptomatic patients presented with tumours of low-grade malignancy. In all patients, tumours were surrounded by a fibrous pseudocapsule. In 5 out of 17 tumours, pseudocapsular invasion was observed, and three of these five tumours broke through the pseudocapsule. To date, these patients have not shown a less favourable outcome than those without pseudocapsular involvement by tumour growth. Multicentricity of RCC was relatively low (4.6 lesions per kidney). In two of the three RN patients, only a single satellite lesion, in the direct vicinity of a RCC, was found in one kidney. Six tumours (1.8-5.5 cm) were enucleated by NSS. During a mean follow-up of 30 months, renal function in these patients was well preserved. In our patients, RCCs grew slowly, were of low grade, had a dense fibrous pseudocapsule and were thus good candidates for NSS.

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

    Directory of Open Access Journals (Sweden)

    Tali Gidalevitz

    2009-03-01

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

  14. Fibrinolytic Activity of Recombinant Mutant Streptokinase

    Directory of Open Access Journals (Sweden)

    Mahboobeh Mobarrez

    2015-04-01

    Full Text Available Background: Streptokinase is a bacterial protein produced by different beta hemolytic streptococci and widely used in thrombolytic treatment. The main disadvantage of using streptokinase is antibody formation which causes allergic reaction to neutralize effects of streptokinase therapy. Aim of this study was investigate of recombinant mutant streptokinase fibrinolytic activity.Materials and Methods: In this study recombinant mutant streptokinase without 42 amino acids from the C terminal region was purified by affinity S-Tag column chromatography and its fibrinolytic activity was studied.Results: The concentration of expressed and purified protein was 10 mg/ml. Its enzyme activity was assayed using zymography, radial caseinolytic activity and fibrin plate test methods and estimated quantitatively by casein digestion method compared to a commercial form.Conclusion: It was found that this product had the more volume and more enzymatic activity.

  15. Characterization of a Legionella micdadei mip mutant

    DEFF Research Database (Denmark)

    O'Connell, W A; Bangsborg, Jette Marie; Cianciotto, N P

    1995-01-01

    The pathogenesis of Legionella micdadei is dependent upon its ability to infect alveolar phagocytes. To better understand the basis of intracellular infection by this organism, we examined the importance of its Mip surface protein. In Legionella pneumophila, Mip promotes infection of both human m...... Mip. Although unimpaired in its ability to grow in bacteriologic media, this Mip mutant was defective in its capacity to infect U937 cells, a human macrophage-like cell line. Most significantly, the Mip- organism displayed a 24-fold reduction in survivability immediately after its entry...... into the phagocyte. Similarly, the mutant was less able to parasitize Hartmannella amoebae. Taken together, these data argue that Mip specifically potentiates intracellular growth by L. micdadei....

  16. A new neurological rat mutant "mutilated foot".

    OpenAIRE

    Jacobs, J M; Scaravilli, F; Duchen, L W; Mertin, J

    1981-01-01

    A new autosomal recessive mutant rat (mutilated foot) with a neurological disorder is described. Affected animals become ataxic and the feet, generally of the hind limbs, are mutilated. Quantitative studies show a severe reduction in numbers of sensory ganglion cells and fibres, including unmyelinated fibres. The numbers of ventral root fibres, particularly those of small diameter, are also reduced. Markedly decreased numbers of spindles are found in the limb muscles. These quantitative abnor...

  17. Courtship song analysis of Drosophila muscle mutants.

    Science.gov (United States)

    Chakravorty, Samya; Wajda, Mathew P; Vigoreaux, Jim O

    2012-01-01

    As part of the mating ritual, males of Drosophila species produce species-specific courtship songs through wing vibrations generated by the thoracic musculature. While previous studies have shown that indirect flight muscles (IFM) are neurally activated during courtship song production, the precise role of these muscles in song production has not been investigated. Fortunately, IFM mutants abound in Drosophila melanogaster and studies spanning several decades have shed light on the role of muscle proteins in IFM-powered flight. Analysis of courtship songs in these mutants offers the opportunity to uncover the role of the IFM in a behavior distinct than flight and subject to different evolutionary selection regimes. Here, we describe protocols for the recording and analysis of courtship behavior and mating song of D. melanogaster muscle transgenic and mutant strains. To record faint acoustic signal of courtship songs, an insulated mating compartment was used inside a recording device (INSECTAVOX) equipped with a modified electret microphone, a low-noise power supply, and noise filters. Songs recorded in the INSECTAVOX are digitized using Goldwave, whose several features enable extraction of critical song parameters, including carrier frequencies for pulse song and sine song. We demonstrate the utility of this approach by showing that deletion of the N-terminal region of the myosin regulatory light chain, a mutation known to decrease wing beat frequency and flight power, affects courtship song parameters. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Adrenal Cortex-Sparing Surgery for Bilateral Multiple Pheochromocytomas in a Patient with Von Hippel-Lindau Disease

    Directory of Open Access Journals (Sweden)

    Tarık Esen

    2012-01-01

    Full Text Available Pheochromocytomas can be a part of familial neoplastic syndromes, in which case they tend to be multiple and involve both adrenal glands. Therefore, sparing adrenocortical function represents a major concern while dealing with these hereditary lesions. Herein, we describe the clinical characteristics and the management strategy of a patient with von Hippel-Lindau (VHL disease who had multiple, bilateral pheochromocytomas as well as bilateral renal masses, pancreatic masses, and a paracaval mass. Only a portion of the left adrenal gland has remained in situ after two consecutive open surgeries and a percutaneous radiofrequency ablation which have been performed to treat the various components of this syndrome. No adrenal or extra-adrenal pheochromocytoma recurrences have been detected during a follow-up period of more than 2 years. Pancreatic and adrenal functions were normal throughout the postoperative period and never necessitated any replacement therapy. Adrenal cortex-sparing surgery is a valid option for VHL disease patients who present with synchronous bilateral adrenal pheochromocytomas.

  19. Parkin mutant in the fly is largely rescued by metal-responsive transcription factor (MTF-1)

    OpenAIRE

    Saini, N.; Georgiev, O; Schaffner, W

    2011-01-01

    The gene for Parkin, an E3 ubiquitin ligase, is mutated in some familial forms of Parkinson's disease, a severe neurodegenerative disorder. A homozygous mutant of the Drosophila ortholog of human parkin is viable but results in severe motoric impairment including an inability to fly, female and male sterility, and a decreased lifespan. Here we show that a double mutant of the genes for Parkin and the metal-responsive transcription factor MTF-1 is not viable. MTF-1, which is conserved from ins...

  20. DISEASES

    DEFF Research Database (Denmark)

    Pletscher-Frankild, Sune; Pallejà, Albert; Tsafou, Kalliopi

    2015-01-01

    Text mining is a flexible technology that can be applied to numerous different tasks in biology and medicine. We present a system for extracting disease-gene associations from biomedical abstracts. The system consists of a highly efficient dictionary-based tagger for named entity recognition...... of human genes and diseases, which we combine with a scoring scheme that takes into account co-occurrences both within and between sentences. We show that this approach is able to extract half of all manually curated associations with a false positive rate of only 0.16%. Nonetheless, text mining should...... not stand alone, but be combined with other types of evidence. For this reason, we have developed the DISEASES resource, which integrates the results from text mining with manually curated disease-gene associations, cancer mutation data, and genome-wide association studies from existing databases...

  1. Agrobacterium rhizogenes mutants that fail to bind to plant cells.

    OpenAIRE

    Crews, J L; Colby, S; Matthysse, A G

    1990-01-01

    Transposon insertion mutants of Agrobacterium rhizogenes were screened to obtain mutant bacteria that failed to bind to carrot suspension culture cells. A light microscope binding assay was used. The bacterial isolates that were reduced in binding to carrot cells were all avirulent on Bryophyllum diagremontiana leaves and on carrot root disks. The mutants did not appear to be altered in cellulose production. The composition of the medium affected the ability of the parent and mutant bacteria ...

  2. Neuregulin-1 mutant mice indicate motor and sensory deficits, indeed few references for schizophrenia endophenotype model.

    Science.gov (United States)

    Schneider, Silvia; Götz, Katja; Birchmeier, Carmen; Schwegler, Herbert; Roskoden, Thomas

    2017-03-30

    Neuregulins (Nrg) are a gene family that binds to tyrosine kinase receptors of the ErbB family. The protein of Nrg1 is to be involved in heart formation, migration of neurons, axonal pathfinding and synaptic function. A relation between Nrg1 and schizophrenia is assumed. Chronic impairment in schizophrenia is characterized by different positive and negative symptoms. Detectable markers of this disease in human and in animal models are activity, social behavior and sensory processing. In this study we compared heterozygous Nrg1 mutant mice in behavior and quantification of dopaminergic and serotoninergic neurons with wild type-like littermates. In the Nrg1 mutant mice the epidermal growth factor-like domain is replaced by the neomycin resistance gene. We found significant differences in locomotor and pain perception behavior. No differences were found in specific schizophrenia social interaction and prepulse inhibition behavior. The number of dopaminergic and serotoninergic neurons did not differ in the investigated regions ventral tegmental area, substantia nigra, periaqueductal grey and raphe nuclei. In conclusion, this analyzed Nrg1 mutant mice model did not serve as a complete schizophrenia model. Particular aspects of schizophrenia disease in locomotor and sensory behavior deficits could represent in this Nrg1 mutant mice. Beside several different models could Nrg1 deficiency represent an endophenotype of schizophrenia disease. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Pheochromocytoma complicated by cyanotic congenital heart disease: a case report.

    Science.gov (United States)

    Yamamoto, Keiko; Namba, Noriyuki; Kubota, Takuo; Usui, Takeshi; Takahashi, Kunihiko; Kitaoka, Taichi; Fujiwara, Makoto; Hori, Yumiko; Kogaki, Shigetoyo; Oue, Takaharu; Morii, Eiichi; Ozono, Keiichi

    2016-04-01

    Coincidental cyanotic congenital heart disease and pheochromocytoma is uncommon, although some cases have been reported. We describe a girl aged 15 yr and 11 mo with pheochromocytoma and tricuspid atresia treated by performing the Fontan surgery. The patient did not have any specific symptoms of syndrome related to pheochromoytoma or a family history of pheochromocytoma. During cardiac catheterization, her blood pressure increased markedly, and an α-blocker was administered. Catecholamine hypersecretion was observed in the blood and urine, and abdominal computed tomography revealed a tumor in the right adrenal gland. Scintigraphy showed marked accumulation of (123)I-metaiodobenzylguanidine in the tumor, which led to a diagnosis of pheochromocytoma. We did not detect any germline mutations in the RET, VHL, SDHB, SDHD, TMEM127, or MAX genes. This patient had experienced mild systemic hypoxia since birth, which may have contributed to the development of pheochromocytoma.

  4. A Novel Two-Step Method for Screening Shade Tolerant Mutant Plants via Dwarfism

    Science.gov (United States)

    Li, Wei; Katin-Grazzini, Lorenzo; Krishnan, Sanalkumar; Thammina, Chandra; El-Tanbouly, Rania; Yer, Huseyin; Merewitz, Emily; Guillard, Karl; Inguagiato, John; McAvoy, Richard J.; Liu, Zongrang; Li, Yi

    2016-01-01

    When subjected to shade, plants undergo rapid shoot elongation, which often makes them more prone to disease and mechanical damage. Shade-tolerant plants can be difficult to breed; however, they offer a substantial benefit over other varieties in low-light areas. Although perennial ryegrass (Lolium perenne L.) is a popular species of turf grasses because of their good appearance and fast establishment, the plant normally does not perform well under shade conditions. It has been reported that, in turfgrass, induced dwarfism can enhance shade tolerance. Here we describe a two-step procedure for isolating shade tolerant mutants of perennial ryegrass by first screening for dominant dwarf mutants, and then screening dwarf plants for shade tolerance. The two-step screening process to isolate shade tolerant mutants can be done efficiently with limited space at early seedling stages, which enables quick and efficient isolation of shade tolerant mutants, and thus facilitates development of shade tolerant new cultivars of turfgrasses. Using the method, we isolated 136 dwarf mutants from 300,000 mutagenized seeds, with 65 being shade tolerant (0.022%). When screened directly for shade tolerance, we recovered only four mutants from a population of 150,000 (0.003%) mutagenized seeds. One shade tolerant mutant, shadow-1, was characterized in detail. In addition to dwarfism, shadow-1 and its sexual progeny displayed high degrees of tolerance to both natural and artificial shade. We showed that endogenous gibberellin (GA) content in shadow-1 was higher than wild-type controls, and shadow-1 was also partially GA insensitive. Our novel, simple and effective two-step screening method should be applicable to breeding shade tolerant cultivars of turfgrasses, ground covers, and other economically important crop plants that can be used under canopies of existing vegetation to increase productivity per unit area of land. PMID:27752260

  5. A novel two-step method for screening shade tolerant mutant plants via dwarfism

    Directory of Open Access Journals (Sweden)

    Wei Li

    2016-10-01

    Full Text Available When subjected to shade, plants undergo rapid shoot elongation, which often makes them more prone to disease and mechanical damage. Shade-tolerant plants can be difficult to breed; however, they offer a substantial benefit over other varieties in low-light areas. Although perennial ryegrass (Lolium perenne L. is a popular species of turf grasses because of their good appearance and fast establishment, the plant normally does not perform well under shade conditions. It has been reported that, in turfgrass, induced dwarfism can enhance shade tolerance. Here we describe a two-step procedure for isolating shade tolerant mutants of perennial ryegrass by first screening for dominant dwarf mutants, and then screening dwarf plants for shade tolerance. The two-step screening process to isolate shade tolerant mutants can be done efficiently with limited space at early seedling stages, which enables quick and efficient isolation of shade tolerant mutants, and thus facilitates development of shade tolerant new cultivars of turfgrasses. Using the method, we isolated 136 dwarf mutants from 300,000 mutagenized seeds, with 65 being shade tolerant (0.022%. When screened directly for shade tolerance, we recovered only four mutants from a population of 150,000 (0.003% mutagenized seeds. One shade tolerant mutant, shadow-1, was characterized in detail. In addition to dwarfism, shadow-1 and its sexual progeny displayed high degrees of tolerance to both natural and artificial shade. We showed that endogenous gibberellin (GA content in shadow-1 was higher than wild-type controls, and shadow-1 was also partially GA insensitive. Our novel, simple and effective two-step screening method should be applicable to breeding shade tolerant cultivars of turfgrasses, ground covers, and other economically important crop plants that can be used under canopies of existing vegetation to increase productivity per unit area of land.

  6. Isolation and Characterization of Sexual Sporulation Mutants of Aspergillus nidulans

    NARCIS (Netherlands)

    Swart, K.; Heemst, van D.; Slakhorst, M.; Debets, A.J.M.; Heyting, C.

    2001-01-01

    For the genetic dissection of sexual sporulation in Aspergillus nidulans, we started a collection of ascosporeless mutants. After mutagenization of conidiospores with high doses of UV, we isolated 20 mutants with defects in ascospore formation. We crossed these mutants in two successive rounds with

  7. Isolation and characterization of gallium resistant Pseudomonas aeruginosa mutants

    NARCIS (Netherlands)

    García-Contreras, R; Lira-Silva, E; Jasso-Chávez, R; Hernández-González, I.L.; Maeda, T.; Hashimoto, T.; Boogerd, F.C.; Sheng, L; Wood, TK; Moreno-Sánchez, R

    2013-01-01

    Pseudomonas aeruginosa PA14 cells resistant to the novel antimicrobial gallium nitrate (Ga) were developed using transposon mutagenesis and by selecting spontaneous mutants. The mutants showing the highest growth in the presence of Ga were selected for further characterization. These mutants showed

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

    African Journals Online (AJOL)

    STORAGESEVER

    2009-12-15

    Dec 15, 2009 ... The amounts of protoplasts obtained in the developed mutants of M. mucedo MMM1 (U.V. irradiated mutant) and MMM2 (ethyl methyl sulfonate treated mutant) which are very effective decolourisers were. 5.23 x 106 and 5.65 x 106 protoplasts/ml respectively. Among the 385 colonies isolated after ...

  9. Characterization of a novel curled-cotyledons mutant in soybean ...

    African Journals Online (AJOL)

    ... some organelles degradation, and membranous multilamellar appear at different stages. Protein and amino acid contents in seeds of mutant are higher than those of the wild type, especially methionine and cysteine. These results suggest that the curled-cotyledons mutant is a novel cotyledon development mutant, which ...

  10. Histone deacetylase inhibitors correct the cholesterol storage defect in most Niemann-Pick C1 mutant cells.

    Science.gov (United States)

    Pipalia, Nina H; Subramanian, Kanagaraj; Mao, Shu; Ralph, Harold; Hutt, Darren M; Scott, Samantha M; Balch, William E; Maxfield, Frederick R

    2017-04-01

    Niemann-Pick C (NPC) disease is an autosomal recessive disorder that leads to excessive storage of cholesterol and other lipids in late endosomes and lysosomes. The large majority of NPC disease is caused by mutations in NPC1, a large polytopic membrane protein that functions in late endosomes. There are many disease-associated mutations in NPC1, and most patients are compound heterozygotes. The most common mutation, NPC1I1061T, has been shown to cause endoplasmic reticulum-associated degradation of the NPC1 protein. Treatment of patient-derived NPC1I1061T fibroblasts with histone deacetylase inhibitors (HDACis) vorinostat or panobinostat increases expression of the mutant NPC1 protein and leads to correction of the cholesterol storage. Here, we show that several other human NPC1 mutant fibroblast cell lines can also be corrected by vorinostat or panobinostat and that treatment with vorinostat extends the lifetime of the NPC1I1061T protein. To test effects of HDACi on a large number of NPC1 mutants, we engineered a U2OS cell line to suppress NPC1 expression by shRNA and then transiently transfected these cells with 60 different NPC1 mutant constructs. The mutant NPC1 did not significantly reduce cholesterol accumulation, but approximately 85% of the mutants showed reduced cholesterol accumulation when treated with vorinostat or panobinostat. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  11. Spontaneous Remission in an Older Patient with Relapsed FLT3 ITD Mutant AML

    Directory of Open Access Journals (Sweden)

    Pankit Vachhani

    2016-01-01

    Full Text Available Spontaneous remission (SR of acute myeloid leukemia (AML is a very rare phenomenon. AML characterized by FLT3 internal tandem duplication (FLT3 ITD is typically associated with an aggressive clinical course with rapid progression, relapse, and short overall survival in the absence of transplantation. We report here the first case of SR of FLT3 ITD mutant AML in the literature. Our patient was an elderly woman with relapsed NPM1 and FLT3 ITD mutant AML whose disease underwent SR for a brief duration without precipitating cause. We review the potential immune mechanisms underlying SR in AML and discuss the implications for novel immunotherapeutic approaches for FLT3 mutant AML.

  12. NRAS mutant melanoma: biological behavior and future strategies for therapeutic management

    Science.gov (United States)

    Fedorenko, Inna V.; Gibney, Geoffrey T.; Smalley, Keiran S. M.

    2014-01-01

    The recent years have seen a significant shift in the expectations for the therapeutic management of disseminated melanoma. The clinical success of BRAF targeted therapy suggest that long-term disease control may one day be a reality for genetically defined sub-groups of melanoma patients. Despite this progress, few advances have been made in developing targeted therapeutic strategies for the 50% of patients whose melanomas are BRAF wild-type. The most significant sub-group of BRAF wild-type tumors is the 15–20% of all melanomas that harbor activating NRAS mutations. Emerging preclinical and clinical evidence suggests that NRAS mutant melanomas have patterns of signal transduction and biological behavior that is distinct from BRAF mutant melanomas. This overview will discuss the unique clinical and prognostic behavior of NRAS mutant melanoma and will summarize the emerging data on how NRAS-driven signaling networks can be translated into novel therapeutic strategies. PMID:23069660

  13. Towards a "Golden Standard" for computing globin stability: Stability and structure sensitivity of myoglobin mutants

    DEFF Research Database (Denmark)

    Kepp, Kasper Planeta

    2015-01-01

    Fast and accurate computation of protein stability is increasingly important for e.g. protein engineering and protein misfolding diseases, but no consensus methods exist for important proteins such as globins, and performance may depend on the type of structural input given. This paper reports...... performances reveal room for improvement, but I-Mutant 2.0 is proficient for this purpose, as further validated against a data set of related cytochrome c like proteins. The results also emphasize the importance of high-quality crystal structures and reveal structure-dependent effects even in the near...... benchmarking of six protein stability calculators (POPMUSIC 2.1, I-Mutant 2.0, I-Mutant 3.0, CUPSAT, SDM, and mCSM) against 134 experimental stability changes for mutations of sperm-whale myoglobin. Six different high-resolution structures were used to test structure sensitivity that may impair protein...

  14. Temperature-sensitive retinoid isomerase activity of RPE65 mutants associated with Leber Congenital Amaurosis.

    Science.gov (United States)

    Li, Songhua; Hu, Jane; Jin, Robin J; Aiyar, Ashok; Jacobson, Samuel G; Bok, Dean; Jin, Minghao

    2015-08-01

    RPE65 is a membrane-associated retinoid isomerase involved in the visual cycle responsible for sustaining vision. Many mutations in the human RPE65 gene are associated with distinct forms of retinal degenerative diseases. The pathogenic mechanisms for most of these mutations remain poorly understood. Here, we show that three Leber congenital amaurosis -associated RPE65 mutants (R91W, Y249C and R515W) undergo rapid proteasomal degradation mediated by the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13) in cultured human retinal pigment epithelium (RPE) cells. These mutant proteins formed cytosolic inclusion bodies or high molecular weight complexes via disulfide bonds. The mutations are mapped on non-active sites but severely reduced isomerase activity of RPE65. At 30°C, however, the enzymatic function and membrane-association of the mutant RPE65s are significantly rescued possibly due to proper folding. In addition, PSMD13 displayed a drastically decreased effect on degradation of the mutant proteins in the cells grown at 30°C. These results suggest that PSMD13 plays a critical role in regulating pathogenicity of the mutations and the molecular basis for the PSMD13-mediated rapid degradation and loss of function of the mutants is misfolding of RPE65. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  15. Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening.

    Science.gov (United States)

    Verissimo, Carla S; Overmeer, René M; Ponsioen, Bas; Drost, Jarno; Mertens, Sander; Verlaan-Klink, Ingrid; Gerwen, Bastiaan van; van der Ven, Marieke; Wetering, Marc van de; Egan, David A; Bernards, René; Clevers, Hans; Bos, Johannes L; Snippert, Hugo J

    2016-11-15

    Colorectal cancer (CRC) organoids can be derived from almost all CRC patients and therefore capture the genetic diversity of this disease. We assembled a panel of CRC organoids carrying either wild-type or mutant RAS, as well as normal organoids and tumor organoids with a CRISPR-introduced oncogenic KRAS mutation. Using this panel, we evaluated RAS pathway inhibitors and drug combinations that are currently in clinical trial for RAS mutant cancers. Presence of mutant RAS correlated strongly with resistance to these targeted therapies. This was observed in tumorigenic as well as in normal organoids. Moreover, dual inhibition of the EGFR-MEK-ERK pathway in RAS mutant organoids induced a transient cell-cycle arrest rather than cell death. In vivo drug response of xenotransplanted RAS mutant organoids confirmed this growth arrest upon pan-HER/MEK combination therapy. Altogether, our studies demonstrate the potential of patient-derived CRC organoid libraries in evaluating inhibitors and drug combinations in a preclinical setting.

  16. Multiple HIV-1 infection of cells and the evolutionary dynamics of cytotoxic T lymphocyte escape mutants.

    Science.gov (United States)

    Wodarz, Dominik; Levy, David N

    2009-09-01

    Cytotoxic T lymphocytes (CTL) are an important branch of the immune system, killing virus-infected cells. Many viruses can mutate so that infected cells are not killed by CTL anymore. This escape can contribute to virus persistence and disease. A prominent example is HIV-1. The evolutionary dynamics of CTL escape mutants in vivo have been studied experimentally and mathematically, assuming that a cell can only be infected with one HIV particle at a time. However, according to data, multiple virus particles frequently infect the same cell, a process called coinfection. Here, we study the evolutionary dynamics of CTL escape mutants in the context of coinfection. A mathematical model suggests that an intermediate strength of the CTL response against the wild-type is most detrimental for an escape mutant, minimizing overall virus load and even leading to its extinction. A weaker or, paradoxically, stronger CTL response against the wild-type both lead to the persistence of the escape mutant and higher virus load. It is hypothesized that an intermediate strength of the CTL response, and thus the suboptimal virus suppression observed in HIV-1 infection, might be adaptive to minimize the impact of existing CTL escape mutants on overall virus load.

  17. Phospholipid Bicelles Improve the Conformational Stability of Rhodopsin Mutants Associated with Retinitis Pigmentosa.

    Science.gov (United States)

    Dong, Xiaoyun; Ramon, Eva; Herrera-Hernández, María Guadalupe; Garriga, Pere

    2015-08-11

    Mutations in the visual photoreceptor rhodopsin are the cause of the retinal degenerative disease retinitis pigmentosa. Some naturally occurring mutations can lead to protein conformational instability. Two such mutations, N55K and G90V, in the first and second transmembrane helices of the receptor, have been associated with sector and classical retinitis pigmentosa, respectively, and showed enhanced thermal sensitivity. We have carefully analyzed the effect of phospholipid bicelles on the stability and ligand binding properties of these two mutants and compared it with those of the detergent-solubilized samples. We have used a phospholipid bilayer consisting of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC). We find that DMPC/DHPC bicelles dramatically increase the thermal stability of the rhodopsin mutants G90V and N55K. The chromophore stability and regeneration of the mutants were also increased in bicelles when compared to their behavior in a dodecyl maltoside detergent solution. The retinal release process was slowed in bicelles, and chromophore entry, after illumination, was improved for the G90V mutant but not for N55K. Furthermore, fluorescence spectroscopy measurements showed that bicelles allowed more exogenous retinal binding to the photoactivated G90V mutant than in a detergent solution. In contrast, N55K could not reposition any chromophore either in the detergent or in bicelles. The results demonstrate that DMPC/DHPC bicelles can counteract the destabilizing effect of the disease-causing mutations and can modulate the structural changes in the ensuing receptor photoactivation in a distinct specific manner for different retinitis pigmentosa mutant phenotypes.

  18. Mutant HSP70 Reverses Autoimmune Depigmentation in Vitiligo

    Science.gov (United States)

    Mosenson, Jeffrey A.; Zloza, Andrew; Nieland, John D.; Garrett-Mayer, Elizabeth; Eby, Jonathan M.; Huelsmann, Erica J.; Kumar, Previn; Denman, Cecele J.; Lacek, Andrew T.; Kohlhapp, Frederick J.; Alamiri, Ahmad; Hughes, Tasha; Bines, Steven D.; Kaufman, Howard L.; Overbeck, Andreas; Mehrotra, Shikhar; Hernandez, Claudia; Nishimura, Michael I.; Guevara-Patino, Jose A.; Le Poole, I. Caroline

    2013-01-01

    Vitiligo is an autoimmune disease characterized by destruction of melanocytes, leaving 0.5% of the population with progressive depigmentation. Current treatments offer limited efficacy. We report that modified inducible heat shock protein 70 (HSP70i) prevents T cell–mediated depigmentation. HSP70i is the molecular link between stress and the resultant immune response. We previously showed that HSP70i induces an inflammatory dendritic cell (DC) phenotype and is necessary for depigmentation in vitiligo mouse models. Here, we observed a similar DC inflammatory phenotype in vitiligo patients. In a mouse model of depigmentation, DNA vaccination with a melanocyte antigen and the carboxyl terminus of HSP70i was sufficient to drive autoimmunity. Mutational analysis of the HSP70i substrate-binding domain established the peptide QPGVLIQVYEG as invaluable for DC activation, and mutant HSP70i could not induce depigmentation. Moreover, mutant HSP70iQ435A bound human DCs and reduced their activation, as well as induced a shift from inflammatory to tolerogenic DCs in mice. HSP70iQ435A-encoding DNA applied months before spontaneous depigmentation prevented vitiligo in mice expressing a transgenic, melanocyte-reactive T cell receptor. Furthermore, use of HSP70iQ435A therapeutically in a different, rapidly depigmenting model after loss of differentiated melanocytes resulted in 76% recovery of pigmentation. Treatment also prevented relevant T cells from populating mouse skin. In addition, ex vivo treatment of human skin averted the disease-related shift from quiescent to effector T cell phenotype. Thus, HSP70iQ435A DNA delivery may offer potent treatment opportunities for vitiligo. PMID:23447019

  19. Arabinose Kinase-Deficient Mutant of Arabidopsis thaliana.

    Science.gov (United States)

    Dolezal, O; Cobbett, C S

    1991-08-01

    A mutant of Arabidopsis thaliana that is sensitive to exogenous l-arabinose has been isolated. Comparisons of growth of the wild type, mutant, and F1 and F2 progeny of crosses showed the arabinose-sensitive phenotype is semidominant and segregates as a single Mendelian locus. Crosses of the mutant to marker strains showed the mutation is linked to the eceriferum-2 locus on chromosome 4. In vivo incorporation of exogenous labeled l-arabinose into ethanol-insoluble polysaccharides was greatly reduced in the mutant with a concomitant accumulation of free labeled arabinose. Enzyme assays of crude plant extracts demonstrated a defect in arabinose kinase activity in the mutant.

  20. disease

    African Journals Online (AJOL)

    Objective. To undertake an economic evaluation of the administration and monitoring costs of the two different forms of heparin in patients with unstable coronary artery disease (DCAD). Study design. Equivalent efficacy was found for low- molecular-weight heparin (LMWH) and for unfraction- ated heparin (UFH) in the ...

