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Sample records for vhl gene inactivation

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

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    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.

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

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    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.

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

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    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.

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

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    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.

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

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    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.

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

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    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

  7. VHL Manifestations

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    ... 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 ...

  8. Playing Tag with HIF: The VHL Story

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    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.

  9. 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.

  10. 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.

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

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    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.

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

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    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....

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

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    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.

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

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    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

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

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    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

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

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    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

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

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    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.

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

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    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.

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

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    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.

  20. 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...

  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. 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)

  3. 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.

  4. 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.

  5. Inactivation Effect of Antibiotic-Resistant Gene Using Chlorine Disinfection

    Directory of Open Access Journals (Sweden)

    Takashi Furukawa

    2017-07-01

    Full Text Available The aim of this study was to elucidate the inactivation effects on the antibiotic-resistance gene (vanA of vancomycin-resistant enterococci (VRE using chlorination, a disinfection method widely used in various water treatment facilities. Suspensions of VRE were prepared by adding VRE to phosphate-buffered saline, or the sterilized secondary effluent of a wastewater treatment plant. The inactivation experiments were carried out at several chlorine concentrations and stirring time. Enterococci concentration and presence of vanA were determined. The enterococci concentration decreased as chlorine concentrations and stirring times increased, with more than 7.0 log reduction occurring under the following conditions: 40 min stirring at 0.5 mg Cl2/L, 20 min stirring at 1.0 mg Cl2/L, and 3 min stirring at 3.0 mg Cl2/L. In the inactivation experiment using VRE suspended in secondary effluent, the culturable enterococci required much higher chlorine concentration and longer treatment time for complete disinfection than the cases of suspension of VRE. However, vanA was detected in all chlorinated suspensions of VRE, even in samples where no enterococcal colonies were present on the medium agar plate. The chlorine disinfection was not able to destroy antibiotic-resistance genes, though it can inactivate and decrease bacterial counts of antibiotic-resistant bacteria (ARB. Therefore, it was suggested that remaining ARB and/or antibiotic-resistance gene in inactivated bacterial cells after chlorine disinfection tank could be discharged into water environments.

  6. Somatic mutation analysis of the SDHB, SDHC, SDHD, and RET genes in the clinical assessment of sporadic and hereditary pheochromocytoma.

    Science.gov (United States)

    Weber, Alexander; Hoffmann, Michael M; Neumann, Hartmut P H; Erlic, Zoran

    2012-08-01

    Systemic analysis of somatic mutations of other susceptibility genes in syndromic tumors as well as apparently sporadic tumors in well-characterized specimens is lacking. Its clinical relevance has not been studied. Our objective was to determine the frequency of second allele inactivation in syndromic tumors and determine the frequency and potential clinical impact of somatic mutations and loss of heterozygosity (LOH) of the known susceptibility genes in syndromic and sporadic tumors. Nine tumor specimens from clinically characterized VHL mutation, five from SDHB mutation, four from SDHD mutation, two from RET mutation carriers, and eight from apparently sporadic cases were analyzed. Tumor DNA mutation screening of the SDHx, VHL, and RET genes and LOH analyses of the SDHx and VHL genes were performed. The Yates-corrected chi-squared test was used for comparison of the clinical data and the molecular-genetic results. Second allele inactivation in tumors was identified in 83% of VHL, 80% of SDHB, and 50% of SDHD specimen. High prevalence of VHL (6/6, p=0.024) and SDHB (7/7, p=0.018) somatic mutations has been identified in the sporadic group compared to all others. In the group of the VHL tumors the SDHB somatic events were significantly lower (2/6; p=0.045). In 18/19 (95%) of cases, we were able to demonstrate the presence of at least two concomitant affected susceptibility genes. We conclude that LOH is the most prevalent second allele-inactivating event. SDHB and VHL somatic mutation might play a role in the sporadic forms of tumor development. There is no clinical impact of mutation screening or LOH analysis of tumor specimens.

  7. 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.

  8. 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

  9. 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

  10. 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...

  11. 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.

  12. 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.

  13. 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

  14. Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells

    National Research Council Canada - National Science Library

    Olaf Voets; Frans Tielen; Edo Elstak; Julian Benschop; Max Grimbergen; Jan Stallen; Richard Janssen; Andre van Marle; Christian Essrich

    2017-01-01

    .... Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines...

  15. Metazoan Ribosome Inactivating Protein encoding genes acquired by Horizontal Gene Transfer.

    Science.gov (United States)

    Lapadula, Walter J; Marcet, Paula L; Mascotti, María L; Sanchez-Puerta, M Virginia; Juri Ayub, Maximiliano

    2017-05-12

    Ribosome inactivating proteins (RIPs) are RNA N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of 28S rRNA. These enzymes are widely distributed among plants and their presence has also been confirmed in several bacterial species. Recently, we reported for the first time in silico evidence of RIP encoding genes in metazoans, in two closely related species of insects: Aedes aegypti and Culex quinquefasciatus. Here, we have experimentally confirmed the presence of these genes in mosquitoes and attempted to unveil their evolutionary history. A detailed study was conducted, including evaluation of taxonomic distribution, phylogenetic inferences and microsynteny analyses, indicating that mosquito RIP genes derived from a single Horizontal Gene Transfer (HGT) event, probably from a cyanobacterial donor species. Moreover, evolutionary analyses show that, after the HGT event, these genes evolved under purifying selection, strongly suggesting they play functional roles in these organisms.

  16. 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...

  17. 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

  18. 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.

  19. 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

  20. 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...

  1. CHM gene molecular analysis and X-chromosome inactivation pattern determination in two families with choroideremia.

    Science.gov (United States)

    Perez-Cano, Hector J; Garnica-Hayashi, Rosa E; Zenteno, Juan C

    2009-10-01

    Choroideremia is an X-linked recessive retinal dystrophy characterized by progressive loss of the photoreceptor, the retinal pigment epithelium, and the choriocapillaris layers which ultimately can result in blindness by the fifth decade of life. The disease is caused by mutations in the gene CHM, which encodes a protein involved in the regulation of intracellular vesicular traffic. Typically, hemizygous males are affected by the disease and female carriers are asymptomatic with only a diffuse mottled pattern of hyperpigmentation on funduscopy. Uncommon instances of fully affected females have been described previously and these cases are proposed to arise from an skewed Lyonization mechanism preferentially inactivating the X chromosome carrying the normal CHM allele. In this work, the clinical and molecular features of two Mexican families with choroideremia are described. A novel and a previously described CHM mutation were identified. X-chromosome inactivation assays were performed in a total of 12 heterozygous carriers from the two families. In an affected female from family A, a random X-inactivation pattern was demonstrated; on the other hand, in a female carrier from family B displaying a conspicuous pattern of pigment epithelium mottling at the peripheral retina, a skewed X-inactivation pattern was found. However, the X-chromosome preferentially inactivated in this female was the one carrying the mutated allele. Our results add to the genotypic spectrum in choroideremia and does not support a correlation between X-inactivation status and abnormal retinal phenotype in heterozygous female carriers from these two families.

  2. Inactivation of RAD52 and HDF1 DNA repair genes leads to ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Biosciences; Volume 42; Issue 2. Inactivation of RAD52 and HDF1 DNA repair genes leads to premature chronological aging and cellular instability. SILVIA MERCADO-SÁENZ BEATRIZ LÓPEZ-DÍAZ FRANCISCO SENDRA-PORTERO MANUEL MARTÍNEZ-MORILLO MIGUEL J RUIZ-GÓMEZ.

  3. Epigenetic inactivation of tumour suppressor coding and non-coding genes in human cancer: an update.

    Science.gov (United States)

    Llinàs-Arias, Pere; Esteller, Manel

    2017-09-01

    Cancer cells undergo many different alterations during their transformation, including genetic and epigenetic events. The controlled division of healthy cells can be impaired through the downregulation of tumour suppressor genes. Here, we provide an update of the mechanisms in which epigenetically altered coding and non-coding tumour suppressor genes are implicated. We will highlight the importance of epigenetics in the different molecular pathways that lead to enhanced and unlimited capacity of division, genomic instability, metabolic shift, acquisition of mesenchymal features that lead to metastasis, and tumour plasticity. We will briefly describe these pathways, focusing especially on genes whose epigenetic inactivation through DNA methylation has been recently described, as well as on those that are well established as being epigenetically silenced in cancer. A brief perspective of current clinical therapeutic approaches that can revert epigenetic inactivation of non-coding tumour suppressor genes will also be given. © 2017 The Authors.

  4. Gene inactivation by CRISPR-Cas9 in Nicotiana tabacum BY-2 suspension cells

    Directory of Open Access Journals (Sweden)

    Sébastien eMercx

    2016-02-01

    Full Text Available Plant suspension cells are interesting hosts for the heterologous production of pharmacological proteins such as antibodies. They have the advantage to facilitate the containment and the application of good manufacturing practices (GMPs. Furthermore, antibodies can be secreted to the extracellular medium, which makes the purification steps much simpler. However, improvements are still to be made regarding the quality and the production yield. For instance, the inactivation of proteases and the humanization of glycosylation are both important targets which require either gene silencing or gene inactivation. To this purpose, CRISPR-Cas9 is a very promising technique which has been used recently in a series of plant species, but not yet in plant suspension cells. Here, we sought to use the CRISPR-Cas9 system for gene inactivation in Nicotiana tabacum BY-2 suspension cells. We transformed a transgenic line expressing a red fluorescent protein (mCherry with a binary vector containing genes coding for Cas9 and three guide RNAs targeting mCherry restriction sites, as well as a bialaphos-resistant (bar gene for selection. To demonstrate gene inactivation in the transgenic lines, the mCherry gene was PCR-amplified and analyzed by electrophoresis. Seven out of 20 transformants displayed a shortened fragment, indicating that a deletion occurred between two target sites. We also analyzed the transformants by restriction fragment length polymorphism and observed that the three targeted restriction sites were hit. DNA sequencing of the PCR fragments confirmed either deletion between two target sites or single nucleotide deletion. We therefore conclude that CRISPR-Cas9 can be used in N. tabacum BY2 cells.

  5. Promoter hypermethylation-mediated inactivation of multiple Slit-Robo pathway genes in cervical cancer progression

    Directory of Open Access Journals (Sweden)

    Mansukhani Mahesh

    2006-05-01

    Full Text Available Abstract Background Cervical Cancer (CC exhibits highly complex genomic alterations. These include hemizygous deletions at 4p15.3, 10q24, 5q35, 3p12.3, and 11q24, the chromosomal sites of Slit-Robo pathway genes. However, no candidate tumor suppressor genes at these regions have been identified so far. Slit family of secreted proteins modulates chemokine-induced cell migration of distinct somatic cell types. Slit genes mediate their effect by binding to its receptor Roundabout (Robo. These genes have shown to be inactivated by promoter hypermethylation in a number of human cancers. Results To test whether Slit-Robo pathway genes are targets of inactivation at these sites of deletion, we examined promoter hypermethylation of SLIT1, SLIT2, SLIT3, ROBO1, and ROBO3 genes in invasive CC and its precursor lesions. We identified a high frequency of promoter hypermethylation in all the Slit-Robo genes resulting in down regulated gene expression in invasive CC, but the inhibitors of DNA methylation and histone deacetylases (HDACs in CC cell lines failed to effectively reactivate the down-regulated expression. These results suggest a complex mechanism of inactivation in the Slit-Robo pathway in CC. By analysis of cervical precancerous lesions, we further show that promoter hypermethylation of Slit-Robo pathway occurs early in tumor progression. Conclusion Taken together, these findings suggest that epigenetic alterations of Slit-Robo pathway genes (i play a role in CC development, (ii further delineation of molecular basis of promoter methylation-mediated gene regulation provides a potential basis for epigenetic-based therapy in advanced stage CC, and (iii form epigenetic signatures to identify precancerous lesions at risk to progression.

  6. Promoter hypermethylation-mediated inactivation of multiple Slit-Robo pathway genes in cervical cancer progression.

    Science.gov (United States)

    Narayan, Gopeshwar; Goparaju, Chandra; Arias-Pulido, Hugo; Kaufmann, Andreas M; Schneider, Achim; Dürst, Matthias; Mansukhani, Mahesh; Pothuri, Bhavana; Murty, Vundavalli V

    2006-05-15

    Cervical Cancer (CC) exhibits highly complex genomic alterations. These include hemizygous deletions at 4p15.3, 10q24, 5q35, 3p12.3, and 11q24, the chromosomal sites of Slit-Robo pathway genes. However, no candidate tumor suppressor genes at these regions have been identified so far. Slit family of secreted proteins modulates chemokine-induced cell migration of distinct somatic cell types. Slit genes mediate their effect by binding to its receptor Roundabout (Robo). These genes have shown to be inactivated by promoter hypermethylation in a number of human cancers. To test whether Slit-Robo pathway genes are targets of inactivation at these sites of deletion, we examined promoter hypermethylation of SLIT1, SLIT2, SLIT3, ROBO1, and ROBO3 genes in invasive CC and its precursor lesions. We identified a high frequency of promoter hypermethylation in all the Slit-Robo genes resulting in down regulated gene expression in invasive CC, but the inhibitors of DNA methylation and histone deacetylases (HDACs) in CC cell lines failed to effectively reactivate the down-regulated expression. These results suggest a complex mechanism of inactivation in the Slit-Robo pathway in CC. By analysis of cervical precancerous lesions, we further show that promoter hypermethylation of Slit-Robo pathway occurs early in tumor progression. Taken together, these findings suggest that epigenetic alterations of Slit-Robo pathway genes (i) play a role in CC development, (ii) further delineation of molecular basis of promoter methylation-mediated gene regulation provides a potential basis for epigenetic-based therapy in advanced stage CC, and (iii) form epigenetic signatures to identify precancerous lesions at risk to progression.

  7. 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.

  8. Reversible Gene Drive Mechanism Utilizing Trana Inactivating Paramutatlons In Insects (paramutale 0.9)

    Energy Technology Data Exchange (ETDEWEB)

    2017-09-06

    The paramutate software package Is a tool to calculate the genotyplc and phenotyplc propagation of a gene drive that can be silenced with a homologuus trans-Inactivating panmutatlon, ln dlptera or other species with a slmUar-acttng pl RNA system/Plwi pathway. Method of SolaUon: paramutate uses rults of Mendelian, gene drive (I.e.. stimulated conversion), and maternal Inheritance to compute the propaptlon of a notional gene drive construct and Its trans-lnactlvat1n1 paramutatlon, throu1b a panmlctlc, fixed-size population reproducing In synchronous generations.

  9. Inactivation of the olfactory marker protein (OMP) gene in river dolphins and other odontocete cetaceans.

    Science.gov (United States)

    Springer, Mark S; Gatesy, John

    2017-04-01

    Various toothed whales (Odontoceti) are unique among mammals in lacking olfactory bulbs as adults and are thought to be anosmic (lacking the olfactory sense). At the molecular level, toothed whales have high percentages of pseudogenic olfactory receptor genes, but species that have been investigated to date retain an intact copy of the olfactory marker protein gene (OMP), which is highly expressed in olfactory receptor neurons and may regulate the temporal resolution of olfactory responses. One hypothesis for the retention of intact OMP in diverse odontocete lineages is that this gene is pleiotropic with additional functions that are unrelated to olfaction. Recent expression studies provide some support for this hypothesis. Here, we report OMP sequences for representatives of all extant cetacean families and provide the first molecular evidence for inactivation of this gene in vertebrates. Specifically, OMP exhibits independent inactivating mutations in six different odontocete lineages: four river dolphin genera (Platanista, Lipotes, Pontoporia, Inia), sperm whale (Physeter), and harbor porpoise (Phocoena). These results suggest that the only essential role of OMP that is maintained by natural selection is in olfaction, although a non-olfactory role for OMP cannot be ruled out for lineages that retain an intact copy of this gene. Available genome sequences from cetaceans and close outgroups provide evidence of inactivating mutations in two additional genes (CNGA2, CNGA4), which imply further pseudogenization events in the olfactory cascade of odontocetes. Selection analyses demonstrate that evolutionary constraints on all three genes (OMP, CNGA2, CNGA4) have been greatly reduced in Odontoceti, but retain a signature of purifying selection on the stem Cetacea branch and in Mysticeti (baleen whales). This pattern is compatible with the 'echolocation-priority' hypothesis for the evolution of OMP, which posits that negative selection was maintained in the common

  10. 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.

  11. Chromosomal gene inactivation in the green sulfur bacterium Chlorobium tepidum by natural transformation

    DEFF Research Database (Denmark)

    Frigaard, N-U; Bryant, D A

    2001-01-01

    Conditions for inactivating chromosomal genes of Chlorobium tepidum by natural transformation and homologous recombination were established. As a model, mutants unable to perform nitrogen fixation were constructed by interrupting nifD with various antibiotic resistance markers. Growth of wild...

  12. Inactivation of the retinoblastoma gene yields a mouse model of malignant colorectal cancer.

    Science.gov (United States)

    Parisi, T; Bronson, R T; Lees, J A

    2015-11-26

    The retinoblastoma gene (Rb) is mutated at significant frequency in various human epithelial tumors, including colorectal cancer, and is strongly associated with metastatic disease. However, sole inactivation of Rb in the mouse has so far failed to yield epithelial cancers. Here, we specifically inactivate Rb and/or p53 in the urogenital epithelium and the intestine. We find that the loss of both tumor suppressors is unable to yield tumors in the transitional epithelium lining the bladder, kidneys and ureters. Instead, these mice develop highly metastatic tumors of neuroendocrine, not epithelial, origin within the urogenital tract to give prostate cancer in the males and vaginal tumors in the females. Additionally, we discovered that the sole inactivation of Rb in the intestine was sufficient to induce formation of metastatic colorectal adenocarcinomas. These tumors closely mirror the human disease in regard to the age of onset, histological appearance, invasiveness and metastatic potential. Like most human colorectal carcinomas, our murine Rb-deficient tumors demonstrate genomic instability and they show activation of β-catenin. Deregulation of the Wnt/β-catenin pathway is specific to the intestinal tumors, as genomic instability but not activation of β-catenin was observed in the neuroendocrine tumors. To date, attempts to generate genetically engineered mouse models of colorectal cancer tumors have yielded mostly cancer of the small intestine, which rarely occurs in humans. Our system provides the opportunity to accurately model and study colorectal cancer in the mouse via a single gene mutation.

  13. The Self-Inactivating KamiCas9 System for the Editing of CNS Disease Genes.

    Science.gov (United States)

    Merienne, Nicolas; Vachey, Gabriel; de Longprez, Lucie; Meunier, Cécile; Zimmer, Virginie; Perriard, Guillaume; Canales, Mathieu; Mathias, Amandine; Herrgott, Lucas; Beltraminelli, Tim; Maulet, Axelle; Dequesne, Thomas; Pythoud, Catherine; Rey, Maria; Pellerin, Luc; Brouillet, Emmanuel; Perrier, Anselme L; du Pasquier, Renaud; Déglon, Nicole

    2017-09-19

    Neurodegenerative disorders are a major public health problem because of the high frequency of these diseases. Genome editing with the CRISPR/Cas9 system is making it possible to modify the sequence of genes linked to these disorders. We designed the KamiCas9 self-inactivating editing system to achieve transient expression of the Cas9 protein and high editing efficiency. In the first application, the gene responsible for Huntington's disease (HD) was targeted in adult mouse neuronal and glial cells. Mutant huntingtin (HTT) was efficiently inactivated in mouse models of HD, leading to an improvement in key markers of the disease. Sequencing of potential off-targets with the constitutive Cas9 system in differentiated human iPSC revealed a very low incidence with only one site above background level. This off-target frequency was significantly reduced with the KamiCas9 system. These results demonstrate the potential of the self-inactivating CRISPR/Cas9 editing for applications in the context of neurodegenerative diseases. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  14. Inactivation of Stigmatella aurantiaca CsgA gene impares rippling formation

    Directory of Open Access Journals (Sweden)

    Milosevic-Đeric Ana

    2015-01-01

    Full Text Available Stigmatella aurantiaca fruiting body development depends on cell-cell interactions. One type of the signaling molecule stigmolone isolated from S. aurantiaca cells acts to help cells to stay together in the aggregation phase. Another gene product involved in intercellular signaling in S. aurantiaca is the csgA homolog of Myxococcus xanthus. In close relative M. xanthus C signal the product of the csgA gene is required for rippling, aggregation and sporulation. Isolation of homologous gene in S. aurantiaca implicates a probable role of CsgA in intercellular communication. Inactivation of the gene by insertion mutagenesis caused alterations in S. aurantiaca fruiting. The motility behavior of the cells during development was changed as well as their ability to stay more closely together in the early stages of development. Inactivation of the csgA gene completely abolished rippling of the cells. This indicates the crucial role of the CsgA protein in regulating this rhythmic behavior.

  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. Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells.

    Science.gov (United States)

    Voets, Olaf; Tielen, Frans; Elstak, Edo; Benschop, Julian; Grimbergen, Max; Stallen, Jan; Janssen, Richard; van Marle, Andre; Essrich, Christian

    2017-01-01

    Phenotypic assays using human primary cells are highly valuable tools for target discovery and validation in drug discovery. Expression knockdown (KD) of such targets in these assays allows the investigation of their role in models of disease processes. Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines. Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient gene inactivation in two human primary cell types, normal human lung fibroblasts and human bronchial epithelial cells. The effects of gene inactivation were studied in the TGF-β-induced fibroblast to myofibroblast transition assay (FMT) and the epithelial to mesenchymal transition assay (EMT), which are SMAD3 dependent and reflect pathogenic mechanisms observed in fibrosis. Co-transduction (co-TD) of AdV Cas9 with SMAD3-targeting guide RNAs (gRNAs) resulted in fast and efficient genome editing judged by insertion/deletion (indel) formation, as well as significant reduction of SMAD3 protein expression and nuclear translocation. This led to phenotypic changes downstream of SMAD3 inhibition, including substantially decreased alpha smooth muscle actin and fibronectin 1 expression, which are markers for FMT and EMT, respectively. A direct comparison between co-TD of separate Cas9 and gRNA AdV, versus TD with a single "all-in-one" Cas9/gRNA AdV, revealed that both methods achieve similar levels of indel formation. These data demonstrate that AdV CRISPR/Cas9 is a useful and efficient tool for protein KD in human primary cell phenotypic assays. The use of AdV CRISPR/Cas9 may offer significant advantages over the current existing tools and should enhance target discovery and validation opportunities.

  17. Highly efficient gene inactivation by adenoviral CRISPR/Cas9 in human primary cells

    Science.gov (United States)

    Tielen, Frans; Elstak, Edo; Benschop, Julian; Grimbergen, Max; Stallen, Jan; Janssen, Richard; van Marle, Andre; Essrich, Christian

    2017-01-01

    Phenotypic assays using human primary cells are highly valuable tools for target discovery and validation in drug discovery. Expression knockdown (KD) of such targets in these assays allows the investigation of their role in models of disease processes. Therefore, efficient and fast modes of protein KD in phenotypic assays are required. The CRISPR/Cas9 system has been shown to be a versatile and efficient means of gene inactivation in immortalized cell lines. Here we describe the use of adenoviral (AdV) CRISPR/Cas9 vectors for efficient gene inactivation in two human primary cell types, normal human lung fibroblasts and human bronchial epithelial cells. The effects of gene inactivation were studied in the TGF-β-induced fibroblast to myofibroblast transition assay (FMT) and the epithelial to mesenchymal transition assay (EMT), which are SMAD3 dependent and reflect pathogenic mechanisms observed in fibrosis. Co-transduction (co-TD) of AdV Cas9 with SMAD3-targeting guide RNAs (gRNAs) resulted in fast and efficient genome editing judged by insertion/deletion (indel) formation, as well as significant reduction of SMAD3 protein expression and nuclear translocation. This led to phenotypic changes downstream of SMAD3 inhibition, including substantially decreased alpha smooth muscle actin and fibronectin 1 expression, which are markers for FMT and EMT, respectively. A direct comparison between co-TD of separate Cas9 and gRNA AdV, versus TD with a single “all-in-one” Cas9/gRNA AdV, revealed that both methods achieve similar levels of indel formation. These data demonstrate that AdV CRISPR/Cas9 is a useful and efficient tool for protein KD in human primary cell phenotypic assays. The use of AdV CRISPR/Cas9 may offer significant advantages over the current existing tools and should enhance target discovery and validation opportunities. PMID:28800587

  18. 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....

  19. Genetic Inactivation of COPI Coatomer Separately Inhibits Vesicular Stomatitis Virus Entry and Gene Expression

    Science.gov (United States)

    Burdeinick-Kerr, Rebeca

    2012-01-01

    Viruses coopt cellular membrane transport to invade cells, establish intracellular sites of replication, and release progeny virions. Recent genome-wide RNA interference (RNAi) screens revealed that genetically divergent viruses require biosynthetic membrane transport by the COPI coatomer complex for efficient replication. Here we found that disrupting COPI function by RNAi inhibited an early stage of vesicular stomatitis virus (VSV) replication. To dissect which replication stage(s) was affected by coatomer inactivation, we used visual and biochemical assays to independently measure the efficiency of viral entry and gene expression in hamster (ldlF) cells depleted of the temperature-sensitive ε-COP subunit. We show that ε-COP depletion for 12 h caused a primary block to virus internalization and a secondary defect in viral gene expression. Using brefeldin A (BFA), a chemical inhibitor of COPI function, we demonstrate that short-term (1-h) BFA treatments inhibit VSV gene expression, while only long-term (12-h) treatments block virus entry. We conclude that prolonged coatomer inactivation perturbs cellular endocytic transport and thereby indirectly impairs VSV entry. Our results offer an explanation of why COPI coatomer is frequently identified in screens for cellular factors that support cell invasion by microbial pathogens. PMID:22072764

  20. X chromosome inactivation: new players in the initiation of gene silencing [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Ines Pinheiro

    2017-03-01

    Full Text Available X chromosome inactivation (XCI is a dosage compensation process that was adopted by female mammals to balance gene dosage between XX females and XY males. XCI starts with the upregulation of the non-coding RNA Xist, after which most X-linked genes are silenced and acquire a repressive chromatin state. Even though the chromatin marks of the inactive X have been fairly well described, the mechanisms responsible for the initiation of XCI remain largely unknown. In this review, we discuss recent developments that revealed unexpected factors playing a role in XCI and that might be of crucial importance to understand the mechanisms responsible for the very first steps of this chromosome-wide gene-silencing event.

  1. Revising the taxonomic distribution, origin and evolution of ribosome inactivating protein genes.

    Directory of Open Access Journals (Sweden)

    Walter J Lapadula

    Full Text Available Ribosome inactivating proteins are enzymes that depurinate a specific adenine residue in the alpha-sarcin-ricin loop of the large ribosomal RNA, being ricin and Shiga toxins the most renowned examples. They are widely distributed in plants and their presence has also been confirmed in a few bacterial species. According to this taxonomic distribution, the current model about the origin and evolution of RIP genes postulates that an ancestral RIP domain was originated in flowering plants, and later acquired by some bacteria via horizontal gene transfer. Here, we unequivocally detected the presence of RIP genes in fungi and metazoa. These findings, along with sequence and phylogenetic analyses, led us to propose an alternative, more parsimonious, hypothesis about the origin and evolutionary history of the RIP domain, where several paralogous RIP genes were already present before the three domains of life evolved. This model is in agreement with the current idea of the Last Universal Common Ancestor (LUCA as a complex, genetically redundant organism. Differential loss of paralogous genes in descendants of LUCA, rather than multiple horizontal gene transfer events, could account for the complex pattern of RIP genes across extant species, as it has been observed for other genes.

  2. Sexy transgenes: the impact of gene transfer and gene inactivation technologies on the understanding of mammalian sex determination.

    Science.gov (United States)

    Vaiman, Daniel

    2003-06-01

    Amongst the various developmental pathways ending in a sound mammal, sex determination presents the peculiarity of a choice between two equally viable options: female or male. Therefore, destroying a 'male-determining gene' or a 'female-determining gene' should generally not be lethal. Genetic sex determination is divided into two consecutive steps: construction of the bipotential gonad, and then sex determination per se. The genes involved in the first step are in fact involved in the development of various body compartments, and their mutation is generally far from innocuous. From transgenic and inactivation studies carried out on the laboratory mouse, a complete picture of the two steps is beginning to emerge, where the gonad itself and the necessary ducts are shown to evolve in a very coordinate way, with well-defined sex-specificities. Compared with testis determination, the ovarian side of the picture is still relatively empty, but this situation can change rapidly as candidate ovarian genes for inactivation studies are beginning to be identified.

  3. Re-expression of an inactivated variable surface glycoprotein gene in Trypanosoma equiperdum.

    Science.gov (United States)

    Buck, G A; Jacquemot, C; Baltz, T; Eisen, H

    1984-12-01

    Variable surface glycoprotein (VSG) genes in African trypanosomes are often activated by the duplicative transposition of a silent basic copy (BC) gene into an unlinked telomerically located expression site, producing an active expression-linked copy (ELC) of that gene. However, some BC genes that are already linked to a telomere are activated without apparent duplication or transposition. We have recently shown that an active VSG ELC can be inactivated in situ, apparently without rearrangement. To explain these observations it has been suggested that VSG genes that are associated with chromosome telomeres are activated by chromosome end exchanges that occur at a considerable distance upstream from the genes themselves and place them cis to a unique VSG expression element. In an attempt to test this model we derived five VSG-1 expressing variants from BoTat-2, a VSG-2 expressing variant of Trypanosoma equiperdum which carries an inactive residual VSG-1 ELC (R-ELC) as well as the active VSG-2 ELC near unlinked chromosome telomeres. We examined the fates of the VSG-2 ELC and the VSG-1 R-ELC in these variants. All five had maintained the VSG-1 R-ELC; three in a reactivated form and two in an inactive state. The latter two variants carried new, active VSG-1 ELCs: one in the site that had previously contained the VSG-2 ELC and one in a previously unidentified site. The VSG-2 ELC was lost in all five of the variants. The results are not consistent with the simple chromosome end exchange model, which predicts that the VSG-2 ELC would be inactivated but not deleted when the VSG-1 R-ELC was reactivated.

  4. 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...

  5. An X-linked homologue of the autosomal inprinted gene ZNF127 escapes X inactivation

    Energy Technology Data Exchange (ETDEWEB)

    Longstreet, M.; Nicholls, R.D.; Willard, H.F. [Case Western Reserve Univ., Cleveland, OH (United States)] [and others

    1994-09-01

    The ZNF127 gene has been shown to be subject to parental imprinting in both humans and the mouse and maps to within the Prader-Willi/Angelman Syndrome critical region on chromosome 15. We have cloned two X-linked related loci, one of which, ZNFXp is a transcribed gene while the other, ZNFXq, is an untranscribed pseudogene. ZNFXp is 83.6% identical to ZNFXq and 65.4% identical to ZNF127 over 1.4 kb of open reading frame they share in common, Like ZNF127, the predicted protein sequence of ZNFXp contains a C{sub 3}HC{sub 4} zinc finger domain and C{sub 3}H zinc finger-like motifs. Whereas ZNF127 has three C{sub 3}H motifs, ZNFXp has four. A strong CpG island is located within 1 kb 5{prime} of the predicted amino terminus of ZNFXp. Expression of ZNFXp has been detected from mouse/human somatic cell hybrids containing either an active (n=2) or an inactive (n=4) chromosome, and thus escapes X inactivation. Probes made from the 3{prime} UTR of ZNFXp detect a number of related loci in both human and murine DNA, none of which is the ZNF127 locus on chromosome 15. None of the detectable murine bands shows dosage differences between males and females as would be expected for X-linked loci. This raises the possibility that ZNFXp inserted into the human X chromosome after its divergence from a common ancestor with the murine X. We have mapped ZNFXp to Xp11.4 by Southern blotting and PCR of hybrid DNAs and by fluorescence in situ hybridization (FISH). ZNFXq maps within the X Inactivation Center (XIC) region on Xq13.2, approximately 300 kb distal to the XIST gene. We find it intriguing, and perhaps significant, that two members of this gene family are subject to epigenetic regulation -- one autosomal imprinting, and the other escape from X inactivation. These results could imply an evolutionary and mechanistic relationship between these two processes.

  6. Escape of X-linked miRNA genes from meiotic sex chromosome inactivation.

    Science.gov (United States)

    Sosa, Enrique; Flores, Luis; Yan, Wei; McCarrey, John R

    2015-11-01

    Past studies have indicated that transcription of all X-linked genes is repressed by meiotic sex chromosome inactivation (MSCI) during the meiotic phase of spermatogenesis in mammals. However, more recent studies have shown an increase in steady-state levels of certain X-linked miRNAs in pachytene spermatocytes, suggesting that either synthesis of these miRNAs increases or that degradation of these miRNAs decreases dramatically in these cells. To distinguish between these possibilities, we performed RNA-FISH to detect nascent transcripts from multiple miRNA genes in various spermatogenic cell types. Our results show definitively that Type I X-linked miRNA genes are subject to MSCI, as are all or most X-linked mRNA genes, whereas Type II and III X-linked miRNA genes escape MSCI by continuing ongoing, active transcription in primary spermatocytes. We corroborated these results by co-localization of RNA-FISH signals with both a corresponding DNA-FISH signal and an immunofluorescence signal for RNA polymerase II. We also found that X-linked miRNA genes that escape MSCI locate non-randomly to the periphery of the XY body, whereas genes that are subject to MSCI remain located within the XY body in pachytene spermatocytes, suggesting that the mechanism of escape of X-linked miRNA genes from MSCI involves their relocation to a position outside of the repressive chromatin domain associated with the XY body. The fact that Type II and III X-linked miRNA genes escape MSCI suggests an immediacy of function of the encoded miRNAs specifically required during the meiotic stages of spermatogenesis. © 2015. Published by The Company of Biologists Ltd.

  7. 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.

  8. Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene.

    Science.gov (United States)

    Nuez, B; Michalovich, D; Bygrave, A; Ploemacher, R; Grosveld, F

    1995-05-25

    Erythroid Krüppel-like factor (EKLF) was originally isolated from erythroid cell RNA by differential screening and shown to be erythroid-specific, although a low level of EKLF was found in mast cell lines. EKLF contains three zinc-fingers homologous to those found in the Krüppel family of transcription factors. Because it binds the sequence CCACACCCT, EKLF may affect erythroid development as a result of its ability to bind to the CAC box in the promoter of the beta-globin gene. Mutation of this element leads to reduced beta-globin expression and it appears to mediate the effect of the globin locus control region on the promoter. Here we inactivate the EKLF gene through insertion of a lacZ reporter gene by homologous recombination in embryonic stem (ES) cells. Heterozygous EKLF+/- mice show that the reporter gene is expressed in a developmentally specific manner in all types of erythroblasts in the fetal liver and adult bone marrow. Homozygous EKLF-/- mice appear normal during the embryonic stage of haematopoiesis in the yolk sac, but develop a fatal anaemia during early fetal life when haematopoiesis has switched to the fetal liver. Enucleated erythrocytes are formed but these do not contain the proper amount of haemoglobin. We conclude that the transcription factor EKLF is essential for the final steps of definitive erythropoiesis in fetal liver.

  9. 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

  10. 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.

  11. Inactivation and inducible oncogenic mutation of p53 in gene targeted pigs.

    Directory of Open Access Journals (Sweden)

    Simon Leuchs

    Full Text Available Mutation of the tumor suppressor p53 plays a major role in human carcinogenesis. Here we describe gene-targeted porcine mesenchymal stem cells (MSCs and live pigs carrying a latent TP53(R167H mutant allele, orthologous to oncogenic human mutant TP53(R175H and mouse Trp53(R172H, that can be activated by Cre recombination. MSCs carrying the latent TP53(R167H mutant allele were analyzed in vitro. Homozygous cells were p53 deficient, and on continued culture exhibited more rapid proliferation, anchorage independent growth, and resistance to the apoptosis-inducing chemotherapeutic drug doxorubicin, all characteristic of cellular transformation. Cre mediated recombination activated the latent TP53(R167H allele as predicted, and in homozygous cells expressed mutant p53-R167H protein at a level ten-fold greater than wild-type MSCs, consistent with the elevated levels found in human cancer cells. Gene targeted MSCs were used for nuclear transfer and fifteen viable piglets were produced carrying the latent TP53(R167H mutant allele in heterozygous form. These animals will allow study of p53 deficiency and expression of mutant p53-R167H to model human germline, or spontaneous somatic p53 mutation. This work represents the first inactivation and mutation of the gatekeeper tumor suppressor gene TP53 in a non-rodent mammal.

  12. A new method for rapid identification of ansamycin compounds by inactivating KLM gene clusters in potential ansamycin-producing actinomyces.

    Science.gov (United States)

    Wang, G; Zhang, H; Sun, G; Wu, L; Zhang, J; Wang, Y

    2012-02-01

    In this study, we explored the possibility of construction of a 'universal targeting vector' by Red/ET recombination to inactivate L gene encoding 3-amino-5-hydroxybenzoic acid (AHBA)-oxidoreductase in AHBA biosynthetic gene cluster to facilitate the detection of ansamycins production in actinomycetes. Based on the conserved regions of linked AHBA synthase (K), oxidoreductase (L) and phosphatase (M) gene clusters, degenerate primers were designed and PCR was performed to detect KLM gene clusters within 33 AHBA synthase gene-positive actinomycetes strains. Among them, 22 KLM gene cluster-positive strains were identified. A 'universal targeting vector' was further constructed using the 50-nt homologous sequences chosen from four strains internal L gene in KLM gene clusters through Red/ET recombination. The L gene from nine of the KLM gene cluster-positive actinomycetes strains was inactivated by insertion of a kanamycin (Km) resistance marker into its internal region from the 'universal targeting vector'. By comparison of the metabolites produced in parent strains with those in L gene-inactivated mutants, we demonstrated the possible ansamycins production produced by these strains. One strain (4089) was proved to be a geldanamycin producer. Three strains (3-20, 7-32 and 8-32) were identified as potential triene-ansamycins producers. Another strain (3-27) was possible to be a streptovaricin C producer. Strains 24-100 and 4-124 might be served as ansamitocin-like producers. The results confirmed the feasibility that a 'universal targeting vector' could be constructed through Red/ET recombination using the conserved regions of KLM gene clusters to detect ansamycins production in actinomycetes. The 'universal targeting vector' provides a rapid approach in certain degree to detect the potential ansamycin producers from the 22 KLM gene cluster-positive actinomycetes strains. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

  13. Glucocorticoid receptor gene inactivation in dopamine-innervated areas selectively decreases behavioral responses to amphetamine

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    Sebastien eParnaudeau

    2014-02-01

    Full Text Available The meso-cortico-limbic system, via dopamine release, encodes the rewarding and reinforcing properties of natural rewards. It is also activated in response to abused substances and is believed to support drug-related behaviors. Dysfunctions of this system lead to several psychiatric conditions including feeding disorders and drug addiction. These disorders are also largely influenced by environmental factors and in particular stress exposure. Stressors activate the corticotrope axis ultimately leading to glucocorticoid hormone (GCs release. GCs bind the glucocorticoid receptor (GR a transcription factor ubiquitously expressed including within the meso-cortico-limbic tract. While the GR within dopamine-innervated areas drives cocaine’s behavioral responses, its implication in responses to other psychostimulants such as amphetamine has never been clearly established. Moreover, while extensive work has been made to uncover the role of this receptor in addicted behaviors, its contribution to the rewarding and reinforcing properties of food has yet to be investigated. Using mouse models carrying GR gene inactivation in either dopamine neurons or in dopamine-innervated areas, we found that GR in dopamine responsive neurones is essential to properly build amphetamine-induced conditioned place preference and locomotor sensitization. c-Fos quantification in the nucleus accumbens further confirmed defective neuronal activation following amphetamine injection. These diminished neuronal and behavioral responses to amphetamine may involve alterations in glutamate transmission as suggested by the decreased MK801-elicited hyperlocomotion and by the hyporeactivity to glutamate of a subpopulation of medium spiny neurons. In contrast, GR inactivation did not affect rewarding and reinforcing properties of food suggesting that responding for natural reward under basal conditions is preserved in these mice.

  14. 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.

  15. Inactivation of tumor suppressor genes and cancer therapy: An evolutionary game theory approach.

    Science.gov (United States)

    Khadem, Heydar; Kebriaei, Hamed; Veisi, Zahra

    2017-06-01

    Inactivation of alleles in tumor suppressor genes (TSG) is one of the important issues resulting in evolution of cancerous cells. In this paper, the evolution of healthy, one and two missed allele cells is modeled using the concept of evolutionary game theory and replicator dynamics. The proposed model also takes into account the interaction rates of the cells as designing parameters of the system. Different combinations of the equilibrium points of the parameterized nonlinear system is studied and categorized into some cases. In each case, the interaction rates' values are suggested in a way that the equilibrium points of the replicator dynamics are located on an appropriate region of the state space. Based on the suggested interaction rates, it is proved that the system doesn't have any undesirable interior equilibrium point as well. Therefore, the system will converge to the desirable region, where there is a scanty level of cancerous cells. In addition, the proposed conditions for interaction rates guarantee that, when a trajectory of the system reaches the boundaries, then it will stay there forever which is a desirable property since the equilibrium points have been already located on the boundaries, appropriately. The simulation results show the effectiveness of the suggestions in the elimination of the cancerous cells in different scenarios. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. 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

  17. 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...

  18. Differential expression of ribosome-inactivating protein genes during somatic embryogenesis in spinach (Spinacia oleracea).

    Science.gov (United States)

    Kawade, Kensuke; Ishizaki, Takuma; Masuda, Kiyoshi

    2008-10-01

    Root segments from spinach (Spinacia oleracea L. cv. Jiromaru) seedlings form embryogenic callus (EC) that responded to exogenous GA(3) by accumulating a 31-kDa glycoprotein [BP31 or S. oleracea ribosome-inactivating protein (EC 3.2.2.22) (SoRIP1)] in association with the expression of embryogenic potential. Microsequencing of this protein revealed significant similarity with type 1 RIPs. We identified cDNAs for SoRIP1 and S. oleracea RIP2 (SoRIP2), a novel RIP having a consensus shiga/ricin toxic domain and performed a comparative analysis of the expression of SoRIPs during somatic embryogenesis. Western blotting and quantitative polymerase chain reaction analyses revealed that the expression of SoRIP1 in calli increased remarkably in association with the acquisition of embryogenic potential, although the expression in somatic embryos decreased moderately with their development. However, the expression of SoRIP2 in calli remained low and constant but increased markedly with the development of somatic embryos. Treatment of callus with GA(3) and/or ABA for 24 h, or with ABA for a longer period, failed to stimulate the expression of either gene. Immunohistochemistry showed that SoRIP1 preferentially accumulated in the proembryos and peripheral meristem of somatic embryos early in development. Appreciable expression of SoRIP2 was not detected in the callus, but intense expression was found in the epidermis of somatic embryos. These results suggest that the expression of spinach RIP genes is differentially regulated in a development-dependent fashion during somatic embryogenesis in spinach.

  19. Transcriptional control of two ribosome-inactivating protein genes expressed in spinach (Spinacia oleracea) embryos.

    Science.gov (United States)

    Kawade, Kensuke; Masuda, Kiyoshi

    2009-05-01

    SoRIP1 and SoRIP2 are ribosome-inactivating protein (RIP: EC 3.2.2.22) genes identified in spinach (Spinacia oleracea). They are differentially expressed in a development-dependent manner during spinach somatic embryogenesis. Here, we isolated genomic clones of SoRIP1 and SoRIP2. These two RIP genes have different genomic organization. Phylogenetic analysis of predicted amino acid sequences of RIPs in Caryophyllales plants revealed that they are divided into two major subfamilies, corresponding to SoRIP1 and SoRIP2. To gain further insight into the transcriptional control of SoRIP1 and SoRIP2, we obtained their 5'-flanking sequences by inverse PCR. Comparison of two 5'-flanking sequences revealed the characteristic cis elements in each region that confer differential transcriptional control. In the 5'-flanking region of SoRIP1, we found several motifs with functions related to embryonic development. The 5'-flanking region of SoRIP2 contains some defense-responsive motifs. Expression of SoRIP1 was detected in various tissues. In particular, SoRIP1 was highly expressed in the early immature fruits, and immunohistochemistry showed that SoRIP1 accumulated in the peripheral region of the immature embryo, with weaker expression in internal cells. During fruit development, the expression of SoRIP2 was low. However, the accumulation of SoRIP2 was conspicuous in the epidermis of the immature embryo. The expression of SoRIP2, but not SoRIP1, in leaves was induced by salicylic acid treatment. This differential transcriptional regulation of SoRIP1 and SoRIP2 suggests that the corresponding proteins may have different functions, one being related to embryonic development and the other to embryo defense.

  20. To Screen Inactivation Mutation of Exon 1 of FSHR Gene in Polycystic Ovarian Syndrome: A South Indian Cohort Study

    Science.gov (United States)

    Sekar, Nishu; Yeole, Samiksha; Pradeep, Rashmi; Prabhu, Yogamaya D.; Renu, Kaviyarasi; Ramgir, Shalaka S.; Abilash, V. G.

    2017-11-01

    Polycystic ovary syndrome is an endocrine disorder. Irregular menstrual cycle, acne, facial hair and elevated androgen levels are the most common signs for PCOS. PCOS has an estimated prevalence of 4-12% among reproductive age women, thus making it a forerunner in female infertility. FSHR plays an important role in FSH signaling pathway making it an important gene for PCOS. In this study, we aim to focus on any association between the FSHR gene and PCOS. Our study was to evaluate any polymorphism of exon 1 of FSHR gene associated with PCOS.PCR-RFLP technique was performed on the PCOS samples. Hormonal changes were found in the patients. Exon 1 inactivation mutation of FSHR gene was not observed in the patient sample. A study of this association needs to be done using large sample size.

  1. 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

  2. The inactivation of the sortilin gene leads to a partial disruption of prosaposin trafficking to the lysosomes

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Jibin; Racicott, Jesse [Department of Anatomy and Cell Biology, McGill University, Montreal (Canada); Morales, Carlos R., E-mail: carlos.morales@mcgill.ca [Department of Anatomy and Cell Biology, McGill University, Montreal (Canada)

    2009-11-01

    Lysosomes are intracellular organelles which contain enzymes and activator proteins involved in the digestion and recycling of a variety of cellular and extracellular substances. We have identified a novel sorting receptor, sortilin, which is involved in the lysosomal trafficking of the sphingolipid activator proteins, prosaposin and GM{sub 2}AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing prosaposin. In addition, the nonciliated cells are known to endocytose luminal prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of prosaposin in the lysosomes. When luminal prosaposin was excluded from the efferent ducts, the level of prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin

  3. Inactivation of genes encoding plastoglobuli-like proteins in Synechocystis sp. PCC 6803 leads to a light-sensitive phenotype.

    Science.gov (United States)

    Cunningham, Francis X; Tice, Ashley B; Pham, Christina; Gantt, Elisabeth

    2010-03-01

    Plastoglobulins (PGL) are the predominant proteins of lipid globules in the plastids of flowering plants. Genes encoding proteins similar to plant PGL are also present in algae and cyanobacteria but in no other organisms, suggesting an important role for these proteins in oxygenic photosynthesis. To gain an understanding of the core and fundamental function of PGL, the two genes that encode PGL-like polypeptides in the cyanobacterium Synechocystis sp. PCC 6803 (pgl1 and pgl2) were inactivated individually and in combination. The resulting mutants were able to grow under photoautotrophic conditions, dividing at rates that were comparable to that of the wild-type (WT) under low-light (LL) conditions (10 microeinsteins x m(-2) x s(-1)) but lower than that of the WT under moderately high-irradiance (HL) conditions (150 microeinsteins x m(-2) x s(-1)). Under HL, each Deltapgl mutant had less chlorophyll, a lower photosystem I (PSI)/PSII ratio, more carotenoid per unit of chlorophyll, and very much more myxoxanthophyll (a carotenoid symptomatic of high light stress) per unit of chlorophyll than the WT. Large, heterogeneous inclusion bodies were observed in cells of mutants inactivated in pgl2 or both pgl2 and pgl1 under both LL and HL conditions. The mutant inactivated in both pgl genes was especially sensitive to the light environment, with alterations in pigmentation, heterogeneous inclusion bodies, and a lower PSI/PSII ratio than the WT even for cultures grown under LL conditions. The WT cultures grown under HL contained 2- to 3-fold more PGL1 and PGL2 per cell than cultures grown under LL conditions. These and other observations led us to conclude that the PGL-like polypeptides of Synechocystis play similar but not identical roles in some process relevant to the repair of photooxidative damage.

  4. 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.

  5. Inactivation of the Odontogenic ameloblast-associated gene affects the integrity of the junctional epithelium and gingival healing.

    Science.gov (United States)

    Wazen, R M; Moffatt, P; Ponce, K J; Kuroda, S; Nishio, C; Nanci, A

    2015-09-28

    Odontogenic ameloblast-associated (ODAM) belongs to the secretory calcium-binding phosphoprotein (SCPP) gene cluster. It is expressed by the epithelial ameloblasts during the accrued mineralisation of enamel and by cells of the junctional epithelium (JE), a specialised portion of the gingiva that plays a critical role in periodontal health. In both cases, ODAM localises at the interface between the cells and the tooth surface. It is also present among the cells of the JE, and is distinctively highly expressed in many epithelial tumours. ODAM has been proposed to be a matricellular protein implicated in the adhesion of epithelial cells to tooth surfaces, and possibly in mediating cell status. To gain further understanding of the role of ODAM, we have created an Odam knockout (KO) mouse by deleting coding exons 2-6. Inactivation of the gene was verified by Southern blot, PCR, real-time qPCR and loss of immunostaining for the protein. Young Odam KO mice showed no readily apparent phenotype. No significant differences were observed in enamel volume and density, rod-interrod organisation, and its attrition. However, in older animals, the JE presented some detachment, an increase in inflammatory infiltrate, and apical down-growth. In addition, its regeneration was delayed following a gingivectomy challenge. Our results indicate that inactivation of Odam expression has no dramatic consequence on enamel but the phenotype in older animals replicates some JE changes seen during human periodontal disease. Altogether, our results suggest that ODAM plays a role in maintaining integrity of the JE.

  6. Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers

    Science.gov (United States)

    Veigl, Martina L.; Kasturi, Lakshmi; Olechnowicz, Joseph; Ma, AiHong; Lutterbaugh, James D.; Periyasamy, Sumudra; Li, Guo-Min; Drummond, James; Modrich, Paul L.; Sedwick, W. David; Markowitz, Sanford D.

    1998-01-01

    Mutations of DNA mismatch repair genes, including the hMLH1 gene, have been linked to human colon and other cancers in which defective DNA repair is evidenced by the associated instability of DNA microsatellite sequences (MSI). Germ-line hMLH1 mutations are causally associated with inherited MSI colon cancer, and somatic mutations are causally associated with sporadic MSI colon cancer. Previously however, we demonstrated that in many sporadic MSI colon cancers hMLH1 and all other DNA mismatch repair genes are wild type. To investigate this class of tumors further, we examined a group of MSI cancer cell lines, most of which were documented as established from antecedent MSI-positive malignant tumors. In five of six such cases we found that hMLH1 protein was absent, even though hMLH1-coding sequences were wild type. In each such case, absence of hMLH1 protein was associated with the methylation of the hMLH1 gene promoter. Furthermore, in each case, treatment with the demethylating agent 5-azacytidine induced expression of the absent hMLH1 protein. Moreover, in single cell clones, hMLH1 expression could be turned on, off, and on again by 5-azacytidine exposure, washout, and reexposure. This epigenetic inactivation of hMLH1 additionally accounted for the silencing of both maternal and paternal tumor hMLH1 alleles, both of which could be reactivated by 5-azacytidine. In summary, substantial numbers of human MSI cancers appear to arise by hMLH1 silencing via an epigenetic mechanism that can inactivate both of the hMLH1 alleles. Promoter methylation is intimately associated with this epigenetic silencing mechanism. PMID:9671741

  7. 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.

  8. Cigarette smoking, von Hippel-Lindau gene mutations and sporadic renal cell carcinoma

    NARCIS (Netherlands)

    Dijk, B.A.C. van; Schouten, L.J.; Oosterwijk, E.; Hulsbergen van de; Kaa, C.A.; Kiemeney, L.A.L.M.; Goldbohm, R.A.; Schalken, J.A.; Brandt, P.A. van den

    2006-01-01

    We investigated whether smoking is associated with mutations in the Von Hippel-Lindau (VHL) gene in 337 cases of sporadic renal cell carcinoma (RCC) among 120 852 people followed for 11.3 years; the findings suggest that smoking causes RCC independently of VHL gene mutations. © 2006 Cancer Research.

  9. Inactivating Mutation screening of Exon 6 and Exon 10E of FSHR gene in women with Polycystic Ovarian Syndrome in Vellore population

    Science.gov (United States)

    Sekar, Nishu; Sapre, Madhura; Kale, Vaikhari; Prabhu, Yogamaya D.; Renu, Kaviyarasi; Ramgir, Shalaka S.; Abilash, V. G.

    2017-11-01

    Polycystic Ovarian syndrome (PCOS) is a major cause of infertility in females of reproducing age and is typified by oligo-anovulation, hyperandrogenism, hirsutism and polycystic ovaries. FSHR gene located on chromosome 2 p21 is responsible for the normal follicular development and any deletion or mutation in the gene affects the interaction of FSH with its receptor. Thus, it becomes the candidate gene for PCOS study. Inactivating mutation in FSHR gene limits the receptor’s function by creating a complete block, changing the receptor-ligand complex or the basic hormone signal transduction.To screen the inactivating mutations in Exon 6 and Exon 10E of FSHR gene in women diagnosed with PCOS.PCR-RFLP analysis indicated that there were no inactivating mutations found in Exon 6 and Exon 10E. Variations in hormone levels were seen amongst the PCOS patients. There were no inactivating mutations found in FSHR gene of the women diagnosed with PCOS according to the Rotterdam criteria in Vellore population.

  10. Inactivation of antibiotic resistance genes in municipal wastewater effluent by chlorination and sequential UV/chlorination disinfection

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yingying; Zhuang, Yao; Geng, Jinju, E-mail: jjgeng@nju.edu.cn; Ren, Hongqiang, E-mail: hqren@nju.edu.cn; Zhang, Yan; Ding, Lili; Xu, Ke

    2015-04-15

    This study investigated disinfection methods including chlorination, ultraviolet (UV) irradiation and sequential UV/chlorination treatment on the inactivation of antibiotic resistance genes (ARGs). ARGs including sul1, tetX, tetG, intI1, and 16S rRNA genes in municipal wastewater treatment plant (MWTP) effluent were examined. The results indicated a positive correlation between the removal of ARGs and chlorine dosage (p = 0.007–0.014, n = 6),as well as contact time (p = 0.0001, n = 10). Greater free chlorine (FC) dosage leads to higher removal for all the genes and the maximum removal (1.30–1.49 logs) could be achieved at FC dosage of 30 mg L{sup −1}. The transformation kinetic data for ARGs removal (log C{sub 0} / C) followed the second-order reaction kinetic model with FC dosage (R{sup 2} = 0.6829–0.9999) and contact time (R{sup 2} = 0.7353–8634), respectively. Higher ammonia nitrogen (NH{sub 3}–N) concentration was found to lead to lower removal of ARGs at the same chlorine dosage. When the applied Cl{sub 2}:NH{sub 3}–N ratio was over 7.6:1, a significant reduction of ARGs (1.20–1.49 logs) was achieved. By using single UV irradiation, the log removal values of tetX and 16Ss rRNA genes were 0.58 and 0.60, respectively, while other genes were 0.36–0.40 at a fluence of 249.5 mJ cm{sup −2}, which was observed to be less effective than chlorination. With sequential UV/chlorination treatment, 0.006 to 0.31 log synergy values of target genes were observed under different operation parameters. - Highlights: • Chlorine is more effective than UV irradiation in removing ARGs from MWTP effluent. • The chlorination reaction followed the second-order reaction kinetic model. • Higher NH{sub 3}–N contents result in lower ARGs removal in the chlorination process. • FC is more effective than CC on the inactivation of ARGs. • UV irradiation followed by chlorination shows high efficiency in removing ARGs.

  11. Improved immunogenicity of Newcastle disease virus inactivated vaccine following DNA vaccination using Newcastle disease virus hemagglutinin-neuraminidase and fusion protein genes.

    Science.gov (United States)

    Firouzamandi, Masoumeh; Moeini, Hassan; Hosseini, Davood; Bejo, Mohd Hair; Omar, Abdul Rahman; Mehrbod, Parvaneh; Ideris, Aini

    2016-03-01

    The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.

  12. Inactivation of antibiotic resistance genes in municipal wastewater effluent by chlorination and sequential UV/chlorination disinfection.

    Science.gov (United States)

    Zhang, Yingying; Zhuang, Yao; Geng, Jinju; Ren, Hongqiang; Zhang, Yan; Ding, Lili; Xu, Ke

    2015-04-15

    This study investigated disinfection methods including chlorination, ultraviolet (UV) irradiation and sequential UV/chlorination treatment on the inactivation of antibiotic resistance genes (ARGs). ARGs including sul1, tetX, tetG, intI1, and 16S rRNA genes in municipal wastewater treatment plant (MWTP) effluent were examined. The results indicated a positive correlation between the removal of ARGs and chlorine dosage (p=0.007-0.014, n=6),as well as contact time (p=0.0001, n=10). Greater free chlorine (FC) dosage leads to higher removal for all the genes and the maximum removal (1.30-1.49 logs) could be achieved at FC dosage of 30 mg L(-1). The transformation kinetic data for ARGs removal (log C0/C) followed the second-order reaction kinetic model with FC dosage (R(2)=0.6829-0.9999) and contact time (R(2)=0.7353-8634), respectively. Higher ammonia nitrogen (NH3-N) concentration was found to lead to lower removal of ARGs at the same chlorine dosage. When the applied Cl2:NH3-N ratio was over 7.6:1, a significant reduction of ARGs (1.20-1.49 logs) was achieved. By using single UV irradiation, the log removal values of tetX and 16Ss rRNA genes were 0.58 and 0.60, respectively, while other genes were 0.36-0.40 at a fluence of 249.5 mJ cm(-2), which was observed to be less effective than chlorination. With sequential UV/chlorination treatment, 0.006 to 0.31 log synergy values of target genes were observed under different operation parameters. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Evaluation of gene expression SAP5, LIP9, and PLB2 of Candida albicans biofilms after photodynamic inactivation.

    Science.gov (United States)

    Freire, Fernanda; de Barros, Patrícia Pimentel; da Silva Ávila, Damara; Brito, Graziella Nuernberg Back; Junqueira, Juliana Campos; Jorge, Antonio Olavo Cardoso

    2015-07-01

    With the increasing number of strains of Candida ssp. resistant to antifungal agents, the accomplishment of researches that evaluate the effects of new therapeutic methods, like photodynamic inactivation (PDI), becomes important and necessary. Thus, the objective of this study was to verify the effects of the PDI on Candida albicans biofilms, evaluating their effects on the expression of the gene hydrolytic enzymes aspartyl proteinase (SAP5), lipase (LIP9), and phospholipase (PLB2). Clinical strains of C. albicans (n = 9) isolated from patient bearers of the virus HIV and a pattern strain ATCC 18804 were used. The quantification of gene expression was related to the production of hydrolytic enzymes using the quantitative polymerase chain reaction (qPCR) assay. For PDI, we used laser-aluminum-gallium arsenide low power (red visible, 660 nm) as a light source and the methylene blue at 300 μM as a photosensitizer. We assessed two experimental groups for each strain: (a) PDI: sensitization with methylene blue and laser irradiation and (b) control: without sensitization with methylene blue and light absence. The PDI decreased gene expression in 60 % of samples for gene SAP5 and 50 % of the samples decreased expression of LIP9 and PLB2. When we compared the expression profile for of each gene between the treated and control group, a decrease in all gene expression was observed, however no statistically significant difference (Tukey's test/p = 0.12). It could be concluded that PDI (photosensitization with methylene blue followed by low-level laser irradiation) showed a slight reduction on the expression of hydrolytic enzymes of C. albicans, without statistical significance.

  14. Disparate chromatin landscapes and kinetics of inactivation impact differential regulation of p53 target genes.

    Science.gov (United States)

    Gomes, Nathan P; Espinosa, Joaquín M

    2010-09-01

    The p53 transcription factor regulates the expression of genes involved in cellular responses to stress, including cell cycle arrest and apoptosis. The p53 transcriptional program is extremely malleable, with target gene expression varying in a stress- and cell type-specific fashion. The molecular mechanisms underlying differential p53 target gene expression remain elusive. Here we provide evidence for gene-specific mechanisms affecting expression of three important p53 target genes. First we show that transcription of the apoptotic gene PUMA is regulated through intragenic chromatin boundaries, as revealed by distinct histone modification territories that correlate with binding of the insulator factors CTCF, Cohesins and USF1/2. Interestingly, this mode of regulation produces an evolutionary conserved long non-coding RNA of unknown function. Second, we demonstrate that the kinetics of transcriptional competence of the cell cycle arrest gene p21 and the apoptotic gene FAS are markedly different in vivo, as predicted by recent biochemical dissection of their core promoter elements in vitro. After a pulse of p53 activity in cells, assembly of the transcriptional apparatus on p21 is rapidly reversed, while FAS transcriptional activation is more sustained. Collectively these data add to a growing list of p53-autonomous mechanisms that impact differential regulation of p53 target genes.

  15. Inactivation of RAD52 and HDF1 DNA repair genes leads to ...

    Indian Academy of Sciences (India)

    Silvia Mercado-Sáenz

    2017-04-18

    Apr 18, 2017 ... respiration. As a consequence, low glucose leads to increase both replicative and chronological life span (Breitenbach et al. 2012). It has been reported that glycolytic/fermentative genes are upregulated in Sch9Δ, Ras2Δ and Tor1Δ mutants, while mitochondrial genes are down-regulated. In this way, it has.

  16. Association of Exon 10A and 10B inactivating mutation of follicle stimulating hormone receptor gene (FSHR) and Polycystic Ovarian Syndrome in Vellore cohort

    Science.gov (United States)

    Sekar, Nishu; Kulkarni, Rucha; Ozalkar, Sharvari; Prabhu, Yogamaya D.; Renu, Kaviyarasi; Ramgir, Shalaka S.; Abilash, V. G.

    2017-11-01

    Polycystic ovarian syndrome is the most common heterogenous endocrine disorder in women. Follicle stimulating hormone receptor is associated with normal development as well as maturation of follicles and triggers estrogen production in granulosa cells of the ovary. Inactivating mutation in FSHR gene correlated with reduction of ovarian function in women is due to damage to receptor function. This study aims to investigate whether inactivating mutations, in follicle stimulating hormone receptor gene is related to polycystic ovarian morphology in women with PCOS. Genomic DNA isolated from 15 subjects from Sandhya Hospital, Vellore (10 patients with PCOS and 5 healthy controls) was taken for this study. Patient data included a clinical report, hormonal levels, and ovarian morphological details. DNA isolation was followed by DNA amplification by polymerase chain reaction using Exon 10 A and Exon 10 B primers. The PCR-RFLP analysis was performed using Dde1 restriction enzyme. Here we discuss inactivating mutation found in Exon 10 of FSHR gene in patients with PCOS.The absence of inactivating mutation was observed through PCR-RFLP study on Exon 10A and Exon 10B.

  17. Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations

    Directory of Open Access Journals (Sweden)

    Li Yongchao

    2012-01-01

    Full Text Available Abstract Background The model bacterium Clostridium cellulolyticum efficiently degrades crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H2 and CO2, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels production. Therefore genetic engineering will likely be required to improve the ethanol yield. Plasmid transformation, random mutagenesis and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism, hindering genetic engineering. Results The first targeted gene inactivation system was developed for C. cellulolyticum, based on a mobile group II intron originating from the Lactococcus lactis L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous L-lactate dehydrogenase (Ccel_2485; ldh and L-malate dehydrogenase (Ccel_0137; mdh genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain, resulting in a substantial shift in fermentation toward ethanol production. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products, corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutant's tricarboxylic acid pathway. Conclusions The efficient intron-based gene inactivation system produced the first non-random, targeted mutations in C. cellulolyticum. As a key component of the genetic toolbox

  18. Inactivation of a Pleurotus ostreatus versatile peroxidase-encoding gene (mnp2) results in reduced lignin degradation.

    Science.gov (United States)

    Salame, Tomer M; Knop, Doriv; Levinson, Dana; Mabjeesh, Sameer J; Yarden, Oded; Hadar, Yitzhak

    2014-01-01

    Lignin biodegradation by white-rot fungi is pivotal to the earth's carbon cycle. Manganese peroxidases (MnPs), the most common extracellular ligninolytic peroxidases produced by white-rot fungi, are considered key in ligninolysis. Pleurotus ostreatus, the oyster mushroom, is a preferential lignin degrader occupying niches rich in lignocellulose such as decaying trees. Here, we provide direct, genetically based proof for the functional significance of MnP to P. ostreatus ligninolytic capacity under conditions mimicking its natural habitat. When grown on a natural lignocellulosic substrate of cotton stalks under solid-state culture conditions, gene and isoenzyme expression profiles of its short MnP and versatile peroxidase (VP)-encoding gene family revealed that mnp2 was predominately expressed. mnp2, encoding the versatile short MnP isoenzyme 2 was disrupted. Inactivation of mnp2 resulted in three interrelated phenotypes, relative to the wild-type strain: (i) reduction of 14% and 36% in lignin mineralization of stalks non-amended and amended with Mn(2+), respectively; (ii) marked reduction of the bioconverted lignocellulose sensitivity to subsequent bacterial hydrolyses; and (iii) decrease in fungal respiration rate. These results may serve as the basis to clarify the roles of the various types of fungal MnPs and VPs in their contribution to white-rot decay of wood and lignocellulose in various ecosystems. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  19. Evolution of the MC5R gene in placental mammals with evidence for its inactivation in multiple lineages that lack sebaceous glands.