  1. Distribution of soluble amino acids in maize endosperm mutants

    Directory of Open Access Journals (Sweden)

    Toro Alejandro Alberto

    2003-01-01

    Full Text Available For human nutrition the main source of vegetable proteins are cereal and legume seeds. The content of total soluble amino acids in mature endosperm of wild-type, opaque and floury maize (Zea mays L. mutants were determined by HPLC. The total absolute concentration of soluble amino acids among the mutants varied depending on the mutant. The o11 and o13 mutants exhibited the highest average content, whereas o10, fl3 and fl1 exhibited the lowest average content. In general, the mutants exhibited similar concentrations of total soluble amino acids when compared to the wild-type lines, with the clear exception of mutants o11 and fl1, with the o11 mutant exhibiting a higher concentration of total soluble amino acids when compared to its wild-type counterpart W22 and the fl1 mutant a lower concentration when compared to its wild-type counterpart Oh43. For methionine, the mutants o2 and o11 and wild-type Oh43 exhibited the highest concentrations of this amino acid. Significant differences were not observed between mutants for other amino acids such as lysine and threonine. The high lysine concentrations obtained originally for these mutants may be due to the amino acids incorporated into storage proteins, but not those present in the soluble form.

  2. Using of AFLP to evaluate gamma-irradiated amaranth mutants

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    Labajová Mária

    2013-01-01

    Full Text Available To determine which of several gamma-irradiated mutants of amaranth Ficha cultivar and K-433 hybrid are most genetically similar to their non-irradiated control genotypes, we performed amplified fragment length polymorphism (AFLP based analysis. A total of 40 selective primer combinations were used in reported analyses. First analyses of gamma-irradiated amaranth mutant lines were done used the AFLP. In the study, primers with the differentiation ability for all analysed mutant lines are reported. The very specific changes in the mutant lines´ non-coding regions based on AFLP length polymorphism were analysed. Mutant lines of the Ficha cultivar (C15, C26, C27, C82, C236 shared a genetic dissimilarity of 0,11 and their ISSR profiles are more similar to the Ficha than those of K-433 hybrid mutant lines. The K-433 mutant lines (D54, D279, D282 shared genetic dissimilarity of 0,534 but are more distinct to their control plant as a whole, as those of the Ficha mutant lines. Different AFLP fingerprints patters of the mutant lines when compared to the Ficha cultivar and K-433 hybrid AFLP profiles may be a consequence of the complex response of the intergenic space of mutant lines to the gamma-radiance. Although a genetic polymorphism was detected within accessions, the AFLP markers successfully identified all the accessions. The AFLP results are discussed by a combination of biochemical characteristics of mutant lines and their control genotypes.

  3. Ribosylurea accumulates in yeast urc4 mutants.

    Science.gov (United States)

    Björnberg, O; Vodnala, M; Domkin, V; Hofer, A; Rasmussen, A; Andersen, G; Piskur, J

    2010-06-01

    Yeast Saccharomyces (Lachancea) kluyveri urc4 mutants, unable to grow on uracil, biotransformed (14)C(2)-uracil into two labeled compounds, as detected by high performance liquid chromatography (HPLC). These two compounds could also be obtained following organic synthesis of ribosylurea. This finding demonstrates that in the URC pyrimidine degradation pathway, the opening of the uracil ring takes place when uracil is attached to the ribose moiety. Ribosylurea has not been reported in the cell metabolism before and the two observed compounds likely represent an equilibrium mixture of the pyranosyl and furanosyl forms.

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

    Science.gov (United States)

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

    2013-08-01

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

  5. Auxin physiology of the tomato mutant diageotropica

    Science.gov (United States)

    Daniel, S. G.; Rayle, D. L.; Cleland, R. E.

    1989-01-01

    The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxin-induced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.

  6. Indy mutants: live long and prosper

    Directory of Open Access Journals (Sweden)

    Stewart eFrankel

    2012-02-01

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

  7. Mutant TDP-43 deregulates AMPK activation by PP2A in ALS models.

    Directory of Open Access Journals (Sweden)

    Nirma D Perera

    Full Text Available Bioenergetic abnormalities and metabolic dysfunctionoccur in amyotrophic lateral sclerosis (ALS patients and genetic mouse models. However, whether metabolic dysfunction occurs earlyin ALS pathophysiology linked to different ALS genes remains unclear.Here, we investigatedAMP-activated protein kinase (AMPK activation, which is a key enzyme induced by energy depletion and metabolic stress, inneuronal cells and mouse models expressing mutantsuperoxide dismutase 1 (SOD1or TAR DNA binding protein 43 (TDP-43 linked to ALS.AMPKphosphorylation was sharply increased in spinal cords of transgenic SOD1G93A mice at disease onset and accumulated incytoplasmic granules in motor neurons, but not in pre-symptomatic mice. AMPK phosphorylation also occurred in peripheraltissues, liver and kidney, in SOD1G93A mice at disease onset, demonstrating that AMPK activation occurs late and is not restricted to motor neurons. Conversely, AMPK activity was drastically diminished in spinal cords and brains of presymptomatic and symptomatictransgenic TDP-43A315T mice and motor neuronal cells expressing different TDP-43 mutants. We show that mutant TDP-43 induction of the AMPK phosphatase,protein phosphatase 2A (PP2A, is associated with AMPK inactivation in these ALS models. Furthermore, PP2A inhibition by okadaic acid reversed AMPK inactivation by mutant TDP-43 in neuronal cells. Our results suggest that mutant SOD1 and TDP-43 exert contrasting effects on AMPK activation which may reflect key differences in energy metabolism and neurodegeneration in spinal cords of SOD1G93A and TDP-43A315T mice. While AMPK activation in motor neurons correlateswith progressionin mutant SOD1-mediated disease, AMPK inactivation mediated by PP2Ais associated withmutant TDP-43-linked ALS.

  8. Generation and screening of a comprehensive Mycobacterium avium subsp. paratuberculosis transposon mutant bank

    Directory of Open Access Journals (Sweden)

    Govardhan eRathnaiah

    2014-10-01

    Full Text Available Mycobacterium avium subsp. paratuberculosis (MAP is the etiologic agent of Johne’s Disease in ruminants. This enteritis has significant economic impact and worldwide distribution. Vaccination is one of the most cost effective infectious disease control measures. Unfortunately, current vaccines reduce clinical disease and shedding, but are of limited efficacy and do not provide long-term protective immunity. Several strategies have been followed to mine the MAP genome for virulence determinants that could be applied to vaccine and diagnostic assay development. In this study, a comprehensive mutant bank of 13,536 MAP K-10 Tn5367 mutants (P > 95% was constructed and screened in vitro for phenotypes related to virulence. This strategy was designated to maximize identification of genes important to MAP pathogenesis without relying on studies of other mycobacterial species that may not translate into similar effects in MAP. This bank was screened for mutants with colony morphology alterations, susceptibility to D-cycloserine, impairment in siderophore production or secretion, reduced cell association, and decreased biofilm and clump formation. Mutants with interesting phenotypes were analyzed by PCR, Southern blotting and DNA sequencing to determine transposon insertion sites. These insertion sites mapped upstream from the MAP1152-MAP1156 cluster, internal to either the Mod operon gene MAP1566 or within the coding sequence of lsr2, and several intergenic regions. Growth curves in broth cultures, invasion assays and kinetics of survival and replication in primary bovine macrophages were also determined. The ability of vectors carrying Tn5370 to generate stable MAP mutants was also investigated.

  9. Mitochondrial mutant cells are hypersensitive to ionizing radiation, phleomycin and mitomycin C

    Energy Technology Data Exchange (ETDEWEB)

    Kulkarni, Rohan; Reither, Adrian; Thomas, Robert A. [Department of Biological Sciences, Wayne State University, 5047 Gullen Mall, Suite 1370, Detroit, MI 48202 (United States); Tucker, James D., E-mail: jtucker@biology.biosci.wayne.edu [Department of Biological Sciences, Wayne State University, 5047 Gullen Mall, Suite 1370, Detroit, MI 48202 (United States)

    2009-04-26

    Mitochondrial DNA (mtDNA) is an important contributor to the ATP-generating oxidative phosphorylation complex. Single nucleotide mutations in mitochondrial genes involved in ATP synthesis result in a broad range of diseases. Leber optic atrophy and Leigh's syndrome are two such diseases arising from point mutations in the mitochondrial genome. Here, ionizing radiation, phleomycin and mitomycin C (MMC) were used to induce structural chromosomal aberrations in Leber's and Leigh's cells to investigate how these mitochondrial mutations affect the cell's DNA repair processes. Because of the energy deprivation that results from mitochondrial mutations, we hypothesized that these mutant cells would demonstrate hypersensitivity when exposed to oxidative and genotoxic stress and we also expected that these cells would not be able to repair nuclear DNA damage as efficiently as normal cells. As a consequence, these mutant cells are expected to show increased levels of DNA damage, longer cell cycle delays and increased levels of cell death. Following acute radiation exposure these mutant cells showed an increase in the number of chromosomal aberrations and decreased mitotic indices when compared with normal human lymphoblastoid cells with wild-type mtDNA. When exposed to phleomycin or MMC, the mitochondrial mutant cells again showed hypersensitivity and decreased mitotic indices compared to normal cells. These results suggest that Leber's and Leigh's cells have an impaired ability to cope with oxidative and genotoxic stress. These observations may help explain the role of ATP generation in understanding the enhanced sensitivity of mitochondrial mutant cells to cancer therapeutic agents and to adverse environmental exposure, suggesting that individuals with mtDNA mutations may be at a greater risk for cancer and other diseases that result from an accumulation of nuclear DNA damage.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2017-03-01

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

  11. Kinetic partitioning during de novo septin filament assembly creates a critical G1 "window of opportunity" for mutant septin function.

    Science.gov (United States)

    Schaefer, Rachel M; Heasley, Lydia R; Odde, David J; McMurray, Michael A

    2016-09-16

    Septin proteins form highly conserved cytoskeletal filaments composed of hetero-oligomers with strict subunit stoichiometry. Mutations within one hetero-oligomerization interface (the "G" interface) bias the mutant septin toward conformations that are incompatible with filament assembly, causing disease in humans and, in budding yeast cells, temperature-sensitive defects in cytokinesis. We previously found that, when the amount of other hetero-oligomerization partners is limiting, wild-type and G interface-mutant alleles of a given yeast septin "compete" along parallel but distinct folding pathways for occupancy of a limited number of positions within septin hetero-octamers. Here, we synthesize a mathematical model that outlines the requirements for this phenomenon: if a wild-type septin traverses a folding pathway that includes a single rate-limiting folding step, the acquisition by a mutant septin of additional slow folding steps creates an initially large disparity between wild-type and mutant in the cellular concentrations of oligomerization-competent monomers. When the 2 alleles are co-expressed, this kinetic disparity results in mutant exclusion from hetero-oligomers, even when the folded mutant monomer is oligomerization-competent. To test this model experimentally, we first visualize the kinetic delay in mutant oligomerization in living cells, and then narrow or widen the "window of opportunity" for mutant septin oligomerization by altering the length of the G1 phase of the yeast cell cycle, and observe the predicted exacerbation or suppression, respectively, of mutant cellular phenotypes. These findings reveal a fundamental kinetic principle governing in vivo assembly of multiprotein complexes, independent of the ability of the subunits to associate with each other.

  12. Neurobehavioral Mutants Identified in an ENU Mutagenesis Project

    Energy Technology Data Exchange (ETDEWEB)

    Cook, Melloni N. [University of Memphis; Dunning, Jonathan P [University of Memphis; Wiley, Ronald G [Vanderbilt University and Veterans Administration, Nashville, TN; Chesler, Elissa J [ORNL; Johnson, Dabney K [ORNL; Goldowitz, Daniel [University of Tennessee Health Science Center, Memphis

    2007-01-01

    We report on a behavioral screening test battery that successfully identified several neurobehavioral mutants among a large-scale ENU-mutagenized mouse population. Large numbers of ENU mutagenized mice were screened for abnormalities in central nervous system function based on abnormal performance in a series of behavior tasks. We developed and employed a high-throughput screen of behavioral tasks to detect behavioral outliers. Twelve mutant pedigrees, representing a broad range of behavioral phenotypes, have been identified. Specifically, we have identified two open field mutants (one displaying hyper-locomotion, the other hypo-locomotion), four tail suspension mutants (all displaying increased immobility), one nociception mutant (displaying abnormal responsiveness to thermal pain), two prepulse inhibition mutants (displaying poor inhibition of the startle response), one anxiety-related mutant (displaying decreased anxiety in the light/dark test), and one learning and memory mutant (displaying reduced response to the conditioned stimulus) These findings highlight the utility of a set of behavioral tasks used in a high throughput screen to identify neurobehavioral mutants. Further analysis (i.e., behavioral and genetic mapping studies) of mutants is in progress with the ultimate goal of identification of novel genes and mouse models relevant to human disorders as well as the identification of novel therapeutic targets.

  13. Forward genetic screen for auxin-deficient mutants by cytokinin.

    Science.gov (United States)

    Wu, Lei; Luo, Pan; Di, Dong-Wei; Wang, Li; Wang, Ming; Lu, Cheng-Kai; Wei, Shao-Dong; Zhang, Li; Zhang, Tian-Zi; Amakorová, Petra; Strnad, Miroslav; Novák, Ondřej; Guo, Guang-Qin

    2015-07-06

    Identification of mutants with impairments in auxin biosynthesis and dynamics by forward genetic screening is hindered by the complexity, redundancy and necessity of the pathways involved. Furthermore, although a few auxin-deficient mutants have been recently identified by screening for altered responses to shade, ethylene, N-1-naphthylphthalamic acid (NPA) or cytokinin (CK), there is still a lack of robust markers for systematically isolating such mutants. We hypothesized that a potentially suitable phenotypic marker is root curling induced by CK, as observed in the auxin biosynthesis mutant CK-induced root curling 1 / tryptophan aminotransferase of Arabidopsis 1 (ckrc1/taa1). Phenotypic observations, genetic analyses and biochemical complementation tests of Arabidopsis seedlings displaying the trait in large-scale genetic screens showed that it can facilitate isolation of mutants with perturbations in auxin biosynthesis, transport and signaling. However, unlike transport/signaling mutants, the curled (or wavy) root phenotypes of auxin-deficient mutants were significantly induced by CKs and could be rescued by exogenous auxins. Mutants allelic to several known auxin biosynthesis mutants were re-isolated, but several new classes of auxin-deficient mutants were also isolated. The findings show that CK-induced root curling provides an effective marker for discovering genes involved in auxin biosynthesis or homeostasis.

  14. Mutant Parkin impairs mitochondrial function and morphology in human fibroblasts.

    Directory of Open Access Journals (Sweden)

    Anne Grünewald

    Full Text Available BACKGROUND: Mutations in Parkin are the most common cause of autosomal recessive Parkinson disease (PD. The mitochondrially localized E3 ubiquitin-protein ligase Parkin has been reported to be involved in respiratory chain function and mitochondrial dynamics. More recent publications also described a link between Parkin and mitophagy. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the impact of Parkin mutations on mitochondrial function and morphology in a human cellular model. Fibroblasts were obtained from three members of an Italian PD family with two mutations in Parkin (homozygous c.1072delT, homozygous delEx7, compound-heterozygous c.1072delT/delEx7, as well as from two relatives without mutations. Furthermore, three unrelated compound-heterozygous patients (delEx3-4/duplEx7-12, delEx4/c.924C>T and delEx1/c.924C>T and three unrelated age-matched controls were included. Fibroblasts were cultured under basal or paraquat-induced oxidative stress conditions. ATP synthesis rates and cellular levels were detected luminometrically. Activities of complexes I-IV and citrate synthase were measured spectrophotometrically in mitochondrial preparations or cell lysates. The mitochondrial membrane potential was measured with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide. Oxidative stress levels were investigated with the OxyBlot technique. The mitochondrial network was investigated immunocytochemically and the degree of branching was determined with image processing methods. We observed a decrease in the production and overall concentration of ATP coinciding with increased mitochondrial mass in Parkin-mutant fibroblasts. After an oxidative insult, the membrane potential decreased in patient cells but not in controls. We further determined higher levels of oxidized proteins in the mutants both under basal and stress conditions. The degree of mitochondrial network branching was comparable in mutants and

  15. [Establishment of a mutant Lumican transgenic mouse model].

    Science.gov (United States)

    Song, Yanzheng; Zhao, Yanyan; Zhang, Fengju; Yu, Yanqiu; Ma, Ling

    2014-01-01

    Pathological myopia (PM) is a hereditary ocular disease leading to severe loss of visual acuity and blindness. Lumican gene (LUM) is one of those candidate genes of PM. The purpose of this study was to establish a mutant Lumican transgenic mouse model, and to prepare for the further study of the pathogenesis of PM. Experimental study. Mutation of LUM gene was created by site-directed mutagenesis. Recombinant DNA techniques were used for the construction of the pRP. EX3d-EF1A>LUM/flag>IRES/hrGFP transgene. The gene fragments were microinjected into the zygote male pronuclei of BDF1 mice, and then the zygote cells alive were transplanted into the oviduct of acceptor pregnant female ICR mice. The F0 generation transgenic mice obtained were named C57-TgN (LUM)CCMU. Genome DNA from mice tail was detected by PCR and Western blotting. Six of 31 F0 generation mice were positive transgenic mice. The western blotting study showed that the flag-tag was expressed in the mouse tail tissue. Sixty-eight of 128 mice (F1 to F3 generation) were positive transgenic mice, the positive rate is 53.13%. The mutant Lumican (cDNA 596T>C) transgenic mouse model has been established. This model will provide fundamental conditions for studies of the pathogenesis of PM. Also it will be the basis of further studies about the effect of Lumican mutation on the development of PM and structure and function of the extra cellular matrix.

  16. Screening of Mycobacterium avium subsp. paratuberculosis Mutants for Attenuation in a Bovine Monocyte-Derived Macrophage Model

    Directory of Open Access Journals (Sweden)

    Elise A Lamont

    2014-06-01

    Full Text Available Vaccination remains a major tool for prevention and progression of Johne’s disease, a chronic enteritis of ruminants worldwide. Currently there is only one licensed vaccine within the United States and two vaccines licensed internationally against Johne’s disease. All licensed vaccines reduce fecal shedding of Mycobacterium avium subsp. paratuberculosis (MAP and delay disease progression. However, there are no available vaccines that prevent disease onset. A joint effort by the Johne’s Disease Integrated Program (JDIP, a USDA-funded consortium, and USDA- APHIS/VS sought to identify transposon insertion mutant strains as vaccine candidates in part of a three phase study. The focus of the Phase I study was to evaluate MAP mutant attenuation in a well-defined in vitro bovine monocyte-derived macrophage (MDM model. Attenuation was determined by colony forming unit (CFUs counts and slope estimates. Based on CFU counts alone, the MDM model did not identify any mutant that significantly differed from the wild-type control, MAP K-10. Slope estimates using mixed models approach identified six mutants as being attenuated. These were enrolled in protection studies involving murine and baby goat vaccination-challenge models. MDM based approach identified trends in attenuation but this did not correlate with protection in a natural host model. These results suggest the need for alternative strategies for Johne’s disease vaccine candidate screening and evaluation.

  17. Google: a narrativa de uma marca mutante

    Directory of Open Access Journals (Sweden)

    Elizete de Azevedo Kreutz

    2010-01-01

    Full Text Available As marcas mutantes já fazem parte de nossa realidade, embora ainda não totalmente percebidas e/ou aceitas como tal. O presente artigo busca refletir sobre a relevância dessas novas estratégias de comunicação e branding, identificando suas principais características. Para isso, utilizamos o método de estudo de caso, o Google, ancorado nos métodos de pesquisa bibliográfica e de internet. A escolha foi intencional, posto que a organização é referência em sua categoria, mecanismo de busca, e reflete essa estratégia comunicacional contemporânea. Como resultado, as informações obtidas nos possibilitam compreender essa tendência de comportamento de marca que busca a interação com seus públicos.

  18. Studies on mutant breeding of Hibiscus syriacus

    Energy Technology Data Exchange (ETDEWEB)

    Song, Hi Sup; Kim, Jin Kyu; Lee, Ki Un; Kim, Young Taik

    1997-01-01

    Hibiscus has been known as a national flower of Korea. Hibiscus has such a characteristic of self-incompatibility that all the plant exist as natural hybrids and have heterogeneous genes. Many domestic 91 varieties of Hibiscus syriacus were collected. Radiosensitivity of H. Syriacus irradiated with {gamma}-ray was investigated in plant cuttings. The plant height was reduced by 45% in 5KR irradiated group, compared to control group. The radiation dose of 5KR could be recommended for mutation breeding of Hibiscus cuttings. Radiosensitivity of {gamma}-ray irradiated Hibiscus seed were investigated. The germination rate, survival rate and plant height was better in the 4KR irradiation plot than control. The radiation dose of 10{approx}12KR are recommended for mutation breeding of Hibiscus. Promising mutant lines were selected form the varieties of Hwarang, Wolsan no. 176, Ilpyondansim, Emille, Hanol, Yongkwang, Saeyongkwang, Chungmu, Imjinhong, Arang, Hungdansim-1 and Hongdansim-2. (author). 66 refs., 16 tabs., 13 figs.

  19. Mutant WD-repeat protein in triple-A syndrome.

    Science.gov (United States)

    Tullio-Pelet, A; Salomon, R; Hadj-Rabia, S; Mugnier, C; de Laet, M H; Chaouachi, B; Bakiri, F; Brottier, P; Cattolico, L; Penet, C; Bégeot, M; Naville, D; Nicolino, M; Chaussain, J L; Weissenbach, J; Munnich, A; Lyonnet, S

    2000-11-01

    Triple-A syndrome (MIM 231550; also known as Allgrove syndrome) is an autosomal recessive disorder characterized by adrenocorticotropin hormone (ACTH)-resistant adrenal insufficiency, achalasia of the oesophageal cardia and alacrima. Whereas several lines of evidence indicate that triple-A syndrome results from the abnormal development of the autonomic nervous system, late-onset progressive neurological symptoms (including cerebellar ataxia, peripheral neuropathy and mild dementia) suggest that the central nervous system may be involved in the disease as well. Using fine-mapping based on linkage disequilibrium in North African inbred families, we identified a short ancestral haplotype on chromosome 12q13 (<1 cM), sequenced a BAC contig encompassing the triple-A minimal region and identified a novel gene (AAAS) encoding a protein of 547 amino acids that is mutant in affected individuals. We found five homozygous truncating mutations in unrelated patients and ascribed the founder effect in North African families to a single splice-donor site mutation that occurred more than 2,400 years ago. The predicted product of AAAS, ALADIN (for alacrima-achalasia-adrenal insufficiency neurologic disorder), belongs to the WD-repeat family of regulatory proteins, indicating a new disease mechanism involved in triple-A syndrome. The expression of the gene in both neuroendocrine and cerebral structures points to a role in the normal development of the peripheral and central nervous systems.

  20. Mutant presenilin2 promotes apoptosis through the p53/miR-34a axis in neuronal cells.

    Science.gov (United States)

    Li, Liu-Hong; Tu, Qiu-Yun; Deng, Xiao-Hua; Xia, Jian; Hou, De-Ren; Guo, Ke; Zi, Xiao-Hong

    2017-05-01

    Neurodegenerative disorders have attracted attention in last decades due to their high incidence in the world. The p53/miR-34a axis triggers apoptosis and suppresses viability in multiple types of cells, but little is known about its role in neurodegenerative diseases. In this study, we showed that presenilin (PS)-2, a major gene associated with familial Alzheimer's disease (AD) could trigger the apoptosis through the p53/miR-34a axis in PC12 cells. First we found that PC12 cell viability was downregulated by PS-2 and mutant PS-2 overexpression, especially by mutant PS-2 overexpression. Then, we established a mutant PS-2-overexpressing PC12 cell line and confirmed that mutant PS-2 induced not only p53 but also miR-34a expression. The transfection of miR-34a inhibitor reversed PS-2-induced effects on cellular viability and apoptosis. Mutant PS-2 overexpression promoted caspase-3 expression, reduced Sirt1 and Bcl-2 expression, all of which were miR-34a downstream genes related with cell apoptosis. Moreover, mutant PS-2 also activated the p53/miR-34a axis and induced apoptosis in AD transgenic mice brain. These results implied that mutant PS-2 might promote the apoptosis of neuronal cells through triggering the p53/miR-34a axis. Altogether our results provide a novel insight into neurodegenerative disease and deepen our understandings of AD pathogenic processes. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer

    Science.gov (United States)

    2016-11-01

    AWARD NUMBER: W81XWH-14-1-0177 TITLE: Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer PRINCIPAL INVESTIGATOR: Katerina...5a. CONTRACT NUMBER Cellular Plasticity and Heterogeneity of EGFR Mutant Lung Cancer 5b. GRANT NUMBER W81XWH-14-1-0177 5c. PROGRAM ELEMENT NUMBER...epigenomic landscape of EGFR mutant SCLCs and their corresponding pre- treatment LUADs. These are very rare specimens. Through our Yale rebiopsy program

  2. Mutant-specific gene programs in the zebrafish

    OpenAIRE

    Weber, Gerhard J.; Choe, Sung E; Dooley, Kimberly A.; Paffett-Lugassy, Noëlle N.; Zhou, Yi; Zon, Leonard I.

    2005-01-01

    Hematopoiesis involves the production of stem cells, followed by the orchestrated differentiation of the blood lineages. Genetic screens in zebrafish have identified mutants with defects that disrupt specific stages of hematopoiesis and vasculogenesis, including the cloche, spadetail (tbx16), moonshine (tif1g), bloodless, and vlad tepes (gata1) mutants. To better characterize the blood program, gene expression profiling was carried out in these mutants and in scl-morphants (scl mo). Distinct ...

  3. Mutant p53 in Cancer: New Functions and Therapeutic Opportunities

    Science.gov (United States)

    Muller, Patricia A.J.; Vousden, Karen H.

    2014-01-01

    Many different types of cancer show a high incidence of TP53 mutations, leading to the expression of mutant p53 proteins. There is growing evidence that these mutant p53s have both lost wild-type p53 tumor suppressor activity and gained functions that help to contribute to malignant progression. Understanding the functions of mutant p53 will help in the development of new therapeutic approaches that may be useful in a broad range of cancer types. PMID:24651012

  4. Bilateral papillopathy as a presenting sign of pheochromocytoma associated with von Hippel–Lindau disease

    Directory of Open Access Journals (Sweden)

    Shah V

    2014-03-01

    Full Text Available Veeral Shah, Leonid Zlotcavitch, Angela M Herro, Sander R Dubovy, Zohar Yehoshua, Byron L LamBascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, FL, USAAbstract: A 7-year-old girl presented with decreased vision in both eyes for 1 month. Examination showed visual acuity of 20/50 and 20/60, no afferent pupillary defect, cecocentral scotomas, and bilateral optic disc edema with extensive peripapillary and macular exudates. Magnetic resonance imaging showed multiple cortical and subcortical white matter lesions. Both the laboratory workup and the systemic examination were unrevealing. However, on follow-up, the patient showed episodic elevations of blood pressure as high as 240/160. Further workup revealed elevated urine catecholamines and a right supra-adrenal mass proven to be a pheochromocytoma by histopathologic analysis. The paroxysmal hypertension resolved, and the visual acuity, visual fields, fundus exam, and neuroimaging improved. The patient was lost to follow-up until age 18 when she developed shortness of breath and was found to have multiple pulmonary metastases identified as pheochromocytoma by biopsy. Genetic testing identified a 3p25-26 (c.482 G>A VHL gene chromosomal mutation consistent with von Hippel–Lindau disease genotype. Multiple peripheral retinal vascular dilations and small retinal capillary hemangioblastomas were also found. This case highlights the importance of recognizing the lability of blood pressure often seen with pheochromocytomas, which may mask the underlying cause of hypertensive papillopathy and retinopathy, a diagnosis of low clinical suspicion in the pediatric population. The case also underscores the importance of thorough systemic workup, including genotyping to detect conditions where pheochromocytoma may be the presenting sign of the disease, such as multiple endocrine neoplasia 2A and 2B, von Hippel–Lindau disease, von Recklinghausen disease, tuberous sclerosis, and

  5. Counteracting quasispecies adaptability: extinction of a ribavirin-resistant virus mutant by an alternative mutagenic treatment.

    Science.gov (United States)

    Perales, Celia; Agudo, Rubén; Domingo, Esteban

    2009-01-01

    Lethal mutagenesis, or virus extinction promoted by mutagen-induced elevation of mutation rates of viruses, may meet with the problem of selection of mutagen-resistant variants, as extensively documented for standard, non-mutagenic antiviral inhibitors. Previously, we characterized a mutant of foot-and-mouth disease virus that included in its RNA-dependent RNA polymerase replacement M296I that decreased the sensitivity of the virus to the mutagenic nucleoside analogue ribavirin. Replacement M296I in the viral polymerase impedes the extinction of the mutant foot-and-mouth disease virus by elevated concentrations of ribavirin. In contrast, wild type virus was extinguished by the same ribavirin treatment and, interestingly, no mutants resistant to ribavirin were selected from the wild type populations. Decreases of infectivity and viral load of the ribavirin-resistant M296I mutant were attained with a combination of the mutagen 5-fluorouracil and the non-mutagenic inhibitor guanidine hydrocloride. However, extinction was achieved with a sequential treatment, first with ribavirin, and then with a minimal dose of 5-fluorouracil in combination with guanidine hydrochloride. Both, wild type and ribavirin-resistant mutant M296I exhibited equal sensitivity to this combination, indicating that replacement M296I in the polymerase did not confer a significant cross-resistance to 5-fluorouracil. We discuss these results in relation to antiviral designs based on lethal mutagenesis. (i) When dominant in the population, a mutation that confers partial resistance to a mutagenic agent can jeopardize virus extinction by elevated doses of the same mutagen. (ii) A wild type virus, subjected to identical high mutagenic treatment, need not select a mutagen-resistant variant, and the population can be extinguished. (iii) Extinction of the mutagen-resistant variant can be achieved by a sequential treatment of a high dose of the same mutagen, followed by a combination of another mutagen with

  6. Counteracting quasispecies adaptability: extinction of a ribavirin-resistant virus mutant by an alternative mutagenic treatment.

    Directory of Open Access Journals (Sweden)

    Celia Perales

    Full Text Available BACKGROUND: Lethal mutagenesis, or virus extinction promoted by mutagen-induced elevation of mutation rates of viruses, may meet with the problem of selection of mutagen-resistant variants, as extensively documented for standard, non-mutagenic antiviral inhibitors. Previously, we characterized a mutant of foot-and-mouth disease virus that included in its RNA-dependent RNA polymerase replacement M296I that decreased the sensitivity of the virus to the mutagenic nucleoside analogue ribavirin. METHODOLOGY AND PRINCIPAL FINDINGS: Replacement M296I in the viral polymerase impedes the extinction of the mutant foot-and-mouth disease virus by elevated concentrations of ribavirin. In contrast, wild type virus was extinguished by the same ribavirin treatment and, interestingly, no mutants resistant to ribavirin were selected from the wild type populations. Decreases of infectivity and viral load of the ribavirin-resistant M296I mutant were attained with a combination of the mutagen 5-fluorouracil and the non-mutagenic inhibitor guanidine hydrocloride. However, extinction was achieved with a sequential treatment, first with ribavirin, and then with a minimal dose of 5-fluorouracil in combination with guanidine hydrochloride. Both, wild type and ribavirin-resistant mutant M296I exhibited equal sensitivity to this combination, indicating that replacement M296I in the polymerase did not confer a significant cross-resistance to 5-fluorouracil. We discuss these results in relation to antiviral designs based on lethal mutagenesis. CONCLUSIONS: (i When dominant in the population, a mutation that confers partial resistance to a mutagenic agent can jeopardize virus extinction by elevated doses of the same mutagen. (ii A wild type virus, subjected to identical high mutagenic treatment, need not select a mutagen-resistant variant, and the population can be extinguished. (iii Extinction of the mutagen-resistant variant can be achieved by a sequential treatment of a

  7. Large-scale RNA interference screening in mammalian cells identifies novel regulators of mutant huntingtin aggregation.

    Directory of Open Access Journals (Sweden)

    Tomoyuki Yamanaka

    Full Text Available In polyglutamine (polyQ diseases including Huntington's disease (HD, mutant proteins containing expanded polyQ stretch form aggregates in neurons. Genetic or RNAi screenings in yeast, C. elegans or Drosophila have identified multiple genes modifying polyQ aggregation, a few of which are confirmed effective in mammals. However, the overall molecular mechanism underlying polyQ protein aggregation in mammalian cells still remains obscure. We here perform RNAi screening in mouse neuro2a cells to identify mammalian modifiers for aggregation of mutant huntingtin, a causative protein of HD. By systematic cell transfection and automated cell image analysis, we screen ∼ 12000 shRNA clones and identify 111 shRNAs that either suppress or enhance mutant huntingtin aggregation, without altering its gene expression. Classification of the shRNA-targets suggests that genes with various cellular functions such as gene transcription and protein phosphorylation are involved in modifying the aggregation. Subsequent analysis suggests that, in addition to the aggregation-modifiers sensitive to proteasome inhibition, some of them, such as a transcription factor Tcf20, and kinases Csnk1d and Pik3c2a, are insensitive to it. As for Tcf20, which contains polyQ stretches at N-terminus, its binding to mutant huntingtin aggregates is observed in neuro2a cells and in HD model mouse neurons. Notably, except Pik3c2a, the rest of the modifiers identified here are novel. Thus, our first large-scale RNAi screening in mammalian system identifies previously undescribed genetic players that regulate mutant huntingtin aggregation by several, possibly mammalian-specific mechanisms.

  8. Progranulin is neurotrophic in vivo and protects against a mutant TDP-43 induced axonopathy.

    Directory of Open Access Journals (Sweden)

    Angela S Laird

    Full Text Available Mislocalization, aberrant processing and aggregation of TAR DNA-binding protein 43 (TDP-43 is found in the neurons affected by two related diseases, amyotrophic lateral sclerosis (ALS and frontotemporal lobe dementia (FTLD. These TDP-43 abnormalities are seen when TDP-43 is mutated, such as in familial ALS, but also in FTLD, caused by null mutations in the progranulin gene. They are also found in many patients with sporadic ALS and FTLD, conditions in which only wild type TDP-43 is present. The common pathological hallmarks and symptomatic cross over between the two diseases suggest that TDP-43 and progranulin may be mechanistically linked. In this study we aimed to address this link by establishing whether overexpression of mutant TDP-43 or knock-down of progranulin in zebrafish embryos results in motor neuron phenotypes and whether human progranulin is neuroprotective against such phenotypes. Mutant TDP-43 (A315T mutation induced a motor axonopathy characterized by short axonal outgrowth and aberrant branching, similar, but more severe, than that induced by mutant SOD1. Knockdown of the two zebrafish progranulin genes, grna and grnb, produced a substantial decrease in axonal length, with knockdown of grna alone producing a greater decrease in axonal length than grnb. Progranulin overexpression rescued the axonopathy induced by progranulin knockdown. Interestingly, progranulin also rescued the mutant TDP-43 induced axonopathy, whilst it failed to affect the mutant SOD1-induced phenotype. TDP-43 was found to be nuclear in all conditions described. The findings described here demonstrate that progranulin is neuroprotective in vivo and may have therapeutic potential for at least some forms of motor neuron degeneration.

  9. Growth and development of maize that contains mutant tubulin genes

    Energy Technology Data Exchange (ETDEWEB)

    Susan M. Wick

    2004-07-23

    Mutant maize plants containing a Mu transposon disrupting one of the five beta tubulin genes of interest were followed for several generations and hybridized with each other to produce plants containing disruptions in both copies of a single gene or disruption of more than one tubulin gene. Seedlings of some of these plants were grown under chilling conditions for a few weeks. After DOE funding ended, plants have been assessed to see whether mutant are more or less tolerant to chilling. Other mutant plants will be assessed for their male and female fertility relative to non-mutant siblings or other close relatives.

  10. Sphingolipid synthesis deficiency in a mutant of Bacteroides levii

    Energy Technology Data Exchange (ETDEWEB)

    Brumleve, B.; Lev, M.

    1986-05-01

    Bacteroides levii, an anaerobic bacterium, synthesizes two sphingolipids; the sphingomyelin analogue, ceramide phosphorylethanolamine (CPE), and also ceramide phosphorylglycerol (CPG). The first enzyme in the sphingolipid pathway, 3-ketodihydro-sphingosine (3KDS) synthase, has been partially purified previously. To study subsequent steps in the pathways, mutants defective in sphingolipid synthesis were derived by ethyl methanesulfonate and nitrosoguanidine mutagenesis. Extracts of the mutant, 1075BB, show synthase activity although the cells do not synthesize CPE or CPG. The mutant differs from the wild type in that: (1) synthase activity was much diminished in the mutant, (2) sphingolipid synthesis does not occur in the mutant as evidenced by the absence of spots at sites where CPE and CPG migrate following two-dimensional thin layer chromatography, (3) incorporation of uniformly-labelled (/sup 14/C)serine carbon or (/sup 14/C)3KDS into sphingolipids was not observed in the mutant, (4) following incubation with (/sup 14/C)3KDS, radioactivity corresponding to dihydrosphingosine (DHS) and ceramide were observed in the mutant; no (/sup 14/C)DHS was detected in the wild type, and (5) enhanced incorporation of (/sup 14/C)serine carbon into two lipids not containing phosphorus was found in the mutant. The authors conclude, therefore, that this mutant, 1075BB, has a metabolic block at the terminal biosynthetic steps of sphingolipid synthesis.

  11. Standardized, systemic phenotypic analysis of Slc12a1I299F mutant mice.

    Science.gov (United States)

    Kemter, Elisabeth; Rathkolb, Birgit; Becker, Lore; Bolle, Ines; Busch, Dirk H; Dalke, Claudia; Elvert, Ralf; Favor, Jack; Graw, Jochen; Hans, Wolfgang; Ivandic, Boris; Kalaydjiev, Svetoslav; Klopstock, Thomas; Rácz, Ildikó; Rozman, Jan; Schrewe, Anja; Schulz, Holger; Zimmer, Andreas; Fuchs, Helmut; Gailus-Durner, Valérie; Hrabe de Angelis, Martin; Wolf, Eckhard; Aigner, Bernhard

    2014-08-02

    Type I Bartter syndrome is a recessive human nephropathy caused by loss-of-function mutations in the SLC12A1 gene coding for the Na+-K+-2Cl- cotransporter NKCC2. We recently established the mutant mouse line Slc12a1I299F exhibiting kidney defects highly similar to the late-onset manifestation of this hereditary human disease. Besides the kidney defects, low blood pressure and osteopenia were revealed in the homozygous mutant mice which were also described in humans. Beside its strong expression in the kidney, NKCC2 has been also shown to be expressed in other tissues in rodents i.e. the gastrointestinal tract, pancreatic beta cells, and specific compartments of the ear, nasal tissue and eye. To examine if, besides kidney defects, further organ systems and/or metabolic pathways are affected by the Slc12a1I299F mutation as primary or secondary effects, we describe a standardized, systemic phenotypic analysis of the mutant mouse line Slc12a1I299F in the German Mouse Clinic. Slc12a1I299F homozygous mutant mice and Slc12a1I299F heterozygous mutant littermates as controls were tested at the age of 4-6 months. Beside the already published changes in blood pressure and bone metabolism, a significantly lower body weight and fat content were found as new phenotypes for Slc12a1I299F homozygous mutant mice. Small additional effects included a mild erythropenic anemia in homozygous mutant males as well as a slight hyperalgesia in homozygous mutant females. For other functions, such as immunology, lung function and neurology, no distinct alterations were observed. In this systemic analysis no clear primary effects of the Slc12a1I299F mutation appeared for the organs other than the kidneys where Slc12a1 expression has been described. On the other hand, long-term effects additional and/or secondary to the kidney lesions might also appear in humans harboring SLC12A1 mutations.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

    in patients with autosomal-dominant hypocalcemia type 2 (ADH2), an inherited disorder of hypocalcemia, low parathyroid hormone (PTH), and hyperphosphatemia. We have generated knockin mice harboring the point mutation GNA11 c.C178T (p.Arg60Cys) identified in ADH2 patients. The mutant mice faithfully replicated....... This animal model of ADH2 provides insights into molecular mechanism of this G protein-related disease and potential paths toward new lines of therapy....

  13. Recovery from impaired muscle growth arises from prolonged postnatal accretion of myonuclei in Atrx mutant mice.

    Science.gov (United States)

    Huh, Michael S; Young, Kevin G; Yan, Keqin; Price-O'Dea, Tina; Picketts, David J

    2017-01-01

    Reduced muscle mass due to pathological development can occur through several mechanisms, including the loss or reduced proliferation of muscle stem cells. Muscle-specific ablation of the α-thalassemia mental retardation syndrome mutant protein, Atrx, in transgenic mice results in animals with a severely reduced muscle mass at three weeks of age; yet this muscle mass reduction resolves by adult age. Here, we explore the cellular mechanism underlying this effect. Analysis of Atrx mutant mice included testing for grip strength and rotorod performance. Muscle fiber length, fiber volume and numbers of myofiber-associated nuclei were determined from individual EDL or soleus myofibers isolated at three, five, or eight weeks. Myofibers from three week old Atrx mutant mice are smaller with fewer myofiber-associated nuclei and reduced volume compared to control animals, despite similar fiber numbers. Nonetheless, the grip strength of Atrx mutant mice was comparable to control mice when adjusted for body weight. Myofiber volume remained smaller at five weeks, becoming comparable to controls by 8 weeks of age. Concomitantly, increased numbers of myofiber-associated nuclei and Ki67+ myoblasts indicated that the recovery of muscle mass likely arises from the prolonged accretion of new myonuclei. This suggests that under disease conditions the muscle satellite stem cell niche can remain in a prolonged active state, allowing for the addition of a minimum number of myonuclei required to achieve a normal muscle size.

  14. Uric Acid Accumulation in an Arabidopsis Urate Oxidase Mutant Impairs Seedling Establishment by Blocking Peroxisome Maintenance[W

    Science.gov (United States)

    Hauck, Oliver K.; Scharnberg, Jana; Escobar, Nieves Medina; Wanner, Gerhard; Giavalisco, Patrick; Witte, Claus-Peter

    2014-01-01

    Purine nucleotides can be fully catabolized by plants to recycle nutrients. We have isolated a urate oxidase (uox) mutant of Arabidopsis thaliana that accumulates uric acid in all tissues, especially in the developing embryo. The mutant displays a reduced germination rate and is unable to establish autotrophic growth due to severe inhibition of cotyledon development and nutrient mobilization from the lipid reserves in the cotyledons. The uox mutant phenotype is suppressed in a xanthine dehydrogenase (xdh) uox double mutant, demonstrating that the underlying cause is not the defective purine base catabolism, or the lack of UOX per se, but the elevated uric acid concentration in the embryo. Remarkably, xanthine accumulates to similar levels in the xdh mutant without toxicity. This is paralleled in humans, where hyperuricemia is associated with many diseases whereas xanthinuria is asymptomatic. Searching for the molecular cause of uric acid toxicity, we discovered a local defect of peroxisomes (glyoxysomes) mostly confined to the cotyledons of the mature embryos, which resulted in the accumulation of free fatty acids in dry seeds. The peroxisomal defect explains the developmental phenotypes of the uox mutant, drawing a novel link between uric acid and peroxisome function, which may be relevant beyond plants. PMID:25052714

  15. Technical Report on the Development of Mutant Paracoccus strain and Optimization of Medium Composition for the Mass Production of Astaxanthin

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Jong Il; Lee, Ju Woon; Kim, Jae Hun; Song, Beom Seok

    2010-08-15

    Astaxanthin is used to role of provitamin A, and it is stronger antioxidant activity than vitamin E (100-500 times higher activity) and other carotenoids (10-fold). Furthermore, astaxanthin is also used as a nutraceutical and a medicinal ingredient against degenerative diseases such as cancer, heart disease, and skin related illness. The objective of this study was develop a carotenoid-hyperproducing mutant of Paracoccus N81106 using gamma irradiation and optimized medium composition. A mutant of Paracoccus having higher carotenoid content was isolated, and the production medium was optimized using response surface methodology. These results support that astaxanthin with strong antioxidant activity could be economically produced using the mutant and will be helpful for the related industry

  16. Age-dependent memory loss, synaptic pathology and altered brain plasticity in the Drosophila mutant cardinal accumulating 3-hydroxykynurenine.

    Science.gov (United States)

    Savvateeva, E; Popov, A; Kamyshev, N; Bragina, J; Heisenberg, M; Senitz, D; Kornhuber, J; Riederer, P

    2000-01-01

    A search for Drosophila mutants with phenotypes similar to human diseases might help to unravel evolutionary conserved genes implicated in polygenic human disorders. Among these are neurodegenerative diseases, characterized by a late onset disturbance of memory, synaptic and glial pathology, structural brain impairments and altered content of the intermediates of the kynurenine pathway, the modulators of glutamate excito- and oxidative toxicity. This pathway is conserved in insects, in rodents, and in humans. We tested the Drosophila mutants cardinal (3-hydroxykynurenine excess) and cinnabar (kynurenic acid excess) for age-dependent changes in memory, synaptic pathology, structural brain plasticity and glial immunoreactivity. The mutant cardinal demonstrated a decline in learning and memory from the 12th to the 29th day of life in a paradigm of conditioned courtship suppression. Memory decline was accompanied by a sharp decrease in immunoreactivity to the synaptic cysteine string protein, and alterations in volumetric parameters of the mushroom bodies, the brain structures implicated in memory.

  17. FUS Mutant Human Motoneurons Display Altered Transcriptome and microRNA Pathways with Implications for ALS Pathogenesis

    Directory of Open Access Journals (Sweden)

    Riccardo De Santis

    2017-11-01

    Full Text Available The FUS gene has been linked to amyotrophic lateral sclerosis (ALS. FUS is a ubiquitous RNA-binding protein, and the mechanisms leading to selective motoneuron loss downstream of ALS-linked mutations are largely unknown. We report the transcriptome analysis of human purified motoneurons, obtained from FUS wild-type or mutant isogenic induced pluripotent stem cells (iPSCs. Gene ontology analysis of differentially expressed genes identified significant enrichment of pathways previously associated to sporadic ALS and other neurological diseases. Several microRNAs (miRNAs were also deregulated in FUS mutant motoneurons, including miR-375, involved in motoneuron survival. We report that relevant targets of miR-375, including the neural RNA-binding protein ELAVL4 and apoptotic factors, are aberrantly increased in FUS mutant motoneurons. Characterization of transcriptome changes in the cell type primarily affected by the disease contributes to the definition of the pathogenic mechanisms of FUS-linked ALS.

  18. Therapeutic targeting of p53: all mutants are equal, but some mutants are more equal than others.

    Science.gov (United States)

    Sabapathy, Kanaga; Lane, David P

    2017-09-26

    TP53, which encodes the tumour-suppressor protein p53, is the most frequently mutated gene across all cancer types. The presence of mutant p53 predisposes to cancer development, promotes the survival of cancer cells, and is associated with ineffective therapeutic responses and unfavourable prognoses. Despite these effects, no drug that abrogates the oncogenic functions of mutant p53 has yet been approved for the treatment of cancer. Current investigational therapeutic strategies are mostly aimed at restoring the wild-type activity of mutant p53, based on the assumption that all p53 mutants are functionally equal. Our increasing knowledge of mutant forms of p53, however, supports the antithetical hypothesis that not all p53 mutants have equivalent cellular effects; hence, a judicious approach to therapeutic targeting of mutant p53 is required. In this Review, we propose a categorization of the major classes of p53 mutants based on their functionality in tumour suppression and response to therapy. The emerging picture is that the mutations across TP53 form a 'rainbow of mutants', with varying degrees of functionality and different pathobiological consequences, necessitating the use of diverse therapeutic strategies to selectively target specific classes of mutation. The utility of this knowledge of TP53 mutations in developing selective therapeutic options, and in facilitating clinical decision-making is discussed.

  19. Prevacuolar compartment morphology in vps mutants of Saccharomyces cerevisiae.

    Science.gov (United States)

    Hedman, Jamie M; Eggleston, Matthew D; Attryde, Amanda L; Marshall, Pamela A

    2007-10-01

    Over 60 genes have been identified that affect protein sorting to the lysosome-like vacuole in Saccharomyces cerevisiae. Cells with mutations in these vacuolar protein sorting (vps) genes fall into seven general classes based upon their vacuolar morphology. Class A mutants have a morphologically wild type vacuole, while Class B mutants have a fragmented vacuole. There is no discernable vacuolar structure in Class C mutants. Class D mutants have a slightly enlarged vacuole, but Class E mutants have a normal looking vacuole with an enlarged prevacuolar compartment (PVC), which is analogous to the mammalian late endosome. Class F mutants have a wild type appearing vacuole as well as fragmented vacuolar structures. vps mutants have also been found with a tubulo-vesicular vacuole structure. vps mutant morphology is pertinent, as mutants of the same class may work together and/or have a block in the same general step in the vacuolar protein sorting pathway. We probed PVC morphology and location microscopically in live cells of several null vps mutants using a GFP fusion protein of Nhx1p, an Na(+)/H(+) exchanger normally localized to the PVC. We show that cell strains deleted for VPS proteins that have been previously shown to work together, regardless of VPS Class, have the same PVC morphology. Cell strains lacking VPS genes that have not been implicated in the same pathway show different PVC morphologies, even if the mutant strains are in the same VPS Class. These new studies indicate that PVC morphology is another tier of classification that may more accurately identify proteins that function together in vacuolar protein sorting than the original vps mutation classes.

  20. Brucellosis vaccines: assessment of Brucella melitensis lipopolysaccharide rough mutants defective in core and O-polysaccharide synthesis and export.

    Directory of Open Access Journals (Sweden)

    David González

    Full Text Available BACKGROUND: The brucellae are facultative intracellular bacteria that cause brucellosis, one of the major neglected zoonoses. In endemic areas, vaccination is the only effective way to control this disease. Brucella melitensis Rev 1 is a vaccine effective against the brucellosis of sheep and goat caused by B. melitensis, the commonest source of human infection. However, Rev 1 carries a smooth lipopolysaccharide with an O-polysaccharide that elicits antibodies interfering in serodiagnosis, a major problem in eradication campaigns. Because of this, rough Brucella mutants lacking the O-polysaccharide have been proposed as vaccines. METHODOLOGY/PRINCIPAL FINDINGS: To examine the possibilities of rough vaccines, we screened B. melitensis for lipopolysaccharide genes and obtained mutants representing all main rough phenotypes with regard to core oligosaccharide and O-polysaccharide synthesis and export. Using the mouse model, mutants were classified into four attenuation patterns according to their multiplication and persistence in spleens at different doses. In macrophages, mutants belonging to three of these attenuation patterns reached the Brucella characteristic intracellular niche and multiplied intracellularly, suggesting that they could be suitable vaccine candidates. Virulence patterns, intracellular behavior and lipopolysaccharide defects roughly correlated with the degree of protection afforded by the mutants upon intraperitoneal vaccination of mice. However, when vaccination was applied by the subcutaneous route, only two mutants matched the protection obtained with Rev 1 albeit at doses one thousand fold higher than this reference vaccine. These mutants, which were blocked in O-polysaccharide export and accumulated internal O-polysaccharides, stimulated weak anti-smooth lipopolysaccharide antibodies. CONCLUSIONS/SIGNIFICANCE: The results demonstrate that no rough mutant is equal to Rev 1 in laboratory models and question the notion that

  1. Isolation and characterization of stable mutants of Streptomyces ...

    Indian Academy of Sciences (India)

    Daunorubicin and its derivative doxorubicin are antitumour anthracycline antibiotics produced by Streptomyces peucetius. In this study we report isolation of stable mutants of S. peucetius blocked in different steps of the daunorubicin biosynthesis pathway. Mutants were screened on the basis of colony colour since producer ...

  2. Differentially expressed genes in white egg 2 mutant of silkworm ...

    African Journals Online (AJOL)

    These pathways were related to amino acid metabolism, sugar metabolism, and series of major physiological metabolism. Our results hopefully shed light on the further study of molecular mechanism of white egg 2 mutant. Key words: Bombyx mori, white egg 2 mutant, microarray, embryo, differentially expressed gene.

  3. Genomic diversity among Basmati rice ( Oryza sativa L) mutants ...

    African Journals Online (AJOL)

    Genomic diversity among Basmati rice ( Oryza sativa L) mutants obtained through 60 Co gamma radiations using AFLP markers. ... In order to obtain new varieties of rice with improved agronomic and grain characteristics, gamma radiation (60Co) has been used to generate novel mutants of the Basmati rice. In this study ...

  4. Enhanced production of glucose oxidase from UV- mutant of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-01-19

    Jan 19, 2009 ... UV rays were used as mutagen in wild type strain of Aspergillus niger for enhanced production of glucose oxidase. After mutangenization and selection, mutant A. niger strains, resistant to 2-deoxy-D- glucose were obtained. The mutants showed 1.57 and 1.98 fold increase in activities of extra and intra.

  5. Comparison of lignin deposition in three ectopic lignification mutants.

    Science.gov (United States)

    Rogers, Louisa A; Dubos, Christian; Surman, Christine; Willment, Janet; Cullis, Ian F; Mansfield, Shawn D; Campbell, Malcolm M

    2005-10-01

    The Arabidopsis thaliana mutants de-etiolated3 (det3), pom-pom1 (pom1) and ectopic lignification1 (eli1) all deposit lignins in cells where these polymers would not normally be found. Comparison of these mutants provides an opportunity to determine if the shared mutant phenotype arose by perturbing a common regulatory mechanism in each of the mutants. The mutants were compared using a combination of genetics, histochemistry, chemical profiling, transcript profiling using both Northern blots and microarrays, and bioinformatics. The subset of cells that ectopically lignified was shared between all three mutants, but clear differences in cell wall chemistry were evident between the mutants. Northern blot analysis of lignin biosynthetic genes over diurnal and circadian cycles revealed that transcript abundance of several key genes was clearly altered in all three mutants. Microarray analysis suggests that changes in the expression of specific members of the R2R3-MYB and Dof transcription factor families may contribute to the ectopic lignification phenotypes. This comparative analysis provides a suite of hypotheses that can be tested to examine the control of lignin biosynthesis.

  6. Molecularly targeted therapies for p53-mutant cancers.

    Science.gov (United States)

    Zhao, Dekuang; Tahaney, William M; Mazumdar, Abhijit; Savage, Michelle I; Brown, Powel H

    2017-11-01

    The tumor suppressor p53 is lost or mutated in approximately half of human cancers. Mutant p53 not only loses its anti-tumor transcriptional activity, but also often acquires oncogenic functions to promote tumor proliferation, invasion, and drug resistance. Traditional strategies have been taken to directly target p53 mutants through identifying small molecular compounds to deplete mutant p53, or to restore its tumor suppressive function. Accumulating evidence suggest that cancer cells with mutated p53 often exhibit specific functional dependencies on secondary genes or pathways to survive, providing alternative targets to indirectly treat p53-mutant cancers. Targeting these genes or pathways, critical for survival in the presence of p53 mutations, holds great promise for cancer treatment. In addition, mutant p53 often exhibits novel gain-of-functions to promote tumor growth and metastasis. Here, we review and discuss strategies targeting mutant p53, with focus on targeting the mutant p53 protein directly, and on the progress of identifying genes and pathways required in p53-mutant cells.

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

    African Journals Online (AJOL)

    FKOLADE

    2016-11-09

    Nov 9, 2016 ... for crop improvement, hence the need to broaden the genetic base of any crop. This study was done in order to further enhance this in cowpea. While assessing diversity and phylogenetic relationship with other mutants and their parents, each unique mutant was also characterized. Randomly amplified ...

  8. Cadmium-Sensitive Mutants of Arabidopsis thaliana1

    Science.gov (United States)

    Howden, Ross; Cobbett, Christopher S.

    1992-01-01

    A screening procedure for identifying Cd-sensitive mutants of Arabidopsis thaliana is described. With this procedure, two Cd-sensitive mutants were isolated. These represent independent mutations in the same locus, referred to as CAD1. Genetic analysis has shown that the sensitive phenotype is recessive to the wild type and segregates as a single Mendelian locus. Crosses of the mutant to marker strains showed that the mutation is closely linked to the tt3 locus on chromosome 5. In addition to Cd, the mutants are also significantly more sensitive to mercuric ions and only slightly more sensitive to Cu and Zn, while being no more sensitive than the wild type to Mn, thus indicating a degree of specificity in the mechanism affected by the mutation. Undifferentiated callus tissue is also Cd sensitive, suggesting that the mutant phenotype is expressed at the cellular level. Both wild-type and mutant plants showed increased sensitivity to Cd in the presence of buthionine sulfoximine, an inhibitor of the biosynthesis of the cadmium-binding (γ-glutamylcysteine)n-glycine peptides, suggesting that the mutant is still able to synthesize these peptides. However, the effects of a cad1 mutation and buthionine sulfoximine together on cadmium sensitivity are essentially nonadditive, indicating that they may affect different aspects of the same detoxification mechanism. Assays of Cd uptake by intact plants indicate that the mutant is deficient in its ability to sequester Cd. Images Figure 1 Figure 7 PMID:16652930

  9. Differentially expressed genes in white egg 2 mutant of silkworm ...

    African Journals Online (AJOL)

    use

    2011-12-21

    Dec 21, 2011 ... egg 2 (w-2) has the same phenotypes as white egg 1 and white egg 3 mutants with white egg color, but its mechanism is more complicated than white egg 1 and white egg 3 mutants based on recent report (Tatematsu et al., 2011) which suggest that the silkworm w-2 locus existed multi-allelic mutations.

  10. Characterization of human glucocerebrosidase from different mutant alleles

    NARCIS (Netherlands)

    Ohashi, T.; Hong, C. M.; Weiler, S.; Tomich, J. M.; Aerts, J. M.; Tager, J. M.; Barranger, J. A.

    1991-01-01

    Human cDNA was mutagenized to duplicate six naturally occurring mutations in the gene for glucocere-brosidase. The mutant genes were expressed in NIH 3T3 cells. The abnormal human enzymes were purified by immunoaffinity chromatography and characterized. The Asn370----Ser mutant protein differed from

  11. Photosynthetic characterization of a rolled leaf mutant of rice ( Oryza ...

    African Journals Online (AJOL)

    A new rolling leaf rice mutant was identified which showed an apparently straighter longitudinal shape normal transverse rolling characters at all developing stages. The chlorophyll contents per fresh weight of this mutant leaves were lower than those of wild-type. The electron transfer rate (ETR) and photochemical ...