    Science.gov (United States)

    Springer, Mark S; Gatesy, John

    2017-12-19

    MC5R is one of five melanocortin receptor genes found in placental mammals. MC5R plays an important role in energy homeostasis and is also expressed in the terminal differentiation of sebaceous glands. Among placental mammals there are multiple lineages that either lack or have degenerative sebaceous glands including Cetacea (whales, dolphins, and porpoises), Hippopotamidae (hippopotamuses), Sirenia (manatees and dugongs), Proboscidea (elephants), Rhinocerotidae (rhinos), and Heterocephalus glaber (naked mole rat). Given the loss or diminution of sebaceous glands in these taxa, we procured MC5R sequences from publicly available genomes and transcriptomes, supplemented by a newly generated sequence for Choeropsis liberiensis (pygmy hippopotamus), to determine if this gene remains intact or is inactivated in association with loss/reduction of sebaceous glands. Our data set includes complete MC5R sequences for 114 placental mammal species including two individuals of Mammuthus primigenius (woolly mammoth) from Oimyakon and Wrangel Island. Complete loss or inactivation of the MC5R gene occurs in multiple placental lineages that have lost sebaceous glands (Cetacea, West Indian manatee, African elephant, white rhinoceros) or are characterized by unusual skin (pangolins, aardvarks). Both M. primigenius individuals share inactivating mutations with the African elephant even though sebaceous glands have been reported in the former. MC5R remains intact in hippopotamuses and the naked mole rat, although slightly elevated dN/dS ratios in these lineages allow for the possibility that the accumulation of inactivating mutations in MC5R may lag behind the relaxation of purifying selection. For Cetacea and Hippopotamidae, the absence of shared inactivating mutations in two different skin genes (MC5R, PSORS1C2) is consistent with the hypothesis that semi-aquatic lifestyles were acquired independently in these clades following divergence from a common ancestor. Copyright © 2017

  20. Efficient inactivation of symbiotic nitrogen fixation related genes in Lotus japonicus using CRISPR-Cas9

    Directory of Open Access Journals (Sweden)

    Longxiang Wang

    2016-08-01

    Full Text Available The targeted genome editing technique, CRISPR/Cas9 system, has been widely used to modify genes of interest in a predictable and precise manner. In this study, we describe the CRISPR/Cas9-mediated efficient editing of representative SNF (symbiotic nitrogen fixation related genes in the model legume Lotus japonicus via Agrobacterium-mediated stable or hairy root transformation. We first predicted nine endogenous U6 genes in Lotus and then demonstrated the efficacy of the LjU6-1 gene promoter in driving expression of single guide RNAs (sgRNAs by using a split yellow fluorescence protein (YFP reporter system to restore the fluorescence in Arabidopsis protoplasts. Next, we chose a customized sgRNA targeting SYMRK (symbiosis receptor kinase loci and achieved ~35% mutagenic efficiency in 20 T0 transgenic plants, two of them containing biallelic homozygous mutations with a 2-bp deletion near the PAM region. We further designed two sgRNAs targeting three homologous leghemoglobin loci (LjLb1, LjLb2, LjLb3 for testing the possibility of generating multi-gene knockouts. 20 out of 70 hairy root transgenic plants exhibited white nodules, with at least two LjLbs disrupted in each plant. Compared with the constitutively active CaMV 35S promoter, the nodule-specific LjLb2 promoter was also effective in gene editing in nodules by hairy root transformation. Triple mutant knockout of LjLbs was also obtained by stable transformation using two sgRNAs. Collectively, these studies demonstrate that the CRISPR/Cas9 system should greatly facilitate functional analyses of SNF related genes in Lotus japonicus.

  1. Inactivation of expression of two genes in Saccharomyces cerevisiae with the external guide sequence methodology

    OpenAIRE

    Cheng, Xudong; Ko, Jae-hyeong; Altman, Sidney

    2011-01-01

    The artificial inhibition of expression of genes in Saccharomyces cerevisiae is not a widespread, useful phenomenon. The external guide sequence (EGS) technology, which is well-proven in bacteria and mammalian cells in tissue culture and in mice, can also be utilized in yeast. The TOP2 and SRG1 genes can be inhibited by ∼30% with EGSs in vivo. Results in vitro also show convenient cleavage of the relevant transcripts by RNase P and appropriate EGSs. The feasible constructs shown to date have ...

  2. 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.

  3. Inactivation of expression of several genes in a variety of bacterial species by EGS technology.

    Science.gov (United States)

    Shen, Ning; Ko, Jae-hyeong; Xiao, Gaoping; Wesolowski, Donna; Shan, Ge; Geller, Bruce; Izadjoo, Mina; Altman, Sidney

    2009-05-19

    The expression of gene products in bacteria can be inhibited by the use of RNA external guide sequences (EGSs) that hybridize to a target mRNA. Endogenous RNase P cleaves the mRNA in the complex, making it inactive. EGSs participate in this biochemical reaction as the data presented here show. They promote mRNA cleavage at the expected site and sometimes at other secondary sites. Higher-order structure must affect these reactions if the cleavage does not occur at the defined site, which has been determined by techniques based on their ability to find sites that are accessible to the EGS oligonucleotides. Sites defined by a random EGS technique occur as expected. Oligonucleotides made up primarily of defined or random nucleotides are extremely useful in inhibiting expression of the gyrA and rnpA genes from several different bacteria or the cat gene that determines resistance to chloramphenicol in Escherichia coli. An EGS made up of a peptide-phosphorodiamidate morpholino oligonucleotide (PPMO) does not cleave at the same site as an unmodified RNA EGS for reasons that are only partly understood. However, PPMO-EGSs are useful in inhibiting the expression of targeted genes from Gram-negative and Gram-positive organisms during ordinary growth in broth and may provide a basis for broad-spectrum antibiotics.

  4. Heritable targeted inactivation of myostatin gene in yellow catfish (Pelteobagrus fulvidraco using engineered zinc finger nucleases.

    Directory of Open Access Journals (Sweden)

    Zhangji Dong

    Full Text Available Yellow catfish (Pelteobagrus fulvidraco is one of the most important freshwater aquaculture species in China. However, its small size and lower meat yield limit its edible value. Myostatin (MSTN is a negative regulator of mammalian muscle growth. But, the function of Mstn in fish remains elusive. To explore roles of mstn gene in fish growth and create a strain of yellow catfish with high amount of muscle mass, we performed targeted disruption of mstn in yellow catfish using engineered zinc-finger nucleases (ZFNs. Employing zebrafish embryos as a screening system to identify ZFN activity, we obtained one pair of ZFNs that can edit mstn in yellow catfish genome. Using the ZFNs, we successfully obtained two founders (Founder July29-7 and Founder July29-8 carrying mutated mstn gene in their germ cells. The mutated mstn allele inherited from Founder July29-7 was a null allele (mstn(nju6 containing a 4 bp insertion, predicted to encode function null Mstn. The mutated mstn inherited from Founder July29-8 was a complex type of mutation (mstn(nju7, predicted to encode a protein lacking two amino acids in the N-terminal secretory signal of Mstn. Totally, we obtained 6 mstn(nju6/+ and 14 mstn(nju7/+ yellow catfish. To our best knowledge, this is the first endogenous gene knockout in aquaculture fish. Our result will help in understanding the roles of mstn gene in fish.

  5. Heritable targeted inactivation of myostatin gene in yellow catfish (Pelteobagrus fulvidraco) using engineered zinc finger nucleases.

    Science.gov (United States)

    Dong, Zhangji; Ge, Jiachun; Li, Kui; Xu, Zhiqiang; Liang, Dong; Li, Jingyun; Li, Junbo; Jia, Wenshuang; Li, Yuehua; Dong, Xiaohua; Cao, Shasha; Wang, Xiaoxiao; Pan, Jianlin; Zhao, Qingshun

    2011-01-01

    Yellow catfish (Pelteobagrus fulvidraco) is one of the most important freshwater aquaculture species in China. However, its small size and lower meat yield limit its edible value. Myostatin (MSTN) is a negative regulator of mammalian muscle growth. But, the function of Mstn in fish remains elusive. To explore roles of mstn gene in fish growth and create a strain of yellow catfish with high amount of muscle mass, we performed targeted disruption of mstn in yellow catfish using engineered zinc-finger nucleases (ZFNs). Employing zebrafish embryos as a screening system to identify ZFN activity, we obtained one pair of ZFNs that can edit mstn in yellow catfish genome. Using the ZFNs, we successfully obtained two founders (Founder July29-7 and Founder July29-8) carrying mutated mstn gene in their germ cells. The mutated mstn allele inherited from Founder July29-7 was a null allele (mstn(nju6)) containing a 4 bp insertion, predicted to encode function null Mstn. The mutated mstn inherited from Founder July29-8 was a complex type of mutation (mstn(nju7)), predicted to encode a protein lacking two amino acids in the N-terminal secretory signal of Mstn. Totally, we obtained 6 mstn(nju6/+) and 14 mstn(nju7/+) yellow catfish. To our best knowledge, this is the first endogenous gene knockout in aquaculture fish. Our result will help in understanding the roles of mstn gene in fish.

  6. Metastasis Suppressor Gene Inactivates Actin-Based Mechanism of Tumor Cell Motility | Center for Cancer Research

    Science.gov (United States)

    Metastasis is responsible for up to 90 percent of all cancer-related deaths. Though proteins derived from nearly a dozen metastasis suppressor genes have been discovered over the past 15 years, strategies for exploiting the proteins in metastasis-prevention therapies has been hampered by the lack of knowledge regarding the mechanisms underlying the proteins’ interactions with other proteins.

  7. Inactivation of MarR gene homologs increases susceptibility to antimicrobials in Bacteroides fragilis.

    Science.gov (United States)

    Silva, Clara Maria Guimarães; Silva, Déborah Nascimento Dos Santos; Costa, Scarlathe Bezerra da; Almeida, Juliana Soares de Sá; Boente, Renata Ferreira; Teixeira, Felipe Lopes; Domingues, Regina Maria Cavalcanti Pilotto; Lobo, Leandro Araujo

    Bacteroides fragilis is the strict anaerobic bacteria most commonly found in human infections, and has a high mortality rate. Among other virulence factors, the remarkable ability to acquire resistance to a variety of antimicrobial agents and to tolerate nanomolar concentrations of oxygen explains in part their success in causing infection and colonizing the mucosa. Much attention has been given to genes related to multiple drug resistance derived from plasmids, integrons or transposon, but such genes are also detected in chromosomal systems, like the mar (multiple antibiotic resistance) locus, that confer resistance to a range of drugs. Regulators like MarR, that control expression of the locus mar, also regulate resistance to organic solvents, disinfectants and oxygen reactive species are important players in these events. Strains derived from the parental strain 638R, with mutations in the genes hereby known as marRI (BF638R_3159) and marRII (BF638R_3706) were constructed by gene disruption using a suicide plasmid. Phenotypic response of the mutant strains to hydrogen peroxide, cell survival assay against exposure to oxygen, biofilm formation, resistance to bile salts and resistance to antibiotics was evaluated. The results showed that the mutant strains exhibit statistically significant differences in their response to oxygen stress, but no changes were observed in survival when exposed to bile salts. Biofilm formation was not affected by either gene disruption. Both mutant strains however, became more sensitive to multiple antimicrobial drugs tested. This indicates that as observed in other bacterial species, MarR are an important resistance mechanism in B. fragilis. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  8. A systematic study of gene mutations in urothelial carcinoma; inactivating mutations in TSC2 and PIK3R1.

    Directory of Open Access Journals (Sweden)

    Gottfrid Sjödahl

    Full Text Available BACKGROUND: Urothelial carcinoma (UC is characterized by frequent gene mutations of which activating mutations in FGFR3 are the most frequent. Several downstream targets of FGFR3 are also mutated in UC, e.g., PIK3CA, AKT1, and RAS. Most mutation studies of UCs have been focused on single or a few genes at the time or been performed on small sample series. This has limited the possibility to investigate co-occurrence of mutations. METHODOLOGY/PRINCIPAL FINDINGS: We performed mutation analyses of 16 genes, FGFR3, PIK3CA, PIK3R1 PTEN, AKT1, KRAS, HRAS, NRAS, BRAF, ARAF, RAF1, TSC1, TSC2, APC, CTNNB1, and TP53, in 145 cases of UC. We show that FGFR3 and PIK3CA mutations are positively associated. In addition, we identified PIK3R1 as a target for mutations. We demonstrate a negative association at borderline significance between FGFR3 and RAS mutations, and show that these mutations are not strictly mutually exclusive. We show that mutations in BRAF, ARAF, RAF1 rarely occurs in UC. Our data emphasize the possible importance of APC signaling as 6% of the investigated tumors either showed inactivating APC or activating CTNNB1 mutations. TSC1, as well as TSC2, that constitute the mTOR regulatory tuberous sclerosis complex were found to be mutated at a combined frequency of 15%. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate a significant association between FGFR3 and PIK3CA mutations in UC. Moreover, the identification of mutations in PIK3R1 further emphasizes the importance of the PI3-kinase pathway in UC. The presence of TSC2 mutations, in addition to TSC1 mutations, underlines the involvement of mTOR signaling in UC.

  9. Conditional inactivation of MRG15 gene function limits survival during larval and adult stages of Drosophila melanogaster.

    Science.gov (United States)

    Zhang, Hongjun; Li, Yishi; Yang, Junsheng; Tominaga, Kaoru; Pereira-Smith, Olivia M; Tower, John

    2010-11-01

    The mammalian MRG15 gene encodes a chromodomain protein predicted to bind to chromatin via methylated histone tails. Human MORF4 encodes a related but truncated protein that is capable of promoting cellular senescence in a subset of human tumor cell lines. Drosophila contains a single homolog of human MRG15, called DmMRG15. Null mutation of MRG15 is embryonic-lethal in mice and Drosophila, making the study of MRG15 requirements in adults difficult. In these studies the DmMRG15 gene was over-expressed in Drosophila, during developmental stages and in adults, using a doxycycline-regulated system (Tet-on). In addition an inverted-repeated construct was designed to inactivate DmMRG15 via the RNAi pathway, and RNAi constructs were expressed using both the Tet-on system and Geneswitch system. The DmMRG15 protein was readily expressed in adult flies in a doxycycline-dependent manner. A truncated form of DmMRG15 (called DmMT1) was designed to mimic the structure of human MORF4, and expression of this mutant protein or the inverted-repeat constructs inhibited fertility in females. Conditional expression of the DmMRG15 inverted-repeat constructs during larval development or in adults caused reductions in survival. These experiments indicate that Drosophila DmMRG15 gene function is required for female fertility, larval survival and adult life span, and provide reagents that should be useful for further dissecting the role of DmMRG15 in cell proliferation and aging. Copyright © 2010 Elsevier Inc. All rights reserved.

  10. Inactivation of expression of two genes in Saccharomyces cerevisiae with the external guide sequence methodology.

    Science.gov (United States)

    Cheng, Xudong; Ko, Jae-Hyeong; Altman, Sidney

    2011-03-01

    The artificial inhibition of expression of genes in Saccharomyces cerevisiae is not a widespread, useful phenomenon. The external guide sequence (EGS) technology, which is well-proven in bacteria and mammalian cells in tissue culture and in mice, can also be utilized in yeast. The TOP2 and SRG1 genes can be inhibited by ∼30% with EGSs in vivo. Results in vitro also show convenient cleavage of the relevant transcripts by RNase P and appropriate EGSs. The feasible constructs shown to date have an EGS covalently linked to M1 RNA, the RNA subunit of RNase P from Escherichia coli. Greater efficiency in cleavage of transcripts can be fashioned using more than one EGS targeted to different sites in a transcript and stronger promoters controlling the EGS constructs.

  11. Heritable Targeted Inactivation of Myostatin Gene in Yellow Catfish (Pelteobagrus fulvidraco) Using Engineered Zinc Finger Nucleases

    OpenAIRE

    Zhangji Dong; Jiachun Ge; Kui Li; Zhiqiang Xu; Dong Liang; Jingyun Li; Junbo Li; Wenshuang Jia; Yuehua Li; Xiaohua Dong; Shasha Cao; Xiaoxiao Wang; Jianlin Pan; Qingshun Zhao

    2011-01-01

    Yellow catfish (Pelteobagrus fulvidraco) is one of the most important freshwater aquaculture species in China. However, its small size and lower meat yield limit its edible value. Myostatin (MSTN) is a negative regulator of mammalian muscle growth. But, the function of Mstn in fish remains elusive. To explore roles of mstn gene in fish growth and create a strain of yellow catfish with high amount of muscle mass, we performed targeted disruption of mstn in yellow catfish using engineered zinc-...

  12. Prevalence of von Hippel-Lindau gene mutations in sporadic renal cell carcinoma: results from The Netherlands cohort study.

    NARCIS (Netherlands)

    Houwelingen, K.P. van; Dijk, B.A. van; Hulsbergen- van de Kaa, C.A.; Schouten, L.J.; Gorissen, H.; Schalken, J.A.; Brandt, P.A. van den; Oosterwijk, E.

    2005-01-01

    BACKGROUND: Biallelic von Hippel-Lindau (VHL) gene defects, a rate-limiting event in the carcinogenesis, occur in approximately 75% of sporadic clear-cell Renal Cell Carcinoma (RCC). We studied the VHL mutation status in a large population-based case group. METHODS: Cases were identified within the

  13. Multiple phenotypes in adult mice following inactivation of the Coxsackievirus and Adenovirus Receptor (Car gene.

    Directory of Open Access Journals (Sweden)

    Ahmad Pazirandeh

    Full Text Available To determine the normal function of the Coxsackievirus and Adenovirus Receptor (CAR, a protein found in tight junctions and other intercellular complexes, we constructed a mouse line in which the CAR gene could be disrupted at any chosen time point in a broad spectrum of cell types and tissues. All knockouts examined displayed a dilated intestinal tract and atrophy of the exocrine pancreas with appearance of tubular complexes characteristic of acinar-to-ductal metaplasia. The mice also exhibited a complete atrio-ventricular block and abnormal thymopoiesis. These results demonstrate that CAR exerts important functions in the physiology of several organs in vivo.

  14. Glucose-induced repression of PPARalpha gene expression in pancreatic beta-cells involves PP2A activation and AMPK inactivation

    DEFF Research Database (Denmark)

    Ravnskjaer, Kim; Boergesen, Michael; Dalgaard, Louise T

    2006-01-01

    , the mechanism underlying this transcriptional repression by glucose remains unclear. Here we report that glucose-induced repression of PPARalpha gene expression in INS-1E cells is independent of beta-cell excitation and insulin secretion but requires activation of protein phosphatase 2A in a process involving...... but not AMPKalpha1 using RNAi suppressed PPARalpha expression, thereby mimicking the effect of glucose. These results indicate that activation of protein phosphatase 2A and subsequent inactivation of AMPK is necessary for glucose repression of PPARalpha expression in pancreatic beta-cells....... inactivation of the AMP-activated protein kinase (AMPK). Pharmacological activation of AMPK at high glucose concentrations interferes with glucose repression of PPARalpha and PPARalpha target genes in INS-1E cells as well as in rat islets. Specific knock-down of the catalytic AMPK-subunit AMPKalpha2...

  15. An inactivating mutation in the SOD 1 gene causes familial amyotrophic lateral sclerosis

    Energy Technology Data Exchange (ETDEWEB)

    Pramatarova, A.; Rouleau, G.A. [Montreal General Hospital Research Institute (Canada); Goto, J. [Univ. of Tokyo (Japan)] [and others

    1994-09-01

    Amyotrophic lateral sclerosis (ALS) is characterized by highly selective death of large motor neurons in the cerebral cortex and spinal cord. The familial form of ALS (FALS) accounts for approximately 10% of the cases and is transmitted in an autosomal dominant manner. Recently the defective gene causing chromosome 21-linked FALS was shown to be the Cu/Zn superoxide dismutase (SOD 1). However, the precise mechanism of neurotoxicity seen in FALS with SOD 1 mutations is still unknown. Until now all SOD 1 mutations reported were single base pair substitutions (missense). We have identified a nonsense mutation in exon 5 of the SOD 1 gene in a FALS kindred. This two base pair deletion provokes a frameshift and a predicted premature truncation of the protein. The region affected has a very important structural and functional role: it contains part of the active loop and is involved in dimer contact. We would predict that the loss of these structures would impair the functioning of the enzyme.

  16. Genetic inactivation of the Fanconi anemia gene FANCC identified in the hepatocellular carcinoma cell line HuH-7 confers sensitivity towards DNA-interstrand crosslinking agents

    Directory of Open Access Journals (Sweden)

    Bassermann Florian

    2010-05-01

    Full Text Available Abstract Background Inactivation of the Fanconi anemia (FA pathway through defects in one of 13 FA genes occurs at low frequency in various solid cancer entities among the general population. As FA pathway inactivation confers a distinct hypersensitivity towards DNA interstrand-crosslinking (ICL-agents, FA defects represent rational targets for individualized therapeutic strategies. Except for pancreatic cancer, however, the prevalence of FA defects in gastrointestinal (GI tumors has not yet been systematically explored. Results A panel of GI cancer cell lines was screened for FA pathway inactivation applying FANCD2 monoubiquitination and FANCD2/RAD51 nuclear focus formation and a newly identified FA pathway-deficient cell line was functionally characterized. The hepatocellular carcinoma (HCC line HuH-7 was defective in FANCD2 monoubiquitination and FANCD2 nuclear focus formation but proficient in RAD51 focus formation. Gene complementation studies revealed that this proximal FA pathway inactivation was attributable to defective FANCC function in HuH-7 cells. Accordingly, a homozygous inactivating FANCC nonsense mutation (c.553C > T, p.R185X was identified in HuH-7, resulting in partial transcriptional skipping of exon 6 and leading to the classic cellular FA hypersensitivity phenotype; HuH-7 cells exhibited a strongly reduced proliferation rate and a pronounced G2 cell cycle arrest at distinctly lower concentrations of ICL-agents than a panel of non-isogenic, FA pathway-proficient HCC cell lines. Upon retroviral transduction of HuH-7 cells with FANCC cDNA, FA pathway functions were restored and ICL-hypersensitivity abrogated. Analyses of 18 surgical HCC specimens yielded no further examples for genetic or epigenetic inactivation of FANCC, FANCF, or FANCG in HCC, suggesting a low prevalence of proximal FA pathway inactivation in this tumor type. Conclusions As the majority of HCC are chemoresistant, assessment of FA pathway function in HCC could

  17. Anti-tumor effects of inactivated Sendai virus particles with an IL-2 gene on angiosarcoma.

    Science.gov (United States)

    Takehara, Yuki; Satoh, Takahiro; Nishizawa, Aya; Saeki, Kazumi; Nakamura, Masataka; Masuzawa, Mikio; Kaneda, Yasufumi; Katayama, Ichiro; Yokozeki, Hiroo

    2013-10-01

    Cutaneous angiosarcoma is a life-threatening tumor that is resistant to conventional therapies. The therapeutic effects of Sendai virus particles (hemagglutinating virus of Japan envelope: HVJ-E) carrying IL-2 gene (HVJ-E/IL-2) were examined in a mouse model of angiosarcoma. Intra-tumoral injection of HVJ-E/IL-2 effectively inhibited the growth of angiosarcoma cells (ISOS-1) inoculated in mice and improved tumor-free rates. HVJ-E/IL-2 stimulated local accumulation of CD8 (+) T cells and NK cells and reduced regulatory T cells in regional lymph nodes. Notably, the prevalence of myeloid-derived suppressor cells was lower in HVJ-E/IL-2-treated mice than in HVJ-E-treated mice. HVJ-E/IL-2 treatment promoted IFN-γ production from CD8 (+) T cells in response to tumor cells, more significantly than HVJ-E treatment. Greatly improved tumor-free rates were obtained when sunitinib, a tyrosine kinase inhibitor, was administered in combination with HVJ-E/IL-2. Immunogene therapy with HVJ-E/IL-2 with or without sunitinib could be a promising therapeutic option for cutaneous angiosarcoma. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Targeted Inactivation of DNA Photolyase Genes in Medaka Fish (Oryzias latipes).

    Science.gov (United States)

    Ishikawa-Fujiwara, Tomoko; Shiraishi, Eri; Fujikawa, Yoshihiro; Mori, Toshio; Tsujimura, Tohru; Todo, Takeshi

    2017-01-01

    Proteins of the cryptochrome/photolyase family (CPF) exhibit sequence and structural conservation, but their functions are divergent. Photolyase is a DNA repair enzyme that catalyzes the light-dependent repair of ultraviolet (UV)-induced photoproducts, whereas cryptochrome acts as a photoreceptor or circadian clock protein. Two types of DNA photolyase exist: CPD photolyase, which repairs cyclobutane pyrimidine dimers (CPDs), and 6-4 photolyase, which repairs 6-4 pyrimidine-pyrimidone photoproducts (6-4PPs). Although the Cry-DASH protein is classified as a cryptochrome, it also has light-dependent DNA repair activity. To determine the significance of the three light-dependent repair enzymes in recovering from solar UV-induced DNA damage at the organismal level, we generated mutants in each gene in medaka using the CRISPR genome editing technique. The light-dependent repair activity of the mutants was examined in vitro in cultured cells and in vivo in skin tissue. Light-dependent repair of CPD was lost in the CPD photolyase-deficient mutant, whereas weak repair activity against 6-4PPs persisted in the 6-4 photolyase-deficient mutant. These results suggest the existence of a heretofore unknown 6-4PP repair pathway and thus improve our understanding of the mechanisms of defense against solar UV in vertebrates. © 2016 The Authors. Photochemistry and Photobiology published by Wiley Periodicals, Inc. on behalf of American Society for Photobiology.

  19. Ocular findings in adult subjects with an inactivating mutation in GH releasing hormone receptor gene.

    Science.gov (United States)

    Faro, Augusto C N; Pereira-Gurgel, Virginia M; Salvatori, Roberto; Campos, Viviane C; Melo, Gustavo B; Oliveira, Francielle T; Oliveira-Santos, Alecia A; Oliveira, Carla R P; Pereira, Francisco A; Hellström, Ann; Oliveira-Neto, Luís A; Valença, Eugenia H O; Aguiar-Oliveira, Manuel H

    2017-06-01

    Ocular function is fundamental for environmental adaptation and survival capacity. Growth factors are necessary for a mature eyeball, needed for adequate vision. However, the consequences of the deficiency of circulating growth hormone (GH) and its effector insulin-like growth factor I (IGF-I) on the physical aspects of the human eye are still debated. A model of untreated isolated GH deficiency (IGHD), with low but measurable serum GH, may clarify this issue. The aim of this study was to assess the ocular aspects of adult IGHD individuals who have never received GH therapy. Cross sectional study. Setting: University Hospital, Federal University of Sergipe, Brazil. Twenty-five adult (13 males, mean age 50.1years, range 26 to 70years old) IGHD subjects homozygous for a null mutation (c.57+1G>A) in the GHRH receptor gene, and 28 (15 males, mean age 51.1years, range 26 to 67years old) controls were submitted to an endocrine and ophthalmological assessment. Forty-six IGHD and 50 control eyes were studied. Visual acuity, intraocular pressure, refraction (spherical equivalent), ocular axial length (AL), anterior chamber depth (ACD), lens thickness (LT), vitreous depth (VD), mean corneal curvature (CC) and central corneal thickness (CCT). IGHD subjects exhibited unmeasurable serum IGF-I levels, similar visual acuity, intraocular pressure and LT, higher values of spherical equivalent and CC, and lower measures of AL, ACD, VD and CCT in comparison to controls, but within their respective normal ranges. While mean stature in IGHD group was 78% of the control group, mean head circumference was 92% and axial AL was 96%. These observations suggest mild ocular effects in adult subjects with severe IGF-I deficiency due to non-treated IGHD. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. The SKINT1-like gene is inactivated in hominoids but not in all primate species: implications for the origin of dendritic epidermal T cells.

    Directory of Open Access Journals (Sweden)

    Rania Hassan Mohamed

    Full Text Available Dendritic epidermal T cells, which express an invariant Vγ5Vδ1 T-cell receptor and account for 95% of all resident T cells in the mouse epidermis, play a critical role in skin immune surveillance. These γδ T cells are generated by positive selection in the fetal thymus, after which they migrate to the skin. The development of dendritic epidermal T cells is critically dependent on the Skint1 gene expressed specifically in keratinocytes and thymic epithelial cells, suggesting an indispensable role for Skint1 in the selection machinery for specific intraepithelial lymphocytes. Phylogenetically, rodents have functional SKINT1 molecules, but humans and chimpanzees have a SKINT1-like (SKINT1L gene with multiple inactivating mutations. In the present study, we analyzed SKINT1L sequences in representative primate species and found that all hominoid species have a common inactivating mutation, but that Old World monkeys such as olive baboons, green monkeys, cynomolgus macaques and rhesus macaques have apparently functional SKINT1L sequences, indicating that SKINT1L was inactivated in a common ancestor of hominoids. Interestingly, the epidermis of cynomolgus macaques contained a population of dendritic-shaped γδ T cells expressing a semi-invariant Vγ10/Vδ1 T-cell receptor. However, this population of macaque T cells differed from rodent dendritic epidermal T cells in that their Vγ10/Vδ1 T-cell receptors displayed junctional diversity and expression of Vγ10 was not epidermis-specific. Therefore, macaques do not appear to have rodent-type dendritic epidermal T cells despite having apparently functional SKINT1L. Comprehensive bioinformatics analysis indicates that SKINT1L emerged in an ancestor of placental mammals but was inactivated or lost multiple times in mammalian evolution and that Skint1 arose by gene duplication in a rodent lineage, suggesting that authentic dendritic epidermal T cells are presumably unique to rodents.

  1. The SKINT1-like gene is inactivated in hominoids but not in all primate species: implications for the origin of dendritic epidermal T cells.

    Science.gov (United States)

    Mohamed, Rania Hassan; Sutoh, Yoichi; Itoh, Yasushi; Otsuka, Noriyuki; Miyatake, Yukiko; Ogasawara, Kazumasa; Kasahara, Masanori

    2015-01-01

    Dendritic epidermal T cells, which express an invariant Vγ5Vδ1 T-cell receptor and account for 95% of all resident T cells in the mouse epidermis, play a critical role in skin immune surveillance. These γδ T cells are generated by positive selection in the fetal thymus, after which they migrate to the skin. The development of dendritic epidermal T cells is critically dependent on the Skint1 gene expressed specifically in keratinocytes and thymic epithelial cells, suggesting an indispensable role for Skint1 in the selection machinery for specific intraepithelial lymphocytes. Phylogenetically, rodents have functional SKINT1 molecules, but humans and chimpanzees have a SKINT1-like (SKINT1L) gene with multiple inactivating mutations. In the present study, we analyzed SKINT1L sequences in representative primate species and found that all hominoid species have a common inactivating mutation, but that Old World monkeys such as olive baboons, green monkeys, cynomolgus macaques and rhesus macaques have apparently functional SKINT1L sequences, indicating that SKINT1L was inactivated in a common ancestor of hominoids. Interestingly, the epidermis of cynomolgus macaques contained a population of dendritic-shaped γδ T cells expressing a semi-invariant Vγ10/Vδ1 T-cell receptor. However, this population of macaque T cells differed from rodent dendritic epidermal T cells in that their Vγ10/Vδ1 T-cell receptors displayed junctional diversity and expression of Vγ10 was not epidermis-specific. Therefore, macaques do not appear to have rodent-type dendritic epidermal T cells despite having apparently functional SKINT1L. Comprehensive bioinformatics analysis indicates that SKINT1L emerged in an ancestor of placental mammals but was inactivated or lost multiple times in mammalian evolution and that Skint1 arose by gene duplication in a rodent lineage, suggesting that authentic dendritic epidermal T cells are presumably unique to rodents.

  2. Body Mass Index and von Hippel-Lindau Gene Mutations in Clear-cell Renal Cancer: Results of the Netherlands Cohort Study on Diet and Cancer

    NARCIS (Netherlands)

    Smits, K.M.; Schouten, L.J.; Hudak, E.; Verhage, B.; Dijk, B.A.C. van; Hulsbergen - Kaa, C.A. van de; Goldbohm, R.A.; Oosterwijk, E.; Brandt, P.A. van den

    2010-01-01

    Purpose: Body mass index (BMI) is an important risk factor for clear-cell renal cancer (cc-RCC). A common molecular alteration in cc-RCC is loss-of-function of the von Hippel-Lindau (VHL) gene. We evaluated the association between BMI and VHL mutations in cc-RCC by using data from the Netherlands

  3. Gene Inactivation in the Cyanobacterium Synechococcus sp. PCC 7002 and the Green Sulfur Bacterium Chlorobium tepidum Using In Vitro-Made DNA Constructs and Natural Transformation

    DEFF Research Database (Denmark)

    Frigaard, Niels-Ulrik; Sakuragi, Yumiko; Bryant, Donald A

    2004-01-01

    Inactivation of a chromosomal gene is a useful approach to study the function of the gene in question and can be used to produce a desired phenotype in the organism. This chapter describes how to generate such mutants of the cyanobacterium Synechococcus sp. PCC 7002 and the green sulfur bacterium...... Chlorobium tepidum by natural transformation with synthetic DNA constructs. Two alternative methods to generate the DNA constructs, both performed entirely in vitro and based on the polymerase chain reaction (PCR), are also presented. These methods are ligation of DNA fragments with T4 DNA ligase...

  4. Inactivation of the tumor suppressor genes causing the hereditary syndromes predisposing to head and neck cancer via promoter hypermethylation in sporadic head and neck cancers.

    Science.gov (United States)

    Smith, Ian M; Mithani, Suhail K; Mydlarz, Wojciech K; Chang, Steven S; Califano, Joseph A

    2010-01-01

    Fanconi anemia (FA) and dyskeratosis congenita (DC) are rare inherited syndromes that cause head and neck squamous cell cancer (HNSCC). Prior studies of inherited forms of cancer have been extremely important in elucidating tumor suppressor genes inactivated in sporadic tumors. Here, we studied whether sporadic tumors have epigenetic silencing of the genes causing the inherited forms of HNSCC. Using bisulfite sequencing, we investigated the incidence of promoter hypermethylation of the 17 Fanconi- and DC-associated genes in sporadic HNSCC. Genes that only showed methylation in the tumor patients were chosen for quantitative methylation-specific PCR (qMSP) in a set of 45 tumor and 16 normal patients. Three gene promoters showed differences in methylation: FancB (FAAP95, FA core complex), FancJ (BRIP1, DNA Helicase/ATPase), and DKC1 (dyskeratin). Bisulfite sequencing revealed that only FancB and DKC1 showed no methylation in normal patients, yet the presence of promoter hypermethylation in tumor patients. On qMSP, 1/16 (6.25%) of the normal mucosal samples from non-cancer patients and 14/45 (31.1%) of the tumor patients demonstrated hypermethylation of the FancB locus (p < 0.05). These results suggest that inactivation of FancB may play a role in the pathogenesis of sporadic HNSCC.

  5. Gene and protein analysis reveals that p53 pathway is functionally inactivated in cytogenetically normal Acute Myeloid Leukemia and Acute Promyelocytic Leukemia.

    Science.gov (United States)

    Abramowitz, Julia; Neuman, Tzahi; Perlman, Riki; Ben-Yehuda, Dina

    2017-03-24

    Mechanisms that inactivate the p53 pathway in Acute Myeloid Leukemia (AML), other than rare mutations, are still not well understood. We performed a bioinformatics study of the p53 pathway function at the gene expression level on our collection of 1153 p53-pathway related genes. Publically available Affymetrix data of 607 de-novo AML patients at diagnosis were analyzed according to the patients cytogenetic, FAB and molecular mutations subtypes. We further investigated the functional status of the p53 pathway in cytogenetically normal AML (CN-AML) and Acute Promyelocytic Leukemia (APL) patients using bioinformatics, Real-Time PCR and immunohistochemistry. We revealed significant and differential alterations of p53 pathway-related gene expression in most of the AML subtypes. We found that p53 pathway-related gene expression was not correlated with the accepted grouping of AML subtypes such as by cytogenetically-based prognosis, morphological stage or by the type of molecular mutation. Our bioinformatic analysis revealed that p53 is not functional in CN-AML and APL blasts at inducing its most important functional outcomes: cell cycle arrest, apoptosis, DNA repair and oxidative stress defense. We revealed transcriptional downregulation of important p53 acetyltransferases in both CN-AML and APL, accompanied by increased Mdmx protein expression and inadequate Chk2 protein activation. Our bioinformatic analysis demonstrated that p53 pathway is differentially inactivated in different AML subtypes. Focused gene and protein analysis of p53 pathway in CN-AML and APL patients imply that functional inactivation of p53 protein can be attributed to its impaired acetylation. Our analysis indicates the need in further accurate evaluation of p53 pathway functioning and regulation in distinct subtypes of AML.

  6. 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...

  7. 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.

  8. 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

  9. 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.

  10. Inactivation efficiency of plasmid-encoded antibiotic resistance genes during water treatment with chlorine, UV, and UV/H2O2.

    Science.gov (United States)

    Yoon, Younggun; Chung, Hay Jung; Wen Di, Doris Yoong; Dodd, Michael C; Hur, Hor-Gil; Lee, Yunho

    2017-10-15

    This study assessed the inactivation efficiency of plasmid-encoded antibiotic resistance genes (ARGs) both in extracellular form (e-ARG) and present within Escherichia coli (intracellular form, i-ARG) during water treatment with chlorine, UV (254 nm), and UV/H2O2. A quantitative real-time PCR (qPCR) method was used to quantify the ARG damage to ampR (850 bp) and kanR (806 bp) amplicons, both of which are located in the pUC4K plasmid. The plate count and flow cytometry methods were also used to determine the bacterial inactivation parameters, such as culturability and membrane damage, respectively. In the first part of the study, the kinetics of E. coli inactivation and ARG damage were determined in phosphate buffered solutions. The ARG damage occurred much more slowly than E. coli inactivation in all cases. To achieve 4-log reduction of ARG concentration at pH 7, the required chlorine exposure and UV fluence were 33-72 (mg × min)/L for chlorine and 50-130 mJ/cm2 for UV and UV/H2O2. After increasing pH from 7 to 8, the rates of ARG damage decreased for chlorine, while they did not vary for UV and UV/H2O2. The i-ARGs mostly showed lower rates of damage compared to the e-ARGs due to the protective roles of cellular components against oxidants and UV. The contribution of OH radicals to i-ARG damage was negligible in UV/H2O2 due to significant OH radical scavenging by cellular components. In all cases, the ARG damage rates were similar for ampR versus kanR, except for the chlorination of e-ARGs, in which the damage to ampR occurred faster than that to kanR. Chlorine and UV dose-dependent ARG inactivation levels determined in a wastewater effluent matrix could be reasonably explained by the kinetic data obtained from the phosphate buffered solutions and the expected oxidant (chlorine and OH radicals) demands by water matrix components. These results can be useful in optimizing chlorine and UV-based disinfection systems to achieve ARG inactivation. Copyright

  11. 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

    Hypoxia-inducible factor 2 alpha (HIF2α), prolyl hydroxylase domain protein 2 (PHD2), and the von Hippel Lindau tumor suppressor protein (pVHL) are three principal proteins in the oxygen-sensing pathway. Under normoxic conditions, a conserved proline in HIF2α is hydroxylated by PHD2 in an oxygen-dependent manner, and then pVHL binds and promotes the degradation of HIF2α. However, the crystal structure of the HIF2α-pVHL complex has not yet been established, and this has limited research on the interaction between HIF and pVHL. Here, we constructed a structural model of a 23-residue HIF2α peptide (528-550)-pVHL-ElonginB-ElonginC complex by using homology modeling and molecular dynamics simulations. We also applied these methods to HIF2α mutants (HYP531PRO, F540L, A530 V, A530T, and G537R) to reveal structural defects that explain how these mutations weaken the interaction with pVHL. Homology modeling and molecular dynamics simulations were used to construct a three-dimensional (3D) structural model of the HIF2α-VHL complex. Subsequently, MolProbity, an active validation tool, was used to analyze the reliability of the model. Molecular mechanics energies combined with the generalized Born and surface area continuum solvation (MM-GBSA) and solvated interaction energy (SIE) methods were used to calculate the binding free energy between HIF2a and pVHL, and the stability of the simulation system was evaluated by using root mean square deviation (RMSD) analysis. We also determined the secondary structure of the system by using the definition of secondary structure of proteins (DSSP) algorithm. Finally, we investigated the structural significance of specific point mutations known to have clinical implications. We established a reliable structural model of the HIF2α-pVHL complex, which is similar to the crystal structure of HIF1α in 1LQB. Furthermore, we compared the structural model of the HIF2α-pVHL complex and the HIF2α (HYP531P, F540L, A530V, A530T, and G537

  12. Inactivation of mrcA gene derepresses the basal-level expression of L1 and L2 β-lactamases in Stenotrophomonas maltophilia.

    Science.gov (United States)

    Lin, Cheng-Wen; Lin, Hsin-Chieh; Huang, Yi-Wei; Chung, Tung-Ching; Yang, Tsuey-Ching

    2011-09-01

    To characterize the relationship between inactivation of the mrcA gene and β-lactamase expression and β-lactams resistance in Stenotrophomonas maltophilia KJ and to investigate the involvement of ampR, ampN-ampG, ampD(I) and creBC in this. The mrcA deletion mutant KJΔmrcA was constructed to investigate the role of this putative penicillin-binding protein 1a (PBP1a) in β-lactamase expression and β-lactam resistance. The ΔampR, ΔampNG, ΔampDI and ΔcreBC alleles were introduced into KJΔmrcA, and KJΔDIΔBC and KJΔDIΔmrcAΔBC were also constructed for comparison. All the mutants and their corresponding parent strains were assayed for β-lactamase activities and MICs of β-lactams. Inactivation of mrcA caused basal L1/L2 β-lactamase production to increase by ∼100-fold, but made little difference to cefuroxime-induced β-lactamase activity and the MICs of β-lactams. The ΔmrcA-derived basal β-lactamase hyperproduction was ampR and ampN-ampG dependent. Simultaneous inactivation of ampD(I) and mrcA did not augment β-lactamase production over and above that seen in an ampD(I) mutant alone. Furthermore, we could find no evidence for a role of the creBC two-component regulatory system in β-lactamase hyperproduction in a ΔampD(I) or ΔmrcA background. Inactivation of mrcA, predicted to encode PBP1a, causes basal L1/L2 β-lactamase hyperproduction in S. maltophilia.

  13. Fumarate Mediates a Chronic Proliferative Signal in Fumarate Hydratase-Inactivated Cancer Cells by Increasing Transcription and Translation of Ferritin Genes.

    Science.gov (United States)

    Kerins, Michael John; Vashisht, Ajay Amar; Liang, Benjamin Xi-Tong; Duckworth, Spencer Jordan; Praslicka, Brandon John; Wohlschlegel, James Akira; Ooi, Aikseng

    2017-06-01

    Germ line mutations of the gene encoding the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) cause a hereditary cancer syndrome known as hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC-associated tumors harbor biallelic FH inactivation that results in the accumulation of the TCA cycle metabolite fumarate. Although it is known that fumarate accumulation can alter cellular signaling, if and how fumarate confers a growth advantage remain unclear. Here we show that fumarate accumulation confers a chronic proliferative signal by disrupting cellular iron signaling. Specifically, fumarate covalently modifies cysteine residues on iron regulatory protein 2 (IRP2), rendering it unable to repress ferritin mRNA translation. Simultaneously, fumarate increases ferritin gene transcription by activating the NRF2 (nuclear factor [erythroid-derived 2]-like 2) transcription factor. In turn, increased ferritin protein levels promote the expression of the promitotic transcription factor FOXM1 (Forkhead box protein M1). Consistently, clinical HLRCC tissues showed increased expression levels of both FOXM1 and its proliferation-associated target genes. This finding demonstrates how FH inactivation can endow cells with a growth advantage. Copyright © 2017 American Society for Microbiology.

  14. Ras-induced epigenetic inactivation of the RRAD (Ras-related associated with diabetes) gene promotes glucose uptake in a human ovarian cancer model.

    Science.gov (United States)

    Wang, Yan; Li, Guiling; Mao, Fengbiao; Li, Xianfeng; Liu, Qi; Chen, Lin; Lv, Lu; Wang, Xin; Wu, Jinyu; Dai, Wei; Wang, Guan; Zhao, Enfeng; Tang, Kai-Fu; Sun, Zhong Sheng

    2014-05-16

    RRAD (Ras-related associated with diabetes) is a small Ras-related GTPase that is frequently inactivated by DNA methylation of the CpG island in its promoter region in cancer tissues. However, the role of the methylation-induced RRAD inactivation in tumorigenesis remains unclear. In this study, the Ras-regulated transcriptome and epigenome were profiled by comparing T29H (a Ras(V12)-transformed human ovarian epithelial cell line) with T29 (an immortalized but non-transformed cell line) through reduced representation bisulfite sequencing and digital gene expression. We found that Ras(V12)-mediated oncogenic transformation was accompanied by RRAD promoter hypermethylation and a concomitant loss of RRAD expression. In addition, we found that the RRAD promoter was hypermethylated, and its transcription was reduced in ovarian cancer versus normal ovarian tissues. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine resulted in demethylation in the RRAD promoter and restored RRAD expression in T29H cells. Additionally, treatment with farnesyltransferase inhibitor FTI277 resulted in restored RRAD expression and inhibited DNA methytransferase expression and activity in T29H cells. By employing knockdown and overexpression techniques in T29 and T29H, respectively, we found that RRAD inhibited glucose uptake and lactate production by repressing the expression of glucose transporters. Finally, RRAD overexpression in T29H cells inhibited tumor formation in nude mice, suggesting that RRAD is a tumor suppressor gene. Our results indicate that Ras(V12)-mediated oncogenic transformation induces RRAD epigenetic inactivation, which in turn promotes glucose uptake and may contribute to ovarian cancer tumorigenesis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Dicty_cDB: VHL343 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available region, section 4; and merlin (NF2) gene, exons 2 through 16 and complete cds. 34 4.0 3 AC133850 |AC133850.1...ence. 34 4.0 6 AY123429 |AY123429.1 Papio anubis anubis neurofibromatosis type 2

  16. Caffeine mediates sustained inactivation of breast cancer-associated myofibroblasts via up-regulation of tumor suppressor genes.

    Science.gov (United States)

    Al-Ansari, Mysoon M; Aboussekhra, Abdelilah

    2014-01-01

    Active cancer-associated fibroblasts (CAFs) or myofibroblasts play important roles not only in the development and progression of breast carcinomas, but also in their prognosis and treatment. Therefore, targeting these cells through suppressing their supportive procarcinogenic paracrine effects is mandatory for improving the current therapies that are mainly targeting tumor cells. To this end, we investigated the effect of the natural and pharmacologically safe molecule, caffeine, on CAF cells and their various procarcinogenic effects. We have shown here that caffeine up-regulates the tumor suppressor proteins p16, p21, p53 and Cav-1, and reduces the expression/secretion of various cytokines (IL-6, TGF-β, SDF-1 and MMP-2), and down-regulates α-SMA. Furthermore, caffeine suppressed the migratory/invasiveness abilities of CAF cells through PTEN-dependent Akt/Erk1/2 inactivation. Moreover, caffeine reduced the paracrine pro-invasion/-migration effects of CAF cells on breast cancer cells. These results indicate that caffeine can inactivate breast stromal myofibroblasts. This has been confirmed by showing that caffeine also suppresses the paracrine pro-angiogenic effect of CAF cells through down-regulating HIF-1αand its downstream effector VEGF-A. Interestingly, these effects were sustained in absence of caffeine. The present findings provide a proof of principle that breast cancer myofibroblasts can be inactivated, and thereby caffeine may provide a safe and effective prevention against breast tumor growth/recurrence through inhibition of the procarcinogenic effects of active stromal fibroblasts.

  17. 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...

  18. Oncogene activation and tumor suppressor gene inactivation find their sites of expression in the changes in time and space of the age-adjusted cancer incidence rate.

    Science.gov (United States)

    Kodama, M; Kodama, T; Murakami, M

    2000-01-01

    The purpose of the present investigation is to elucidate the relation between the distribution pattern of the age-adjusted incidence rate (AAIR) changes in time and space of 15 tumors of bothe sexes and the locations of centers of centripetal-(oncogene type) and centrifugal-(tumoe suppressor gene type) forces. The fitness of the observed log AAIR data sets to the oncogene type- and the tumor suppressor gene type-equilibrium models and the locations of 2 force centers were calculated by applying the least square method of Gauss to log AAIR pair data series with and without topological data manipulations, which are so designed as to let log AAIR pair data series fit to 2 variant (x, y) frameworks, the Rect-coordinates and the Para-coordinates. The 2 variant (x, y) coordinates are defined each as an (x, y) framework with its X axis crossed at a right angle to the regression line of the original log AAIR data (the Rect-coordinates) and as another framework with its X axis run in parallel with the regression line of the original log AAIR pair data series (the Para-coordinates). The fitness test of log AAIR data series to either the oncogene activation type equilibrium model (r = -1.000) or the tumor suppressor gene inactivation type (r = 1.000) was conducted for each of the male-female type pair data and the female-male type data, for each of log AAIR changes in space and log AAIR changes in time, and for each of the 3 (x, y) frameworks in a given neoplasia of both sexes. The results obtained are given as follows: 1) The positivity rates of the fitness test to the oncogene type equilibrium model and the tumor suppressor gene type model were each 63.3% and 56.7% with the log AAIR changes in space, and 73.3% and 73.3% with log AAIR changes in time, as tested in 15 human neoplasias of both sexes. 2) Evidence was presented to indicate that the clearance of oncogene activation and tumor suppressor gene inactivation is the sine qua non premise of carciniogenesis. 3) The r

  19. Caffeine Mediates Sustained Inactivation of Breast Cancer-Associated Myofibroblasts via Up-Regulation of Tumor Suppressor Genes

    Science.gov (United States)

    Al-Ansari, Mysoon M.; Aboussekhra, Abdelilah

    2014-01-01

    Background Active cancer-associated fibroblasts (CAFs) or myofibroblasts play important roles not only in the development and progression of breast carcinomas, but also in their prognosis and treatment. Therefore, targeting these cells through suppressing their supportive procarcinogenic paracrine effects is mandatory for improving the current therapies that are mainly targeting tumor cells. To this end, we investigated the effect of the natural and pharmacologically safe molecule, caffeine, on CAF cells and their various procarcinogenic effects. Methodology/Principal Findings We have shown here that caffeine up-regulates the tumor suppressor proteins p16, p21, p53 and Cav-1, and reduces the expression/secretion of various cytokines (IL-6, TGF-β, SDF-1 and MMP-2), and down-regulates α-SMA. Furthermore, caffeine suppressed the migratory/invasiveness abilities of CAF cells through PTEN-dependent Akt/Erk1/2 inactivation. Moreover, caffeine reduced the paracrine pro-invasion/−migration effects of CAF cells on breast cancer cells. These results indicate that caffeine can inactivate breast stromal myofibroblasts. This has been confirmed by showing that caffeine also suppresses the paracrine pro-angiogenic effect of CAF cells through down-regulating HIF-1αand its downstream effector VEGF-A. Interestingly, these effects were sustained in absence of caffeine. Conclusion/Significance The present findings provide a proof of principle that breast cancer myofibroblasts can be inactivated, and thereby caffeine may provide a safe and effective prevention against breast tumor growth/recurrence through inhibition of the procarcinogenic effects of active stromal fibroblasts. PMID:24595168

  20. Caffeine mediates sustained inactivation of breast cancer-associated myofibroblasts via up-regulation of tumor suppressor genes.

    Directory of Open Access Journals (Sweden)

    Mysoon M Al-Ansari

    Full Text Available BACKGROUND: Active cancer-associated fibroblasts (CAFs or myofibroblasts play important roles not only in the development and progression of breast carcinomas, but also in their prognosis and treatment. Therefore, targeting these cells through suppressing their supportive procarcinogenic paracrine effects is mandatory for improving the current therapies that are mainly targeting tumor cells. To this end, we investigated the effect of the natural and pharmacologically safe molecule, caffeine, on CAF cells and their various procarcinogenic effects. METHODOLOGY/PRINCIPAL FINDINGS: We have shown here that caffeine up-regulates the tumor suppressor proteins p16, p21, p53 and Cav-1, and reduces the expression/secretion of various cytokines (IL-6, TGF-β, SDF-1 and MMP-2, and down-regulates α-SMA. Furthermore, caffeine suppressed the migratory/invasiveness abilities of CAF cells through PTEN-dependent Akt/Erk1/2 inactivation. Moreover, caffeine reduced the paracrine pro-invasion/-migration effects of CAF cells on breast cancer cells. These results indicate that caffeine can inactivate breast stromal myofibroblasts. This has been confirmed by showing that caffeine also suppresses the paracrine pro-angiogenic effect of CAF cells through down-regulating HIF-1αand its downstream effector VEGF-A. Interestingly, these effects were sustained in absence of caffeine. CONCLUSION/SIGNIFICANCE: The present findings provide a proof of principle that breast cancer myofibroblasts can be inactivated, and thereby caffeine may provide a safe and effective prevention against breast tumor growth/recurrence through inhibition of the procarcinogenic effects of active stromal fibroblasts.

  1. 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.

  2. Effect of constitutive inactivation of the myostatin gene on the gain in muscle strength during postnatal growth in two murine models.

    Science.gov (United States)

    Stantzou, Amalia; Ueberschlag-Pitiot, Vanessa; Thomasson, Remi; Furling, Denis; Bonnieu, Anne; Amthor, Helge; Ferry, Arnaud

    2017-02-01

    The effect of constitutive inactivation of the gene encoding myostatin on the gain in muscle performance during postnatal growth has not been well characterized. We analyzed 2 murine myostatin knockout (KO) models, (i) the Lee model (KO Lee ) and (ii) the Grobet model (KO Grobet ), and measured the contraction of tibialis anterior muscle in situ. Absolute maximal isometric force was increased in 6-month-old KO Lee and KO Grobet mice, as compared to wild-type mice. Similarly, absolute maximal power was increased in 6-month-old KO Lee mice. In contrast, specific maximal force (relative maximal force per unit of muscle mass was decreased in all 6-month-old male and female KO mice, except in 6-month-old female KO Grobet mice, whereas specific maximal power was reduced only in male KO Lee mice. Genetic inactivation of myostatin increases maximal force and power, but in return it reduces muscle quality, particularly in male mice. Muscle Nerve 55: 254-261, 2017. © 2016 Wiley Periodicals, Inc.

  3. SLIT2, a human homologue of the Drosophila Slit2 gene, has tumor suppressor activity and is frequently inactivated in lung and breast cancers.

    Science.gov (United States)

    Dallol, Ashraf; Da Silva, Nancy Fernandes; Viacava, Paolo; Minna, John D; Bieche, Ivan; Maher, Eamonn R; Latif, Farida

    2002-10-15

    Slit2 plays a vital role in axon guidance by signaling through Robo receptors. Recent evidence suggests that Slit2 protein may function in other settings because human and Xenopus Slit2 has been shown to inhibit leukocyte chemotaxis. SLIT2 protein is a putative ligand for the ROBO receptors. We recently demonstrated that ROBO1 is inactivated by promoter region hypermethylation in cancers; furthermore, tumor suppressor activity has not been shown. Thus, the importance of ROBO1 inactivation in human cancer is uncertain. Therefore, we investigated the status of SLIT2 located at 4p15.2 in lung and breast cancers. Although somatic SLIT2 mutations were not detected, epigenetic inactivation was common. SLIT2 promoter methylation was detected in 59% of breast cancer, 77% of non-small cell lung cancer, and 55% of small cell lung cancer cell lines. In these tumor lines, SLIT2 expression was restored by treatment with a demethylating agent. SLIT2 promoter methylation was detected in 43% of breast cancer, 53% of non-small cell lung cancer, and 36% of small cell lung cancer primary tumors. The majority of methylated tumors demonstrated allelic loss at 4p15.2. In addition, SLIT2 expression was down-regulated in methylated breast tumors, relative to normal control, as demonstrated by quantitative real-time reverse transcription-PCR. Overexpression of SLIT2 suppressed >70% of colony growth in each of three breast tumor lines (with either absent or low SLIT2 expression). Because SLIT2 is primarily a secreted protein, SLIT2-conditioned medium suppressed the growth of several breast cancer lines (with absent or weak SLIT2expression) by 26-51% but had no significant effect on a breast tumor cell line that expresses normal levels of SLIT2. These findings demonstrate that SLIT2 is frequently inactivated in lung and breast cancer by promoter region hypermethylation and allele loss and is an excellent candidate for the lung and breast tumor suppressor gene previously mapped to 4p15.2.

  4. Rapid and efficient CRISPR/Cas9 gene inactivation in human neurons during human pluripotent stem cell differentiation and direct reprogramming.

    Science.gov (United States)

    Rubio, Alicia; Luoni, Mirko; Giannelli, Serena G; Radice, Isabella; Iannielli, Angelo; Cancellieri, Cinzia; Di Berardino, Claudia; Regalia, Giulia; Lazzari, Giovanna; Menegon, Andrea; Taverna, Stefano; Broccoli, Vania

    2016-11-18

    The CRISPR/Cas9 system is a rapid and customizable tool for gene editing in mammalian cells. In particular, this approach has widely opened new opportunities for genetic studies in neurological disease. Human neurons can be differentiated in vitro from hPSC (human Pluripotent Stem Cells), hNPCs (human Neural Precursor Cells) or even directly reprogrammed from fibroblasts. Here, we described a new platform which enables, rapid and efficient CRISPR/Cas9-mediated genome targeting simultaneously with three different paradigms for in vitro generation of neurons. This system was employed to inactivate two genes associated with neurological disorder (TSC2 and KCNQ2) and achieved up to 85% efficiency of gene targeting in the differentiated cells. In particular, we devised a protocol that, combining the expression of the CRISPR components with neurogenic factors, generated functional human neurons highly enriched for the desired genome modification in only 5 weeks. This new approach is easy, fast and that does not require the generation of stable isogenic clones, practice that is time consuming and for some genes not feasible.

  5. 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.

  6. X chromosome-linked and mitochondrial gene control of Leber hereditary optic neuropathy: Evidence from segregation analysis for dependence on X chromosome inactivation

    Energy Technology Data Exchange (ETDEWEB)

    Xiangdong Bu; Rotter, J.I. (Cedars-Sinai Medical Center, Los Angeles, CA (United States) Univ. of California, Los Angeles (United States))

    1991-09-15

    Leber hereditary optic neuropathy (LHON) has been shown to involve mutation(s) of mitochondrial DNA, yet there remain several confusing aspects of its inheritance not explained by mitochondrial inheritance alone, including male predominance, reduced penetrance, and a later age of onset in females. By extending segregation analysis methods to disorders that involve both a mitochondrial and a nuclear gene locus, the authors show that the available pedigree data for LHON are most consistent with a two-locus disorder, with one responsible gene being mitochondrial and the other nuclear and X chromosome-linked. Furthermore, they have been able to extend the two-locus analytic method and demonstrate that a proportion of affected females are likely heterozygous at the X chromosome-linked locus and are affected due to unfortunate X chromosome inactivation, thus providing an explanation for the later age of onset in females. The estimated penetrance for a heterozygous female is 0.11{plus minus}0.02. The calculated frequency of the X chromosome-linked gene for LHON is 0.l08. Among affected females, 60% are expected to be heterozygous, and the remainder are expected to be homozygous at the responsible X chromosome-linked locus.

  7. Transformation of blackgram (Vigna mungo (L.) Hepper) by barley chitinase and ribosome-inactivating protein genes towards improving resistance to Corynespora leaf spot fungal disease.

    Science.gov (United States)

    Chopra, Rajan; Saini, Raman

    2014-12-01

    Blackgram (Vigna mungo (L.) Hepper), an important grain legume crop, is sensitive to many fungal pathogens including Corynespora cassiicola, the causal agent of corynespora leaf spot disease. In the present study, plasmid pGJ42 harboring neomycin phosphotransferase (nptII) a selectable marker gene, the barley antifungal genes chitinase (AAA56786) and ribosome-inactivating protein (RIP; AAA32951) were used for the transformation, to develop fungal resistance for the first time in blackgram. The presence and integration of transgene into the blackgram genome was confirmed by PCR and Southern analysis with an overall transformation frequency of 10.2 %. Kanamycin selection and PCR analysis of T0 progeny revealed the inheritance of transgene in Mendelian fashion (3:1). Transgenic plants (T1), evaluated for fungal resistance by in vitro antifungal assay, arrested the growth of C. cassiicola up to 25-40 % over the wild-type plants. In fungal bio-assay screening, the transgenic plants (T1) sprayed with C. cassiicola spores showed a delay in onset of disease along with their lesser extent in terms of average number of diseased leaves and reduced number and size of lesions. The percent disease protection among different transformed lines varies in the range of 27-47 % compare to control (untransformed) plants. These results demonstrate potentiality of chitinase and RIP from a heterologous source in developing fungal disease protection in blackgram and can be helpful in increasing the production of blackgram.

  8. Disease Control in Animals Using Molecular Technology by Inactivation of ASO, RNAi and ss-siRNA Genes

    Directory of Open Access Journals (Sweden)

    Muhamad Ali

    2014-03-01

    Full Text Available Globalization causes high mobility of human and livestock, hence increase the transmission of infectious diseases, including avian influenza, severe acute respiratory syndrome (SARS, and swine influenza. Therefore, prevention of those diseases is required. Vaccines are effective to prevent infectious diseases; however, their development takes a long time and they cannot provide immediate protection in pandemic cases. This paper describes several gene silencing technologies including antisense oligonucleotide (ASO, RNA interference (RNAi and single strand-small interfering RNA (ss-siRNA for controlling diseases. The primary mechanism of these technologies is inhibition of gene expression, typically by causing the destruction of specific RNA molecule of the pathogen. The use of gene silencing technologies is expected to give new alternative that is more effective in eradication of infectious diseases in animals before threaten human being.

  9. Over-expression of XIST, the Master Gene for X Chromosome Inactivation, in Females With Major Affective Disorders

    Directory of Open Access Journals (Sweden)

    Baohu Ji

    2015-08-01

    Research in context: Due to lack of biological markers, diagnosis and treatment of psychiatric disorders are subjective. There is utmost urgency to identify biomarkers for clinics, research, and drug development. We found that XIST and KDM5C gene expression may be used as a biological marker for diagnosis of major affective disorders in a significantly large subset of female patients from the general population. Our studies show that over-expression of XIST and some X-linked escapee genes may be a common mechanism for development of psychiatric disorders between the patients with rare genetic diseases (XXY or XXX and the general population of female psychiatric patients.

  10. Combined Inactivation and Expression Strategy To Study Gene Function under Physiological Conditions: Application to Identification of New Escherichia coli Adhesins

    OpenAIRE

    Roux, Agnès; Beloin, Christophe; Ghigo, Jean-Marc

    2005-01-01

    In bacteria, whereas disruption methods have been improved recently, the use of plasmid complementation strategies are still subject to limitations, such as cloning difficulties, nonphysiological levels of gene expression, or a requirement for antibiotics as plasmid selection pressure. Moreover, because of the pleiotropic modifications of cell physiology often induced by plasmid-based complementation, these strategies may introduce biases when biological process such as adhesion or biofilm fo...

  11. Inactivation of the Huntington's disease gene (Hdh impairs anterior streak formation and early patterning of the mouse embryo

    Directory of Open Access Journals (Sweden)

    Conlon Ronald A

    2005-08-01

    Full Text Available Abstract Background Huntingtin, the HD gene encoded protein mutated by polyglutamine expansion in Huntington's disease, is required in extraembryonic tissues for proper gastrulation, implicating its activities in nutrition or patterning of the developing embryo. To test these possibilities, we have used whole mount in situ hybridization to examine embryonic patterning and morphogenesis in homozygous Hdhex4/5 huntingtin deficient embryos. Results In the absence of huntingtin, expression of nutritive genes appears normal but E7.0–7.5 embryos exhibit a unique combination of patterning defects. Notable are a shortened primitive streak, absence of a proper node and diminished production of anterior streak derivatives. Reduced Wnt3a, Tbx6 and Dll1 expression signify decreased paraxial mesoderm and reduced Otx2 expression and lack of headfolds denote a failure of head development. In addition, genes initially broadly expressed are not properly restricted to the posterior, as evidenced by the ectopic expression of Nodal, Fgf8 and Gsc in the epiblast and T (Brachyury and Evx1 in proximal mesoderm derivatives. Despite impaired posterior restriction and anterior streak deficits, overall anterior/posterior polarity is established. A single primitive streak forms and marker expression shows that the anterior epiblast and anterior visceral endoderm (AVE are specified. Conclusion Huntingtin is essential in the early patterning of the embryo for formation of the anterior region of the primitive streak, and for down-regulation of a subset of dynamic growth and transcription factor genes. These findings provide fundamental starting points for identifying the novel cellular and molecular activities of huntingtin in the extraembryonic tissues that govern normal anterior streak development. This knowledge may prove to be important for understanding the mechanism by which the dominant polyglutamine expansion in huntingtin determines the loss of neurons in

  12. Inactivation of polyketide synthase and related genes results in the loss of complex lipids in Mycobacterium tuberculosis H37Rv

    OpenAIRE

    Waddell, S J; Chung, G A; Gibson, K J C; Everett, M J; Minnikin, D E; Besra, G S; Butcher, P D

    2005-01-01

    Aims: Phthiocerol dimycocerosate (PDIM) waxes and other lipids are necessary for successful Mycobacterium tuberculosis infection, although the exact role of PDIM in host-pathogen interactions remains unclear. In this study, we investigated the contribution of tesA, drrB, pks6 and pks11 genes in complex lipid biosynthesis in M. tuberculosis. Methods and \\ud \\ud Results: Four mutants were selected from M. tuberculosis H37Rv transposon mutant library. The transposon insertion sites were confirme...

  13. Hopping into a hot seat: Role of DNA structural features on IS5-mediated gene activation and inactivation under stress.