  12. Decreased cariogenicity of a mutant of Streptococcus mutans

    NARCIS (Netherlands)

    Stoppelaar, J.D.; König, K.G.; Plasschaert, A.J.M.; Hoeven, J.S. van der

    A strain of Streptococcus mutans was treated with a mutagenic agent. This resulted in isolation of a mutant which, compared to the original strain, had lost the ability to form sticky deposits on hard surfaces in sucrose medium. Apart from colonial morphology, the mutant had not changed in any other

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

    Directory of Open Access Journals (Sweden)

    Cong Tian

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

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

    Science.gov (United States)

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

    2016-01-01

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

  15. Accelerated Course of Experimental Autoimmune Encephalomyelitis in PD-1-Deficient Central Nervous System Myelin Mutants

    Science.gov (United States)

    Kroner, Antje; Schwab, Nicholas; Ip, Chi Wang; Ortler, Sonja; Göbel, Kerstin; Nave, Klaus-Armin; Mäurer, Mathias; Martini, Rudolf; Wiendl, Heinz

    2009-01-01

    It is assumed that the onset and course of autoimmune inflammatory central nervous system (CNS) disorders (eg, multiple sclerosis) are influenced by factors that afflict immune regulation as well as CNS vulnerability. We challenged this concept experimentally by investigating how genetic alterations that affect myelin (primary oligodendrocyte damage in PLPtg mice) and/or T-cell regulation (deficiency of PD-1) influence both the onset and course of an experimental autoimmune CNS inflammatory disease [MOG35-55-induced experimental autoimmune encephalomyelitis (EAE)]. We observed that double pathology was associated with a significantly earlier onset of disease, a slight increase in the neurological score, an increase in the number of infiltrating cells, and enhanced axonal degeneration compared with wild-type mice and the respective, single mutant controls. Double-mutant PLPtg/PD-1−/− mice showed an increased production of interferon-γ by CNS immune cells at the peak of disease. Neither PD-1 deficiency nor oligodendropathy led to detectable spread of antigenic MHC class I- or class II-restricted epitopes during EAE. However, absence of PD-1 clearly increased the propensity of T lymphocytes to expand, and the number of clonal expansions reliably reflected the severity of the EAE disease course. Our data show that the interplay between immune dysregulation and myelinopathy results in a stable exacerbation of actively induced autoimmune CNS inflammation, suggesting that the combination of several pathological issues contributes significantly to disease susceptibility or relapses in human disease. PMID:19443704

  16. Mutants of Cre recombinase with improved accuracy

    Science.gov (United States)

    Eroshenko, Nikolai; Church, George M.

    2013-01-01

    Despite rapid advances in genome engineering technologies, inserting genes into precise locations in the human genome remains an outstanding problem. It has been suggested that site-specific recombinases can be adapted towards use as transgene delivery vectors. The specificity of recombinases can be altered either with directed evolution or via fusions to modular DNA-binding domains. Unfortunately, both wildtype and altered variants often have detectable activities at off-target sites. Here we use bacterial selections to identify mutations in the dimerization surface of Cre recombinase (R32V, R32M, and 303GVSdup) that improve the accuracy of recombination. The mutants are functional in bacteria, in human cells, and in vitro (except for 303GVSdup, which we did not purify), and have improved selectivity against both model off-target sites and the entire E. coli genome. We propose that destabilizing binding cooperativity may be a general strategy for improving the accuracy of dimeric DNA-binding proteins. PMID:24056590

  17. Enhanced oligomerization of the alpha-synuclein mutant by the Cu,Zn-superoxide dismutase and hydrogen peroxide system.

    Science.gov (United States)

    Kang, Jung Hoon; Kim, Kyung Sik

    2003-02-28

    The alpha-synuclein is a major component of Lewy bodies that are found in the brains of patients with Parkinson's disease (PD). Also, two point mutations in this protein, A53T and A30P, are associated with rare familial forms of the disease. We investigated whether there are differences in the Cu,Zn-SOD and hydrogen peroxide system mediated-protein modification between the wild-type and mutant alpha-synucleins. When alpha-synuclein was incubated with both Cu,Zn-SOD and H2O2, then the amount of A53T mutant oligomerization increased relative to that of the wild-type protein. This process was inhibited by radical scavenger, spin-trapping agent, and copper chelator. These results suggest that the oligomerization of alpha-synuclein is mediated by the generation of the hydroxyl radical through the metal-catalyzed reaction. The dityrosine formation of the A53T mutant protein was enhanced relative to that of the wild-type protein. Antioxidant molecules, carnosine, and anserine effectively inhibited the wild-type and mutant proteins' oligomerization. Therefore, these compounds may be explored as potential therapeutic agents for PD patients. The present experiments, in part, may provide an explanation for the association between PD and the alpha-synuclein mutant.

  18. Analysis of mutant SOD1 electrophoretic mobility by Blue Native gel electrophoresis; evidence for soluble multimeric assemblies.

    Directory of Open Access Journals (Sweden)

    Hilda H Brown

    Full Text Available Mutations in superoxide dismutase 1 (SOD1 cause familial forms of amyotrophic lateral sclerosis (fALS. Disease causing mutations have diverse consequences on the activity and half-life of the protein, ranging from complete inactivity and short half-life to full activity and long-half-life. Uniformly, disease causing mutations induce the protein to misfold and aggregate and such aggregation tendencies are readily visualized by over-expression of the proteins in cultured cells. In the present study we have investigated the potential of using immunoblotting of proteins separated by Blue-Native gel electrophoresis (BNGE as a means to identify soluble multimeric forms of mutant protein. We find that over-expressed wild-type human SOD1 (hSOD1 is generally not prone to form soluble high molecular weight entities that can be separated by BNGE. For ALS mutant SOD1, we observe that for all mutants examined (A4V, G37R, G85R, G93A, and L126Z, immunoblots of BN-gels separating protein solubilized by digitonin demonstrated varied amounts of high molecular weight immunoreactive entities. These entities lacked reactivity to ubiquitin and were partially dissociated by reducing agents. With the exception of the G93A mutant, these entities were not reactive to the C4F6 conformational antibody. Collectively, these data demonstrate that BNGE can be used to assess the formation of soluble multimeric assemblies of mutant SOD1.

  19. Low protein to carbohydrate ratio diet delays onset of Parkinsonism like phenotype in Drosophila melanogaster parkin null mutants.

    Science.gov (United States)

    Bajracharya, Rijan; Ballard, J William O

    2016-12-01

    Dietary management plays a key role in the treatment of many diseases. However, no prospective studies have critically investigated the potential for dietary modification to delay the onset, or slow the progression, of Parkinson's Disease (PD). To study whether manipulating the Protein to Carbohydrate (P:C) ratio in the diet could affect the progression of PD, we compared Drosophila melanogaster parkin null mutants and their experimental controls fed with diets differing in their P:C ratio. We considered lifespan and feeding behaviors as well as motor and cellular functions on the 1:2 and 1:16 P:C diets. We observe that parkin mutants have a longer lifespan when fed the 1:16 P:C compared to those fed the 1:2 P:C diet. Parkin mutants fed the 1:16 P:C diet have delayed climbing deficit, increased resistance to starvation. Mutant flies fed the 1:16 P:C diet also have improved mitochondrial functions as evidenced by increased respiratory control ratio and mitochondrial membrane potential and decreased reactive oxygen species production and superoxide activity especially in old parkin mutants. Combined, these results suggest that dietary management has potential to delay the progression of PD. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  20. Dystonia-4 (DYT4)-associated TUBB4A mutants exhibit disorganized microtubule networks and inhibit neuronal process growth.

    Science.gov (United States)

    Watanabe, Natsumi; Itakaoka, Misa; Seki, Yoich; Morimoto, Takako; Homma, Keiichi; Miyamoto, Yuki; Yamauchi, Junji

    2018-01-01

    Dystonia-1 (DYT1) is an autosomal dominant early-onset torsion form of dystonia, a neurological disease affecting movement. DYT1 is the prototypic hereditary dystonia and is caused by the mutation of the tor1a gene. The gene product has chaperone functions important for the control of protein folding and stability. Dystonia-4 (DYT4) is another autosomal dominant dystonia that is characterized by onset in the second to third decade of progressive laryngeal dysphonia. DYT4 is associated with the mutation of the tubb4a gene, although it remains to be understood how disease-associated mutation affects biochemical as well as cell biological properties of the gene product as the microtubule component (a tubulin beta subunit). Herein we demonstrate that DYT4-associated TUBB4A missense mutants (Arg2-to-Gly or Ala271-to-Thr) form disorganized tubulin networks in cells. Transfected mutants are indeed expressed in cytoplasmic regions, as observed in wild-type transfectants. However, mutant proteins do not exhibit typical radial tubulin networks. Rather, they have diminished ability to interact with tubulin alpha subunits. Processes do not form in sufficient amounts in cells of the N1E-115 neuronal cell line expressing each of these mutants as compared to parental cells. Together, DYT4-associated TUBB4A mutants themselves form aberrant tubulin networks and inhibit neuronal process growth, possibly explaining progress through the pathological states at cellular levels. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Induction and characterization of Arabidopsis mutants by Ion beam

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Y. H.; Choi, J. D.; Park, J. Y.; Lee, J. R.; Sohn, H. S. [Gyeongbuk Institute for Bio Industry, Andong (Korea, Republic of)

    2008-03-15

    This study was conducted to search the proper conditions and times for irradiating proton beam to seeds generally used for induction of mutant. Arabidopsis as model plants has good characters that is a short generation time, producing a lot of seeds, sequenced genome, developed maker. This points were the best materials for plant breeding for this study. The data of inducing mutants of Arabidopsis is used to be applicate to crops have more longer generation that is the final goals of this study. The goals of this project were to inducing and characterizing arabidopsis mutants by the proton ion beam and {gamma}-ray. As well as, the purpose of this study was securing more than 10 lines of arabidopsis mutants in this project and also to know the changed DNA structure of the mutants using the basic data for applying to the more study

  2. Arabinose Kinase-Deficient Mutant of Arabidopsis thaliana 1

    Science.gov (United States)

    Dolezal, Olan; Cobbett, Christopher S.

    1991-01-01

    A mutant of Arabidopsis thaliana that is sensitive to exogenous l-arabinose has been isolated. Comparisons of growth of the wild type, mutant, and F1 and F2 progeny of crosses showed the arabinose-sensitive phenotype is semidominant and segregates as a single Mendelian locus. Crosses of the mutant to marker strains showed the mutation is linked to the eceriferum-2 locus on chromosome 4. In vivo incorporation of exogenous labeled l-arabinose into ethanol-insoluble polysaccharides was greatly reduced in the mutant with a concomitant accumulation of free labeled arabinose. Enzyme assays of crude plant extracts demonstrated a defect in arabinose kinase activity in the mutant. ImagesFigure 2Figure 3 PMID:16668327

  3. Isolation and characterization of gallium resistant Pseudomonas aeruginosa mutants.

    Science.gov (United States)

    García-Contreras, Rodolfo; Lira-Silva, Elizabeth; Jasso-Chávez, Ricardo; Hernández-González, Ismael L; Maeda, Toshinari; Hashimoto, Takahiro; Boogerd, Fred C; Sheng, Lili; Wood, Thomas K; Moreno-Sánchez, Rafael

    2013-12-01

    Pseudomonas aeruginosa PA14 cells resistant to the novel antimicrobial gallium nitrate (Ga) were developed using transposon mutagenesis and by selecting spontaneous mutants. The mutants showing the highest growth in the presence of Ga were selected for further characterization. These mutants showed 4- to 12-fold higher Ga minimal inhibitory growth concentrations and a greater than 8-fold increase in the minimum biofilm eliminating Ga concentration. Both types of mutants produced Ga resistant biofilms whereas the formation of wild-type biofilms was strongly inhibited by Ga. The gene interrupted in the transposon mutant was hitA, which encodes a periplasmic iron binding protein that delivers Fe³⁺ to the HitB iron permease; complementation of the mutant with the hitA gene restored the Ga sensitivity. This hitA mutant showed a 14-fold decrease in Ga internalization versus the wild-type strain, indicating that the HitAB system is also involved in the Ga uptake. Ga uptake in the spontaneous mutant was also lower, although no mutations were found in the hitAB genes. Instead, this mutant harbored 64 non-silent mutations in several genes including those of the phenazine pyocyanin biosynthesis. The spontaneous mutant produced 2-fold higher pyocyanin basal levels than the wild-type; the addition of this phenazine to wild-type cultures protected them from the Ga bacteriostatic effect. The present data indicate that mutations affecting Ga transport and probably pyocyanin biosynthesis enable cells to develop resistance to Ga. Copyright © 2013 Elsevier GmbH. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Fei Yuan

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

  5. Properties of astrocytes cultured from GFAP over-expressing and GFAP mutant mice

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Woosung [Waisman Center and Department of Comparative Biosciences, University of Wisconsin-Madison, 1500 Highland Ave, Rm 713, Madison, WI 53705 (United States); Messing, Albee, E-mail: messing@waisman.wisc.edu [Waisman Center and Department of Comparative Biosciences, University of Wisconsin-Madison, 1500 Highland Ave, Rm 713, Madison, WI 53705 (United States)

    2009-04-15

    Alexander disease is a fatal leukoencephalopathy caused by dominantly-acting coding mutations in GFAP. Previous work has also implicated elevations in absolute levels of GFAP as central to the pathogenesis of the disease. However, identification of the critical astrocyte functions that are compromised by mis-expression of GFAP has not yet been possible. To provide new tools for investigating the nature of astrocyte dysfunction in Alexander disease, we have established primary astrocyte cultures from two mouse models of Alexander disease, a transgenic that over-expresses wild type human GFAP, and a knock-in at the endogenous mouse locus that mimics a common Alexander disease mutation. We find that mutant GFAP, as well as excess wild type GFAP, promotes formation of cytoplasmic inclusions, disrupts the cytoskeleton, decreases cell proliferation, increases cell death, reduces proteasomal function, and compromises astrocyte resistance to stress.

  6. Genome-wide transcriptional response of an avian pathogenic Escherichia coli (APEC) pst mutant.

    Science.gov (United States)

    Crépin, Sébastien; Lamarche, Martin G; Garneau, Philippe; Séguin, Julie; Proulx, Julie; Dozois, Charles M; Harel, Josée

    2008-11-28

    Avian pathogenic E. coli (APEC) are associated with extraintestinal diseases in poultry. The pstSCAB-phoU operon belongs to the Pho regulon and encodes the phosphate specific transport (Pst) system. A functional Pst system is required for full virulence in APEC and other bacteria and contributes to resistance of APEC to serum, to cationic antimicrobial peptides and acid shock. The global mechanisms contributing to the attenuation and decreased resistance of the APEC pst mutant to environmental stresses have not been investigated at the transcriptional level. To determine the global effect of a pst mutation on gene expression, we compared the transcriptomes of APEC strain chi7122 and its isogenic pst mutant (K3) grown in phosphate-rich medium. Overall, 470 genes were differentially expressed by at least 1.5-fold. Interestingly, the pst mutant not only induced systems involved in phosphate acquisition and metabolism, despite phosphate availability, but also modulated stress response mechanisms. Indeed, transcriptional changes in genes associated with the general stress responses, including the oxidative stress response were among the major differences observed. Accordingly, the K3 strain was less resistant to reactive oxygen species (ROS) than the wild-type strain. In addition, the pst mutant demonstrated reduced expression of genes involved in lipopolysaccharide modifications and coding for cell surface components such as type 1 and F9 fimbriae. Phenotypic tests also established that the pst mutant was impaired in its capacity to produce type 1 fimbriae, as demonstrated by western blotting and agglutination of yeast cells, when compared to wild-type APEC strain chi7122. Overall, our data elucidated the effects of a pst mutation on the transcriptional response, and further support the role of the Pho regulon as part of a complex network contributing to phosphate homeostasis, adaptive stress responses, and E. coli virulence.

  7. Modelling the spread of HIV immune escape mutants in a vaccinated population.

    Science.gov (United States)

    Fryer, Helen R; McLean, Angela R

    2011-12-01

    Because cytotoxic T-lymphocytes (CTLs) have been shown to play a role in controlling human immunodeficiency virus (HIV) infection and because CTL-based simian immunodeficiency virus (SIV) vaccines have proved effective in non-human primates, one goal of HIV vaccine design is to elicit effective CTL responses in humans. Such a vaccine could improve viral control in patients who later become infected, thereby reducing onwards transmission and enhancing life expectancy in the absence of treatment. The ability of HIV to evolve mutations that evade CTLs and the ability of these 'escape mutants' to spread amongst the population poses a challenge to the development of an effective and robust vaccine. We present a mathematical model of within-host evolution and between-host transmission of CTL escape mutants amongst a population receiving a vaccine that elicits CTL responses to multiple epitopes. Within-host evolution at each epitope is represented by the outgrowth of escape mutants in hosts who restrict the epitope and their reversion in hosts who do not restrict the epitope. We use this model to investigate how the evolution and spread of escape mutants could affect the impact of a vaccine. We show that in the absence of escape, such a vaccine could markedly reduce the prevalence of both infection and disease in the population. However the impact of such a vaccine could be significantly abated by CTL escape mutants, especially if their selection in hosts who restrict the epitope is rapid and their reversion in hosts who do not restrict the epitope is slow. We also use the model to address whether a vaccine should span a broad or narrow range of CTL epitopes and target epitopes restricted by rare or common HLA types. We discuss the implications and limitations of our findings. © 2011 Fryer, McLean.

  8. Guanylate cyclase 2C agonism corrects CFTR mutants.

    Science.gov (United States)

    Arora, Kavisha; Huang, Yunjie; Mun, Kyushik; Yarlagadda, Sunitha; Sundaram, Nambirajan; Kessler, Marco M; Hannig, Gerhard; Kurtz, Caroline B; Silos-Santiago, Inmaculada; Helmrath, Michael; Palermo, Joseph J; Clancy, John P; Steinbrecher, Kris A; Naren, Anjaparavanda P

    2017-10-05

    Cystic fibrosis (CF) is a genetic disorder in which epithelium-generated fluid flow from the lung, intestine, and pancreas is impaired due to mutations disrupting CF transmembrane conductance regulator (CFTR) channel function. CF manifestations of the pancreas and lung are present in the vast majority of CF patients, and 15% of CF infants are born with obstructed gut or meconium ileus. However, constipation is a significantly underreported outcome of CF disease, affecting 47% of the CF patients, and management becomes critical in the wake of increasing life span of CF patients. In this study, we unraveled a potentially novel molecular role of a membrane-bound cyclic guanosine monophosphate-synthesizing (cGMP-synthesizing) intestinal enzyme, guanylate cyclase 2C (GCC) that could be targeted to ameliorate CF-associated intestinal fluid deficit. We demonstrated that GCC agonism results in functional rescue of murine F508del/F508del and R117H/R117H Cftr and CFTR mutants in CF patient-derived intestinal spheres. GCC coexpression and activation facilitated processing and ER exit of F508del CFTR and presented a potentially novel rescue modality in the intestine, similar to the CF corrector VX-809. Our findings identify GCC as a biological CFTR corrector and potentiator in the intestine.

  9. Fecal corticosterone levels in RCAN1 mutant mice.

    Science.gov (United States)

    Rakowski-Anderson, Tammy; Wong, Helen; Rothermel, Beverly; Cain, Peter; Lavilla, Carmencita; Pullium, Jennifer K; Hoeffer, Charles

    2012-04-01

    Regulator of calcineurin 1 (RCAN1) is related to the expression of human neurologic disorders such as Down syndrome, Alzheimer disease, and chromosome 21q deletion syndrome. We showed here that RCAN1-knockout mice exhibit reduced innate anxiety as indicated by the elevated-plus maze. To examine whether glucocorticoids contribute to this phenotype, we measured fecal corticosterone in male wildtype and RCAN1-knockout mice and in male and female transgenic mice with neuronal overexpression of RCAN1 (Tg-RCAN1(TG)). We found no difference in fecal corticosterone levels of RCAN1-knockout mice and their wildtype littermates. As expected, we found differences between sexes in fecal corticosterone levels. In addition, we found higher levels of excreted corticosterone in Tg-RCAN1(TG) female mice as compared with female wildtype mice. Our data indicate normal diurnal corticosterone production in RCAN1 mutant mice and do not suggest a causal role in either the cognitive or anxiety phenotypes exhibited by RCAN1-knockout mice.

  10. PedonnanceofE3rly MatUring MutantS Derived from ''SuPa'~ Rice ...

    African Journals Online (AJOL)

    sig"ificant di.ffere-"~s between the mutants and their'parent for 'ail the maraders testeiiexcept 1 (j()(J grains weight and ~ipe weight The mutants .... reported in earlier rice improvement programmes

  11. Transposon insertion libraries for the characterization of mutants from the kiwifruit pathogen Pseudomonas syringae pv. actinidiae

    Science.gov (United States)

    Mesarich, Carl H.; Rees-George, Jonathan; Gardner, Paul P.; Ghomi, Fatemeh Ashari; Gerth, Monica L.; Andersen, Mark T.; Rikkerink, Erik H. A.; Fineran, Peter C.

    2017-01-01

    Pseudomonas syringae pv. actinidiae (Psa), the causal agent of kiwifruit canker, is one of the most devastating plant diseases of recent times. We have generated two mini-Tn5-based random insertion libraries of Psa ICMP 18884. The first, a ‘phenotype of interest’ (POI) library, consists of 10,368 independent mutants gridded into 96-well plates. By replica plating onto selective media, the POI library was successfully screened for auxotrophic and motility mutants. Lipopolysaccharide (LPS) biosynthesis mutants with ‘Fuzzy-Spreader’-like morphologies were also identified through a visual screen. The second, a ‘mutant of interest’ (MOI) library, comprises around 96,000 independent mutants, also stored in 96-well plates, with approximately 200 individuals per well. The MOI library was sequenced on the Illumina MiSeq platform using Transposon-Directed Insertion site Sequencing (TraDIS) to map insertion sites onto the Psa genome. A grid-based PCR method was developed to recover individual mutants, and using this strategy, the MOI library was successfully screened for a putative LPS mutant not identified in the visual screen. The Psa chromosome and plasmid had 24,031 and 1,236 independent insertion events respectively, giving insertion frequencies of 3.65 and 16.6 per kb respectively. These data suggest that the MOI library is near saturation, with the theoretical probability of finding an insert in any one chromosomal gene estimated to be 97.5%. However, only 47% of chromosomal genes had insertions. This surprisingly low rate cannot be solely explained by the lack of insertions in essential genes, which would be expected to be around 5%. Strikingly, many accessory genes, including most of those encoding type III effectors, lacked insertions. In contrast, 94% of genes on the Psa plasmid had insertions, including for example, the type III effector HopAU1. These results suggest that some chromosomal sites are rendered inaccessible to transposon insertion, either

  12. Cyclin D1 genotype and expression in sporadic hemangioblastomas.

    NARCIS (Netherlands)

    Gijtenbeek, J.M.M.; Sprenger, S.H.E.; Franke, B.; Wesseling, P.; Jeuken, J.W.M.

    2005-01-01

    Central nervous system (CNS) hemangioblastomas are highly-vascularized tumors occurring in sporadic form or as a manifestation of von Hippel-Lindau disease (VHL). The VHL protein (pVHL) regulates various target genes, one of which is the CCND1 gene, encoding cyclin D1, a protein that plays a

  13. Retinal hemangioblastoma

    DEFF Research Database (Denmark)

    Binderup, Marie Louise Mølgaard; Stendell, Anne-Sophie; Galanakis, Michael

    2017-01-01

    an asymptomatic first hemangioblastoma (80% vs 20%). Overall, 76% (41 of 54) of the vHL patients had a family history of vHL, while none of the patients without vHL did. Despite the rarity of the disease, on average more than eight new tumours are diagnosed each year due to multiple tumour development in v...

  14. Induction of mutant resistant to alternaria blotch of apple by gamma-ray irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Yoshioka, Toji [Hokuriku National Agricultural Experiment Station, Joetsu, Niigata (Japan); Ito, Yuji [National Inst. of Agrobiological Resources, Tsukuba, Ibaraki (Japan); Masuda, Tetsuo [National Institute of Fruit Tree Science, Morioka, Iwate (Japan)

    2000-07-01

    Apple cultivars resistant to Alternaria blotch disease have been produced by cross-breeding, but it is difficult to produce resistance by crossing without changing the properties of cultivar because the gene composition of the cultivar tree is almost heterozygous. This study aimed to investigate the resistant mutation in Alternaria blotch susceptible and semiresistant cultivars. The resistance to Alternaria blotch pathogen or AM toxin is classified into the following three groups: 1) highly sensitive group including Indo, Redgold and Starking delicious, 2) semi-resistant group including Fuji, Orin and Golden delicious and 3) resistant group including Gala and Tsugaru. After gamma ray exposure of 80 Gy (at 5 Gy/hour), AM-toxin insensitive clones were selected in the VM{sub 6} generation. These selected mutants could be rooted and habituated under field conditions. The degree of disease resistance was assessed by AM toxin treatment and Alternaria blotch fungi spore inoculation test. The leaves of these mutants were changed to variegated at high temperature, suggesting that some mutation related to chloroplast might have occurred. Alternaria blotch resistant strains could be produced by exposing to {gamma}ray and selecting with AM toxin in shoot-tip culture system, but the functional effects of the AM toxin in Alternaria blotch and also the mechanism in the mutant lines were still unclear. (M.N.)

  15. Lamin A/C mutants disturb sumo1 localization and sumoylation in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Émilie Boudreau

    Full Text Available A-type lamins A and C are nuclear intermediate filament proteins in which mutations have been implicated in multiple disease phenotypes commonly known as laminopathies. A few studies have implicated sumoylation in the regulation of A-type lamins. Sumoylation is a post-translational protein modification that regulates a wide range of cellular processes through the attachment of small ubiquitin-related modifier (sumo to various substrates. Here we showed that laminopathy mutants result in the mislocalization of sumo1 both in vitro (C2C12 cells overexpressing mutant lamins A and C and in vivo (primary myoblasts and myopathic muscle tissue from the Lmna(H222P/H222P mouse model. In C2C12 cells, we showed that the trapping of sumo1 in p.Asp192Gly, p.Gln353Lys, and p.Arg386Lys aggregates of lamin A/C correlated with an increased steady-state level of sumoylation. However, lamin A and C did not appear to be modified by sumo1. Our results suggest that mutant lamin A/C alters the dynamics of sumo1 and thus misregulation of sumoylation may be contributing to disease progression in laminopathies.

  16. Mapping pathological phenotypes in Reelin mutant mice

    Directory of Open Access Journals (Sweden)

    Caterina eMichetti

    2014-09-01

    Full Text Available Autism Spectrum Disorders (ASD are neurodevelopmental disorders with multifactorial origin characterized by social communication and behavioural perseveration deficits. Several studies showed an association between the reelin gene mutation and increased risk of ASD and a reduced reelin expression in some brain regions of ASD subjects, suggesting a role for reelin deficiency in ASD etiology. Reelin is a large extracellular matrix glycoprotein playing important roles during development of the central nervous system. To deeply investigate the role of reelin dysfunction as vulnerability factor in ASD, we investigated the behavioural, neurochemical and brain morphological features of reeler male mice. We recently reported a genotype-dependent deviation in ultrasonic vocal repertoire and a general delay in motor development in reeler pups. We now report that adult male heterozygous reeler mice did not show social behaviour and communication deficits during male-female social interactions. Wildtype and heterozygous mice also showed a typical light/dark locomotor activity profile, with a peak during the central interval of the dark phase. However, when faced with a mild stressful stimulus (a saline injection only heterozygous mice showed an over response to stress. At the end of the behavioural studies, we conducted high performance liquid chromatography and magnetic resonance imaging and spectroscopy to investigate whether reelin mutation influences brain monoamine and metabolites levels in regions involved in ASD. Low levels of dopamine in cortex and high levels of glutamate and taurine in hippocampus were detected in heterozygous mice, in line with clinical data collected on ASD children. Altogether, our data detected subtle but relevant neurochemical abnormalities in reeler mice supporting this mutant line, particularly male subjects, as a valid experimental model to estimate the contribution played by reelin deficiency in the global ASD

  17. Cp/Heph mutant mice have iron-induced neurodegeneration diminished by deferiprone

    Science.gov (United States)

    Zhao, Liangliang; Hadziahmetovic, Majda; Wang, Chenguang; Xu, Xueying; Song, Ying; Jinnah, H.A.; Wodzinska, Jolanta; Iacovelli, Jared; Wolkow, Natalie; Krajacic, Predrag; Weissberger, Alyssa Cwanger; Connelly, John; Spino, Michael; Lee, Michael K.; Connor, James; Giasson, Benoit; Harris, Z. Leah; Dunaief, Joshua L.

    2016-01-01

    Brain iron accumulates in several neurodegenerative diseases and can cause oxidative damage, but mechanisms of brain iron homeostasis are incompletely understood. Patients with mutations in the cellular iron-exporting ferroxidase ceruloplasmin (Cp) have brain iron accumulation causing neurodegeneration. Here, we assessed the brains of mice with combined mutation of Cp and its homolog hephaestin. Compared to single mutants, brain iron accumulation was accelerated in double mutants in the cerebellum, substantia nigra, and hippocampus. Iron accumulated within glia, while neurons were iron deficient. There was loss of both neurons and glia. Mice developed ataxia and tremor, and most died by 9 months. Treatment with the oral iron chelator deferiprone diminished brain iron levels, protected against neuron loss, and extended lifespan. Ferroxidases play important, partially overlapping roles in brain iron homeostasis by facilitating iron export from glia, making iron available to neurons. PMID:26303407

  18. A method for rapidly screening functionality of actin mutants and tagged actins

    Directory of Open Access Journals (Sweden)

    Rommelaere Heidi

    2004-01-01

    Full Text Available Recombinant production and biochemical analysis of actin mutants has been hampered by the fact that actin has an absolute requirement for the eukaryotic chaperone CCT to reach its native state. We therefore have developed a method to rapidly screen the folding capacity and functionality of actin variants, by combining in vitro expression of labelled actin with analysis on native gels, band shift assays or copolymerization tests. Additionally, we monitor, using immuno-fluorescence, incorporation of actin variants in cytoskeletal structures in transfected cells. We illustrate the method by two examples. In one we show that tagged versions of actin do not always behave native-like and in the other we study some of the molecular defects of three &bgr;-actin mutants that have been associated with diseases.

  19. The ataxic Cacna1a-mutant mouse rolling nagoya: an overview of neuromorphological and electrophysiological findings.

    Science.gov (United States)

    Plomp, Jaap J; van den Maagdenberg, Arn M J M; Kaja, Simon

    2009-09-01

    Homozygous rolling Nagoya natural mutant mice display a severe ataxic gait and frequently roll over to their side or back. The causative mutation resides in the Cacna1a gene, encoding the pore-forming alpha(1) subunit of Ca(v)2.1 type voltage-gated Ca(2+) channels. These channels are crucially involved in neuronal Ca(2+) signaling and in neurotransmitter release at many central synapses and, in the periphery, at the neuromuscular junction. We here review the behavioral, histological, biochemical, and neurophysiological studies on this mouse mutant and discuss its usefulness as a model of human neurological diseases associated with Ca(v)2.1 dysfunction.