    Directory of Open Access Journals (Sweden)

    M Zafri Humayun

    Full Text Available Insertion sequence elements (IS elements are proposed to play major roles in shaping the genetic and phenotypic landscapes of prokaryotic cells. Recent evidence has raised the possibility that environmental stress conditions increase IS hopping into new sites, and often such hopping has the phenotypic effect of relieving the stress. Although stress-induced targeted mutations have been reported for a number of E. coli genes, the glpFK (glycerol utilization and the cryptic bglGFB (β-glucoside utilization systems are among the best characterized where the effects of IS insertion-mediated gene activation are well-characterized at the molecular level. In the glpFK system, starvation of cells incapable of utilizing glycerol leads to an IS5 insertion event that activates the glpFK operon, and enables glycerol utilization. In the case of the cryptic bglGFB operon, insertion of IS5 (and other IS elements into a specific region in the bglG upstream sequence has the effect of activating the operon in both growing cells, and in starving cells. However, a major unanswered question in the glpFK system, the bgl system, as well as other examples, has been why the insertion events are promoted at specific locations, and how the specific stress condition (glycerol starvation for example can be mechanistically linked to enhanced insertion at a specific locus. In this paper, we show that a specific DNA structural feature (superhelical stress-induced duplex destabilization, SIDD is associated with "stress-induced" IS5 insertion in the glpFK, bglGFB, flhDC, fucAO and nfsB systems. We propose a speculative mechanistic model that links specific environmental conditions to the unmasking of an insertional hotspot in the glpFK system. We demonstrate that experimentally altering the predicted stability of a SIDD element in the nfsB gene significantly impacts IS5 insertion at its hotspot.

  14. Non-homologous End-joining Genes are not Inactivated in Human Radiation-induced Sarcomas with Genomic Instability

    OpenAIRE

    Sandrine H., LEFEVRE; Arnaud, COQUELLE; Nathalie, GONIN LAURENT; Andrej, COR; Nicolas, VOGT; Laurent, CHAUVEINC; Philippe, ANRACT; Bernard, DUTRILLAUX; Sylvie, CHEVILLARD; Bernard, MALFOY; Institut for Histology and Embryology, Medical Faculty; Institut Curie, Service de Radiotherapie; Hopital Cochin. Service de Chirurgie Orthopedique et Oncologique; CEA, DSV DRR; Institut Curie-CNRS-UPMC UMR7147, CEA LRC38

    2005-01-01

    DNA double-strand break (DSB) repair pathways are implicated in the maintenance of genomic stability. However the alterations of these pathways, as may occur in human tumor cells with strong genomic instability, remain poorly characterized. We analyzed the loss of heterozygosity (LOH) and the presence of mutations for a series of genes implicated in DSB repair by non-homologous end-joining in five radiation-induced sarcomas devoid of both active Tp53 and Rb1. LOH was recurrently observed for ...

  15. Inactivation of polyketide synthase and related genes results in the loss of complex lipids in Mycobacterium tuberculosis H37Rv.

    Science.gov (United States)

    Waddell, S J; Chung, G A; Gibson, K J C; Everett, M J; Minnikin, D E; Besra, G S; Butcher, P D

    2005-01-01

    Phthiocerol dimycocerosate (PDIM) waxes and other lipids are necessary for successful Mycobacterium tuberculosis infection, although the exact role of PDIM in host-pathogen interactions remains unclear. In this study, we investigated the contribution of tesA, drrB, pks6 and pks11 genes in complex lipid biosynthesis in M. tuberculosis. Four mutants were selected from M. tuberculosis H37Rv transposon mutant library. The transposon insertion sites were confirmed to be within the M. tuberculosis open reading frames for tesA (a probable thioesterase), drrB (predicted ABC transporter), pks11 (putative chalcone synthase) and pks6 (polyketide synthase). The first three of these transposon mutants were unable to generate PDIM and the fourth lacked novel polar lipids. Mycobacterium tuberculosis can be cultivated in vitro without the involvement of certain lipid synthesis genes, which may be necessary for in vivo pathogenicity. The use of transposon mutants is a new functional genomic approach for the eventual definition of the mycobacterial 'lipidome'.

  16. Expression of inactive glutathione peroxidase 4 leads to embryonic lethality, and inactivation of the Alox15 gene does not rescue such knock-in mice.

    Science.gov (United States)

    Brütsch, Simone Hanna; Wang, Chi Chiu; Li, Lu; Stender, Hannelore; Neziroglu, Nilgün; Richter, Constanze; Kuhn, Hartmut; Borchert, Astrid

    2015-02-01

    Glutathione peroxidases (Gpx) and lipoxygenases (Alox) are functional counterplayers in the metabolism of hydroperoxy lipids that regulate cellular redox homeostasis. Gpx4 is a moonlighting protein that has been implicated not only as an enzyme in anti-oxidative defense, gene expression regulation, and programmed cell death, but also as a structural protein in spermatogenesis. Homozygous Gpx4 knock-out mice are not viable, but molecular reasons for intrauterine lethality are not completely understood. This study was aimed at investigating whether the lack of catalytic activity or the impaired function as structural protein is the dominant reason for embryonic lethality. We further explored whether the pro-oxidative enzyme mouse 12/15 lipoxygenase (Alox15) plays a major role in embryonic lethality of Gpx4-deficient mice. To achieve these goals, we first created knock-in mice, which express a catalytically inactive Gpx4 mutant (Sec46Ala). As homozygous Gpx4-knock-out mice Sec46Ala-Gpx4(+/+) knock-in animals are not viable but undergo intrauterine resorption between embryonic day 6 and 7 (E6-7). In contrast, heterozygous knock-in mice (Sec46Ala-Gpx4(-/+)) are viable, fertile and do not show major phenotypic alterations. Interestingly, homozygous Alox15 deficiency did not rescue the U46A-Gpx4(+/+) mice from embryonic lethality. In fact, when heterozygous U46A-Gpx4(-/+) mice were stepwise crossed into an Alox15-deficent background, no viable U46A-Gpx4(+/+)+Alox15(-/-) individuals were obtained. However, we were able to identify U46A-Gpx4(+/+)+Alox15(-/-) embryos in the state of resorption around E7. These data suggest that the lack of catalytic activity is the major reason for the embryonic lethality of Gpx4(-/-) mice and that systemic inactivation of the Alox15 gene does not rescue homozygous knock-in mice expressing catalytically silent Gpx4.

  17. Development of antibiotic resistance and up-regulation of the antimutator gene pfpI in mutator Pseudomonas aeruginosa due to inactivation of two DNA oxidative repair genes (mutY, mutM)

    DEFF Research Database (Denmark)

    Mandsberg, Lotte Frigaard; Macia, Maria D.; Bergmann, Kirsten R.

    2011-01-01

    Prevention and correction of oxidative DNA lesions in Pseudomonas aeruginosa is ensured by the DNA oxidative repair system (GO). Single inactivation of mutT, mutY and mutM involved in GO led to elevated mutation rates (MRs) that correlated to increased development of resistance to antibiotics...... showed only a fivefold increase, whereas the single mutant PAOMMgm (mutM) showed a nonsignificant increase in MR compared with PAO1 and the single mutants. Mutations in the regulator nfxB leading to hyperexpression of MexCD-OprJ efflux pump were found as the mechanism of resistance to ciprofloxacin...... in the double mutant. A better fitness of the mutator compared with PAO1 was found in growth competition experiments in the presence of ciprofloxacin at concentrations just below minimal inhibitory concentration. Up-regulation of the antimutator gene pfpI, that has been shown to provide protection to oxidative...

  18. Cephem Potentiation by Inactivation of Nonessential Genes Involved in Cell Wall Biogenesis of beta-Lactamase-Producing Escherichia coli

    DEFF Research Database (Denmark)

    Baker, Kristin R.; Sigurdardottir, Helga Høeg; Jana, Bimal

    2017-01-01

    was transferred by conjugation to seven isogenic deletion mutants exhibiting cephem hypersusceptibility. The effect of each mutation was evaluated by comparing the MICs in the wild type and the mutant harboring the same plasmid. Mutation of two genes encoding proteins involved in cell wall biosynthesis, dap......F and mrcB, restored susceptibility to cefoxitin (FOX) and reduced the MICs of cefotaxime and ceftazidime, respectively, from the resistant to the intermediate category according to clinical breakpoints. The same mutants harboring a CTX-M-1-encoding plasmid fell into the intermediate or susceptible category...... cost in a ΔmrcB mutant, whereas a ΔdapF mutant had a 3-fold longer lag phase than the wild type, suggesting that drugs targeting DapF may display antimicrobial activity, in addition to synergizing with selected cephems. DapF appeared to be a potential FOX helper drug target candidate, since dap...

  19. Variable phenotypic penetrance of thrombosis in adult mice after tissue-selective and temporally controlled Thbd gene inactivation.

    Science.gov (United States)

    van Mens, Thijs E; Liang, Hai-Po H; Basu, Sreemanti; Hernandez, Irene; Zogg, Mark; May, Jennifer; Zhan, Min; Yang, Qiuhui; Foeckler, Jamie; Kalloway, Shawn; Sood, Rashmi; Karlson, Caren Sue; Weiler, Hartmut

    2017-06-27

    Thrombomodulin (Thbd) exerts pleiotropic effects on blood coagulation, fibrinolysis, and complement system activity by facilitating the thrombin-mediated activation of protein C and thrombin-activatable fibrinolysis inhibitor and may have additional thrombin- and protein C (pC)-independent functions. In mice, complete Thbd deficiency causes embryonic death due to defective placental development. In this study, we used tissue-selective and temporally controlled Thbd gene ablation to examine the function of Thbd in adult mice. Selective preservation of Thbd function in the extraembryonic ectoderm and primitive endoderm via the Meox2Cre-transgene enabled normal intrauterine development of Thbd-deficient (Thbd -/- ) mice to term. Half of the Thbd -/- offspring expired perinatally due to thrombohemorrhagic lesions. Surviving Thbd -/- animals only rarely developed overt thrombotic lesions, exhibited low-grade compensated consumptive coagulopathy, and yet exhibited marked, sudden-onset mortality. A corresponding pathology was seen in mice in which the Thbd gene was ablated after reaching adulthood. Supplementation of activated PC by transgenic expression of a partially Thbd-independent murine pC zymogen prevented the pathologies of Thbd -/- mice. However, Thbd -/- females expressing the PC transgene exhibited pregnancy-induced morbidity and mortality with near-complete penetrance. These findings suggest that Thbd function in nonendothelial embryonic tissues of the placenta and yolk sac affects through as-yet-unknown mechanisms the penetrance and severity of thrombosis after birth and provide novel opportunities to study the role of the natural Thbd-pC pathway in adult mice and during pregnancy.

  20. Microsatellite instability in pediatric high grade glioma is associated with genomic profile and differential target gene inactivation.

    Directory of Open Access Journals (Sweden)

    Marta Viana-Pereira

    Full Text Available High grade gliomas (HGG are one of the leading causes of cancer-related deaths in children, and there is increasing evidence that pediatric HGG may harbor distinct molecular characteristics compared to adult tumors. We have sought to clarify the role of microsatellite instability (MSI in pediatric versus adult HGG. MSI status was determined in 144 patients (71 pediatric and 73 adults using a well established panel of five quasimonomorphic mononucleotide repeat markers. Expression of MLH1, MSH2, MSH6 and PMS2 was determined by immunohistochemistry, MLH1 was assessed for mutations by direct sequencing and promoter methylation using MS-PCR. DNA copy number profiles were derived using array CGH, and mutations in eighteen MSI target genes studied by multiplex PCR and genotyping. MSI was found in 14/71 (19.7% pediatric cases, significantly more than observed in adults (5/73, 6.8%; p = 0.02, Chi-square test. MLH1 expression was downregulated in 10/13 cases, however no mutations or promoter methylation were found. MSH6 was absent in one pediatric MSI-High tumor, consistent with an inherited mismatch repair deficiency associated with germline MSH6 mutation. MSI was classed as Type A, and associated with a remarkably stable genomic profile. Of the eighteen classic MSI target genes, we identified mutations only in MSH6 and DNAPKcs and described a polymorphism in MRE11 without apparent functional consequences in DNA double strand break detection and repair. This study thus provides evidence for a potential novel molecular pathway in a proportion of gliomas associated with the presence of MSI.

  1. MUTATIONS IN THE VHL GENE FRIOM POTASSIUM BROMATE-INDUCED RAT CLEAR CELL RENAL TUMORS

    Science.gov (United States)

    Potassium bromate (KBrO3) is a rat renal carcinogen and a major drinking water disinfection by-product in water disinfected with ozone. Clear cell renal tumors, the most common form of human renal epithelial neoplasm, are rare in animals but are inducible by KBrO3 in F344 rats. ...

  2. Inactivation Data.xlsx

    Data.gov (United States)

    U.S. Environmental Protection Agency — The data set is a spreadsheet that contains results of inactivation experiments that were conducted to to determine the effectiveness of chlorine in inactivating B....

  3. The glucose metabolite methylglyoxal inhibits expression of the glucose transporter genes by inactivating the cell surface glucose sensors Rgt2 and Snf3 in yeast.

    Science.gov (United States)

    Roy, Adhiraj; Hashmi, Salman; Li, Zerui; Dement, Angela D; Cho, Kyu Hong; Kim, Jeong-Ho

    2016-03-01

    Methylglyoxal (MG) is a cytotoxic by-product of glycolysis. MG has inhibitory effect on the growth of cells ranging from microorganisms to higher eukaryotes, but its molecular targets are largely unknown. The yeast cell-surface glucose sensors Rgt2 and Snf3 function as glucose receptors that sense extracellular glucose and generate a signal for induction of expression of genes encoding glucose transporters (HXTs). Here we provide evidence that these glucose sensors are primary targets of MG in yeast. MG inhibits the growth of glucose-fermenting yeast cells by inducing endocytosis and degradation of the glucose sensors. However, the glucose sensors with mutations at their putative ubiquitin-acceptor lysine residues are resistant to MG-induced degradation. These results suggest that the glucose sensors are inactivated through ubiquitin-mediated endocytosis and degraded in the presence of MG. In addition, the inhibitory effect of MG on the glucose sensors is greatly enhanced in cells lacking Glo1, a key component of the MG detoxification system. Thus the stability of these glucose sensors seems to be critically regulated by intracellular MG levels. Taken together, these findings suggest that MG attenuates glycolysis by promoting degradation of the cell-surface glucose sensors and thus identify MG as a potential glycolytic inhibitor. © 2016 Roy, Hashmi, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  4. Inactivation of the gbpA gene of Streptococcus mutans alters structural and functional aspects of plaque biofilm which are compensated by recombination of the gtfB and gtfC genes.

    Science.gov (United States)

    Hazlett, K R; Mazurkiewicz, J E; Banas, J A

    1999-08-01

    Inactivation of the gbpA gene of Streptococcus mutans increases virulence in a gnotobiotic rat model and also promotes in vivo accumulation of organisms in which gtfB and gtfC have recombined to reduce virulence (K. R. O. Hazlett, S. M. Michalek, and J. A. Banas, Infect. Immun. 66:2180-2185, 1998). These changes in virulence were hypothesized to result from changes in plaque structure. We have utilized an in vitro plaque model to test the hypothesis that the absence of GbpA alters S. mutans plaque structure and that the presence of gtfBC recombinant organisms within a gbpA background restores a wild-type (wt)-like plaque structure. When grown in the presence of sucrose within hydroxyapatite-coated wells, the wt S. mutans plaque consisted primarily of large aggregates which did not completely coat the hydroxyapatite surface, whereas the gbpA mutant plaque consisted of a uniform layer of smaller aggregates which almost entirely coated the hydroxyapatite. If 25% of the gbpA mutants used as inoculum were also gtfBC recombinants (gbpA/25%gtfBC), a wt-like plaque was formed. These changes in plaque structure correlated with differences in susceptibility to ampicillin; gbpA plaque organisms were more susceptible than organisms in either the wt or gbpA/25%gtfBC plaques. These data allow the conclusion that GbpA contributes to S. mutans plaque biofilm development. Since the changes in plaque structure detailed in this report correlate well with previously observed changes in virulence, it seems likely that S. mutans biofilm structure influences virulence. A potential model for this influence, which can account for the gtfBC recombination compensating gbpA inactivation, is that the ratio of glucan to glucan-binding protein is a critical factor in plaque development.

  5. Inactivation of allergens and toxins.

    Science.gov (United States)

    Morandini, Piero

    2010-11-30

    Plants are replete with thousands of proteins and small molecules, many of which are species-specific, poisonous or dangerous. Over time humans have learned to avoid dangerous plants or inactivate many toxic components in food plants, but there is still room for ameliorating food crops (and plants in general) in terms of their allergens and toxins content, especially in their edible parts. Inactivation at the genetic rather than physical or chemical level has many advantages and classical genetic approaches have resulted in significant reduction of toxin content. The capacity, offered by genetic engineering, of turning off (inactivating) specific genes has opened up the possibility of altering the plant content in a far more precise manner than previously available. Different levels of intervention (genes coding for toxins/allergens or for enzymes, transporters or regulators involved in their metabolism) are possible and there are several tools for inactivating genes, both direct (using chemical and physical mutagens, insertion of transposons and other genetic elements) and indirect (antisense RNA, RNA interference, microRNA, eventually leading to gene silencing). Each level/strategy has specific advantages and disadvantages (speed, costs, selectivity, stability, reversibility, frequency of desired genotype and regulatory regime). Paradigmatic examples from classical and transgenic approaches are discussed to emphasize the need to revise the present regulatory process. Reducing the content of natural toxins is a trade-off process: the lesser the content of natural toxins, the higher the susceptibility of a plant to pests and therefore the stronger the need to protect plants. As a consequence, more specific pesticides like Bt are needed to substitute for general pesticides. Copyright © 2010 Elsevier B.V. All rights reserved.

  6. 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.

  7. 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

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    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. 

  10. Inactivation of GDP-fucose transporter gene (Slc35c1) in CHO cells by ZFNs, TALENs and CRISPR-Cas9 for production of fucose-free antibodies.

    Science.gov (United States)

    Chan, Kah Fai; Shahreel, Wahyu; Wan, Corrine; Teo, Gavin; Hayati, Noor; Tay, Shi Jie; Tong, Wen Han; Yang, Yuansheng; Rudd, Pauline M; Zhang, Peiqing; Song, Zhiwei

    2016-03-01

    Removal of core fucose from N-glycans attached to human IgG1 significantly enhances its affinity for the receptor FcγRIII and thereby dramatically improves its antibody-dependent cellular cytotoxicity activity. While previous works have shown that inactivation of fucosyltransferase 8 results in mutants capable of producing fucose-free antibodies, we report here the use of genome editing techniques, namely ZFNs, TALENs and the CRISPR-Cas9, to inactivate the GDP-fucose transporter (SLC35C1) in Chinese hamster ovary (CHO) cells. A FACS approach coupled with a fucose-specific lectin was developed to rapidly isolate SLC35C1-deficient cells. Mass spectrometry analysis showed that both EPO-Fc produced in mutants arising from CHO-K1 and anti-Her2 antibody produced in mutants arising from a pre-existing antibody-producing CHO-HER line lacked core fucose. Lack of functional SLC35C1 in these cells does not affect cell growth or antibody productivity. Our data demonstrate that inactivating Slc35c1 gene represents an alternative approach to generate CHO cells for production of fucose-free antibodies. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Inactivation of the gbpA Gene of Streptococcus mutans Increases Virulence and Promotes In Vivo Accumulation of Recombinations between the Glucosyltransferase B and C Genes

    Science.gov (United States)

    Hazlett, Karsten R. O.; Michalek, Suzanne M.; Banas, Jeffrey A.

    1998-01-01

    Glucan-binding protein A (GbpA) of Streptococcus mutans has been hypothesized to promote sucrose-dependent adherence and the cohesiveness of plaque and therefore to contribute to caries formation. We have analyzed the adherence properties and virulence of isogenic gbpA mutants relative to those of wild-type S. mutans. Contrary to expectations, the gbpA mutant strains displayed enhanced sucrose-dependent adherence in vitro and enhanced cariogenicity in vivo. In vitro, S. mutans was grown in the presence of [3H]thymidine and sucrose within glass vials. When grown with constant rotation, significantly higher levels of gbpA mutant organisms than of wild type remained adherent to the vial walls. Postgrowth vortexing of rotated cultures significantly decreased adherence of wild-type organisms, whereas the adherence of gbpA mutant organisms was unaffected. In the gnotobiotic rat model, the gbpA mutant strain was hypercariogenic though the colonization levels were not significantly different from those of the wild type. The gbpA mutant strain became enriched in vivo with organisms that had undergone a recombination involving the gtfB and gtfC genes. The incidence of gtfBC recombinant organisms increased as a function of dietary sucrose availability and was inversely correlated with caries development. We propose that the absence of GbpA elevates the cariogenic potential of S. mutans by altering the structure of plaque. However, the hypercariogenic plaque generated by gbpA mutant organisms may be suboptimal for S. mutans, leading to the accumulation of gtfBC recombinants whose reduced glucosyltransferase activity restores a less cariogenic plaque structure. PMID:9573105

  12. Silence of the fathers: early X inactivation.

    Science.gov (United States)

    Cheng, Mimi K; Disteche, Christine M

    2004-08-01

    X chromosome inactivation is the mammalian answer to the dilemma of dosage compensation between males and females. The study of this fascinating form of chromosome-wide gene regulation has yielded surprising insights into early development and cellular memory. In the past few months, three papers reported unexpected findings about the paternal X chromosome (X(p)). All three studies agree that the X(p) is imprinted to become inactive earlier than ever suspected during embryonic development. Although apparently incomplete, this early form of inactivation insures dosage compensation throughout development. Silencing of the X(p) persists in cells of extraembryonic tissues, but it is erased and followed by random X inactivation in cells of the embryo proper. These findings challenge several aspects of the current view of X inactivation during early development and may have profound impact on studies of pluripotency and epigenetics.

  13. 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

  14. Familial pheochromocytoma associated with a novel mutation in the von Hippel-Lindau gene

    Energy Technology Data Exchange (ETDEWEB)

    Gross, D.J.; Avishai, N.; Meiner, V.; Abeliovich, D.; Filon, D. [Hadassah Univ. Hospital and the Hebrew Univ.-Hadassah Medical School, Jerusalem (Israel)] [and others

    1996-01-01

    We report a three generation, 25 member kindred with familial pheochromocytoma. Seven subjects of generations I and II had pheochromocytoma, in five of the seven, the tumors were bilateral, and in two of the seven, the tumors were both adrenal and extraadrenal. One patient also had a carotid body chemodectoma, and one patient had a malignant adrenal tumor and abdominal paraganglioma. In the patient with the chemodectoma, a cerebellar hemagioblastoma became manifest 25 yr after his initial diagnosis with pheochromocytoma, leading only then to a clinical diagnosis of von Hippel-Lindau disease (VHL). A mutational analysis of the VHL gene revealed a novel nucleotide 709 G{r_arrow}T transversion present in all affected subjects and in four presymptomatic children. In familial pheochromocytoma the diagnosis of VHL should be considered, even when the formal criteria for diagnosis of the syndrome are lacking. 16 refs., 2 figs.

  15. Carotenoid and vitamin intake, von Hippel-Lindau gene mutations and sporadic renal cell carcinoma.

    NARCIS (Netherlands)

    Dijk, B.A.C. van; Schouten, L.J.; Oosterwijk, E.; Hulsbergen- van de Kaa, C.A.; Kiemeney, L.A.L.M.; Goldbohm, R.A.; Schalken, J.A.; Brandt, P.A. van den

    2008-01-01

    OBJECTIVE: We investigated whether dietary carotenoid and vitamin intake and supplemental vitamin use were inversely associated with RCC risk and with Von Hippel-Lindau (VHL)-gene mutations in clear-cell renal cell carcinoma (RCC). METHODS: The Netherlands Cohort Study on diet and cancer (NLCS)

  16. Carotenoid and vitamin intake, von Hippel-Lindau gene mutations and sporadic renal cell carcinoma

    NARCIS (Netherlands)

    Dijk, B.A.C. van; Schouten, L.J.; Oosterwijk, E.; Hulsbergen van de; Kaa, C.A.; Kiemeney, L.A.L.M.; Goldbohm, R.A.; Schalken, J.A.; Brandt, P.A. van den

    2008-01-01

    Objective: We investigated whether dietary carotenoid and vitamin intake and supplemental vitamin use were inversely associated with RCC risk and with Von Hippel-Lindau (VHL)-gene mutations in clear-cell renal cell carcinoma (RCC). Methods: The Netherlands Cohort Study on diet and cancer (NLCS)

  17. Inactivation of rabies virus.

    Science.gov (United States)

    Wu, Guanghui; Selden, David; Fooks, Anthony R; Banyard, Ashley

    2017-05-01

    Rabies virus is a notifiable pathogen that must be handled in high containment facilities where national and international guidelines apply. For the effective inactivation of rabies virus, a number of reagents were tested. Virkon S (1%) solution caused more than 4log reduction of rabies virus in culture medium supplemented with 10% foetal calf serum within 1min. Isopropyl alcohol (70%) treatment resulted in >3log reduction of rabies virus within 20s when applied at a ratio of 19:1, making it a suitable agent for surface decontamination whereas 70% ethanol was ineffective. Rabies virus (from 102.33 to 103ffu/ml) was also inactivated when cell cultures were fixed with 3% or 4% paraformaldehyde for 30min. Regardless of inactivation procedure, when taking inactivated virus preparations out of a biological containment envelope, proof of inocuity must be demonstrated to cover any possible error/deviation from procedure. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  18. A genetic mechanism for emergence of races in Fusarium oxysporum f. sp. lycopersici: inactivation of avirulence gene AVR1 by transposon insertion.

    Directory of Open Access Journals (Sweden)

    Keigo Inami

    Full Text Available Compatible/incompatible interactions between the tomato wilt fungus Fusarium oxysporum f. sp. lycopersici (FOL and tomato Solanum lycopersicum are controlled by three avirulence genes (AVR1-3 in FOL and the corresponding resistance genes (I-I3 in tomato. The three known races (1, 2 and 3 of FOL carry AVR genes in different combinations. The current model to explain the proposed order of mutations in AVR genes is: i FOL race 2 emerged from race 1 by losing the AVR1 and thus avoiding host resistance mediated by I (the resistance gene corresponding to AVR1, and ii race 3 emerged when race 2 sustained a point mutation in AVR2, allowing it to evade I2-mediated resistance of the host. Here, an alternative mechanism of mutation of AVR genes was determined by analyses of a race 3 isolate, KoChi-1, that we recovered from a Japanese tomato field in 2008. Although KoChi-1 is race 3, it has an AVR1 gene that is truncated by the transposon Hormin, which belongs to the hAT family. This provides evidence that mobile genetic elements may be one of the driving forces underlying race evolution. KoChi-1 transformants carrying a wild type AVR1 gene from race 1 lost pathogenicity to cultivars carrying I, showing that the truncated KoChi-1 avr1 is not functional. These results imply that KoChi-1 is a new race 3 biotype and propose an additional path for emergence of FOL races: Race 2 emerged from race 1 by transposon-insertion into AVR1, not by deletion of the AVR1 locus; then a point mutation in race 2 AVR2 resulted in emergence of race 3.

  19. Loss of the Birt-Hogg-Dubé gene product folliculin induces longevity in a hypoxia-inducible factor-dependent manner.

    Science.gov (United States)

    Gharbi, Hakam; Fabretti, Francesca; Bharill, Puneet; Rinschen, Markus M; Brinkkötter, Sibylle; Frommolt, Peter; Burst, Volker; Schermer, Bernhard; Benzing, Thomas; Müller, Roman-Ulrich

    2013-08-01

    Signaling through the hypoxia-inducible factor hif-1 controls longevity, metabolism, and stress resistance in Caenorhabditis elegans. Hypoxia-inducible factor (HIF) protein levels are regulated through an evolutionarily conserved ubiquitin ligase complex. Mutations in the VHL gene, encoding a core component of this complex, cause a multitumor syndrome and renal cell carcinoma in humans. In the nematode, deficiency in vhl-1 promotes longevity mediated through HIF-1 stabilization. However, this longevity assurance pathway is not yet understood. Here, we identify folliculin (FLCN) as a novel interactor of the hif-1/vhl-1 longevity pathway. FLCN mutations cause Birt-Hogg-Dubé syndrome in humans, another tumor syndrome with renal tumorigenesis reminiscent of the VHL disease. Loss of the C. elegans ortholog of FLCN F22D3.2 significantly increased lifespan and enhanced stress resistance in a hif-1-dependent manner. F22D3.2, vhl-1, and hif-1 control longevity by a mechanism distinct from insulin-like signaling. Daf-16 deficiency did not abrogate the increase in lifespan mediated by flcn-1. These findings define FLCN as a player in HIF-dependent longevity signaling and connect organismal aging, stress resistance, and regulation of longevity with the formation of renal cell carcinoma. © 2013 John Wiley & Sons Ltd and the Anatomical Society.

  20. Yes-associated protein and WW-containing transcription regulator 1 regulate the expression of sex-determining genes in Sertoli cells, but their inactivation does not cause sex reversal.

    Science.gov (United States)

    Levasseur, Adrien; Paquet, Marilène; Boerboom, Derek; Boyer, Alexandre

    2017-07-01

    Yes-associated protein (YAP) and WW-containing transcription regulator 1 (WWTR1) are two functionally redundant transcriptional regulators that are downstream effectors of the Hippo signaling pathway, and that act as major regulators of cell growth and differentiation. To elucidate their role in Sertoli cells, primary Sertoli cell culture from Yapflox/flox; Wwtr1flox/flox animals were infected with a Cre recombinase-expressing adenovirus. Concomitant inactivation of Yap and Wwtr1 resulted in a decrease in the mRNA levels of the male sex differentiation genes Dhh, Dmrt1, Sox9, and Wt1, whereas those of genes involved in female differentiation (Wnt4, Rspo1, and Foxl2) were induced. SOX9, FOXL2, and WNT4 proteins were regulated in the same manner as their mRNAs in response to loss of YAP and WWTR1. To further characterize the role of YAP and WWTR1 in Sertoli cells, we generated a mouse model (Yapflox/flox; Wwtr1flox/flox; Amhcre/+) in which Yap and Wwtr1 were conditionally deleted in Sertoli cells. An increase in the number of apoptotic cells was observed in the seminiferous tubules of 4 dpp mutant mice, leading to a reduction in testis weights and a decrease in the number of Sertoli cells in adult animals. Gene expression analyses of testes from 4 dpp Yapflox/flox; Wwtr1flox/flox; Amhcre/+ mice showed that Sertoli cell differentiation is initially altered, as Dhh, Dmrt1, and Sox9 mRNA levels were downregulated, whereas Wnt4 mRNA levels were increased. However, expression of these genes was not changed in older animals. Together, these results suggest a novel role of the Hippo signaling pathway in the mechanisms of sex differentiation. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Inactivation and Gene Expression of a Virulent WastewaterEscherichia coliStrain and the Nonvirulent CommensalEscherichia coliDSM1103 Strain upon Solar Irradiation

    KAUST Repository

    Aljassim, Nada I.

    2017-03-06

    This study examined the decay kinetics and molecular responses of two Escherichia coli strains upon solar irradiation. The first is E. coli PI-7, a virulent and antibiotic-resistant strain that was isolated from wastewater and carries the emerging NDM-1 antibiotic resistance gene. The other strain, E. coli DSM1103, displayed lower virulence and antibiotic resistance than E. coli PI-7. In a buffer solution, E. coli PI-7 displayed a longer lag phase prior to decay and a longer half-life compared with E. coli DSM1103 (6.64 ± 0.63 h and 2.85 ± 0.46 min vs 1.33 ± 0.52 h and 2.04 ± 0.36 min). In wastewater, both E. coli strains decayed slower than they did in buffer. Although solar irradiation remained effective in reducing the numbers of both strains by more than 5-log10 in <24 h, comparative genomics and transcriptomics revealed differences in the genomes and overall regulation of genes between the two E. coli strains. A wider arsenal of genes related to oxidative stress, cellular repair and protective mechanisms were upregulated in E. coli PI-7. Subpopulations of E. coli PI-7 expressed genes related to dormancy and persister cell formation during the late decay phase, which may have accounted for its prolonged persistence. Upon prolonged solar irradiation, both E. coli strains displayed upregulation of genes related to horizontal gene transfer and antibiotic resistance. Virulence functions unique to E. coli PI-7 were also upregulated. Our findings collectively indicated that, whereas solar irradiation is able to reduce total cell numbers, viable E. coli remained and expressed genes that enable survival despite solar treatment. There remains a need for heightened levels of concern regarding risks arising from the dissemination of E. coli that may remain viable in wastewater after solar irradiation.

  2. Alcohol consumption and mutations or promoter hypermethylation of the von Hippel-Lindau gene in renal cell carcinoma.

    NARCIS (Netherlands)

    Schouten, L.J.; Dijk, B.A.C. van; Oosterwijk, E.; Engeland, M. van; Hulsbergen- van de Kaa, C.A.; Kiemeney, L.A.L.M.; Goldbohm, R.A.; Kester, A.; Vogel, S de; Schalken, J.A.; Brandt, P.A. van den

    2008-01-01

    Alcohol consumption has been associated with a decreased risk for renal cell cancer in several studies. We investigated whether alcohol is associated with (epi)genetic changes of the von Hippel-Lindau (VHL) gene in renal cell cancer. The Netherlands Cohort Study (NLCS) on Diet and Cancer started in

  3. Alcohol consumption and mutations or promoter hypermethylation of the von Hippel-Lindau gene in renal cell carcinoma

    NARCIS (Netherlands)

    Schouten, L.J.; Dijk, B.A.C.van; Oosterwijk, E.; Engeland, M. van; Hulsbergen - Kaa, C.A. van de; Kiemeney, L.A.L.M.; Goldbohm, R.A.; Kester, A.; Vogel, S. de; Schalken, J.A.; Brandt, P.A. van den

    2008-01-01

    Alcohol consumption has been associated with a decreased risk for renal cell cancer in several studies. We investigated whether alcohol is associated with (epi)genetic changes of the von Hippel-Lindau (VHL) gene in renal cell cancer. The Netherlands Cohort Study (NLCS) on Diet and Cancer started in

  4. Inactivation of the DNA repair genes mutS, mutL or the anti-recombination gene mutS2 leads to activation of vitamin B1 biosynthesis genes.

    Directory of Open Access Journals (Sweden)

    Kenji Fukui

    Full Text Available Oxidative stress generates harmful reactive oxygen species (ROS that attack biomolecules including DNA. In living cells, there are several mechanisms for detoxifying ROS and repairing oxidatively-damaged DNA. In this study, transcriptomic analyses clarified that disruption of DNA repair genes mutS and mutL, or the anti-recombination gene mutS2, in Thermus thermophilus HB8, induces the biosynthesis pathway for vitamin B(1, which can serve as an ROS scavenger. In addition, disruption of mutS, mutL, or mutS2 resulted in an increased rate of oxidative stress-induced mutagenesis. Co-immunoprecipitation and pull-down experiments revealed previously-unknown interactions of MutS2 with MutS and MutL, indicating that these proteins cooperatively participate in the repair of oxidatively damaged DNA. These results suggested that bacterial cells sense the accumulation of oxidative DNA damage or absence of DNA repair activity, and signal the information to the transcriptional regulation machinery for an ROS-detoxifying system.

  5. The effect of 1,25 dihydroxyvitamin D3 treatment on the mRNA levels of β catenin target genes in mice with colonic inactivation of both APC alleles.

    Science.gov (United States)

    DeWitt, Marsha; Johnson, Robert L; Snyder, Paul; Fleet, James C

    2015-04-01

    In colon cancer, adenomatous polyposis coli (APC) inactivating gene mutations increase nuclear β-catenin levels and stimulate proliferation. In vitro, 1,25 dihydroxyvitamin D (1,25(OH)2D), suppresses β-catenin-mediated gene transcription by inducing vitamin D receptor (VDR)-β-catenin interactions. We examined whether acute treatment with 1,25(OH)2D could suppress β-catenin-mediated gene transcription in the hyperplastic colonic lesions of mice with colon-specific deletion of both APC gene alleles (CAC; APC(Δ580/Δ580)). At four weeks of age, CAC; APC(Δ580/Δ580) and control mice were injected with vehicle or 1,25(OH)2D (1μg/kg body weight) once a day for three days and then killed six hours after the last injection. mRNA levels of β-catenin target genes were elevated in the colon of CAC; APC(Δ580/Δ580) mice. 1,25(OH)2D increased 25 hydroxyvitamin D-24 hydroxylase mRNA levels in the colon of CAC; APC(Δ580/Δ580) and control mice indicating the treatments activated the VDR. However, 1,25(OH)2D had no effect on either β-catenin target gene mRNA levels or the proliferation index in CAC; APC(Δ580/Δ580) or control mice. VDR mRNA and protein levels were lower (-65% and -90%) in the colon of CAC; APC(Δ580/Δ580) mice compared to control mice, suggesting loss of colon responsiveness to vitamin D. Consistent with this, vitamin D-induced expression of transient receptor potential cation channel, subfamily V, member 6 mRNA was reduced in the colon of CAC; APC(Δ580/Δ580) mice. Our data show that short term exposure to 1,25(OH)2D does not suppress colonic β-catenin signaling in vivo. This article is part of a special issue entitled '17th Vitamin D Workshop'. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. The effect of age at exposure on the inactivating mechanisms and relative contributions of key tumor suppressor genes in radiation-induced mouse T-cell lymphomas

    Energy Technology Data Exchange (ETDEWEB)

    Sunaoshi, Masaaki [Radiobiology for Children' s Health Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Department of Biological Sciences, College of Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512 (Japan); Amasaki, Yoshiko; Hirano-Sakairi, Shinobu; Blyth, Benjamin J. [Radiobiology for Children' s Health Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Morioka, Takamitsu [Radiobiology for Children' s Health Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Radiation Effect Accumulation and Prevention Project, Fukushima Project Headquarters, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Kaminishi, Mutsumi [Radiobiology for Children' s Health Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Shang, Yi [Radiation Effect Accumulation and Prevention Project, Fukushima Project Headquarters, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Nishimura, Mayumi; Shimada, Yoshiya [Radiobiology for Children' s Health Program, Research Center for Radiation Protection, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Radiation Effect Accumulation and Prevention Project, Fukushima Project Headquarters, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Tachibana, Akira [Department of Biological Sciences, College of Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki 310-8512 (Japan); and others

    2015-09-15

    Highlights: • T-cell lymphoma incidence, latency and weight did not change with age at exposure. • Lymphomas had frequent loss of heterozygosity on chromosomes 4, 11 and 19. • These lesions targeted the Cdkn2a, Ikaros and Pten tumor suppressor genes. • Age at exposure may influence which tumor suppressor genes are lost in each tumor. • The mechanisms of tumor suppressor gene loss were different at each locus. - Abstract: Children are considered more sensitive to radiation-induced cancer than adults, yet any differences in genomic alterations associated with age-at-exposure and their underlying mechanisms remain unclear. We assessed genome-wide DNA copy number and mutation of key tumor suppressor genes in T-cell lymphomas arising after weekly irradiation of female B6C3F1 mice with 1.2 Gy X-rays for 4 consecutive weeks starting during infancy (1 week old), adolescence (4 weeks old) or as young adults (8 weeks old). Although T-cell lymphoma incidence was similar, loss of heterozygosity at Cdkn2a on chromosome 4 and at Ikaros on chromosome 11 was more frequent in the two older groups, while loss at the Pten locus on chromosome 19 was more frequent in the infant-irradiated group. Cdkn2a and Ikaros mutation/loss was a common feature of the young adult-irradiation group, with Ikaros frequently (50%) incurring multiple independent hits (including deletions and mutations) or suffering a single hit predicted to result in a dominant negative protein (such as those lacking exon 4, an isoform we have designated Ik12, which lacks two DNA binding zinc-finger domains). Conversely, Pten mutations were more frequent after early irradiation (60%) than after young adult-irradiation (30%). Homozygous Pten mutations occurred without DNA copy number change after irradiation starting in infancy, suggesting duplication of the mutated allele by chromosome mis-segregation or mitotic recombination. Our findings demonstrate that while deletions on chromosomes 4 and 11 affecting Cdkn2

  7. Inactivation of the Kluyveromyces lactis KlPDA1 gene leads to loss of pyruvate dehydrogenase activity, impairs growth on glucose and triggers aerobic alcoholic fermentation.

    Science.gov (United States)

    Zeeman, A M; Luttik, M A; Thiele, C; van Dijken, J P; Pronk, J T; Steensma, H Y

    1998-12-01

    The KlPDA1 gene, encoding the E1alpha subunit of the mitochondrial pyruvate-dehydrogenase (PDH) complex was isolated from a Kluyveromyces lactis genomic library by screening with a 1.1 kb internal fragment of the Saccharomyces cerevisiae PDA1 gene. The predicted amino acid sequence encoded by KlPDA1 showed 87% similarity and 79% identity to its S. cerevisiae counterpart. Disruption of KIPDA1 resulted in complete absence of PDH activity in cell extracts. The maximum specific growth rate on glucose of null mutants was 3.5-fold lower than that of the wild-type, whereas growth on ethanol was unaffected. Wild-type K. lactis CBS 2359 exhibits a Crabtree-negative phenotype, i.e. no ethanol was produced in aerobic batch cultures grown on glucose. In contrast, substantial amounts of ethanol and acetaldehyde were produced in aerobic cultures of an isogenic Klpda1 null mutant. A wild-type specific growth rate was restored after introduction of an intact KlPDA1 gene but not, as previously found for S. cerevisiae pda1 mutants, by cultivation in the presence of leucine. The occurrence of aerobic fermentation and slow growth of the Klpda1 null mutant indicate that, although present, the enzymes of the PDH bypass (pyruvate decarboxylase, acetaldehyde dehydrogenase and acetyl-CoA synthetase) could not efficiently replace the PDH complex during batch cultivation on glucose. Only at relatively low growth rates (D = 0.10 h(-1)) in aerobic, glucose-limited chemostat cultures, could the PDH bypass completely replace the PDH complex, thus allowing fully respiratory growth. This resulted in a lower biomass yield [g biomass (g glucose)-1] than in the wild-type due to a higher consumption of ATP in the PDH bypass compared to the formation of acetyl-CoA via the PDH complex.

  8. Thermal Inactivation of Viruses

    Science.gov (United States)

    1977-10-01

    S) VIKU3FS THERMAL RESISTANCE FOODS FLUIDS FOOD PROCESSING FOOD PRESERVATION CONTAMINATION HEAT VIRAL NUCLEIC ACIDS ao’.AjUsTNACT (Conilnum...an rm**— •**» It nmc +nmy m>d Id+atttr by M«o* fmbm) A review of the literature pertaining to thermal inactivation of virus in fluid media» fluid...vacuum packaged in cans or in flexible pouches , frozen to ca. -40 C, and irradiated within a temperature range of -40 C to -8 C to obtain the

  9. Female meiotic sex chromosome inactivation in chicken.

    Directory of Open Access Journals (Sweden)

    Sam Schoenmakers

    2009-05-01

    Full Text Available During meiotic prophase in male mammals, the heterologous X and Y chromosomes remain largely unsynapsed, and meiotic sex chromosome inactivation (MSCI leads to formation of the transcriptionally silenced XY body. In birds, the heterogametic sex is female, carrying Z and W chromosomes (ZW, whereas males have the homogametic ZZ constitution. During chicken oogenesis, the heterologous ZW pair reaches a state of complete heterologous synapsis, and this might enable maintenance of transcription of Z- and W chromosomal genes during meiotic prophase. Herein, we show that the ZW pair is transiently silenced, from early pachytene to early diplotene using immunocytochemistry and gene expression analyses. We propose that ZW inactivation is most likely achieved via spreading of heterochromatin from the W on the Z chromosome. Also, persistent meiotic DNA double-strand breaks (DSBs may contribute to silencing of Z. Surprisingly, gammaH2AX, a marker of DSBs, and also the earliest histone modification that is associated with XY body formation in mammalian and marsupial spermatocytes, does not cover the ZW during the synapsed stage. However, when the ZW pair starts to desynapse, a second wave of gammaH2AX accumulates on the unsynapsed regions of Z, which also show a reappearance of the DSB repair protein RAD51. This indicates that repair of meiotic DSBs on the heterologous part of Z is postponed until late pachytene/diplotene, possibly to avoid recombination with regions on the heterologously synapsed W chromosome. Two days after entering diplotene, the Z looses gammaH2AX and shows reactivation. This is the first report of meiotic sex chromosome inactivation in a species with female heterogamety, providing evidence that this mechanism is not specific to spermatogenesis. It also indicates the presence of an evolutionary force that drives meiotic sex chromosome inactivation independent of the final achievement of synapsis.

  10. The effects of Capn1 gene inactivation on skeletal muscle growth, development, and atrophy, and the compensatory role of other proteolytic systems.

    Science.gov (United States)

    Kemp, C M; Oliver, W T; Wheeler, T L; Chishti, A H; Koohmaraie, M

    2013-07-01

    Myofibrillar protein turnover is a key component of muscle growth and degeneration, requiring proteolytic enzymes to degrade the skeletal muscle proteins. The objective of this study was to investigate the role of the calpain proteolytic system in muscle growth development using μ-calpain knockout (KO) mice in comparison with control wild-type (WT) mice, and evaluate the subsequent effects of silencing this gene on other proteolytic systems. No differences in muscle development between genotypes were observed during the early stages of growth due to the up regulation of other proteolytic systems. The KO mice showed significantly greater m-calpain protein abundance (P muscle fibers (P muscle. Also, expression of proteins associated with muscle regeneration (neural cell adhesion molecule and myoD) were both reduced in the mature KO mice (P muscle regeneration and, therefore, less muscle damage. These findings indicate the concerted action of proteolytic systems to ensure muscle protein homeostasis in vivo. Furthermore, these data contribute to the existing evidence of the importance of the calpain system's involvement in muscle growth, development, and atrophy. Collectively, these data suggest that there are opportunities to target the calpain system to promote the growth and/or restoration of skeletal muscle mass.

  11. Inactivation of pyocyanin synthesis genes has no effect on the virulence of Pseudomonas aeruginosa PAO1 toward the silkworm, Bombyx mori.

    Science.gov (United States)

    Chieda, Yuuka; Iiyama, Kazuhiro; Lee, Jae Man; Kusakabe, Takahiro; Yasunaga-Aoki, Chisa; Shimizu, Susumu

    2008-01-01

    The contribution of pyocyanin to the virulence of Pseudomonas aeruginosa against the silkworm Bombyx mori was studied. First, purified pyocyanin was injected into the hemocoel of B. mori. Acute toxicity was observed only when a high dose of pyocyanin was injected. The lethal dose 50% value of pyocyanin was found to be 9.52 microg per larva. Next, mutant strains of phzM and phzS, which encode putative phenazine-specific methytransferase and flavin-containing monooxygenase, respectively, were created, and their virulence was compared with that of the PAO1 parent strain. Although the ability to produce pyocyanin was completely lost in the phz-mutant strains, they maintained the same level of virulence as the PAO1 parent strain. In addition, the complementation of the corresponding gene in trans in the mutant strains did not have any effect on the virulence of those mutant strains. These results indicated that pyocyanin does not act as a virulence factor in B. mori after invasion, which was different from the results obtained in other Lepidopteran host models.

  12. Inactivation of the Rcan2 gene in mice ameliorates the age- and diet-induced obesity by causing a reduction in food intake.

    Directory of Open Access Journals (Sweden)

    Xiao-yang Sun

    Full Text Available Obesity is a serious international health problem that increases the risk of several diet-related chronic diseases. The genetic factors predisposing to obesity are little understood. Rcan2 was originally identified as a thyroid hormone-responsive gene. In the mouse, two splicing variants that harbor distinct tissue-specific expression patterns have been identified: Rcan2-3 is expressed predominately in the brain, whereas Rcan2-1 is expressed in the brain and other tissues such as the heart and skeletal muscle. Here, we show that Rcan2 plays an important role in the development of age- and diet-induced obesity. We found that although the loss of Rcan2 function in mice slowed growth in the first few weeks after birth, it also significantly ameliorated age- and diet-induced obesity in the mice by causing a reduction in food intake rather than increased energy expenditure. Rcan2 expression was most prominent in the ventromedial, dorsomedial and paraventricular hypothalamic nuclei governing energy balance. Fasting and refeeding experiment showed that only Rcan2-3 mRNA expression is up-regulated in the hypothalamus by fasting, and loss of Rcan2 significantly attenuates the hyperphagic response to starvation. Using double-mutant (Lep(ob/ob Rcan2(-/- mice, we were also able to demonstrate that Rcan2 and leptin regulate body weight through different pathways. Our findings indicate that there may be an Rcan2-dependent mechanism which regulates food intake and promotes weight gain through a leptin-independent pathway. This study provides novel information on the control of body weight in mice and should improve our understanding of the mechanisms of obesity in humans.

  13. Inactivation of the spxA1 or spxA2 gene of Streptococcus mutans decreases virulence in the rat caries model.

    Science.gov (United States)

    Galvão, L C C; Rosalen, P L; Rivera-Ramos, I; Franco, G C N; Kajfasz, J K; Abranches, J; Bueno-Silva, B; Koo, H; Lemos, J A

    2017-04-01

    In oral biofilms, the major environmental challenges encountered by Streptococcus mutans are acid and oxidative stresses. Previously, we showed that the transcriptional regulators SpxA1 and SpxA2 are involved in general stress survival of S. mutans with SpxA1 playing a primary role in activation of antioxidant and detoxification strategies whereas SpxA2 serves as a back up activator of oxidative stress genes. We have also found that spxA1 mutant strains (∆spxA1 and ∆spxA1∆spxA2) are outcompeted by peroxigenic oral streptococci in vitro and have impaired abilities to colonize the teeth of rats fed a highly cariogenic diet. Here, we show that the Spx proteins can also exert regulatory roles in the expression of additional virulence attributes of S. mutans. Competence activation is significantly impaired in Δspx strains and the production of mutacin IV and V is virtually abolished in ΔspxA1 strains. Unexpectedly, the ∆spxA2 strain showed increased production of glucans from sucrose, without affecting the total amount of bacteria within biofilms when compared with the parent strain. By using the rat caries model, we showed that the capacity of the ΔspxA1 and ΔspxA2 strains to cause caries on smooth tooth surfaces is significantly impaired. The ∆spxA2 strain also formed fewer lesions on sulcal surfaces. This report reveals that global regulation via Spx contributes to the cariogenic potential of S. mutans and highlights that animal models are essential in the characterization of bacterial traits implicated in virulence. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Inactivation of Microorganisms

    Science.gov (United States)

    Alzamora, Stella Maris; Guerrero, Sandra N.; Schenk, Marcela; Raffellini, Silvia; López-Malo, Aurelio

    Minimal processing techniques for food preservation allow better retention of product flavor, texture, color, and nutrient content than comparable conventional treatments. A wide range of novel alternative physical factors have been intensely investigated in the last two decades. These physical factors can cause inactivation of microorganisms at ambient or sublethal temperatures (e.g., high hydrostatic pressure, pulsed electric fields, ultrasound, pulsed light, and ultraviolet light). These technologies have been reported to reduce microorganism population in foods while avoiding the deleterious effects of severe heating on quality. Among technologies, high-energy ultrasound (i.e., intensities higher than 1 W/cm2, frequencies between 18 and 100 kHz) has attracted considerable interest for food preservation applications (Mason et al., 1996; Povey and Mason, 1998).

  15. Nuclear expression of hypoxia-inducible factor-1alpha in clear cell renal cell carcinoma is involved in tumor progression.

    Science.gov (United States)

    Di Cristofano, Claudio; Minervini, Andrea; Menicagli, Michele; Salinitri, Giuseppe; Bertacca, Gloria; Pefanis, Gerasimos; Masieri, Lorenzo; Lessi, Francesca; Collecchi, Paola; Minervini, Riccardo; Carini, Marco; Bevilacqua, Generoso; Cavazzana, Andrea

    2007-12-01

    The most frequent genomic abnormality in clear cell renal cell carcinoma (cc-RCC) is inactivation of Von Hippel-Lindau gene (VHL). pVHL19 is a ligase promoting proteosomal degradation of hypoxia-inducible factor-1alpha (HIF-1alpha); pVHL30 is associated with microtubules. VHL exert its oncogenetic action both directly and through HIF-1alpha activation. TNM classification is unable to define a correct prognostic evaluation of intracapsular cc-RCC. The nucleo-cytoplasmic trafficking in VHL/HIF-1alpha pathway could be relevant in understanding the molecular pathogenesis of renal carcinogenesis. This study analyzes VHL/HIF-1alpha proteins in a large series of intracapsular cc-RCCs, correlating their expression and cellular localization with prognosis. Two anti-pVHL (clones Ig32 and Ig33) and 1 anti-HIF-1alpha were used on tissue microarrays from 136 intracapsular cc-RCCs (mean follow-up: 74 mo). Clone 32 recognizes both pVHLs, whereas clone 33 only pVHL30. Results were matched with clinicopathologic variables and tumor-specific survival (TSS). A strong cytoplasmic positivity was found for all antibodies in the largest part of cases, associated to a strong nuclear localization in the case of HIF-1alpha. All pVHL-negative cases were associated with high HIF-1alpha expression. pVHL negativity and HIF-1alpha nuclear positivity significantly correlated with shorter TSS. In multivariate analysis both pVHL negativity and HIF-1alpha nuclear expression were independent predictors of TSS. The localization of the proteins well matches with their role and with the supposed tumor molecular pathways. The correlation with prognosis of VHL/HIF-1alpha alterations confirms the relevance of their molecular pathway and of the cellular trafficking of their products in the pathogenesis of renal cancer.

  16. X-changing information on X inactivation

    Energy Technology Data Exchange (ETDEWEB)

    Barakat, Tahsin Stefan; Jonkers, Iris; Monkhorst, Kim [Department of Reproduction and Development, Room Ee 09-71, Erasmus MC, PO Box 2040, 3000 CA Rotterdam (Netherlands); Gribnau, Joost, E-mail: j.gribnau@erasmusmc.nl [Department of Reproduction and Development, Room Ee 09-71, Erasmus MC, PO Box 2040, 3000 CA Rotterdam (Netherlands)

    2010-03-10

    In female somatic cells of mammalian species one X chromosome is inactivated to ensure dosage equality of X-encoded genes between females and males, during development and adulthood. X chromosome inactivation (XCI) involves various epigenetic mechanisms, including RNA mediated gene silencing in cis, DNA methylation, and changes in chromatin modifications and composition. XCI therefore provides an attractive paradigm to study epigenetic gene regulation in a more general context. The XCI process starts with counting of the number of X chromosomes present in a nucleus, and initiation of XCI follows if this number exceeds one per diploid genome. Recently, X-encoded RNF12 has been identified as a dose-dependent activator of XCI. In addition, other factors, including the pluripotency factors OCT4, SOX2 and Nanog, have been implicated to play a role in suppression of initiation of XCI. In this review, we highlight and explain these new and old findings in the context of a stochastic model for X chromosome counting and XCI initiation.

  17. X Inactivation and Progenitor Cancer Cells

    Directory of Open Access Journals (Sweden)

    Ruben Agrelo

    2011-04-01

    Full Text Available In mammals, silencing of one of the two X chromosomes is necessary to achieve dosage compensation. The 17 kb non-coding RNA called Xist triggers X inactivation. Gene silencing by Xist can only be achieved in certain contexts such as in cells of the early embryo and in certain hematopoietic progenitors where silencing factors are present. Moreover, these epigenetic contexts are maintained in cancer progenitors in which SATB1 has been identified as a factor related to Xist-mediated chromosome silencing.

  18. Effects of inactivated Enterococcus faecalis on the proliferation and osteogenic induction of osteoblasts.

    Science.gov (United States)

    Tong, Zhongchun; Ma, Jinglei; Tan, Jiali; Huang, Lijia; Ling, Junqi

    2016-12-01

    The present study aimed to evaluate the effects of Enterococcus faecalis, inactivated by the common intracanal irrigants sodium hypochlorite (NaOCl) and chlorhexidine (CHX), on osteoblasts. E. faecalis was inactivated with 2% CHX or 5.25% NaOCl. Subsequently, the Cell Counting kit‑8 assay was used to examine the effects of CHX‑ and NaOCl-inactivated E. faecalis on MC3T3‑E1 osteoblast cell proliferation. Alizarin red staining was used to determine osteoblast mineralization, and osteogenic induction was quantified by determining the optical density of the dye solution. The relative expression levels of osteogenic genes were detected after 1, 4, 7 and 14 days of stimulation with CHX‑ and NaOCl-inactivated E. faecalis by reverse transcription‑quantitative polymerase chain reaction. The results indicated that CHX‑inactivated E. faecalis inhibited osteoblast proliferation, whereas NaOCl‑inactivated E. faecalis did not suppress cell proliferation. Various concentrations of CHX‑ and NaOCl‑inactivated E. faecalis induced different degrees of osteoblast mineralization. The expression levels of osteocalcin, alkaline phosphatase, runt‑related transcription factor 2, osteopontin and osterix were upregulated in cells following stimulation with 107 and 105 colony‑forming units/ml E. faecalis inactivated by CHX and NaOCl; the upregulation of these osteogenic genes occurred at various time points. In conclusion, the present study demonstrated that CHX‑inactivated E. faecalis exerted more of an effect on osteoblast proliferation compared with NaOCl‑inactivated E. faecalis. In addition, CHX‑ and NaOCl‑inactivated E. faecalis was able to induce mineralization and relevant osteogenic gene expression in osteoblast cells.

  19. Effects of inactivated Enterococcus faecalis on the proliferation and osteogenic induction of osteoblasts

    Science.gov (United States)

    Tong, Zhongchun; Ma, Jinglei; Tan, Jiali; Huang, Lijia; Ling, Junqi

    2016-01-01

    The present study aimed to evaluate the effects of Enterococcus faecalis, inactivated by the common intracanal irrigants sodium hypochlorite (NaOCl) and chlorhexidine (CHX), on osteoblasts. E. faecalis was inactivated with 2% CHX or 5.25% NaOCl. Subsequently, the Cell Counting kit-8 assay was used to examine the effects of CHX- and NaOCl-inactivated E. faecalis on MC3T3-E1 osteoblast cell proliferation. Alizarin red staining was used to determine osteoblast mineralization, and osteogenic induction was quantified by determining the optical density of the dye solution. The relative expression levels of osteogenic genes were detected after 1, 4, 7 and 14 days of stimulation with CHX- and NaOCl-inactivated E. faecalis by reverse transcription-quantitative polymerase chain reaction. The results indicated that CHX-inactivated E. faecalis inhibited osteoblast proliferation, whereas NaOCl-inactivated E. faecalis did not suppress cell proliferation. Various concentrations of CHX- and NaOCl-inactivated E. faecalis induced different degrees of osteoblast mineralization. The expression levels of osteocalcin, alkaline phosphatase, runt-related transcription factor 2, osteopontin and osterix were upregulated in cells following stimulation with 107 and 105 colony-forming units/ml E. faecalis inactivated by CHX and NaOCl; the upregulation of these osteogenic genes occurred at various time points. In conclusion, the present study demonstrated that CHX-inactivated E. faecalis exerted more of an effect on osteoblast proliferation compared with NaOCl-inactivated E. faecalis. In addition, CHX- and NaOCl-inactivated E. faecalis was able to induce mineralization and relevant osteogenic gene expression in osteoblast cells. PMID:27840919

  20. X chromosome inactivation in women with alcoholism.

    Science.gov (United States)

    Manzardo, Ann M; Henkhaus, Rebecca; Hidaka, Brandon; Penick, Elizabeth C; Poje, Albert B; Butler, Merlin G

    2012-08-01

    All female mammals with 2 X chromosomes balance gene expression with males having only 1 X by inactivating one of their X chromosomes (X chromosome inactivation [XCI]). Analysis of XCI in females offers the opportunity to investigate both X-linked genetic factors and early embryonic development that may contribute to alcoholism. Increases in the prevalence of skewing of XCI in women with alcoholism could implicate biological risk factors. The pattern of XCI was examined in DNA isolated in blood from 44 adult women meeting DSM-IV criteria for an alcohol use disorder and 45 control women with no known history of alcohol abuse or dependence. XCI status was determined by analyzing digested and undigested polymerase chain reaction (PCR) products of the polymorphic androgen receptor (AR) gene located on the X chromosome. Subjects were categorized into 3 groups based upon the degree of XCI skewness: random (50:50 to 64:36%), moderately skewed (65:35 to 80:20%), and highly skewed (>80:20%). XCI status from informative women with alcoholism was found to be random in 59% (n = 26), moderately skewed in 27% (n = 12), or highly skewed in 14% (n = 6). Control subjects showed 60, 29, and 11%, respectively. The distribution of skewed XCI observed among women with alcoholism did not differ statistically from that of control subjects (χ(2) test = 0.14, 2 df, p = 0.93). Our data did not support an increase in XCI skewness among women with alcoholism or implicate early developmental events associated with embryonic cell loss or unequal (nonrandom) expression of X-linked gene(s) or defects in alcoholism among women. Copyright © 2012 by the Research Society on Alcoholism.

  1. Prevalence of von Hippel-Lindau gene mutations in sporadic renal cell carcinoma: results from the Netherlands cohort study

    Directory of Open Access Journals (Sweden)

    Schalken Jack A

    2005-06-01

    Full Text Available Abstract Background Biallelic von Hippel-Lindau (VHL gene defects, a rate-limiting event in the carcinogenesis, occur in approximately 75% of sporadic clear-cell Renal Cell Carcinoma (RCC. We studied the VHL mutation status in a large population-based case group. Methods Cases were identified within the Netherlands cohort study on diet and cancer, which includes 120,852 men and women. After 11.3 years of follow-up, 337 incident cases with histologically confirmed epithelial cancers were identified. DNA was isolated from paraffin material collected from 51 pathology laboratories and revised by one pathologist, leaving material from 235 cases. VHL mutational status was assessed by SSCP followed by direct sequencing, after testing SSCP as a screening tool in a subsample. Results The number of mutations was significantly higher for clear-cell RCC compared to other histological types. We observed 131 mutations in 114 out of 187 patients (61% with clear-cell RCC. The majority of mutations were truncating mutations (47%. The mean tumor size was 72.7 mm for mutated tumors compared to 65.3 mm for wildtype tumors (p = 0.06. No statistically significant differences were observed for nuclear grade, TNM distribution or stage. In other histological types, we observed 8 mutations in 7 out of 48 patients (15%, 1 mutation in 1 of 6 oncocytoma, 3 mutations in 2 of 7 chromophobe RCC, 2 mutations in 2 of 30 papillary RCC, no mutations in 1 collecting duct carcinoma and 2 mutations in 2 of 4 unclassified RCC. Conclusion VHL mutations were detected in 61% of sporadic clear-cell RCC. VHL mutated and wildtype clear-cell RCC did not differ with respect to most parameters.

  2. Phenylbutazone radicals inactivate creatine kinase.

    Science.gov (United States)

    Miura, T; Muraoka, S; Fujimoto, Y

    2001-02-01

    Creatine kinase (CK) was used as a marker molecule to examine the side effect of damage to tissues by phenylbutazone (PB), an effective drug to treat rheumatic and arthritic diseases, with horseradish peroxidase and hydrogen peroxide (HRP-H(2)O2). PB inactivated CK during its interaction with HRP-H(2) O(2), and inactivated CK in rat heart homogenate. PB carbon-centered radicals were formed during the interaction of PB with HRP-H(2)O2. The CK efficiently reduced electron spin resonance signals of the PB carbon-centered radicals. The spin trap agent 2-methyl-2-nitrosopropane strongly prevented CK inactivation. These results show that CK was inactivated through interaction with PB carbon-centered radicals. Sulfhydryl groups and tryptophan residues in CK were lost during the interaction of PB with HRP-H(2)O2, suggesting that cysteine and tryptophan residues are oxidized by PB carbon-centered radicals. Other enzymes, including alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, but not lactate dehydrogenase, were also inactivated. Sulfhydryl enzymes seem to be sensitive to attack by PB carbon-centered radicals. Inhibition of SH enzymes may explain some of the deleterious effects induced by PB.

  3. Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis.

    Science.gov (United States)

    Yu, Jian; Ni, Min; Xu, Jian; Zhang, Hongyu; Gao, Baomei; Gu, Jianren; Chen, Jianguo; Zhang, Lisheng; Wu, Mengchao; Zhen, Sushen; Zhu, Jingde

    2002-11-15

    Hepatocellular carcinoma (HCC) presents one of the major health threats in China today. A better understanding of the molecular genetics underlying malignant transformation of hepatocytes is critical to success in the battle against this disease. The methylation state of C5 of the cytosine in the CpG di-nucleotide that is enriched within or near the promoter region of over 50 % of the polymerase II genes has a drastic effect on transcription of these genes. Changes in the methylation profile of the promoters represent an alternative to genetic lesions as causative factors for the tumor-specific aberrant expression of the genes. We have used the methylation specific PCR method in conjunction with DNA sequencing to assess the methylation state of the promoter CpG islands of twenty genes. Aberrant expression of these genes have been attributed to the abnormal methylation profile of the corresponding promoter CpG islands in human tumors. While the following sixteen genes remained the unmethylated in all tumor and normal tissues: CDH1, APAF1, hMLH1, BRCA1, hTERC, VHL, RARbeta, TIMP3, DAPK1, SURVIVIN, p14ARF, RB1, p15INK4b, APC, RASSF1c and PTEN, varying degrees of tumor specific hypermethylation were associated with the p16INK4a, RASSF1a, CASP8 and CDH13 genes. For instance, the p16INK4a was highly methylated in HCC (17/29, 58.6%) and less significantly methylated in non-cancerous tissue (4/29. 13.79%). The RASSF1a was fully methylated in all tumor tissues (29/29, 100%), and less frequently methylated in corresponding non-cancerous tissue (24/29, 82.75%). Furthermore, co-existence of methylated with unmethylated DNA in some cases suggested that both genetic and epigenetic (CpG methylation) mechanisms may act in concert to inactivate the p16INK4a and RASSF1a in HCC. Finally, we found a significant association of cirrhosis with hypermethylation of the p16INK4a and hypomethylation of the CDH13 genes. For the first time, the survey was carried out on such an extent that it

  4. Methylation profiling of twenty promoter-CpG islands of genes which may contribute to hepatocellular carcinogenesis

    Directory of Open Access Journals (Sweden)

    Zhang Lisheng

    2002-11-01

    Full Text Available Abstract Background Hepatocellular carcinoma (HCC presents one of the major health threats in China today. A better understanding of the molecular genetics underlying malignant transformation of hepatocytes is critical to success in the battle against this disease. The methylation state of C5 of the cytosine in the CpG di-nucleotide that is enriched within or near the promoter region of over 50 % of the polymerase II genes has a drastic effect on transcription of these genes. Changes in the methylation profile of the promoters represent an alternative to genetic lesions as causative factors for the tumor-specific aberrant expression of the genes. Methods We have used the methylation specific PCR method in conjunction with DNA sequencing to assess the methylation state of the promoter CpG islands of twenty genes. Aberrant expression of these genes have been attributed to the abnormal methylation profile of the corresponding promoter CpG islands in human tumors. Results While the following sixteen genes remained the unmethylated in all tumor and normal tissues: CDH1, APAF1, hMLH1, BRCA1, hTERC, VHL, RARβ, TIMP3, DAPK1, SURVIVIN, p14ARF, RB1, p15INK4b, APC, RASSF1c and PTEN, varying degrees of tumor specific hypermethylation were associated with the p16INK4a , RASSF1a, CASP8 and CDH13 genes. For instance, the p16INK4a was highly methylated in HCC (17/29, 58.6% and less significantly methylated in non-cancerous tissue (4/29. 13.79%. The RASSF1a was fully methylated in all tumor tissues (29/29, 100%, and less frequently methylated in corresponding non-cancerous tissue (24/29, 82.75%. Conclusions Furthermore, co-existence of methylated with unmethylated DNA in some cases suggested that both genetic and epigenetic (CpG methylation mechanisms may act in concert to inactivate the p16INK4a and RASSF1a in HCC. Finally, we found a significant association of cirrhosis with hypermethylation of the p16INK4a and hypomethylation of the CDH13 genes. For the

  5. Reduced rates of gene loss, gene silencing, and gene mutation in Dnmt1-deficient embryonic stem cells

    NARCIS (Netherlands)

    Chan, M.F.; van Amerongen, R.; Nijjar, T.; Cuppen, E.; Jones, P.A.; Laird, P.W.

    2001-01-01

    Tumor suppressor gene inactivation is a crucial event in oncogenesis. Gene inactivation mechanisms include events resulting in loss of heterozygosity (LOH), gene mutation, and transcriptional silencing. The contribution of each of these different pathways varies among tumor suppressor genes and by

  6. Human male meiotic sex chromosome inactivation.

    Directory of Open Access Journals (Sweden)

    Marieke de Vries

    Full Text Available In mammalian male gametogenesis the sex chromosomes are distinctive in both gene activity and epigenetic strategy. At first meiotic prophase the heteromorphic X and Y chromosomes are placed in a separate chromatin domain called the XY body. In this process, X,Y chromatin becomes highly phosphorylated at S139 of H2AX leading to the repression of gonosomal genes, a process known as meiotic sex chromosome inactivation (MSCI, which has been studied best in mice. Post-meiotically this repression is largely maintained. Disturbance of MSCI in mice leads to harmful X,Y gene expression, eventuating in spermatocyte death and sperm heterogeneity. Sperm heterogeneity is a characteristic of the human male. For this reason we were interested in the efficiency of MSCI in human primary spermatocytes. We investigated MSCI in pachytene spermatocytes of seven probands: four infertile men and three fertile controls, using direct and indirect in situ methods. A considerable degree of variation in the degree of MSCI was detected, both between and within probands. Moreover, in post-meiotic stages this variation was observed as well, indicating survival of spermatocytes with incompletely inactivated sex chromosomes. Furthermore, we investigated the presence of H3K9me3 posttranslational modifications on the X and Y chromatin. Contrary to constitutive centromeric heterochromatin, this heterochromatin marker did not specifically accumulate on the XY body, with the exception of the heterochromatic part of the Y chromosome. This may reflect the lower degree of MSCI in man compared to mouse. These results point at relaxation of MSCI, which can be explained by genetic changes in sex chromosome composition during evolution and candidates as a mechanism behind human sperm heterogeneity.

  7. MO-DE-207B-05: Predicting Gene Mutations in Renal Cell Carcinoma Based On CT Imaging Features: Validation Using TCGA-TCIA Datasets

    Energy Technology Data Exchange (ETDEWEB)

    Chen, X; Zhou, Z; Thomas, K; Wang, J [UT Southwestern Medical Center, Dallas, TX (United States)

    2016-06-15

    Purpose: The goal of this work is to investigate the use of contrast enhanced computed tomographic (CT) features for the prediction of mutations of BAP1, PBRM1, and VHL genes in renal cell carcinoma (RCC). Methods: For this study, we used two patient databases with renal cell carcinoma (RCC). The first one consisted of 33 patients from our institution (UT Southwestern Medical Center, UTSW). The second one consisted of 24 patients from the Cancer Imaging Archive (TCIA), where each patient is connected by a unique identi?er to the tissue samples from the Cancer Genome Atlas (TCGA). From the contrast enhanced CT image of each patient, tumor contour was first delineated by a physician. Geometry, intensity, and texture features were extracted from the delineated tumor. Based on UTSW dataset, we completed feature selection and trained a support vector machine (SVM) classifier to predict mutations of BAP1, PBRM1 and VHL genes. We then used TCIA-TCGA dataset to validate the predictive model build upon UTSW dataset. Results: The prediction accuracy of gene expression of TCIA-TCGA patients was 0.83 (20 of 24), 0.83 (20 of 24), and 0.75 (18 of 24) for BAP1, PBRM1, and VHL respectively. For BAP1 gene, texture feature was the most prominent feature type. For PBRM1 gene, intensity feature was the most prominent. For VHL gene, geometry, intensity, and texture features were all important. Conclusion: Using our feature selection strategy and models, we achieved predictive accuracy over 0.75 for all three genes under the condition of using patient data from one institution for training and data from other institutions for testing. These results suggest that radiogenomics can be used to aid in prognosis and used as convenient surrogates for expensive and time consuming gene assay procedures.

  8. Lack of X inactivation associated with maternal X isodisomy: Evidence for a counting mechanism prior to X inactivation during human embryogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Migeon, B.R.; Torchia, B.S.; Fu, S. [Johns Hopkins Univ. School of Medicine, Baltimore, MD (United States)] [and others

    1996-01-01

    We have previously reported functional disomy for X-linked genes in females with tiny ring X chromosomes and a phenotype significantly more abnormal than Turner syndrome. In such cases the disomy results from failure of these X chromosomes to inactivate because they lack DNA sequences essential for cis X inactivation. Here we describe a novel molecular mechanism for functional X disomy that is associated with maternal isodisomy. In this case, the severe mental retardation and multiple congenital abnormalities in a female with a mosaic 45,X/46,X,del(X) (q21.3-qter)/46X,r(X) karyotype are associated with overexpression of the genes within Xpter to Xq21.31 in many of her cells. Her normal X, ring X, and deleted linear X chromosomes originate from the same maternal X chromosome, and all are transcriptionally active. None expresses X inactive specific transcript (XIST), although the locus and region of the putative X inactivation center (XIC) are present on both normal and linear deleted X chromosomes. To our knowledge, this is the first report of a functional maternal X isodisomy, and the largest X chromosome to escape inactivation. In addition, these results (1) show that cis inactivation does not invariably occur in human females with two X chromosomes, even when the XIC region is present on both of them; (2) provide evidence for a critical time prior to the visible onset of X inactivation in the embryo when decisions about X inactivation are made; and (3) support the hypothesis that the X chromosome counting mechanism involves chromosomal imprinting, occurs prior to the onset of random inactivation, and is required for subsequent inactivation of the chromosome. 41 refs., 4 figs., 2 tabs.

  9. Influenza Vaccine, Inactivated or Recombinant

    Science.gov (United States)

    ... die from flu, and many more are hospitalized.Flu vaccine can:keep you from getting flu, make flu ... What is inactivated or recombinant influenza vaccine?A dose of flu vaccine is recommended every flu season. Children 6 months through 8 years of age may need two ...

  10. Genetic architecture of skewed X inactivation in the laboratory mouse.

    Directory of Open Access Journals (Sweden)

    John D Calaway

    Full Text Available X chromosome inactivation (XCI is the mammalian mechanism of dosage compensation that balances X-linked gene expression between the sexes. Early during female development, each cell of the embryo proper independently inactivates one of its two parental X-chromosomes. In mice, the choice of which X chromosome is inactivated is affected by the genotype of a cis-acting locus, the X-chromosome controlling element (Xce. Xce has been localized to a 1.9 Mb interval within the X-inactivation center (Xic, yet its molecular identity and mechanism of action remain unknown. We combined genotype and sequence data for mouse stocks with detailed phenotyping of ten inbred strains and with the development of a statistical model that incorporates phenotyping data from multiple sources to disentangle sources of XCI phenotypic variance in natural female populations on X inactivation. We have reduced the Xce candidate 10-fold to a 176 kb region located approximately 500 kb proximal to Xist. We propose that structural variation in this interval explains the presence of multiple functional Xce alleles in the genus Mus. We have identified a new allele, Xce(e present in Mus musculus and a possible sixth functional allele in Mus spicilegus. We have also confirmed a parent-of-origin effect on X inactivation choice and provide evidence that maternal inheritance magnifies the skewing associated with strong Xce alleles. Based on the phylogenetic analysis of 155 laboratory strains and wild mice we conclude that Xce(a is either a derived allele that arose concurrently with the domestication of fancy mice but prior the derivation of most classical inbred strains or a rare allele in the wild. Furthermore, we have found that despite the presence of multiple haplotypes in the wild Mus musculus domesticus has only one functional Xce allele, Xce(b. Lastly, we conclude that each mouse taxa examined has a different functional Xce allele.

  11. Global survey of escape from X inactivation by RNA-sequencing in mouse.

    Science.gov (United States)

    Yang, Fan; Babak, Tomas; Shendure, Jay; Disteche, Christine M

    2010-05-01

    X inactivation equalizes the dosage of gene expression between the sexes, but some genes escape silencing and are thus expressed from both alleles in females. To survey X inactivation and escape in mouse, we performed RNA sequencing in Mus musculus x Mus spretus cells with complete skewing of X inactivation, relying on expression of single nucleotide polymorphisms to discriminate allelic origin. Thirteen of 393 (3.3%) mouse genes had significant expression from the inactive X, including eight novel escape genes. We estimate that mice have significantly fewer escape genes compared with humans. Furthermore, escape genes did not cluster in mouse, unlike the large escape domains in human, suggesting that expression is controlled at the level of individual genes. Our findings are consistent with the striking differences in phenotypes between female mice and women with a single X chromosome--a near normal phenotype in mice versus Turner syndrome and multiple abnormalities in humans. We found that escape genes are marked by the absence of trimethylation at lysine 27 of histone H3, a chromatin modification associated with genes subject to X inactivation. Furthermore, this epigenetic mark is developmentally regulated for some mouse genes.

  12. The eag domain regulates hERG channel inactivation gating via a direct interaction

    Science.gov (United States)

    Gustina, Ahleah S.

    2013-01-01

    Human ether-á-go-go (eag)-related gene (hERG) potassium channel kinetics are characterized by rapid inactivation upon depolarization, along with rapid recovery from inactivation and very slow closing (deactivation) upon repolarization. These factors combine to create a resurgent hERG current, where the current amplitude is paradoxically larger with repolarization than with depolarization. Previous data showed that the hERG N-terminal eag domain regulated deactivation kinetics by making a direct interaction with the C-terminal region of the channel. A primary mechanism for fast inactivation depends on residues in the channel pore; however, inactivation was also shown to be slower after deletion of a large N-terminal region. The mechanism for N-terminal region regulation of inactivation is unclear. Here, we investigated the contributions of the large N-terminal domains (amino acids 1–354), including the eag domain (amino acids 1–135), to hERG channel inactivation kinetics and steady-state inactivation properties. We found that N-deleted channels lacking just the eag domain (Δ2–135) or both the eag domain and the adjacent proximal domain (Δ2–354) had less rectifying current–voltage (I-V) relationships, slower inactivation, faster recovery from inactivation, and lessened steady-state inactivation. We coexpressed genetically encoded N-terminal fragments for the eag domain (N1–135) or the eag domain plus the proximal domain (N1–354) with N-deleted hERG Δ2–135 or hERG Δ2–354 channels and found that the resulting channels had more rectifying I-V relationships, faster inactivation, slower recovery from inactivation, and increased steady-state inactivation, similar to those properties measured for wild-type (WT) hERG. We also found that the eag domain–containing fragments regulated the time to peak and the voltage at the peak of a resurgent current elicited with a ramp voltage protocol. The eag domain–containing fragments effectively converted N

  13. Independent evolution of transcriptional inactivation on sex chromosomes in birds and mammals.

    Directory of Open Access Journals (Sweden)

    Alexandra M Livernois

    Full Text Available X chromosome inactivation in eutherian mammals has been thought to be tightly controlled, as expected from a mechanism that compensates for the different dosage of X-borne genes in XX females and XY males. However, many X genes escape inactivation in humans, inactivation of the X in marsupials is partial, and the unrelated sex chromosomes of monotreme mammals have incomplete and gene-specific inactivation of X-linked genes. The bird ZW sex chromosome system represents a third independently evolved amniote sex chromosome system with dosage compensation, albeit partial and gene-specific, via an unknown mechanism (i.e. upregulation of the single Z in females, down regulation of one or both Zs in males, or a combination. We used RNA-fluorescent in situ hybridization (RNA-FISH to demonstrate, on individual fibroblast cells, inactivation of 11 genes on the chicken Z and 28 genes on the X chromosomes of platypus. Each gene displayed a reproducible frequency of 1Z/1X-active and 2Z/2X-active cells in the homogametic sex. Our results indicate that the probability of inactivation is controlled on a gene-by-gene basis (or small domains on the chicken Z and platypus X chromosomes. This regulatory mechanism must have been exapted independently to the non-homologous sex chromosomes in birds and mammals in response to an over-expressed Z or X in the homogametic sex, highlighting the universal importance that (at least partial silencing plays in the evolution on amniote dosage compensation and, therefore, the differentiation of sex chromosomes.

  14. Essential Bacillus subtilis genes

    DEFF Research Database (Denmark)

    Kobayashi, K.; Ehrlich, S.D.; Albertini, A.

    2003-01-01

    To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximate to4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were...

  15. Inactivation of rabies virus by hydrogen peroxide.

    Science.gov (United States)

    Abd-Elghaffar, Asmaa A; Ali, Amal E; Boseila, Abeer A; Amin, Magdy A

    2016-02-03

    Development of safe and protective vaccines against infectious pathogens remains a challenge. Inactivation of rabies virus is a critical step in the production of vaccines and other research reagents. Beta-propiolactone (βPL); the currently used inactivating agent for rabies virus is expensive and proved to be carcinogenic in animals. This study aimed to investigate the ability of hydrogen peroxide (H2O2) to irreversibly inactivate rabies virus without affecting its antigenicity and immunogenicity in pursuit of finding safe, effective and inexpensive alternative inactivating agents. H2O2 3% rapidly inactivated a Vero cell adapted fixed rabies virus strain designated as FRV/K within 2h of exposure without affecting its antigenicity or immunogenicity. No residual infectious virus was detected and the H2O2-inactivated vaccine proved to be safe and effective when compared with the same virus harvest inactivated with the classical inactivating agent βPL. Mice immunized with H2O2-inactivated rabies virus produced sufficient level of antibodies and were protected when challenged with lethal CVS virus. These findings reinforce the idea that H2O2 can replace βPL as inactivating agent for rabies virus to reduce time and cost of inactivation process. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Sex-differential selection and the evolution of X inactivation strategies.

    Directory of Open Access Journals (Sweden)

    Tim Connallon

    2013-04-01

    Full Text Available X inactivation--the transcriptional silencing of one X chromosome copy per female somatic cell--is universal among therian mammals, yet the choice of which X to silence exhibits considerable variation among species. X inactivation strategies can range from strict paternally inherited X inactivation (PXI, which renders females haploid for all maternally inherited alleles, to unbiased random X inactivation (RXI, which equalizes expression of maternally and paternally inherited alleles in each female tissue. However, the underlying evolutionary processes that might account for this observed diversity of X inactivation strategies remain unclear. We present a theoretical population genetic analysis of X inactivation evolution and specifically consider how conditions of dominance, linkage, recombination, and sex-differential selection each influence evolutionary trajectories of X inactivation. The results indicate that a single, critical interaction between allelic dominance and sex-differential selection can select for a broad and continuous range of X inactivation strategies, including unequal rates of inactivation between maternally and paternally inherited X chromosomes. RXI is favored over complete PXI as long as alleles deleterious to female fitness are sufficiently recessive, and the criteria for RXI evolution is considerably more restrictive when fitness variation is sexually antagonistic (i.e., alleles deleterious to females are beneficial to males relative to variation that is deleterious to both sexes. Evolutionary transitions from PXI to RXI also generally increase mean relative female fitness at the expense of decreased male fitness. These results provide a theoretical framework for predicting and interpreting the evolution of chromosome-wide expression of X-linked genes and lead to several useful predictions that could motivate future studies of allele-specific gene expression variation.

  17. Demeanor of rivaroxaban in activated/inactivated FXa

    Directory of Open Access Journals (Sweden)

    Kaname Seki

    2017-03-01

    Full Text Available Activated factor X (FXa plays an important role in thrombin generation and inflammation. Factor X is not converted constitutively to FXa, but only after intrinsic clotting factors are activated and/or cellular injury occurs. Although rivaroxaban is one of direct FXa inhibitors, its function in the inactivated coagulation cascade is unclear. In human umbilical vein endothelial cells that natively express protease-activated receptor-1 and -2, high dose rivaroxaban did not alter gene transcripts including pro-inflammatory genes in DNA microarray. Upon FXa stimulation, the expressions of pro-inflammatory genes such as monocyte chemoattractant protein-1 (MCP-1, intracellular adhesion molecule-1, and interleukin-8 were maximally increased at 4 h after stimulation, and were suppressed by rivaroxaban. To confirm these results, quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA for MCP-1 were performed. FXa evoked the expression of MCP-1 maximally at 4 h after stimulation, whereas MCP-1 displayed a different temporal activation in ELISA. Interestingly, rivaroxaban inhibited both time courses of MCP-1 expression. These results suggest that rivaroxaban may not influence gene modulation in the inactivated coagulation state, but can attenuate the endothelial damage evoked by FXa and pro-inflammatory cytokine genes.

  18. Effects of track structure and cell inactivation on the calculation of heavy ion mutation rates in mammalian cells

    Science.gov (United States)

    Cucinotta, F. A.; Wilson, J. W.; Shavers, M. R.; Katz, R.

    1996-01-01

    It has long been suggested that inactivation severely effects the probability of mutation by heavy ions in mammalian cells. Heavy ions have observed cross sections of inactivation that approach and sometimes exceed the geometric size of the cell nucleus in mammalian cells. In the track structure model of Katz the inactivation cross section is found by summing an inactivation probability over all impact parameters from the ion to the sensitive sites within the cell nucleus. The inactivation probability is evaluated using the dose-response of the system to gamma-rays and the radial dose of the ions and may be equal to unity at small impact parameters for some ions. We show how the effects of inactivation may be taken into account in the evaluation of the mutation cross sections from heavy ions in the track structure model through correlation of sites for gene mutation and cell inactivation. The model is fit to available data for HPRT mutations in Chinese hamster cells and good agreement is found. The resulting calculations qualitatively show that mutation cross sections for heavy ions display minima at velocities where inactivation cross sections display maxima. Also, calculations show the high probability of mutation by relativistic heavy ions due to the radial extension of ions track from delta-rays in agreement with the microlesion concept. The effects of inactivation on mutations rates make it very unlikely that a single parameter such as LET or Z*2/beta(2) can be used to specify radiation quality for heavy ion bombardment.

  19. Paternal X inactivation does not correlate with X chromosome evolutionary strata in marsupials.

    Science.gov (United States)

    Rodríguez-Delgado, Claudia L; Waters, Shafagh A; Waters, Paul D

    2014-12-24

    X chromosome inactivation is the transcriptional silencing of one X chromosome in the somatic cells of female mammals. In eutherian mammals (e.g. humans) one of the two X chromosomes is randomly chosen for silencing, with about 15% (usually in younger evolutionary strata of the X chromosome) of genes escaping this silencing. In contrast, in the distantly related marsupial mammals the paternally derived X is silenced, although not as completely as the eutherian X. A chromosome wide examination of X inactivation, using RNA-seq, was recently undertaken in grey short-tailed opossum (Monodelphis domestica) brain and extraembryonic tissues. However, no such study has been conduced in Australian marsupials, which diverged from their American cousins ~80 million years ago, leaving a large gap in our understanding of marsupial X inactivation. We used RNA-seq data from blood or liver of a family (mother, father and daughter) of tammar wallabies (Macropus eugenii), which in conjunction with available genome sequence from the mother and father, permitted genotyping of 42 expressed heterozygous SNPs on the daughter's X. These 42 SNPs represented 34 X loci, of which 68% (23 of the 34) were confirmed as inactivated on the paternally derived X in the daughter's liver; the remaining 11 X loci escaped inactivation. Seven of the wallaby loci sampled were part of the old X evolutionary stratum, of which three escaped inactivation. Three loci were classified as part of the newer X stratum, of which two escaped inactivation. A meta-analysis of previously published opossum X inactivation data revealed that 5 of 52 genes in the old X stratum escaped inactivation. We demonstrate that chromosome wide inactivation of the paternal X is common to an Australian marsupial representative, but that there is more escape from inactivation than reported for opossum (32% v 14%). We also provide evidence that, unlike the human X chromosome, the location of loci within the oldest evolutionary stratum on

  20. Mechanistic insight into digoxin inactivation by Eggerthella lenta augments our understanding of its pharmacokinetics.

    Science.gov (United States)

    Haiser, Henry J; Seim, Kristen L; Balskus, Emily P; Turnbaugh, Peter J

    2014-01-01

    The human gut microbiota plays a key role in pharmacology, yet the mechanisms responsible remain unclear, impeding efforts toward personalized medicine. We recently identified a cytochrome-encoding operon in the common gut Actinobacterium Eggerthella lenta that is transcriptionally activated by the cardiac drug digoxin. These genes represent a predictive microbial biomarker for the inactivation of digoxin. Gnotobiotic mouse experiments revealed that increased protein intake can limit microbial drug inactivation. Here, we present a biochemical rationale for how the proteins encoded by this operon might inactivate digoxin through substrate promiscuity. We discuss digoxin signaling in eukaryotic systems, and consider the possibility that endogenous digoxin-like molecules may have selected for microbial digoxin inactivation. Finally, we highlight the diverse contributions of gut microbes to drug metabolism, present a generalized approach to studying microbe-drug interactions, and argue that mechanistic studies will pave the way for the clinical application of this work.

  1. Characterization of the BPI-like gene from a subtracted cDNA library of large yellow croaker (Pseudosciaena crocea) and induced expression by formalin-inactivated Vibrio alginolyticus and Nocardia seriolae vaccine challenges.

    Science.gov (United States)

    Huang, Yanqing; Lou, Huifang; Wu, Xinzhong; Chen, Yanxia

    2008-12-01

    One expressed sequence tag (EST 64LF004 clone), which is from the subtracted cDNA library of the head kidney of large yellow croaker (Pseudosciaena crocea) stimulated with peptidoglycan (PG) by suppression subtractive hybridization (SSH) method, was cloned using RACE-PCR. The full length cDNA, which possesses typical structural features of a signal peptide, a conserved LPS binding domain and two bactericidal permeability-increasing (BPI) motifs as in higher vertebrates, was identified as a novel homologue, namely of the large yellow croaker BPI-like molecule (Pc-BPI-L). Phylogenetic analysis showed this Pc-BPI-L of large yellow croaker as the most ancestral branch in bony fish clade. The recombinant Pc-BPI-L protein expressed in the Tn-5B1-4 insect cells was successfully produced and confirmed to have the predicted size of 52 kDa by Western blot analysis. At the message level, Pc-BPI-L mRNA was ubiquitously expressed in all tissues examined. Following formalin-inactivated Vibrio alginolyticus and Nocardia seriolae treatment, Pc-BPI-L message was differentially up-regulated in primary immune organs. These results indicate that Pc-BPI-L might be involved in the immune response to bacterial infection.

  2. Inactivation of the gene katA or sodA affects the transient entry into the viable but non-culturable response of Staphylococcus aureus in natural seawater at low temperature.

    Science.gov (United States)

    Masmoudi, Salma; Denis, Michel; Maalej, Sami

    2010-12-01

    We have investigated the fate of Staphylococcus aureus by starving the cells and maintaining them in natural seawater at 22 and 4 °C. At 22 °C, cells developed a long-term survival state where about 0.037% of the initial population remained culturable over more than 7 months, whereas at 4 °C, bacteria lost culturability and transiently entered into the viable but non-culturable state (VBNC). However, after 22 days of entry into the VBNC state, the number of viable cells detected via the direct viable count method decreased significantly. We show here that mutational inactivation of catalase (KatA) or superoxide dismutase (SodA) rendered strains hypersensitive to seawater stress at 4 °C and consequently, part of the seawater lethality on S. aureus at low temperature is mediated by reactive oxygen species (ROS) during microcosm-survival process. Shifting the temperature from 4 to 22 °C of totally non-culturable wild-type cells induced a partial recovery of the population. However, deficiencies in catalase or superoxide dismutase prevent resuscitation ability. Copyright © 2010 Elsevier Ltd. All rights reserved.

  3. Photodynamic Inactivation of Mammalian Viruses and Bacteriophages

    Directory of Open Access Journals (Sweden)

    Liliana Costa

    2012-06-01

    Full Text Available Photodynamic inactivation (PDI has been used to inactivate microorganisms through the use of photosensitizers. The inactivation of mammalian viruses and bacteriophages by photosensitization has been applied with success since the first decades of the last century. Due to the fact that mammalian viruses are known to pose a threat to public health and that bacteriophages are frequently used as models of mammalian viruses, it is important to know and understand the mechanisms and photodynamic procedures involved in their photoinactivation. The aim of this review is to (i summarize the main approaches developed until now for the photodynamic inactivation of bacteriophages and mammalian viruses and, (ii discuss and compare the present state of the art of mammalian viruses PDI with phage photoinactivation, with special focus on the most relevant mechanisms, molecular targets and factors affecting the viral inactivation process.

  4. Inhibition of TNF-α-induced MUC5AC mucin gene expression and production by wogonin through the inactivation of NF-κB signaling in airway epithelial cells.

    Science.gov (United States)

    Sikder, Md Asaduzzaman; Lee, Hyun Jae; Mia, Md Zakaria; Park, Su Hyun; Ryu, Jiho; Kim, Jang-Hyun; Min, Sang Yeon; Hong, Jang-Hee; Seok, Jeong Ho; Lee, Choong Jae

    2014-01-01

    In this study, we investigated whether wogonin significantly affects MUC5AC mucin gene expression and production in human airway epithelial cells. Confluent NCI-H292 cells were pretreated with wogonin for 30 min and then stimulated with tumor necrosis factor-α (TNF-α) for 24 h or the indicated periods. The MUC5AC mucin gene expression and mucin protein production were measured by RT-PCR and ELISA, respectively. We found that incubation of NCI-H292 cells with wogonin significantly inhibited mucin production and down-regulated MUC5AC gene expression induced by TNF-α in a dose-dependent fashion. To elucidate the action mechanism of wogonin, effect of wogonin on TNF-α-induced NF-κB signaling pathway was investigated by western blot analysis. Wogonin inhibited NF-κB activation induced by TNF-α. Inhibition of IKK by wogonin led to the suppression of IκB phosphorylation and degradation, p65 nuclear translocation and NF-κB-regulated gene expression. This, in turn, led to the down-regulation of MUC5AC protein production in NCI-H292 cells. Wogonin also inhibited the gene products involved in cell survival (Bcl-2) and proliferation (cyclooxygenase-2). These results suggest that wogonin inhibits the NF-κB signaling pathway, which may explain its role in the inhibition of MUC5AC mucin gene expression and production. Copyright © 2013 John Wiley & Sons, Ltd.

  5. The allele frequency of two single nucleotide polymorphisms in the von Hippel-Lindau (VHL tumor suppressor gene in the Taiwanese population

    Directory of Open Access Journals (Sweden)

    Wen-Chung Wang

    2011-10-01

    Conclusion: We found that the G allele frequency at these two loci in the Taiwanese population is much lower than that in people from Western countries. This phenomenon may be attributed to ethnic effects.

  6. The probability to initiate X chromosome inactivation is determined by the X to autosomal ratio and X chromosome specific allelic properties

    NARCIS (Netherlands)

    Monkhorst, Kim; de Hoon, Bas; Jonkers, Iris; Mulugeta Achame, Eskeatnaf; Monkhorst, Wouter; Hoogerbrugge, Jos; Rentmeester, Eveline; Westerhoff, Hans V; Grosveld, Frank; Grootegoed, J Anton; Gribnau, Joost

    2009-01-01

    BACKGROUND: In female mammalian cells, random X chromosome inactivation (XCI) equalizes the dosage of X-encoded gene products to that in male cells. XCI is a stochastic process, in which each X chromosome has a probability to be inactivated. To obtain more insight in the factors setting up this

  7. Inactivation of norovirus on dry copper alloy surfaces.

    Science.gov (United States)

    Warnes, Sarah L; Keevil, C William

    2013-01-01

    Noroviruses (family Caliciviridae) are the primary cause of viral gastroenteritis worldwide. The virus is highly infectious and touching contaminated surfaces can contribute to infection spread. Although the virus was identified over 40 years ago the lack of methods to assess infectivity has hampered the study of the human pathogen. Recently the murine virus, MNV-1, has successfully been used as a close surrogate. Copper alloys have previously been shown to be effective antimicrobial surfaces against a range of bacteria and fungi. We now report rapid inactivation of murine norovirus on alloys, containing over 60% copper, at room temperature but no reduction of infectivity on stainless steel dry surfaces in simulated wet fomite and dry touch contamination. The rate of inactivation was initially very rapid and proportional to copper content of alloy tested. Viral inactivation was not as rapid on brass as previously observed for bacteria but copper-nickel alloy was very effective. The use of chelators and quenchers of reactive oxygen species (ROS) determined that Cu(II) and especially Cu(I) ions are still the primary effectors of toxicity but quenching superoxide and hydroxyl radicals did not confer protection. This suggests Fenton generation of ROS is not important for the inactivation mechanism. One of the targets of copper toxicity was the viral genome and a reduced copy number of the gene for a viral encoded protein, VPg (viral-protein-genome-linked), which is essential for infectivity, was observed following contact with copper and brass dry surfaces. The use of antimicrobial surfaces containing copper in high risk closed environments such as cruise ships and care facilities could help to reduce the spread of this highly infectious and costly pathogen.

  8. Inactivation of norovirus on dry copper alloy surfaces.

    Directory of Open Access Journals (Sweden)

    Sarah L Warnes

    Full Text Available Noroviruses (family Caliciviridae are the primary cause of viral gastroenteritis worldwide. The virus is highly infectious and touching contaminated surfaces can contribute to infection spread. Although the virus was identified over 40 years ago the lack of methods to assess infectivity has hampered the study of the human pathogen. Recently the murine virus, MNV-1, has successfully been used as a close surrogate. Copper alloys have previously been shown to be effective antimicrobial surfaces against a range of bacteria and fungi. We now report rapid inactivation of murine norovirus on alloys, containing over 60% copper, at room temperature but no reduction of infectivity on stainless steel dry surfaces in simulated wet fomite and dry touch contamination. The rate of inactivation was initially very rapid and proportional to copper content of alloy tested. Viral inactivation was not as rapid on brass as previously observed for bacteria but copper-nickel alloy was very effective. The use of chelators and quenchers of reactive oxygen species (ROS determined that Cu(II and especially Cu(I ions are still the primary effectors of toxicity but quenching superoxide and hydroxyl radicals did not confer protection. This suggests Fenton generation of ROS is not important for the inactivation mechanism. One of the targets of copper toxicity was the viral genome and a reduced copy number of the gene for a viral encoded protein, VPg (viral-protein-genome-linked, which is essential for infectivity, was observed following contact with copper and brass dry surfaces. The use of antimicrobial surfaces containing copper in high risk closed environments such as cruise ships and care facilities could help to reduce the spread of this highly infectious and costly pathogen.

  9. Inactivation of poliovirus by chloramine-T.

    Science.gov (United States)

    Gowda, N M; Trieff, N M; Stanton, G J

    1981-01-01

    Since concern has recently been expressed about the presence of genotoxic substances due to chlorination of water and wastewater, chloramine-T (CAT) is proposed as an alternative disinfectant to chlorine. The viricidal properties of chlorine and CAT were compared. Kinetics of inactivation of poliovirus type 2 by chlorine and CAT in chlorine demand-free water were investigated by using a kinetic apparatus. Inactivation of the virus by chlorine and CAT occurred in two steps. The initial linear part of the inactivation curve followed a pseudo-first-order reaction with the virus. An obvious dose-response relationship was demonstrated with CAT. The rate of inactivation of the virus by CAT was faster in acid medium than in alkaline medium. Inactivation kinetic studies were performed at different temperatures, and the kinetic, Arrhenius, and thermodynamic parameters were evaluated. The rate of inactivation of poliovirus type 2 by chlorine was faster than that by CAT under identical conditions. A mechanism for the viral inactivation in acid conditions was proposed which led to a rate equation consistent with the experimental results. The results indicate that CAT may be an effective viricide against poliovirus type 2 in an acid medium. PMID:6271058

  10. Expression of genes from the human active and inactive X chromosomes.

    OpenAIRE

    Brown, C J; Carrel, L; Willard, H F

    1997-01-01

    X-chromosome inactivation results in the cis-limited inactivation of many, but not all, of the genes on one of the pair of X chromosomes in mammalian females. In addition to the genes from the pseudoautosomal region, which have long been anticipated to escape inactivation, genes from several other regions of the human X chromosome have now been shown to escape inactivation and to be expressed from both the active and inactive X chromosomes. The growing number of genes escaping inactivation em...

  11. Monitoring the effect of gene silencing by RNA interference in human CD34+ cells injected into newborn RAG2-/- gammac-/- mice: functional inactivation of p53 in developing T cells

    NARCIS (Netherlands)

    Gimeno, Ramon; Weijer, Kees; Voordouw, Arie; Uittenbogaart, Christel H.; Legrand, Nicolas; Alves, Nuno L.; Wijnands, Erwin; Blom, Bianca; Spits, Hergen

    2004-01-01

    Tumor suppressor p53 plays an important role in regulating cell cycle progression and apoptosis. Here we applied RNA interference to study the role of p53 in human hematopoietic development in vivo. An siRNA construct specifically targeting the human tumor-suppressor gene p53 was introduced into

  12. Inactivation of pea genes by RNAi supports the involvement of two similar O-methyltransferases in the biosynthesis of (+)-pisatin and of chiral intermediates with a configuration opposite that found in (+)-pisatin.

    Science.gov (United States)

    Kaimoyo, Evans; VanEtten, Hans D

    2008-01-01

    (+)-Pisatin, the major phytoalexin of pea (Pisum sativum L.), is believed to be synthesized via two chiral intermediates, (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanone [(-)-sophorol] and (-)-7,2'-dihydroxy-4',5'-methylenedioxyisoflavanol [(-)-DMDI]; both have an opposite C-3 absolute configuration to that found at C-6a in (+)-pisatin. The expression of isoflavone reductase (IFR), which converts 7,2'-dihydroxy-4',5'-methylenedioxyisoflavone (DMD) to (-)-sophorol, sophorol reductase (SOR), which converts (-)-sophorol to (-)-DMDI, and hydroxymaackiain-3-O-methyltransferase (HMM), believed to be the last step of (+)-pisatin biosynthesis, were inactivated by RNA-mediated genetic interference (RNAi) in pea hairy roots. Some hairy root lines containing RNAi constructs of IFR and SOR accumulated DMD or (-)-sophorol, respectively, and were deficient in (+)-pisatin biosynthesis supporting the involvement of chiral intermediates with a configuration opposite to that found in (+)-pisatin in the biosynthesis of (+)-pisatin. Pea proteins also converted (-)-DMDI to an achiral isoflavene suggesting that an isoflavene might be the intermediate through which the configuration is changed to that found in (+)-pisatin. Hairy roots containing RNAi constructs of HMM also were deficient in (+)-pisatin biosynthesis, but did not accumulate (+)-6a-hydroxymaackiain, the proposed precursor to (+)-pisatin. Instead, 2,7,4'-trihydroxyisoflavanone (TIF), daidzein, isoformononetin, and liquiritigenin accumulated. HMM has a high amino acid similarity to hydroxyisoflavanone-4'-O-methyltransferase (HI4'OMT), an enzyme that methylates TIF, an early intermediate in the isoflavonoid pathway. The accumulation of these four compounds is consistent with the blockage of the synthesis of (+)-pisatin at the HI4'OMT catalyzed step resulting in the accumulation of liquiritigenin and TIF and the diversion of the pathway to produce daidzein and isoformononetin, compounds not normally made by pea. Previous

  13. Gene

    Data.gov (United States)

    U.S. Department of Health & Human Services — Gene integrates information from a wide range of species. A record may include nomenclature, Reference Sequences (RefSeqs), maps, pathways, variations, phenotypes,...

  14. Mutations Inactivating Herpes Simplex Virus 1 MicroRNA miR-H2 Do Not Detectably Increase ICP0 Gene Expression in Infected Cultured Cells or Mouse Trigeminal Ganglia.

    Science.gov (United States)

    Pan, Dongli; Pesola, Jean M; Li, Gang; McCarron, Seamus; Coen, Donald M

    2017-01-15

    Herpes simplex virus 1 (HSV-1) latency entails the repression of productive ("lytic") gene expression. An attractive hypothesis to explain some of this repression involves inhibition of the expression of ICP0, a lytic gene activator, by a viral microRNA, miR-H2, which is completely complementary to ICP0 mRNA. To test this hypothesis, we engineered mutations that disrupt miR-H2 without affecting ICP0 in HSV-1. The mutant virus exhibited drastically reduced expression of miR-H2 but showed wild-type levels of infectious virus production and no increase in ICP0 expression in lytically infected cells, which is consistent with the weak expression of miR-H2 relative to the level of ICP0 mRNA in that setting. Following corneal inoculation of mice, the mutant was not significantly different from wild-type virus in terms of infectious virus production in the trigeminal ganglia during acute infection, mouse mortality, or the rate of reactivation from explanted latently infected ganglia. Critically, the mutant was indistinguishable from wild-type virus for the expression of ICP0 and other lytic genes in acutely and latently infected mouse trigeminal ganglia. The latter result may be related to miR-H2 being less effective in inhibiting ICP0 expression in transfection assays than a host microRNA, miR-138, which has previously been shown to inhibit lytic gene expression in infected ganglia by targeting ICP0 mRNA. Additionally, transfected miR-138 reduced lytic gene expression in infected cells more effectively than miR-H2. While this study provides little support for the hypothesis that miR-H2 promotes latency by inhibiting ICP0 expression, the possibility remains that miR-H2 might target other genes during latency. Herpes simplex virus 1 (HSV-1), which causes a variety of diseases, can establish lifelong latent infections from which virus can reactivate to cause recurrent disease. Latency is the most biologically interesting and clinically vexing feature of the virus. Ever since

  15. Induction of ovarian leiomyosarcomas in mice by conditional inactivation of Brca1 and p53.

    Directory of Open Access Journals (Sweden)

    Bridget A Quinn

    2009-12-01

    Full Text Available Approximately one out of every ten cases of epithelial ovarian cancer (EOC is inherited. The majority of inherited cases of EOC result from mutations in the breast cancer associated gene 1 (BRCA1. In addition to mutation of BRCA1, mutation of the p53 gene is often found in patients with inherited breast and ovarian cancer syndrome.We investigated the role of loss of function of BRCA1 and p53 in ovarian cancer development using mouse models with conditionally expressed alleles of Brca1 and/or p53. Our results show that ovary-specific Cre-recombinase-mediated conditional inactivation of both Brca1(LoxP/LoxP and p53(LoxP/LoxP resulted in ovarian or reproductive tract tumor formation in 54% of mice, whereas conditional inactivation of either allele alone infrequently resulted in tumors (< or =5% of mice. In mice with conditionally inactivated Brca1(LoxP/LoxP and p53(LoxP/LoxP, ovarian tumors arose after long latency with the majority exhibiting histological features consistent with high grade leiomyosarcomas lacking expression of epithelial, follicular or lymphocyte markers. In addition, tumors with conditional inactivation of both Brca1(LoxP/LoxP and p53(LoxP/LoxP exhibited greater genomic instability compared to an ovarian tumor with inactivation of only p53(LoxP/LoxP.Although conditional inactivation of both Brca1 and p53 results in ovarian tumorigenesis, our results suggest that additional genetic alterations or alternative methods for targeting epithelial cells of the ovary or fallopian tube for conditional inactivation of Brca1 and p53 are required for the development of a mouse model of Brca1-associated inherited EOC.

  16. Role of p53 and CDKN2A Inactivation in Human Squamous Cell Carcinomas

    Directory of Open Access Journals (Sweden)

    Alessia Pacifico

    2007-01-01

    Several studies have shown that human SCCs harbour unique mutations in the p53 gene as well as inactivation of the CDKN2A gene. While mutations in the p53 gene are induced by UV radiation and represent tumor initiating events, the majority of alterations detected in the CDKN2A gene do not appear to be UV-dependent. In conclusion, in addition to p53 mutations, silencing of the CDKN2A gene might play a significant role in SCC development.

  17. Inactivating mutations in NPC1L1 and protection from coronary heart disease

    NARCIS (Netherlands)

    Stitziel, Nathan O.; Won, Hong-Hee; Morrison, Alanna C.; Peloso, Gina M.; Do, Ron; Lange, Leslie A.; Fontanillas, Pierre; Gupta, Namrata; Duga, Stefano; Goel, Anuj; Farrall, Martin; Saleheen, Danish; Ferrario, Paola; König, Inke; Asselta, Rosanna; Merlini, Piera A.; Marziliano, Nicola; Notarangelo, Maria Francesca; Schick, Ursula; Auer, Paul; Assimes, Themistocles L.; Reilly, Muredach; Wilensky, Robert; Rader, Daniel J.; Hovingh, G. Kees; Meitinger, Thomas; Kessler, Thorsten; Kastrati, Adnan; Laugwitz, Karl-Ludwig; Siscovick, David; Rotter, Jerome I.; Hazen, Stanley L.; Tracy, Russell; Cresci, Sharon; Spertus, John; Jackson, Rebecca; Schwartz, Stephen M.; Natarajan, Pradeep; Crosby, Jacy; Muzny, Donna; Ballantyne, Christie; Rich, Stephen S.; O'Donnell, Christopher J.; Abecasis, Goncalo; Sunyaev, Shamil; Nickerson, Deborah A.; Buring, Julie E.; Ridker, Paul M.; Chasman, Daniel I.; Austin, Erin; Ye, Zi; Kullo, Iftikhar J.; Weeke, Peter E.; Shaffer, Christian M.; Bastarache, Lisa A.; Denny, Joshua C.; Roden, Dan M.; Palmer, Colin; Deloukas, Panos; Lin, Dan-Yu; Tang, Zheng-zheng; Erdmann, Jeanette; Schunkert, Heribert; Danesh, John; Marrugat, Jaume; Elosua, Roberto; Ardissino, Diego; McPherson, Ruth; Watkins, Hugh; Reiner, Alex P.; Wilson, James G.; Altshuler, David; Gibbs, Richard A.; Lander, Eric S.; Boerwinkle, Eric; Gabriel, Stacey; Kathiresan, Sekar

    2014-01-01

    Ezetimibe lowers plasma levels of low-density lipoprotein (LDL) cholesterol by inhibiting the activity of the Niemann-Pick C1-like 1 (NPC1L1) protein. However, whether such inhibition reduces the risk of coronary heart disease is not known. Human mutations that inactivate a gene encoding a drug

  18. Inactivation of C4orf26 in toothless placental mammals.

    Science.gov (United States)

    Springer, Mark S; Starrett, James; Morin, Phillip A; Lanzetti, Agnese; Hayashi, Cheryl; Gatesy, John

    2016-02-01

    Previous studies have reported inactivated copies of six enamel-related genes (AMBN, AMEL, AMTN, ENAM, KLK4, MMP20) and one dentin-related gene (DSPP) in one or more toothless vertebrates and/or vertebrates with enamelless teeth, thereby providing evidence that these genes are enamel or tooth-specific with respect to their critical functions that are maintained by natural selection. Here, we employ available genome sequences for edentulous and enamelless mammals to evaluate the enamel specificity of four genes (WDR72, SLC24A4, FAM83H, C4orf26) that have been implicated in amelogenesis imperfecta, a condition in which proper enamel formation is abrogated during tooth development. Coding sequences for WDR72, SCL24A4, and FAM83H are intact in four edentulous taxa (Chinese pangolin, three baleen whales) and three taxa (aardvark, nine-banded armadillo, Hoffmann's two-toed sloth) with enamelless teeth, suggesting that these genes have critical functions beyond their involvement in tooth development. By contrast, genomic data for C4orf26 reveal inactivating mutations in pangolin and bowhead whale as well as evidence for deletion of this gene in two minke whale species. Hybridization capture of exonic regions and PCR screens provide evidence for inactivation of C4orf26 in eight additional baleen whale species. However, C4orf26 is intact in all three species with enamelless teeth that were surveyed, as well as in 95 additional mammalian species with enamel-capped teeth. Estimates of selection intensity suggest that dN/dS ratios on branches leading to taxa with enamelless teeth are similar to the dN/dS ratio on branches leading to taxa with enamel-capped teeth. Based on these results, we conclude that C4orf26 is tooth-specific, but not enamel-specific, with respect to its essential functions that are maintained by natural selection. A caveat is that an alternative splice site variant, which translates exon 3 in a different reading frame, is putatively functional in

  19. Generation of Biotechnology-Derived Flavobacterium columnare Ghosts by PhiX174 Gene E-Mediated Inactivation and the Potential as Vaccine Candidates against Infection in Grass Carp

    Directory of Open Access Journals (Sweden)

    Wenxing Zhu

    2012-01-01

    Full Text Available Flavobacterium columnare is a bacterial pathogen causing high mortality rates for many freshwater fish species. Fish vaccination with a safe and effective vaccine is a potential approach for prevention and control of fish disease. Here, in order to produce bacterial ghost vaccine, a specific Flavobacterium lysis plasmid pBV-E-cat was constructed by cloning PhiX174 lysis gene E and the cat gene with the promoter of F. columnare into the prokaryotic expression vector pBV220. The plasmid was successfully electroporated into the strain F. columnare G4cpN22 after curing of its endogenous plasmid. F. columnare G4cpN22 ghosts (FCGs were generated for the first time by gene E-mediated lysis, and the vaccine potential of FCG was investigated in grass carp (Ctenopharyngodon idellus by intraperitoneal route. Fish immunized with FCG showed significantly higher serum agglutination titers and bactericidal activity than fish immunized with FKC or PBS. Most importantly, after challenge with the parent strain G4, the relative percent survival (RPS of fish in FCG group (70.9% was significantly higher than FKC group (41.9%. These results showed that FCG could confer immune protection against F. columnare infection. As a nonliving whole cell envelope preparation, FCG may provide an ideal alternative to pathogen-based vaccines against columnaris in aquaculture.

  20. Microbial Inactivation by Ultrasound Assisted Supercritical Fluids

    Science.gov (United States)

    Benedito, Jose; Ortuño, Carmen; Castillo-Zamudio, Rosa Isela; Mulet, Antonio

    A method combining supercritical carbon dioxide (SC-CO2) and high power ultrasound (HPU) has been developed and tested for microbial/enzyme inactivation purposes, at different process conditions for both liquid and solid matrices. In culture media, using only SC-CO2, the inactivation rate of E. coli and S. cerevisiae increased with pressure and temperature; and the total inactivation (7-8 log-cycles) was attained after 25 and 140 min of SC-CO2 (350 bar, 36 °C) treatment, respectively. Using SC-CO2+HPU, the time for the total inactivation of both microorganisms was reduced to only 1-2 min, at any condition selected. The SC-CO2+HPU inactivation of both microorganisms was slower in juices (avg. 4.9 min) than in culture media (avg. 1.5 min). In solid samples (chicken, turkey ham and dry-cured pork cured ham) treated with SC-CO2 and SC-CO2+HPU, the inactivation rate of E. coli increased with temperature. The application of HPU to the SC-CO2 treatments accelerated the inactivation rate of E. coli and that effect was more pronounced in treatments with isotonic solution surrounding the solid food samples. The application of HPU enhanced the SC-CO2 inactivation mechanisms of microorganisms, generating a vigorous agitation that facilitated the CO2 solubilization and the mass transfer process. The cavitation generated by HPU could damage the cell walls accelerating the extraction of vital constituents and the microbial death. Thus, using the combined technique, reasonable industrial processing times and mild process conditions could be used which could result into a cost reduction and lead to the minimization in the food nutritional and organoleptic changes.

  1. Photodynamic-induced inactivation of Propionibacterium acnes

    Science.gov (United States)

    Koenig, Karsten; Teschke, M.; Eick, Stephen G.; Pfister, W.; Meyer, Herbert; Halbhuber, Karl-Juergen

    1998-05-01

    We report on photodynamically induced inactivation of the skin bacterium Propionibacterium acnes (P. acnes) using endogenous as well as exogenous photosensitizers and red light sources. P. acnes is involved in the pathogenesis of the skin disease acne vulgaris. The skin bacterium is able to synthesize the metal-free fluorescent porphyrins protoporphyrin IX (PP) and coproporphyrin (CP) as shown by in situ spectrally-resolved detection of natural autofluorescence of human skin and bacteria colonies. These naturally occurring intracellular porphyrins act as efficient endogenous photosensitizers. Inactivation of P. acnes suspensions was achieved by irradiation with He-Ne laser light in the red spectral region (632.8 nm). We monitored the photodynamically-induced death of single bacteria using a fluorescent viability kit in combination with confocal laser scanning microscopy. In addition, the photo-induced inactivation was calculated by CFU (colony forming units) determination. We found 633 nm-induced inactivation (60 mW, 0.12 cm2 exposure area, 1 hour irradiation) of 72% in the case of non-incubated bacteria based on the destructive effect of singlet oxygen produced by red light excited endogenous porphyrins and subsequent energy transfer to molecular oxygen. In order to achieve a nearly complete inactivation within one exposure procedure, the exogenous photosensitizer Methylene Blue (Mb) was added. Far red exposure of Mb-labeled bacteria using a krypton ion laser at 647 nm and 676 nm resulted in 99% inactivation.

  2. Role of Merlin/NF2 inactivation in tumor biology.

    Science.gov (United States)

    Petrilli, A M; Fernández-Valle, C

    2016-02-04

    Merlin (Moesin-ezrin-radixin-like protein, also known as schwannomin) is a tumor suppressor protein encoded by the neurofibromatosis type 2 gene NF2. Loss of function mutations or deletions in NF2 cause neurofibromatosis type 2 (NF2), a multiple tumor forming disease of the nervous system. NF2 is characterized by the development of bilateral vestibular schwannomas. Patients with NF2 can also develop schwannomas on other cranial and peripheral nerves, as well as meningiomas and ependymomas. The only potential treatment is surgery/radiosurgery, which often results in loss of function of the involved nerve. There is an urgent need for chemotherapies that slow or eliminate tumors and prevent their formation in NF2 patients. Interestingly NF2 mutations and merlin inactivation also occur in spontaneous schwannomas and meningiomas, as well as other types of cancer including mesothelioma, glioma multiforme, breast, colorectal, skin, clear cell renal cell carcinoma, hepatic and prostate cancer. Except for malignant mesotheliomas, the role of NF2 mutation or inactivation has not received much attention in cancer, and NF2 might be relevant for prognosis and future chemotherapeutic approaches. This review discusses the influence of merlin loss of function in NF2-related tumors and common human cancers. We also discuss the NF2 gene status and merlin signaling pathways affected in the different tumor types and the molecular mechanisms that lead to tumorigenesis, progression and pharmacological resistance.

  3. Assaying the Stability and Inactivation of AAV Serotype 1 Vectors.

    Science.gov (United States)

    Howard, Douglas B; Harvey, Brandon K

    2017-02-01

    Adeno-associated virus (AAV) vectors are a commonplace tool for gene delivery ranging from cell culture to human gene therapy. One feature that makes AAV a desirable vector is its stability, in regard to both the duration of transgene expression and retention of infectivity as a viral particle. This study examined the stability of AAV serotype 1 (AAV1) vectors under different conditions. First, transducibility after storage at 4°C decreased 20% over 7 weeks. Over 10 freeze-thaw cycles, the resulting transduction efficiency became variable at 60-120% of a single thaw. Using small stainless steel slugs to mimic a biosafety cabinet or metal lab bench surface, it was found that an AAV1 vector can be reconstituted after 6 days of storage at room temperature. The stability of AAV is a desired feature, but effective decontamination procedures must be available for safety and experimental integrity. Multiple disinfectants commonly used in the laboratory for ability to inactivate an AAV1 vector were tested, and it was found that autoclaving, 0.25% peracetic acid, iodine, or 10% Clorox bleach completely prevented AAV-mediated transgene expression. These data suggest that peracetic acid should be used for inactivating AAV1 vectors on metal-based surfaces or instruments in order to avoid inadvertent transgene expression in human cells or cross-contamination of instruments.

  4. Ribosome-Inactivating Proteins: From Plant Defense to Tumor Attack

    Directory of Open Access Journals (Sweden)

    Maria Serena Fabbrini

    2010-11-01

    Full Text Available Ribosome-inactivating proteins (RIPs are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a single adenine (A4324 in rat and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells. Ricin, the plant RIP prototype that comprises a catalytic A subunit linked to a galactose-binding lectin B subunit to allow cell surface binding and toxin entry in most mammalian cells, shows a potency in the picomolar range. The most promising way to exploit plant RIPs as weapons against cancer cells is either by designing molecules in which the toxic domains are linked to selective tumor targeting domains or directly delivered as suicide genes for cancer gene therapy. Here, we will provide a comprehensive picture of plant RIPs and discuss successful designs and features of chimeric molecules having therapeutic potential.

  5. Inactivation of pathogenic bacteria in food matrices: high pressure processing, photodynamic inactivation and pressure-assisted photodynamic inactivation

    Science.gov (United States)

    Cunha, A.; Couceiro, J.; Bonifácio, D.; Martins, C.; Almeida, A.; Neves, M. G. P. M. S.; Faustino, M. A. F.; Saraiva, J. A.

    2017-09-01

    Traditional food processing methods frequently depend on the application of high temperature. However, heat may cause undesirable changes in food properties and often has a negative impact on nutritional value and organoleptic characteristics. Therefore, reducing the microbial load without compromising the desirable properties of food products is still a technological challenge. High-pressure processing (HPP) can be classified as a cold pasteurization technique, since it is a non-thermal food preservation method that uses hydrostatic pressure to inactivate spoilage microorganisms. At the same time, it increases shelf life and retains the original features of food. Photodynamic inactivation (PDI) is also regarded as promising approach for the decontamination of food matrices. In this case, the inactivation of bacterial cells is achieved by the cytotoxic effects of reactive oxygens species (ROS) produced from the combined interaction of a photosensitizer molecule, light and oxygen. This short review examines some recent developments on the application of HPP and PDI with food-grade photosensitizers for the inactivation of listeriae, taken as a food pathogen model. The results of a proof-of-concept trial of the use of high-pressure as a coadjutant to increase the efficiency of photodynamic inactivation of bacterial endospores is also addressed.

  6. Low pH inactivation for xenotropic gamma retrovirus in recombinant human TNF-α receptor immunoglobulin G and mechanism of inactivation.

    Science.gov (United States)

    Ma, Rong; Cui, Xiaolan

    2014-01-01

    CHO-derived recombinant proteins for human therapeutic are used commonly. There are noninfectious endogenous retroviruses in CHO cells. Validation study for inactivation process is required. Murine xenotropic gamma retrovirus (X-MulV) is a model virus in validation study. In our previous study, optimum conditions for X-MulV inactivation were sifted. In this study, we performed a further research on low pH inactivation for evaluation of X-MulV clearance in manufacturing of recombinant human TNF-α receptor immunoglobulin G fusion proteins (rhTNF-α) for injection. Cell-based infectivity assay was used for the evaluation of X-MulV clearance. RhTNF-α were spiked with X-MulV and were inactivated at pH 3.60 ∼ 3.90, 25 ± 2 °C, and 0 ∼ 240 min, respectively. Samples incubated at the conditions for 15 ∼ 180 min were not inactivated effectively. For 4 h incubation, log10 reductions were achieved 5.0 log10. Biological activity of rhTNF-α incubated at pH 3.60, 25 °C for 4 h, which was assayed on murine L929 fibroblasts cells, was not affected by low pH. Env gene of X-MulV, which was detected by conventional PCR method for the first time, was not detected after incubation at pH 3.60, and it may be the mechanism of low pH inactivation. Copyright © 2013. Published by Elsevier Ltd.

  7. X-inactivation pattern in multiple tissues from two Leber's hereditary optic neuropathy (LHON) patients.

    Science.gov (United States)

    Pegoraro, Elena; Vettori, Andrea; Valentino, Maria L; Molon, Annamaria; Mostacciuolo, Maria L; Howell, Neil; Carelli, Valerio

    2003-05-15

    The more frequent manifestation of ophthalmological abnormalities in males, relative to females, is an unexplained feature of Leber's hereditary optic neuropathy (LHON) that suggests an X-linked modifying gene acting in concert with the pathogenic LHON mitochondrial DNA (mtDNA) mutation. In addition, segregation analysis of the optic neuropathy in LHON pedigrees was compatible with the presence of a recessive-modifying gene on chromosome X. According to this two-locus model, females would be affected only if homozygous or if they were susceptible to skewed X-inactivation. Attempts both to localize the putative LHON-modifying gene by linkage analysis and to find an excess of skewed X-inactivation in affected females were unsuccessful, although the inactivation pattern was only studied in DNA isolated from blood cells. We had the opportunity to analyze a wide range of tissues at autopsy, including the optic nerves and the retina, from two LHON female patients. We found no evidence of skewed X-inactivation in the affected tissues, thus weakening further the hypothesized involvement of a specific X chromosome locus in the pathophysiological expression of LHON. Copyright 2003 Wiley-Liss, Inc.

  8. Reversible Heat-Induced Inactivation of Chimeric β-Glucuronidase in Transgenic Plants1

    Science.gov (United States)

    Almoguera, Concepción; Rojas, Anabel; Jordano, Juan

    2002-01-01

    We compared the expression patterns in transgenic tobacco (Nicotiana tabacum) of two chimeric genes: a translational fusion to β-glucuronidase (GUS) and a transcriptional fusion, both with the same promoter and 5′-flanking sequences of Ha hsp17.7 G4, a small heat shock protein (sHSP) gene from sunflower (Helianthus annuus). We found that immediately after heat shock, the induced expression from the two fusions in seedlings was similar, considering chimeric mRNA or GUS protein accumulation. Surprisingly, we discovered that the chimeric GUS protein encoded by the translational fusion was mostly inactive in such conditions. We also found that this inactivation was fully reversible. Thus, after returning to control temperature, the GUS activity was fully recovered without substantial changes in GUS protein accumulation. In contrast, we did not find differences in the in vitro heat inactivation of the respective GUS proteins. Insolubilization of the chimeric GUS protein correlated with its inactivation, as indicated by immunoprecipitation analyses. The inclusion in another chimeric gene of the 21 amino-terminal amino acids from a different sHSP lead to a comparable reversible inactivation. That effect not only illustrates unexpected post-translational problems, but may also point to sequences involved in interactions specific to sHSPs and in vivo heat stress conditions. PMID:12011363

  9. Detailed analysis of X chromosome inactivation in a 49,XXXXX pentasomy

    Directory of Open Access Journals (Sweden)

    Menezes Albert N

    2009-10-01

    Full Text Available Abstract Background Pentasomy X (49,XXXXX has been associated with a severe clinical condition, presumably resulting from failure or disruption of X chromosome inactivation. Here we report that some human X chromosomes from a patient with 49,XXXXX pentasomy were functionally active following isolation in inter-specific (human-rodent cell hybrids. A comparison with cytogenetic and molecular findings provided evidence that more than one active X chromosome was likely to be present in the cells of this patient, accounting for her abnormal phenotype. Results 5-bromodeoxyuridine (BrdU-pulsed cultures showed different patterns among late replicating X chromosomes suggesting that their replication was asynchronic and likely to result in irregular inactivation. Genotyping of the proband and her mother identified four maternal and one paternal X chromosomes in the proband. It also identified the paternal X chromosome haplotype (P, indicating that origin of this X pentasomy resulted from two maternal, meiotic non-disjunctions. Analysis of the HUMANDREC region of the androgen receptor (AR gene in the patient's mother showed a skewed inactivation pattern, while a similar analysis in the proband showed an active paternal X chromosome and preferentially inactivated X chromosomes carrying the 173 AR allele. Analyses of 33 cell hybrid cell lines selected in medium containing hypoxanthine, aminopterin and thymidine (HAT allowed for the identification of three maternal X haplotypes (M1, M2 and MR and showed that X chromosomes with the M1, M2 and P haplotypes were functionally active. In 27 cell hybrids in which more than one X haplotype were detected, analysis of X inactivation patterns provided evidence of preferential inactivation. Conclusion Our findings indicated that 12% of X chromosomes with the M1 haplotype, 43.5% of X chromosomes with the M2 haplotype, and 100% of the paternal X chromosome (with the P haplotype were likely to be functionally active in the

  10. Activation of X Chromosome Inactivation

    NARCIS (Netherlands)

    C.M. Maduro (Cheryl)

    2016-01-01

    markdownabstractIn mammals, males are the heterogametic sex having an X chromosome and a Y chromosome whereas females have two X chromosomes. Despite originating from an ancient homologous autosomal pair, the X and Y chromosome now differ greatly in size and gene content after ~180 MY of evolution.

  11. Kinetics of Hydrothermal Inactivation of Endotoxins ▿

    Science.gov (United States)

    Li, Lixiong; Wilbur, Chris L.; Mintz, Kathryn L.

    2011-01-01

    A kinetic model was established for the inactivation of endotoxins in water at temperatures ranging from 210°C to 270°C and a pressure of 6.2 × 106 Pa. Data were generated using a bench scale continuous-flow reactor system to process feed water spiked with endotoxin standard (Escherichia coli O113:H10). Product water samples were collected and quantified by the Limulus amebocyte lysate assay. At 250°C, 5-log endotoxin inactivation was achieved in about 1 s of exposure, followed by a lower inactivation rate. This non-log-linear pattern is similar to reported trends in microbial survival curves. Predictions and parameters of several non-log-linear models are presented. In the fast-reaction zone (3- to 5-log reduction), the Arrhenius rate constant fits well at temperatures ranging from 120°C to 250°C on the basis of data from this work and the literature. Both biphasic and modified Weibull models are comparable to account for both the high and low rates of inactivation in terms of prediction accuracy and the number of parameters used. A unified representation of thermal resistance curves for a 3-log reduction and a 3 D value associated with endotoxin inactivation and microbial survival, respectively, is presented. PMID:21193667

  12. Hyperactivated Wnt signaling induces synthetic lethal interaction with Rb inactivation by elevating TORC1 activities.

    Directory of Open Access Journals (Sweden)

    Tianyi Zhang

    2014-05-01

    Full Text Available Inactivation of the Rb tumor suppressor can lead to increased cell proliferation or cell death depending on specific cellular context. Therefore, identification of the interacting pathways that modulate the effect of Rb loss will provide novel insights into the roles of Rb in cancer development and promote new therapeutic strategies. Here, we identify a novel synthetic lethal interaction between Rb inactivation and deregulated Wg/Wnt signaling through unbiased genetic screens. We show that a weak allele of axin, which deregulates Wg signaling and increases cell proliferation without obvious effects on cell fate specification, significantly alters metabolic gene expression, causes hypersensitivity to metabolic stress induced by fasting, and induces synergistic apoptosis with mutation of fly Rb ortholog, rbf. Furthermore, hyperactivation of Wg signaling by other components of the Wg pathway also induces synergistic apoptosis with rbf. We show that hyperactivated Wg signaling significantly increases TORC1 activity and induces excessive energy stress with rbf mutation. Inhibition of TORC1 activity significantly suppressed synergistic cell death induced by hyperactivated Wg signaling and rbf inactivation, which is correlated with decreased energy stress and decreased induction of apoptotic regulator expression. Finally the synthetic lethality between Rb and deregulated Wnt signaling is conserved in mammalian cells and that inactivation of Rb and APC induces synergistic cell death through a similar mechanism. These results suggest that elevated TORC1 activity and metabolic stress underpin the evolutionarily conserved synthetic lethal interaction between hyperactivated Wnt signaling and inactivated Rb tumor suppressor.

  13. MYC Inactivation Elicits Oncogene Addiction through Both Tumor Cell-Intrinsic and Host-Dependent Mechanisms.

    Science.gov (United States)

    Felsher, Dean W

    2010-06-01

    Tumorigenesis is generally caused by genetic changes that activate oncogenes or inactivate tumor suppressor genes. The targeted inactivation of oncogenes can be associated with tumor regression through the phenomenon of oncogene addiction. One of the most common oncogenic events in human cancer is the activation of the MYC oncogene. The inactivation of MYC may be a general and effective therapy for human cancer. Indeed, it has been experimentally shown that the inactivation of MYC can result in dramatic and sustained tumor regression in lymphoma, leukemia, osteosarcoma, hepatocellular carcinoma, squamous carcinoma, and pancreatic carcinoma through a multitude of mechanisms, including proliferative arrest, terminal differentiation, cellular senescence, induction of apoptosis, and the shutdown of angiogenesis. Cell-autonomous and cell-dependent mechanisms have both been implicated, and recent results suggest a critical role for autocrine factors, including thrombospondin-1 and TGF-β. Hence, targeting the inactivation of MYC appears to elicit oncogene addiction and, thereby, tumor regression through both tumor cell-intrinsic and host-dependent mechanisms.