  20. Reversal of a full-length mutant huntingtin neuronal cell phenotype by chemical inhibitors of polyglutamine-mediated aggregation

    Directory of Open Access Journals (Sweden)

    MacDonald Marcy E

    2005-01-01

    Full Text Available Abstract Background Huntington's disease (HD is an inherited neurodegenerative disorder triggered by an expanded polyglutamine tract in huntingtin that is thought to confer a new conformational property on this large protein. The propensity of small amino-terminal fragments with mutant, but not wild-type, glutamine tracts to self-aggregate is consistent with an altered conformation but such fragments occur relatively late in the disease process in human patients and mouse models expressing full-length mutant protein. This suggests that the altered conformational property may act within the full-length mutant huntingtin to initially trigger pathogenesis. Indeed, genotype-phenotype studies in HD have defined genetic criteria for the disease initiating mechanism, and these are all fulfilled by phenotypes associated with expression of full-length mutant huntingtin, but not amino-terminal fragment, in mouse models. As the in vitro aggregation of amino-terminal mutant huntingtin fragment offers a ready assay to identify small compounds that interfere with the conformation of the polyglutamine tract, we have identified a number of aggregation inhibitors, and tested whether these are also capable of reversing a phenotype caused by endogenous expression of mutant huntingtin in a striatal cell line from the HdhQ111/Q111 knock-in mouse. Results We screened the NINDS Custom Collection of 1,040 FDA approved drugs and bioactive compounds for their ability to prevent in vitro aggregation of Q58-htn 1–171 amino terminal fragment. Ten compounds were identified that inhibited aggregation with IC50 HdhQ111/Q111 striatal cells. Conclusions At least some compounds identified as aggregation inhibitors also prevent a neuronal cellular phenotype caused by full-length mutant huntingtin, suggesting that in vitro fragment aggregation can act as a proxy for monitoring the disease-producing conformational property in HD. Thus, identification and testing of compounds that

  1. The parkin Mutant Phenotype in the Fly Is Largely Rescued by Metal-Responsive Transcription Factor (MTF-1) ▿ †

    OpenAIRE

    Saini, Nidhi; Georgiev, Oleg; Schaffner, Walter

    2011-01-01

    The gene for Parkin, an E3 ubiquitin ligase, is mutated in some familial forms of Parkinson's disease, a severe neurodegenerative disorder. A homozygous mutant of the Drosophila ortholog of human parkin is viable but results in severe motoric impairment including an inability to fly, female and male sterility, and a decreased life span. We show here that a double mutant of the genes for Parkin and the metal-responsive transcription factor 1 (MTF-1) is not viable. MTF-1, which is conserved fro...

  2. Avirulent mutants of Macrophomina phaseolina and Aspergillus ...

    Indian Academy of Sciences (India)

    Indian Institute of Chemical Biology, 4, Raja S C Mullick Road, Jadavpur, Calcutta 700 032, India. *Corresponding author (Fax, 91-33-4730284; Email, iichbio@giascl01.vsnl.net.in). To evaluate the role of phaseolinone, a phytotoxin produced by Macrophomina phaseolina, in disease initiation, three nontoxigenic avirulent ...

  3. Functional Characterization of Human ProNGF and NGF Mutants: Identification of NGF P61SR100E as a "Painless" Lead Investigational Candidate for Therapeutic Applications.

    Directory of Open Access Journals (Sweden)

    Francesca Malerba

    Full Text Available Nerve Growth Factor (NGF holds a great therapeutic promise for Alzheimer's disease, diabetic neuropathies, ophthalmic diseases, dermatological ulcers. However, the necessity for systemic delivery has hampered the clinical applications of NGF due to its potent pro-nociceptive action. A "painless" human NGF (hNGF R100E mutant has been engineered. It has equal neurotrophic potency to hNGF but a lower nociceptive activity. We previously described and characterized the neurotrophic and nociceptive properties also of the hNGF P61S and P61SR100E mutants, selectively detectable against wild type hNGF. However, the reduced pain-sensitizing potency of the "painless" hNGF mutants has not been quantified.Aiming at the therapeutic application of the "painless" hNGF mutants, we report on the comparative functional characterization of the precursor and mature forms of the mutants hNGF R100E and hNGF P61SR100E as therapeutic candidates, also in comparison to wild type hNGF and to hNGF P61S. The mutants were assessed by a number of biochemical, biophysical methods and assayed by cellular assays. Moreover, a highly sensitive ELISA for the detection of the P61S-tagged mutants in biological samples has been developed. Finally, we explored the pro-nociceptive effects elicited by hNGF mutants in vivo, demonstrating an expanded therapeutic window with a ten-fold increase in potency.This structure-activity relationship study has led to validate the concept of developing painless NGF as a therapeutic, targeting the NGF receptor system and supporting the choice of hNGF P61S R100E as the best candidate to advance in clinical development. Moreover, this study contributes to the identification of the molecular determinants modulating the properties of the hNGF "painless" mutants.

  4. Functional Characterization of Human ProNGF and NGF Mutants: Identification of NGF P61SR100E as a "Painless" Lead Investigational Candidate for Therapeutic Applications.

    Science.gov (United States)

    Malerba, Francesca; Paoletti, Francesca; Bruni Ercole, Bruno; Materazzi, Serena; Nassini, Romina; Coppi, Elisabetta; Patacchini, Riccardo; Capsoni, Simona; Lamba, Doriano; Cattaneo, Antonino

    2015-01-01

    Nerve Growth Factor (NGF) holds a great therapeutic promise for Alzheimer's disease, diabetic neuropathies, ophthalmic diseases, dermatological ulcers. However, the necessity for systemic delivery has hampered the clinical applications of NGF due to its potent pro-nociceptive action. A "painless" human NGF (hNGF R100E) mutant has been engineered. It has equal neurotrophic potency to hNGF but a lower nociceptive activity. We previously described and characterized the neurotrophic and nociceptive properties also of the hNGF P61S and P61SR100E mutants, selectively detectable against wild type hNGF. However, the reduced pain-sensitizing potency of the "painless" hNGF mutants has not been quantified. Aiming at the therapeutic application of the "painless" hNGF mutants, we report on the comparative functional characterization of the precursor and mature forms of the mutants hNGF R100E and hNGF P61SR100E as therapeutic candidates, also in comparison to wild type hNGF and to hNGF P61S. The mutants were assessed by a number of biochemical, biophysical methods and assayed by cellular assays. Moreover, a highly sensitive ELISA for the detection of the P61S-tagged mutants in biological samples has been developed. Finally, we explored the pro-nociceptive effects elicited by hNGF mutants in vivo, demonstrating an expanded therapeutic window with a ten-fold increase in potency. This structure-activity relationship study has led to validate the concept of developing painless NGF as a therapeutic, targeting the NGF receptor system and supporting the choice of hNGF P61S R100E as the best candidate to advance in clinical development. Moreover, this study contributes to the identification of the molecular determinants modulating the properties of the hNGF "painless" mutants.

  5. Signature-tagged mutagenesis of Pasteurella multocida identifies mutants displaying differential virulence characteristics in mice and chickens.

    Science.gov (United States)

    Harper, Marina; Boyce, John D; Wilkie, Ian W; Adler, Ben

    2003-09-01

    Pasteurella multocida is the causative agent of fowl cholera in birds. Signature-tagged mutagenesis (STM) was used to identify potential virulence factors in a mouse septicemia disease model and a chicken fowl cholera model. A library of P. multocida mutants was constructed with a modified Tn916 and screened for attenuation in both animal models. Mutants identified by the STM screening were confirmed as attenuated by competitive growth assays in both chickens and mice. Of the 15 mutants identified in the chicken model, only 5 were also attenuated in mice, showing for the first time the presence of host-specific virulence factors and indicating the importance of screening for attenuation in the natural host.

  6. The oncolytic activity of Newcastle disease virus in clear cell renal carcinoma cells in normoxic and hypoxic conditions: the interplay between von Hippel-Lindau and interferon-β signaling.

    Science.gov (United States)

    Ch'ng, Wei-Choong; Stanbridge, Eric J; Yusoff, Khatijah; Shafee, Norazizah

    2013-07-01

    Viral-mediated oncolysis is a promising cancer therapeutic approach offering an increased efficacy with less toxicity than the current therapies. The complexity of solid tumor microenvironments includes regions of hypoxia. In these regions, the transcription factor, hypoxia inducible factor (HIF), is active and regulates expression of many genes that contribute to aggressive malignancy, radio-, and chemo-resistance. To investigate the oncolytic efficacy of a highly virulent (velogenic) Newcastle disease virus (NDV) in the presence or absence of HIF-2α, renal cell carcinoma (RCC) cell lines with defective or reconstituted wild-type (wt) von Hippel-Lindau (VHL) activity were used. We show that these RCC cells responded to NDV by producing only interferon (IFN)-β, but not IFN-α, and are associated with increased STAT1 phosphorylation. Restoration of wt VHL expression enhanced NDV-induced IFN-β production, leading to prolonged STAT1 phosphorylation and increased cell death. Hypoxia augmented NDV oncolytic activity regardless of the cells' HIF-2α levels. These results highlight the potential of oncolytic NDV as a potent therapeutic agent in the killing of hypoxic cancer cells.

  7. Mutant Resources for the Functional Analysis of the Rice Genome

    National Research Council Canada - National Science Library

    Nili Wang Tuan Long Wen Yao Lizhong Xiong Qifa Zhang Changyin Wu

    2013-01-01

    .... In order to systematically assign functions to all predicted genes in the rice genome, a large number of rice mutant lines, including those created by T-DNA insertion, Ds/dSpm tagging, Tos17 tagging...

  8. Hole poking and motor coordination in lurcher mutant mice.

    Science.gov (United States)

    Lalonde, R; Joyal, C C; Guastavino, J M; Botez, M I

    1993-07-01

    Lurcher mutant mice, a cerebellar mutant displaying ataxia and equilibrium deficits, had fewer hole pokes in a 16-hole matrix than normal mice. Lurcher mutants also took longer to reach a platform from a grid and to begin to climb a grid from the floor. However, the lurchers climbed as high as normal mice on the grid and their exploratory patterns of the holeboard were similar in many respects to normal mice, such as the ratio of center to peripheral hole exploration. In a wooden beam test, although lurchers did not differ from normal mice in terms of the amount of time spent on the beam or in the distance travelled, the mutants were found more often in unstable positions.

  9. Enhanced Cellulase Production by a Mutant of Sclerotium rolfsii.

    Science.gov (United States)

    Sadana, J C; Shewale, J G; Deshpande, M V

    1979-10-01

    A mutant of Sclerotium rolfsii CPC 142 that secretes about two times more filter paper-degrading activity in NM-2 growth medium in submerged cultures than the parent strain was obtained by ultraviolet mutagenesis of crushed sclerotia. The production of endo-beta-glucanase in the mutant was affected to a lesser extent. With the parent strain, the addition of 3% rice bran to NM-2 medium was essential for optimal formation of cellulase, including filter paper-degrading activity. However, with the mutant the addition of rice bran to NM-2 medium increased the formation of endo-beta-glucanase but not filter paper-degrading or cellobiase activity. An altered control mechanism for the production of filter paper-degrading enzymes is suggested. The genome(s) controlling the cellulase complex of enzymes in the UV-8 mutant is not under coordinate control.

  10. Enhanced Cellulase Production by a Mutant of Sclerotium rolfsii†

    Science.gov (United States)

    Sadana, J. C.; Shewale, J. G.; Deshpande, M. V.

    1979-01-01

    A mutant of Sclerotium rolfsii CPC 142 that secretes about two times more filter paper-degrading activity in NM-2 growth medium in submerged cultures than the parent strain was obtained by ultraviolet mutagenesis of crushed sclerotia. The production of endo-β-glucanase in the mutant was affected to a lesser extent. With the parent strain, the addition of 3% rice bran to NM-2 medium was essential for optimal formation of cellulase, including filter paper-degrading activity. However, with the mutant the addition of rice bran to NM-2 medium increased the formation of endo-β-glucanase but not filter paper-degrading or cellobiase activity. An altered control mechanism for the production of filter paper-degrading enzymes is suggested. The genome(s) controlling the cellulase complex of enzymes in the UV-8 mutant is not under coordinate control. Images PMID:16345449

  11. Globulin gene expression in embryos of maize viviparous mutants

    Energy Technology Data Exchange (ETDEWEB)

    Kriz, A.R.; Wallace, M.S.; Paiva, R. (Univ. of Illinois, Urbana (USA))

    1990-02-01

    Expression of genes encoding the major Zea mays embryo globulins was examined in the maize precocious germination viviparous (vp) mutants. Comparison of globulin protein profiles of precociously germinating mutant embryos with those of normally germinating mature embryos revealed substantial differences with respect to the proteins encoded by the Glb1 gene. Analysis of Glb1 transcript levels in vp/vp embryos suggests that these mutants do not fully switch from a program of embryo maturation to one of germination. These preliminary studies indicate that the vp mutants provide an excellent system for the study of embryo maturation in maize. We also provide evidence for the positive regulation of Glb1 expression by the plant growth regulator abscisic acid.

  12. Phosphoribosylpyrophosphate synthetase of Escherichia coli, Identification of a mutant enzyme

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne; Nygaard, Per

    1982-01-01

    , stimulated the mutant enzyme. The activity of PRib-PP synthetase in crude extract was higher in the mutant than in the parent. When starved for purines an accumulation of PRib-PP was observed in the parent strain, while the pool decreased in the mutant. During pyrimidine starvation derepression of PRib....... Kinetic analysis of the mutant PRib-PP synthetase revealed an apparent Km for ATP and ribose 5-phosphate of 1.0 mM and 240 μM respectively, compared to 60 μM and 45 μM respectively for the wild-type enzyme. ADP, which inhibits the wild-type enzyme at a concentration of 0.5 mM ribose 5-phosphate...

  13. Characterization of Glutamine-Requiring Mutants of Pseudomonas aeruginosa

    NARCIS (Netherlands)

    Janssen, Dick B.; Joosten, Han M.L.J.; Herst, Patricia M.; Drift, Chris van der

    1982-01-01

    Revertants were isolated from a glutamine-requiring mutant of Pseudomonas aeruginosa PAO. One strain showed thermosensitive glutamine requirement and formed thermolabile glutamine synthetase, suggesting the presence of a mutation in the structural gene for glutamine synthetase. The mutation

  14. Characterization of Pasteurella multocida mutants of low virulence.

    Science.gov (United States)

    Lee, M D; Glisson, J R; Wooley, R E; Brown, J

    1990-01-01

    Ten temperature-sensitive mutants of the Clemson University (CU) vaccine strain of Pasteurella multocida have been developed and were characterized by phenotypic attributes such as carbohydrate fermentation, antibiotic resistance, and membrane protein profiles. Some mutants were found to have lost the ability to utilize some substrates, notably xylose and gluconate, whereas others were able to ferment additional carbohydrates such as arabinose and rhamnose. CU was found to be resistant to sulfisoxazole, of intermediate resistance to bacitracin, and sensitive to rifampin; the sensitivity to these three antibiotics varied among the mutant strains, but 60% were resistant to rifampin. Membrane protein profiles demonstrated some changes in major bands, and there was variation in 50% of the mutants in proteins in the 31 kilodalton range. All strains were assayed for the presence of several virulence factors, and many were found to produce siderophore and to exhibit some degree of complement resistance.

  15. Selection of mutants of capsicum annuum induced by gamma ray

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Y. I.; Lee, Y. B. [Korea Atomic Energy Research Institute, Taejeon (Korea, Republic of); Lee, E. K. [Chungnam National Univ., Taejeon (Korea, Republic of)

    1998-06-01

    For induction and selection of mutations of Capsicum annuum L., dry seeds of pure lines No.1 and No.2 were irradiated with gamma ray of 150Gy, 200Gy and 250Gy. Various mutants were selected such as showing early maturity, short plant height, long fruit and chlorophyll mutations. Mutation frequency of No.1 line was 3.4% in the dose of 150Gy, while the frequency of No.2 line was 2.7% in the dose of 250Gy. For selection of resistant mutant to amino acid analog, the optimum concentration of 5-methyltryptophan (5-MT) and S-(2-aminoethyl)-L-cysteine were 25 ppm and 30 ppm, respectively. Four resistant mutant lines to 5-MT were selected among 400 mutant lines.

  16. Mutant spastin proteins promote deficits in axonal transport through an isoform-specific mechanism involving casein kinase 2 activation.

    Science.gov (United States)

    Leo, Lanfranco; Weissmann, Carina; Burns, Matthew; Kang, Minsu; Song, Yuyu; Qiang, Liang; Brady, Scott T; Baas, Peter W; Morfini, Gerardo

    2017-06-15

    Mutations of various genes cause hereditary spastic paraplegia (HSP), a neurological disease involving dying-back degeneration of upper motor neurons. From these, mutations in the SPAST gene encoding the microtubule-severing protein spastin account for most HSP cases. Cumulative genetic and experimental evidence suggests that alterations in various intracellular trafficking events, including fast axonal transport (FAT), may contribute to HSP pathogenesis. However, the mechanisms linking SPAST mutations to such deficits remain largely unknown. Experiments presented here using isolated squid axoplasm reveal inhibition of FAT as a common toxic effect elicited by spastin proteins with different HSP mutations, independent of microtubule-binding or severing activity. Mutant spastin proteins produce this toxic effect only when presented as the tissue-specific M1 isoform, not when presented as the ubiquitously-expressed shorter M87 isoform. Biochemical and pharmacological experiments further indicate that the toxic effects of mutant M1 spastins on FAT involve casein kinase 2 (CK2) activation. In mammalian cells, expression of mutant M1 spastins, but not their mutant M87 counterparts, promotes abnormalities in the distribution of intracellular organelles that are correctable by pharmacological CK2 inhibition. Collectively, these results demonstrate isoform-specific toxic effects of mutant M1 spastin on FAT, and identify CK2 as a critical mediator of these effects. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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

    Directory of Open Access Journals (Sweden)

    Khoa D Tran

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

  18. The Fd-GOGAT1 mutant gene lc7 confers resistance to Xanthomonas oryzae pv. Oryzae in rice.

    Science.gov (United States)

    Chen, Honglin; Li, Chunrong; Liu, Liping; Zhao, Jiying; Cheng, Xuzhen; Jiang, Guanghuai; Zhai, Wenxue

    2016-05-23

    Disease resistance is an important goal of crop improvement. The molecular mechanism of resistance requires further study. Here, we report the identification of a rice leaf color mutant, lc7, which is defective in chlorophyll synthesis and photosynthesis but confers resistance to Xanthomonas oryzae pv. Oryzae (Xoo). Map-based cloning revealed that lc7 encodes a mutant ferredoxin-dependent glutamate synthase1 (Fd-GOGAT1). Fd-GOGAT1 has been proposed to have great potential for improving nitrogen-use efficiency, but its function in bacterial resistance has not been reported. The lc7 mutant accumulates excessive levels of ROS (reactive oxygen species) in the leaves, causing the leaf color to become yellow after the four-leaf stage. Compared to the wild type, lc7 mutants have a broad-spectrum high resistance to seven Xoo strains. Differentially expressed genes (DEGs) and qRT-PCR analysis indicate that many defense pathways that are involved in this broad-spectrum resistance are activated in the lc7 mutant. These results suggest that Fd-GOGAT1 plays an important role in broad-spectrum bacterial blight resistance, in addition to modulating nitrogen assimilation and chloroplast development.

  19. [Surgical treatment of a pheocromocytoma bilateral in a 5 year old patient with the von Hippel-Lindau disease].

    Science.gov (United States)

    Blanco, J A; Blanco, D; Alastrue, A; Castellví, A; Isnard, R M; Pintos, G; Mangas, A; Roig, N; Casasa, J M

    2004-01-01

    The disease of Von Hippel Lindau (VHL) is hereditary and causes a predisposition to the development of tumours. Organs such as the cerebellum, the pancreas, the kidney, the suprarenal glands and the retina are more usually affected by this disease. We present the case of a 5-year-old patient who suffers from asiymptomatic high blood pressure. In the family antecedents, it is relevant the case of the father, with pheocromocytoma bilateral, which led us to carry out a genetic study of his two sons. Our patient, the younger; presented a mutation of the VHL gene in the short arm of the chromosome 3. In one of the periodic controls, it could be detected high blood pressure of 160/100 mm. Hg, clinically asymptomatic. The other child did not present a genetic mutation and has no disease. The presence of high catecholamines, the detection of a 3 cm left suprarenal mass through the ecography, the TAC that did not show a right suprarenal pathology and the MBIG scintigraphy confirmed the diagnostic of pheocromocytoma. The RNM showed another 0.8-cm mass which confirmed a pheocromocytoma bilateral. We started the treatment against high blood pressure with fenoxibenzamine and diltiazem, and we controlled this problem. We also prepared the pre-and-post operation anesthetic strategy, which is so important for the surgical success. The operation started by a laparoscopic, we made left adrenalectomy and we had to reconvert to laparotomy to make partial right adrenalectomy. Six months after the operation, the patient is free from symptomatology and follows a treatment with glucocorticoides with smaller and smaller doses. The case is exceptional because it embodies the following characteristics: early diagnostic age, family affectation and discovery of asymptomatic high blood pressure. It needed an appropriate preanesthetic and anesthetic preparation, which gave way to an operation without complications. The postoperation was also stable and presented no complications.

  20. Uv- and Gamma-Radiation Sensitive Mutants of Arabidopsis Thaliana

    OpenAIRE

    Jiang, C Z; Yen, C. N.; Cronin, K; Mitchell, D.; Britt, A B

    1997-01-01

    Arabidopsis seedlings repair UV-induced DNA damage via light-dependent and -independent pathways. The mechanism of the ``dark repair'' pathway is still unknown. To determine the number of genes required for dark repair and to investigate the substrate-specificity of this process we isolated mutants with enhanced sensitivity to UV radiation in the absence of photoreactivating light. Seven independently derived UV sensitive mutants were isolated from an EMS-mutagenized population. These fell in...

  1. [The behavioral development of the mutant "staggerer" mouse].

    Science.gov (United States)

    Guastavino, J M

    1978-01-01

    The behavioural study, in particular rearing environmental conditions, of the mutant mouse staggerer has shown that such animals may live more than 90 days. (he behavioural diagnosis of this mutation has been possible from the second week of life, using specific tests. A typical "bat posture" permits one to recognize the mutant from the normal Mouse. Locomotory and feeding behaviours also present late and various qualitatige particularities.

  2. Structure prediction of subtilisin BPN' mutants using molecular dynamics methods

    OpenAIRE

    Heiner, Andreas P.; Berendsen, Herman J.C.; van Gunsteren, Wilfred F.

    2017-01-01

    In this paper we describe the achievements and pitfalls encountered in doing structure predictions of protein mutants using molecular dynamics simulation techniques in which properties of atoms are slowly changed as a function of time. Basically the method consists of a thermodynamic integration (slow growth) calculation used for free energy determination, but aimed at structure prediction; this allows for a fast determination of the mutant structure. We compared the calculated structure of t...

  3. Fate of peptides in peptidase mutants of Lactococcus lactis

    NARCIS (Netherlands)

    Kunji, E.R S; Mierau, I; Poolman, B.; Konings, W.N; Venema, G; Kok, J.

    The utilization of exogenous peptides was studied in mutants of Lactococcus lactis in which combinations of the peptidase genes pepN, pepC, pepO, pepX and pepT were deleted, Multiple mutants lacking PepN, PepC, PepT plus PepX could not grow on peptides such as Leu-Gly-Gly, Gly-Phe-Leu, Leu-Gly-Pro,

  4. Induction of cell death and gain-of-function properties of connexin26 mutants predict severity of skin disorders and hearing loss.

    Science.gov (United States)

    Press, Eric R; Shao, Qing; Kelly, John J; Chin, Katrina; Alaga, Anton; Laird, Dale W

    2017-06-09

    Connexin26 (Cx26) is a gap junction protein that oligomerizes in the cell to form hexameric transmembrane channels called connexons. Cell surface connexons dock between adjacent cells to allow for gap junctional intercellular communication. Numerous autosomal dominant mutations in the Cx26-encoding GJB2 gene lead to many skin disorders and sensorineural hearing loss. Although some insights have been gained into the pathogenesis of these diseases, it is not fully understood how distinct GJB2 mutations result in hearing loss alone or in skin pathologies with comorbid hearing loss. Here we investigated five autosomal dominant Cx26 mutants (N14K, D50N, N54K, M163V, and S183F) linked to various syndromic or nonsyndromic diseases to uncover the molecular mechanisms underpinning these disease links. We demonstrated that when gap junction-deficient HeLa cells expressed the N14K and D50N mutants, they undergo cell death. The N54K mutant was retained primarily within intracellular compartments and displayed dominant or transdominant properties on wild-type Cx26 and coexpressed Cx30 and Cx43. The S183F mutant formed some gap junction plaques but was largely retained within the cell and exhibited only a mild transdominant reduction in gap junction communication when co-expressed with Cx30. The M163V mutant, which causes only hearing loss, exhibited impaired gap junction function and showed no transdominant interactions. These findings suggest that Cx26 mutants that promote cell death or exert transdominant effects on other connexins in keratinocytes will lead to skin diseases and hearing loss, whereas mutants having reduced channel function but exhibiting no aberrant effects on coexpressed connexins cause only hearing loss. Moreover, cell death-inducing GJB2 mutations lead to more severe syndromic disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Ozone-Sensitive Arabidopsis Mutants with Deficiencies in Photorespiratory Enzymes.

    Science.gov (United States)

    Saji, Shoko; Bathula, Srinivas; Kubo, Akihiro; Tamaoki, Masanori; Aono, Mitsuko; Sano, Tomoharu; Tobe, Kazuo; Timm, Stefan; Bauwe, Hermann; Nakajima, Nobuyoshi; Saji, Hikaru

    2017-05-01

    An ozone-sensitive mutant was isolated from T-DNA-tagged lines of Arabidopsis thaliana. The T-DNA was inserted at a locus on chromosome 3, where two genes encoding glycolate oxidases, GOX1 and GOX2, peroxisomal enzymes involved in photorespiration, reside contiguously. The amounts of the mutant's foliar transcripts for these genes were reduced, and glycolate oxidase activity was approximately 60% of that of the wild-type plants. No difference in growth and appearance was observed between the mutant and the wild-type plants under normal conditions with ambient air under a light intensity of 100 µmol photons m-2 s-1. However, signs of severe damage, such as chlorosis and ion leakage from the tissue, rapidly appeared in mutant leaves in response to ozone treatment at a concentration of 0.2 µl l-1 under a higher light intensity of 350 µmol photons m-2 s-1 that caused no such symptoms in the wild-type plant. The mutant also exhibited sensitivity to sulfur dioxide and long-term high-intensity light. Arabidopsis mutants with deficiencies in other photorespiratory enzymes such as glutamate:glyoxylate aminotransferase and hydroxypyruvate reductase also exhibited ozone sensitivities. Therefore, photorespiration appears to be involved in protection against photooxidative stress caused by ozone and other abiotic factors under high-intensity light. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  6. pH dependency of sclerotial development and pathogenicity revealed by using genetically defined oxalate-minus mutants of Sclerotinia sclerotiorum.

    Science.gov (United States)

    Xu, Liangsheng; Xiang, Meichun; White, David; Chen, Weidong

    2015-08-01

    The devastating plant pathogen Sclerotinia sclerotiorum produces copious (up to 50 mM) amounts of oxalic acid, which, for over a quarter century, has been claimed as the pathogenicity determinant based on UV-induced mutants that concomitantly lost oxalate production and pathogenicity. Such a claim was made without fulfilling the molecular Koch's postulates because the UV mutants are genetically undefined and harbour a developmental defect in sclerotial production. Here, we generated oxalate-minus mutants of S. sclerotiorum using two independent mutagenesis techniques, and tested the resulting mutants for growth at different pHs and for pathogenicity on four host plants. The oxalate-minus mutants accumulated fumaric acid, produced functional sclerotia and have reduced ability to acidify the environment. The oxalate-minus mutants retained pathogenicity on plants, but their virulence varied depending on the pH and buffering capacity of host tissue. Acidifying the host tissue enhanced virulence of the oxalate-minus mutants, whereas supplementing with oxalate did not. These results suggest that it is low pH, not oxalic acid itself, that establishes the optimum conditions for growth, reproduction, pathogenicity and virulence expression of S. sclerotiorum. Exonerating oxalic acid as the primary pathogenicity determinant will stimulate research into identifying additional candidates as pathogenicity factors towards better understanding and managing Sclerotinia diseases. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  7. Genetics of peripheral vestibular dysfunction: lessons from mutant mouse strains.

    Science.gov (United States)

    Jones, Sherri M; Jones, Timothy A

    2014-03-01

    A considerable amount of research has been published about genetic hearing impairment. Fifty to sixty percent of hearing loss is thought to have a genetic cause. Genes may also play a significant role in acquired hearing loss due to aging, noise exposure, or ototoxic medications. Between 1995 and 2012, over 100 causative genes have been identified for syndromic and nonsyndromic forms of hereditary hearing loss. Mouse models have been extremely valuable in facilitating the discovery of hearing loss genes and in understanding inner ear pathology due to genetic mutations or elucidating fundamental mechanisms of inner ear development. Whereas much is being learned about hereditary hearing loss and the genetics of cochlear disorders, relatively little is known about the role genes may play in peripheral vestibular impairment. Here we review the literature with regard to genetics of vestibular dysfunction and discuss what we have learned from studies using mutant mouse models and direct measures of peripheral vestibular neural function. Several genes are considered that when mutated lead to varying degrees of inner ear vestibular dysfunction due to deficits in otoconia, stereocilia, hair cells, or neurons. Behavior often does not reveal the inner ear deficit. Many of the examples presented are also known to cause human disorders. Knowledge regarding the roles of particular genes in the operation of the vestibular sensory apparatus is growing, and it is clear that gene products co-expressed in the cochlea and vestibule may play different roles in the respective end organs. The discovery of new genes mediating critical inner ear vestibular function carries the promise of new strategies in diagnosing, treating, and managing patients as well as predicting the course and level of morbidity in human vestibular disease. American Academy of Audiology.