  14. Independent clonal origin of multiple uterine leiomyomas that was determined by X chromosome inactivation and microsatellite analysis

    DEFF Research Database (Denmark)

    Canevari, Renata A; Pontes, Anaglória; Rosa, Fabíola E

    2005-01-01

    OBJECTIVE: In an attempt to clarify the clonality and genetic relationships that are involved in the tumorigenesis of uterine leiomyomas, we used a total of 43 multiple leiomyomas from 14 patients and analyzed the allelic status with 15 microsatellite markers and X chromosome inactivation analysis....... STUDY DESIGN: We have used a set of 15 microsatellite polymorphism markers mapped on 3q, 7p, 11, and 15q by automated analysis. The X chromosome inactivation was evaluated by the methylation status of the X-linked androgen receptor gene. RESULTS: Loss of heterozygosity analysis showed a different...... pattern in 7 of the 8 cases with allelic loss for at least 1 of 15 microsatellite markers that were analyzed. A similar loss of heterozygosity findings at 7p22-15 was detected in 3 samples from the same patient. X chromosome inactivation analysis demonstrated the same inactivated allele in all tumors...

  15. Inactivation of glutathione peroxidase by benzaldehyde.

    Science.gov (United States)

    Tabatabaie, T; Floyd, R A

    1996-12-01

    Chronic benzaldehyde exposure is known to cause central nervous system (CNS) disturbances. Previous studies have shown that benzaldehyde causes the formation of reactive oxygen species (ROS) in rat synaptosomal fractions. Benzaldehyde has also been implicated in ROS formation in the CNS of rats treated with toluene. We have found that benzaldehyde effectively inactivates the antioxidant enzyme glutathione peroxidase (Ki approximately 15 microM), but has no effect on the other antioxidant enzymes tested: catalase, superoxide dismutase, and glutathione reductase. This effect has been found to be specific to benzaldehyde since other structurally related and unrelated aldehydes tested were found to be devoid of inactivating capacity toward glutathione peroxidase. Since glutathione peroxidase is the main enzyme responsible for removal of hydrogen peroxide and organic hydroperoxides in brain, its inactivation by benzaldehyde may be a main contributor to the observed ROS formation and the observed neurotoxicity caused by either benzaldehyde or toluene exposure.

  16. Comparison of the immunogenicity of two inactivated recombinant rabies viruses overexpressing the glycoprotein.

    Science.gov (United States)

    Navid, Muhammad Tariq; Li, Yingying; Zhou, Ming; Cui, Min; Fu, Zhen F; Tang, Lijun; Zhao, Ling

    2016-10-01

    Two recombinant rabies viruses overexpressing their glycoprotein (G) were compared in this study, with the overexpressed G inserted between P and M genes (named LBNSE-PM-G), and between the G and L genes (named LBNSE-GL-G), respectively. LBNSE-PM-G produced more G protein and induced stronger apoptosis than LBNSE-GL-G in infected cells, while the amount of virion-incorporated G in LBNSE-PM-G was less than in LBNSE-GL-G. Mice immunized with inactivated LBNSE-PM-G produced lower titers of virus-neutralizing antibody, and this recombinant conferred worse protection than LBNSE-GL-G. Our results suggest that over expressed G gene inserted between G and L, but not between P and M, enhanced the immunogenicity when used as an inactivated rabies vaccine.

  17. phoU Inactivation in Pseudomonas aeruginosa Enhances Accumulation of ppGpp and Polyphosphate

    Science.gov (United States)

    de Almeida, Luiz Gustavo; Ortiz, Julia Helena; Schneider, René P.

    2015-01-01

    Inorganic polyphosphate (polyP) is a linear polymer composed of several molecules of orthophosphate (Pi) linked by energy-rich phosphoanhydride bonds. In Pseudomonas aeruginosa, Pi is taken up by the ABC transporter Pst, encoded by an operon consisting of five genes. The first four genes encode proteins involved in the transport of Pi and the last gene of the operon, phoU, codes for a protein which exact function is unknown. We show here that the inactivation of phoU in P. aeruginosa enhanced Pi removal from the medium and polyP accumulation. The phoU mutant also accumulated high levels of the alarmone guanosine tetraphosphate (ppGpp), which in turn increased the buildup of polyP. In addition, phoU inactivation had several pleiotropic effects, such as reduced growth rate and yield and increased sensitivity to antibiotics and stresses. However, biofilm formation was not affected by the phoU mutation. PMID:25710363

  18. Kinetics of Hydrothermal Inactivation of Endotoxins ▿

    OpenAIRE

    Li, Lixiong; Wilbur, Chris L.; Mintz, Kathryn L.

    2011-01-01

    A kinetic model was established for the inactivation of endotoxins in water at temperatures ranging from 210°C to 270°C and a pressure of 6.2 × 106 Pa. Data were generated using a bench scale continuous-flow reactor system to process feed water spiked with endotoxin standard (Escherichia coli O113:H10). Product water samples were collected and quantified by the Limulus amebocyte lysate assay. At 250°C, 5-log endotoxin inactivation was achieved in about 1 s of exposure, followed by a lower ina...

  19. Inactivation of adenovirus types 5 and 6 by Virkon S.

    Science.gov (United States)

    McCormick, Lisa; Maheshwari, Gargi

    2004-10-01

    Throughout the pharmaceutical industry, adenovirus-based products are being developed for human use as vaccine vectors and gene therapy delivery vehicles. The implementation of effective decontamination procedures is critical to the successful manufacture of these products to minimize the risk of personnel exposure and prevent product cross-contamination in the manufacturing facility. In this investigation, we have conducted small-scale decontamination studies to determine the efficacy of Virkon S on the inactivation of adenovirus types 5 and 6 in suspension. Virkon S is a commercially available oxidative disinfectant used against a variety of bacteria, spores, fungi, and viruses. A cytotoxicity-based endpoint dilution assay was used to quantify adenovirus potency before and after Virkon S treatment. We show that the level of organic content in the inactivation sample matrix has a significant impact on Virkon S activity. The potency of adenovirus types 5 and 6 was reduced by greater than six logs upon a five minute exposure to the appropriate concentration of Virkon S. Based on these results, we propose that Virkon S liquid decontamination procedures for adenovirus types 5 and 6 use 0.9% Virkon S for contact times greater than five minutes.

  20. Management of Gene Variants of Unknown Significance

    DEFF Research Database (Denmark)

    Alosi, Daniela; Bisgaard, Marie Luise; Hemmingsen, Sophie Nowak

    2017-01-01

    (IHC); 3) Assessment of the variant’s impact on protein structure and function, using multiple databases, in silico algorithms, and reports of functional studies. Results: Only one family member had clinical signs of vHL with early-onset RCC. IHC analysis showed no VHL protein expressed in the tumor...

  1. X-chromosome inactivation and escape

    Indian Academy of Sciences (India)

    2015-11-06

    Nov 6, 2015 ... She predicted many of the features of X inactivation, for e.g., .... feature that locks silencing, i.e. DNA methylation at CpG islands of X-linked ..... 1996 XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J. Cell Biol. 132, 259–275.

  2. Inactivation of Bacillus atrophaeus by OH radicals

    Science.gov (United States)

    Ono, Ryo; Yonetamari, Kenta; Tokumitsu, Yusuke; Yonemori, Seiya; Yasuda, Hachiro; Mizuno, Akira

    2016-08-01

    The inactivation of Bacillus atrophaeus by OH radicals is measured. This study aims to evaluate the bactericidal effects of OH radicals produced by atmospheric-pressure nonthermal plasma widely used for plasma medicine; however, in this study, OH radicals are produced by vacuum ultraviolet (VUV) photolysis of water vapor instead of plasma to allow the production of OH radicals with almost no other reactive species. A 172 nm VUV light from a Xe2 excimer lamp irradiates a He-H2O mixture flowing in a quartz tube to photodissociate H2O to produce OH, H, O, HO2, H2O2, and O3. The produced reactive oxygen species (ROS) flow out of the quartz tube nozzle to the bacteria on an agar plate and cause inactivation. The inactivation by OH radicals among the six ROS is observed by properly setting the experimental conditions with the help of simulations calculating the ROS densities. A 30 s treatment with approximately 0.1 ppm OH radicals causes visible inactivation.

  3. Bioinactivation: Software for modelling dynamic microbial inactivation.

    Science.gov (United States)

    Garre, Alberto; Fernández, Pablo S; Lindqvist, Roland; Egea, Jose A

    2017-03-01

    This contribution presents the bioinactivation software, which implements functions for the modelling of isothermal and non-isothermal microbial inactivation. This software offers features such as user-friendliness, modelling of dynamic conditions, possibility to choose the fitting algorithm and generation of prediction intervals. The software is offered in two different formats: Bioinactivation core and Bioinactivation SE. Bioinactivation core is a package for the R programming language, which includes features for the generation of predictions and for the fitting of models to inactivation experiments using non-linear regression or a Markov Chain Monte Carlo algorithm (MCMC). The calculations are based on inactivation models common in academia and industry (Bigelow, Peleg, Mafart and Geeraerd). Bioinactivation SE supplies a user-friendly interface to selected functions of Bioinactivation core, namely the model fitting of non-isothermal experiments and the generation of prediction intervals. The capabilities of bioinactivation are presented in this paper through a case study, modelling the non-isothermal inactivation of Bacillus sporothermodurans. This study has provided a full characterization of the response of the bacteria to dynamic temperature conditions, including confidence intervals for the model parameters and a prediction interval of the survivor curve. We conclude that the MCMC algorithm produces a better characterization of the biological uncertainty and variability than non-linear regression. The bioinactivation software can be relevant to the food and pharmaceutical industry, as well as to regulatory agencies, as part of a (quantitative) microbial risk assessment. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Pulsed electric field inactivation in a microreactor

    NARCIS (Netherlands)

    Fox, M.B.

    2006-01-01

    Pulsed electric fields (PEF) is a novel, non-thermal pasteurization method which uses short, high electric field pulses to inactivate microorganisms. The advantage of a pasteurization method like PEF compared to regular heat pasteurization is that the taste, flavour, texture and nutritional value

  5. Inactivation of human norovirus using chemical sanitizers

    Science.gov (United States)

    The porcine gastric mucin binding magnetic bead (PGM-MB) assay was used to evaluate the ability of chlorine, chlorine dioxide, peroxyacetic acid, hydrogen peroxide, and trisodium phosphate to inactivate human norovirus within 10 percent stool filtrate. One min free chlorine treatments at concentrat...

  6. Photodynamic Inactivation of Food Pathogen Listeria monocytogenes

    Directory of Open Access Journals (Sweden)

    Irina Buchovec

    2010-01-01

    Full Text Available The aim of this study is to examine the possibility to inactivate food pathogen Listeria monocytogenes by nonthermal antimicrobial treatment – photosensitization. L. monocytogenes was incubated with 5-aminolevulinic acid (ALA (7.5 mM for 0–2 h to produce endogenous photosensitizers and then illuminated with visible light. The LED-based light source used for the illumination of L. monocytogenes emitted light at λ=400 nm with energy density of 20 mW/cm2. The illumination time varied from 0 to 20 min, and a total energy dose reached 0–24 J/cm2. The obtained results reveal that L. monocytogenes can effectively produce endogenous porphyrins after incubation with 7.5 mM ALA. Subsequent illumination of cells with visible light significantly decreased their viability in vitro (4 log. After adhesion of Listeria to the surface of packaging material and following photosensitization, the surface-attached bacterial population was inactivated by 3.7 log. In addition, most resistant Listeria biofilms are susceptible to this treatment. Their inactivation reached 3.1 log under certain experimental conditions. The cells and biofilms of Gram-positive bacteria L. monocytogenes ATCL3C 7644 could be effectively inactivated by ALA-based photosensitization in the solution as well as adhered onto the surface of packaging material in a nonthermal way.

  7. Inactivation of prion infectivity by ionizing rays

    Energy Technology Data Exchange (ETDEWEB)

    Gominet, M. [Ionisos, ZI les Chatinieres, F01120 Dagneux (France); Vadrot, C.; Austruy, G. [Paris V University, Central Pharmacy of Hospitals, 4 avenue de l' Observatoire, F-75006, Paris (France); Darbord, J.C. [Paris V University, Central Pharmacy of Hospitals, 4 avenue de l' Observatoire, F-75006, Paris (France)], E-mail: darbord@pharmacie.univ-paris5.fr

    2007-11-15

    Inactivation of prion deposits on medical devices or prion contamination in pharmaceutical raw materials is considered as impossible by using gamma irradiation. Early, the guideline WHO/CDS/CSR/APH/2000 has described irradiation as an ineffective process. But, in 2003, S. Miekka et al. noted radiation inactivation of prions in a particular application to purify human albumin, shown by the physical denaturation of the infectious protein (PrP). The aim of our study was to determine the inactivation of prions with a scrapie model (strain C506M3) by irradiating standardised preparations. Results: Gamma irradiation was partially effective, showing a 4-5 log reduction on exposure to 50 kGy. A characteristic effect-dose curve was not observed (25, 50 and 100 kGy), only an increase in the incubation period of the murine disease (229 days with 25 kGy to 290 days with 100 kGy) compared with 170 days without irradiation. Since the inactivation was not a total one, the observed effect is significant. It is proposed that further work be undertaken with the model to investigate the application of gamma radiation known levels of prion contamination.

  8. Targeted inactivation of transcription factors by overexpression of their truncated forms in plants.

    Science.gov (United States)

    Seo, Pil Joon; Hong, Shin-Young; Ryu, Jae Yong; Jeong, Eun-Young; Kim, Sang-Gyu; Baldwin, Ian T; Park, Chung-Mo

    2012-10-01

    Transcription factors are central constituents of gene regulatory networks that control diverse aspects of plant development and environmental adaptability. Therefore they have been explored for decades as primary targets for agricultural biotechnology. A gene of interest can readily be introduced into many crop plants, whereas targeted gene inactivation is practically difficult in many cases. Here, we developed an artificial small interfering peptide (a-siPEP) approach, which is based on overexpression of specific protein domains, and evaluated its application for the targeted inactivation of transcription factors in the dicot model, Arabidopsis, and monocot model, Brachypodium. We designed potential a-siPEPs of two representative MADS box transcription factors, SUPPRESSOR OF OVEREXPRESSOR OF CONSTANS 1 (SOC1) and AGAMOUS (AG), and a MYB transcription factor, LATE ELONGATED HYPOCOTYL (LHY). Transgenic plants overproducing the a-siPEPs displayed phenotypes comparable to those of gene-deficient mutants. The a-siPEPs attenuate nuclear import and DNA-binding of target transcription factors. Our data demonstrate that the a-siPEP tool is an efficient genetic means of inactivating specific transcription factors in plants. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  9. Inactivation of Escherichia coli by citral.

    Science.gov (United States)

    Somolinos, M; García, D; Condón, S; Mackey, B; Pagán, R

    2010-06-01

    The aim was to evaluate (i) the resistance of Escherichia coli BJ4 to citral in a buffer system as a function of citral concentration, treatment medium pH, storage time and initial inoculum size, (ii) the role of the sigma factor RpoS on citral resistance of E. coli, (iii) the role of the cell envelope damage in the mechanism of microbial inactivation by citral and (iiii) possible synergistic effects of mild heat treatment and pulsed electric fields (PEF) treatment combined with citral. The initial inoculum size greatly affected the efficacy of citral against E. coli cells. Exposure to 200 microl l(-1) of citral at pH 4.0 for 24 h at 20 degrees C caused the inactivation of more than 5 log(10) cycles of cells starting at an inoculum size of 10(6) or 10(7) CFU ml(-1), whereas increasing the cell concentration to 10(9) CFU ml(-1) caused citral at pH 4.0 than pH 7.0. The rpoS null mutant strain E. coli BJ4L1 was less resistant to citral than the wild-type strain. Occurrence of sublethal injury to both the cytoplasmic and outer membranes was demonstrated by adding sodium chloride or bile salts to the recovery media. The majority of sublethally injured cells by citral required energy and lipid synthesis for repair. A strongly synergistic lethal effect was shown by mild heat treatment combined with citral but the presence of citral during the application of a PEF treatment did not show any advantage. This work confirms that cell envelope damage is an important event in citral inactivation of bacteria, and it describes the key factors on the inactivation of E. coli cells by citral. Knowledge about the mechanism of microbial inactivation by citral helps establish successful combined preservation treatments.

  10. Female-biased expression of long non-coding RNAs in domains that escape X-inactivation in mouse

    Directory of Open Access Journals (Sweden)

    Lu Lu

    2010-11-01

    Full Text Available Abstract Background Sexual dimorphism in brain gene expression has been recognized in several animal species. However, the relevant regulatory mechanisms remain poorly understood. To investigate whether sex-biased gene expression in mammalian brain is globally regulated or locally regulated in diverse brain structures, and to study the genomic organisation of brain-expressed sex-biased genes, we performed a large scale gene expression analysis of distinct brain regions in adult male and female mice. Results This study revealed spatial specificity in sex-biased transcription in the mouse brain, and identified 173 sex-biased genes in the striatum; 19 in the neocortex; 12 in the hippocampus and 31 in the eye. Genes located on sex chromosomes were consistently over-represented in all brain regions. Analysis on a subset of genes with sex-bias in more than one tissue revealed Y-encoded male-biased transcripts and X-encoded female-biased transcripts known to escape X-inactivation. In addition, we identified novel coding and non-coding X-linked genes with female-biased expression in multiple tissues. Interestingly, the chromosomal positions of all of the female-biased non-coding genes are in close proximity to protein-coding genes that escape X-inactivation. This defines X-chromosome domains each of which contains a coding and a non-coding female-biased gene. Lack of repressive chromatin marks in non-coding transcribed loci supports the possibility that they escape X-inactivation. Moreover, RNA-DNA combined FISH experiments confirmed the biallelic expression of one such novel domain. Conclusion This study demonstrated that the amount of genes with sex-biased expression varies between individual brain regions in mouse. The sex-biased genes identified are localized on many chromosomes. At the same time, sexually dimorphic gene expression that is common to several parts of the brain is mostly restricted to the sex chromosomes. Moreover, the study uncovered

  11. Effects of Bacterial Inactivation Methods on Downstream Proteomic Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Andy; Merkley, Eric D.; Clowers, Brian H.; Hutchison, Janine R.; Kreuzer, Helen W.

    2015-05-01

    Inactivation of pathogenic microbial samples is often necessary for the protection of researchers and to comply with local and federal regulations. By its nature, biological inactivation causes changes to microbial samples, potentially affecting observed experimental results. While inactivation induced damage to materials such as DNA has been evaluated, the effect of various inactivation strategies on proteomic data, to our knowledge, has not been discussed. To this end, we inactivated samples of Yersinia pestis and Escherichia coli by autoclave, ethanol, or irradiation treatment to determine how inactivation changes liquid chromatography tandem mass spectrometry data quality as well as apparent protein content of cells. Proteomic datasets obtained from aliquots of samples inactivated by different methods were highly similar, with Pearson correlation coefficients ranging from 0.822 to 0.985 and 0.816 to 0.985 for E. coli and Y. pestis, respectively, suggesting that inactivation had only slight impacts on the set of proteins identified. In addition, spectral quality metrics such as distributions of various database search algorithm scores remained constant across inactivation methods, indicating that inactivation does not appreciably degrade spectral quality. Though overall changes resulting from inactivation were small, there were detectable trends. For example, one-sided Fischer exact tests determined that periplasmic proteins decrease in observed abundance after sample inactivation by autoclaving (α = 1.71x10-2 for E. coli, α = 4.97x10-4 for Y. pestis) and irradiation (α = 9.43x10-7 for E. coli, α = 1.21x10-5 for Y. pestis) when compared to controls that were not inactivated. Based on our data, if sample inactivation is necessary, we recommend inactivation with ethanol treatment with secondary preference given to irradiation.

  12. Sfp-type PPTase inactivation promotes bacterial biofilm formation and ability to enhance wheat drought tolerance

    Directory of Open Access Journals (Sweden)

    Salme eTimmusk

    2015-05-01

    Full Text Available Paenibacillus polymyxa is a common soil bacterium with broad range of practical applications. An important group of secondary metabolites in P. polymyxa are nonribosomal peptide and polyketide derived metabolites (NRP/PK. Modular nonribosomal peptide synthetases catalyse main steps in the biosynthesis of the complex secondary metabolites. Here we report on the inactivation of an A26 sfp-type phosphopantetheinyl transferase. The inactivation of the gene resulted in loss of NRP/PK production. In contrast to the former Bacillus spp. model the mutant strain compared to wild type showed greatly enhanced biofilm formation ability. Its biofilm promotion is directly mediated by NRP/PK, as exogenous addition of the wild type metabolite extracts restores its biofilm formation level. Wheat inoculation with bacteria that had lost their sfp-type PPTase gene resulted in two times higher plant survival and about three times increased biomass under severe drought stress compared to wild type.

  13. Immunogenicity of a cell culture-derived inactivated vaccine against a common virulent isolate of grass carp reovirus.

    Science.gov (United States)

    Zeng, Weiwei; Wang, Qing; Wang, Yingying; Zhao, Changchen; Li, Yingying; Shi, Chunbin; Wu, Shuqin; Song, Xinjian; Huang, Qiwen; Li, Shoujun

    2016-07-01

    Grass carp (Ctenopharyngodon idella) hemorrhagic disease, caused by grass carp reovirus (GCRV), is emerging as a serious problem in grass carp aquaculture. There is no available antiviral therapy and vaccination is the primary method of disease control. In the present study, the immunological effects and protective efficacy of an inactivated HuNan1307 vaccine in grass carp were evaluated. The GCRV isolate HuNan1307 was produced by replication onto the grass carp PSF cell line, and inactivated with 1% β-propiolactone for 60 h at 4 °C. Grass carp were injected with inactivated GCRV vaccine, followed by challenge with the isolate HuNan1307. The results showed that the minimum dosage of the inactivated vaccine was 10(5.5) TCID50/0.2 mL to induce immune protection. All grass carp immunized with the inactivated vaccine produced a high titer of serum antibodies and GCRV-specific neutralizing antibody. Moreover, the inactivated vaccine injection increased the expression of 6 immune-related genes in the spleen and head kidney, which indicated that a immune response was induced by the HuNan1307 vaccine. In addition, grass carp immunized with the inactivated vaccine showed a survival rate above 80% after the viral challenge, equal to that of grass carp immunized with a commercial attenuated vaccine, and the protection lasted at least for one year. The data in this study suggested that the inactivated HuNan1307 vaccine may represent an efficient method to induce immunity against GCRV infection and the induced disease in grass carp. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Quantitative reverse transcription PCR to determine the inactivation of Human Rotavirus by chlorine.

    Science.gov (United States)

    Xue, Bin; Li, Chenyu; Zhang, Bin; Zhao, Tianyu; Shen, Zhiqiang; Qiu, Zhigang; Jin, Min; Wang, Jingfeng; Li, Junwen

    2017-06-01

    Human rotaviruses (HRVs) are the major cause of acute diarrhea in infants and young children. Here, a real-time reverse transcription polymerase chain reaction assay targeting the rotaviral VP4 gene (VP4-RT-qPCR) was established to evaluate the inactivation of HRV upon chlorine disinfection, based on a previous report that damage to the 1227-2354bp region of the VP4 gene was associated with eliminated HRV infectivity by chlorine. In this study, inactivation of HRV by 0.6mg/L free chlorine was assessed in phosphate buffered saline (PBS; pH 7.2), and tap and river water samples, using both TCID50 and RT-qPCR (VP2- and VP4-RT-qPCR) assays, respectively. Among the samples tested, the VP2-RT-qPCR method did not show significant inactivation after chlorine disinfection; however, the reduction in VP4-RT-qPCR signal was correlated with decreased HRV infectivity. Moreover, the higher sensitivity of the VP4-RT-qPCR assay allowed for assessment of chlorine HRV inactivation at longer exposure times compared with the conventional TCID50 assay. Collectively, these results indicated that the VP4-RT-qPCR assay is a rapid, sensitive, and reliable tool to detect infectious HRV following chlorine inactivation, and highlights the potential for further development of qPCR/RT-qPCR assays to provide information regarding viral infectivity from drinking water plants. Copyright © 2017 Elsevier GmbH. All rights reserved.

  15. Inactivation of a Norovirus by High-Pressure Processing▿

    Science.gov (United States)

    Kingsley, David H.; Holliman, Daniel R.; Calci, Kevin R.; Chen, Haiqiang; Flick, George J.

    2007-01-01

    Murine norovirus (strain MNV-1), a propagable norovirus, was evaluated for susceptibility to high-pressure processing. Experiments with virus stocks in Dulbecco's modified Eagle medium demonstrated that at room temperature (20°C) the virus was inactivated over a pressure range of 350 to 450 MPa, with a 5-min, 450-MPa treatment being sufficient to inactivate 6.85 log10 PFU of MNV-1. The inactivation of MNV-1 was enhanced when pressure was applied at an initial temperature of 5°C; a 5-min pressure treatment of 350 MPa at 30°C inactivated 1.15 log10 PFU of virus, while the same treatment at 5°C resulted in a reduction of 5.56 log10 PFU. Evaluation of virus inactivation as a function of treatment times ranging from 0 to 150 s and 0 to 900 s at 5°C and 20°C, respectively, indicated that a decreasing rate of inactivation with time was consistent with Weibull or log-logistic inactivation kinetics. The inactivation of MNV-1 directly within oyster tissues was demonstrated; a 5-min, 400-MPa treatment at 5°C was sufficient to inactivate 4.05 log10 PFU. This work is the first demonstration that norovirus can be inactivated by high pressure and suggests good prospects for inactivation of nonpropagable human norovirus strains in foods. PMID:17142353

  16. Chromatin inactivation precedes de novo dna methylation during the progressive epigenetic silencing of the rassf1a promoter

    Energy Technology Data Exchange (ETDEWEB)

    Strunnikova Maria; Schagdarsurengin, Undraga; Kehlen, Astrid; Garbe, James C.; Stampfer, Martha R.; Dammann, Reinhard

    2005-02-23

    Epigenetic inactivation of the RASSF1A tumor suppressor by CpG island methylation was frequently detected in cancer. However, the mechanisms of this aberrant DNA methylation are unknown. In the RASSF1A promoter, we characterized four Sp1 sites, which are frequently methylated in cancer. We examined the functional relationship between DNA methylation, histone modification, Sp1 binding, and RASSF1A expression in proliferating human mammary epithelial cells. With increasing passages, the transcription of RASSF1A was dramatically silenced. This inactivation was associated with deacetylation and lysine 9 trimethylation of histone H3 and an impaired binding of Sp1 at the RASSF1A promoter. In mammary epithelial cells that had overcome a stress-associated senescence barrier, a spreading of DNA methylation in the CpG island promoter was observed. When the RASSF1A-silenced cells were treated with inhibitors of DNA methyltransferase and histone deacetylase, binding of Sp1 and expression of RASSF1 A reoccurred. In summary, we observed that histone H3 deacetylation and H3 lysine 9 trimethylation occur in the same time window as gene inactivation and precede DNA methylation. Our data suggest that in epithelial cells, histone inactivation may trigger de novo DNA methylation of the RASSF1A promoter and this system may serve as a model for CpG island inactivation of tumor suppressor genes.

  17. Cathepsin D inactivates cysteine proteinase inhibitors, cystatins.

    Science.gov (United States)

    Lenarcic, B; Kos, J; Dolenc, I; Lucovnik, P; Krizaj, I; Turk, V

    1988-07-29

    The formation of inactive complexes in excess molar amounts of human cathepsins H and L with their protein inhibitors human stefin A, human stefin B and chicken cystatin at pH 5.6 has been shown by measurement of enzyme activity coupled with reverse-phase HPLC not to involve covalent cleavage of the inhibitors. Inhibition must be the direct result of binding. On the contrary the interaction of cystatins with aspartic proteinase cathepsin D at pH 3.5 for 60 min followed by HPLC resulted in their inactivation accompanied by peptide bond cleavage at several sites, preferentially those involving hydrophobic amino acid residues. The released peptides do not inhibit papain and cathepsin L. These results explain reported elevated levels of cysteine proteinases and lead to the proposal that cathepsin D exerts an important function, through inactivation of cystatins, in the increased activities of cysteine proteinases in human diseases including muscular distrophy.

  18. Distinct mutations led to inactivation of type 1 fimbriae expression in Shigella spp.

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    Verónica Bravo

    Full Text Available Shigella spp. are responsible for bacillary dysentery in humans. The acquisition or the modification of the virulence plasmid encoding factors promoting entry of bacteria into and dissemination within epithelial cells was a critical step in the evolution of these bacteria from their Escherichia coli ancestor(s. Incorporation of genomic islands (GI and gene inactivation also shaped interactions between these pathogens and their human host. Sequence analysis of the GI inserted next to the leuX tRNA gene in S. boydii, S. dysenteriae, S. flexneri, S. sonnei and enteroinvasive E. coli (EIEC suggests that this region initially carried the fec, yjhATS and fim gene clusters. The fim cluster encoding type I fimbriae is systematically inactivated in both reference strains and clinical isolates and distinct mutations are responsible for this inactivation in at least three phylogenetic groups. To investigate consequences of the presence of fimbriae on the outcome of the interaction of Shigella with host cells, we used a S. flexneri strain harboring a plasmid encoding the E. coli fim operon. Production of fimbriae by this recombinant strain increased the ability of bacteria to adhere to and enter into epithelial cells and had no effect on their ability to disseminate from cell to cell. The observations that production of type I fimbriae increases invasion of epithelial cells and that independent mutations abolish fimbriae production in Shigella suggest that these mutations correspond to pathoadaptive events.

  19. Distinct Mutations Led to Inactivation of Type 1 Fimbriae Expression in Shigella spp.

    Science.gov (United States)

    Bravo, Verónica; Puhar, Andrea; Sansonetti, Philippe; Parsot, Claude; Toro, Cecilia S.

    2015-01-01

    Shigella spp. are responsible for bacillary dysentery in humans. The acquisition or the modification of the virulence plasmid encoding factors promoting entry of bacteria into and dissemination within epithelial cells was a critical step in the evolution of these bacteria from their Escherichia coli ancestor(s). Incorporation of genomic islands (GI) and gene inactivation also shaped interactions between these pathogens and their human host. Sequence analysis of the GI inserted next to the leuX tRNA gene in S. boydii, S. dysenteriae, S. flexneri, S. sonnei and enteroinvasive E. coli (EIEC) suggests that this region initially carried the fec, yjhATS and fim gene clusters. The fim cluster encoding type I fimbriae is systematically inactivated in both reference strains and clinical isolates and distinct mutations are responsible for this inactivation in at least three phylogenetic groups. To investigate consequences of the presence of fimbriae on the outcome of the interaction of Shigella with host cells, we used a S. flexneri strain harboring a plasmid encoding the E. coli fim operon. Production of fimbriae by this recombinant strain increased the ability of bacteria to adhere to and enter into epithelial cells and had no effect on their ability to disseminate from cell to cell. The observations that production of type I fimbriae increases invasion of epithelial cells and that independent mutations abolish fimbriae production in Shigella suggest that these mutations correspond to pathoadaptive events. PMID:25811616

  20. E-cadherin inactivation in lobular carcinoma in situ of the breast: an early event in tumorigenesis

    NARCIS (Netherlands)

    Vos, C. B.; Cleton-Jansen, A. M.; Berx, G.; de Leeuw, W. J.; ter Haar, N. T.; van Roy, F.; Cornelisse, C. J.; Peterse, J. L.; van de Vijver, M. J.

    1997-01-01

    In breast cancer, inactivating point mutations in the E-cadherin gene are frequently found in invasive lobular carcinoma (ILC) but never in invasive ductal carcinoma (IDC). Lobular carcinoma in situ (LCIS) adjacent to ILC has previously been shown to lack E-cadherin expression, but whether LCIS

  1. Analysis of the viral replication cycle of adenovirus serotype 2 after inactivation by free chlorine.

    Science.gov (United States)

    Gall, Aimee M; Shisler, Joanna L; Mariñas, Benito J

    2015-04-07

    Free chlorine is effective at inactivating a wide range of waterborne viral pathogens including human adenovirus (HAdV), but the mechanisms by which free chlorine inactivates HAdV and other human viruses remain to be elucidated. Such advances in fundamental knowledge are key for development of new disinfection technologies and novel sensors to detect infectious viruses in drinking water. We developed and tested a quantitative assay to analyze several steps in the HAdV replication cycle upon increasing free chlorine exposure. We used quantitative polymerase chain reaction (qPCR) to detect HAdV genomic DNA as a means to quantify attachment and genome replication of untreated and treated virions. Also, we used quantitative reverse-transcription PCR (RT-qPCR) to quantify the transcription of E1A (first early protein) and hexon mRNA. We compared these replication cycle events to virus inactivation kinetics to determine what stage of the virus replication cycle was inhibited as a function of free chlorine exposure. We observed that adenovirus inactivated at levels up to 99.99% by free chlorine still attached to host cells; however, viral DNA synthesis and early E1A and late hexon gene transcription were inhibited. We conclude that free chlorine exposure interferes with a replication cycle event occurring postbinding but prior to early viral protein synthesis.

  2. Loss of Atrx affects trophoblast development and the pattern of X-inactivation in extraembryonic tissues.

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    David Garrick

    2006-04-01

    Full Text Available ATRX is an X-encoded member of the SNF2 family of ATPase/helicase proteins thought to regulate gene expression by modifying chromatin at target loci. Mutations in ATRX provided the first example of a human genetic disease associated with defects in such proteins. To better understand the role of ATRX in development and the associated abnormalities in the ATR-X (alpha thalassemia mental retardation, X-linked syndrome, we conditionally inactivated the homolog in mice, Atrx, at the 8- to 16-cell stage of development. The protein, Atrx, was ubiquitously expressed, and male embryos null for Atrx implanted and gastrulated normally but did not survive beyond 9.5 days postcoitus due to a defect in formation of the extraembryonic trophoblast, one of the first terminally differentiated lineages in the developing embryo. Carrier female mice that inherit a maternal null allele should be affected, since the paternal X chromosome is normally inactivated in extraembryonic tissues. Surprisingly, however, some carrier females established a normal placenta and appeared to escape the usual pattern of imprinted X-inactivation in these tissues. Together these findings demonstrate an unexpected, specific, and essential role for Atrx in the development of the murine trophoblast and present an example of escape from imprinted X chromosome inactivation.

  3. Chlorine Inactivation of Spores of Encephalitozoon spp.

    OpenAIRE

    Johnson, C. H.; Marshall, M. M.; DeMaria, L. A.; Moffet, J. M.; Korich, D. G.

    2003-01-01

    This report is an extension of a preliminary investigation on the use of chlorine to inactivate spores of Encephalitozoon intestinalis and to investigate the effect of chlorine on two other species, E cuniculi and E. hellem, associated with human infection. The 50% tissue culture infective doses of these three species were also determined. On the basis of the results obtained, it appears that chlorination of water is an effective means of controlling spores of these organisms in the aquatic e...

  4. Hyaluronan decreases surfactant inactivation in vitro.

    Science.gov (United States)

    Lu, Karen W; Goerke, Jon; Clements, John A; Taeusch, H William

    2005-02-01

    Hyaluronan (HA) is an anionic polymer and a constituent of alveolar fluid that can bind proteins, phospholipids, and water. Previous studies have established that nonionic polymers improve the surface activity of pulmonary surfactants by decreasing inactivation of surfactant. In this work, we investigate whether HA can also have beneficial effects when added to surfactants. We used a modified pulsating bubble surfactometer to measure mixtures of several commercially available pulmonary surfactants or native calf surfactant with and without serum inactivation. Surface properties such as equilibrium surface tension, minimum and maximum surface tensions on compression and expansion of a surface film, and degree of surface area reduction required to reach a surface tension of 10 mN/m were measured. In the presence of serum, addition of HA dramatically improved the surface activities of all four surfactants and in some cases in the absence of serum as well. These results indicate that HA reduces inactivation of surfactants caused by serum and add evidence that endogenous HAs may interact with alveolar surfactant under normal and abnormal conditions.

  5. Rapid inactivation of SARS-like coronaviruses.

    Energy Technology Data Exchange (ETDEWEB)

    Kapil, Sanjay (Kansas State University, Manhattan, KS); Oberst, R. D. (Kansas State University, Manhattan, KS); Bieker, Jill Marie; Tucker, Mark David; Souza, Caroline Ann; Williams, Cecelia Victoria

    2004-03-01

    Chemical disinfection and inactivation of viruses is largely understudied, but is very important especially in the case of highly infectious viruses. The purpose of this LDRD was to determine the efficacy of the Sandia National Laboratories developed decontamination formulations against Bovine Coronavirus (BCV) as a surrogate for the coronavirus that causes Severe Acute Respiratory Syndrome (SARS) in humans. The outbreak of SARS in late 2002 resulted from a highly infectious virus that was able to survive and remain infectious for extended periods. For this study, preliminary testing with Escherichia coli MS-2 (MS-2) and Escherichia coli T4 (T4) bacteriophages was conducted to develop virucidal methodology for verifying the inactivation after treatment with the test formulations following AOAC germicidal methodologies. After the determination of various experimental parameters (i.e. exposure, concentration) of the formulations, final testing was conducted on BCV. All experiments were conducted with various organic challenges (horse serum, bovine feces, compost) for results that more accurately represent field use condition. The MS-2 and T4 were slightly more resistant than BCV and required a 2 minute exposure while BCV was completely inactivated after a 1 minute exposure. These results were also consistent for the testing conducted in the presence of the various organic challenges indicating that the test formulations are highly effective for real world application.

  6. Antithrombin inactivation by neutrophil elastase requires heparin.

    Science.gov (United States)

    Jordan, R E; Nelson, R M; Kilpatrick, J; Newgren, J O; Esmon, P C; Fournel, M A

    1989-09-11

    In certain thrombotic states, large declines in the levels of functional circulating antithrombin occur, which may reflect the highly active nature of the endothelial surface in suppressing excessive amounts of activated coagulation enzymes. Alternatively, we have recently observed an unexpected and paradoxical in vitro functioning of heparin that could result in the inactivation of antithrombin in pathologic conditions. Specifically, antithrombin was rendered nonfunctional as an inhibitor of clotting enzymes as a result of a limited, heparin-dependent cleavage by neutrophil elastase. This inactivation occurred only in the presence of the active anticoagulant heparin fraction, which suggested that the heparin-antithrombin complex was the substrate for elastase attack. Interestingly, neutrophil elastase was found to bind tightly to heparin and heparin-like materials. Neutrophil elastase has been previously linked to nonspecific proteinolysis occurring in inflammatory thrombotic reactions. This affinity of both antithrombin and elastase for heparin suggests a novel mechanism of potential specificity. An important component of this hypothesis is the localization of the elastase/antithrombin reaction away from the high circulating levels of elastase inhibitors. The proposed inactivation of antithrombin on the vascular surface would likely occur only in pathologic states associated with neutrophil sequestration and activation. Nevertheless, this mechanism could lead to a localized reversal of the nonthrombogenic nature of the endothelium and potentially lead to significant reductions of functional antithrombin in certain disease states.

  7. Normal X-inactivation mosaicism in corneas of heterozygous FlnaDilp2/+ female mice--a model of human Filamin A (FLNA diseases

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    Douvaras Panagiotis

    2012-02-01

    Full Text Available Abstract Background Some abnormalities of mouse corneal epithelial maintenance can be identified by the atypical mosaic patterns they produce in X-chromosome inactivation mosaics and chimeras. Human FLNA/+ females, heterozygous for X-linked, filamin A gene (FLNA mutations, display a range of disorders and X-inactivation mosaicism is sometimes quantitatively unbalanced. FlnaDilp2/+ mice, heterozygous for an X-linked filamin A (Flna nonsense mutation have variable eye, skeletal and other abnormalities, but X-inactivation mosaicism has not been investigated. The aim of this study was to determine whether X-inactivation mosaicism in the corneal epithelia of FlnaDilp2/+ mice was affected in any way that might predict abnormal corneal epithelial maintenance. Results X-chromosome inactivation mosaicism was studied in the corneal epithelium and a control tissue (liver of FlnaDilp2/+ and wild-type (WT female X-inactivation mosaics, hemizygous for the X-linked, LacZ reporter H253 transgene, using β-galactosidase histochemical staining. The corneal epithelia of FlnaDilp2/+ and WT X-inactivation mosaics showed similar radial, striped patterns, implying epithelial cell movement was not disrupted in FlnaDilp2/+ corneas. Corrected stripe numbers declined with age overall (but not significantly for either genotype individually, consistent with previous reports suggesting an age-related reduction in stem cell function. Corrected stripe numbers were not reduced in FlnaDilp2/+ compared with WT X-inactivation mosaics and mosaicism was not significantly more unbalanced in the corneal epithelia or livers of FlnaDilp2/+ than wild-type Flna+/+ X-inactivation mosaics. Conclusions Mosaic analysis identified no major effect of the mouse FlnaDilp2 mutation on corneal epithelial maintenance or the balance of X-inactivation mosaicism in the corneal epithelium or liver.

  8. Inactivation of the Rgg2 transcriptional regulator ablates the virulence of Streptococcus pyogenes.

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    Anastasia A Zutkis

    Full Text Available Streptococcus pyogenes adapts to different niches encountered in the human host via the activity of numerous regulatory proteins including the Rgg family of transcriptional regulators. The S. pyogenes chromosome encodes four Rgg paralogues designated Rgg1 (RopB, Rgg2 (MutR, Rgg3, and Rgg4 (ComR. In order to understand the role of the Rgg2 protein in the regulation of metabolic and virulence-associated properties of S. pyogenes, the rgg2 gene was inactivated in the M1 serotype strain SF370. Inactivation of rgg2 increased the growth yield of S. pyogenes in THY broth, increased biofilm formation, and increased production of SIC, which is an important virulence factor that inhibits complement mediated lysis. To identify Rgg2-regulated genes, the transcriptomes of SF370 and the rgg2 mutant strains were compared in the middle-exponential and post-exponential phases of growth. Rgg2 was found to control the expression of dozens of genes primarily in the exponential phase of growth, including genes associated with virulence (sse, scpA, slo, nga, mf-3, DNA transformation, and nucleotide metabolism. Inactivation of rgg2 decreased the ability of S. pyogenes to adhere to epithelial cells. In addition, the mutant strain was more sensitive to killing when incubated with human blood and avirulent in a murine bacteremia model. Finally, inoculation of mice with the avirulent rgg2 mutant of S. pyogenes SF370 conferred complete protection to mice subsequently challenged with the wild-type strain. Restoration of an intact rgg2 gene in mutant strain restored the wild-type phenotypes. Overall, the results demonstrate that Rgg2 is an important regulatory protein in S. pyogenes involved in controlling genes associated with both metabolism and virulence.

  9. Inactivation of the Rgg2 transcriptional regulator ablates the virulence of Streptococcus pyogenes.

    Science.gov (United States)

    Zutkis, Anastasia A; Anbalagan, Srivishnupriya; Chaussee, Michael S; Dmitriev, Alexander V

    2014-01-01

    Streptococcus pyogenes adapts to different niches encountered in the human host via the activity of numerous regulatory proteins including the Rgg family of transcriptional regulators. The S. pyogenes chromosome encodes four Rgg paralogues designated Rgg1 (RopB), Rgg2 (MutR), Rgg3, and Rgg4 (ComR). In order to understand the role of the Rgg2 protein in the regulation of metabolic and virulence-associated properties of S. pyogenes, the rgg2 gene was inactivated in the M1 serotype strain SF370. Inactivation of rgg2 increased the growth yield of S. pyogenes in THY broth, increased biofilm formation, and increased production of SIC, which is an important virulence factor that inhibits complement mediated lysis. To identify Rgg2-regulated genes, the transcriptomes of SF370 and the rgg2 mutant strains were compared in the middle-exponential and post-exponential phases of growth. Rgg2 was found to control the expression of dozens of genes primarily in the exponential phase of growth, including genes associated with virulence (sse, scpA, slo, nga, mf-3), DNA transformation, and nucleotide metabolism. Inactivation of rgg2 decreased the ability of S. pyogenes to adhere to epithelial cells. In addition, the mutant strain was more sensitive to killing when incubated with human blood and avirulent in a murine bacteremia model. Finally, inoculation of mice with the avirulent rgg2 mutant of S. pyogenes SF370 conferred complete protection to mice subsequently challenged with the wild-type strain. Restoration of an intact rgg2 gene in mutant strain restored the wild-type phenotypes. Overall, the results demonstrate that Rgg2 is an important regulatory protein in S. pyogenes involved in controlling genes associated with both metabolism and virulence.

  10. Chlorine inactivation of Tubifex tubifex in drinking water and the synergistic effect of sequential inactivation with UV irradiation and chlorine.

    Science.gov (United States)

    Nie, Xiao-Bao; Li, Zhi-Hong; Long, Yuan-Nan; He, Pan-Pan; Xu, Chao

    2017-06-01

    The inactivation of Tubifex tubifex is important to prevent contamination of drinking water. Chlorine is a widely-used disinfectant and the key factor in the inactivation of T. tubifex. This study investigated the inactivation kinetics of chlorine on T. tubifex and the synergistic effect of the sequential use of chlorine and UV irradiation. The experimental results indicated that the Ct (concentration × timereaction) concept could be used to evaluate the inactivation kinetics of T. tubifex with chlorine, thus allowing for the use of a simpler Ct approach for the assessment of T. tubifex chlorine inactivation requirements. The inactivation kinetics of T. tubifex by chlorine was found to be well-fitted to a delayed pseudo first-order Chick-Watson expression. Sequential experiments revealed that UV irradiation and chlorine worked synergistically to effectively inactivate T. tubifex as a result of the decreased activation energy, Ea, induced by primary UV irradiation. Furthermore, the inactivation effectiveness of T. tubifex by chlorine was found to be affected by several drinking water quality parameters including pH, turbidity, and chemical oxygen demand with potassium permanganate (CODMn) concentration. High pH exhibited pronounced inactivation effectiveness and the decrease in turbidity and CODMn concentrations contributed to the inactivation of T. tubifex. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Abnormal X : autosome ratio, but normal X chromosome inactivation in human triploid cultures

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    Norwood Thomas H

    2006-07-01

    Full Text Available Abstract Background X chromosome inactivation (XCI is that aspect of mammalian dosage compensation that brings about equivalence of X-linked gene expression between females and males by inactivating one of the two X chromosomes (Xi in normal female cells, leaving them with a single active X (Xa as in male cells. In cells with more than two X's, but a diploid autosomal complement, all X's but one, Xa, are inactivated. This phenomenon is commonly thought to suggest 1 that normal development requires a ratio of one Xa per diploid autosomal set, and 2 that an early event in XCI is the marking of one X to be active, with remaining X's becoming inactivated by default. Results Triploids provide a test of these ideas because the ratio of one Xa per diploid autosomal set cannot be achieved, yet this abnormal ratio should not necessarily affect the one-Xa choice mechanism for XCI. Previous studies of XCI patterns in murine triploids support the single-Xa model, but human triploids mostly have two-Xa cells, whether they are XXX or XXY. The XCI patterns we observe in fibroblast cultures from different XXX human triploids suggest that the two-Xa pattern of XCI is selected for, and may have resulted from rare segregation errors or Xi reactivation. Conclusion The initial X inactivation pattern in human triploids, therefore, is likely to resemble the pattern that predominates in murine triploids, i.e., a single Xa, with the remaining X's inactive. Furthermore, our studies of XIST RNA accumulation and promoter methylation suggest that the basic features of XCI are normal in triploids despite the abnormal X:autosome ratio.

  12. p53 inactivation upregulates p73 expression through E2F-1 mediated transcription.

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    Chaitali Tophkhane

    Full Text Available While p73 overexpression has been associated with increased apoptosis in cancer tissues, p73 overexpressing tumors appear to be of high grade malignancy. Why this putative tumor suppressor is overexpressed in cancer cells and what the function of overexpressed p73 is in breast cancers are critical questions to be addressed. By investigating the effect of p53 inactivation on p73 expression, we found that both protein and mRNA levels of TAp73 were increased in MCF-7/p53siRNA cells, MCF-7/p53mt135 cells and HCT-116/p53-/- cells, as compared to wild type control, suggesting that p53 inactivation by various forms upregulates p73. We showed that p53 knockdown induced p73 was mainly regulated at the transcriptional level. However, although p53 has a putative binding site in the TAp73 promoter, deletion of this binding site did not affect p53 knockdown mediated activation of TAp73 promoter. Chromatin immuno-precipitation (ChIP data demonstrated that loss of p53 results in enhanced occupancy of E2F-1 in the TAp73 promoter. The responsive sequence of p53 inactivation mediated p73 upregulation was mapped to the proximal promoter region of the TAp73 gene. To test the role of E2F-1 in p53 inactivation mediated regulation of p73 transcription, we found that p53 knockdown enhanced E2F-1 dependent p73 transcription, and mutations in E2F-1 binding sites in the TAp73 promoter abrogated p53 knockdown mediated activation of TAp73 promoter. Moreover, we demonstrated that p21 is a mediator of p53-E2F crosstalk in the regulation of p73 transcription. We concluded that p53 knockdown/inactivation may upregulate TAp73 expression through E2F-1 mediated transcriptional regulation. p53 inactivation mediated upregulation of p73 suggests an intrinsic rescuing mechanism in response to p53 mutation/inactivation. These findings support further analysis of the correlation between p53 status and p73 expression and its prognostic/predictive significance in human cancers.

  13. Normal human adipose tissue functions and differentiation in patients with biallelic LPIN1 inactivating mutations.

    Science.gov (United States)

    Pelosi, Michele; Testet, Eric; Le Lay, Soazig; Dugail, Isabelle; Tang, Xiaoyun; Mabilleau, Guillaume; Hamel, Yamina; Madrange, Marine; Blanc, Thomas; Odent, Thierry; McMullen, Todd P W; Alfò, Marco; Brindley, David N; de Lonlay, Pascale

    2017-12-01

    Lipin-1 is a Mg2+-dependent phosphatidic acid phosphatase (PAP) that in mice is necessary for normal glycerolipid biosynthesis, controlling adipocyte metabolism, and adipogenic differentiation. Mice carrying inactivating mutations in the Lpin1 gene display the characteristic features of human familial lipodystrophy. Very little is known about the roles of lipin-1 in human adipocyte physiology. Apparently, fat distribution and weight is normal in humans carrying LPIN1 inactivating mutations, but a detailed analysis of adipose tissue appearance and functions in these patients has not been available so far. In this study, we performed a systematic histopathological, biochemical, and gene expression analysis of adipose tissue biopsies from human patients harboring LPIN1 biallelic inactivating mutations and affected by recurrent episodes of severe rhabdomyolysis. We also explored the adipogenic differentiation potential of human mesenchymal cell populations derived from lipin-1 defective patients. White adipose tissue from human LPIN1 mutant patients displayed a dramatic decrease in lipin-1 protein levels and PAP activity, with a concomitant moderate reduction of adipocyte size. Nevertheless, the adipose tissue develops without obvious histological signs of lipodystrophy and with normal qualitative composition of storage lipids. The increased expression of key adipogenic determinants such as SREBP1, PPARG, and PGC1A shows that specific compensatory phenomena can be activated in vivo in human adipocytes with deficiency of functional lipin-1. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  14. X-chromosomal inactivation directly influences the phenotypic manifestation of X-linked protoporphyria.

    Science.gov (United States)

    Brancaleoni, V; Balwani, M; Granata, F; Graziadei, G; Missineo, P; Fiorentino, V; Fustinoni, S; Cappellini, M D; Naik, H; Desnick, R J; Di Pierro, E

    2016-01-01

    X-linked protoporphyria (XLP), a rare erythropoietic porphyria, results from terminal exon gain-of-function mutations in the ALAS2 gene causing increased ALAS2 activity and markedly increased erythrocyte protoporphyrin levels. Patients present with severe cutaneous photosensitivity and may develop liver dysfunction. XLP was originally reported as X-linked dominant with 100% penetrance in males and females. We characterized 11 heterozygous females from six unrelated XLP families and show markedly varying phenotypic and biochemical heterogeneity, reflecting the degree of X-chromosomal inactivation of the mutant gene. ALAS2 sequencing identified the specific mutation and confirmed heterozygosity among the females. Clinical history, plasma and erythrocyte protoporphyrin levels were determined. Methylation assays of the androgen receptor and zinc-finger MYM type 3 short tandem repeat polymorphisms estimated each heterozygotes X-chromosomal inactivation pattern. Heterozygotes with equal or increased skewing, favoring expression of the wild-type allele had no clinical symptoms and only slightly increased erythrocyte protoporphyrin concentrations and/or frequency of protoporphyrin-containing peripheral blood fluorocytes. When the wild-type allele was preferentially inactivated, heterozygous females manifested the disease phenotype and had both higher erythrocyte protoporphyrin levels and circulating fluorocytes. These findings confirm that the previous dominant classification of XLP is inappropriate and genetically misleading, as the disorder is more appropriately designated XLP. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Study of the integrated immune response induced by an inactivated EV71 vaccine.

    Directory of Open Access Journals (Sweden)

    Longding Liu

    Full Text Available Enterovirus 71 (EV71, a major causative agent of hand-foot-and-mouth disease (HFMD, causes outbreaks among children in the Asia-Pacific region. A vaccine is urgently needed. Based on successful pre-clinical work, phase I and II clinical trials of an inactivated EV71 vaccine, which included the participants of 288 and 660 respectively, have been conducted. In the present study, the immune response and the correlated modulation of gene expression in the peripheral blood mononuclear cells (PBMCs of 30 infants (6 to 11 months immunized with this vaccine or placebo and consented to join this study in the phase II clinical trial were analyzed. The results showed significantly greater neutralizing antibody and specific T cell responses in vaccine group after two inoculations on days 0 and 28. Additionally, more than 600 functional genes that were up- or down-regulated in PBMCs were identified by the microarray assay, and these genes included 68 genes associated with the immune response in vaccine group. These results emphasize the gene expression profile of the immune system in response to an inactivated EV71 vaccine in humans and confirmed that such an immune response was generated as the result of the positive mobilization of the immune system. Furthermore, the immune response was not accompanied by the development of a remarkable inflammatory response.NCT01391494 and NCT01512706.

  16. Promoter hypermethylation of KLF4 inactivates its tumor suppressor function in cervical carcinogenesis.

    Directory of Open Access Journals (Sweden)

    Wen-Ting Yang

    Full Text Available OBJECTIVE: The KLF4 gene has been shown to be inactivated in cervical carcinogenesis as a tumor suppressor. However, the mechanism of KLF4 silencing in cervical carcinomas has not yet been identified. DNA methylation plays a key role in stable suppression of gene expression. METHODS: The methylation status of the KLF4 promoter CpG islands was analyzed by bisulfite sequencing (BSQ in tissues of normal cervix and cervical cancer. KLF4 gene expression was detected by RT-PCR, immunohistochemistry and western blot. KLF4 promoter methylation in cervical cancer cell line was determined by BSQ and methylation-specific polymerase chain reaction (MS-PCR. Cell proliferation ability was detected by cell growth curve and MTT assay. RESULTS: The methylated allele was found in 41.90% of 24 cervical cancer tissues but only in 11.11% of 11 normal cervix tissues (P<0.005. KLF4 mRNA levels were significantly reduced in cervical cancer tissues compared with normal cervix tissues (P<0.01 and KLF4 mRNA expression showed a significant negative correlation with the promoter hypermethylation (r = -0.486, P = 0.003. Cervical cancer cell lines also showed a significant negative correlation between KLF4 expression and hypermethylation. After treatment with the demethylating agent 5-Azacytidine (5-Aza, the expression of KLF4 in the cervical cancer cell lines at both mRNA and protein levels was drastically increased, the cell proliferation ability was inhibited and the chemosensitivity for cisplatin was significantly increased. CONCLUSION: KLF4 gene is inactivated by methylation-induced silencing mechanisms in a large subset of cervical carcinomas and KLF4 promoter hypermethylation inactivates the gene's function as a tumor suppressor in cervical carcinogenesis.

  17. Modeling the pressure inactivation dynamics of Escherichia coli

    Directory of Open Access Journals (Sweden)

    Yamamoto K.

    2005-01-01

    Full Text Available Escherichia coli, as a model microorganism, was treated in phosphate-buffered saline under high hydrostatic pressure between 100 and 300 MPa, and the inactivation dynamics was investigated from the viewpoint of predictive microbiology. Inactivation data were curve fitted by typical predictive models: logistic, Gompertz and Weibull functions. Weibull function described the inactivation curve the best. Two parameters of Weibull function were calculated for each holding pressure and their dependence on holding pressure was obtained by interpolation. With the interpolated parameters, inactivation curves were simulated and compared with the experimental data sets.

  18. Inactivation of virus in solution by cold atmospheric pressure plasma: identification of chemical inactivation pathways

    Science.gov (United States)

    Aboubakr, Hamada A.; Gangal, Urvashi; Youssef, Mohammed M.; Goyal, Sagar M.; Bruggeman, Peter J.

    2016-05-01

    Cold atmospheric pressure plasma (CAP) inactivates bacteria and virus through in situ production of reactive oxygen and nitrogen species (RONS). While the bactericidal and virucidal efficiency of plasmas is well established, there is limited knowledge about the chemistry leading to the pathogen inactivation. This article describes a chemical analysis of the CAP reactive chemistry involved in the inactivation of feline calicivirus. We used a remote radio frequency CAP produced in varying gas mixtures leading to different plasma-induced chemistries. A study of the effects of selected scavengers complemented with positive control measurements of relevant RONS reveal two distinctive pathways based on singlet oxygen and peroxynitrous acid. The first mechanism is favored in the presence of oxygen and the second in the presence of air when a significant pH reduction is induced in the solution by the plasma. Additionally, smaller effects of the H2O2, O3 and \\text{NO}2- produced were also found. Identification of singlet oxygen-mediated 2-imidazolone/2-oxo-His (His  +14 Da)—an oxidative modification of His 262 comprising the capsid protein of feline calicivirus links the plasma induced singlet oxygen chemistry to viral inactivation.

  19. Seemingly neutral polymorphic variants may confer immunity to splicing-inactivating mutations

    DEFF Research Database (Denmark)

    Nielsen, Karsten Bork; Sørensen, Suzette; Cartegni, Luca

    2007-01-01

    The idea that point mutations in exons may affect splicing is intriguing and adds an additional layer of complexity when evaluating their possible effects. Even in the best-studied examples, the molecular mechanisms are not fully understood. Here, we use patient cells, model minigenes, and in vitro...... assays to show that a missense mutation in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD) gene primarily causes exon skipping by inactivating a crucial exonic splicing enhancer (ESE), thus leading to loss of a functional protein and to MCAD deficiency. This ESE functions by antagonizing...... a juxtaposed exonic splicing silencer (ESS) and is necessary to define a suboptimal 3' splice site. Remarkably, a synonymous polymorphic variation in MCAD exon 5 inactivates the ESS, and, although this has no effect on splicing by itself, it makes splicing immune to deleterious mutations in the ESE...

  20. Genetic characterization in symptomatic female DMD carriers: lack of relationship between X-inactivation, transcriptional DMD allele balancing and phenotype

    Directory of Open Access Journals (Sweden)

    Brioschi Simona

    2012-08-01

    Full Text Available Abstract Background Although Duchenne and Becker muscular dystrophies, X-linked recessive myopathies, predominantly affect males, a clinically significant proportion of females manifesting symptoms have also been reported. They represent an heterogeneous group characterized by variable degrees of muscle weakness and/or cardiac involvement. Though preferential inactivation of the normal X chromosome has long been considered the principal mechanism behind disease manifestation in these females, supporting evidence is controversial. Methods Eighteen females showing a mosaic pattern of dystrophin expression on muscle biopsy were recruited and classified as symptomatic (7 or asymptomatic (11, based on the presence or absence of muscle weakness. The causative DMD gene mutations were identified in all cases, and the X-inactivation pattern was assessed in muscle DNA. Transcriptional analysis in muscles was performed in all females, and relative quantification of wild-type and mutated transcripts was also performed in 9 carriers. Dystrophin protein was quantified by immunoblotting in 2 females. Results The study highlighted a lack of relationship between dystrophic phenotype and X-inactivation pattern in females; skewed X-inactivation was found in 2 out of 6 symptomatic carriers and in 5 out of 11 asymptomatic carriers. All females were characterized by biallelic transcription, but no association was found between X-inactivation pattern and allele transcriptional balancing. Either a prevalence of wild-type transcript or equal proportions of wild-type and mutated RNAs was observed in both symptomatic and asymptomatic females. Moreover, very similar levels of total and wild-type transcripts were identified in the two groups of carriers. Conclusions This is the first study deeply exploring the DMD transcriptional behaviour in a cohort of female carriers. Notably, no relationship between X-inactivation pattern and transcriptional behaviour of DMD gene was

  1. Genetic characterization in symptomatic female DMD carriers: lack of relationship between X-inactivation, transcriptional DMD allele balancing and phenotype.

    Science.gov (United States)

    Brioschi, Simona; Gualandi, Francesca; Scotton, Chiara; Armaroli, Annarita; Bovolenta, Matteo; Falzarano, Maria S; Sabatelli, Patrizia; Selvatici, Rita; D'Amico, Adele; Pane, Marika; Ricci, Giulia; Siciliano, Gabriele; Tedeschi, Silvana; Pini, Antonella; Vercelli, Liliana; De Grandis, Domenico; Mercuri, Eugenio; Bertini, Enrico; Merlini, Luciano; Mongini, Tiziana; Ferlini, Alessandra

    2012-08-16

    Although Duchenne and Becker muscular dystrophies, X-linked recessive myopathies, predominantly affect males, a clinically significant proportion of females manifesting symptoms have also been reported. They represent an heterogeneous group characterized by variable degrees of muscle weakness and/or cardiac involvement. Though preferential inactivation of the normal X chromosome has long been considered the principal mechanism behind disease manifestation in these females, supporting evidence is controversial. Eighteen females showing a mosaic pattern of dystrophin expression on muscle biopsy were recruited and classified as symptomatic (7) or asymptomatic (11), based on the presence or absence of muscle weakness. The causative DMD gene mutations were identified in all cases, and the X-inactivation pattern was assessed in muscle DNA. Transcriptional analysis in muscles was performed in all females, and relative quantification of wild-type and mutated transcripts was also performed in 9 carriers. Dystrophin protein was quantified by immunoblotting in 2 females. The study highlighted a lack of relationship between dystrophic phenotype and X-inactivation pattern in females; skewed X-inactivation was found in 2 out of 6 symptomatic carriers and in 5 out of 11 asymptomatic carriers. All females were characterized by biallelic transcription, but no association was found between X-inactivation pattern and allele transcriptional balancing. Either a prevalence of wild-type transcript or equal proportions of wild-type and mutated RNAs was observed in both symptomatic and asymptomatic females. Moreover, very similar levels of total and wild-type transcripts were identified in the two groups of carriers. This is the first study deeply exploring the DMD transcriptional behaviour in a cohort of female carriers. Notably, no relationship between X-inactivation pattern and transcriptional behaviour of DMD gene was observed, suggesting that the two mechanisms are regulated

  2. Photodynamic inactivation of pathogens causing infectious keratitis

    Science.gov (United States)

    Simon, Carole; Wolf, G.; Walther, M.; Winkler, K.; Finke, M.; Hüttenberger, D.; Bischoff, Markus; Seitz, B.; Cullum, J.; Foth, H.-J.

    2014-03-01

    The increasing prevalence of antibiotic resistance requires new approaches also for the treatment of infectious keratitis. Photodynamic Inactivation (PDI) using the photosensitizer (PS) Chlorin e6 (Ce6) was investigated as an alternative to antibiotic treatment. An in-vitro cornea model was established using porcine eyes. The uptake of Ce6 by bacteria and the diffusion of the PS in the individual layers of corneal tissue were investigated by fluorescence. After removal of the cornea's epithelium Ce6-concentrations keratitis patients were tested in liquid culture against different concentrations of Ce6 (1 - 512 μM) using 10 minutes irradiation (E = 18 J/cm2 ). This demonstrated that a complete inactivation of the pathogen strains were feasible whereby SA was slightly more susceptible than PA. 3909 mutants of the Keio collection of Escherichia coli (E.coli) were screened for potential resistance factors. The sensitive mutants can be grouped into three categories: transport mutants, mutants in lipopolysaccharide synthesis and mutants in the bacterial SOS-response. In conclusion PDI is seen as a promising therapy concept for infectious keratitis.

  3. Efficacy of select disinfectants at inactivating Ranavirus.

    Science.gov (United States)

    Bryan, Laura K; Baldwin, Charles A; Gray, Matthew J; Miller, Debra L

    2009-04-06

    Ranavirus can cause disease in reptiles and amphibians. Because survival time outside of a host remains uncertain, equipment must be disinfected to prevent transmission of ranaviruses. However, disinfectant efficacy against amphibian ranaviruses has not been investigated for chlorhexidine (Nolvasan), sodium hypochlorite (bleach), or potassium compounds. Our goal was to determine the efficacy of Nolvasan (0.25, 0.75 and 2.0%), bleach (0.2, 1.0, 3.0 and 5.0%), and Virkon S (1.0%) at inactivating Ranavirus at 1 and 5 min contact durations. Potassium permanganate (KMnO4) (2.0 and 5.0 ppm) was also tested with a 60 min contact time. Nolvasan at 0.75 and 2.0% and bleach at 3.0 and 5.0% concentration were effective for both contact durations. Virkon S was effective for both durations, but KMnO4 was not effective at either concentration. Concentrations of Nolvasan, bleach and Virkon S that are at least 0.75, 3.0 and 1.0%, respectively, are effective at inactivating Ranavirus after 1 min exposure time.

  4. "Slow" Voltage-Dependent Inactivation of CaV2.2 Calcium Channels Is Modulated by the PKC Activator Phorbol 12-Myristate 13-Acetate (PMA.

    Directory of Open Access Journals (Sweden)

    Lei Zhu

    Full Text Available CaV2.2 (N-type voltage-gated calcium channels (Ca2+ channels play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. "Fast" voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of "slow" voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate "slow" inactivation of sodium channels, but little is known about if/how second messengers control "slow" inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA dramatically prolonged recovery from "slow" inactivation, but an inactive control (4α-PMA had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel β-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-β-S reduced the effect of PMA suggesting a role for G proteins in modulating "slow" inactivation. We postulate that the kinetics of recovery from "slow" inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.

  5. Nonrandom X chromosome inactivation in natural killer cells from obligate carriers of X-linked severe combined immunodeficiency

    Energy Technology Data Exchange (ETDEWEB)

    Wengler, G.S.; Parolini, O.; Conley, M.E. (Univ. of Tennessee, Memphis (United States) St. Jude Children' s Research Hospital, Memphis, TN (United States)); Allen, R.C. (Baylor College of Medicine, Houston, TX (United States)); Smith, H. (St. Jude Children' s Research Hospital, Memphis, TN (United States))

    1993-01-15

    X-linked severe combined immunodeficiency (XSCID) is characterized by hypogammaglobulinemia, markedly reduced numbers of T cells, absent mitogen responses, decreased numbers of NK cells, and normal or elevated numbers of B cells. The abnormalities in the NK cell and B cell lineages could be attributed to dependence of these cell lineages on T cells or T cell-derived factors, or to expression of the XSCID gene defect in these cell lineages. In past experiments, the authors have examined X chromosome inactivation patterns in T cells and cultured B cells from female obligate carriers of XSCID and have found that both cell lineages demonstrate nonrandom X chromosome inactivation. This indicates that the gene defect is intrinsic to both of these cell lineages. In the present experiments, a polymerase chain reaction technique was used to evaluate X chromosome inactivation patterns in highly purified populations of freshly isolated NK cells, B cells, CD4[sup +] cells, and CD8[sup +] cells from three obligate carriers of XSCID. All four lymphoid cell populations from these three women exhibited exclusive use of a single X as the active X. In contrast, both X chromosomes were used as the active X in neutrophils and monocytes. These findings indicate that the XSCID gene is expressed in the NK cell lineage as well as in T cells and B cells. This observation makes it highly unlikely that the XSCID gene is involved in Ag receptor gene rearrangements. 21 refs., 4 figs.

  6. [Polyphenolic antioxidants efficiently protect urease from inactivation by ultrasonic cavitation].

    Science.gov (United States)

    Metelitsa, D I; Tarun, E I; Losev, Iu P

    2002-01-01

    Inactivation of urease (25 nM) in aqueous solutions (pH 5.0-6.0) treated with low-frequency ultrasound (LFUS; 27 kHz, 60 Wt/cm2, 36-56 degrees C) or high-frequency ultrasound (HFUS; 2.64 MHz, 1 Wt/cm2, 36 or 56 degrees C) has been characterized quantitatively, using first-order rate constants: kin, aggregate inactivation; kin*, thermal inactivation; and kin* (US), ultrasonic inactivation. Within the range from 1 nM to 10 microM, propyl gallate (PG) decreases approximately threefold the rate of LFUS-induced inactivation of urease (56 degrees C), whereas resorcinol poly-2-disulfide prevents this process at 1 nM or higher concentrations. PG completely inhibits HFUS-induced inactivation of urease at 1 nM (36 degrees C) or 10 nM (56 degrees C). At 0.2-10 microM, human serum albumin (HSA) increases the resistance of urease (at 56 degrees C) treated with HFUS to temperature- and cavitation-induced inactivation. Complexes of gallic acid polydisulfide (GAPDS) with HSA (GAPDS-HSA), formed by conjugation of 1.0 nM PGDS with 0.33 nM HSA, prevent HFUS-induced urease inactivation (56 degrees C).

  7. Scale down of the inactivated polio vaccine production process

    NARCIS (Netherlands)

    Thomassen, Y.E.; Oever, van 't R.; Vinke, C.M.; Spiekstra, A.; Wijffels, R.H.; Pol, van der L.A.; Bakker, W.A.M.

    2013-01-01

    The anticipated increase in the demand for inactivated polio vaccines resulting from the success in the polio eradication program requires an increase in production capacity and cost price reduction of the current inactivated polio vaccine production processes. Improvement of existing production

  8. Suicide inactivation of horseradish peroxidase by excess hydrogen ...

    African Journals Online (AJOL)

    In reactions carried out in sodium acetate buffer, higher inactivation rates were observed when the buffer ion concentration was increased, an indication that peroxidase might be generating reactive radicals from the buffer molecules. Promethazine exerted a modest protective effect against inactivation; however, higher ...

  9. Effects of electrolytes on virus inactivation by acidic solutions.

    Science.gov (United States)

    Nishide, Mitsunori; Tsujimoto, Kazuko; Uozaki, Misao; Ikeda, Keiko; Yamasaki, Hisashi; Koyama, A Hajime; Arakawa, Tsutomu

    2011-06-01

    Acidic pH is frequently used to inactivate viruses. We have previously shown that arginine synergizes with low pH in enhancing virus inactivation. Considering a potential application of the acid inactivation of viruses for the prevention and treatment of superficial virus infection at body surfaces and fixtures, herein we have examined the effects of various electrolytes on the acid-induced inactivation of the herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), the influenza A virus (IAV) and the poliovirus upon their incubation at 30˚C for 5 min. Eight electrolytes, i.e., phosphate, NaCl, glutamate, aspartate, pyrrolidone carboxylate, citrate, malate and acetate were tested. No detectable inactivation of the poliovirus was observed under the conditions examined, reflecting its acid-resistance. HSV-1 and HSV-2 responded similarly to the acid-treatment and electrolytes. Some electrolytes showed a stronger virus inactivation than others at a given pH and concentration. The effects of the electrolytes were virus-dependent, as IAV responded differently from HSV-1 and HSV-2 to these electrolytes, indicating that certain combinations of the electrolytes and a low pH can exert a more effective virus inactivation than other combinations and that their effects are virus-specific. These results should be useful in designing acidic solvents for the inactivation of viruses at various surfaces.

  10. Ebola Virus Inactivation by Detergents Is Annulled in Serum

    NARCIS (Netherlands)

    van Kampen, Jeroen J. A.; Tintu, Andrei; Russcher, Henk; Fraaij, Pieter L. A.; Reusken, Chantal B. E. M.; Rijken, Mikel; van Hellemond, Jaap J.; van Genderen, Perry J. J.; Koelewijn, Rob; de Jong, Menno D.; Haddock, Elaine; Fischer, Robert J.; Munster, Vincent J.; Koopmans, Marion P. G.

    2017-01-01

    Treatment of blood samples from hemorrhagic fever virus (HFV)-infected patients with 0.1% detergents has been recommended for virus inactivation and subsequent safe laboratory testing. However, data on virus inactivation by this procedure are lacking. Here we show the effect of this procedure on

  11. Disruption of diphthamide synthesis genes and resulting toxin resistance as a robust technology for quantifying and optimizing CRISPR/Cas9-mediated gene editing.

    Science.gov (United States)

    Killian, Tobias; Dickopf, Steffen; Haas, Alexander K; Kirstenpfad, Claudia; Mayer, Klaus; Brinkmann, Ulrich

    2017-11-13

    We have devised an effective and robust method for the characterization of gene-editing events. The efficacy of editing-mediated mono- and bi-allelic gene inactivation and integration events is quantified based on colony counts. The combination of diphtheria toxin (DT) and puromycin (PM) selection enables analyses of 10,000-100,000 individual cells, assessing hundreds of clones with inactivated genes per experiment. Mono- and bi-allelic gene inactivation is differentiated by DT resistance, which occurs only upon bi-allelic inactivation. PM resistance indicates integration. The robustness and generalizability of the method were demonstrated by quantifying the frequency of gene inactivation and cassette integration under different editing approaches: CRISPR/Cas9-mediated complete inactivation was ~30-50-fold more frequent than cassette integration. Mono-allelic inactivation without integration occurred >100-fold more frequently than integration. Assessment of gRNA length confirmed 20mers to be most effective length for inactivation, while 16-18mers provided the highest overall integration efficacy. The overall efficacy was ~2-fold higher for CRISPR/Cas9 than for zinc-finger nuclease and was significantly increased upon modulation of non-homologous end joining or homology-directed repair. The frequencies and ratios of editing events were similar for two different DPH genes (independent of the target sequence or chromosomal location), which indicates that the optimization parameters identified with this method can be generalized.

  12. Chlorophyll mediated photodynamic inactivation of blue laser on Streptococcus mutans

    Science.gov (United States)

    Astuti, Suryani Dyah; Zaidan, A.; Setiawati, Ernie Maduratna; Suhariningsih

    2016-03-01

    Photodynamic inactivation is an inactivation method in microbial pathogens that utilize light and photosensitizer. This study was conducted to investigate photodynamic inactivation effects of low intensity laser exposure with various dose energy on Streptococcus mutans bacteria. The photodynamic inactivation was achieved with the addition of chlorophyll as photosensitizers. To determine the survival percentage of Streptococcus mutans bacteria after laser exposure, the total plate count method was used. For this study, the wavelength of the laser is 405 nm and variables of energy doses are 1.44, 2.87, 4.31, 5.74, 7.18, and 8.61 in J/cm2. The results show that exposure to laser with energy dose of 7.18 J/cm2 has the best photodynamic inactivation with a decrease of 78% in Streptococcus

  13. Kinetic modelling of enzyme inactivation : kinetics of heat inactivation of the extracellular proteinase from Pseudomonas fluorescens 22F

    NARCIS (Netherlands)

    Schokker, E.P.

    1997-01-01

    The kinetics of heat inactivation of the extracellular proteinase from Pseudomonas fluorescens 22F was studied. It was established, by making use of kinetic modelling, that heat inactivation in the temperature range 35 - 70 °C was most likely caused

  14. Development of thermostable lyophilized inactivated polio vaccine.

    Science.gov (United States)

    Kraan, Heleen; van Herpen, Paul; Kersten, Gideon; Amorij, Jean-Pierre

    2014-10-01

    The aim of current study was to develop a dried inactivated polio vaccine (IPV) formulation with minimal loss during the drying process and improved stability when compared with the conventional liquid IPV. Extensive excipient screening was combined with the use of a Design of Experiment (DoE) approach in order to achieve optimal results with high probability. Although it was shown earlier that the lyophilization of a trivalent IPV while conserving its antigenicity is challenging, we were able to develop a formulation that showed minimal loss of potency during drying and subsequent storage at higher temperatures. This study showed the potential of a highly stable and safe lyophilized polio vaccine, which might be used in developing countries without the need of a cold-chain.

  15. Inactivation of Coxiella burnetti by gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Scott, G.H.; McCaul, T.F.; Williams, J.C.

    1989-01-01

    The gamma radiation inactivation kinetics for Coxiella burnetii at - 79 C were exponential. The radiation dose needed to reduce the number of infective C. burnetii by 90% varied from 0-64 to 1.2 kGy depending on the phase of hte micro-organism, purity of the culture and composition of suspending menstruum. The viability of preparations containing C. burnetti was completely abolished by 10 kGy without diminishing antigenicity or ability to elicit a protective immune response in vaccinated mice. Immunocytochemical examinations using monoclonal antibodies and electron microscopy demonstrated that radiation doses of 20 kGy did not alter cell-wall morphology or cell-surface antigenic epitopes.

  16. Inactivation of Coxiella burnetii by gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Scott, G.H.; McCaul, T.F. (Army Medical Research Inst. of Infectious Diseases, Fort Detrick, Frederick, MD (USA)); Williams, J.C. (National Inst. of Allergy and Infectious Diseases, Bethesda, MD (USA))

    1989-12-01

    The gamma radiation inactivation kinetics for Coxiella burnetii at - 79{sup 0}C were exponential. The radiation dose needed to reduce the number of infective C. burnetii by 90% varied from 0.64 to 1.2 kGy depending on the phase of the micro-organism, purity of the culture and composition of suspending menstruum. The viability of preparations containing 10{sup 11} C. burnetii ml{sup -1} was completely abolished by 10 kGy without diminishing antigenicity or ability to elicit a protective immune response in vaccinated mice. Immunocytochemical examinations using monoclonal antibodies and electron microscopy demonstrated that radiation doses of 20 kGy did not alter cell-wall morphology or cell-surface antigenic epitopes. (author).

  17. Esterase resistant to inactivation by heavy metals

    KAUST Repository

    El, Dorry Hamza

    2014-09-25

    EstATII is an esterase that a halotolerant, thermophilic and resistant to a spectrum of heavy metals including toxic concentration of metals. It was isolated from the lowest convective layer of the Atlantis II Red Sea brine pool. The Atlantis II brine pool is an extreme environment that possesses multiple harsh conditions such as; high temperature, salinity, pH and high concentration of metals, including toxic heavy metals. A fosmid metagenomic library using DNA isolated from the lowest convective layer this pool was used to identify EstATII. Polynucleotides encoding EstATII and similar esterases are disclosed and can be used to make EstATII. EstATII or compositions or apparatuses that contain it may be used in various processes employing lipases/esterases especially when these processes are performed under harsh conditions that inactivate other kinds of lipases or esterases.

  18. Mechanisms of Inactivation of Dry Escherichia coli by High-Pressure Carbon Dioxide

    Science.gov (United States)

    Chen, Yuan Yao; Temelli, Feral

    2017-01-01

    ABSTRACT High-pressure carbon dioxide processing is a promising technology for nonthermal food preservation. However, few studies have determined the lethality of high-pressure CO2 on dry bacterial cells, and the mechanism of inactivation remains unknown. This study explored the mechanisms of inactivation by using Escherichia coli AW1.7 and mutant strains differing in heat and acid resistance, in membrane composition based on disruption of the locus of heat resistance, and in genes coding for glutamate decarboxylases and cyclopropane fatty acid synthase. The levels of lethality of treatments with liquid, gaseous, and supercritical CO2 were compared. The cell counts of E. coli AW1.7 and mutants with a water activity (aW) of 1.0 were reduced by more than 3 log10 (CFU/ml) after supercritical CO2 treatment at 35°C for 15 min; increasing the pressure generally enhanced inactivation, except for E. coli AW1.7 ΔgadAB. E. coli AW1.7 Δcfa was more susceptible than E. coli AW1.7 after treatment at 10 and 40 MPa; other mutations did not affect survival. Dry cells of E. coli were resistant to treatments with supercritical and liquid CO2 at any temperature. Treatments with gaseous CO2 at 65°C were more bactericidal than those with supercritical CO2 or treatments at 65°C only. Remarkably, E. coli AW1.7 was more susceptible than E. coli AW1.7 Δcfa when subjected to the gaseous CO2 treatment. This study identified CO2-induced membrane fluidization and permeabilization as causes of supercritical mediated microbial inactivation, and diffusivity was a dominant factor for gaseous CO2. IMPORTANCE The safety of dry foods is of increasing concern for public health. Desiccated microorganisms, including pathogens, remain viable over long periods of storage and generally tolerate environmental insults that are lethal to the same organisms at high water activity. This study explored the use of high-pressure carbon dioxide to determine its lethality for dried Escherichia coli and to

  19. Mechanisms of Inactivation of Dry Escherichia coli by High-Pressure Carbon Dioxide.

    Science.gov (United States)

    Chen, Yuan Yao; Temelli, Feral; Gänzle, Michael G

    2017-05-15

    High-pressure carbon dioxide processing is a promising technology for nonthermal food preservation. However, few studies have determined the lethality of high-pressure CO 2 on dry bacterial cells, and the mechanism of inactivation remains unknown. This study explored the mechanisms of inactivation by using Escherichia coli AW1.7 and mutant strains differing in heat and acid resistance, in membrane composition based on disruption of the locus of heat resistance, and in genes coding for glutamate decarboxylases and cyclopropane fatty acid synthase. The levels of lethality of treatments with liquid, gaseous, and supercritical CO 2 were compared. The cell counts of E. coli AW1.7 and mutants with a water activity (a W ) of 1.0 were reduced by more than 3 log 10 (CFU/ml) after supercritical CO 2 treatment at 35°C for 15 min; increasing the pressure generally enhanced inactivation, except for E. coli AW1.7 Δ gadAB E. coli AW1.7 Δ cfa was more susceptible than E. coli AW1.7 after treatment at 10 and 40 MPa; other mutations did not affect survival. Dry cells of E. coli were resistant to treatments with supercritical and liquid CO 2 at any temperature. Treatments with gaseous CO 2 at 65°C were more bactericidal than those with supercritical CO 2 or treatments at 65°C only. Remarkably, E. coli AW1.7 was more susceptible than E. coli AW1.7 Δ cfa when subjected to the gaseous CO 2 treatment. This study identified CO 2 -induced membrane fluidization and permeabilization as causes of supercritical mediated microbial inactivation, and diffusivity was a dominant factor for gaseous CO 2 IMPORTANCE The safety of dry foods is of increasing concern for public health. Desiccated microorganisms, including pathogens, remain viable over long periods of storage and generally tolerate environmental insults that are lethal to the same organisms at high water activity. This study explored the use of high-pressure carbon dioxide to determine its lethality for dried Escherichia coli and to

  20. A key inactivation factor of HeLa cell viability by a plasma flow

    Energy Technology Data Exchange (ETDEWEB)

    Sato, Takehiko; Yokoyama, Mayo [Institute of Fluid Science, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai 980-8577 (Japan); Johkura, Kohei, E-mail: sato@ifs.tohoku.ac.jp [Department of Histology and Embryology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621 (Japan)

    2011-09-21

    Recently, a plasma flow has been applied to medical treatment using effects of various kinds of stimuli such as chemical species, charged particles, heat, light, shock wave and electric fields. Among them, the chemical species are known to cause an inactivation of cell viability. However, the mechanisms and key factors of this event are not yet clear. In this study, we focused on the effect of H{sub 2}O{sub 2} in plasma-treated culture medium because it is generated in the culture medium and it is also chemically stable compared with free radicals generated by the plasma flow. To elucidate the significance of H{sub 2}O{sub 2}, we assessed the differences in the effects of plasma-treated medium and H{sub 2}O{sub 2}-added medium against inactivation of HeLa cell viability. These two media showed comparable effects on HeLa cells in terms of the survival ratios, morphological features of damage processes, permeations of H{sub 2}O{sub 2} into the cells, response to H{sub 2}O{sub 2} decomposition by catalase and comprehensive gene expression. The results supported that among chemical species generated in a plasma-treated culture medium, H{sub 2}O{sub 2} is one of the main factors responsible for inactivation of HeLa cell viability. (fast track communication)

  1. X-linked sideroblastic anemia associated with a novel ALAS2 mutation and unfortunate skewed X-chromosome inactivation patterns.

    Science.gov (United States)

    Aivado, Manuel; Gattermann, Norbert; Rong, Astrid; Giagounidis, Aristoteles A N; Prall, Wolf C; Czibere, Akos; Hildebrandt, Barbara; Haas, Rainer; Bottomley, Sylvia S

    2006-01-01

    Historically X-linked sideroblastic anemia, with rare exceptions, was thought to be manifested only in males. Since the discovery of the erythroid-specific isoform of 5-aminolevulinate synthase (ALAS2) and the cloning of its gene (ALAS2) 15 years ago, mutation analysis has revealed that clinical expression of this X-linked disorder is prevalent in females as well. However, presence of the disease in both genders within affected kindreds appears to be very uncommon. We report a unique family with the disorder in three women who have had widely disparate clinical courses. The anemia is associated with a previously unrecognized ALAS2 mutation (Arg436Trp) and is unresponsive to pyridoxine. To clarify the varied clinical courses of the patients, X-chromosome inactivation patterns were examined in hematopoietic and non-hematopoietic cells. We observed inactivation patterns supporting the conclusions that one daughter has a mild phenotype at age 31 because of moderate constitutive skewed X-chromosome inactivation, another daughter with clinical onset at age 16 is severely affected due to extreme constitutive X-skewing, whereas the mother developed progressive anemia in the fifth decade as she acquired an age-related non-random X-inactivation in hematopoietic cells. In addition, we observed random X-inactivation in reticulocytes of all three women that contrasted with a markedly skewed inactivation pattern in bone marrow erythroid cells. This discordance is attributable to apoptosis of erythroid precursors derived from progenitor cells with an active X-chromosome bearing the ALAS2 mutation. The features of the disorder in this family are also instructive in regard to the differential diagnosis of sideroblastic anemias in women.

  2. The amidase domain of lipoamidase specifically inactivates lipoylated proteins in vivo.

    Directory of Open Access Journals (Sweden)

    Maroya D Spalding

    Full Text Available BACKGROUND: In the 1950s, Reed and coworkers discovered an enzyme activity in Streptococcus faecalis (Enterococcus faecalis extracts that inactivated the Escherichia. coli and E. faecalis pyruvate dehydrogenase complexes through cleavage of the lipoamide bond. The enzyme that caused this lipoamidase activity remained unidentified until Jiang and Cronan discovered the gene encoding lipoamidase (Lpa through the screening of an expression library. Subsequent cloning and characterization of the recombinant enzyme revealed that lipoamidase is an 80 kDa protein composed of an amidase domain containing a classic Ser-Ser-Lys catalytic triad and a carboxy-terminal domain of unknown function. Here, we show that the amidase domain can be used as an in vivo probe which specifically inactivates lipoylated enzymes. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated whether Lpa could function as an inducible probe of alpha-ketoacid dehydrogenase inactivation using E. coli as a model system. Lpa expression resulted in cleavage of lipoic acid from the three lipoylated proteins expressed in E. coli, but did not result in cleavage of biotin from the sole biotinylated protein, the biotin carboxyl carrier protein. When expressed in lipoylation deficient E. coli, Lpa is not toxic, indicating that Lpa does not interfere with any other critical metabolic pathways. When truncated to the amidase domain, Lpa retained lipoamidase activity without acquiring biotinidase activity, indicating that the carboxy-terminal domain is not essential for substrate recognition or function. Substitution of any of the three catalytic triad amino acids with alanine produced inactive Lpa proteins. CONCLUSIONS/SIGNIFICANCE: The enzyme lipoamidase is active against a broad range of lipoylated proteins in vivo, but does not affect the growth of lipoylation deficient E. coli. Lpa can be truncated to 60% of its original size with only a partial loss of activity, resulting in a smaller probe that can

  3. Trading direct for indirect defense? Phytochrome B inactivation in tomato attenuates direct anti-herbivore defenses whilst enhancing volatile-mediated attraction of predators.

    Science.gov (United States)

    Cortés, Leandro E; Weldegergis, Berhane T; Boccalandro, Hernán E; Dicke, Marcel; Ballaré, Carlos L

    2016-12-01

    Under conditions of competition for light, which lead to the inactivation of the photoreceptor phytochrome B (phyB), the growth of shade-intolerant plants is promoted and the accumulation of direct anti-herbivore defenses is down-regulated. Little is known about the effects of phyB on emissions of volatile organic compounds (VOCs), which play a major role as informational cues in indirect defense. We investigated the effects of phyB on direct and indirect defenses in tomato (Solanum lycopersicum) using two complementary approaches to inactivate phyB: illumination with a low red to far-red ratio, simulating competition, and mutation of the two PHYB genes present in the tomato genome. Inactivation of phyB resulted in low levels of constitutive defenses and down-regulation of direct defenses induced by methyl jasmonate (MeJA). Interestingly, phyB inactivation also had large effects on the blends of VOCs induced by MeJA. Moreover, in two-choice bioassays using MeJA-induced plants, the predatory mirid bug Macrolophus pygmaeus preferred VOCs from plants in which phyB was inactivated over VOCs from control plants. These results suggest that, in addition to repressing direct defense, phyB inactivation has consequences for VOC-mediated tritrophic interactions in canopies, presumably attracting predators to less defended plants, where they are likely to find more abundant prey. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  4. Non-random X chromosome inactivation in an affected twin in a monozygotic twin pair discordant for Wiedemann-Beckwith syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Oestavik, R.E.; Eiklid, K.; Oerstavik, K.H. [Ulleval Univ. Hospital, Oslo (Norway)] [and others

    1995-03-27

    Wiedemann-Beckwith syndrome (WBS) is a syndrome including exomphalos, macroglossia, and generalized overgrowth. The locus has been assigned to 11p15, and genomic imprinting may play a part in the expression of one or more genes involved. Most cases are sporadic. An excess of female monozygotic twins discordant for WBS have been reported, and it has been proposed that this excess could be related to the process of X chromosome inactivation. We have therefore studied X chromosome inactivation in 13-year-old monozygotic twin girls who were discordant for WBS. In addition, both twins had Tourette syndrome. The twins were monochorionic and therefore the result of a late twinning process. This has also been the case in previously reported discordant twin pairs with information on placentation. X chromosome inactivation was determined in DNA from peripheral blood cells by PCR analysis at the androgen receptor locus. The affected twin had a completely skewed X inactivation, where the paternal allele was on the active X chromosome in all cells. The unaffected twin had a moderately skewed X inactivation in the same direction, whereas the mother had a random pattern. Further studies are necessary to establish a possible association between the expression of WBS and X chromosome inactivation. 18 refs., 2 figs., 1 tab.

  5. Virus-specific thermostability and heat inactivation profiles of alphaviruses.

    Science.gov (United States)

    Park, So Lee; Huang, Yan-Jang S; Hsu, Wei-Wen; Hettenbach, Susan M; Higgs, Stephen; Vanlandingham, Dana L

    2016-08-01

    Serological diagnosis is a critical component for disease surveillance and is important to address the increase in incidence and disease burden of alphaviruses, such as the chikungunya (CHIKV) and Ross River (RRV) viruses. The gold standard for serological diagnosis is the plaque reduction neutralization test (PRNT), which demonstrates the neutralizing capacity of serum samples after the removal of complement activity and adventitious viruses. This procedure is normally performed following inactivation of the virus at 56°C for 30min. Although this protocol has been widely accepted for the inactivation of envelope RNA viruses, recent studies have demonstrated that prolonged heat inactivation is required to completely inactivate two alphaviruses, Western equine encephalitis virus and CHIKV. Incomplete inactivation of viruses poses a laboratory biosafety risk and can also lead to spurious test results. Despite its importance in ensuring the safety of laboratory personnel as well as test integrity, systematic investigation on the thermostability of alphaviruses has not been performed. In this study, the temperature tolerance and heat inactivation profiles of RRV, Barmah Forest, and o'nyong-nyong viruses were determined. Variations in thermostability were observed within the Semliki forest serocomplex. Therefore, evidence-based heat inactivation procedures for alphaviruses are recommended. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Blue cone monochromatism in a female due to skewed X-inactivation

    DEFF Research Database (Denmark)

    Frederiksen, Anja Lisbeth; Duno, Morten; Welinder, Lotte Gjesing

    2013-01-01

    Blue cone monochromatism (BCM) is a rare cone dystrophy with recessive X-linked inheritance and therefore diagnosed in males whereas females are clinically unaffected. We present a female with clinically manifested BCM. The diagnosis was genetically verified with the identification of one single...... red-green OPN1LW/MW hybrid gene harboring a point mutation c.607C>G, p.Cys203Arg that associates with BCM and in addition a completely biased X-inactivation in DNA isolated from full blood and buccal mucosa. The present case illustrates that females may develop symptoms of recessive X-linked eye...

  7. Relative Inactivation by Staphylococcus aureus of Eight Cephalosporin Antibiotics

    Science.gov (United States)

    Fong, Ignatius W.; Engelking, Elin R.; Kirby, William M. M.

    1976-01-01

    These studies extend the recent observation that cefazolin is inactivated to a greater extent than cephaloridine by some strains of penicillinase-producing Staphylococcus aureus, whereas cephalothin undergoes little if any inactivation. In Mueller-Hinton broth (inoculum, 3 × 106) 100 recently isolated strains had minimal inhibitory concentrations (MICs) ≤ 2 μg/ml for cephalothin and cephaloridine, whereas in Trypticase soy broth (TSB) 50% had MICs > 2 μg/ml and 10% (designated “resistant” strains) were >8 μg/ml for cephaloridine but remained ≤2 μg/ml for cephalothin. A large inoculum (3 × 107) of strains with high MICs in TSB almost completely inactivated 50 μg of cefazolin per ml in 6 h, with progressively less inactivation, in the following order, of cephaloridine, cephalexin, cephradine, cephapirin, and cefamandole; cefoxitin and cephalothin underwent little if any inactivation. The greater inactivation in TSB than in Mueller-Hinton broth appeared to be due to a greater production of β-lactamases by each colony-forming unit, since the inoculum size in the two broths was not significantly different. In contrast, “susceptible” strains (MICs ≤ 2 μg/ml in both broths) inactivated cephaloridine more than cefazolin, and equal amounts of powdered bacterial extracts confirmed the fact that qualitatively different β-lactamases were produced by the susceptible and resistant strains. Disk diffusion tests were unreliable in separating the two groups of staphylococci. The clinical significance of inactivation by strains with high MICs is not known but, unless susceptibility can be clearly established, cephalothin appears preferable for severe staphylococcal infections, since it undergoes little if any inactivation by any strains of staphylococci. PMID:938023

  8. ICA-105574 interacts with a common binding site to elicit opposite effects on inactivation gating of EAG and ERG potassium channels.

    Science.gov (United States)

    Garg, Vivek; Stary-Weinzinger, Anna; Sanguinetti, Michael C

    2013-04-01

    Rapid and voltage-dependent inactivation greatly attenuates outward currents in ether-a-go-go-related gene (ERG) K(+) channels. In contrast, inactivation of related ether-a-go-go (EAG) K(+) channels is very slow and minimally reduces outward currents. ICA-105574 (ICA, or 3-nitro-N-[4-phenoxyphenyl]-benzamide) has opposite effects on inactivation of these two channel types. Although ICA greatly attenuates ERG inactivation by shifting its voltage dependence to more positive potentials, it enhances the rate and extent of EAG inactivation without altering its voltage dependence. Here, we investigate whether the inverse functional response to ICA in EAG and ERG channels is related to differences in ICA binding site or to intrinsic mechanisms of inactivation. Molecular modeling coupled with site-directed mutagenesis suggests that ICA binds in a channel-specific orientation to a hydrophobic pocket bounded by the S5/pore helix/S6 of one subunit and S6 of an adjacent subunit. ICA is a mixed agonist of mutant EAG and EAG/ERG chimera channels that inactivate by a combination of slow and fast mechanisms. With the exception of three residues, the specific amino acids that form the putative binding pocket for ICA in ERG are conserved in EAG. Mutations introduced into EAG to replicate the ICA binding site in ERG did not alter the functional response to ICA. Together these findings suggest that ICA binds to the same site in EAG and ERG channels to elicit opposite functional effects. The resultant agonist or antagonist activity is determined solely by channel-specific differences in the mechanisms of inactivation gating.

  9. Thermoradiation inactivation of naturally occurring organisms in soil

    Science.gov (United States)

    Reynolds, M. C.; Lindell, K. F.; David, T. J.

    1973-01-01

    Samples of soil collected from Kennedy Space Center near spacecraft assembly facilities were found to contain microorganisms very resistant to conventional sterilization techniques. The inactivation behavior of the naturally occurring spores in soil was investigated using dry heat and ionizing radiation, first separately, then in combination. Dry heat inactivation rates of spores were determined for 105 and 125 C. Radiation inactivation rates were determined for dose rates of 660 and 76 krad/hr at 25 C. Simultaneous combinations of heat and radiation were then investigated at 105, 110, 115, 120, and 125 C. Combined treatment was found to be highly synergistic requiring greatly reduced radiation doses to accomplish sterilization.

  10. The pituitary-thyroid axis set point in women is uninfluenced by X chromosome inactivation pattern? A twin study

    DEFF Research Database (Denmark)

    Brix, Thomas H; Hansen, Pia S; Kyvik, Kirsten O

    2010-01-01

    The pituitary-thyroid axis (PTA) set point is determined by a combination of genetic and environmental factors. However, despite considerable efforts to characterize the background, the causative genes as well as environmental factors are not well established. Theoretically, as shown for autoimmune...... thyroid disease, the pattern of X chromosome inactivation (XCI) could offer a novel explanation for the observed variability of the PTA set point in women....

  11. Conditional ablation of Raptor in the male germline causes infertility due to meiotic arrest and impaired inactivation of sex chromosomes.

    Science.gov (United States)

    Xiong, Mengneng; Zhu, Zhiping; Tian, Suwen; Zhu, Ruping; Bai, Shun; Fu, Kaiqiang; Davis, James G; Sun, Zheng; Baur, Joseph A; Zheng, Ke; Ye, Lan

    2017-09-01

    Rapamycin is a clinically important drug that is used in transplantation and cancer therapy but which causes a number of side effects, including male infertility. Its canonical target, mammalian target of rapamycin complex 1 (mTORC1), plays a key role in metabolism and binds chromatin; however, its precise role in the male germline has not been elucidated. Here, we inactivate the core component, Raptor, to show that mTORC1 function is critical for male meiosis and the inactivation of sex chromosomes. Disruption of the Raptor gene impairs chromosomal synapsis and prevents the efficient spreading of silencing factors into the XY chromatin. Accordingly, mRNA for XY-linked genes remains inappropriately expressed in Raptor-deficient mice. Molecularly, the failure to suppress gene expression corresponded with deficiencies in 2 repressive chromatin markers, H3K9 dimethylation and H3K9 trimethylation, in the XY body. Together, these results demonstrate that mTORC1 has an essential role in the meiotic progression and silencing of sex chromosomes in the male germline, which may explain the infertility that has been associated with such inhibitors as rapamycin.-Xiong, M., Zhu, Z., Tian, S., Zhu, R., Bai, S., Fu, K., Davis, J. G., Sun, Z., Baur, J. A., Zheng, K., Ye, L. Conditional ablation of Raptor in the male germline causes infertility due to meiotic arrest and impaired inactivation of sex chromosomes. © FASEB.

  12. Effective immunotherapy of weakly immunogenic solid tumours using a combined immunogene therapy and regulatory T-cell inactivation.

    LENUS (Irish Health Repository)

    Whelan, M C

    2012-01-31

    Obstacles to effective immunotherapeutic anti-cancer approaches include poor immunogenicity of the tumour cells and the presence of tolerogenic mechanisms in the tumour microenvironment. We report an effective immune-based treatment of weakly immunogenic, growing solid tumours using a locally delivered immunogene therapy to promote development of immune effector responses in the tumour microenvironment and a systemic based T regulatory cell (Treg) inactivation strategy to potentiate these responses by elimination of tolerogenic or immune suppressor influences. As the JBS fibrosarcoma is weakly immunogenic and accumulates Treg in its microenvironment with progressive growth, we used this tumour model to test our combined immunotherapies. Plasmids encoding GM-CSF and B7-1 were electrically delivered into 100 mm(3) tumours; Treg inactivation was accomplished by systemic administration of anti-CD25 antibody (Ab). Using this approach, we found that complete elimination of tumours was achieved at a level of 60% by immunogene therapy, 25% for Treg inactivation and 90% for combined therapies. Moreover, we found that these responses were immune transferable, systemic, tumour specific and durable. Combined gene-based immune effector therapy and Treg inactivation represents an effective treatment for weakly antigenic solid growing tumours and that could be considered for clinical development.

  13. Aβ seeds resist inactivation by formaldehyde.

    Science.gov (United States)

    Fritschi, Sarah K; Cintron, Amarallys; Ye, Lan; Mahler, Jasmin; Bühler, Anika; Baumann, Frank; Neumann, Manuela; Nilsson, K Peter R; Hammarström, Per; Walker, Lary C; Jucker, Mathias

    2014-10-01

    Cerebral β-amyloidosis can be exogenously induced by the intracerebral injection of brain extracts containing aggregated β-amyloid (Aβ) into young, pre-depositing Aβ precursor protein- (APP) transgenic mice. Previous work has shown that the induction involves a prion-like seeding mechanism in which the seeding agent is aggregated Aβ itself. Here we report that the β-amyloid-inducing activity of Alzheimer's disease (AD) brain tissue or aged APP-transgenic mouse brain tissue is preserved, albeit with reduced efficacy, after formaldehyde fixation. Moreover, spectral analysis with amyloid conformation-sensitive luminescent conjugated oligothiophene dyes reveals that the strain-like properties of aggregated Aβ are maintained in fixed tissues. The resistance of Aβ seeds to inactivation and structural modification by formaldehyde underscores their remarkable durability, which in turn may contribute to their persistence and spread within the body. The present findings can be exploited to establish the relationship between the molecular structure of Aβ aggregates and the variable clinical features and disease progression of AD even in archived, formalin-fixed autopsy material.

  14. Inactivation of RNA viruses by gamma irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Nonomiya, Takashi; Morimoto, Akinori; Iwatsuki, Kazuo; Tsutsumi, Takamasa (Ministry of Agriculture, Forestry and fisheries, Yokohama, Kanagawa (Japan). Animal Quarantine Service); Ito, Hitoshi; Yamashiro, Tomio; Ishigaki, Isao

    1992-09-01

    Four kinds of RNA viruses, Bluetongue virus (BT), Bovine Virus Diarrhea-Mucosal Disease virus (BVD[center dot]MD), Bovine Respiratory Syncytial virus (RS), Vesicular Stmatitis virus (VS), were subjected to various doses of gamma irradiation to determine the lethal doses. The D[sub 10] values, which are the dose necessary to decimally reduce infectivity, ranged from 1.5 to 3.4 kGy under frozen condition at dry-ice temperature, and they increased to 2.6 to 5.0 kGy under frozen condition at dry-ice temperature. Serum neutralzing antibody titer of Infectious Bovine Rhinotracheitis (IBR) was not adversely changed by the exposure to 36 kGy of gamma-rays under frozen condition. Analysis of electrophoresis patterns of the bovine serum also reveales that the serum proteins were not remarkably affected, even when exposed to 36 kGy of gamma radiation under frozen condition. The results suggested that gamma irradiation under frozen condition is an effective means for inactivating both DNA and RNA viruses without adversely affecting serum proteins and neutralizing antibody titer. (author).

  15. CHLORINE INACTIVATION OF CATEGORY "A" BIO-TERRORISM AGENTS

    Science.gov (United States)

    This poster presents information on the inactivation of select bioterrorist agents. Information will be presented on chlorine disinfection of vegetative cells of Brucella suis, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis and endos...

  16. Use of genetic algorithms for high hydrostatic pressure inactivation ...

    African Journals Online (AJOL)

    Jane

    2011-10-24

    ) for high hydrostatic pressure (HHP) inactivation of Bacillus cereus spores, Bacillus subtilis spores and cells,. Staphylococcus aureus and Listeria monocytogenes, all in milk buffer, were used to demonstrate the utility of ...

  17. Inactivation Strategies for Clostridium perfringens Spores and Vegetative Cells.

    Science.gov (United States)

    Talukdar, Prabhat K; Udompijitkul, Pathima; Hossain, Ashfaque; Sarker, Mahfuzur R

    2017-01-01

    Clostridium perfringens is an important pathogen to human and animals and causes a wide array of diseases, including histotoxic and gastrointestinal illnesses. C. perfringens spores are crucial in terms of the pathogenicity of this bacterium because they can survive in a dormant state in the environment and return to being live bacteria when they come in contact with nutrients in food or the human body. Although the strategies to inactivate C. perfringens vegetative cells are effective, the inactivation of C. perfringens spores is still a great challenge. A number of studies have been conducted in the past decade or so toward developing efficient inactivation strategies for C. perfringens spores and vegetative cells, which include physical approaches and the use of chemical preservatives and naturally derived antimicrobial agents. In this review, different inactivation strategies applied to control C. perfringens cells and spores are summarized, and the potential limitations and challenges of these strategies are discussed. Copyright © 2016 American Society for Microbiology.

  18. Inactivation of rabies diagnostic reagents by gamma radiation

    Energy Technology Data Exchange (ETDEWEB)

    Gamble, W.C.; Chappell, W.A.; George, E.H.

    1980-11-01

    Treatment of CVS-11 rabies adsorbing suspensions and street rabies infected mouse brains with gamma radiation resulted in inactivated reagents that are safer to distribute and use. These irradiated reagents were as sensitive and reactive as the nonirradiated control reagents.

  19. Biocontrol interventions for inactivation of foodborne pathogens on produce

    Science.gov (United States)

    Post-harvest interventions for control of foodborne pathogens on minimally processed foods are crucial for food safety. Biocontrol interventions have the primary objective of developing novel antagonists in combinations with physical and chemical interventions to inactivate pathogenic microbes. Ther...

  20. Enzyme inactivation kinetics: Coupled effects of temperature and moisture content

    NARCIS (Netherlands)

    Perdana, J.A.; Fox, M.B.; Schutyser, M.A.I.; Boom, R.M.

    2012-01-01

    Enzymes are often dried for stability reasons and to facilitate handling. However, they are often susceptible to inactivation during drying. It is generally known that temperature and moisturecontent influence the enzymeinactivation kinetics. However, the coupledeffect of both variables on

  1. Enterococcus faecalis and pathogenic streptococci inactivate daptomycin by releasing phospholipids.

    Science.gov (United States)

    Ledger, Elizabeth V K; Pader, Vera; Edwards, Andrew M

    2017-10-01

    Daptomycin is a lipopeptide antibiotic with activity against Gram-positive bacteria. We showed previously that Staphylococcus aureus can survive daptomycin exposure by releasing membrane phospholipids that inactivate the antibiotic. To determine whether other pathogens possess this defence mechanism, phospholipid release and daptomycin activity were measured after incubation of Staphylococcus epidermidis, group A or B streptococci, Streptococcus gordonii or Enterococcus faecalis with the antibiotic. All bacteria released phospholipids in response to daptomycin, which resulted in at least partial inactivation of the antibiotic. However, E. faecalis showed the highest levels of lipid release and daptomycin inactivation. As shown previously for S. aureus, phospholipid release by E. faecalis was inhibited by the lipid biosynthesis inhibitor platensimycin. In conclusion, several pathogenic Gram-positive bacteria, including E. faecalis, inactivate daptomycin by releasing phospholipids, which may contribute to the failure of daptomycin to resolve infections caused by these pathogens.

  2. Evaluation of the Efficacy of Inactivated Oil-Emulsion Newcastle ...

    African Journals Online (AJOL)

    Evaluation of the Efficacy of Inactivated Oil-Emulsion Newcastle Disease Komarov Vaccine against Clinical Disease, Lesions and Immune Response, Following Challenge with Velogenic Newcastle Disease Virus in Laying Chickens.

  3. 21 CFR 610.11a - Inactivated influenza vaccine, general safety test.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Inactivated influenza vaccine, general safety test... Inactivated influenza vaccine, general safety test. For inactivated influenza vaccine, the general safety test... subcutaneous or intraperitoneal injection of 5.0 milliliters of inactivated influenza vaccine into each guinea...

  4. Shaker IR T449 mutants separate C- from U-type inactivation.

    Science.gov (United States)

    Jamieson, Quentin; Jones, Stephen W

    2014-04-01

    Previous studies demonstrated that slow inactivation of the Shaker potassium channel can be made ~100-fold faster or slower by point mutations at a site in the outer pore (T449). However, the discovery that two forms of slow inactivation coexist in Shaker raises the question of which inactivation process is affected by mutation. Equivalent mutations in K(V)2.1, a channel exhibiting only U-type inactivation, have minimal effects on inactivation, suggesting that mutation of Shaker T449 acts on C-type inactivation alone, a widely held yet untested hypothesis. This study reexamines mutations at Shaker T449, confirming that T449A speeds inactivation and T449Y/V slow it. T449Y and T449V exhibit U-type inactivation that is enhanced by high extracellular potassium, in contrast to C-type inactivation in T449A which is inhibited by high potassium. Automated parameter estimation for a 12-state Markov model suggests that U-type inactivation occurs mainly from closed states upon weak depolarization, but primarily from the open state at positive voltages. The model also suggests that WT channels, which in this study exhibit mostly C-type inactivation, recover from inactivation through closed-inactivated states, producing voltage-dependent recovery. This suggests that both C-type and U-type inactivation involve both open-inactivated and closed-inactivated states.

  5. Non-thermal plasma for inactivated-vaccine preparation.

    Science.gov (United States)

    Wang, Guomin; Zhu, Ruihao; Yang, Licong; Wang, Kaile; Zhang, Qian; Su, Xia; Yang, Bing; Zhang, Jue; Fang, Jing

    2016-02-17

    Vaccines are of great importance in controlling the spread of infectious diseases in poultry farming. The safety and efficacy of vaccines are also essential. To explore the feasibility of a novel technology (non-thermal plasma) in inactivated vaccine preparation, an alternating current atmospheric pressure non-thermal plasma (NTP) jet with Ar/O2/N2 as the operating gas was used to inactivate a Newcastle disease virus (NDV, LaSota) strain and H9N2 avian influenza virus (AIV, A/Chicken/Hebei/WD/98) for vaccine preparation. The results showed that complete inactivation could be achieved with 2 min of NTP treatment for both NDV and AIV. Moreover, a proper NTP treatment time is needed for inactivation of a virus without destruction of the antigenic determinants. Compared to traditional formaldehyde-inactivated vaccine, the vaccine made from NDV treated by NTP for 2 min (NTP-2 min-NDV-vaccine) could induce a higher NDV-specific antibody titer in specific pathogen-free (SPF) chickens, and the results of a chicken challenge experiment showed that NTP-2 min-NDV-vaccine could protect SPF chickens from a lethal NDV challenge. Vaccines made from AIV treated by NTP for 2 min (NTP-2 min-AIV-vaccine) also showed a similar AIV-specific antibody titer compared with traditional AIV vaccines prepared using formaldehyde inactivation. Studies of the morphological changes of the virus, chemical analysis of NDV allantoic fluid and optical emission spectrum analysis of NTP suggested that reactive oxygen species and reactive nitrogen species produced by NTP played an important role in the virus inactivation process. All of these results demonstrated that it could be feasible to use non-thermal NTP as an alternative strategy to prepare inactivated vaccines for Newcastle disease and avian influenza. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Stochastic and deterministic model of microbial heat inactivation.

    Science.gov (United States)

    Corradini, Maria G; Normand, Mark D; Peleg, Micha

    2010-03-01

    Microbial inactivation is described by a model based on the changing survival probabilities of individual cells or spores. It is presented in a stochastic and discrete form for small groups, and as a continuous deterministic model for larger populations. If the underlying mortality probability function remains constant throughout the treatment, the model generates first-order ("log-linear") inactivation kinetics. Otherwise, it produces survival patterns that include Weibullian ("power-law") with upward or downward concavity, tailing with a residual survival level, complete elimination, flat "shoulder" with linear or curvilinear continuation, and sigmoid curves. In both forms, the same algorithm or model equation applies to isothermal and dynamic heat treatments alike. Constructing the model does not require assuming a kinetic order or knowledge of the inactivation mechanism. The general features of its underlying mortality probability function can be deduced from the experimental survival curve's shape. Once identified, the function's coefficients, the survival parameters, can be estimated directly from the experimental survival ratios by regression. The model is testable in principle but matching the estimated mortality or inactivation probabilities with those of the actual cells or spores can be a technical challenge. The model is not intended to replace current models to calculate sterility. Its main value, apart from connecting the various inactivation patterns to underlying probabilities at the cellular level, might be in simulating the irregular survival patterns of small groups of cells and spores. In principle, it can also be used for nonthermal methods of microbial inactivation and their combination with heat.

  7. Pathogen Inactivation Technologies for Cellular Blood Components: an Update

    Science.gov (United States)

    Schlenke, Peter

    2014-01-01

    Summary Nowadays patients receiving blood components are exposed to much less transfusion-transmitted infectious diseases than three decades before when among others HIV was identified as causative agent for the acquired immunodeficiency syndrome and the transmission by blood or coagulation factors became evident. Since that time the implementation of measures for risk prevention and safety precaution was socially and politically accepted. Currently emerging pathogens like arboviruses and the well-known bacterial contamination of platelet concentrates still remain major concerns of blood safety with important clinical consequences, but very rarely with fatal outcome for the blood recipient. In contrast to the well-established pathogen inactivation strategies for fresh frozen plasma using the solvent-detergent procedure or methylene blue and visible light, the bench-to-bedside translation of novel pathogen inactivation technologies for cell-containing blood components such as platelets and red blood cells are still underway. This review summarizes the pharmacological/toxicological assessment and the inactivation efficacy against viruses, bacteria, and protozoa of each of the currently available pathogen inactivation technologies and highlights the impact of the results obtained from several randomized clinical trials and hemovigilance data. Until now in some European countries pathogen inactivation technologies are in in routine use for single-donor plasma and platelets. The invention and adaption of pathogen inactivation technologies for red blood cell units and whole blood donations suggest the universal applicability of these technologies and foster a paradigm shift in the manufacturing of safe blood. PMID:25254027

  8. Thermal inactivation kinetics of β-galactosidase during bread baking.

    Science.gov (United States)

    Zhang, Lu; Chen, Xiao Dong; Boom, Remko M; Schutyser, Maarten A I

    2017-06-15

    In this study, β-galactosidase was utilized as a model enzyme to investigate the mechanism of enzyme inactivation during bread baking. Thermal inactivation of β-galactosidase was investigated in a wheat flour/water system at varying temperature-moisture content combinations, and in bread during baking at 175 or 205°C. In the wheat flour/water system, the thermostability of β-galactosidase increased with decreased moisture content, and a kinetic model was accurately fitted to the corresponding inactivation data (R 2 =0.99). Interestingly, the residual enzyme activity in the bread crust (about 30%) was hundredfold higher than that in the crumb (about 0.3%) after baking, despite the higher temperature in the crust throughout baking. This result suggested that the reduced moisture content in the crust increased the thermostability of the enzyme. Subsequently, the kinetic model reasonably predicted the enzyme inactivation in the crumb using the same parameters derived from the wheat flour/water system. However, the model predicted a lower residual enzyme activity in the crust compared with the experimental result, which indicated that the structure of the crust may influence the enzyme inactivation mechanism during baking. The results reported can provide a quantitative understanding of the thermal inactivation kinetics of enzyme during baking, which is essential to better retain enzymatic activity in bakery products supplemented with heat-sensitive enzymes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Kinetic analysis of Legionella inactivation using ozone in wastewater.

    Science.gov (United States)

    Li, Jun; Li, Kunquan; Zhou, Yan; Li, Xuebin; Tao, Tao

    2017-02-01

    Legionella inactivation using ozone was studied in wastewater using kinetic analysis and modeling. The experimental results indicate that the relationship between the ozone concentration, germ concentration, and chemical oxygen demand (COD) can be used to predict variations in germ and COD concentrations. The ozone reaction with COD and inactivation of Legionella occurred simultaneously, but the reaction with COD likely occurred at a higher rate than the inactivation, as COD is more easily oxidized by ozone than Legionella. Higher initial COD concentrations resulted in a lower inactivation rate and higher lnN/N0. Higher temperature led to a higher inactivation efficiency. The relationship of the initial O3 concentration and Legionella inactivation rate was not linear, and thus, the Ct value required for a 99.99% reduction was not constant. The initial O3 concentration was more important than the contact time, and a reduction of the initial O3 concentration could not be compensated by increasing the contact time. The Ct values were compared over a narrow range of initial concentrations; the Ct values could only be contrasted when the initial O3 concentrations were very similar. A higher initial O3 concentration led to a higher inflection point value for the lnN/N0 vs C0t curve. Energy consumption using a plasma corona was lower than when using boron-doped diamond electrodes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Oxidation of multiple methionine residues impairs rapid sodium channel inactivation

    Science.gov (United States)

    Kassmann, Mario; Hansel, Alfred; Leipold, Enrico; Birkenbeil, Jan; Lu, Song-Qing; Hoshi, Toshinori; Heinemann, Stefan H.

    2010-01-01

    Reactive oxygen species (ROS) readily oxidize the sulfur-containing amino acids cysteine and methionine (Met). The impact of Met oxidation on the fast inactivation of the skeletal muscle sodium channel NaV1.4 expressed in human embryonic kidney cells was studied by applying the Met-preferring oxidant chloramine-T (ChT) or by irradiating the ROS-producing dye Lucifer Yellow in the patch pipettes. Both interventions dramatically slowed down inactivation of the sodium channels. Replacement of Met in the Ile-Phe-Met inactivation motif with Leu (M1305L) strongly attenuated the oxidizing effect on inactivation but did not eliminate it completely. Mutagenesis of conserved Met residues in the intracellular linkers connecting the membrane-spanning segments of the channel (M1469L and M1470L) also markedly diminished the oxidation sensitivity of the channel, while that of other conserved Met residues (442, 1139, 1154, 1316) were without any noticeable effect. The results of mutagenesis of results, assays of other NaV channel isoforms (NaV1.2, NaV1.5, NaV1.7) and the kinetics of the oxidation-induced removal of inactivation collectively indicate that multiple Met target residues need to be oxidized to completely impair inactivation. This arrangement using multiple Met residues confers a finely graded oxidative modulation of NaV channels and allows organisms to adapt to a variety of oxidative stress conditions, such as ischemic reperfusion. PMID:18369661

  11. Thermal Inactivation of Feline Calicivirus in Pet Food Processing.

    Science.gov (United States)

    Haines, J; Patel, M; Knight, A I; Corley, D; Gibson, G; Schaaf, J; Moulin, J; Zuber, S

    2015-12-01

    Extrusion is the most common manufacturing process used to produce heat-treated dry dog and cat food (pet food) for domestic use and international trade. Due to reoccurring outbreaks of notifiable terrestrial animal diseases and their impact on international trade, experiments were undertaken to demonstrate the effectiveness of heat-treated extruded pet food on virus inactivation. The impact of extrusion processing in a pet food matrix on virus inactivation has not been previously reported and very few inactivation studies have examined the thermal inactivation of viruses in complex food matrices. The feline calicivirus vaccine strain FCV F-9 was used as a surrogate model RNA virus pathogen. Small-scale heat inactivation experiments using animal-derived pet food raw materials showed that a > 4 log10 reduction (log10 R) in infectivity occurred at 70 °C prior to reaching the minimum extrusion manufacturing operating temperature of 100 °C. As anticipated, small-scale pressure studies at extrusion pressure (1.6 MPa) showed no apparent effect on FCV F-9 inactivation. Additionally, FCV F-9 was shown not to survive the acidic conditions used to produce pet food palatants of animal origin that are typically used as a coating after the extrusion process.

  12. High pressure inactivation of Brettanomyces bruxellensis in red wine.

    Science.gov (United States)

    van Wyk, Sanelle; Silva, Filipa V M

    2017-05-01

    Brettanomyces bruxellensis ("Brett") is a major spoilage concern for the wine industry worldwide, leading to undesirable sensory properties. Sulphur dioxide, is currently the preferred method for wine preservation. However, due to its negative effects on consumers, the use of new alternative non-thermal technologies are increasingly being investigated. The aim of this study was to determine and model the effect of high pressure processing (HPP) conditions and yeast strain on the inactivation of "Brett" in Cabernet Sauvignon wine. Processing at 200 MPa for 3 min resulted in 5.8 log reductions. However higher pressure is recommended to achieve high throughput in the wine industry, for example >6.0 log reductions were achieved after 400 MPa for 5 s. The inactivation of B. bruxellensis is pressure and time dependent, with increased treatment time and pressure leading to increased yeast inactivation. It was also found that yeast strain had a significant effect on HPP inactivation, with AWRI 1499 being the most resistant strain. The Weibull model successfully described the HPP "Brett" inactivation. HPP is a viable alternative for the inactivation of B. bruxellensis in wine, with the potential to reduce the industry's reliance on sulphur dioxide. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Viral inactivation in hemotherapy: systematic review on inactivators with action on nucleic acids

    Directory of Open Access Journals (Sweden)

    Patricia Marial Sobral

    2012-01-01

    Full Text Available The aim of this study was to conduct a systematic review on the photoinactivators used in hemotherapy, with action on viral genomes. The SciELO, Science Direct, PubMed and Lilacs databases were searched for articles. The inclusion criterion was that these should be articles on inactivators with action on genetic material that had been published between 2000 and 2010. The key words used in identifying such articles were "hemovigilance", "viral inactivation", "photodynamics", "chemoprevention" and "transfusion safety". Twenty-four articles on viral photoinactivation were found with the main photoinactivators covered being: methylene blue, amotosalen HCl, S-303 frangible anchor linker effector (FRALE, riboflavin and inactin. The results showed that methylene blue has currently been studied least, because it diminishes coagulation factors and fibrinogen. Riboflavin has been studied most because it is a photoinactivator of endogenous origin and has few collateral effects. Amotosalen HCl is effective for platelets and is also used on plasma, but may cause changes both to plasma and to platelets, although these are not significant for hemostasis. S-303 FRALE may lead to neoantigens in erythrocytes and is less indicated for red-cell treatment; in such cases, PEN 110 is recommended. Thus, none of the methods for pathogen reduction is effective for all classes of agents and for all blood components, but despite the high cost, these photoinactivators may diminish the risk of blood-transmitted diseases.

  14. A History of the Discovery of Random X Chromosome Inactivation in the Human Female and its Significance

    Directory of Open Access Journals (Sweden)

    Sophia Balderman

    2011-07-01

    Full Text Available Genetic determinants of sex in placental mammals developed by the evolution of primordial autosomes into the male and female sex chromosomes. The Y chromosome determines maleness by the action of the gene SRY, which encodes a protein that initiates a sequence of events prompting the embryonic gonads to develop into testes. The X chromosome in the absence of a Y chromosome results in a female by permitting the conversion of the embryonic gonads into ovaries. We trace the historical progress that resulted in the discovery that one X chromosome in the female is randomly inactivated in early embryogenesis, accomplishing approximate equivalency of X chromosome gene dosage in both sexes. This event results in half of the somatic cells in a tissue containing proteins encoded by the genes of the maternal X chromosome and half having proteins encoded by the genes of the paternal X chromosome, on average, accounting for the phenotype of a female heterozygote with an X chromosome mutation. The hypothesis of X chromosome inactivation as a random event early in embryogenesis was first described as a result of studies of variegated coat color in female mice. Similar results were found in women using the X chromosome-linked gene, glucose-6-phosphate dehydrogenase, studied in red cells. The random inactivation of the X chromosome-bearing genes for isoenzyme types A and B of glucose-6-phosphate dehydrogenase was used to establish the clonal origin of neoplasms in informative women with leiomyomas. Behind these discoveries are the stories of the men and women scientists whose research enlightened these aspects of X chromosome function and their implication for medicine.

  15. DNMT3AR882H mutant and Tet2 inactivation cooperate in the deregulation of DNA methylation control to induce lymphoid malignancies in mice

    DEFF Research Database (Denmark)

    Scourzic, L; Couronné, L; Pedersen, Marianne Terndrup

    2016-01-01

    in DNA methylation affecting tumor suppressor genes and local hypomethylation affecting genes involved in the Notch pathway. Our data confirm the transformation potential of DNMT3A(R882H) Tet2(-/-) progenitors and represent the first cooperative model in mice involving Tet2 inactivation driving lymphoid...

  16. Laser-induced inactivation of Plasmodium falciparum.

    Science.gov (United States)

    LeBlanc, Danielle; Story, Robert; Gross, Eitan

    2012-08-08

    Haemozoin crystals, produced by Plasmodium during its intra-erythrocytic asexual reproduction cycle, can generate UV light via the laser-induced, non-linear optical process of third harmonic generation (THG). In the current study the feasibility of using haemozoin, constitutively stored in the parasite's food vacuole, to kill the parasite by irradiation with a near IR laser was evaluated. Cultured Plasmodium parasites at different stages of development were irradiated with a pulsed NIR laser and the viability of parasites at each stage was evaluated from their corresponding growth curves using the continuous culture method. Additional testing for germicidal effects of haemozoin and NIR laser was performed by adding synthetic haemozoin crystals to Escherichia coli in suspension. Cell suspensions were then irradiated with the laser and small aliquots taken and spread on agar plates containing selective agents to determine cell viability (CFU). Parasites in the late-trophozoites form as well as trophozoites in early-stage of DNA synthesis were found to be the most sensitive to the treatment with -4-log reduction in viability after six passes through the laser beam; followed by parasites in ring phase (-2-log reduction). A -1-log reduction in E. coli viability was obtained following a 60 min irradiation regimen of the bacteria in the presence of 1 μM synthetic haemozoin and a -2-log reduction in the presence of 10 μM haemozoin. Minimal (≤ 15%) cell kill was observed in the presence of 10 μM haemin. Laser-induced third-harmonic generation by haemozoin can be used to inactivate Plasmodium. This result may have clinical implications for treating severe malaria symptoms by irradiating the patient's blood through the skin or through dialysis tubing with a NIR laser.

  17. Laser-induced inactivation of Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    LeBlanc Danielle

    2012-08-01

    Full Text Available Abstract Background Haemozoin crystals, produced by Plasmodium during its intra-erythrocytic asexual reproduction cycle, can generate UV light via the laser-induced, non-linear optical process of third harmonic generation (THG. In the current study the feasibility of using haemozoin, constitutively stored in the parasite’s food vacuole, to kill the parasite by irradiation with a near IR laser was evaluated. Methods Cultured Plasmodium parasites at different stages of development were irradiated with a pulsed NIR laser and the viability of parasites at each stage was evaluated from their corresponding growth curves using the continuous culture method. Additional testing for germicidal effects of haemozoin and NIR laser was performed by adding synthetic haemozoin crystals to Escherichia coli in suspension. Cell suspensions were then irradiated with the laser and small aliquots taken and spread on agar plates containing selective agents to determine cell viability (CFU. Results Parasites in the late-trophozoites form as well as trophozoites in early-stage of DNA synthesis were found to be the most sensitive to the treatment with ~4-log reduction in viability after six passes through the laser beam; followed by parasites in ring phase (~2-log reduction. A ~1-log reduction in E. coli viability was obtained following a 60 min irradiation regimen of the bacteria in the presence of 1 μM synthetic haemozoin and a ~2-log reduction in the presence of 10 μM haemozoin. Minimal (≤15% cell kill was observed in the presence of 10 μM haemin. Conclusions Laser-induced third-harmonic generation by haemozoin can be used to inactivate Plasmodium. This result may have clinical implications for treating severe malaria symptoms by irradiating the patient’s blood through the skin or through dialysis tubing with a NIR laser.

  18. Inactivation of a putative efflux pump (LmrB) in Streptococcus mutans results in altered biofilm structure and increased exopolysaccharide synthesis: implications for biofilm resistance.

    Science.gov (United States)

    Liu, Jia; Zhang, Jianying; Guo, Lihong; Zhao, Wei; Hu, Xiaoli; Wei, Xi

    2017-07-01

    Efflux pumps are a mechanism associated with biofilm formation and resistance. There is limited information regarding efflux pumps in Streptococcus mutans, a major pathogen in dental caries. The aim of this study was to investigate potential roles of a putative efflux pump (LmrB) in S. mutans biofilm formation and susceptibility. Upon lmrB inactivation and antimicrobial exposure, the biofilm structure and expression of other efflux pumps were examined using confocal laser scanning microscopy (CLSM) and qRT-PCR. lmrB inactivation resulted in biofilm structural changes, increased EPS formation and EPS-related gene transcription (p biofilms.

  19. Giardia, Entamoeba, and Trichomonas Enzymes Activate Metronidazole (Nitroreductases) and Inactivate Metronidazole (Nitroimidazole Reductases) ▿ †

    Science.gov (United States)

    Pal, Dibyarupa; Banerjee, Sulagna; Cui, Jike; Schwartz, Aaron; Ghosh, Sudip K.; Samuelson, John

    2009-01-01

    Infections with Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis, which cause diarrhea, dysentery, and vaginitis, respectively, are each treated with metronidazole. Here we show that Giardia, Entamoeba, and Trichomonas have oxygen-insensitive nitroreductase (ntr) genes which are homologous to those genes that have nonsense mutations in metronidazole-resistant Helicobacter pylori isolates. Entamoeba and Trichomonas also have nim genes which are homologous to those genes expressed in metronidazole-resistant Bacteroides fragilis isolates. Recombinant Giardia, Entamoeba, and Trichomonas nitroreductases used NADH rather than the NADPH used by Helicobacter, and two recombinant Entamoeba nitroreductases increased the metronidazole sensitivity of transformed Escherichia coli strains. Conversely, the recombinant nitroimidazole reductases (NIMs) of Entamoeba and Trichmonas conferred very strong metronidazole resistance to transformed bacteria. The Ehntr1 gene of the genome project HM-1:IMSS strain of Entamoeba histolytica had a nonsense mutation, and the same nonsense mutation was present in 3 of 22 clinical isolates of Entamoeba. While ntr and nim mRNAs were variably expressed by cultured Entamoeba and Trichomonas isolates, there was no relationship to metronidazole sensitivity. We conclude that microaerophilic protists have bacterium-like enzymes capable of activating metronidazole (nitroreductases) and inactivating metronidazole (NIMs). While Entamoeba and Trichomonas displayed some of the changes (nonsense mutations and gene overexpression) associated with metronidazole resistance in bacteria, these changes did not confer metronidazole resistance to the microaerophilic protists examined here. PMID:19015349

  20. Giardia, Entamoeba, and Trichomonas enzymes activate metronidazole (nitroreductases) and inactivate metronidazole (nitroimidazole reductases).

    Science.gov (United States)

    Pal, Dibyarupa; Banerjee, Sulagna; Cui, Jike; Schwartz, Aaron; Ghosh, Sudip K; Samuelson, John

    2009-02-01

    Infections with Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis, which cause diarrhea, dysentery, and vaginitis, respectively, are each treated with metronidazole. Here we show that Giardia, Entamoeba, and Trichomonas have oxygen-insensitive nitroreductase (ntr) genes which are homologous to those genes that have nonsense mutations in metronidazole-resistant Helicobacter pylori isolates. Entamoeba and Trichomonas also have nim genes which are homologous to those genes expressed in metronidazole-resistant Bacteroides fragilis isolates. Recombinant Giardia, Entamoeba, and Trichomonas nitroreductases used NADH rather than the NADPH used by Helicobacter, and two recombinant Entamoeba nitroreductases increased the metronidazole sensitivity of transformed Escherichia coli strains. Conversely, the recombinant nitroimidazole reductases (NIMs) of Entamoeba and Trichmonas conferred very strong metronidazole resistance to transformed bacteria. The Ehntr1 gene of the genome project HM-1:IMSS strain of Entamoeba histolytica had a nonsense mutation, and the same nonsense mutation was present in 3 of 22 clinical isolates of Entamoeba. While ntr and nim mRNAs were variably expressed by cultured Entamoeba and Trichomonas isolates, there was no relationship to metronidazole sensitivity. We conclude that microaerophilic protists have bacterium-like enzymes capable of activating metronidazole (nitroreductases) and inactivating metronidazole (NIMs). While Entamoeba and Trichomonas displayed some of the changes (nonsense mutations and gene overexpression) associated with metronidazole resistance in bacteria, these changes did not confer metronidazole resistance to the microaerophilic protists examined here.