  8. Laminin alpha2 deficiency and muscular dystrophy; genotype-phenotype correlation in mutant mice

    DEFF Research Database (Denmark)

    Guo, L T; Zhang, X U; Kuang, W

    2003-01-01

    Deficiency of laminin alpha2 is the cause of one of the most severe muscular dystrophies in humans and other species. It is not yet clear how particular mutations in the laminin alpha2 chain gene affect protein expression, and how abnormal levels or structure of the protein affect disease. Animal...... the human laminin alpha2 chain gene in skeletal muscle. The dy(3K)/dy(3K) experimental mutant mice are completely deficient in laminin alpha2; the dy/dy spontaneous mutant mice have small amounts of apparently normal laminin; and the dy(W)/dy(W) mice express even smaller amounts of a truncated laminin alpha......2, lacking domain VI. Interestingly, all mutants lack laminin alpha2 in peripheral nerve. We have demonstrated previously, that overexpression of the human laminin alpha2 in skeletal muscle in dy(2J)/dy(2J) and dy(W)/dy(W) mice under the control of a striated muscle-specific creatine kinase promoter...

  9. Natural history of acute and chronic hepatitis B: The role of HBV genotypes and mutants.

    Science.gov (United States)

    Lin, Chih-Lin; Kao, Jia-Horng

    2017-06-01

    Molecular epidemiologic studies reveal remarkable differences in the geographical distribution of hepatitis B virus (HBV) genotypes. The frequency of mutants among HBV genotypes also varies. The role of HBV genotypes/mutants in the pathogenesis of HBV infection and natural history of HBV infection has been extensively investigated. The distribution of HBV genotypes in acute hepatitis B patients reflects the predominant genotypes in a given geographic area. In chronic hepatitis B patients, genotype C and D have a higher frequency of basal core promoter A1762T/G1764A mutations than genotype A and B. HBV genotypes C, D and F carry a higher lifetime risk of cirrhosis and HCC development than genotype A and B. HBV pre-S/S gene mutations were associated with immune escape of hepatitis B immunoglobulin or vaccine-induced immunity. Mutations in the pre-S, core promoter and X regions correlate with an increased risk of cirrhosis and HCC. In summary, HBV genotypes and mutants are associated with the disease progression and long-term outcome of HBV infection. They may serve as viral genetic markers for risk stratification of chronic hepatitis B patients in clinical practice. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Accumulation and aggregate formation of mutant superoxide dismutase 1 in canine degenerative myelopathy.

    Science.gov (United States)

    Nakamae, S; Kobatake, Y; Suzuki, R; Tsukui, T; Kato, S; Yamato, O; Sakai, H; Urushitani, M; Maeda, S; Kamishina, H

    2015-09-10

    Canine degenerative myelopathy (DM) is an adult-onset progressive neurodegenerative disorder that has recently been linked to mutations in the superoxide dismutase 1 (SOD1) gene. We generated a polyclonal antibody against canine SOD1 to further characterize the mutant SOD1 protein and its involvement in DM pathogenesis. This antibody (SYN3554) was highly specific to canine SOD1 and had the ability to reveal distinct cytoplasmic aggregates in cultured cells expressing canine mutant SOD1 and also in the spinal neurons of symptomatic homozygotes. A similar staining pattern was observed in asymptomatic homozygotes. SOD1 aggregates were not detected in the spinal neurons of heterozygotes; the accumulation of SOD1 was also detected in the reactive astrocytes of homozygotes and heterozygotes to a similar extent. Our results support the hypothesis that the cytoplasmic accumulation and aggregate formation of the mutant SOD1 protein, especially in astrocytes, are closely associated with the pathogenesis of DM. Therefore, this disease is regarded as a spontaneous large-animal model of SOD1-mediated amyotrophic lateral sclerosis in humans. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  11. Cerebellar Purkinje cells die by apoptosis in the shaker mutant rat.

    Science.gov (United States)

    Erekat, Nour S

    2017-02-15

    Cerebellar Purkinje cells (PCs) die in humans leading to a variety of diseases including ataxia and essential tremor. The etiology underlying PC death is poorly understood. Shaker mutant rat is a unique animal model of progressive PC degeneration that is compartmentalized and of adult-onset. In shaker mutant rats, hereditary degeneration of at risk PCs occurs between 7 and 14 postnatal weeks of age as a natural phenotypic expression of the shaker mutation and at earlier or later ages depending upon experimental conditions in restricted anterior (ADC) and posterior (PDC) vermal degeneration compartments. Secure PCs in a flocculonodular survival compartment (FNSC) always survive. In this study, we investigated DNA fragmentation and active caspase-3 expression characteristic of apoptosis using immunofluorescence and fluorescence microscopy. We also sought to confirm the occurrence of apoptosis in at risk PCs using transmission electron microscope. Purkinje neurons were the only cerebellar cells labeled for TUNEL and immunoreactive to active caspase-3 in the shaker mutant. Active caspase-3 expression observed was spatially and temporally congruous with the pattern of calbindin immunolabeling of degenerating PCs that is characterized by dendritic atrophy, shrinkage of PC cell bodies and the appearance of PC axonal torpedoes. DNA fragmentation, detected by TUNEL, as well as ultrastructural morphological changes characteristic for apoptosis, provided additional evidence for the occurrence of apoptosis in at risk PCs. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Generation and Standardized, Systemic Phenotypic Analysis of Pou3f3L423P Mutant Mice.

    Science.gov (United States)

    Kumar, Sudhir; Rathkolb, Birgit; Kemter, Elisabeth; Sabrautzki, Sibylle; Michel, Dian; Adler, Thure; Becker, Lore; Beckers, Johannes; Busch, Dirk H; Garrett, Lillian; Hans, Wolfgang; Hölter, Sabine M; Horsch, Marion; Klingenspor, Martin; Klopstock, Thomas; Rácz, Ildikó; Rozman, Jan; Vargas Panesso, Ingrid Liliana; Vernaleken, Alexandra; Zimmer, Andreas; Fuchs, Helmut; Gailus-Durner, Valérie; Hrabě de Angelis, Martin; Wolf, Eckhard; Aigner, Bernhard

    2016-01-01

    Increased levels of blood plasma urea were used as phenotypic parameter for establishing novel mouse models for kidney diseases on the genetic background of C3H inbred mice in the phenotype-driven Munich ENU mouse mutagenesis project. The phenotypically recessive mutant line HST011 was established and further analyzed. The causative mutation was detected in the POU domain, class 3 transcription factor 3 (Pou3f3) gene, which leads to the amino acid exchange Pou3f3L423P thereby affecting the conserved homeobox domain of the protein. Pou3f3 homozygous knockout mice are published and show perinatal death. Line Pou3f3L423P is a viable mouse model harboring a homozygous Pou3f3 mutation. Standardized, systemic phenotypic analysis of homozygous mutants was carried out in the German Mouse Clinic. Main phenotypic changes were low body weight and a state of low energy stores, kidney dysfunction and secondary effects thereof including low bone mineralization, multiple behavioral and neurological defects including locomotor, vestibular, auditory and nociceptive impairments, as well as multiple subtle changes in immunological parameters. Genome-wide transcriptome profiling analysis of kidney and brain of Pou3f3L423P homozygous mutants identified significantly regulated genes as compared to wild-type controls.

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

    Science.gov (United States)

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

    2016-12-07

    The vertebrate Scube (Signal peptide, CUB, and EGF-like domain-containing protein) family consists of three independent members, Scube1-3, which encode secreted cell surface-associated membrane glycoproteins. Limited information about the general function of this gene family is available, and their roles during adulthood. Here, we present the first Scube3 mutant mouse line (Scube3N294K/N294K), which clearly shows phenotypic alterations by carrying a missense mutation in exon 8, and thus contributes to our understanding of SCUBE3 functions. We performed a detailed phenotypic characterization in the German Mouse Clinic (GMC). Scube3N294K/N294K mutants showed morphological abnormalities of the skeleton, alterations of parameters relevant for bone metabolism, changes in renal function, and hearing impairments. These findings correlate with characteristics of the rare metabolic bone disorder Paget disease of bone (PDB), associated with the chromosomal region of human SCUBE3 In addition, alterations in energy metabolism, behavior, and neurological functions were detected in Scube3N294K/N294K mice. The Scube3N294K/N294K mutant mouse line may serve as a new model for further studying the effect of impaired SCUBE3 gene function. Copyright © 2016 Fuchs et al.

  14. Generation and Standardized, Systemic Phenotypic Analysis of Pou3f3L423P Mutant Mice.

    Directory of Open Access Journals (Sweden)

    Sudhir Kumar

    Full Text Available Increased levels of blood plasma urea were used as phenotypic parameter for establishing novel mouse models for kidney diseases on the genetic background of C3H inbred mice in the phenotype-driven Munich ENU mouse mutagenesis project. The phenotypically recessive mutant line HST011 was established and further analyzed. The causative mutation was detected in the POU domain, class 3 transcription factor 3 (Pou3f3 gene, which leads to the amino acid exchange Pou3f3L423P thereby affecting the conserved homeobox domain of the protein. Pou3f3 homozygous knockout mice are published and show perinatal death. Line Pou3f3L423P is a viable mouse model harboring a homozygous Pou3f3 mutation. Standardized, systemic phenotypic analysis of homozygous mutants was carried out in the German Mouse Clinic. Main phenotypic changes were low body weight and a state of low energy stores, kidney dysfunction and secondary effects thereof including low bone mineralization, multiple behavioral and neurological defects including locomotor, vestibular, auditory and nociceptive impairments, as well as multiple subtle changes in immunological parameters. Genome-wide transcriptome profiling analysis of kidney and brain of Pou3f3L423P homozygous mutants identified significantly regulated genes as compared to wild-type controls.

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

    Directory of Open Access Journals (Sweden)

    Helmut Fuchs

    2016-12-01

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

  16. Heterodimerization of Two Pathological Mutants Enhances the Activity of Human Phosphomannomutase2.

    Directory of Open Access Journals (Sweden)

    Giuseppina Andreotti

    Full Text Available The most frequent disorder of glycosylation is due to mutations in the gene encoding phosphomannomutase2 (PMM2-CDG. For this disease, which is autosomal and recessive, there is no cure at present. Most patients are composite heterozygous and carry one allele encoding an inactive mutant, R141H, and one encoding a hypomorphic mutant. Phosphomannomutase2 is a dimer. We reproduced composite heterozygosity in vitro by mixing R141H either with the wild type protein or the most common hypomorphic mutant F119L and compared the quaternary structure, the activity and the stability of the heterodimeric enzymes. We demonstrated that the activity of R141H/F119L heterodimers in vitro, which reproduces the protein found in patients, has the same activity of wild type/R141H, which reproduces the protein found in healthy carriers. On the other hand the stability of R141H/F119L appears to be reduced both in vitro and in vivo. These findings suggest that a therapy designed to enhance protein stability such as those based on pharmacological chaperones or modulation of proteostasis could be beneficial for PMM2-CDG patients carrying R141H/F119L genotype as well as for other genotypes where protein stability rather than specific activity is affected by mutations.

  17. Pharmacological NAD-Boosting Strategies Improve Mitochondrial Homeostasis in Human Complex I-Mutant Fibroblasts.

    Science.gov (United States)

    Felici, Roberta; Lapucci, Andrea; Cavone, Leonardo; Pratesi, Sara; Berlinguer-Palmini, Rolando; Chiarugi, Alberto

    2015-06-01

    Mitochondrial disorders are devastating genetic diseases for which efficacious therapies are still an unmet need. Recent studies report that increased availability of intracellular NAD obtained by inhibition of the NAD-consuming enzyme poly(ADP-ribose) polymerase (PARP)-1 or supplementation with the NAD-precursor nicotinamide riboside (NR) ameliorates energetic derangement and symptoms in mouse models of mitochondrial disorders. Whether these pharmacological approaches also improve bioenergetics of human cells harboring mitochondrial defects is unknown. It is also unclear whether the same signaling cascade is prompted by PARP-1 inhibitors and NR supplementation to improve mitochondrial homeostasis. Here, we show that human fibroblasts mutant for the NADH dehydrogenase (ubiquinone) Fe-S protein 1 (NDUFS1) subunit of respiratory complex I have similar ATP, NAD, and mitochondrial content compared with control cells, but show reduced mitochondrial membrane potential. Interestingly, mutant cells also show increased transcript levels of mitochondrial DNA but not nuclear DNA respiratory complex subunits, suggesting activation of a compensatory response. At variance with prior work in mice, however, NR supplementation, but not PARP-1 inhibition, increased intracellular NAD content in NDUFS1 mutant human fibroblasts. Conversely, PARP-1 inhibitors, but not NR supplementation, increased transcription of mitochondrial transcription factor A and mitochondrial DNA-encoded respiratory complexes constitutively induced in mutant cells. Still, both NR and PARP-1 inhibitors restored mitochondrial membrane potential and increased organelle content as well as oxidative activity of NDUFS1-deficient fibroblasts. Overall, data provide the first evidence that in human cells harboring a mitochondrial respiratory defect exposure to NR or PARP-1, inhibitors activate different signaling pathways that are not invariantly prompted by NAD increases, but equally able to improve energetic

  18. Exploring the mechanism of zanamivir resistance in a neuraminidase mutant: a molecular dynamics study.

    Directory of Open Access Journals (Sweden)

    Nanyu Han

    Full Text Available It is critical to understand the molecular basis of the drug resistance of influenza viruses to efficiently treat this infectious disease. Recently, H1N1 strains of influenza A carrying a mutation of Q136K in neuraminidase were found. The new strain showed a strong Zanamivir neutralization effect. In this study, normal molecular dynamics simulations and metadynamics simulations were employed to explore the mechanism of Zanamivir resistance. The wild-type neuraminidase contained a 3(10 helix before the 150 loop, and there was interaction between the 150 and 430 loops. However, the helix and the interaction between the two loops were disturbed in the mutant protein due to interaction between K136 and nearby residues. Hydrogen-bond network analysis showed weakened interaction between the Zanamivir drug and E276/D151 on account of the electrostatic interaction between K136 and D151. Metadynamics simulations showed that the free energy landscape was different in the mutant than in the wild-type neuraminidase. Conformation with the global minimum of free energy for the mutant protein was different from the wild-type conformation. While the drug fit completely into the active site of the wild-type neuraminidase, it did not match the active site of the mutant variant. This study indicates that the altered hydrogen-bond network and the deformation of the 150 loop are the key factors in development of Zanamivir resistance. Furthermore, the Q136K mutation has a variable effect on conformation of different N1 variants, with conformation of the 1918 N1 variant being more profoundly affected than that of the other N1 variants studied in this paper. This observation warrants further experimental investigation.

  19. The parkin mutant phenotype in the fly is largely rescued by metal-responsive transcription factor (MTF-1).

    Science.gov (United States)

    Saini, Nidhi; Georgiev, Oleg; Schaffner, Walter

    2011-05-01

    The gene for Parkin, an E3 ubiquitin ligase, is mutated in some familial forms of Parkinson's disease, a severe neurodegenerative disorder. A homozygous mutant of the Drosophila ortholog of human parkin is viable but results in severe motoric impairment including an inability to fly, female and male sterility, and a decreased life span. We show here that a double mutant of the genes for Parkin and the metal-responsive transcription factor 1 (MTF-1) is not viable. MTF-1, which is conserved from insects to mammals, is a key regulator of heavy metal homeostasis and detoxification and plays additional roles in other stress conditions, notably oxidative stress. In contrast to the synthetic lethality of the double mutant, elevated expression of MTF-1 dramatically ameliorates the parkin mutant phenotype, as evidenced by a prolonged life span, motoric improvement including short flight episodes, and female fertility. At the cellular level, muscle and mitochondrial structures are substantially improved. A beneficial effect is also seen with a transgene encoding human MTF-1. We propose that Parkin and MTF-1 provide complementary functions in metal homeostasis, oxidative stress and other cellular stress responses. Our findings also raise the possibility that MTF-1 gene polymorphisms in humans could affect the severity of Parkinson's disease.

  20. The parkin Mutant Phenotype in the Fly Is Largely Rescued by Metal-Responsive Transcription Factor (MTF-1) ▿ †

    Science.gov (United States)

    Saini, Nidhi; Georgiev, Oleg; Schaffner, Walter

    2011-01-01

    The gene for Parkin, an E3 ubiquitin ligase, is mutated in some familial forms of Parkinson's disease, a severe neurodegenerative disorder. A homozygous mutant of the Drosophila ortholog of human parkin is viable but results in severe motoric impairment including an inability to fly, female and male sterility, and a decreased life span. We show here that a double mutant of the genes for Parkin and the metal-responsive transcription factor 1 (MTF-1) is not viable. MTF-1, which is conserved from insects to mammals, is a key regulator of heavy metal homeostasis and detoxification and plays additional roles in other stress conditions, notably oxidative stress. In contrast to the synthetic lethality of the double mutant, elevated expression of MTF-1 dramatically ameliorates the parkin mutant phenotype, as evidenced by a prolonged life span, motoric improvement including short flight episodes, and female fertility. At the cellular level, muscle and mitochondrial structures are substantially improved. A beneficial effect is also seen with a transgene encoding human MTF-1. We propose that Parkin and MTF-1 provide complementary functions in metal homeostasis, oxidative stress and other cellular stress responses. Our findings also raise the possibility that MTF-1 gene polymorphisms in humans could affect the severity of Parkinson's disease. PMID:21383066

  1. Serum response factor: positive and negative regulation of an epithelial gene expression network in the destrin mutant cornea.

    Science.gov (United States)

    Kawakami-Schulz, Sharolyn V; Verdoni, Angela M; Sattler, Shannon G; Jessen, Erik; Kao, Winston W-Y; Ikeda, Akihiro; Ikeda, Sakae

    2014-04-15

    Increased angiogenesis, inflammation, and proliferation are hallmarks of diseased tissues, and in vivo models of these disease phenotypes can provide insight into disease pathology. Dstn(corn1) mice, deficient for the actin depolymerizing factor destrin (DSTN), display an increase of serum response factor (SRF) that results in epithelial hyperproliferation, inflammation, and neovascularization in the cornea. Previous work demonstrated that conditional ablation of Srf from the corneal epithelium of Dstn(corn1) mice returns the cornea to a wild-type (WT) like state. This result implicated SRF as a major regulator of genes that contributes to abnormal phenotypes in Dstn(corn1) cornea. The purpose of this study is to identify gene networks that are affected by increased expression of Srf in the Dstn(corn1) cornea. Microarray analysis led to characterization of gene expression changes that occur when conditional knockout of Srf rescues mutant phenotypes in the cornea of Dstn(corn1) mice. Comparison of gene expression values from WT, Dstn(corn1) mutant, and Dstn(corn1) rescued cornea identified >400 differentially expressed genes that are downstream from SRF. Srf ablation had a significant effect on genes associated with epithelial cell-cell junctions and regulation of actin dynamics. The majority of genes affected by SRF are downregulated in the Dstn(corn1) mutant cornea, suggesting that increased SRF negatively affects transcription of SRF gene targets. ChIP-seq analysis on Dstn(corn1) mutant and WT tissue revealed that, despite being present in higher abundance, SRF binding is significantly decreased in the Dstn(corn1) mutant cornea. This study uses a unique model combining genetic and genomic approaches to identify genes that are regulated by SRF. These findings expand current understanding of the role of SRF in both normal and abnormal tissue homeostasis.

  2. In vitro exploration of latent prothrombin mutants conveying antithrombin resistance.

    Science.gov (United States)

    Tamura, Shogo; Murata-Kawakami, Moe; Takagi, Yuki; Suzuki, Sachiko; Katsumi, Akira; Takagi, Akira; Kojima, Tetsuhito

    2017-09-20

    Antithrombin resistance (ATR) prothrombinemia is an inherited thrombophilic disorder caused by missense mutations in prothrombin gene (F2) at Arg596 of the sodium-binding region. Previously, prothrombin mutants Yukuhashi (Arg596Leu), Belgrade (Arg596Gln), and Padua 2 (Arg596Trp) were reported as ATR-prothrombins possessing a risk of familial venous thrombosis. To identify additional F2 mutations causing the ATR-phenotype, we investigated the coagulant properties of recombinant prothrombins mutated at amino acid residues within the sodium-binding region by single nucleotide substitutions (Thr540, Arg541, Glu592, and Lys599). We constructed expression vectors of prothrombin mutants, established stably transfected HEK293 cells, and isolated the recombinant prothrombin proteins. We evaluated procoagulant activity and ATR-phenotypes of those mutants in reconstituted plasma by mixing with prothrombin deficient plasma. The secreted quantity of all prothrombin mutants was the same as that of the wild-type prothrombin. Procoagulant activity of each mutant varied from 1.7% to 79.5% in a one-stage clotting assay and from 2.0% to 104.5% in a two-stage chromogenic assay. Most prothrombin mutants tested presented with a severe ATR-phenotype. To estimate the thrombosis risk of these mutations, we determined the residual clotting activity (RCA) after 30min inactivation with antithrombin. RCA scores, normalized to the wild-type, revealed that prothrombin mutants Lys599Arg (5.35) and Glu592Gln (4.71) had high scores, which were comparable with prothrombins Yukuhashi (4.36) and Belgrade (5.19). Mutation of prothrombin at the sodium-binding site caused ATR-phenotypes. Of those tested, Lys599Arg and Glu592Gln may possess a thrombosis risk as large as the known pathogenic prothrombins Yukuhashi and Belgrade. Copyright © 2017. Published by Elsevier Ltd.

  3. NRAS-mutant melanoma: current challenges and future prospect

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    Muñoz-Couselo E

    2017-08-01

    Full Text Available Eva Muñoz-Couselo,1,2 Ester Zamora Adelantado,1,2 Carolina Ortiz,1,2 Jesús Soberino García,3 José Perez-Garcia31Medical Oncology Department, Vall d’Hebron Hospital, Barcelona, Spain; 2Vall d’Hebron Institute of Oncology (VHIO, Barcelona, Spain; 3Baselga Institute of Oncology, Hospital Quirón, Barcelona, SpainAbstract: Melanoma is one of the most common cutaneous cancers worldwide. Activating mutations in RAS oncogenes are found in a third of all human cancers and NRAS mutations are found in 15%–20% of melanomas. The NRAS-mutant subset of melanoma is more aggressive and associated with poorer outcomes, compared to non-NRAS-mutant melanoma. Although immune checkpoint inhibitors and targeted therapies for BRAF-mutant melanoma are transforming the treatment of metastatic melanoma, the ideal treatment for NRAS-mutant melanoma remains unknown. Despite promising preclinical data, current therapies for NRAS-mutant melanoma remain limited, showing a modest increase in progression-free survival but without any benefit in overall survival. Combining MEK inhibitors with agents inhibiting cell cycling and the PI3K–AKT pathway appears to provide additional benefit; in particular, a strategy of MEK inhibition and CDK4/6 inhibition is likely to be a viable treatment option in the future. Patients whose tumors had NRAS mutations had better response to immunotherapy and better outcomes than patients whose tumors had other genetic subtypes, suggesting that immune therapies – especially immune checkpoint inhibitors – may be particularly effective as treatment options for NRAS-mutant melanoma. Improved understanding of NRAS-mutant melanoma will be essential to develop new treatment strategies for this subset of patients with melanoma.Keywords: metastatic melanoma, NRAS mutation, MEK inhibitor, immunotherapy, trametinib, binimetinib

  4. Kinetic partitioning during de novo septin filament assembly creates a critical G1 “window of opportunity” for mutant septin function

    Science.gov (United States)

    Schaefer, Rachel M.; Heasley, Lydia R.; Odde, David J.; McMurray, Michael A.

    2016-01-01

    ABSTRACT Septin proteins form highly conserved cytoskeletal filaments composed of hetero-oligomers with strict subunit stoichiometry. Mutations within one hetero-oligomerization interface (the “G” interface) bias the mutant septin toward conformations that are incompatible with filament assembly, causing disease in humans and, in budding yeast cells, temperature-sensitive defects in cytokinesis. We previously found that, when the amount of other hetero-oligomerization partners is limiting, wild-type and G interface-mutant alleles of a given yeast septin “compete” along parallel but distinct folding pathways for occupancy of a limited number of positions within septin hetero-octamers. Here, we synthesize a mathematical model that outlines the requirements for this phenomenon: if a wild-type septin traverses a folding pathway that includes a single rate-limiting folding step, the acquisition by a mutant septin of additional slow folding steps creates an initially large disparity between wild-type and mutant in the cellular concentrations of oligomerization-competent monomers. When the 2 alleles are co-expressed, this kinetic disparity results in mutant exclusion from hetero-oligomers, even when the folded mutant monomer is oligomerization-competent. To test this model experimentally, we first visualize the kinetic delay in mutant oligomerization in living cells, and then narrow or widen the “window of opportunity” for mutant septin oligomerization by altering the length of the G1 phase of the yeast cell cycle, and observe the predicted exacerbation or suppression, respectively, of mutant cellular phenotypes. These findings reveal a fundamental kinetic principle governing in vivo assembly of multiprotein complexes, independent of the ability of the subunits to associate with each other. PMID:27398993

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

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    Lisa K. Mullany

    2015-10-01

    Full Text Available Evolutionary Action analyses of The Cancer Gene Atlas data sets show that many specific p53 missense and gain-of-function mutations are selectively overrepresented and functional in high-grade serous ovarian cancer (HGSC. As homozygous alleles, p53 mutants are differentially associated with specific loss of heterozygosity (R273; chromosome 17; copy number variation (R175H; chromosome 9; and up-stream, cancer-related regulatory pathways. The expression of immune-related cytokines was selectively related to p53 status, showing for the first time that specific p53 mutants impact, and are related to, the immune subtype of ovarian cancer. Although the majority (31% of HGSCs exhibit loss of heterozygosity, a significant number (24% maintain a wild-type (WT allele and represent another HGSC subtype that is not well defined. Using human and mouse cell lines, we show that specific p53 mutants differentially alter endogenous WT p53 activity; target gene expression; and responses to nutlin-3a, a small molecular that activates WT p53 leading to apoptosis, providing “proof of principle” that ovarian cancer cells expressing WT and mutant alleles represent a distinct ovarian cancer subtype. We also show that siRNA knock down of endogenous p53 in cells expressing homozygous mutant alleles causes apoptosis, whereas cells expressing WT p53 (or are heterozygous for WT and mutant p53 alleles are highly resistant. Therefore, despite different gene regulatory pathways associated with specific p53 mutants, silencing mutant p53 might be a suitable, powerful, global strategy for blocking ovarian cancer growth in those tumors that rely on mutant p53 functions for survival. Knowing p53 mutational status in HGSC should permit new strategies tailored to control this disease.

  6. Relationship between enzyme properties and disease progression in Canavan disease.

    Science.gov (United States)

    Zano, Stephen; Wijayasinghe, Yasanandana S; Malik, Radhika; Smith, Joshua; Viola, Ronald E

    2013-01-01

    Canavan disease (CD) is a fatal neurological disorder caused by defects in the gene that encodes for a critical metabolic enzyme. The enzyme aspartoacylase catalyzes the deacetylation of N-acetylaspartate to produce acetate required for fatty acid biosynthesis in the brain. The loss of aspartoacylase activity leads to the demyelination and disrupted brain development that is found in CD patients. Sixteen different clinical mutants of aspartoacylase have been cloned, expressed and purified to examine their properties and the relationship between enzyme properties and disease phenotype. In contrast to numerous cell culture studies that reported virtually complete loss of function, each of these purified mutant enzymes was found to have measureable catalytic activity. However, the activities of these mutants are diminished, by as little as three-fold to greater than 100-fold when compared to the native enzyme. Many of these mutated enzyme forms show decreased thermal stability and an increased propensity for denaturation upon exposure to urea, but only four of the 16 mutants examined showed both diminished thermal and diminished conformational stability. Significantly, each of these lower stability mutants are responsible for the more severe phenotypes of CD, while patients with milder forms of CD have aspartoacylase mutants with generally high catalytic activity and with either good thermal or good conformational stability. These results suggest that the loss of catalytic function and the accumulation of N-acetylaspartate in Canavan disease is at least partially a consequence of the decreased protein stability caused by these mutations.

  7. Developmental Biology: We Are All Walking Mutants.

    Science.gov (United States)

    Trainor, Paul A

    2016-01-01

    What is Developmental Biology? Developmental Biology is a discipline that evolved from the collective fields of embryology, morphology, and anatomy, which firmly established that structure underpins function. In its simplest terms, Developmental Biology has come to describe how a single cell becomes a completely formed organism. However, this definition of Developmental Biology is too narrow. Developmental Biology describes the properties of individual cells; their organization into tissues, organs, and organisms; their homeostasis, regeneration, aging, and ultimately death. Developmental Biology provides a context for cellular reprogramming, stem cell biology, regeneration, tissue engineering, evolutionary development and ecology, and involves the reiterated use of the same cellular mechanisms and signaling pathways throughout the lifespan of an organism. Using neural crest cells as an example, this review explores the contribution of Developmental Biology to our understanding of development, evolution, and disease. © 2016 Elsevier Inc. All rights reserved.

  8. Differing von Hippel Lindau genotype in paired primary and metastatic tumors in patients with clear cell renal cell carcinoma

    Directory of Open Access Journals (Sweden)

    Susan A.J. Vaziri

    2012-05-01

    Full Text Available In sporadic clear cell renal cell carcinoma (CCRCC, the von Hippel Lindau (VHL gene is inactivated by mutation or methylation in the majority of primary (P tumors. Due to differing effects of wild-type (WT and mutant (MT VHL gene on downstream signaling pathways regulating angiogenesis, VHL gene status could impact clinical outcome. In CCRCC, comparative genomic hybridization (CGH analysis studies have reported genetic differences between paired P and metastatic (M tumors. We thus sequenced the VHL gene in paired tumor specimens from 10 patients to determine a possible clonal relationship between the P tumor and M lesion(s in patients with CCRCC. Using paraffin embedded specimens, genomic DNA from microdissected samples (>80% tumor of paired P tumor and M lesions from all 10 patients, as well as in normal tissue from 6 of these cases, was analyzed. The DNA was used for PCR-based amplification of each of the 3 exons of the VHL gene. Sequences derived from amplified samples were compared to the wild-type VHL gene sequence (GeneBank Accession No. AF010238. Methylation status of the VHL gene was determined using VHL methylation-specific PCR primers after DNA bisulfite modification. In 4/10 (40% patients the VHL gene status differed between the P tumor and the M lesion. As expected, when the VHL gene was mutated in both the P tumor and M lesion, the mutation was identical. Further, while the VHL genotype differed between the primary tumor in different kidneys or multiple metastatic lesions in the same patient, the VHL germline genotype in the normal adjacent tissue was always wild-type irrespective of the VHL gene status in the P tumor. These results demonstrate for the first time that the VHL gene status can be different between paired primary and metastatic tissue in patients with CCRCC.

  9. PIK3CA mutant tumors depend on oxoglutarate dehydrogenase

    Science.gov (United States)

    Ilic, Nina; Birsoy, Kıvanç; Aguirre, Andrew J.; Kory, Nora; Pacold, Michael E.; Singh, Shambhavi; Moody, Susan E.; DeAngelo, Joseph D.; Spardy, Nicole A.; Freinkman, Elizaveta; Weir, Barbara A.; Cowley, Glenn S.; Root, David E.; Asara, John M.; Vazquez, Francisca; Widlund, Hans R.; Sabatini, David M.; Hahn, William C.

    2017-01-01

    Oncogenic PIK3CA mutations are found in a significant fraction of human cancers, but therapeutic inhibition of PI3K has only shown limited success in clinical trials. To understand how mutant PIK3CA contributes to cancer cell proliferation, we used genome scale loss-of-function screening in a large number of genomically annotated cancer cell lines. As expected, we found that PIK3CA mutant cancer cells require PIK3CA but also require the expression of the TCA cycle enzyme 2-oxoglutarate dehydrogenase (OGDH). To understand the relationship between oncogenic PIK3CA and OGDH function, we interrogated metabolic requirements and found an increased reliance on glucose metabolism to sustain PIK3CA mutant cell proliferation. Functional metabolic studies revealed that OGDH suppression increased levels of the metabolite 2-oxoglutarate (2OG). We found that this increase in 2OG levels, either by OGDH suppression or exogenous 2OG treatment, resulted in aspartate depletion that was specifically manifested as auxotrophy within PIK3CA mutant cells. Reduced levels of aspartate deregulated the malate–aspartate shuttle, which is important for cytoplasmic NAD+ regeneration that sustains rapid glucose breakdown through glycolysis. Consequently, because PIK3CA mutant cells exhibit a profound reliance on glucose metabolism, malate–aspartate shuttle deregulation leads to a specific proliferative block due to the inability to maintain NAD+/NADH homeostasis. Together these observations define a precise metabolic vulnerability imposed by a recurrently mutated oncogene. PMID:28396387

  10. Characterization of Leber Congenital Amaurosis-associated NMNAT1 Mutants.

    Science.gov (United States)

    Sasaki, Yo; Margolin, Zachary; Borgo, Benjamin; Havranek, James J; Milbrandt, Jeffrey

    2015-07-10

    Leber congenital amaurosis 9 (LCA9) is an autosomal recessive retinal degeneration condition caused by mutations in the NAD(+) biosynthetic enzyme NMNAT1. This condition leads to early blindness but no other consistent deficits have been reported in patients with NMNAT1 mutations despite its central role in metabolism and ubiquitous expression. To study how these mutations affect NMNAT1 function and ultimately lead to the retinal degeneration phenotype, we performed detailed analysis of LCA-associated NMNAT1 mutants, including the expression, nuclear localization, enzymatic activity, secondary structure, oligomerization, and promotion of axonal and cellular integrity in response to injury. In many assays, most mutants produced results similar to wild type NMNAT1. Indeed, NAD(+) synthetic activity is unlikely to be a primary mechanism underlying retinal degeneration as most LCA-associated NMNAT1 mutants had normal enzymatic activity. In contrast, the secondary structure of many NMNAT1 mutants was relatively less stable as they lost enzymatic activity after heat shock, whereas wild type NMNAT1 retains significant activity after this stress. These results suggest that LCA-associated NMNAT1 mutants are more vulnerable to stressful conditions that lead to protein unfolding, a potential contributor to the retinal degeneration observed in this syndrome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Development of Bacillus subtilis mutants to produce tryptophan in pigs.

    Science.gov (United States)

    Bjerre, Karin; Cantor, Mette D; Nørgaard, Jan V; Poulsen, Hanne D; Blaabjerg, Karoline; Canibe, Nuria; Jensen, Bent B; Stuer-Lauridsen, Birgitte; Nielsen, Bea; Derkx, Patrick M F

    2017-02-01

    To generate tryptophan-overproducing Bacillus subtilis strains for in situ use in pigs, to reduce the feed cost for farmers and nitrogen pollution. A novel concept has been investigated-to generate B. subtilis strains able to produce tryptophan (Trp) in situ in pigs. Mutagenesis by UV was combined with selection on Trp and purine analogues in an iterative process. Two mutants from different wild types were obtained, mutant 1 (M1) produced 1 mg Trp/l and mutant 2 (M2) 14 mg Trp/l. Genome sequence analysis revealed that M1 had three single nuclear polymorphisms (SNPs) and M2 had two SNPs compared to the wild type strains. In both mutants SNPs were found in genes regulating tryptophan synthesis. Reverse transcription PCR confirmed up-regulation of the tryptophan synthesis genes in both mutants, the expression was up to 3 times higher in M2 than in M1. Tryptophan-excreting B. subtilis strains were obtained with UV-mutagenesis and analogue selection and can be used in animal feed applications.

  12. Analysis of AtCry1 and Mutants

    Science.gov (United States)

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

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

  13. Molecular and Genetic Analysis of Collagen Type IV Mutant Mouse Models of Spontaneous Intracerebral Hemorrhage Identify Mechanisms for Stroke Prevention

    Science.gov (United States)

    Jeanne, Marion; Jorgensen, Jeff; Gould, Douglas B.

    2015-01-01

    Background Collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) form heterotrimers critical for vascular basement membrane stability and function. Patients with COL4A1 or COL4A2 mutations suffer from diverse cerebrovascular diseases including cerebral microbleeds, porencephaly and fatal intracerebral hemorrhage (ICH). However, the pathogenic mechanisms remain unknown and there is a lack of effective treatment. Methods and Results Using Col4a1 and Col4a2 mutant mouse models, we investigated the genetic complexity and cellular mechanisms underlying the disease. We found that Col4a1 mutations cause abnormal vascular development, which triggers small vessel disease, recurrent hemorrhagic strokes and age-related macro-angiopathy. We showed that allelic heterogeneity, genetic context and environmental factors, such as acute exercise or anticoagulant medication, modulated disease severity and contributed to phenotypic heterogeneity. We found that intracellular accumulation of mutant collagen in vascular endothelial cells and pericytes was a key triggering factor of ICH. Finally, we showed that treatment of mutant mice with a FDA-approved chemical chaperone resulted in a decreased collagen intracellular accumulation and a significant reduction of ICH severity. Conclusions Our data are the first to show therapeutic prevention in vivo of ICH due to Col4a1 mutation, and imply that a mechanism-based therapy promoting protein folding might also prevent ICH in patients with COL4A1 and COL4A2 mutations. PMID:25753534

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

    Science.gov (United States)

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

    2014-01-01

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

  15. Metabolic and Phenotypic Differences between Mice Producing a Werner Syndrome Helicase Mutant Protein and Wrn Null Mice

    Science.gov (United States)

    Aumailley, Lucie; Garand, Chantal; Dubois, Marie Julie; Johnson, F. Brad; Marette, André; Lebel, Michel

    2015-01-01

    Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-family DNA helicase, WRN. Mice lacking part of the helicase domain of the WRN orthologue exhibit many phenotypic features of WS, including metabolic abnormalities and a shorter mean life span. In contrast, mice lacking the entire Wrn protein (i.e. Wrn null mice) do not exhibit a premature aging phenotype. In this study, we used a targeted mass spectrometry-based metabolomic approach to identify serum metabolites that are differentially altered in young Wrn helicase mutant and Wrn null mice. An antibody-based quantification of 43 serum cytokines and markers of cardiovascular disease risk complemented this study. We found that Wrn helicase mutants exhibited elevated and decreased levels, respectively, of the anti-inflammatory cytokine IL-10 and the pro-inflammatory cytokine IL-18. Wrn helicase mutants also exhibited an increase in serum hydroxyproline and plasminogen activator inhibitor-1, markers of extracellular matrix remodeling of the vascular system and inflammation in aging. We also observed an abnormal increase in the ratio of very long chain to short chain lysophosphatidylcholines in the Wrn helicase mutants underlying a peroxisome perturbation in these mice. Remarkably, the Wrn mutant helicase protein was mislocalized to the endoplasmic reticulum and the peroxisomal fractions in liver tissues. Additional analyses with mouse embryonic fibroblasts indicated a severe defect of the autophagy flux in cells derived from Wrn helicase mutants compared to wild type and Wrn null animals. These results indicate that the deleterious effects of the helicase-deficient Wrn protein are mediated by the dysfunction of several cellular organelles. PMID:26447695

  16. Generation and Characterization of dickkopf3 Mutant Mice

    Science.gov (United States)

    del Barco Barrantes, Ivan; Montero-Pedrazuela, Ana; Guadaño-Ferraz, Ana; Obregon, Maria-Jesus; Martinez de Mena, Raquel; Gailus-Durner, Valérie; Fuchs, Helmut; Franz, Tobias J.; Kalaydjiev, Svetoslav; Klempt, Martina; Hölter, Sabine; Rathkolb, Birgit; Reinhard, Claudia; Morreale de Escobar, Gabriella; Bernal, Juan; Busch, Dirk H.; Wurst, Wolfgang; Wolf, Eckhard; Schulz, Holger; Shtrom, Svetlana; Greiner, Erich; Hrabé de Angelis, Martin; Westphal, Heiner; Niehrs, Christof

    2006-01-01

    dickkopf (dkk) genes encode a small family of secreted Wnt antagonists, except for dkk3, which is divergent and whose function is poorly understood. Here, we describe the generation and characterization of dkk3 mutant mice. dkk3-deficient mice are viable and fertile. Phenotypic analysis shows no major alterations in organ morphology, physiology, and most clinical chemistry parameters. Since Dkk3 was proposed to function as thyroid hormone binding protein, we have analyzed deiodinase activities, as well as thyroid hormone levels. Mutant mice are euthyroid, and the data do not support a relationship of dkk3 with thyroid hormone metabolism. Altered phenotypes in dkk3 mutant mice were observed in the frequency of NK cells, immunoglobulin M, hemoglobin, and hematocrit levels, as well as lung ventilation. Furthermore, dkk3-deficient mice display hyperactivity. PMID:16508007

  17. Some enzyme activities of acetate mutants of Yarrowia lypolytica

    Energy Technology Data Exchange (ETDEWEB)

    Robak, M.; Wojtatowicz, M.; Rymowicz, W. [Akademia Rolnicza, Wroclaw (Poland)

    1994-12-31

    Activity at the following enzymes: CS (oxaloacetate-lyase citrate), AH (citrate (isocitrate) hydrolyase), ICDH (threo-Ds-isocitrate: NADP oxidoreductase) and ICL (threo-Ds-isocitrate glyoxyglate-lyase) was measured at subsequent stages of citrate fermentation on glucose by wild type strain `Y, lipolytica A-101` and 2 acetate defective mutants, in order to recognize metabolic disorders in those mutants, which resulted in markedly improved homogeneity of citric acid production. Mutants did not show significant changes in activity of TCA cycle enzymes and ICL. Thus suggests that the control of citric:isocitric acid ratio is more difficult and it can also depend on transportation systems of both acids. (author). 19 refs, 3 figs, 2 tabs.

  18. The swimming activity of the staggerer mutant mouse.

    Science.gov (United States)

    Goodall, G; Guastavino, J M; Gheusi, G

    1986-09-01

    Four experiments investigated the swimming behaviour of staggerer mutant mice. The results partially confirmed previous reports that a mouse's swimming is unaffected by the staggerer mutation. In terms of speed and distance there are indeed no measurable differences between normal and staggerer mice, when first placed in the water. The stagger's resistance was however shown to be much lower than a normal's and the genetic difference was also associated with different styles of swimming. Furthermore, whereas the normal mouse's swimming behaviour evolves with increased time in the water, the staggerer's remains constant. The differences are interpreted on the basis of abnormal novelty reactions by the staggerer mutants. Thus, swimming appears to be a better tool for investigating the higher-level cognitive functions of this mutant than terrestrial locomotion. Copyright © 1986. Published by Elsevier B.V.

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

    Science.gov (United States)

    Lalonde, R; Strazielle, C

    2011-04-15

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

  20. Biochemical Analysis of Two Single Mutants that Give Rise to a Polymorphic G6PD A-Double Mutant

    Directory of Open Access Journals (Sweden)

    Edson Jiovany Ramírez-Nava

    2017-10-01

    Full Text Available Glucose-6-phosphate dehydrogenase (G6PD is a key regulatory enzyme that plays a crucial role in the regulation of cellular energy and redox balance. Mutations in the gene encoding G6PD cause the most common enzymopathy that drives hereditary nonspherocytic hemolytic anemia. To gain insights into the effects of mutations in G6PD enzyme efficiency, we have investigated the biochemical, kinetic, and structural changes of three clinical G6PD variants, the single mutations G6PD A+ (Asn126AspD and G6PD Nefza (Leu323Pro, and the double mutant G6PD A− (Asn126Asp + Leu323Pro. The mutants showed lower residual activity (≤50% of WT G6PD and displayed important kinetic changes. Although all Class III mutants were located in different regions of the three-dimensional structure of the enzyme and were not close to the active site, these mutants had a deleterious effect over catalytic activity and structural stability. The results indicated that the G6PD Nefza mutation was mainly responsible for the functional and structural alterations observed in the double mutant G6PD A−. Moreover, our study suggests that the G6PD Nefza and G6PD A− mutations affect enzyme functions in a similar fashion to those reported for Class I mutations.

  1. Genetic interactions among homologous recombination mutants in Candida albicans.

    Science.gov (United States)

    Bellido, Alberto; Andaluz, Encarnación; Gómez-Raja, Jonathan; Álvarez-Barrientos, Alberto; Larriba, Germán

    2015-01-01

    rad52-ΔΔ and, to a lesser extent, rad51-ΔΔ deletants of Candidaalbicans displayed slow growth and aberrant filamentous morphology whereas rad59-ΔΔ mutants, both by growth rate and morphology resembled wild type. In this study, we have constructed pair-wise double deletants to analyze genetic interactions among these homologous recombination (HR) proteins that affect growth and morphology traits. When grown in liquid YPD medium, double mutant rad51-ΔΔ rad59-ΔΔ exhibited growth rates, cell and colony morphologies, and plating efficiencies that were not significantly different from those observed for rad51-ΔΔ. The same was true for rad52-ΔΔ rad59-ΔΔ compared to rad52-ΔΔ. Slow growth and decreased plating efficiency were caused, at least in part, by a decreased viability, as deduced from FUN1 staining. Flow cytometry and microscopic studies of filamentous mutant populations revealed major changes in cell ploidy, size and morphology, whereas DAPI staining identified complex nuclear rearrangements in yeast and filamentous cells. These phenotypes were not observed in the rad59-ΔΔ mutant populations. Our results show that abolishing Rad51 functions induces the appearance of a subpopulation of aberrant yeast and filamentous forms with increased cell size and ploidy. The size of this complex subpopulation was exacerbated in rad52-ΔΔ mutants. The combination of filamentous cell morphology and viability phenotypes was reflected on the colony morphology of the respective mutants. We conclude that the rad52 mutation is epistatic to rad51 for all the morphological traits analyzed. We discuss these results in the light of the several functions of these recombination genes. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Cellular androgen content influences enzalutamide agonism of F877L mutant androgen receptor

    Science.gov (United States)

    Coleman, Daniel J.; Van Hook, Kathryn; King, Carly J.; Schwartzman, Jacob; Lisac, Robert; Urrutia, Joshua; Sehrawat, Archana; Woodward, Josha; Wang, Nicholas J.; Gulati, Roman; Thomas, George V.; Beer, Tomasz M.; Gleave, Martin; Korkola, James E.; Gao, Lina; Heiser, Laura M.; Alumkal, Joshi J.

    2016-01-01

    Prostate cancer is the most commonly diagnosed and second-most lethal cancer among men in the United States. The vast majority of prostate cancer deaths are due to castration-resistant prostate cancer (CRPC) – the lethal form of the disease that has progressed despite therapies that interfere with activation of androgen receptor (AR) signaling. One emergent resistance mechanism to medical castration is synthesis of intratumoral androgens that activate the AR. This insight led to the development of the AR antagonist enzalutamide. However, resistance to enzalutamide invariably develops, and disease progression is nearly universal. One mechanism of resistance to enzalutamide is an F877L mutation in the AR ligand-binding domain that can convert enzalutamide to an agonist of AR activity. However, mechanisms that contribute to the agonist switch had not been fully clarified, and there were no therapies to block AR F877L. Using cell line models of castration-resistant prostate cancer (CRPC), we determined that cellular androgen content influences enzalutamide agonism of mutant F877L AR. Further, enzalutamide treatment of AR F877L-expressing cell lines recapitulated the effects of androgen activation of F877L AR or wild-type AR. Because the BET bromodomain inhibitor JQ-1 was previously shown to block androgen activation of wild-type AR, we tested JQ-1 in AR F877L-expressing CRPC models. We determined that JQ-1 suppressed androgen or enzalutamide activation of mutant F877L AR and suppressed growth of mutant F877L AR CRPC tumors in vivo, demonstrating a new strategy to treat tumors harboring this mutation. PMID:27276681

  3. Global gene profiling of aging lungs in Atp8b1 mutant mice.

    Science.gov (United States)

    Soundararajan, Ramani; Stearns, Timothy M; Czachor, Alexander; Fukumoto, Jutaro; Turn, Christina; Westermann-Clark, Emma; Breitzig, Mason; Tan, Lee; Lockey, Richard F; King, Benjamin L; Kolliputi, Narasaiah

    2016-09-29

    Recent studies implicate cardiolipin oxidation in several age-related diseases. Atp8b1 encoding Type 4 P-type ATPases is a cardiolipin transporter. Mutation in Atp8b1 gene or inflammation of the lungs impairs the capacity of Atp8b1 to clear cardiolipin from lung fluid. However, the link between Atp8b1 mutation and age-related gene alteration is unknown. Therefore, we investigated how Atp8b1 mutation alters age-related genes. We performed Affymetrix gene profiling of lungs isolated from young (7-9 wks, n=6) and aged (14 months, 14 M, n=6) C57BL/6 and Atp8b1 mutant mice. In addition, Ingenuity Pathway Analysis (IPA) was performed. Differentially expressed genes were validated by quantitative real-time PCR (qRT-PCR). Global transcriptome analysis revealed 532 differentially expressed genes in Atp8b1 lungs, 157 differentially expressed genes in C57BL/6 lungs, and 37 overlapping genes. IPA of age-related genes in Atp8b1 lungs showed enrichment of Xenobiotic metabolism and Nrf2-mediated signaling pathways. The increase in Adamts2 and Mmp13 transcripts in aged Atp8b1 lungs was validated by qRT-PCR. Similarly, the decrease in Col1a1 and increase in Cxcr6 transcripts was confirmed in both Atp8b1 mutant and C57BL/6 lungs. Based on transcriptome profiling, our study indicates that Atp8b1 mutant mice may be susceptible to age-related lung diseases.

  4. p53 increases caspase-6 expression and activation in muscle tissue expressing mutant huntingtin.

    Science.gov (United States)

    Ehrnhoefer, Dagmar E; Skotte, Niels H; Ladha, Safia; Nguyen, Yen T N; Qiu, Xiaofan; Deng, Yu; Huynh, Khuong T; Engemann, Sabine; Nielsen, Signe M; Becanovic, Kristina; Leavitt, Blair R; Hasholt, Lis; Hayden, Michael R

    2014-02-01

    Activation of caspase-6 in the striatum of both presymptomatic and affected persons with Huntington's disease (HD) is an early event in the disease pathogenesis. However, little is known about the role of caspase-6 outside the central nervous system (CNS) and whether caspase activation might play a role in the peripheral phenotypes, such as muscle wasting observed in HD. We assessed skeletal muscle tissue from HD patients and well-characterized mouse models of HD. Cleavage of the caspase-6 specific substrate lamin A is significantly increased in skeletal muscle obtained from HD patients as well as in muscle tissues from two different HD mouse models. p53, a transcriptional activator of caspase-6, is upregulated in neuronal cells and tissues expressing mutant huntingtin. Activation of p53 leads to a dramatic increase in levels of caspase-6 mRNA, caspase-6 activity and cleavage of lamin A. Using mouse embryonic fibroblasts (MEFs) from YAC128 mice, we show that this increase in caspase-6 activity can be mitigated by pifithrin-α (pifα), an inhibitor of p53 transcriptional activity, but not through the inhibition of p53's mitochondrial pro-apoptotic function. Remarkably, the p53-mediated increase in caspase-6 expression and activation is exacerbated in cells and tissues of both neuronal and peripheral origin expressing mutant huntingtin (Htt). These findings suggest that the presence of the mutant Htt protein enhances p53 activity and lowers the apoptotic threshold, which activates caspase-6. Furthermore, these results suggest that this pathway is activated both within and outside the CNS in HD and may contribute to both loss of CNS neurons and muscle atrophy.

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

    Directory of Open Access Journals (Sweden)

    Cristina d'Abramo

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

  6. Virulence determinants of Salmonella Gallinarum biovar Pullorum identified by PCR signature-tagged mutagenesis and the spiC mutant as a candidate live attenuated vaccine.

    Science.gov (United States)

    Geng, Shizhong; Jiao, Xinan; Barrow, Paul; Pan, Zhiming; Chen, Xiang

    2014-01-31

    Salmonella Gallinarum biovar Pullorum (S. Gallinarum biovar Pullorum) is the causative agent of pullorum disease (PD) in chickens which results in considerable economic losses to the poultry industries in developing countries. PCR-Signature Tagged Mutagenesis was used to identify virulence determinants of S. Gallinarum biovar Pullorum and novel attenuated live vaccine candidates for use against this disease. A library of 1800 signature-tagged S. Gallinarum biovar Pullorum mutants was constructed and screened for virulence-associated genes in chickens. The attenuation of 10 mutants was confirmed by in vivo and in vitro competitive index (CI) studies. The transposons were found to be located in SPI-1 (2/10 mutants), SPI-2 (3/10), the virulence plasmid (1/10) and non-SPI genes (4/10). One highly attenuated spiC mutant persisted in spleen and liver for less than 10 days and induced high levels of circulating antibody and protective immunity against oral challenge in young broiler chickens. The spiC mutant is a potential new vaccine candidate for use with chickens against this disease. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. An unfolded protein response is the initial cellular response to the expression of mutant matrilin-3 in a mouse model of multiple epiphyseal dysplasia.

    Science.gov (United States)

    Nundlall, Seema; Rajpar, M Helen; Bell, Peter A; Clowes, Christopher; Zeeff, Leo A H; Gardner, Benjamin; Thornton, David J; Boot-Handford, Raymond P; Briggs, Michael D

    2010-11-01

    Multiple epiphyseal dysplasia (MED) can result from mutations in matrilin-3, a structural protein of the cartilage extracellular matrix. We have previously shown that in a mouse model of MED the tibia growth plates were normal at birth but developed a progressive dysplasia characterised by the intracellular retention of mutant matrilin-3 and abnormal chondrocyte morphology. By 3 weeks of age, mutant mice displayed a significant decrease in chondrocyte proliferation and dysregulated apoptosis. The aim of this current study was to identify the initial post-natal stages of the disease. We confirmed that the disease phenotype is seen in rib and xiphoid cartilage and, like tibia growth plate cartilage is characterised by the intracellular retention of mutant matrilin-3. Gene expression profiling showed a significant activation of classical unfolded protein response (UPR) genes in mutant chondrocytes at 5 days of age, which was still maintained by 21 days of age. Interestingly, we also noted the upregulation of arginine-rich, mutated in early stage of tumours (ARMET) and cysteine-rich with EGF-like domain protein 2 (CRELD2) are two genes that have only recently been implicated in the UPR. This endoplasmic reticulum (ER) stress and UPR did not lead to increased chondrocyte apoptosis in mutant cartilage by 5 days of age. In an attempt to alleviate ER stress, mutant mice were fed with a chemical chaperone, 4-sodium phenylbutyrate (SPB). SPB at the dosage used had no effect on chaperone expression at 5 days of age but modestly decreased levels of chaperone proteins at 3 weeks. However, this did not lead to increased secretion of mutant matrilin-3 and in the long term did not improve the disease phenotype. We performed similar studies with a mouse model of Schmid metaphyseal chondrodysplasia, but again this treatment did not improve the phenotype.

  8. RFLP mapping of the barley homeotic mutant lax-a.

    Science.gov (United States)

    Laurie, D A; Pratchett, N; Allen, R L; Hantke, S S

    1996-07-01

    The lax-a homeotic mutant of barley has flowers in which lodicules are replaced by stamens (giving five stamens per flower). RFLP mapping of an F2 population from a Bonus lax-a (1) x H. spontaneum cross showed that the mutation was on the short arm of chromosome 7(5H), closely linked to the centromere. An additional F2 population was used to show that the lax-a mutation gave the five-stamen phenotype in all flowers of 6-rowed spikes and that hoods were elevated and reduced in size in lax-a/Hooded double-mutant plants.

  9. Mutant E. coli strain with increased succinic acid production

    Science.gov (United States)

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    2001-09-25

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  10. Forward and reverse genetics: The LORE1 retrotransposon insertion mutants

    DEFF Research Database (Denmark)

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

    2014-01-01

    The endogenous Lotus retrotransposon 1 (LORE1) transposes in the germ line of Lotus japonicus plants that carry an active element. This feature of LORE1 has been exploited for generation of a large non-transgenic insertion mutant population, where insertions have been annotated using next......-generation sequencing approaches. The LORE1 mutant lines are freely available and can be ordered online. Endogenous retrotransposons are also active in many other plant species. Based on the methods developed for LORE1 mutagenesis, it should be simple to establish similar systems in other species, once an appropriate...

  11. Mutant E. coli strain with increased succinic acid production

    Science.gov (United States)

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    1998-01-01

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  12. Characterization of Sugar Insensitive (sis) Mutants of Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Gibson, Susan I.

    2009-06-08

    Despite the fact that soluble sugar levels have been postulated to play an important role in the control of a wide variety of plant metabolic and developmental pathways, the mechanisms by which plants respond to soluble sugar levels remain poorly understood. Plant responses to soluble sugar levels are also important in bioenergy production, as plant sugar responses are believed to help regulate both carbon fixation and carbon partitioning. For example, accumulation of soluble sugars, such as sucrose and glucose, in source tissues leads to feedback inhibition of photosynthesis, thereby decreasing rates of carbon fixation. Soluble sugar levels can also affect sink strengths, affecting the rates of accumulation of carbon-based compounds into both particular molecular forms (e.g. carbohydrates versus lipids versus proteins) and particular plant organs and tissues. Mutants of Arabidopsis that are defective in the ability to respond to soluble sugar levels were isolated and used as tools to identify some of the factors involved in plant sugar response. These sugar insensitive (sis) mutants were isolated by screening mutagenized seeds for those that were able to germinate and develop relatively normal shoot systems on media containing 0.3 M glucose or 0.3 M sucrose. At these sugar concentrations, wild-type Arabidopsis germinate and produce substantial root systems, but show little to no shoot development. Twenty-eight sis mutants were isolated during the course of four independent mutant screens. Based on a preliminary characterization of all of these mutants, sis3 and sis6 were chosen for further study. Both of these mutations appear to lie in previously uncharacterized loci. Unlike many other sugar-response mutants, sis3 mutants exhibit a wild-type or near wild-type response in all phytohormone-response assays conducted to date. The sis6-1 mutation is unusual in that it appears to be due to overexpression of a gene, rather than representing a loss of function mutation

  13. Clear Plaque Mutants of Lactococcal Phage TP901-1

    DEFF Research Database (Denmark)

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

    2016-01-01

    We report a method for obtaining turbid plaques of the lactococcal bacteriophage TP901-1 and its derivative TP901-BC1034. We have further used the method to isolate clear plaque mutants of this phage. Analysis of 8 such mutants that were unable to lysogenize the host included whole genome...... protein involved in the DNA binding. The conclusion is that cI is the only gene involved in clear plaque formation i.e. the CI protein is the determining factor for the lysogenic pathway and its maintenance in the lactococcal phage TP901-1....

  14. A male sterile pepper (C. annuum L.) mutant.

    Science.gov (United States)

    Daskaloff, S

    1968-08-01

    1. After treatment of dry seeds of red pepperCapsicum annuum L. with X-rays a male-sterile mutant was discovered in the M2. 2. The male-sterile mutant segregates in a ratio of 3.28:1 (χ(2)=3.148, probability 0.07). 3. After an alternative cultivation of male-sterile plants and of a variety with good combining ability relatively good fruit-setting and seed production was obtained. 4. Grafting of male-sterile scions to normal stocks does not affect the male-sterile phenotype.