  1. Green Tea Catechin-Inactivated Viral Vaccine Platform

    Directory of Open Access Journals (Sweden)

    Yun H. Lee

    2017-12-01

    Full Text Available Traditionally, chemical agents such as formalin (FA and β-propiolactone (BPL have long been used for the preparation of inactivated vaccines or toxoids. It has been shown that FA extensively modifies vaccine antigens and thus affects immunogenicity profiles, sometimes compromising the protective efficacy of the vaccines or even exacerbating the disease upon infection. In this study, we show that natural catechins from green tea extracts (GT can be used as an inactivating agent to prepare inactivated viral vaccines. GT treatment resulted in complete and irreversible inactivation of influenza virus as well as dengue virus. In contrast to FA that reacted extensively with multiple amino acids including lysine, a major anchor residue for epitope binding to MHC molecules, GT catechin epigallocatechin-3-gallate (EGCG crosslinked primarily with cysteine residues and thus preserved the major epitopes of the influenza hemagglutinin. In a mouse model, vaccination with GT-inactivated influenza virus (GTi virus elicited higher levels of viral neutralizing antibodies than FA-inactivated virus (FAi virus. The vaccination completely protected the mice from a lethal challenge and restricted the challenge viral replication in the lungs. Of note, the quality of antibody responses of GTi virus was superior to that with FAi virus, in terms of the magnitude of antibody titer, cross-reactivity to hetero-subtypes of influenza viruses, and the avidity to viral antigens. As the first report of using non-toxic natural compounds for the preparation of inactivated viral vaccines, the present results could be translated into a clinically relevant vaccine platform with improved efficacy, safety, productivity, and public acceptance.

  2. Infective and inactivated filamentous phage as carriers for immunogenic peptides.

    Science.gov (United States)

    Samoylova, Tatiana I; Norris, Mandy D; Samoylov, Alexandre M; Cochran, Anna M; Wolfe, Karen G; Petrenko, Valery A; Cox, Nancy R

    2012-07-01

    The focus of this study is on development of vaccines using filamentous phage as a delivery vector for immunogenic peptides. The use of phage as a carrier for immunogenic peptides provides significant benefits such as high immunogenicity, low production costs, and high stability of phage preparations. However, introduction of live recombinant phage into the environment might represent a potential ecological problem. This, for example, may occur when vaccines are used in oral or nasal formulations in field conditions for wild and feral animals. To address this issue, comparative studies of antigenic properties of live and inactivated (non-viable) phage were accomplished. Inactivated phage, if released, will not propagate and will degrade as any other protein. In these experiments, a model phage clone that was previously selected from a phage display library and shown to stimulate production of anti-sperm antibodies with contraceptive properties was used. Multiple methods of phage inactivation were tested, including drying, freezing, autoclaving, heating, and UV irradiation. Under studied conditions, heating at 76°C for 3h, UV irradiation, and autoclaving resulted in complete phage inactivation. Phage samples treated by heat and UV were characterized by spectrophotometry and electron microscopy. To test antigenicity, live and inactivated phage preparations were injected into mice and antibody responses assayed by ELISA. It was found that phage killed by heat causes little to no immune responses, probably due to destruction of phage particles. In contrast, UV-inactivated phage stimulated production of IgG serum antibodies at the levels comparable to live phage. Thus, vaccines formulated to include UV-inactivated filamentous phage might represent environmentally safe alternatives to live phage vaccines. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Distinct Mechanisms of Phenotypic Effects of Inactivation and Prionization of Swi1 Protein in Saccharomyces cerevisiae.

    Science.gov (United States)

    Antonets, K S; Kliver, S F; Polev, D E; Shuvalova, A R; Andreeva, E A; Inge-Vechtomov, S G; Nizhnikov, A A

    2017-10-01

    Prions are proteins that under the same conditions can exist in two or more conformations, and at least one of the conformations has infectious properties. The prionization of a protein is typically accompanied by its functional inactivation due to sequestration of monomers by the prion aggregates. The most of prions has been identified in the yeast Saccharomyces cerevisiae. One of them is [SWI + ], a prion isoform of the Swi1 protein, which is a component of the evolutionarily conserved chromatin remodeling complex SWI/SNF. Earlier, it was shown that the prionization of [SWI + ] induces a nonsense suppression, which leads to weak growth of the [SWI + ] strains containing mutant variants of the SUP35 gene and the nonsense allele ade1-14 UGA on selective medium without adenine. This effect occurs because of [SWI + ] induction that causes a decrease in the amount of the SUP45 mRNA. Strains carrying the SWI1 deletion exhibit significantly higher suppression of the ade1-14 UGA nonsense mutation than the [SWI + ] strains. In the present study, we identified genes whose expression is altered in the background of the SWI1 deletion using RNA sequencing. We found that the ade1-14 UGA suppression in the swi1Δ strains is caused by an increase in the expression of this mutant allele of the ADE1 gene. At the same time, the SUP45 expression level in the swi1Δ strains does not significantly differ from the expression level of this gene in the [swi - ] strains. Thus, we have shown that the phenotypic effects of Swi1 prionization and deletion are mediated by different molecular mechanisms. Based on these data, we have concluded that the prionization of proteins is not only unequal to their inactivation, but also can lead to the acquisition of novel phenotypic effects and functions.

  4. Inactivation of p15INK4b in chronic arsenic poisoning cases

    Directory of Open Access Journals (Sweden)

    Aihua Zhang

    2014-01-01

    Full Text Available Arsenic exposure from burning high arsenic-containing coal has been associated with human skin lesion and cancer. However, the mechanisms of arsenic-related carcinogenesis are not fully understood. Inactivation of critical tumor suppression genes by epigenetic regulation or genetic modification might contribute to arsenic-induced carcinogenicity. This study aims to clarify the correlation between arsenic pollution and functional defect of p15INK4b gene in arsenic exposure residents from a region of Guizhou Province, China. To this end, 103 arsenic exposure residents and 105 control subjects were recruited in this study. The results showed that the exposure group exhibited higher levels of urinary and hair arsenic compared with the control group (55.28 vs 28.87 μg/L, 5.16 vs 1.36 μg/g. Subjects with higher arsenic concentrations are more likely to have p15INK4b methylation and gene deletion (χ2 = 4.28, P = 0.04 and χ2 = 4.31, P = 0.04. We also found that the degree of p15INK4b hypermethylation and gene deletion occurred at higher incidence in the poisoning cases with skin cancer (3.7% and 14.81% in non-skin cancer group, 41.18% and 47.06 in skin cancer group, and were significantly associated with the stage of skin lesions (χ2 = 12.82, P < 0.01 and χ2 = 7.835, P = 0.005. These observations indicate that inactivation of p15INK4b through genetic alteration or epigenetic modification is a common event that is associated with arsenic exposure and the development of arsenicosis.

  5. Immunogenicity and protective effects of inactivated Singapore grouper iridovirus (SGIV) vaccines in orange-spotted grouper, Epinephelus coioides.

    Science.gov (United States)

    Ou-yang, Zhengliang; Wang, Peiran; Huang, Xiaohong; Cai, Jia; Huang, Youhua; Wei, Shina; Ji, Huasong; Wei, Jingguang; Zhou, Yongcan; Qin, Qiwei

    2012-10-01

    Vaccination is one of the best methods against viral diseases. In this study, experimental inactivated Singapore grouper iridovirus (SGIV) vaccines were prepared, and immunogenicity and protection against virus infection of the vaccines were investigated in orange-spotted grouper, Epinephelus coioides. Two kinds of vaccines, including β-propiolactone (BPL) inactivated virus at 4°C for 12 h and formalin inactivated virus at 4°C for 12 d, was highly protective against the challenge at 30-day post-vaccination and produced relative percent of survival rates of 91.7% and 100%, respectively. These effective vaccinations induced potent innate immune responses mediated by pro-inflammatory cytokines and type I interferon (IFN)-stimulated genes (ISGs). It is noteworthy that ISGs, such as Mx and ISG15, were up-regulated only in the effective vaccine groups, which suggested that type I IFN system may be the functional basis of early anti-viral immunity. Moreover, effective vaccination also significantly up-regulated of the expression of MHC class I gene and produced substantial amount of specific serum antibody at 4 weeks post-vaccination. Taken together, our results clearly demonstrated that effective vaccination in grouper induced an early, nonspecific antiviral immunity, and later, a specific immune response involving both humoral and cell-mediated immunity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Human male meiotic sex chromosome inactivation.

    NARCIS (Netherlands)

    Vries, M. de; Vosters, S.; Merkx, G.F.M.; Hauwers, K.W.M. d'; Wansink, D.G.; Ramos, L.; Boer, P. de

    2012-01-01

    In mammalian male gametogenesis the sex chromosomes are distinctive in both gene activity and epigenetic strategy. At first meiotic prophase the heteromorphic X and Y chromosomes are placed in a separate chromatin domain called the XY body. In this process, X,Y chromatin becomes highly

  7. ERADIKASI POLIO DAN IPV (INACTIVATED POLIO VACCINE

    Directory of Open Access Journals (Sweden)

    Gendrowahyuhono Gendrowahyuhono

    2012-09-01

    Full Text Available In the year 1988, World Health Organization (WHO claims that polio viruses should be eradicated after year 2000. However, until year 2010 the world have not been free from polio viruses circulation. So many effort had been achieved and it is estimated that the world will be free from polio virus after the year 2013. Control of poliomyelitis in Indonesia has been commenced since 1982 with routine immunization of polio program and the National Immunization Days (NID has been commenced since 1995,1996,2005 and 2006. When the world is free from polio virus, WHO suggests several alternative effort to maintain the world free from polio viruses : I stop the OPV (Oral Polio Vaccine and no polio immunization, 2 stop OPV and stock pile mOPV (monovalent OPV, 3 use OPV and IPV (Inactivated Polio Vaccine in a certain times, 4 use IPV only in a certain times. IPV has been used routinely in develop countries but has not been used in the developing countries. Several studies in development countries has been conducted, but had not been done in the developing countries. Indonesia collaboration with WHO has conducted the study of IPV in Yogyakarta Province since year 2002 until year 2010. The overall aim of the study is to compile the necessary data that will inform global and national decision-making regarding future polio immunization policies for the OPV cessation era. The data generated from the study will be particularly important to make decisions regarding optimal IPV use in developing tropical countries. It is unlikely that this data can be assembled through other means than through this study. The tentative result of the study shows that OPV immunization coverage in the year 2004 is 99% in four district and 93 % in the Yogyakarta city. Environment surveillance shows that there are 65.7% polio virus detected from 137 sewage samples pre IPV swich, and 4.8% polio virus detected from 83 sewage samples post IPV swich. Survey polio antibody serologis shows

  8. Inactivation of viruses in labile blood derivatives. II. Physical methods

    Energy Technology Data Exchange (ETDEWEB)

    Horowitz, B.; Wiebe, M.E.; Lippin, A.; Vandersande, J.; Stryker, M.H.

    1985-11-01

    The thermal inactivation of viruses in labile blood derivatives was evaluated by addition of marker viruses (VSV, Sindbis, Sendai, EMC) to anti-hemophilic factor (AHF) concentrates. The rate of virus inactivation at 60 degrees C was decreased by at least 100- to 700-fold by inclusion of 2.75 M glycine and 50 percent sucrose, or 3.0 M potassium citrate, additives which contribute to retention of protein biologic activity. Nonetheless, at least 10(4) infectious units of each virus was inactivated within 10 hours. Increasing the temperature from 60 to 70 or 80 degrees C caused a 90 percent or greater loss in AHF activity. An even greater decline in the rate of virus inactivation was observed on heating AHF in the lyophilized state, although no loss in AHF activity was observed after 72 hours of heating at 60 degrees C. Several of the proteins present in lyophilized AHF concentrates displayed an altered electrophoretic mobility as a result of exposure to 60 degrees C for 24 hours. Exposure of lyophilized AHF to irradiation from a cobalt 60 source resulted in an acceptable yield of AHF at 1.0, but not at 2.0, megarads. At 1 megarad, greater than or equal to 6.0 logs of VSV and 3.3 logs of Sindbis virus were inactivated.

  9. Effect of heat on virus inactivation by ammonia.

    Science.gov (United States)

    Burge, W D; Cramer, W N; Kawata, K

    1983-08-01

    The rate of inactivation of bacteriophage f2 and poliovirus 1 (CHAT) by NH3 was strongly influenced by temperature. The process was pseudo-first order at all temperatures and NH3 concentrations. Poliovirus was inactivated at a greater rate than f2, but the change in the rate of inactivation with increasing temperature in the range of approximately 10 to 40 degrees C was greater for f2 than for poliovirus. At higher temperatures, the rate of change was greater for poliovirus. Arrhenius plots of the data were biphasic, indicating that two inactivation processes were occurring, one for the low temperature range and another for the high temperature range. However, the magnitudes of the thermodynamic variables for f2 were low enough, as calculated for the low (10 to 35 degrees C) and high (35 to 60 degrees C) phases, that inactivation could have occurred by breakage of nucleic acid chains. For poliovirus, the sizes indicated possible involvement of nucleic acid at the low temperatures (10 to 40 degrees C) but some unknown mechanism for the high temperatures (40 and 50 degrees C).

  10. Efficacy of chlorine dioxide tablets on inactivation of cryptosporidium oocysts.

    Science.gov (United States)

    Murphy, Jennifer L; Haas, Charles N; Arrowood, Michael J; Hlavsa, Michele C; Beach, Michael J; Hill, Vincent R

    2014-05-20

    The ability of chlorine dioxide (ClO2) to achieve 2-log inactivation of Cryptosporidium in drinking water has been documented. No studies have specifically addressed the effects of ClO2 on C. parvum oocyst infectivity in chlorinated recreational water venues (e.g., pools). The aim of this research was to determine the efficacy of ClO2 as an alternative to existing hyperchlorination protocols that are used to achieve a 3-log inactivation of Cryptosporidium in such venues. To obtain a 3-log inactivation of C. parvum Iowa oocysts, contact times of 105 and 128 min for a solution containing 5 mg/L ClO2 with and without the addition of 2.6 mg/L free chlorine, respectively, were required. Contact times of 294 and 857 min for a solution containing 1.4 mg/L ClO2 with and without the addition of 3.6 mg/L free chlorine, respectively, were required. The hyperchlorination control (21 mg/L free chlorine only) required 455 min for a 3-log inactivation. Use of a solution containing 5 mg/L ClO2 and solutions containing 5 or 1.4 mg/L ClO2 with the addition of free chlorine appears to be a promising alternative to hyperchlorination for inactivating Cryptosporidium in chlorinated recreational water venues, but further studies are required to evaluate safety constraints on use.

  11. Thermal inactivation of the wine spoilage yeasts Dekkera/Brettanomyces.

    Science.gov (United States)

    Couto, José António; Neves, Filipe; Campos, Francisco; Hogg, Tim

    2005-10-25

    The heat resistance of three strains of Dekkera/Brettanomyces (Dekkera anomala PYCC 5,153, Dekkera bruxellensis PYCC 4,801 and Dekkera/Brettanomyces 093) was evaluated at different temperatures between 32.5 and 55 degrees C. Thermal inactivation tests were performed in tartrate buffer solution (pH 4.0) and in wines. In the studies employing buffer as the heating menstruum, measurable thermal inactivation began only at temperatures of 50 degrees C. When heating was performed in wine, significant inactivation begins at 35 degrees C. Subsequent thermal inactivation tests were performed in buffer at various levels of pH, ethanol concentration, and various phenolic acids. Results from experiments in buffer with added ethanol suggest that the greater heat sensitivity shown in wines can be largely attributed to ethanol, although potentiation of this effect might be due to the phenolic content, particularly from ferulic acid. In the range of pH values tested (2.5-4.5), this factor had no influence in the heat inactivation kinetics. Relevant data, in the form of D and Z values calculated in the various environments, potentially useful for the establishment of regimes of thermal control of Dekkera/Brettanomyces yeasts in wine and contaminated equipment is presented.

  12. Inactivation of citrus tristeza virus by gamma ray irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Ieki, Hiroyuki; Yamaguchi, Akira

    1984-12-01

    The total exposure of gamma ray and the intensity of gamma ray per hour for the inactivation of citrus tristeza virus (CTV) and also the effect on citrus tissues are described. The budwoods of Morita navel orange infected with a severe seedling-yellow strain of CTV were irradiated with gamma ray from a /sup 60/Co source for 20 - 52 hours. The buds or small tissue pieces of the irradiated budwoods were subsequently grafted onto Mexcan lime. CTV was easily inactivated by the irradiation from 10 to 18 kR for from 20 to 52 hours. The higher the total exposure, the higher the rate of inactivation. The CTV in the budwoods was almost inactivated after the irradiation with 20 kR. When the total exposure to gamma ray on budwoods was the same, CTV was more efficiently inactivated by the irradiation for long period with low intensity of gamma ray per hour than that for short period with high intensity per hour. Gamma ray irradiation was effective to eliminate CTV from citrus tissues. (Mori, K.).

  13. Comparative innocuity and efficacy of live and inactivated sheeppox vaccines.

    Science.gov (United States)

    Boumart, Zineb; Daouam, Samira; Belkourati, Imane; Rafi, Lamya; Tuppurainen, Eeva; Tadlaoui, Khalid Omari; El Harrak, Mehdi

    2016-06-29

    Sheeppox (SPP) is one of the priorities, high-impact animal diseases in many developing countries, where live attenuated vaccines are routinely used against sheeppox virus (SPPV). In an event of an SPP outbreak, historically disease-free countries would hesitate to use of live vaccines against SPPVdue to the safety and trade reasons. Currently no killed SPPV vaccines are commercially available. In this study, we developed an inactivated Romanian SPPVvaccine and assessed its efficacy and potency in comparison with a live attenuated Romanian SPPV vaccine. Four naïve sheep were vaccinated once with the Romanian SPPV live attenuated vaccine and16 sheep were vaccinated twice with the inactivated vaccine. All sheep in the live vaccine group were included in the challenge trial, which was conducted using a highly virulent Moroccan SPPV field strain. Eight sheep of the inactivated vaccine group were challenged and the remaining sheep were monitored for seroconversion. Experimental animals were closely monitored for the appearance of clinical signs, body temperature and inflammation at the injection site. Two naïve sheep were used as unvaccinated controls. The inactivated Romanian SPPV vaccine was found to be safe and confer a good protection, similar to the live vaccine. Specific antibodies appeared from seven days post vaccination and remained up to nine months. This study showed that the developed inactivated Romanian SPPV vaccine has a potential to replace attenuated vaccine to control and prevent sheep pox in disease-free or endemic countries.

  14. Raman spectroscopy-compatible inactivation method for pathogenic endospores.

    Science.gov (United States)

    Stöckel, S; Schumacher, W; Meisel, S; Elschner, M; Rösch, P; Popp, J

    2010-05-01

    Micro-Raman spectroscopy is a fast and sensitive tool for the detection, classification, and identification of biological organisms. The vibrational spectrum inherently serves as a fingerprint of the biochemical composition of each bacterium and thus makes identification at the species level, or even the subspecies level, possible. Therefore, microorganisms in areas susceptible to bacterial contamination, e.g., clinical environments or food-processing technology, can be sensed. Within the scope of point-of-care-testing also, detection of intentionally released biosafety level 3 (BSL-3) agents, such as Bacillus anthracis endospores, or their products is attainable. However, no Raman spectroscopy-compatible inactivation method for the notoriously resistant Bacillus endospores has been elaborated so far. In this work we present an inactivation protocol for endospores that permits, on the one hand, sufficient microbial inactivation and, on the other hand, the recording of Raman spectroscopic signatures of single endospores, making species-specific identification by means of highly sophisticated chemometrical methods possible. Several physical and chemical inactivation methods were assessed, and eventually treatment with 20% formaldehyde proved to be superior to the other methods in terms of sporicidal capacity and information conservation in the Raman spectra. The latter fact has been verified by successfully using self-learning machines (such as support vector machines or artificial neural networks) to identify inactivated B. anthracis-related endospores with adequate accuracies within the range of the limited model database employed.

  15. Inactivation of complement by Loxosceles reclusa spider venom.

    Science.gov (United States)

    Gebel, H M; Finke, J H; Elgert, K D; Cambell, B J; Barrett, J T

    1979-07-01

    Zymosan depletion of serum complement in guinea pigs rendered them highly resistant to lesion by Loxosceles reclusa spider venom. Guinea pigs deficient in C4 of the complement system are as sensitive to the venom as normal guinea pigs. The injection of 35 micrograms of whole recluse venom intradermally into guinea pigs lowered their complement level by 35.7%. Brown recluse spider venom in concentrations as slight as 0.02 micrograms protein/ml can totally inactivate one CH50 of guinea pig complement in vitro. Bee, scorpion, and other spider venoms had no influence on the hemolytic titer of complement. Fractionation of recluse spider venom by Sephadex G-200 filtration separated the complement-inactivating property of the venom into three major regions which could be distinguished on the basis of heat stability as well as size. None was neutralized by antivenom. Polyacrylamide gel electrophoresis of venom resolved the complement inactivators into five fractions. Complement inactivated by whole venom or the Sephadex fractions could be restored to hemolytic activity by supplements of fresh serum but not by heat-inactivated serum, pure C3, pure C5, or C3 and C5 in combination.

  16. Thermal and high pressure inactivation kinetics of blueberry peroxidase.

    Science.gov (United States)

    Terefe, Netsanet Shiferaw; Delon, Antoine; Versteeg, Cornelis

    2017-10-01

    This study for the first time investigated the stability and inactivation kinetics of blueberry peroxidase in model systems (McIlvaine buffer, pH=3.6, the typical pH of blueberry juice) during thermal (40-80°C) and combined high pressure-thermal processing (0.1-690MPa, 30-90°C). At 70-80°C, the thermal inactivation kinetics was best described by a biphasic model with ∼61% labile and ∼39% stable fractions at temperature between 70 and 75°C. High pressure inhibited the inactivation of the enzyme with no inactivation at pressures as high as 690MPa and temperatures less than 50°C. The inactivation kinetics of the enzyme at 60-70°C, and pressures higher than 500MPa was best described by a first order biphasic model with ∼25% labile fraction and 75% stable fraction. The activation energy values at atmospheric pressure were 548.6kJ/mol and 324.5kJ/mol respectively for the stable and the labile fractions. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

  17. Neurofibromin C terminus-specific antibody (clone NFC) is a valuable tool for the identification of NF1-inactivated GISTs.

    Science.gov (United States)

    Rossi, Sabrina; Gasparotto, Daniela; Cacciatore, Matilde; Sbaraglia, Marta; Mondello, Alessia; Polano, Maurizio; Mandolesi, Alessandra; Gronchi, Alessandro; Reuss, David E; von Deimling, Andreas; Maestro, Roberta; Dei Tos, Angelo Paolo

    2017-09-01

    An increasing body of evidence supports the involvement of NF1 mutations, constitutional or somatic, in the pathogenesis of gastrointestinal stromal tumors (GISTs). Due to the large size of the NF1 locus, the existence of multiple pseudogenes and the wide spectrum of mechanisms of gene inactivation, the analysis of NF1 gene status is still challenging for most laboratories. Here we sought to assess the efficacy of a recently developed neurofibromin-specific antibody (NFC) in detecting NF1-inactivated GISTs. NFC reactivity was analyzed in a series of 98 GISTs. Of these, 29 were 'NF1-associated' (17 with ascertained NF1 mutations and 12 arising in the context of clinically diagnosed Neurofibromatosis type 1 syndrome and thus considered bona fine NF1 inactivated); 38 were 'NF1-unrelated' (either wild-type or carrying non-pathogenic variants of NF1). Thirty-one additional GISTs with no available information on NF1 gene status or with NF1 gene variants of uncertain pathogenic significance were also included in the analysis. Cases were scored as NFC negative when, in the presence of NFC positive internal controls, no cytoplasmic staining was detected in the neoplastic cells. NFC immunoreactivity was lost in 24/29 (83%) NF1-associated GISTs as opposed to only 2/38 (5%) NF1-unrelated GISTs (P=3e-11). NFC staining loss significantly correlated (P=0.007) with the presence of biallelic NF1 inactivation, due essentially to large deletions or truncating mutations. NFC reactivity was instead retained in two cases in which the NF1 alteration was heterozygous and in one case where the pathogenic NF1 variant, although homo/hemizygous, was a missense mutation predicted not to affect neurofibromin half-life. Overall this study provides evidence that NFC is a valuable tool for identifying NF1-inactivated GISTs, thus serving as a surrogate for molecular analysis.Modern Pathology advance online publication, 1 September 2017; doi:10.1038/modpathol.2017.105.

  18. Co-Inactivation of GlnR and CodY Regulators Impacts Pneumococcal Cell Wall Physiology.

    Directory of Open Access Journals (Sweden)

    Calum Johnston

    Full Text Available CodY, a nutritional regulator highly conserved in low G+C Gram-positive bacteria, is essential in Streptococcus pneumoniae (the pneumococcus. A published codY mutant possessed suppressing mutations inactivating the fatC and amiC genes, respectively belonging to iron (Fat/Fec and oligopeptide (Ami ABC permease operons, which are directly repressed by CodY. Here we analyzed two additional published codY mutants to further explore the essentiality of CodY. We show that one, in which the regulator of glutamine/glutamate metabolism glnR had been inactivated by design, had only a suppressor in fecE (a gene in the fat/fec operon, while the other possessed both fecE and amiC mutations. Independent isolation of three different fat/fec suppressors thus establishes that reduction of iron import is crucial for survival without CodY. We refer to these as primary suppressors, while inactivation of ami, which is not essential for survival of codY mutants and acquired after initial fat/fec inactivation, can be regarded as a secondary suppressor. The availability of codY- ami+ cells allowed us to establish that CodY activates competence for genetic transformation indirectly, presumably by repressing ami which is known to antagonize competence. The glnR codY fecE mutant was then found to be only partially viable on solid medium and hypersensitive to peptidoglycan (PG targeting agents such as the antibiotic cefotaxime and the muramidase lysozyme. While analysis of PG and teichoic acid composition uncovered no alteration in the glnR codY fecE mutant compared to wildtype, electron microscopy revealed altered ultrastructure of the cell wall in the mutant, establishing that co-inactivation of GlnR and CodY regulators impacts pneumococcal cell wall physiology. In light of rising levels of resistance to PG-targeting antibiotics of natural pneumococcal isolates, GlnR and CodY constitute potential alternative therapeutic targets to combat this debilitating pathogen, as co-inactivation

  19. 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 ...

  20. 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

  1. Inactivation of Bacillus anthracis in water by photocatalytic, photolytic and sonochemical treatment.

    Science.gov (United States)

    Venieri, Danae; Markogiannaki, Evangelia; Chatzisymeon, Efthalia; Diamadopoulos, Evan; Mantzavinos, Dionissios

    2013-04-01

    Bacillus anthracis is one of the most dangerous and pathogenic bacterial species and its intrusion in aquatic environments is a serious threat to public health. The aim of the present study was to investigate inactivation rates of B. anthracis in water by means of photocatalytic (UVA/TiO2), photolytic (UVC) and sonochemical treatment. The effect of various operating conditions such as bacterial concentration, TiO2 loading, UV irradiation source, ultrasound power and treatment time was examined. The reference strain of B. anthracis proved to be highly resistant during photocatalytic and sonochemical treatment of aquatic samples, even in the presence of hydrogen peroxide solution, which is considered among the chemical disinfectants recommended for B. anthracis removal from aqueous suspensions. UVC irradiation was far more effective, as it achieved total inactivation in short treatment time (10 min) and at high initial concentrations (10(6) CFU mL(-1)). The effectiveness of UVC irradiation is also reinforced by the fact that no photoreactivation occurred even after 72 h of exposure under sunlight after the end of the treatment. Furthermore, the virulence of residual cells was investigated, targeting two genes carried in the plasmids pXO1 and pXO2, respectively, which are required for a fully virulent type. Interestingly, the plasmid pXO2 seems to be lost from the host after photocatalytic and photolytic disinfection, resulting in apathogenic residual strains contained in the treated sample. Overall, our results highlight the importance of B. anthracis efficient inactivation in water, as it shows considerable resistance towards effective and reliable disinfection techniques.

  2. Pokeweed antiviral protein inactivates pokeweed ribosomes; implications for the antiviral mechanism.

    Science.gov (United States)

    Bonness, M S; Ready, M P; Irvin, J D; Mabry, T J

    1994-02-01

    Pokeweed antiviral protein (PAP) and other ribosome-inactivating proteins (RIPs) had previously been thought to be incapable of attacking conspecific ribosomes, thus having no effect on endogenous processes. This assertion conflicts with a model for PAP's in vivo antiviral mechanism in which PAP (a cell wall protein) selectively enters virus-infected cells and disrupts protein synthesis, thus causing local suicide and preventing virus replication. We show here that pokeweed (Phytolacca americana) ribosomes, as well as endod (Phytolacca dodecandra) ribosomes, are indeed highly sensitive to inactivation by conspecific RIPs. Ribosomes isolated from RIP-free pokeweed and endod suspension culture cells were found to be highly active in vitro, as measured by poly(U)-directed polyphenylalanine synthesis. Phytolacca ribosomes challenged with conspecific RIPs generated dose-response curves (IC50 of 1 nM PAP or dodecandrin) very similar to those from wheat germ ribosomes. To determine if Phytolacca cells produce a cytosolic 'anti-RIP' protective element, ribosomes were combined with Phytolacca postribosomal supernatant factors from culture cells, then challenged with conspecific RIPs. Resulting IC50 values of 3-7 nM PAP, PAP-II, PAP-S or dodecandrin indicate that supernatants from these Phytolacca cells lack a ribosomal protective element. This research demonstrates that PAP inactivates pokeweed ribosomes (and is therefore potentially toxic to pokeweed cells) and supports the local suicide model for PAP's in vivo antiviral mechanism. The importance of spatial separation between PAP and ribosomes of cells producing this RIP is emphasized, particularly if crop plants are transformed with the PAP gene to confer antiviral protection.

  3. Inactivation of Heterosigma akashiwo in ballast water by circular orifice plate-generated hydrodynamic cavitation.

    Science.gov (United States)

    Feng, Daolun; Zhao, Jie; Liu, Tian

    2016-01-01

    The discharge of alien ballast water is a well-known, major reason for marine species invasion. Here, circular orifice plate-generated hydrodynamic cavitation was used to inactivate Heterosigma akashiwo in ballast water. In comparison with single- and multihole orifice plates, the conical-hole orifice plate yielded the highest inactivation percentage, 51.12%, and consumed only 6.84% energy (based on a 50% inactivation percentage). Repeating treatment, either using double series-connection or circling inactivation, elevated the inactivation percentage, yet consumed much more energy. The results indicate that conical-hole-generated hydrodynamic cavitation shows great potential as a pre-inactivation method for ballast water treatment.

  4. Microbial inactivation by microwave radiation in the home environment.

    Science.gov (United States)

    Park, Dong-Kyoo; Bitton, Gabriel; Melker, Richard

    2006-12-01

    The study reported here looked at the survival of microorganisms (heterotrophic plate counts, total coliforms, E. coli, and bacterial spores) in a consumer-type microwave oven. Kitchen sponges, scrubbing pads, and syringes were experimentally contaminated with wastewater and subsequently exposed to microwave radiation. At 100 percent power level, it was found that the heterotrophic plate count (i.e., total bacterial count) of the wastewater was reduced by more that 99 percent within 1 to 2 minutes, and the total coliform and E. coli were totally inactivated after 30 seconds of microwave radiation. Bacterial phage MS2 was totally inactivated within 1 to 2 minutes. Spores of Bacillus cereus were more resistant than the other microorganisms tested, and were completely eradicated only after 4-minute irradiation. Similar inactivation rates were obtained in wastewater-contaminated scrubbing pads. Microorganisms attached to plastic syringes were more resistant to microwave irradiation than those associated with kitchen sponges or scrubbing pads. It took 10 minutes for total inactivation of the heterotrophic plate count and 4 minutes for total inactivation of total coliform and E. coli. A 4-log reduction of phage MS2 was obtained after 2 minutes; 97.4 percent reduction was observed after 12 minutes. The authors also observed a higher inactivation of B. cereus spores in syringes placed in a ceramic container than of spores in syringes placed in a glass container. This finding could have some implications for the design of containers to be used in exposure of medical devices to microwave radiation. The article discusses the implications of these findings for consumer safety in the home environment.

  5. Assessing the efficacy of an inactivated chicken anemia virus vaccine.

    Science.gov (United States)

    Zhang, Xinheng; Wu, Boliang; Liu, Yuanjia; Chen, Weiguo; Dai, Zhenkai; Bi, Yingzuo; Xie, Qingmei

    2015-04-15

    Chicken anemia virus (CAV) is an immunosuppressive virus that causes chicken infectious anemia (CIA) which is a highly contagious avian disease. CAV causes major economic losses in the poultry industry worldwide. The current CAV vaccine is a live attenuated strain administered in the drinking water that risks horizontal infection of other chickens. The purpose of this study was to develop a novel vaccine against CAV that can be administered safely using a highly pathogenic isolate inactivated with β-propiolactone hydrolysis that would protect chicks from CAV. Hens were vaccinated twice intramuscularly with a novel CAV GD-G-12 inactivated vaccine and the humoral immune responses of the hens and offspring were monitored by ELISA. A heterologous intramuscular challenge using the CAV strain GD-E-12 was conducted in the chicks hatched from vaccinated or unvaccinated hens. The vaccine strain, GD-G-12, was shown to be highly pathogenic prior to inactivation evidenced by thymic atrophy and bleeding, and weight loss. The inactivated vaccine was considered safe and showed no signs of pathogenicity. High titers of CAV specific antibodies were detected in the vaccinated hens and in their chicks, indicating vertical transfer of maternal antibodies. Furthermore, the chicks hatched from vaccinated hens were resistant to a heterologous CAV challenge and showed no signs of weight loss and thymic atrophy and bleeding. Our studies are proof of principle that inactivated GD-G-12 might be a novel vaccine candidate to prevent CAV infection, and highlight the utility of using an inactivated virus for this vaccine. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Inactivation of VHSV by Percolation and Salt Under Experimental Conditions

    DEFF Research Database (Denmark)

    Skall, Helle Frank; Olesen, Niels Jørgen; Jørgensen, Claus

    2012-01-01

    At the moment the only legal method in Denmark to sanitize wastewater from fish cutting plants is by percolation. To evaluate the inactivation effect of percolation on VHSV an experimental examination was initiated. A column packed with gravel as top- and bottom layer (total of 22 cm) and a mid...... method to sanitize VHSV infected water. Changes in temperature, pH, earth types in the area used for percolation etc. may change the virus reduction, though. As some of the fish cutting plants are also smoking rainbow trout fillets, the question arose whether a brine solution will inactivate VHSV...

  7. Inactivation of VHSV by infiltration and salt under experimental conditions

    DEFF Research Database (Denmark)

    Skall, Helle Frank; Jørgensen, Claus; Olesen, Niels Jørgen

    2014-01-01

    At the moment the only legal method in Denmark to sanitize wastewater from fish cutting plants is by infiltration. To evaluate the inactivation effect of infiltration on VHSV an experimental examination was initiated. A column packed with gravel as top- and bottom layer (total of 22 cm) and a mid...... be a valuable method to sanitize VHSV infected water. Changes in temperature, pH, earth types in the area used for infiltration etc. may change the virus reduction, though. As some of the fish cutting plants are also smoking rainbow trout fillets, the question arose whether a brine solution will inactivate VHSV...

  8. Inactivation of Herpes Simplex Viruses by Nonionic Surfactants

    Science.gov (United States)

    Asculai, Samuel S.; Weis, Margaret T.; Rancourt, Martha W.; Kupferberg, A. B.

    1978-01-01

    Nonionic surface-active agents possessing ether or amide linkages between the hydrophillic and hydrophobic portions of the molecule rapidly inactivated the infectivity of herpes simplex viruses. The activity stemmed from the ability of nonionic surfactants to dissolve lipid-containing membranes. This was confirmed by observing surfactant destruction of mammalian cell plasma membranes and herpes simplex virus envelopes. Proprietary vaginal contraceptive formulations containing nonionic surfactants also inactivated herpes simplex virus infectivity. This observation suggests that nonionic surfactants in appropriate formulation could effectively prevent herpes simplex virus transmission. Images PMID:208460

  9. An insight on bacterial cellular targets of photodynamic inactivation.

    Science.gov (United States)

    Alves, Eliana; Faustino, Maria Af; Neves, Maria Gpms; Cunha, Angela; Tome, Joao; Almeida, Adelaide

    2014-02-01

    The emergence of microbial resistance is becoming a global problem in clinical and environmental areas. As such, the development of drugs with novel modes of action will be vital to meet the threats created by the rise in microbial resistance. Microbial photodynamic inactivation is receiving considerable attention for its potentialities as a new antimicrobial treatment. This review addresses the interactions between photosensitizers and bacterial cells (binding site and cellular localization), the ultrastructural, morphological and functional changes observed at initial stages and during the course of photodynamic inactivation, the oxidative alterations in specific molecular targets, and a possible development of resistance.

  10. Epigenetic inactivation and aberrant transcription of CSMD1 in squamous cell carcinoma cell lines

    Directory of Open Access Journals (Sweden)

    Scholnick Steven B

    2005-09-01

    Full Text Available Abstract Background The p23.2 region of human chromosome 8 is frequently deleted in several types of epithelial cancer and those deletions appear to be associated with poor prognosis. Cub and Sushi Multiple Domains 1 (CSMD1 was positionally cloned as a candidate for the 8p23 suppressor but point mutations in this gene are rare relative to the frequency of allelic loss. In an effort to identify alternative mechanisms of inactivation, we have characterized CSMD1 expression and epigenetic modifications in head and neck squamous cell carcinoma cell lines. Results Only one of the 20 cell lines examined appears to express a structurally normal CSMD1 transcript. The rest express transcripts which either lack internal exons, terminate abnormally or initiate at cryptic promoters. None of these truncated transcripts is predicted to encode a functional CSMD1 protein. Cell lines that express little or no CSMD1 RNA exhibit DNA methylation of a specific region of the CpG island surrounding CSMD1's first exon. Conclusion Correlating methylation patterns and expression suggests that it is modification of the genomic DNA preceding the first exon that is associated with gene silencing and that methylation of CpG dinucleotides further 3' does not contribute to inactivation of the gene. Taken together, the cell line data suggest that epigenetic silencing and aberrant splicing rather than point mutations may be contributing to the reduction in CSMD1 expression in squamous cancers. These mechanisms can now serve as a focus for further analysis of primary squamous cancers.

  11. Dopamine inactivation efficacy related to functional DAT1 and COMT variants influences motor response evaluation.

    Directory of Open Access Journals (Sweden)

    Stephan Bender

    Full Text Available BACKGROUND: Dopamine plays an important role in orienting, response anticipation and movement evaluation. Thus, we examined the influence of functional variants related to dopamine inactivation in the dopamine transporter (DAT1 and catechol-O-methyltransferase genes (COMT on the time-course of motor processing in a contingent negative variation (CNV task. METHODS: 64-channel EEG recordings were obtained from 195 healthy adolescents of a community-based sample during a continuous performance task (A-X version. Early and late CNV as well as motor postimperative negative variation were assessed. Adolescents were genotyped for the COMT Val(158Met and two DAT1 polymorphisms (variable number tandem repeats in the 3'-untranslated region and in intron 8. RESULTS: The results revealed a significant interaction between COMT and DAT1, indicating that COMT exerted stronger effects on lateralized motor post-processing (centro-parietal motor postimperative negative variation in homozygous carriers of a DAT1 haplotype increasing DAT1 expression. Source analysis showed that the time interval 500-1000 ms after the motor response was specifically affected in contrast to preceding movement anticipation and programming stages, which were not altered. CONCLUSIONS: Motor slow negative waves allow the genomic imaging of dopamine inactivation effects on cortical motor post-processing during response evaluation. This is the first report to point towards epistatic effects in the motor system during response evaluation, i.e. during the post-processing of an already executed movement rather than during movement programming.

  12. Dopamine Inactivation Efficacy Related to Functional DAT1 and COMT Variants Influences Motor Response Evaluation

    Science.gov (United States)

    Bender, Stephan; Rellum, Thomas; Freitag, Christine; Resch, Franz; Rietschel, Marcella; Treutlein, Jens; Jennen-Steinmetz, Christine; Brandeis, Daniel; Banaschewski, Tobias; Laucht, Manfred

    2012-01-01

    Background Dopamine plays an important role in orienting, response anticipation and movement evaluation. Thus, we examined the influence of functional variants related to dopamine inactivation in the dopamine transporter (DAT1) and catechol-O-methyltransferase genes (COMT) on the time-course of motor processing in a contingent negative variation (CNV) task. Methods 64-channel EEG recordings were obtained from 195 healthy adolescents of a community-based sample during a continuous performance task (A-X version). Early and late CNV as well as motor postimperative negative variation were assessed. Adolescents were genotyped for the COMT Val158Met and two DAT1 polymorphisms (variable number tandem repeats in the 3′-untranslated region and in intron 8). Results The results revealed a significant interaction between COMT and DAT1, indicating that COMT exerted stronger effects on lateralized motor post-processing (centro-parietal motor postimperative negative variation) in homozygous carriers of a DAT1 haplotype increasing DAT1 expression. Source analysis showed that the time interval 500–1000 ms after the motor response was specifically affected in contrast to preceding movement anticipation and programming stages, which were not altered. Conclusions Motor slow negative waves allow the genomic imaging of dopamine inactivation effects on cortical motor post-processing during response evaluation. This is the first report to point towards epistatic effects in the motor system during response evaluation, i.e. during the post-processing of an already executed movement rather than during movement programming. PMID:22649558

  13. Tumor Suppressor Inactivation in the Pathogenesis of Adult T-Cell Leukemia

    Directory of Open Access Journals (Sweden)

    Christophe Nicot

    2015-01-01

    Full Text Available Tumor suppressor functions are essential to control cellular proliferation, to activate the apoptosis or senescence pathway to eliminate unwanted cells, to link DNA damage signals to cell cycle arrest checkpoints, to activate appropriate DNA repair pathways, and to prevent the loss of adhesion to inhibit initiation of metastases. Therefore, tumor suppressor genes are indispensable to maintaining genetic and genomic integrity. Consequently, inactivation of tumor suppressors by somatic mutations or epigenetic mechanisms is frequently associated with tumor initiation and development. In contrast, reactivation of tumor suppressor functions can effectively reverse the transformed phenotype and lead to cell cycle arrest or death of cancerous cells and be used as a therapeutic strategy. Adult T-cell leukemia/lymphoma (ATLL is an aggressive lymphoproliferative disease associated with infection of CD4 T cells by the Human T-cell Leukemia Virus Type 1 (HTLV-I. HTLV-I-associated T-cell transformation is the result of a multistep oncogenic process in which the virus initially induces chronic T-cell proliferation and alters cellular pathways resulting in the accumulation of genetic defects and the deregulated growth of virally infected cells. This review will focus on the current knowledge of the genetic and epigenetic mechanisms regulating the inactivation of tumor suppressors in the pathogenesis of HTLV-I.

  14. Virion-targeted viral inactivation: new therapy against viral infection.

    Science.gov (United States)

    Okui, N; Kitamura, Y; Kobayashi, N; Sakuma, R; Ishikawa, T; Kitamura, T

    2001-01-01

    scAb, scAbE-Vpr, or scAbE-WXXF, as determined by virion-associated reverse transcriptase assay and by immunoblot analysis, respectively. Because G418-selected HeLa clones carrying the expression plasmid for scAbE-WXXF were obtained much more frequently than those for scAbE-Vpr, scAbE-WXXF was inferred to be less toxic to cells than scAbE-Vpr. The result that scAbE-WXXF with viral incorporation achieved more than a 10-fold reduction in infectivity of the progeny virions than scAb without incorporation suggests that scAbE-WXXF is a potential antiviral molecule, inhibiting replication by neutralization of HIV IN activity both within cells and within virions. Moreover, it is nontoxic to human cells. We termed this gene therapy "virion-"targeted-viral inactivation" and these molecules "packageable antiviral therapeutics." This new gene therapy has the potential for wide application in many viral infectious diseases.

  15. One-step of tryptophan attenuator inactivation and promoter swapping to improve the production of L-tryptophan in Escherichia coli

    Science.gov (United States)

    2012-01-01

    Background L-tryptophan is an aromatic amino acid widely used in the food, chemical and pharmaceutical industries. In Escherichia coli, L-tryptophan is synthesized from phosphoenolpyruvate and erythrose 4-phosphate by enzymes in the shikimate pathway and L-tryptophan branch pathway, while L-serine and phosphoribosylpyrophosphate are also involved in L-tryptophan synthesis. In order to construct a microbial strain for efficient L-tryptophan production from glucose, we developed a one step tryptophan attenuator inactivation and promoter swapping strategy for metabolic flux optimization after a base strain was obtained by overexpressing the tktA, mutated trpE and aroG genes and inactivating a series of competitive steps. Results The engineered E. coli GPT1002 with tryptophan attenuator inactivation and tryptophan operon promoter substitution exhibited 1.67 ~ 9.29 times higher transcription of tryptophan operon genes than the control GPT1001. In addition, this strain accumulated 1.70 g l-1 L-tryptophan after 36 h batch cultivation in 300-mL shake flask. Bioreactor fermentation experiments showed that GPT1002 could produce 10.15 g l-1 L-tryptophan in 48 h. Conclusions The one step inactivating and promoter swapping is an efficient method for metabolic engineering. This method can also be applied in other bacteria. PMID:22380540

  16. RNA-directed gene editing specifically eradicates latent and prevents new HIV-1 infection

    National Research Council Canada - National Science Library

    Wenhui Hu; Rafal Kaminski; Fan Yang; Yonggang Zhang; Laura Cosentino; Fang Li; Biao Luo; David Alvarez-Carbonell; Yoelvis Garcia-Mesa; Jonathan Karn; Xianming Mo; Kamel Khalili

    2014-01-01

    .... We identified highly specific targets within the HIV-1 LTR U3 region that were efficiently edited by Cas9/gRNA, inactivating viral gene expression and replication in latently infected microglial...

  17. Influenza (flu) vaccine (Inactivated or Recombinant): What you need to know

    Science.gov (United States)

    ... taken in its entirety from the CDC Inactivated Influenza Vaccine Information Statement (VIS) www.cdc.gov/vaccines/hcp/vis/vis-statements/flu.html CDC review information for Inactivated Influenza VIS: ...

  18. High pressure processing's potential to inactivate norovirus and other fooodborne viruses

    Science.gov (United States)

    High pressure processing (HPP) can inactivate human norovirus. However, all viruses are not equally susceptible to HPP. Pressure treatment parameters such as required pressure levels, initial pressurization temperatures, and pressurization times substantially affect inactivation. How food matrix ...

  19. X inactivation in Rett syndrome: A preliminary study showing partial preferential inactivation of paternal X with the M27{beta} probe

    Energy Technology Data Exchange (ETDEWEB)

    Camus, P.; Abbadi, N.; Gilgenkrantz, S. [Laboratoire de Genetique, Vandoeuvre les Nancy (France)

    1994-04-15

    Rett syndrome (RS) is a severe progressive neurological disorder occurring exclusively in females. Most cases are sporadic. The few familial cases (less than 1%) cannot be explained by a simple mode of inheritance. Several hypotheses have been proposed: X-linked male lethal mutation, maternal uniparental disomy, fresh mutation on the X chromosome, involvement of mitochondrial DNA and differential inactivation with metabolic interference of X-borne alleles. The authors have examined the pattern of X inactivation in 10 affected girls who were selected according to the clinical criteria previously described and accepted by the French Rett Scientific Committee. The X inactivation pattern was studied by analysis of methylation at the hypervariable locus DXS255 with the M27{beta} probe. The results show a more-or-less skewed inactivation of paternal X in 8 Rett females, and 2 cases of symmetrical inactivation. In control girls, inactivation was symmetrical cases and the maternal X has been preferentially inactivated in the other 2 cases. In no case was a total skewed inactivation observed. Though there was clear evidence for a preferential paternal X inactivation that was statistically significant further studies are necessary to establish a relationship between X inactivation pattern and Rett syndrome.

  20. Inactivation of Ascaris suum by short-chain fatty acids

    Science.gov (United States)

    Ascaris suum eggs were inactivated in distilled water and digested sludge by butanoic, pentanoic and hexanoic acids. The fatty acids (FA) were only effective when protonated and at sufficient concentration. The conjugate bases were not effective at the concentrations evaluated. Predictions from an ...

  1. Testing household disinfectants for the inactivation of helminth eggs ...

    African Journals Online (AJOL)

    2016-10-04

    Oct 4, 2016 ... Keywords: Ascaris, carbolic acid, disinfectant, eggs, inactivation, pit latrine, sanitation, sodium hypochlorite. INTRODUCTION. The lack of ... ronment providing temperature (25°C) and humidity (> 55%) are optimal. ...... The pH of the stomach is strongly acidic, but pH of the gas- trointestinal tract is in the ...

  2. 40 CFR 141.720 - Inactivation toolbox components.

    Science.gov (United States)

    2010-07-01

    ... Cryptosporidium, Giardia lamblia, and virus treatment credits for ultraviolet (UV) light reactors by achieving the... unfiltered systems. UV Dose Table for Cryptosporidium, Giardia lamblia, and Virus Inactivation Credit Log credit Cryptosporidium UV dose (mJ/cm2) Giardia lamblia UV dose (mJ/cm2) VirusUV dose (mJ/cm2) (i) 0.5 1...

  3. Inactivation of human and simian rotaviruses by chlorine dioxide.

    Science.gov (United States)

    Chen, Y S; Vaughn, J M

    1990-01-01

    The inactivation of single-particle stocks of human (type 2, Wa) and simian (SA-11) rotaviruses by chlorine dioxide was investigated. Experiments were conducted at 4 degrees C in a standard phosphate-carbonate buffer. Both virus types were rapidly inactivated, within 20 s under alkaline conditions, when chlorine dioxide concentrations ranging from 0.05 to 0.2 mg/liter were used. Similar reductions of 10(5)-fold in infectivity required additional exposure time of 120 s at 0.2 mg/liter for Wa and at 0.5 mg/liter for SA-11, respectively, at pH 6.0. The inactivation of both virus types was moderate at neutral pH, and the sensitivities to chlorine dioxide were similar. The observed enhancement of virucidal efficiency with increasing pH was contrary to earlier findings with chlorine- and ozone-treated rotavirus particles, where efficiencies decreased with increasing alkalinity. Comparison of 99.9% virus inactivation times revealed ozone to be the most effective virucidal agent among these three disinfectants. PMID:2160222

  4. Application of electrolysis to inactivation of antibacterials in clinical use.

    Science.gov (United States)

    Nakano, Takashi; Hirose, Jun; Kobayashi, Toyohide; Hiro, Naoki; Kondo, Fumitake; Tamai, Hiroshi; Tanaka, Kazuhiko; Sano, Kouichi

    2013-04-01

    Contamination of surface water by antibacterial pharmaceuticals (antibacterials) from clinical settings may affect aquatic organisms, plants growth, and environmental floral bacteria. One of the methods to decrease the contamination is inactivation of antibacterials before being discharged to the sewage system. Recently, we reported the novel method based on electrolysis for detoxifying wastewater containing antineoplastics. In the present study, to clarify whether the electrolysis method is applicable to the inactivation of antibacterials, we electrolyzed solutions of 10 groups of individual antibacterials including amikacin sulfate (AMK) and a mixture (MIX) of some commercial antibacterials commonly prescribed at hospitals, and measured their antibacterial activities. AMK was inactivated in its antibacterial activities and its concentration decreased by electrolysis in a time-dependent manner. Eighty to ninety-nine percent of almost all antibacterials and MIX were inactivated within 6h of electrolysis. Additionally, cytotoxicity was not detected in any of the electrolyzed solutions of antibacterials and MIX by the Molt-4-based cytotoxicity test. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Expression of a ribosome inactivating protein (curcin 2) in Jatropha ...

    Indian Academy of Sciences (India)

    Expression of a ribosome inactivating protein (curcin 2) in Jatropha curcas is induced by stress ... In addition, the 32 kDa band is nearly the molecular weight of curcin 2. ... curcin 2. The presence of this protein molecular marker under stresses may provide an experimental foundation to study the stress proteins in J. curcas.

  6. Significance of cobalamin inactivation in normal and malignant hematopoiesis

    NARCIS (Netherlands)

    A.A.M. Ermens (Anton)

    1990-01-01

    textabstractThis thesis deals with several aspects of the effects of cobalamin inactivation by nitrous oxide on cellular folate metabolism and the consequences on normal and malignant hematopoiesis. Kroes et al. demonstrated the antileukemic effects of nitrous oxide either or not in combination

  7. Inactivation of pathogens on pork by steam-ultrasound treatment

    DEFF Research Database (Denmark)

    Morild, Rikke K; Christiansen, Pia; Sørensen, Anders Morten Hay

    2011-01-01

    The objective of the study was to evaluate a new pathogen inactivation concept that combines application of pressurized steam simultaneously with high-power ultrasound through a series of nozzles. On skin and meat surfaces of pork jowl samples, counts of total viable bacteria were reduced by 1...

  8. Inactivation of bacteria in sewage sludge by gamma radiation.

    Science.gov (United States)

    Pandya, G A; Kapila, S; Kelkar, V B; Negi, S; Modi, V V

    1987-01-01

    The survival of certain bacterial cultures suspended in sewage sludge and exposed to gamma-radiation was studied. The inactivation patterns of most of the organisms were significantly different when irradiation was performed using sewage samples collected in the summer and monsoon seasons. The summer sample collected from the anaerobic digestor afforded significant protection to both Gram negative and Gram positive organisms. This was evident by the increase in dose required to bring about a 6 log cycle reduction in viable count of the bacterial cultures, when suspended in sewage samples instead of phosphate buffer. The observations made using monsoon digestor samples were quite different. This sewage sludge greatly enhanced inactivation by gamma-radiation in most cases. The effects of certain chemicals on the inactivation patterns of two organisms-Salmonella typhi and Shigella flexneri-were examined. Arsenate, mercury and lead salts sensitised S. typhi, while barium acetate and sodium sulphide protected this culture against gamma-radiation. In the case of Sh. flexneri, barium acetate and iodacetamide proved to be radioprotectors. The effects of some chemicals on the inactivation pattern of Sh. flexneri cells irradiated in sludge are also discussed.

  9. Use of genetic algorithms for high hydrostatic pressure inactivation ...

    African Journals Online (AJOL)

    Use of genetic algorithms for high hydrostatic pressure inactivation of microorganisms. ... Depending on the properties of HHP equipment (maximum operating pressure) or the type of the food product (heat-sensitive), it could be possible to select the suitable P-T-t trio among the alternatives. This study reveals that GAs could ...

  10. Inactivation of enveloped virus by laser-driven protein aggregation

    Science.gov (United States)

    Tsen, Shaw-Wei D.; Chapa, Travis; Beatty, Wandy; Tsen, Kong-Thon; Yu, Dong; Achilefu, Samuel

    2012-12-01

    Ultrafast lasers in the visible and near-infrared range have emerged as a potential new method for pathogen reduction of blood products and pharmaceuticals. However, the mechanism of enveloped virus inactivation by this method is unknown. We report the inactivation as well as the molecular and structural effects caused by visible (425 nm) femtosecond laser irradiation on murine cytomegalovirus (MCMV), an enveloped, double-stranded DNA virus. Our results show that laser irradiation (1) caused a 5-log reduction in MCMV titer, (2) did not cause significant changes to the global structure of MCMV virions including membrane and capsid, as assessed by electron microscopy, (3) produced no evidence of double-strand breaks or crosslinking in MCMV genomic DNA, and (4) caused selective aggregation of viral capsid and tegument proteins. We propose a model in which ultrafast laser irradiation induces partial unfolding of viral proteins by disrupting hydrogen bonds and/or hydrophobic interactions, leading to aggregation of closely associated viral proteins and inactivation of the virus. These results provide new insight into the inactivation of enveloped viruses by visible femtosecond lasers at the molecular level, and help pave the way for the development of a new ultrafast laser technology for pathogen reduction.

  11. Inactivation of a transgene due to transposition of insertion ...

    Indian Academy of Sciences (India)

    Agrobacterium strains harbour insertion sequences, which are known to transpose into genomes as well as into Ti plasmids. In this study we report the inactivation of a transgene due to transposition of the A. tumefaciens insertion sequence IS136. The transposition was discovered following transformation of plant tissues, ...

  12. Photoactivated polycationic bioactive chitosan nanoparticles inactivate bacterial endotoxins.

    Science.gov (United States)

    Shrestha, Annie; Cordova, Martha; Kishen, Anil

    2015-05-01

    The current root canal disinfection protocols fail to markedly inactivate bacterial endotoxins from infected root dentin. This study aimed to evaluate the ability of antibacterial photodynamic therapy with chitosan-conjugated rose bengal nanoparticles (CSRBnps) to selectively inactivate endotoxins/lipopolysaccharides (LPSs). Antimicrobial agents such as calcium hydroxide (Ca[OH]2), chitosan nanoparticles (CSnps), CSRBnps, and methylene blue (MB) were assessed for their ability to neutralize LPSs obtained from Pseudomonas aeruginosa in a time-dependent interaction with/without photoactivation (20 and 40 J/cm(2)). The inflammatory potential of the treated/untreated LPSs was assessed on macrophage cells (RAW 267.4) using nitric oxide- and enzyme-linked immunosorbent assay (tumor necrosis factor α and interleukin-6 expression)-based analysis. These antimicrobials were tested directly on macrophage cells for cytotoxicity using the mitochondrial activity assay and light microscopy. The data were analyzed using 1-way analysis of variance and the Tukey test. CSnps were least effective in LPS inactivation. Interluekin-6 expression was reduced only with CSRBnp treatment. CSnps and CSRBnps were completely nontoxic, and MB showed slight toxicity to macrophage cells. Ca(OH)2 was highly cytotoxic (P endotoxins and the subsequent reduction of all tested inflammatory markers from activated macrophages. Antimicrobial CSRBnps in combination with photodynamic therapy showed the potential to effectively inactivate bacterial endotoxins. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  13. Inactivation of dairy manure-borne pathogens by anaerobic digestion

    Science.gov (United States)

    Background: Anaerobic digestion of animal manure has the potential to inactivate enteric pathogens, thereby reducing exposures to livestock and humans when the products of digestion are disposed by land-spreading or irrigation or returned to livestock uses such as bedding. Data on digester effectiv...

  14. Photodynamic inactivation of fibroblasts by a cationic porphyrin

    NARCIS (Netherlands)

    Lambrechts, Saskia A. G.; Schwartz, Kevin R.; Aalders, Maurice C. G.; Dankert, Jacob B.

    2005-01-01

    An important determinant of the clinical applicability and value of antimicrobial photodynamic inactivation (PDI) is the cytotoxicity of the treatment to human cells. We evaluated the in vitro cytotoxicity of PDI to human dermal fibroblasts using 5-phenyl-10,15,20-tris(N-methyl-4-pyridyl)porphyrin

  15. Drying of liquid food droplets : enzyme inactivation and multicomponent diffusion

    NARCIS (Netherlands)

    Meerdink, G.

    1993-01-01

    In this thesis the drying of liquid food droplets is studied from three different points of view: drying kinetics, enzyme inactivation and multicomponent diffusion. Mathematical models are developed and validated experimentally.

    Drying experiments are performed with suspended

  16. Evaluation of the Efficacy of Inactivated Oil-Emulsion Newcastle ...

    African Journals Online (AJOL)

    Since the first recognition of Newcastle disease (ND) in Nigeria, it has been observed to be enzootic despite the intensive vaccination policy, leading to significant economic losses in the poultry industry. This study evaluated the ability of inactivated oil-emulsion ND Komarov vaccine to protect laying chickens from challenge ...

  17. Indicators for suicide substrate inactivation: A kinetic investigation

    Indian Academy of Sciences (India)

    Suicide substrate kinetic pathway and a proposed set of indicators, some theoretical and a few practical ones, that can decisively conclude enzyme inactivation are considered. Steady-state approximation is assumed not only when a catalytic amount of enzyme is used but also for any substrate-enzyme ratio. In each ...

  18. Photodynamic inactivation of Aspergillus flavus mediated by Bidens ...

    African Journals Online (AJOL)

    Occurrence of mycotoxins in food and animal feeds poses major health risks to human and animals, and effective control requires integration of crop management strategies both in the field and during post-harvest storage and processing. Photodynamic inactivation is a novel light-based approach which offers a promising ...

  19. Study of messenger RNA inactivation and protein degradation in an Escherichia coli cell-free expression system

    Directory of Open Access Journals (Sweden)

    Noireaux Vincent

    2010-07-01

    Full Text Available Abstract Background A large amount of recombinant proteins can be synthesized in a few hours with Escherichia coli cell-free expression systems based on bacteriophage transcription. These cytoplasmic extracts are used in many applications that require large-scale protein production such as proteomics and high throughput techniques. In recent years, cell-free systems have also been used to engineer complex informational processes. These works, however, have been limited by the current available cell-free systems, which are not well adapted to these types of studies. In particular, no method has been proposed to increase the mRNA inactivation rate and the protein degradation rate in cell-free reactions. The construction of in vitro informational processes with interesting dynamics requires a balance between mRNA and protein synthesis (the source, and mRNA inactivation and protein degradation (the sink. Results Two quantitative studies are presented to characterize and to increase the global mRNA inactivation rate, and to accelerate the degradation of the synthesized proteins in an E. coli cell-free expression system driven by the endogenous RNA polymerase and sigma factor 70. The E. coli mRNA interferase MazF was used to increase and to adjust the mRNA inactivation rate of the Firefly luciferase (Luc and of the enhanced green fluorescent protein (eGFP. Peptide tags specific to the endogenous E. coli AAA + proteases were used to induce and to adjust the protein degradation rate of eGFP. Messenger RNA inactivation rate, protein degradation rate, maturation time of Luc and eGFP were measured. Conclusions The global mRNA turnover and the protein degradation rate can be accelerated and tuned in a biologically relevant range in a cell-free reaction with quantitative procedures easy to implement. These features broaden the capabilities of cell-free systems with a better control of gene expression. This cell-free extract could find some applications in

  20. Strategy to inactivate Clostridium perfringens spores in meat products.

    Science.gov (United States)

    Akhtar, Saeed; Paredes-Sabja, Daniel; Torres, J Antonio; Sarker, Mahfuzur R

    2009-05-01

    The current study aimed to develop an inactivation strategy for Clostridium perfringens spores in meat through a combination of spore activation at low pressure (100-200 MPa, 7 min) and elevated temperature (80 degrees C, 10 min); spore germination at high temperatures (55, 60 or 65 degrees C); and inactivation of germinated spores with elevated temperatures (80 and 90 degrees C, 10 and 20 min) and high pressure (586 MPa, at 23 and 73 degrees C, 10 min). Low pressures (100-200 MPa) were insufficient to efficiently activate C. perfringens spores for germination. However, C. perfringens spores were efficiently activated with elevated temperature (80 degrees C, 10 min), and germinated at temperatures lethal for vegetative cells (>or= 55 degrees C) when incubated for 60 min with a mixture of L-asparagine and KCl (AK) in phosphate buffer (pH 7) and in poultry meat. Inactivation of spores (approximately 4 decimal reduction) in meat by elevated temperatures (80-90 degrees C for 20 min) required a long germination period (55 degrees C for 60 min). However, similar inactivation level was reached with shorter germination period (55 degrees C for 15 min) when spore contaminated-meat was treated with pressure-assisted thermal processing (568 MPa, 73 degrees C, 10 min). Therefore, the most efficient strategy to inactivate C. perfringens spores in poultry meat containing 50 mM AK consisted: (i) a primary heat treatment (80 degrees C, 10 min) to pasteurize and denature the meat proteins and to activate C. perfringens spores for germination; (ii) cooling of the product to 55 degrees C in about 20 min and further incubation at 55 degrees C for about 15 min for spore germination; and (iii) inactivation of germinated spores by pressure-assisted thermal processing (586 MPa at 73 degrees C for 10 min). Collectively, this study demonstrates the feasibility of an alternative and novel strategy to inactivate C. perfringens spores in meat products formulated with germinants specific for C

  1. Inactivation of the small GTP binding protein Rho induces multinucleate cell formation and apoptosis in murine T lymphoma EL4.

    Science.gov (United States)

    Moorman, J P; Bobak, D A; Hahn, C S

    1996-06-01

    The small G-protein Rho regulates the actin microfilament-dependent cytoskeleton. Exoenzyme C3 of Clostridium botulinum ADP-ribosylates Rho at Asn41, a modification that functionally inactivates Rho. Using a Sindbis virus-based transient gene expression system, we studied the role of Rho in murine EL4 T lymphoma cells. We generated a double subgenomic infectious Sindbis virus (dsSIN:C3) recombinant which expressed C3 in >95% of EL4 cells. This intracellular C3 resulted in modification and inactivation of virtually all endogenous Rho. dsSIN:C3 infection led to the formation of multinucleate cells, likely by inhibiting the actin microfilament-dependent step of cytokinesis. Intriguingly, in spite of the inhibition of cytokinesis, karyokinesis continued, with the result that cells containing a nuclear DNA content as high as 16N (eight nuclei) were observed. In addition, dsSIN:C3-mediated inactivation of Rho was a potent activator of apoptosis in EL4 cells. To discern whether the formation of multinucleate cells was responsible for the activation of apoptosis, 5-fluorouracil (5-FUra) was used to induce cell cycle arrest. As expected, EL4 cells treated with 5-FUra were prevented from forming multinucleate cells upon infection with dsSIN:C3. dsSIN:C3 infection, however, still caused marked apoptosis in 5-FUra-treated cells, indicating that this activation of apoptosis was independent of multinucleate cell formation.

  2. 21 CFR 866.5250 - Complement C2 inhibitor (inactivator) immunological test system.

    Science.gov (United States)

    2010-04-01

    ... Test Systems § 866.5250 Complement C2 inhibitor (inactivator) immunological test system. (a) Identification. A complement C1 inhibitor (inactivator) immunological test system is a device that consists of... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Complement C2 inhibitor (inactivator...

  3. Studies on the inactivation of bovine liver enoyl-CoA hydratase by (methylenecyclopropyl)formyl-CoA: elucidation of the inactivation mechanism and identification of cysteine-114 as the entrapped nucleophile.