  15. Regioselective alkane hydroxylation with a mutant AlkB enzyme

    Science.gov (United States)

    Koch, Daniel J.; Arnold, Frances H.

    2012-11-13

    AlkB from Pseudomonas putida was engineered using in-vivo directed evolution to hydroxylate small chain alkanes. Mutant AlkB-BMO1 hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. Mutant AlkB-BMO2 similarly hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. These biocatalysts are highly active for small chain alkane substrates and their regioselectivity is retained in whole-cell biotransformations.

  16. Functional analysis of a zebrafish myd88 mutant identifies key transcriptional components of the innate immune system

    Directory of Open Access Journals (Sweden)

    Michiel van der Vaart

    2013-05-01

    Toll-like receptors (TLRs are an important class of pattern recognition receptors (PRRs that recognize microbial and danger signals. Their downstream signaling upon ligand binding is vital for initiation of the innate immune response. In human and mammalian models, myeloid differentiation factor 88 (MYD88 is known for its central role as an adaptor molecule in interleukin 1 receptor (IL-1R and TLR signaling. The zebrafish is increasingly used as a complementary model system for disease research and drug screening. Here, we describe a zebrafish line with a truncated version of MyD88 as the first zebrafish mutant for a TLR signaling component. We show that this immune-compromised mutant has a lower survival rate under standard rearing conditions and is more susceptible to challenge with the acute bacterial pathogens Edwardsiella tarda and Salmonella typhimurium. Microarray and quantitative PCR analysis revealed that expression of genes for transcription factors central to innate immunity (including NF-ĸB and AP-1 and the pro-inflammatory cytokine Il1b, is dependent on MyD88 signaling during these bacterial infections. Nevertheless, expression of immune genes independent of MyD88 in the myd88 mutant line was sufficient to limit growth of an attenuated S. typhimurium strain. In the case of infection with the chronic bacterial pathogen Mycobacterium marinum, we show that MyD88 signaling has an important protective role during early pathogenesis. During mycobacterial infection, the myd88 mutant shows accelerated formation of granuloma-like aggregates and increased bacterial burden, with associated lower induction of genes central to innate immunity. This zebrafish myd88 mutant will be a valuable tool for further study of the role of IL1R and TLR signaling in the innate immunity processes underlying infectious diseases, inflammatory disorders and cancer.

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

    Directory of Open Access Journals (Sweden)

    Victoria A Lawson

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

  18. Exploring the elephant: histopathology in high-throughput phenotyping of mutant mice

    Directory of Open Access Journals (Sweden)

    Paul N. Schofield

    2012-01-01

    Full Text Available Recent advances in gene knockout techniques and the in vivo analysis of mutant mice, together with the advent of large-scale projects for systematic mouse mutagenesis and genome-wide phenotyping, have allowed the creation of platforms for the most complete and systematic analysis of gene function ever undertaken in a vertebrate. The development of high-throughput phenotyping pipelines for these and other large-scale projects allows investigators to search and integrate large amounts of directly comparable phenotype data from many mutants, on a genomic scale, to help develop and test new hypotheses about the origins of disease and the normal functions of genes in the organism. Histopathology has a venerable history in the understanding of the pathobiology of human and animal disease, and presents complementary advantages and challenges to in vivo phenotyping. In this review, we present evidence for the unique contribution that histopathology can make to a large-scale phenotyping effort, using examples from past and current programmes at Lexicon Pharmaceuticals and The Jackson Laboratory, and critically assess the role of histopathology analysis in high-throughput phenotyping pipelines.

  19. High-efficiency transduction of the mouse retina by tyrosine-mutant AAV serotype vectors.

    Science.gov (United States)

    Petrs-Silva, Hilda; Dinculescu, Astra; Li, Qiuhong; Min, Seok-Hong; Chiodo, Vince; Pang, Ji-Jing; Zhong, Li; Zolotukhin, Sergei; Srivastava, Arun; Lewin, Alfred S; Hauswirth, William W

    2009-03-01

    Vectors derived from adeno-associated viruses (AAVs) have become important gene delivery tools for the treatment of many inherited ocular diseases in well-characterized animal models. Previous studies have determined that the viral capsid plays an essential role in the cellular tropism and efficiency of transgene expression. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV2 capsid targets the viral particles for ubiquitination and proteasome- mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo. Because the tyrosine residues are highly conserved in other AAV serotypes, in this study we evaluated the intraocular transduction characteristics of vectors containing point mutations in surface- exposed capsid tyrosine residues in AAV serotypes 2, 8, and 9. Several of these novel AAV mutants were found to display a strong and widespread transgene expression in many retinal cells after subretinal or intravitreal delivery compared with their wild-type counterparts. For the first time, we show efficient transduction of the ganglion cell layer by AAV serotype 8 or 9 mutant vectors, thus providing additional tools besides AAV2 for targeting these cells. These enhanced AAV vectors have a great potential for future therapeutic applications for retinal degenerations and ocular neovascular diseases.

  20. Exploring the elephant: histopathology in high-throughput phenotyping of mutant mice.

    Science.gov (United States)

    Schofield, Paul N; Vogel, Peter; Gkoutos, Georgios V; Sundberg, John P

    2012-01-01

    Recent advances in gene knockout techniques and the in vivo analysis of mutant mice, together with the advent of large-scale projects for systematic mouse mutagenesis and genome-wide phenotyping, have allowed the creation of platforms for the most complete and systematic analysis of gene function ever undertaken in a vertebrate. The development of high-throughput phenotyping pipelines for these and other large-scale projects allows investigators to search and integrate large amounts of directly comparable phenotype data from many mutants, on a genomic scale, to help develop and test new hypotheses about the origins of disease and the normal functions of genes in the organism. Histopathology has a venerable history in the understanding of the pathobiology of human and animal disease, and presents complementary advantages and challenges to in vivo phenotyping. In this review, we present evidence for the unique contribution that histopathology can make to a large-scale phenotyping effort, using examples from past and current programmes at Lexicon Pharmaceuticals and The Jackson Laboratory, and critically assess the role of histopathology analysis in high-throughput phenotyping pipelines.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Daniele Vergara

    2017-01-01

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

  3. Copper(II) and the pathological H50Q α-synuclein mutant: Environment meets genetics.

    Science.gov (United States)

    Villar-Piqué, Anna; Rossetti, Giulia; Ventura, Salvador; Carloni, Paolo; Fernández, Claudio O; Outeiro, Tiago Fleming

    2017-01-01

    Copper is one of the metals described to bind the Parkinson disease-related protein α-synuclein (aSyn), and to promote its aggregation. Although histidine at position 50 in the aSyn sequence is one of the most studied copper-anchoring sites, its precise role in copper binding and aSyn aggregation is still unclear. Previous studies suggested that this residue does not significantly affect copper-mediated aSyn aggregation. However, our findings showed that the aggregation of the pathological H50Q aSyn mutant is enhanced by copper hints otherwise. Despite the inexistence of a model for aSyn H50Q-copper complexation, we discuss possible mechanisms by which this metal contributes to the misfolding and self-assembly of this particular aSyn mutant. Considering the genetic association of the H50Q mutation with familial forms of Parkinson disease, and the fact that copper homeostasis is deregulated in this disorder, understanding the interplay between both factors will shed light into the molecular and cellular mechanisms triggering the development and spreading of the aSyn pathology.

  4. Copper(II) and the pathological H50Q α-synuclein mutant: Environment meets genetics

    Science.gov (United States)

    Villar-Piqué, Anna; Rossetti, Giulia; Ventura, Salvador; Carloni, Paolo; Fernández, Claudio O.; Outeiro, Tiago Fleming

    2017-01-01

    ABSTRACT Copper is one of the metals described to bind the Parkinson disease-related protein α-synuclein (aSyn), and to promote its aggregation. Although histidine at position 50 in the aSyn sequence is one of the most studied copper-anchoring sites, its precise role in copper binding and aSyn aggregation is still unclear. Previous studies suggested that this residue does not significantly affect copper-mediated aSyn aggregation. However, our findings showed that the aggregation of the pathological H50Q aSyn mutant is enhanced by copper hints otherwise. Despite the inexistence of a model for aSyn H50Q-copper complexation, we discuss possible mechanisms by which this metal contributes to the misfolding and self-assembly of this particular aSyn mutant. Considering the genetic association of the H50Q mutation with familial forms of Parkinson disease, and the fact that copper homeostasis is deregulated in this disorder, understanding the interplay between both factors will shed light into the molecular and cellular mechanisms triggering the development and spreading of the aSyn pathology. PMID:28289488

  5. Mutant huntingtin gene-dose impacts on aggregate deposition, DARPP32 expression and neuroinflammation in HdhQ150 mice.

    Directory of Open Access Journals (Sweden)

    Douglas Young

    Full Text Available Huntington's disease (HD is an autosomal dominant, progressive and fatal neurological disorder caused by an expansion of CAG repeats in exon-1 of the huntingtin gene. The encoded poly-glutamine stretch renders mutant huntingtin prone to aggregation. HdhQ150 mice genocopy a pathogenic repeat (∼150 CAGs in the endogenous mouse huntingtin gene and model predominantly pre-manifest HD. Treating early is likely important to prevent or delay HD, and HdhQ150 mice may be useful to assess therapeutic strategies targeting pre-manifest HD. This requires appropriate markers and here we demonstrate, that pre-symptomatic HdhQ150 mice show several dramatic mutant huntingtin gene-dose dependent pathological changes including: (i an increase of neuronal intra-nuclear inclusions (NIIs in brain, (ii an increase of extra-nuclear aggregates in dentate gyrus, (iii a decrease of DARPP32 protein and (iv an increase in glial markers of neuroinflammation, which curiously did not correlate with local neuronal mutant huntingtin inclusion-burden. HdhQ150 mice developed NIIs also in all retinal neuron cell-types, demonstrating that retinal NIIs are not specific to human exon-1 R6 HD mouse models. Taken together, the striking and robust mutant huntingtin gene-dose related changes in aggregate-load, DARPP32 levels and glial activation markers should greatly facilitate future testing of therapeutic strategies in the HdhQ150 HD mouse model.

  6. Interaction between environmental and genetic factors modulates schizophrenic endophenotypes in the Snap-25 mouse mutant blind-drunk.

    Science.gov (United States)

    Oliver, Peter L; Davies, Kay E

    2009-12-01

    To understand the pathophysiology of neuropsychiatric disorders such as schizophrenia requires consideration of multiple genetic and non-genetic factors. However, very little is known about the consequences of combining models of synaptic dysfunction with controlled environmental manipulations. Therefore, to generate new insights into gene-environment interactions and complex behaviour, we examined the influence of variable prenatal stress (PNS) on two mouse lines with mutations in synaptosomal-associated protein of 25 kDa (Snap-25): the blind-drunk (Bdr) point mutant and heterozygous Snap-25 knockout mice. Neonatal development was analysed in addition to an assessment of adult behavioural phenotypes relevant to the psychotic, cognitive and negative aspects of schizophrenia. These data show that PNS influenced specific anxiety-related behaviour in all animals. In addition, sensorimotor gating deficits previously noted in Bdr mutants were markedly enhanced by PNS; significantly, these effects could be reversed with the application of anti-psychotic drugs. Moreover, social interaction abnormalities were observed only in Bdr animals from stressed dams but not in wild-type littermates or mutants from non-stressed mothers. These results show for the first time that combining a synaptic mouse point mutant with a controlled prenatal stressor paradigm produces both modified and previously unseen phenotypes, generating new insights into the interactions between genetics and the environment relevant to the study of psychiatric disease.

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

    Directory of Open Access Journals (Sweden)

    Jiou Wang

    2009-01-01

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

  8. Mutant connexin 50 (S276F) inhibits channel and hemichannel ...

    Indian Academy of Sciences (India)

    The mutant and wild-type Cx50 were expressed in equal levels and could efficiently localize to the plasma membrane without transportation and assembly ... Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University, Donghu Road 169#, Wuhan, Hubei 430071, People's Republic of China; Hubei Cancer ...

  9. Evaluation of high yielding mutants of Hordeumvulgare cultivar Izgrev

    Directory of Open Access Journals (Sweden)

    B. Dyulgerova

    2017-06-01

    Full Text Available Abstract. Seeds of Hordeum vulgare L. cultivar Izgrev were treated with different concentrations of sodium azide to induce genetic variability for the selection of genotypes with improved traits. After passing through different stages of selection, 18 promising mutants were selected for further studies. Eighteen mutants and their parent and national standard cultivar Veslets were evaluated in Complete Block Design with four replications. The research was conducted in 2013 – 2014 and 2014 – 2015 growing seasons in the experimental field of the Institute of Agriculture Karnobat, Southeastern Bulgaria. The characters studied included days to heading, plant height, lodging, peduncle length, spike length, awn length, spikelet number per spike, grain number per spike, grain weight per spike, 1000 grains weight and grain yield. Wide variation among mutant lines was observed for different traits. Mutant lines M4/16 and M 3/14 produced significantly greater grain yield than the parent and standard cultivar. Positive changes in lodging tolerance, grain number per spike, grain weight per spike, 1000 grains weightwere also observed. This study showed positive effects in the use of mutation in inducing improvement for grain yield and some yield related traits.

  10. Locating a modifier gene of Ovum mutant through crosses between ...

    Indian Academy of Sciences (India)

    RESEARCH ARTICLE Volume 95 Issue 2 June 2016 pp 297-302 ... mouse; DDK syndrome; ovum mutant; modifier gene; quantitative trait loci ... Previously, some research groups reported that the embryonic mortality deviated from the semilethal rate in backcrosses between heterozygous (Om/+) females and males of other ...

  11. Characterization of a novel curled-cotyledons mutant in soybean ...

    African Journals Online (AJOL)

    ARL

    mutant, the embryo sac becomes smaller and bulbous, and ultrastructure of developing cotyledons exhibits ... has higher protein and oil content, and has altered seed ultrastructure. The purpose of this experiment is to evaluate the features of soybean curled-cotyledons ..... mitochondria of soybean seedling cotyledons.

  12. Enhanced longevity in tau mutant Syrian hamsters, Mesocricetus auratus

    NARCIS (Netherlands)

    Oklejewicz, Malgorzata; Daan, Serge

    The single-gene mutation tau in the Syrian hamster shortens the circadian period by about 20% in the homozygous mutant and simultaneously increases the mass-specific metabolic rate by about 20%. Both effects might be expected to lead to a change in longevity. To test such expectations, the life span

  13. Molecular analysis of sex chromosome-linked mutants in the ...

    Indian Academy of Sciences (India)

    2010-09-06

    Sep 6, 2010 ... In B. mori, polyploid individuals with different sex chromo- some constitutions can be ... RNA was extracted from the ovary, testis, and fat body of a fifth-instar larva, and from the an- tennae of 10 moths. The primer set .... For example, the body of sk. (stick, 4–25.8) mutant larvae is firm to the touch, while that.

  14. Growth properties of Cellulomonas flavigena mutants affected in cellulose utilization.

    Science.gov (United States)

    Béguin, P; Eisen, H

    1978-01-01

    The role of cellobiose metabolism in cellulose utilization by Cellulomonas flavigena was investigated by studying mutants unable to grow on cellobiose or cellulose. The results show that the ability to utilize cellulose is strictly dependent on the ability to utilize cellobiose. PMID:415038

  15. Fusion genetic analysis of jasmonate-signalling mutants in Arabidopsis

    DEFF Research Database (Denmark)

    Jensen, Anders Bøgh; Raventos, D.; Mundy, John Williams

    2002-01-01

    Jasmonates induce plant-defence responses and act to regulate defence-related genes including positive feedback of the lipoxygenase 2 (LOX2) gene involved in jasmonate synthesis. To identify jasmonate-signalling mutants, we used a fusion genetic strategy in which the firefly luciferase (FLUC...

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

    Indian Academy of Sciences (India)

    2012-08-07

    Aug 7, 2012 ... Abstract. The ad-8 gene of Neurospora crassa, in addition to being used for the study of purine biology, has been extensively studied as a model for gene structure, mutagenesis and intralocus recombination. Because of this there is an extensive collection of well- characterized N. crassa ad-8 mutants in the ...

  17. Performance of early maturing mutants derived from 'supa' rice ...

    African Journals Online (AJOL)

    Days to 50% flowering and 1000 grain weight exerted negative direct effect on yield. Changes in grain quality were also observed emphasizing the importance of conducting cooking and taste panel tests. Keywords: Early maturity, grain guality, rice mutants, Oryza saliva, path coefficient. Tanzania J. Agri. Sc. (2001) Vol 4, ...

  18. Isolation and partial characterization of carotenoid mutants of ...

    African Journals Online (AJOL)

    Global Journal of Pure and Applied Sciences ... Three major pigments isolated by high performance liquid chromatography (HPLC) were characterized by their absorption maxima, partition ratios in light ... High performance liquid chromatography was used to compare pigments of the wild-type with those of the mutants.

  19. Dynamics of Mutant Cells in Hierarchical Organized Tissues

    Science.gov (United States)

    Werner, Benjamin; Dingli, David; Lenaerts, Tom; Pacheco, Jorge M.; Traulsen, Arne

    2011-01-01

    Most tissues in multicellular organisms are maintained by continuous cell renewal processes. However, high turnover of many cells implies a large number of error-prone cell divisions. Hierarchical organized tissue structures with stem cell driven cell differentiation provide one way to prevent the accumulation of mutations, because only few stem cells are long lived. We investigate the deterministic dynamics of cells in such a hierarchical multi compartment model, where each compartment represents a certain stage of cell differentiation. The dynamics of the interacting system is described by ordinary differential equations coupled across compartments. We present analytical solutions for these equations, calculate the corresponding extinction times and compare our results to individual based stochastic simulations. Our general compartment structure can be applied to different tissues, as for example hematopoiesis, the epidermis, or colonic crypts. The solutions provide a description of the average time development of stem cell and non stem cell driven mutants and can be used to illustrate general and specific features of the dynamics of mutant cells in such hierarchically structured populations. We illustrate one possible application of this approach by discussing the origin and dynamics of PIG-A mutant clones that are found in the bloodstream of virtually every healthy adult human. From this it is apparent, that not only the occurrence of a mutant but also the compartment of origin is of importance. PMID:22144884

  20. Characterization of mutant cowpea [ Vigna unguiculata (L) Walp ...

    African Journals Online (AJOL)

    Phylogenetic relationship and polymorphism was detected in 10 cowpea lines comprising of leaf, flower and stem mutants, their putative parents and an exotic accession using 10 random ... Genetic distance ranged from 0.05 to 0.30 based on AFLP markers, while it ranged between 0.13 and 0.44 for RAPD markers. Cluster ...

  1. Clustering common bean mutants based on heterotic groupings ...

    African Journals Online (AJOL)

    The objective of this study was to cluster bean mutants from a bean mutation breeding programme, based on heterotic groupings. This was achieved by genotyping 16 bean genotypes, using 21 Simple Sequence Repeats (SSR) bean markers. From the results, three different clusters A, B and C, were obtained suggesting ...

  2. Insulator dysfunction and oncogene activation in IDH mutant gliomas.

    Science.gov (United States)

    Flavahan, William A; Drier, Yotam; Liau, Brian B; Gillespie, Shawn M; Venteicher, Andrew S; Stemmer-Rachamimov, Anat O; Suvà, Mario L; Bernstein, Bradley E

    2016-01-07

    Gain-of-function IDH mutations are initiating events that define major clinical and prognostic classes of gliomas. Mutant IDH protein produces a new onco-metabolite, 2-hydroxyglutarate, which interferes with iron-dependent hydroxylases, including the TET family of 5'-methylcytosine hydroxylases. TET enzymes catalyse a key step in the removal of DNA methylation. IDH mutant gliomas thus manifest a CpG island methylator phenotype (G-CIMP), although the functional importance of this altered epigenetic state remains unclear. Here we show that human IDH mutant gliomas exhibit hypermethylation at cohesin and CCCTC-binding factor (CTCF)-binding sites, compromising binding of this methylation-sensitive insulator protein. Reduced CTCF binding is associated with loss of insulation between topological domains and aberrant gene activation. We specifically demonstrate that loss of CTCF at a domain boundary permits a constitutive enhancer to interact aberrantly with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene. Treatment of IDH mutant gliomaspheres with a demethylating agent partially restores insulator function and downregulates PDGFRA. Conversely, CRISPR-mediated disruption of the CTCF motif in IDH wild-type gliomaspheres upregulates PDGFRA and increases proliferation. Our study suggests that IDH mutations promote gliomagenesis by disrupting chromosomal topology and allowing aberrant regulatory interactions that induce oncogene expression.

  3. Isolation and characterization of Escherichia coli mutants lacking inducible cyanase.

    Science.gov (United States)

    Guilloton, M; Karst, F

    1987-03-01

    To determine the physiological role of cyanate aminohydrolase (cyanase, EC 3.5.5.3) in bacteria, mutants of Escherichia coli K12 devoid of this inducible activity were isolated and their properties investigated. Five independent mutations were localized next to lac; three of them lay between lacY and codA. Thus cyanase activity could depend on the integrity of one gene or set of clustered genes; we propose for this locus the symbol cnt. Growth of the mutant stains was more sensitive to cyanate than growth of wild-type strains. This difference was noticeable in synthetic medium in the presence of low concentrations of cyanate (less than or equal to 1 mM). Higher concentrations inhibited growth of both wild-type and mutant strains. Urea in aqueous solutions dissociates slowly into ammonium cyanate. Accordingly wild-type strains were able to grow on a synthetic medium containing 0.5 M-urea whereas mutants lacking cyanase were not. We conclude that cyanase could play a role in destroying exogenous cyanate originating from the dissociation of carbamoyl compounds such as urea; alternatively cyanate might constitute a convenient nitrogen source for bacteria able to synthesize cyanase in an inducible way.

  4. Development and evaluation of drought resistant mutant germ ...

    African Journals Online (AJOL)

    terms of relative water content, free proline concentration and yield. The yield ... are suitable for boiling and canning. .... definition the permanent wilting point is the soil water content at which plants do not recover turgor overnight, but will recover if water is applied. Selected mutant and control plants were planted in pots in a.

  5. Complementation of sweet corn mutants: a method for grouping ...

    Indian Academy of Sciences (India)

    accumulate sugars at the expense of starch and have low total carbohydrate at the mature kernel stage (Boyer and. Shannon 1984). At 18–21 days after pollination (harvest stage of sweet corn), these mutants have four to eight times higher total sugar than the normal corn (Holder et al. 1974). Due to comparative high sugar ...

  6. A dwarf wheat mutant is associated with increased drought ...

    African Journals Online (AJOL)

    Owner

    'Green revolution' genes encode mutant gibberellin response modulators. Nature 400 (6741):. 256-61. Zhang et al. 1057. Peng J, Carol P, Richards DE, King KE, Cowling RJ, Murphy GP,. Harberd NP (1997). The Arabidopsis GAI gene defines a signaling pathway that negatively regulates gibberellin responses Genes Dev.

  7. Let-7 Sensitizes KRAS Mutant Tumor Cells to Chemotherapy.

    Directory of Open Access Journals (Sweden)

    Xin Dai

    Full Text Available KRAS is the most commonly mutated oncogene in human cancers and is associated with poor prognosis and drug resistance. Let-7 is a family of tumor suppressor microRNAs that are frequently suppressed in solid tumors, where KRAS mutations are highly prevalent. In this study, we investigated the potential use of let-7 as a chemosensitizer. We found that let-7b repletion selectively sensitized KRAS mutant tumor cells to the cytotoxicity of paclitaxel and gemcitabine. Transfection of let-7b mimic downregulated the expression of mutant but not wild-type KRAS. Combination of let-7b mimic with paclitaxel or gemcitabine diminished MEK/ERK and PI3K/AKT signaling concurrently, triggered the onset of apoptosis, and reverted the epithelial-mesenchymal transition in KRAS mutant tumor cells. In addition, let-7b repletion downregulated the expression of β-tubulin III and ribonucleotide reductase subunit M2, two proteins known to mediate tumor resistance to paclitaxel and gemcitabine, respectively. Let-7 may represent a new class of chemosensitizer for the treatment of KRAS mutant tumors.

  8. Enhanced sporulation and toxin production by a mutant derivative of ...

    African Journals Online (AJOL)

    fatima

    based medium. Maximum spore and crystal proteins were produced at 40°C with corn steep liquor as nitrogen source and hydrol as a carbon source. The best mutant MUV7 supported .... containing 10 L of culture medium as described earlier (Ghribi et al.,. 2004). ..... Saccharomyces cerevisiae ITV strain (Ortiz-Muniz et al.,.

  9. nitrosoguanidine-induced cadmium resistant mutants of Aspergillus ...

    Indian Academy of Sciences (India)

    Unknown

    of S. cerevisiae (Inouhe et al 1989) were used for under- standing the molecular genetics of cadmium toxicity and ... The pH of the medium was adjusted to 6⋅4 before autoclaving. Cadmium-resistant mutants after isolation ..... Saccharomyces cerevisiae; Biochem. Biophys. Acta 993 51–55. Kimura M, Otaki N and Imano M ...

  10. Development of a mutant strain of Bacillus subtilis showing ...

    African Journals Online (AJOL)

    Through fermentation experiments, it was confirmed that the mutant strain, TH-49, was not capable of using acetoin accumulated in broth as its energy sources for growth after glucose was consumed. This phenomenon was inconsistent with that the majorities of bacteria accumulate acetoin as stored energy sources and ...

  11. Transcriptional Analysis of serk1 and serk3 coreceptor mutants

    NARCIS (Netherlands)

    Esse, van Wilma; Hove, ten Colette A.; Guzzonato, Francesco; Esse, van Peter; Boekschoten, Mark; Ridder, Lars; Vervoort, Jacques; Vries, de Sacco C.

    2016-01-01

    Somatic embryogenesis receptor kinases (SERKs) are ligand-binding coreceptors that are able to combine with different ligandperceiving receptors such as BRASSINOSTEROID INSENSITIVE1 (BRI1) and FLAGELLIN-SENSITIVE2. Phenotypical analysis of serk single mutants is not straightforward because

  12. Screening of allyl alcohol resistant mutant of Rhizopus oryzae and ...

    African Journals Online (AJOL)

    Ethanol is a main by-product in the fermentation broth of Rhizopus oryzae during the production of high-optical purity L-lactic acid. By screening the lower activity of alcohol dehydrogenase (ADH) mutant, thus decreasing the flux of pyruvic acid to ethanol may be a virtual method for increasing the conversion rate of glucose ...

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

    Science.gov (United States)

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

    2012-07-15

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

  14. A dwarf wheat mutant is associated with increased drought ...

    African Journals Online (AJOL)

    ... was significantly higher than Jingdong 6. Most of the s-dwarf seedlings survived in recovering experiement after water loss. The stalk of s-dwarf seedling also showed reduced gravitropism. This is the first report about a new dwarf wheat mutant associated with increased drought resistance and altered stalk gravitropism.

  15. Ultradian rhythm unmasked in the Pdf clock mutant of Drosophila

    Indian Academy of Sciences (India)

    2014-07-20

    Jul 20, 2014 ... from the mutation. As a result, Pdf01 mutant flies locomote with precise rhythmicity generated by one ultradian oscilla- tor. In future studies, we would like to dissect this system using genetic tools available in Drosophila, for example by silencing a particular set of Pdf-positive neurons. Circadian rhythms are ...

  16. Dynamics of mutant cells in hierarchical organized tissues.

    Directory of Open Access Journals (Sweden)

    Benjamin Werner

    2011-12-01

    Full Text Available Most tissues in multicellular organisms are maintained by continuous cell renewal processes. However, high turnover of many cells implies a large number of error-prone cell divisions. Hierarchical organized tissue structures with stem cell driven cell differentiation provide one way to prevent the accumulation of mutations, because only few stem cells are long lived. We investigate the deterministic dynamics of cells in such a hierarchical multi compartment model, where each compartment represents a certain stage of cell differentiation. The dynamics of the interacting system is described by ordinary differential equations coupled across compartments. We present analytical solutions for these equations, calculate the corresponding extinction times and compare our results to individual based stochastic simulations. Our general compartment structure can be applied to different tissues, as for example hematopoiesis, the epidermis, or colonic crypts. The solutions provide a description of the average time development of stem cell and non stem cell driven mutants and can be used to illustrate general and specific features of the dynamics of mutant cells in such hierarchically structured populations. We illustrate one possible application of this approach by discussing the origin and dynamics of PIG-A mutant clones that are found in the bloodstream of virtually every healthy adult human. From this it is apparent, that not only the occurrence of a mutant but also the compartment of origin is of importance.

  17. Siim Nestor soovitab : Mutant Disco. Azymuth. Klubis Hollywood / Siim Nestor

    Index Scriptorium Estoniae

    Nestor, Siim, 1974-

    2003-01-01

    Mutant Disco klubis Prive 4. juulil. Brasiilia jazz-trio Azmuth klubis BonBon 5. juulil. Pidustuste sarja Hip Hop Cafe sünnipäeva tähistamisest klubis Hollywood 4. juulil, üritusest Ibiza Night 5. juulil

  18. Photophysics and optical switching in green fluorescent protein mutants

    NARCIS (Netherlands)

    Creemers, T.M.H.; Lock, A.J.; Subramaniam, V.; Jovin, T.M.; Völker, S.

    2000-01-01

    We demonstrate by using low-temperature high-resolution spectroscopy that red-shifted mutants of green fluorescent protein are photo- interconverted among three conformations and are, therefore, not photostable 'one-color' systems as previously believed. From our experiments we have further derived

  19. Genetic characterization of glossy-leafed mutant broccoli lines

    Science.gov (United States)

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

  20. Correction of β-thalassemia mutant by base editor in human embryos

    Directory of Open Access Journals (Sweden)

    Puping Liang

    2017-09-01

    Full Text Available Abstract β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB −28 (A>G mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB −28 (A>G mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB −28 (A>G homozygous mutation. Data showed that base editor could precisely correct HBB −28 (A>G mutation in the patient’s primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB −28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.