    Science.gov (United States)

    Dakoji, S; Li, D; Agnihotri, G; Zhou, H Q; Liu, H W

    2001-10-10

    The inhibitory properties of (methylenecyclopropyl)formyl-CoA (MCPF-CoA), a metabolite derived from a natural amino acid, (methylenecyclopropyl)glycine, against bovine liver enoyl-CoA hydratase (ECH) were characterized. We have previously demonstrated that MCPF-CoA specifically targets ECHs, which catalyze the reversible hydration of alpha,beta-unsaturated enoyl-CoA substrates to the corresponding beta-hydroxyacyl-CoA products. Here, we synthesized (R)- and (S)-diastereomers of MCPF-CoA to examine the stereoselectivity of this inactivation. Both compounds were shown to be competent inhibitors for bovine liver ECH with nearly identical second-order inactivation rate constants (k(inact)/K(I)) and partition ratios (k(cat)/k(inact)), indicating that the inactivation is nonstereospecific with respect to ring cleavage. The inhibitor, upon incubation with bovine liver ECH, labels a tryptic peptide, ALGGGXEL, near the active site of the protein, where X is the amino acid that is covalently modified. Cloning and sequence analysis of bovine liver ECH gene revealed the identity of the amino acid residue entrapped by MCPF-CoA as Cys-114 (mature sequence numbering). On the basis of gHMQC (gradient heteronuclear multiple quantum coherence) analysis with [3-(13)C]-labeled MCPF-CoA, the ring cleavage is most likely induced by the nucleophilic attack at the terminal carbon of the exomethylene group (C(2)'). We propose a plausible inactivation mechanism that involves relief of ring strain and is consistent with examples found in the literature. In addition, these studies provide important clues for future design of more efficient and selective inhibitors to control and/or regulate fatty acid metabolism.

  4. Gene decay in archaea

    Directory of Open Access Journals (Sweden)

    M. W. J. van Passel

    2007-01-01

    Full Text Available The gene-dense chromosomes of archaea and bacteria were long thought to be devoid of pseudogenes, but with the massive increase in available genome sequences, whole genome comparisons between closely related species have identified mutations that have rendered numerous genes inactive. Comparative analyses of sequenced archaeal genomes revealed numerous pseudogenes, which can constitute up to 8.6% of the annotated coding sequences in some genomes. The largest proportion of pseudogenes is created by gene truncations, followed by frameshift mutations. Within archaeal genomes, large numbers of pseudogenes contain more than one inactivating mutation, suggesting that pseudogenes are deleted from the genome more slowly in archaea than in bacteria. Although archaea seem to retain pseudogenes longer than do bacteria, most archaeal genomes have unique repertoires of pseudogenes.

  5. Randomized Trials Comparing Inactivated Vaccine after Medium- or High-titer Measles Vaccine with Standard Titer Measles Vaccine after Inactivated Vaccine

    DEFF Research Database (Denmark)

    Aaby, Peter; Ravn, Henrik; Benn, Christine S.

    2016-01-01

    Background: Observational studies have suggested that girls have higher mortality if their most recent immunization is an inactivated vaccine rather than a live vaccine. We therefore reanalyzed 5 randomized trials of early measles vaccine (MV) in which it was possible to compare an inactivated...... vaccines [after medium-titer MV (MTMV) or high-titer MV (HTMV)] and a live standard titer MV (after an initial inactivated vaccine). Methods: The trials were conducted in Sudan, Senegal, The Gambia and Guinea-Bissau. The intervention group received live MTMV or HTMV from 4 to 5 months...... and then an inactivated vaccine from 9 to 10 months of age; the control children received inactivated vaccine/placebo from 4 to 5 months and standard titer MV from 9 to 10 months of age. We compared mortality from 9 months until end of study at 3 to 5 years of age for children who received inactivated vaccine (after MTMV...

  6. The assessment of efficacy of porcine reproductive respiratory syndrome virus inactivated vaccine based on the viral quantity and inactivation methods

    Directory of Open Access Journals (Sweden)

    Lee Byeongchun

    2011-06-01

    Full Text Available Abstract Background There have been many efforts to develop efficient vaccines for the control of porcine reproductive and respiratory syndrome virus (PRRSV. Although inactivated PRRSV vaccines are preferred for their safety, they are weak at inducing humoral immune responses and controlling field PRRSV infection, especially when heterologous viruses are involved. Results In all groups, the sample to positive (S/P ratio of IDEXX ELISA and the virus neutralization (VN titer remained negative until challenge. While viremia did not reduce in the vaccinated groups, the IDEXX-ELISA-specific immunoglobulin G increased more rapidly and to significantly greater levels 7 days after the challenge in all the vaccinated groups compared to the non-vaccinated groups (p 6 PFU/mL PRRSV vaccine-inoculated and binary ethylenimine (BEI-inactivated groups 22 days after challenge (p Conclusions The inactivated vaccine failed to show the humoral immunity, but it showed different immune response after the challenge compared to mock group. Although the 106 PFU/mL-vaccinated and BEI-inactivated groups showed significantly greater VN titers 22 days after challenge, all the groups were already negative for viremia.

  7. Transient inactivation of myostatin induces muscle hypertrophy and overcompensatory growth in zebrafish via inactivation of the SMAD signaling pathway.

    Science.gov (United States)

    Fuentes, Eduardo N; Pino, Katherine; Navarro, Cristina; Delgado, Iselys; Valdés, Juan Antonio; Molina, Alfredo

    2013-12-01

    Myostatin (MSTN) is the main negative regulator of muscle growth and development in vertebrates. In fish, little is known about the molecular mechanisms behind how MSTN inactivation triggers skeletal muscle enhancement, particularly regarding the signaling pathways involved in this process. Moreover, there have not been reports on the biotechnological applications of MSTN and its signal transduction. In this context, zebrafish underwent compensatory growth using fasting and refeeding trials, and MSTN activity was inactivated with dominant negative LAPD76A recombinant proteins during the refeeding period, when a rapid, compensatory muscle growth was observed. Treated fish displayed an overcompensation of growth characterized by higher muscle hypertrophy and growth performance than constantly fed, control fish. Treatment with LAPD76A recombinant proteins triggered inactivation of the SMAD signaling pathway in skeletal muscle, the main signal transduction used by MSTN to achieve its biological actions. Therefore, transient inactivation of MSTN during the compensatory growth of zebrafish led to a decrease in the SMAD signaling pathway in muscle, triggering muscle hypertrophy and finally improving growth performance, thus, zebrafish achieved an overcompensation of growth. The present study shows an attractive strategy for improving muscle growth in a fish species by mixing a classical strategy, such as compensatory growth, and a biotechnological approach, such as the use of recombinant proteins for inhibiting the biological actions of MSTN. The mix of both strategies may represent a method that could be applied in order to improve growth in commercial fish of interest for aquaculture. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Cytolytic T lymphocyte responses to metabolically inactivated stimulator cells. I. Metabolic inactivation impairs both CD and LD antigen signals

    Energy Technology Data Exchange (ETDEWEB)

    Kelso, A.; Boyle, W.

    1982-03-01

    The effects of metabolic inactivation of spleen cells on antigen presentation to precursors of alloreactive cytolytic T lymphocytes (T/sub c/) were examined. By serological methods, populations inactivated by ultraviolet irradiation, glutaraldehyde fixation or plasma membrane isolation were found to retain normal levels of H-2K/D and Ia antigens. However, comparison of the antigen doses required to stimulate secondary T/sub c/ responses in mixed leukocyte culture showed that the inactivated preparations were approximately 10-fold less immunogenic than X-irradiated spleen cells. Their total inability to stimulate primary cytolytic responses pointed to at least a 100-fold impairment of immunogenicity for unprimed T/sub c/ precursors in the case of uv-irradiated and glutaraldehyde-treated stimulator cells, and at least a 10-fold impairment for membrane fragments. Experiments showing that the capacity of cell monolayers to absorb precursor T/sub c/ from unprimed spleen populations was reduced following uv-irradiation or glutaraldehyde treatment provided direct evidence that this loss of immunogenicity was due in part to suboptimal antigen presentation to precursor T/sub c/. It is concluded that, in addition to the traditional view that these treatments damage the ''LD'' signal to helper T lymphocytes, metabolic inactivation also impairs recognition of ''CD'' determinants by precursor T/sub c/.

  9. Drosophila QVR/SSS modulates the activation and C-type inactivation kinetics of Shaker K(+) channels.

    Science.gov (United States)

    Dean, Terry; Xu, Rong; Joiner, William; Sehgal, Amita; Hoshi, Toshinori

    2011-08-03

    The quiver/sleepless (qvr/sss) gene encodes a small, glycosylphosphatidylinositol-anchored protein that plays a critical role in the regulation of sleep in Drosophila. Loss-of-function mutations in qvr/sss severely suppress sleep and effect multiple changes in in situ Shaker K(+) currents, including decreased magnitude, slower time-to-peak, and cumulative inactivation. Recently, we demonstrated that SLEEPLESS (SSS) protein modulates Shaker channel activity, possibly through a direct interaction at the plasma membrane. We show here that SSS accelerates the activation of heterologously expressed Shaker channels with no effect on deactivation or fast N-type inactivation. Furthermore, this SSS-induced acceleration is sensitive to the pharmacological disruption of lipid rafts and sufficiently accounts for the slower time-to-peak of in situ Shaker currents seen in qvr/sss mutants. We also find that SSS decreases the rate of C-type inactivation of heterologously expressed Shaker channels, providing a potential mechanism for the cumulative inactivation phenotype induced by qvr/sss loss-of-function mutations. Kinetic modeling based on the in vitro results suggests that the SSS-dependent regulation of channel kinetics accounts for nearly 40% of the decrease in Shaker current magnitude in flies lacking SSS. Sleep duration in qvr/sss-null mutants is restored to normal by a qvr/sss transgene that fully rescues the Shaker kinetic phenotypes but only partially rescues the decrease in current magnitude. Together, these results suggest that the role of SSS in the regulation of sleep in Drosophila correlates more strongly with the effects of SSS on Shaker kinetics than current magnitude.

  10. Identification, characterization and structure analysis of a type I ribosome-inactivating protein from Sapium sebiferum (Euphorbiaceae)

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Ying [Key Laboratory of Ion Beam Bioengineering and Bioenergy Forest Research Center of State Forestry Administration, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, Anhui (China); School of Life Sciences, University of Science and Technology of China, Hefei 230027, Anhui (China); College of Food and Bioengineering, Henan University of Science and Technology, Luoyang 471023, Henan (China); Mao, Yingji [Key Laboratory of Ion Beam Bioengineering and Bioenergy Forest Research Center of State Forestry Administration, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, Anhui (China); School of Life Sciences, University of Science and Technology of China, Hefei 230027, Anhui (China); Jin, Shan; Hou, Jinyan; Du, Hua [Key Laboratory of Ion Beam Bioengineering and Bioenergy Forest Research Center of State Forestry Administration, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, Anhui (China); Yang, Minglei, E-mail: yml888@mail.ustc.edu.cn [Key Laboratory of Ion Beam Bioengineering and Bioenergy Forest Research Center of State Forestry Administration, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, Anhui (China); Wu, Lifang, E-mail: lfwu@ipp.ac.cn [Key Laboratory of Ion Beam Bioengineering and Bioenergy Forest Research Center of State Forestry Administration, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, Anhui (China); School of Life Sciences, University of Science and Technology of China, Hefei 230027, Anhui (China)

    2015-08-07

    Ribosome-inactivating proteins (RIPs) are N-glycosidases (EC3.2.2.22) that universally inactivate the ribosome, thereby inhibiting protein biosynthesis. In this study, a novel type I RIPs named SEBIN was identified in Sapium sebiferum. Nuclear acid depurine experiment showed that SEBIN had rRNA N-Glycosidase activity. Further experiment indicated that SEBIN significantly inhibited Caenorhabditis elegans development as well as resulted in worm cell apoptosis. This is the first report to evaluate RIPs toxicity using C. elegans. We proposed that SEBIN may impaire C. elegans reproduction in a DNA-damage manner besides traditional protein synthesis inhibition approach. The predicted 3D structure was modeled using threading and ab initio modeling, and the r-RNA binding residue of SEBIN was identified through the protein-ligand docking approach. It showed the amino acid residues, Glu195, Asn81, Ala82, Tyr83, Glu164, Ser163, Ile159 and Arg167, played critical roles in catalytic process. Our results provided the theoretical foundation of structure–function relationships between enzymatic properties, toxicity and structural characterization of SEBIN. - Graphical abstract: Superposition of main chains of ricin (cyan) and SEBIN (brown), and adenine binding site residues of SEBIN. - Highlights: • A Ribosome-inactivating proteins gene (SEBIN) was isolated from Sapium sebiferum. • SEBIN had DNase activity besides widely reported ribosome inactivation via N-glycosidases activity. • SEBIN significantly inhibited Caenorhabditis elegans development in vivo. • SEBIN may impaire C. elegans reproduction in a DNA-damage manner with the aid of mutant strains hus-1 and clk-2. • The possible active sites between SEBIN and the adenine of rRNA were predicted.

  11. Transient inactivation of the medial prefrontal cortex affects both anxiety and decision-making in male Wistar rats

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    Leonie ede Visser

    2011-09-01

    Full Text Available In both humans and rats high levels of anxiety impair decision-making in the Iowa Gambling Task (IGT in male subjects. Expression of the immediate early gene c-fos as marker of neural activity in rat studies indicated a role of the medial prefrontal cortex (prelimbic and infralimbic region; mPFC in mediating the relationship between anxiety and decision-making. To delineate this relationship further and assess the underlying neurobiology in more detail, we inactivated in the present study the mPFC in male rats using a mixture of the GABA-receptor agonists muscimol and baclofen. Rats were exposed to the elevated plus maze (EPM to measure effects on anxiety and to the rodent version of the IGT (r-IGT. Inactivation led to increased levels of anxiety on the EPM, while not affecting general activity. The effect in the r-IGT (trials 61-120 was dependent on levels of performance prior to inactivation (trial 41-60: inactivation of the mPFC hampered task-performance in rats, which already showed a preference for the advantageous option, but not in rats which were still choosing in a random manner. These data suggest that the mPFC becomes more strongly involved as rats have learned task-contingencies, i.e. choose for the best long-term option. Furthermore they suggest, along with the data of our earlier study, that both anxiety and decision-making in rats are mediated through a neural circuitry including at least the mPFC. The data are discussed in relation to recent data of rodent studies on the neural circuitry underlying decision-making.

  12. Cerebral cortex hyperthyroidism of newborn mct8-deficient mice transiently suppressed by lat2 inactivation.

    Directory of Open Access Journals (Sweden)

    Bárbara Núñez

    Full Text Available Thyroid hormone entry into cells is facilitated by transmembrane transporters. Mutations of the specific thyroid hormone transporter, MCT8 (Monocarboxylate Transporter 8, SLC16A2 cause an X-linked syndrome of profound neurological impairment and altered thyroid function known as the Allan-Herndon-Dudley syndrome. MCT8 deficiency presumably results in failure of thyroid hormone to reach the neural target cells in adequate amounts to sustain normal brain development. However during the perinatal period the absence of Mct8 in mice induces a state of cerebral cortex hyperthyroidism, indicating increased brain access and/or retention of thyroid hormone. The contribution of other transporters to thyroid hormone metabolism and action, especially in the context of MCT8 deficiency is not clear. We have analyzed the role of the heterodimeric aminoacid transporter Lat2 (Slc7a8, in the presence or absence of Mct8, on thyroid hormone concentrations and on expression of thyroid hormone-dependent cerebral cortex genes. To this end we generated Lat2-/-, and Mct8-/yLat2-/- mice, to compare with wild type and Mct8-/y mice during postnatal development. As described previously the single Mct8 KO neonates had a transient increase of 3,5,3'-triiodothyronine concentration and expression of thyroid hormone target genes in the cerebral cortex. Strikingly the absence of Lat2 in the double Mct8Lat2 KO prevented the effect of Mct8 inactivation in newborns. The Lat2 effect was not observed from postnatal day 5 onwards. On postnatal day 21 the Mct8 KO displayed the typical pattern of thyroid hormone concentrations in plasma, decreased cortex 3,5,3'-triiodothyronine concentration and Hr expression, and concomitant Lat2 inactivation produced little to no modifications. As Lat2 is expressed in neurons and in the choroid plexus, the results support a role for Lat2 in the supply of thyroid hormone to the cerebral cortex during early postnatal development.

  13. Non-coding RNAs and epigenome: de novo DNA methylation, allelic exclusion and X-inactivation

    Directory of Open Access Journals (Sweden)

    V. A. Halytskiy

    2013-12-01

    Full Text Available Non-coding RNAs are widespread class of cell RNAs. They participate in many important processes in cells – signaling, posttranscriptional silencing, protein biosynthesis, splicing, maintenance of genome stability, telomere lengthening, X-inactivation. Nevertheless, activity of these RNAs is not restricted to posttranscriptional sphere, but cover also processes that change or maintain the epigenetic information. Non-coding RNAs can directly bind to the DNA targets and cause their repression through recruitment of DNA methyltransferases as well as chromatin modifying enzymes. Such events constitute molecular mechanism of the RNA-dependent DNA methylation. It is possible, that the RNA-DNA interaction is universal mechanism triggering DNA methylation de novo. Allelic exclusion can be also based on described mechanism. This phenomenon takes place, when non-coding RNA, which precursor is transcribed from one allele, triggers DNA methylation in all other alleles present in the cell. Note, that miRNA-mediated transcriptional silencing resembles allelic exclusion, because both miRNA gene and genes, which can be targeted by this miRNA, contain elements with the same sequences. It can be assumed that RNA-dependent DNA methylation and allelic exclusion originated with the purpose of counteracting the activity of mobile genetic elements. Probably, thinning and deregulation of the cellular non-coding RNA pattern allows reactivation of silent mobile genetic elements resulting in genome instability that leads to ageing and carcinogenesis. In the course of X-inactivation, DNA methylation and subsequent hete­rochromatinization of X chromosome can be triggered by direct hybridization of 5′-end of large non-coding RNA Xist with DNA targets in remote regions of the X chromosome.

  14. Genes and Gene Therapy

    Science.gov (United States)

    ... correctly, a child can have a genetic disorder. Gene therapy is an experimental technique that uses genes to ... or prevent disease. The most common form of gene therapy involves inserting a normal gene to replace an ...

  15. Epigenetic regulation of steroid inactivating UDP-glucuronosyltransferases by microRNAs in prostate cancer.

    Science.gov (United States)

    Margaillan, Guillaume; Lévesque, Éric; Guillemette, Chantal

    2016-01-01

    Androgens play a central role in prostate cancer progression. Systemic and local androgen bioavailability is controlled by UDP-glucuronosyltransferases conjugating enzymes (UGT), namely UGT2B15, UGT2B17 and UGT2B28. Reporter vector assays in HEK293 cells initially validated in silico-predicted regulatory potential of candidate miRNAs to target UGT transcripts, including miR-376c, miR-409 and miR-494 for UGT2B17, miR-331-5p and miR-376c for UGT2B15 while none were efficient for UGT2B28. miR-376c was shown as the most effective to downregulate UGT2B15 and UGT2B17 through interactions with a site conserved in both UGTs. Ectopic miR-376c expression in prostate cancer cells significantly reduced UGT2B15 and UGT2B17 expression (>32%; P<0.005) with a consequent decrease in dihydrotestosterone glucuronidation (-37%; P<0.001). Consistent with reduced androgen inactivation, ectopic expression of miR-376c changed expression of androgen responsive genes and enhanced cell proliferation with no effect on androgen receptor levels. Sustaining a role of miR-376c in the regulation of androgen-inactivating UGTs, its expression was significantly downregulated in prostatic tumors and further reduced in metastases (P<0.0001), whereas the opposite was observed for UGT2B15/17 (P=0.031). In high-grade tumors (Gleason ≥8), UGT2B15/17 and miR-376c were inversely correlated (r=-0.557; P=0.048) with also a significant relationship in metastases (r=-0.747; P=0.003). In line with a modification in androgen bioavailability, PSA mRNA levels were also negatively correlated to those of UGT2B15/17 (r=-0.573; P=0.01) but positively linked to levels of miR-376c (r=0.577; P=0.039). This study reveals that the androgen-inactivating UGT2B15 and UGT2B17 genes are direct targets of miR-376c and thus may influence steroid metabolism during prostate cancer progression. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Inactivation of Transcriptional Regulators during Within-Household Evolution of Escherichia coli.

    Science.gov (United States)

    Kisiela, Dagmara I; Radey, Matthew; Paul, Sandip; Porter, Stephen; Polukhina, Kseniya; Tchesnokova, Veronika; Shevchenko, Sofiya; Chan, Diana; Aziz, Maliha; Johnson, Timothy J; Price, Lance B; Johnson, James R; Sokurenko, Evgeni V

    2017-07-01

    We analyzed the within-household evolution of two household-associated Escherichia coli strains from pandemic clonal group ST131-H30, using isolates recovered from five individuals within two families, each of which had a distinct strain. Family 1's strain was represented by a urine isolate from the index patient (older sister) with recurrent cystitis and a blood isolate from her younger sister with fatal urosepsis. Family 2's strain was represented by a urine isolate from the index patient (father) with pyelonephritis and renal abscesses, blood and kidney drainage isolates from the daughter with emphysematous pyelonephritis, and urine and fecal isolates from the mother with cystitis. Collectively, the several variants of each family's strain had accumulated a total of 8 (family 1) and 39 (family 2) point mutations; no two isolates were identical. Of the 47 total mutations, 36 resulted in amino acid changes or truncation of coded proteins. Fourteen such mutations (39%) targeted genes encoding transcriptional regulators, and 9 (25%) involved DNA-binding transcription factors (TFs), which significantly exceeded the relative contribution of TF genes to the isolates' genomes (∼6%). At least one-half of the transcriptional regulator mutations were inactivating, based on phenotypic and/or transcriptional analysis. In particular, inactivating mutations in the global regulator LrhA (repressor of type 1 fimbriae and flagella) occurred in the blood isolates from both households and increased the virulence of E. coli strains in a murine sepsis model. The results indicate that E. coli undergoes adaptive evolution between and/or within hosts, generating subpopulations with distinctive phenotypes and virulence potential.IMPORTANCE The clonal evolution of bacterial strains associated with interhost transmission is poorly understood. We characterized the genome sequences of clonal descendants of two Escherichia coli strains, recovered at different time points from multiple

  17. Calcium current-dependent and voltage-dependent inactivation of calcium channels in Helix aspersa

    Science.gov (United States)

    Brown, A. M.; Morimoto, K.; Tsuda, Y.; Wilson, D. L.

    1981-01-01

    1. Inactivation of the Ca channels has been examined in isolated nerve cell bodies of Helix aspersa using the suction pipette method for voltage clamp and internal perfusion. 2. Satisfactory suppression of outward currents was essential. This was achieved over most of the voltage range by substitution of Cs ion for K ion and by the use of TEA intra- and extracellularly and 4-AP extracellularly. A small time- and voltage-dependent non-specific current remained at potentials above +60 mV. 3. In these solutions, Ca current approaches ECa but cannot be detected in the outward direction. The Ca channel appears to be impermeable to Cs and Tris ions. 4. Inactivation of Ca currents occurs as a bi-exponential process. The faster rate is 10-20 times the slower rate and is about one twentieth the rate of activation. The development of inactivation during a single voltage-clamp step and the onset of inactivation produced by prepulses followed after brief intervals by a test pulse, have roughly similar time courses. 5. The rates of inactivation increase monotonically at potentials more positive than about -25 mV. The amount of steady-state inactivation increases with membrane depolarizations to potentials of about +50 mV. At more positive potentials, steady-state inactivation is reduced. 6. Intracellular EGTA slows the faster rate of inactivation of ICa and reduces the amount of steady-state inactivation measured with a standard two pulse protocol. The effect is specifically related to Ca chelation and hydrogen ions are not involved. This component of inactivation is referred to as Ca current-dependent inactivation and is consistent with observations that increased Cai inactivates the Ca channel. The process does not depend upon current flow alone since Ba currents of comparable or greater magnitude have smaller initial rates of inactivation. Furthermore, application of Ba ion intracellularly in large concentrations has no effect on steady-state inactivation. 7. The bi

  18. Photochemical inactivation of pathogenic bacteria in human platelet concentrates.

    Science.gov (United States)

    Lin, L; Londe, H; Janda, J M; Hanson, C V; Corash, L

    1994-05-01

    Platelet concentrates (PC) may be infrequently contaminated with low levels of bacteria that can cause septicemia and death in patients receiving transfusion therapy. We evaluated the efficacy of a photochemical decontamination (PCD) technique using 8-methoxypsoralen (8-MOP) and long wavelength UV light (UVA) to inactivate bacteria in standard therapeutic PC. Twelve phylogenetically distinct pathogenic bacteria, 5 gram-positive and 7 gram-negative organisms, were seeded into PC to a final challenge dose ranging from 10(5) to 10(7) colony-forming units (CFU)/mL. Contaminated PC were treated with 8-MOP (5 micrograms/mL) and 5 J/cm2 of UVA, a PCD treatment regimen found to adequately preserve in vitro platelet function. Greater than 10(5) CFU/mL of all 5 gram-positive (Staphylococcus aureus, Streptococcus epidermidis, Streptococcus pyogenes, Listeria monocytogenes, and Corynebacterium minutissimum) and 2 of the gram-negative (Escherichia coli and Yersinia enterocolitica) organisms were inactivated. The remaining 5 gram-negative organisms were more resistant, with less than 10(1) to 10(3.7) CFU/mL inactivated under these conditions. The inactivation efficiency for this resistant group of gram-negative organisms was improved when PC were resuspended in a synthetic storage medium with reduced plasma protein concentration (15%) and an increased 8-MOP concentration (23.4 micrograms/mL). Illumination with 3 J/cm2 of UVA in this system inactivated greater than 10(5) CFU/mL of 4 resistant gram-negative organisms (Salmonella choleraesuis, Enterobacter cloacae, Serratia marcescens, and Klebsiella pneumoniae) and 10(4.1) CFU/mL of the most resistant gram-negative organism (Pseudomonas aeruginosa). This level of PCD treatment did not adversely affect in vitro platelet function. These results demonstrate that PCD using 8-MOP (5 to 23.4 micrograms/mL) effectively inactivated high levels of pathogenic bacteria in PC with adequate preservation of in vitro platelet properties.

  19. Modeling of human factor Va inactivation by activated protein C

    Directory of Open Access Journals (Sweden)

    Bravo Maria

    2012-05-01

    Full Text Available Abstract Background Because understanding of the inventory, connectivity and dynamics of the components characterizing the process of coagulation is relatively mature, it has become an attractive target for physiochemical modeling. Such models can potentially improve the design of therapeutics. The prothrombinase complex (composed of the protease factor (FXa and its cofactor FVa plays a central role in this network as the main producer of thrombin, which catalyses both the activation of platelets and the conversion of fibrinogen to fibrin, the main substances of a clot. A key negative feedback loop that prevents clot propagation beyond the site of injury is the thrombin-dependent generation of activated protein C (APC, an enzyme that inactivates FVa, thus neutralizing the prothrombinase complex. APC inactivation of FVa is complex, involving the production of partially active intermediates and “protection” of FVa from APC by both FXa and prothrombin. An empirically validated mathematical model of this process would be useful in advancing the predictive capacity of comprehensive models of coagulation. Results A model of human APC inactivation of prothrombinase was constructed in a stepwise fashion by analyzing time courses of FVa inactivation in empirical reaction systems with increasing number of interacting components and generating corresponding model constructs of each reaction system. Reaction mechanisms, rate constants and equilibrium constants informing these model constructs were initially derived from various research groups reporting on APC inactivation of FVa in isolation, or in the presence of FXa or prothrombin. Model predictions were assessed against empirical data measuring the appearance and disappearance of multiple FVa degradation intermediates as well as prothrombinase activity changes, with plasma proteins derived from multiple preparations. Our work integrates previously published findings and through the cooperative

  20. X-chromosome inactivation pattern analysis for the assessment of cell clonality in cats.

    Science.gov (United States)

    Mochizuki, H; Goto-Koshino, Y; Takahashi, M; Fujino, Y; Ohno, K; Tsujimoto, H

    2012-11-01

    X-chromosome inactivation pattern (XCIP) analysis has been widely used to assess cell clonality in various types of neoplasms in humans. In the present study, a polymerase chain reaction-based feline XCIP analysis using the feline androgen receptor gene was developed. To construct the system of the analysis, polymorphism in CAG tandem repeats within the feline androgen receptor gene was explored using somatic DNAs from 50 male and 103 female cats. CAG tandem repeats in exon 1 of the feline androgen receptor gene were found to be polymorphic, containing 15 to 22 CAG repeats. Of the 103 female cats, 70 (68%) were heterozygous for the number of CAG repeats, indicating the possible usefulness of XCIP analysis in cats. Application of the feline XCIP analysis to 3 feline mammary gland adenocarcinoma cell lines revealed distinctly skewed XCIPs in these cell lines, indicating their clonal origins. Twelve (80%) of the 15 primary tissue/cell samples obtained from cats with various neoplastic diseases showed skewed XCIPs. Moreover, bone marrow samples from 3 cats with myelodysplastic syndrome were also found to have skewed XCIPs. The polymerase chain reaction-based XCIP analysis developed in this study can provide information on cell clonality in female cats, potentially facilitating the differential diagnosis of various disorders in cats.

  1. Mouse transplant models for evaluating the oncogenic risk of a self-inactivating XSCID lentiviral vector.

    Directory of Open Access Journals (Sweden)

    Sheng Zhou

    Full Text Available Hematopoietic stem cell gene therapy requires the use of integrating retroviral vectors in order to stably transmit a therapeutic gene to mature blood cells. Human clinical trials have shown that some vector integration events lead to disrupted regulation of proto-oncogenes resulting in disordered hematopoiesis including T-cell leukemia. Newer vectors have been designed to decrease the incidence of these adverse events but require appropriate pre-clinical assays to demonstrate safety. We have used two distinct mouse serial transplant assays to evaluate the safety of a self-inactivating lentiviral vector intended for use in X-linked severe combined immunodeficiency (XSCID gene therapy trials. These experiments entailed 28 months of total follow-up and included 386 mice. There were no cases in which the XSCID lentiviral vector clearly caused hematopoietic malignancies, although a single case of B cell malignancy was observed that contained the lentiviral vector as a likely passenger event. In contrast, a SFFV-DsRed γ-retroviral vector resulted in clonal transformation events in multiple secondary recipients. Non-specific pathology not related to vector insertions was noted including T cell leukemias arising from irradiated recipient cells. Overall, this comprehensive study of mouse transplant safety assays demonstrate the relative safety of the XSCID lentiviral vector but also highlight the limitations of these assays.

  2. Fast- or slow-inactivated state preference of Na+ channel inhibitors: a simulation and experimental study.

    Directory of Open Access Journals (Sweden)

    Robert Karoly

    2010-06-01

    Full Text Available Sodium channels are one of the most intensively studied drug targets. Sodium channel inhibitors (e.g., local anesthetics, anticonvulsants, antiarrhythmics and analgesics exert their effect by stabilizing an inactivated conformation of the channels. Besides the fast-inactivated conformation, sodium channels have several distinct slow-inactivated conformational states. Stabilization of a slow-inactivated state has been proposed to be advantageous for certain therapeutic applications. Special voltage protocols are used to evoke slow inactivation of sodium channels. It is assumed that efficacy of a drug in these protocols indicates slow-inactivated state preference. We tested this assumption in simulations using four prototypical drug inhibitory mechanisms (fast or slow-inactivated state preference, with either fast or slow binding kinetics and a kinetic model for sodium channels. Unexpectedly, we found that efficacy in these protocols (e.g., a shift of the "steady-state slow inactivation curve", was not a reliable indicator of slow-inactivated state preference. Slowly associating fast-inactivated state-preferring drugs were indistinguishable from slow-inactivated state-preferring drugs. On the other hand, fast- and slow-inactivated state-preferring drugs tended to preferentially affect onset and recovery, respectively. The robustness of these observations was verified: i by performing a Monte Carlo study on the effects of randomly modifying model parameters, ii by testing the same drugs in a fundamentally different model and iii by an analysis of the effect of systematically changing drug-specific parameters. In patch clamp electrophysiology experiments we tested five sodium channel inhibitor drugs on native sodium channels of cultured hippocampal neurons. For lidocaine, phenytoin and carbamazepine our data indicate a preference for the fast-inactivated state, while the results for fluoxetine and desipramine are inconclusive. We suggest that

  3. Cometabolic degradation of chlorinated solvents: Bacterial inhibition, inactivation, and recovery

    Energy Technology Data Exchange (ETDEWEB)

    Ely, R.L.; Williamson, K.J.; Hyman, M.R.; Arp, D.J. [Oregon State Univ., Corvallis, OR (United States). Dept. of Civil Engineering

    1995-12-31

    This paper summarizes an approach for quantifying degradation kinetics and bacterial activity changes during cometabolic degradation of chlorinated solvents, results from trichloroethylene (TCE) degradation experiments, and a mathematical model addressing fluctuations in activity caused by enzyme inhibition, inactivation, and respondent enzyme synthesis. Using Nitrosomonas duropaea as a slow-growing exemplar capable of effecting cometabolic transformations, quasi-steady-state ammonia oxidation was established in a small bioreactor. A chlorinated solvent was injected to perturb the system, and bacterial activity and solvent degradation were monitored. At TCE concentrations to about 3.5 mg/L, from slight to nearly complete ammonia monooxygenase (AMO) inactivation occurred without causing immediate cell death. Results suggested cellular injury was limited primarily to AMO, most metabolic systems remained functional, and bacterial recovery processes, independent of cell growth, were initiated while degrading TCE.

  4. Polio endgame: the global introduction of inactivated polio vaccine.

    Science.gov (United States)

    Patel, Manish; Zipursky, Simona; Orenstein, Walt; Garon, Julie; Zaffran, Michel

    2015-05-01

    In 2013, the World Health Assembly endorsed a plan that calls for the ultimate withdrawal of oral polio vaccines (OPV) from all immunization programs globally. The withdrawal would begin in a phased manner with removal of the type 2 component of OPV in 2016 through a global switch from trivalent OPV to bivalent OPV (containing only types 1 and 3). To mitigate risks associated with immunity gaps after OPV type 2 withdrawal, the WHO Strategic Advisory Group of Experts has recommended that all 126 OPV-only using countries introduce at least one dose of inactivated polio vaccine into routine immunization programs by end-2015, before the trivalent OPV-bivalent OPV switch. The introduction of inactivated polio vaccine would reduce risks of reintroduction of type 2 poliovirus by providing some level of seroprotection, facilitating interruption of transmission if outbreaks occur, and accelerating eradication by boosting immunity to types 1 and 3 polioviruses.

  5. Identification of novel RFLPs in the vicinity of CpG islands in Xq28: Application to the analysis of the pattern of X chromosome inactivation

    Energy Technology Data Exchange (ETDEWEB)

    Camerino, G.; Santachiara-Benerecetti, S. (Univ. di Pavia (Italy)); Rocchi, M. (Univ. di Bari (Italy)); Parolini, O.; Notarangelo, L.D. (Univ. di Brecscia (Italy)); Maestrini, E.; Rivella, S.; Tribioli, C.; Toniolo, D.

    1992-01-01

    Probes of CpG islands were cloned from the distal long arm of the human X chromosome; three of them were found to be polymorphic. A HindIII RFLP was identified by the probe 2-25 (DXS606), and it was mapped to the Xq27-Xq28 boundary. Probes 2-19 (DXS605) and 2-55 (DXS707), which identify EcoRI and MspI polymorphisms, respectively, have been mapped to the distal part of Xq28, in the G6PD-RCP/GCP gene region. Probe 2-19 has been further localized about 16 kb from the 3{prime} end of the G6PD gene. The new RFLPs may be useful for the precise mapping of the many disease genes localized in this part of the human X chromosome. Using the methylation-sensitive rare-cutter enzyme EagI in conjunction with the polymorphic EcoRI site, the authors were able to demonstrate that the RFLP may be used both to study randomness of X chromosome inactivation and for carrier detection in X-linked syndromes where nonrandom X inactivation occurs. It is conceivable that the combined use of 2-19 and of the probes described so far (pSPT-PGK and M27{beta}) will make analysis of X inactivation feasible in virtually every female.

  6. Nonthermal Plasma Inactivation of Food-Borne Pathogens

    OpenAIRE

    Misra, N.; Tiwari, B.; Rahavarao, K.; Cullen, Patrick

    2011-01-01

    Non-thermal plasma (NTP) is electrically energized matter, composed of highly reactive species including gas molecules, charged particles in the form of positive ions, negative ions, free radicals, electrons and quanta of electromagnetic radiation (photons) at near-room temperature. NTP is an emerging nonthermal technology with potential applications for decontamination in the food industries. An upsurge in the research activities for plasma based inactivation of food borne pathogens is evide...

  7. Drying of liquid food droplets : enzyme inactivation and multicomponent diffusion

    OpenAIRE

    Meerdink, G.

    1993-01-01

    In this thesis the drying of liquid food droplets is studied from three different points of view: drying kinetics, enzyme inactivation and multicomponent diffusion. Mathematical models are developed and validated experimentally.

    Drying experiments are performed with suspended droplets and with free falling droplets under spray-drying conditions. The experiments with the free falling droplets are performed in a specially designed drying tower using a resonance nozzle. The reso...

  8. Thermal inactivation of eight Salmonella serotypes on dry corn flour.

    OpenAIRE

    VanCauwenberge, J E; Bothast, R J; Kwolek, W F

    1981-01-01

    Dry heat was used to inactivate Salmonella newington, Salmonella typhimurium, Salmonella anatum, Salmonella kentucky, Salmonella cubana, Salmonella seftenberg, Salmonella thompson, and Salmonella tennessee in corn flour at 10 and 15% moisture. The flour was spray inoculated at 10(5) Salmonella cells per g and then stored at 49 degrees C (120 degrees F); viable Salmonella cells were counted on Trypticase (BBL Microbiology Systems) soy agar plates every 30 min for the first 4 h and then at 4-h ...

  9. Mechanism for Mutational Inactivation of the Tumor Suppressor Smad2

    OpenAIRE

    Prunier, Celine; Ferrand, Nathalie; Frottier, Bertrand; Pessah, Marcia; Atfi, Azeddine

    2001-01-01

    Transforming growth factor β (TGF-β) is a potent natural antiproliferative agent that plays an important role in suppressing tumorigenicity. In numerous tumors, loss of TGF-β responsiveness is associated with inactivating mutations that can occur in components of this signaling pathway, such as the tumor suppressor Smad2. Although a general framework for how Smads transduce TGF-β signals has been proposed, the physiological relevance of alterations of Smad2 functions in promoting tumorigenesi...

  10. Significance of cobalamin inactivation in normal and malignant hematopoiesis

    OpenAIRE

    Ermens, Anton

    1990-01-01

    textabstractThis thesis deals with several aspects of the effects of cobalamin inactivation by nitrous oxide on cellular folate metabolism and the consequences on normal and malignant hematopoiesis. Kroes et al. demonstrated the antileukemic effects of nitrous oxide either or not in combination with other drugs interfering with the folate metabolism (132-136). Especially the potentiation of methotrexate activity in the employed rat model for myeloid leukemia (BNML) after preexposure to nitrou...

  11. The efficacy of preservation methods to inactivate foodborne viruses.

    Science.gov (United States)

    Baert, L; Debevere, J; Uyttendaele, M

    2009-05-31

    During the last decade an increased incidence of infections and outbreaks attributed to foodborne viruses, in particular noroviruses (NoV), was observed world wide. The awareness of the presence of viruses on food emphasized the need to acquire knowledge regarding the effect of preservation methods upon viruses. Most foodborne viruses cannot be cultured in the laboratory, which hinders studies of their stability in food. Cultivable surrogate viruses, genetically related to the human infecting strains, are taken as a substitute to define inactivation rates. The last years, the number of survival and inactivation studies using various surrogate viruses increased. In this review, state-of-the-art information regarding the efficacy of preservation methods to reduce the level of viruses on food is compiled. In the first place, the effect of preservation methods establishing microbial growth inhibition (chilling, freezing, acidification, reduced water activity and modified atmosphere packaging) upon foodborne viruses is described. Secondly, the use of preservation methods establishing microbial inactivation such as heat treatment, high hydrostatic pressure processing and irradiation to eliminate viruses is discussed. In the third place, the efficacy of decontamination methods on fresh produce and purification procedures applied on live bivalve shellfish to reduce the viral load is included. These studies indicate that viruses persist well on chilled, acidified, frozen foods and foods packed under modified atmosphere or in dried conditions. Intervention strategies inducing microbial inactivation are required to achieve a 3 log reduction of the level of viruses. Decontamination of fresh produce reduces viruses with a maximum of 1 to 2 log while purification of live bivalves is not adequate to prevent viral outbreaks. It was noted that the effect of a particular food preservation method is dependent upon the virus tested and type of food.

  12. Patulin reduction in apple juice by inactivated Alicyclobacillus spp.

    Science.gov (United States)

    Yuan, Y; Wang, X; Hatab, S; Wang, Z; Wang, Y; Luo, Y; Yue, T

    2014-12-01

    This study aimed to investigate the reduction of patulin (PAT) in apple juice by 12 inactivated Alicyclobacillus strains. The reduction rate of PAT by each strain was determined by high-performance liquid chromatography (HPLC). The results indicated that the removal of PAT was strain specific. Alicyclobacillus acidoterrestris 92 and A. acidoterrestris 96 were the most effective ones among the 12 tested strains in the removal of PAT. Therefore, these two strains were selected to study the effects of incubation time, initial PAT concentration and bacteria powder amount on PAT removal abilities of Alicyclobacillus. The highest PAT reduction rates of 88·8 and 81·6% were achieved after 24-h incubation with initial PAT concentration of 100 μg l(-1) and bacteria powder amount of 40 g l(-1) , respectively. Moreover, it was found that the treatment by these 12 inactivated Alicyclobacillus strains had no negative effect on the quality parameters of apple juice. Similar assays were performed in supermarket apple juice, where inactivated Alicyclobacillus cells could efficiently reduce PAT content. Taken together, these data suggest the possible application of this strategy as a means to detoxify PAT-contaminated juices. Inactivated Alicyclobacillus cells can efficiently reduce patulin concentration in apple juice. It provides a theoretical foundation for recycling of Alicyclobacillus cells from spoiled apple juice to reduce the source of pollution and the cost of juice industry. This is the first report on the use of Alicyclobacillus to remove patulin from apple juice. © 2014 The Society for Applied Microbiology.

  13. Inactivation of murine norovirus by chemical biocides on stainless steel

    Directory of Open Access Journals (Sweden)

    Steinmann Jörg

    2009-07-01

    Full Text Available Abstract Background Human norovirus (NoV causes more than 80% of nonbacterial gastroenteritis in Europe and the United States. NoV transmission via contaminated surfaces may be significant for the spread of viruses. Therefore, measures for prevention and control, such as surface disinfection, are necessary to interrupt the dissemination of human NoV. Murine norovirus (MNV as a surrogate for human NoV was used to study the efficacy of active ingredients of chemical disinfectants for virus inactivation on inanimate surfaces. Methods The inactivating properties of different chemical biocides were tested in a quantitative carrier test with stainless steel discs without mechanical action. Vacuum-dried MNV was exposed to different concentrations of alcohols, peracetic acid (PAA or glutaraldehyde (GDA for 5 minutes exposure time. Detection of residual virus was determined by endpoint-titration on RAW 264.7 cells. Results PAA [1000 ppm], GDA [2500 ppm], ethanol [50% (v/v] and 1-propanol [30% (v/v] were able to inactivate MNV under clean conditions (0.03% BSA on the carriers by ≥ 4 log10 within 5 minutes exposure time, whereas 2-propanol showed a reduced effectiveness even at 60% (v/v. Furthermore, there were no significant differences in virus reduction whatever interfering substances were used. When testing with ethanol, 1- and 2-propanol, results under clean conditions were nearly the same as in the presence of dirty conditions (0.3% BSA plus 0.3% erythrocytes. Conclusion Products based upon PAA, GDA, ethanol and 1-propanol should be used for NoV inactivation on inanimate surfaces. Our data provide valuable information for the development of strategies to control NoV transmission via surfaces.

  14. Inactivation of Bacillus Anthracis Spores Using Carbon Nanotubes

    Science.gov (United States)

    2014-10-30

    AND SUBTITLE 13. SUPPLEMENTARY NOTES 12. DISTRIBUTION AVAILIBILITY STATEMENT 6. AUTHORS 7. PERFORMING ORGANIZATION NAMES AND ADDRESSES 15. SUBJECT...2010 31-May-2014 Approved for Public Release; Distribution Unlimited Final Report: (Life Science Division/Biochemistry) Inactivation of Bacillus ...S) AND ADDRESS (ES) U.S. Army Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 Bacillus Anthracis, Spores, Biofilm, Inhibition

  15. Degradation and inactivation of Shiga toxins by nitrogen gas plasma.

    Science.gov (United States)

    Sakudo, Akikazu; Imanishi, Yuichiro

    2017-12-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) leads to food poisoning by causing hemorrhagic colitis and hemolytic uremic syndrome. Some STEC produce Shiga toxin 1 (Stx1) and/or Shiga toxin 2 (Stx2), a relatively stable protein toxin, necessitating the development of an efficient inactivation method. Here we applied a nitrogen gas plasma apparatus to the inactivation of Stx. Samples of Stx1 and Stx2 were treated with a nitrogen gas plasma generated by a plasma device using a short high-voltage pulse applied by a static induction thyristor power supply at 1.5 kpps (kilo pulse per second). The recovered Stx samples were then analyzed for immunological and biological activities. Immunochromatography demonstrated that Stx1 and Stx2 were degraded by the gas plasma. Quantification by enzyme-linked immunosorbent assay (ELISA) showed that both toxins were efficiently degraded to less than 1/10th of their original concentration within 5 min of treatment. Western blotting further showed the gas plasma treatment degraded the A subunit, which mediates the toxicity of Stx. Moreover, an assay using HEp-2 cells as an index of cytotoxicity showed that gas plasma treatment reduced the toxic activity of Stx. Therefore, nitrogen gas plasma might be an efficient method for the inactivation of Stx.

  16. Influenza Vaccination Strategies: Comparing Inactivated and Live Attenuated Influenza Vaccines

    Science.gov (United States)

    Sridhar, Saranya; Brokstad, Karl A.; Cox, Rebecca J.

    2015-01-01

    Influenza is a major respiratory pathogen causing annual outbreaks and occasional pandemics. Influenza vaccination is the major method of prophylaxis. Currently annual influenza vaccination is recommended for groups at high risk of complications from influenza infection such as pregnant women, young children, people with underlying disease and the elderly, along with occupational groups such a healthcare workers and farm workers. There are two main types of vaccines available: the parenteral inactivated influenza vaccine and the intranasal live attenuated influenza vaccine. The inactivated vaccines are licensed from 6 months of age and have been used for more than 50 years with a good safety profile. Inactivated vaccines are standardized according to the presence of the viral major surface glycoprotein hemagglutinin and protection is mediated by the induction of vaccine strain specific antibody responses. In contrast, the live attenuated vaccines are licensed in Europe for children from 2–17 years of age and provide a multifaceted immune response with local and systemic antibody and T cell responses but with no clear correlate of protection. Here we discuss the immunological immune responses elicited by the two vaccines and discuss future work to better define correlates of protection. PMID:26343192

  17. Raman spectroscopy of Bacillus thuringiensis physiology and inactivation

    Science.gov (United States)

    Morrow, J. B.; Almeida, J.; Cole, K. D.; Reipa, V.

    2012-12-01

    The ability to detect spore contamination and inactivation is relevant to developing and determining decontamination strategy success for food and water safety. This study was conducted to develop a systematic comparison of nondestructive vibrational spectroscopy techniques (Surface-Enhanced Raman Spectroscopy, SERS, and normal Raman) to determine indicators of Bacillus thuringiensis physiology (spore, vegetative, outgrown, germinated and inactivated spore forms). SERS was found to provide better resolution of commonly utilized signatures of spore physiology (dipicolinic acid at 1006 cm-1 and 1387 cm-1) compared to normal Raman and native fluorescence indigenous to vegetative and outgrown cell samples was quenched in SERS experiment. New features including carotenoid pigments (Raman features at 1142 cm-1, 1512 cm-1) were identified for spore cell forms. Pronounced changes in the low frequency region (300 cm-1 to 500 cm-1) in spore spectra occurred upon germination and inactivation (with both free chlorine and by autoclaving) which is relevant to guiding decontamination and detection strategies using Raman techniques.

  18. Pulsed light inactivation of horseradish peroxidase and associated structural changes.

    Science.gov (United States)

    Pellicer, José Antonio; Gómez-López, Vicente M

    2017-12-15

    Pulsed light (PL) is a non-thermal preservation method in which foods are subjected to one or several intense pulses of wide-spectrum light. Peroxidase (POD) is an enzyme that needs to be inactivated or inhibited because of its deleterious effects on the quality of fruits and vegetables. The feasibility of using PL to inactivate POD was tested and results explained based on measurements of UV-vis spectrum, far-UV circular dichroism and tryptophan fluorescence, and the phase-diagram method. PL reduced the activity of POD by more than 95% after applying 128Jcm -2 . There was observed a decrease in the Reinheitzahl value and ellipticity and an increase in tryptophan fluorescence at incremental fluences, as well as linear phase diagrams. The study indicates that the inactivation of POD by PL is an all-or-none process related to loss of helical structure, weak unfolding and ejection of the prostetic group. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Photosensitized inactivation of infectious blood-borne human parasites

    Science.gov (United States)

    Judy, Millard M.; Sogandares-Bernal, Franklin M.; Matthews, James Lester

    1995-05-01

    Blood-borne viruses and protozoan parasites that are infectious to humans pose risk world-wide of infection transmission through blood and blood product transfusion. Blood-borne infectious viruses include human immunodeficiency virus (HIV-I), which causes AIDS; hepatitis C virus, which can cause chronic hepatitis; and cytomegalovirus, which can be dangerous to immunocompromised patients, e.g., the newborn, transplant recipients, and AIDS patients. Infectious blood-borne protozoan parasites include Trypanosoma cruzi, which causes Chagas' disease, endemic throughout Central and South America; the Trypanosoma species causing African sleeping sickness endemic in Central Africa; and Plasmodium falciparum, which causes malignant and increasingly drug- resistant human malaria prevalent throughout the tropics. Some researchers have focused on using photosensitizers to inactivate HIV-I and other viruses in whole blood, packed red cells, and platelet concentrates without compromising blood product function. Our group previously has reported photosensitized in vitro inactivation of P. falciparum and the mouse malaria organism Plasmodium berghei in whole blood using hematoporphyrin derivative (HPD) and of T. cruzi using benzoporphyrin derivatives BPDMA and BPDDA, dihematoporphyrin ether (DHE), and hydroxyethylvinyldeuteroporphyrin (HEVD). These results suggest that continued investigation is warranted to evaluate the potential for photosensitized inactivation of blood-borne parasites in blood banking.

  20. Influenza Vaccination Strategies: Comparing Inactivated and Live Attenuated Influenza Vaccines

    Directory of Open Access Journals (Sweden)

    Saranya Sridhar

    2015-04-01

    Full Text Available Influenza is a major respiratory pathogen causing annual outbreaks and occasional pandemics. Influenza vaccination is the major method of prophylaxis. Currently annual influenza vaccination is recommended for groups at high risk of complications from influenza infection such as pregnant women, young children, people with underlying disease and the elderly, along with occupational groups such a healthcare workers and farm workers. There are two main types of vaccines available: the parenteral inactivated influenza vaccine and the intranasal live attenuated influenza vaccine. The inactivated vaccines are licensed from 6 months of age and have been used for more than 50 years with a good safety profile. Inactivated vaccines are standardized according to the presence of the viral major surface glycoprotein hemagglutinin and protection is mediated by the induction of vaccine strain specific antibody responses. In contrast, the live attenuated vaccines are licensed in Europe for children from 2–17 years of age and provide a multifaceted immune response with local and systemic antibody and T cell responses but with no clear correlate of protection. Here we discuss the immunological immune responses elicited by the two vaccines and discuss future work to better define correlates of protection.

  1. Thermal inactivation kinetics of hepatitis A virus in spinach.

    Science.gov (United States)

    Bozkurt, Hayriye; Ye, Xiaofei; Harte, Federico; D'Souza, Doris H; Davidson, P Michael

    2015-01-16

    Leafy vegetables have been recognized as important vehicles for the transmission of foodborne viral pathogens. To control hepatitis A viral foodborne illness outbreaks associated with mildly heated (e.g., blanched) leafy vegetables such as spinach, generation of adequate thermal processes is important both for consumers and the food industry. Therefore, the objectives of this study were to determine the thermal inactivation behavior of hepatitis A virus (HAV) in spinach, and provide insights on HAV inactivation in spinach for future studies and industrial applications. The D-values calculated from the first-order model (50-72 °C) ranged from 34.40 ± 4.08 to 0.91 ± 0.12 min with a z-value of 13.92 ± 0.87 °C. The calculated activation energy value was 162 ± 11 kJ/mol. Using the information generated in the present study and the thermal parameters of industrial blanching conditions for spinach as a basis (100 °C for 120-180 s), the blanching of spinach in water at 100 °C for 120-180 s under atmospheric conditions will provide greater than 6 log reduction of HAV. The results of this study may be useful to the frozen food industry in designing blanching conditions for spinach to inactivate or control hepatitis A virus outbreaks. Copyright © 2014. Published by Elsevier B.V.

  2. Isolation and characterization of the moxJ, moxG, moxI, and moxR genes of Paracoccus denitrificans: inactivation of moxJ, moxG, and moxR and the resultant effect on methylotrophic growth.

    OpenAIRE

    Van Spanning, R J; Wansell, C W; De Boer, T; Hazelaar, M J; Anazawa, H; Harms, N; Oltmann, L F; Stouthamer, A H

    1991-01-01

    By using the moxF gene encoding the large fragment of methanol dehydrogenase as a probe, a downstream linked chromosomal fragment was isolated from a genomic bank of Paracoccus denitrificans. The nucleotide sequence of the fragment was determined and revealed the 3' part of moxF, four additional open reading frames, and the 5' part of a sixth one. The organization and deduced amino acid sequences of the first three frames downstream from moxF were found to be largely homologous to the moxJ, m...

  3. Method for flow cytometric monitoring of Renibacterium salmoninarum inactivation

    Science.gov (United States)

    Pascho, R.J.; Ongerth, J.E.

    2000-01-01

    The slow growth of Renibacterium salmoninarum limits the usefulness of culture as a research tool. Development of a 2-color flow cytometric assay to quantify the proportions of live and dead R. salmoninarum in a test population is described. Bacteria were simultaneously stained with fluorescein isothiocyanate-conjugated immunoglobulin and exposed to the exclusion dye propidium iodide. Propidium iodide red fluorescence profiles of control groups of untreated and killed R. salmoninarum were compared with those for bacteria exposed to chlorine. Bacterial inactivation was based on mean red fluorescence intensity, and analyzed by high-red fluorescence intensity (HRFI) and curve subtraction (CS) analyses. When the concentration of R. salmoninarum was 8.65 x 106 bacteria ml-1 and the bacteria exposed to chlorine at 1 mg l-1 for periods from 1 to 20 min (high-Rs assessment), the mean red fluorescence intensity of the profile for each chlorine-exposure group was higher than that for the untreated control (p mean red fluorescence intensities of the exposure groups were higher than that for the untreated control only when the R. salmoninarum was exposed to chlorine for at least 1 min (p ??? 0.01). On the basis of red fluorescence intensity, the proportion of dead cells generally increased with the duration of chlorine exposure. Whereas the rates of inactivation derived from the HRFI and CS analyses did not correlate with the duration of exposure in the high-Rs assessment (r2 ??? 0.27), there was a correlation between these estimates and the duration of exposure in the reduced-Rs assessment (r2 ??? 0.92). Because of the rapid loss of culturable R. salmoninarum in both assessments following chlorine exposure, neither the duration of exposure nor the inactivation estimates correlated with bacteriological culture (r2 ??? 0.22). In both assessments, there was a correlation between the estimates of inactivation based upon HRFI and CS analyses (r2 > 0.99). These results suggest that

  4. Exposure to heat-inactivated Trichophyton rubrum resulting in a limited immune response of human keratinocytes.

    Science.gov (United States)

    Huang, Xiao-Qiang; Yi, Jin-Ling; Yin, Song-Chao; Chen, Rong-Zhang; Li, Mei-Rong; Gong, Zi-Jian; Lai, Wei; Chen, Jian

    2013-01-01

    Trichophyton rubrum (T. rubrum) represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested that it may result from the failure of epithelial cells to recognize T. rubrum effectively and initiate effective immune responses. The C-type lectin receptors (CLR) and toll-like receptors (TLR) are the two major pattern recognition receptors (PRRs) that recognize fungal components. Therefore, the purpose of the study was to analyze the expression of those PRRs and the cytokines in HaCaT cells stimulated with heat-inactivated T. rubrum conidia and hyphae, respectively. HaCaT cells were unstimulated or stimulated with heat-inactivated T. rubrum conidia and hyphae (1×10(6) and 1.5×10(5) colony-forming unit (CFU) in 2 ml medium, respectively) for 6, 12 and 24 hours. The mRNA expression of PRRs involved in recognizing fungal pathogen-associated molecular patterns (PAMPs) and signaling molecules were measured by quantitative reverse transcription polymerase chain reaction (RT-PCR). Meanwhile, surface toll-like receptor (TLR) 2, TLR4 and Dectin-1 were analyzed by fluorescence-activated cell sorter (FACS) 24 hours after treatment. The cytokines were detected in cell culture supernatants of HaCaT cells in 12 and 24 hours after treatment. HaCaT cells constitutively expressed mRNA of membrane-bound TLR1, 2, 4 and 6, Dectin1 and DC-SIGN, but not Dectin-2 or Mincle. Heat-killed T. rubrum did not significantly upregulate gene transcriptions of the PRRs of HaCaT cells. Heat-inactivated T. rubrum conidia significantly reduced the surface expression of TLR2 and Dectin-1, and suppressed the secretions of interferon-inducible protein-10 (IP-10) and monocyte chemotactic protein-1 (MCP-1) of HaCaT cells, while heat-killed T. rubrum hyphae significantly induced the secretions of IP-10 and MCP-1. The cell-wall antigens of T. rubrum fail to activate transcriptional expression of PRRs and

  5. Inactivation of B. subtilis spores by low pressure plasma—influence of optical filters and photon/particle fluxes on the inactivation efficiency

    Science.gov (United States)

    Fiebrandt, Marcel; Hillebrand, Bastian; Lackmann, Jan-Wilm; Raguse, Marina; Moeller, Ralf; Awakowicz, Peter; Stapelmann, Katharina

    2018-01-01

    Inactivation experiments were performed with Bacillus subtilis spores in a low pressure double inductively coupled plasma (DICP) system. Argon, nitrogen and oxygen at 5 Pa were used as feed gas to change the emission spectrum in the range of 100 nm to 400 nm, as well as between radical and metastable densities. Optical filters were applied, to block particles and selected wavelengths from the spores. By determining absolute photon fluxes, the sporicidal efficiency of various wavelength ranges was evaluated. The results showed good agreement with other plasma experiments, as well as with monochromatic light inactivation experiments from a synchrotron. The findings indicated that the inactivation rate constants of broadband plasma emission and monochromatic light were identical, and that no synergistic effect exists. Furthermore, the influence of radicals, ions and metastables on the inactivation efficiency was of minor importance in the set-up used, and radiation was the main reason for spore inactivation.

  6. Renal Cell Carcinoma With Chromosome 6p Amplification Including the TFEB Gene: A Novel Mechanism of Tumor Pathogenesis?

    Science.gov (United States)

    Williamson, Sean R; Grignon, David J; Cheng, Liang; Favazza, Laura; Gondim, Dibson D; Carskadon, Shannon; Gupta, Nilesh S; Chitale, Dhananjay A; Kalyana-Sundaram, Shanker; Palanisamy, Nallasivam

    2017-03-01

    Amplification of chromosome 6p has been implicated in aggressive behavior in several cancers, but has not been characterized in renal cell carcinoma (RCC). We identified 9 renal tumors with amplification of chromosome 6p including the TFEB gene, 3 by fluorescence in situ hybridization, and 6 from the Cancer Genome Atlas (TCGA) databases. Patients' ages were 28 to 78 years (median, 61 y). Most tumors were high stage (7/9 pT3a, 2/9 pN1). Using immunohistochemistry, 2/4 were positive for melanocytic markers and cathepsin K. Novel TFEB fusions were reported by TCGA in 2; however, due to a small composition of fusion transcripts compared with full-length transcripts (0.5/174 and 3.3/132 FPKM), we hypothesize that these represent secondary fusions due to amplification. Five specimens (4 TCGA, 1 fluorescence in situ hybridization) had concurrent chromosome 3p copy number loss or VHL deletion. However, these did not resemble clear cell RCC, had negative carbonic anhydrase IX labeling, lacked VHL mutation, and had papillary or unclassified histology (2/4 had gain of chromosome 7 or 17). One tumor each had somatic FH mutation and SMARCB1 mutation. Chromosome 6p amplification including TFEB is a previously unrecognized cytogenetic alteration in RCC, associated with heterogenous tubulopapillary eosinophilic and clear cell histology. The combined constellation of features does not fit cleanly into an existing tumor category (unclassified), most closely resembling papillary or translocation RCC. The tendency for high tumor stage, varied tubulopapillary morphology, and a subset with melanocytic marker positivity suggests the possibility of a unique tumor type, despite some variation in appearance and genetics.

  7. Down regulation of StGA3ox genes in potato results in altered GA content and affect plant and tuber growth characteristics.

    NARCIS (Netherlands)

    Roumeliotis, E.; Kloosterman, B.A.; Oortwijn, M.E.P.; Lange, Theo; Visser, R.G.F.; Bachem, C.W.B.

    2013-01-01

    GA biosynthesis and catabolism has been shown to play an important role in regulating tuberization in potato. Active GAs are inactivated in the stolon tips shortly after induction to tuberization. Overexpression of a GA inactivation gene results in an earlier tuberization phenotype, while reducing

  8. Polymorphism and genetic mapping of the human oxytocin receptor gene on chromosome 3

    Energy Technology Data Exchange (ETDEWEB)

    Michelini, S.; Urbanek, M.; Goldman, D. [National Institute of Health-National Institute of Alcohol Abuse and Alcoholism, Rockville, MD (United States)] [and others

    1995-06-19

    Centrally administered oxytocin has been reported to facilitate affiliative and social behaviors, in functional harmony with its well-known peripheral effects on uterine contraction and milk ejection. The biological effects of oxytocin could be perturbed by mutations occurring in the sequence of the oxytocin receptor gene, and it would be of interest to establish the position of this gene on the human linkage map. Therefore we identified a polymorphism at the human oxytocin receptor gene. A portion of the 3{prime} untranslated region containing a 30 bp CA repeat was amplified by polymerase chain reaction (PCR), revealing a polymorphism with two alleles occurring with frequencies of 0.77 and 0.23 in a sample of Caucasian CEPH parents (n = 70). The CA repeat polymorphism we detected was used to map the human oxytocin receptor to chromosome 3p25-3p26, in a region which contains several important genes, including loci for Von Hippel-Lindau disease (VHL) and renal cell carcinoma. 53 refs., 2 figs., 1 tab.

  9. Brains, Genes and Primates

    Science.gov (United States)

    Belmonte, Juan Carlos Izpisua; Callaway, Edward M.; Churchland, Patricia; Caddick, Sarah J.; Feng, Guoping; Homanics, Gregg E.; Lee, Kuo-Fen; Leopold, David A.; Miller, Cory T.; Mitchell, Jude F.; Mitalipov, Shoukhrat; Moutri, Alysson R.; Movshon, J. Anthony; Okano, Hideyuki; Reynolds, John H.; Ringach, Dario; Sejnowski, Terrence J.; Silva, Afonso C.; Strick, Peter L.; Wu, Jun; Zhang, Feng

    2015-01-01

    One of the great strengths of the mouse model is the wide array of genetic tools that have been developed. Striking examples include methods for directed modification of the genome, and for regulated expression or inactivation of genes. Within neuroscience, it is now routine to express reporter genes, neuronal activity indicators and opsins in specific neuronal types in the mouse. However, there are considerable anatomical, physiological, cognitive and behavioral differences between the mouse and the human that, in some areas of inquiry, limit the degree to which insights derived from the mouse can be applied to understanding human neurobiology. Several recent advances have now brought into reach the goal of applying these tools to understanding the primate brain. Here we describe these advances, consider their potential to advance our understanding of the human brain and brain disorders, discuss bioethical considerations, and describe what will be needed to move forward. PMID:25950631

  10. Molecular Cloning and Characterization of Ecdysone oxidase and 3-dehydroecdysone-3α-reductase Involved in the Ecdysone Inactivation Pathway of Silkworm, Bombyx mori

    Science.gov (United States)

    Sun, Wei; Shen, Yi-Hong; Qi, Deng-Wei; Xiang, Zhong-Huai; Zhang, Ze

    2012-01-01

    Molting hormone (ecdysteroid) is one of the most important hormones in insects. The synthesis and inactivation of the ecdysteroid regulate the developmental process of insects. A major pathway of ecdysone inactivation is that ecdysone is converted to 3-dehydroecdysone, and then further to 3-epiecdysone in insects. Two enzymes (ecdysone oxidase: EO and 3DE-3α-reductase) participate in this pathway. In this study, based on the previously characterized cDNAs in Spodoptera littoralis, we cloned and characterized EO and 3DE-3α-reductase genes in the silkworm, Bombyx mori. The heterologously expressed proteins of the two genes in yeast showed the ecdysone oxidase and 3DE-3α-reductase activities, respectively. Expression of BmEO was only detected in the midgut at transcriptional and translational levels. We also localized EO within the midgut goblet cell cavities. For Bm3DE-3α-reductase gene, RT-PCR and western blot showed that it was expressed in the midgut and the Malpighian tubules. Moreover, we localized 3DE-3α-reductase within the midgut goblet cell cavities and the cytosol of principal cells of the Malpighian tubules. These two genes have similar expression profiles during different developmental stages. Both genes were highly expressed at the beginning of the 5th instar, and remained a relative low level during the feeding stage, and then were highly expressed at the wandering stage. All these results showed that the profiles of the two genes were well correlated with the ecdysteroid titer. The functional characterization of the enzymes participating in ecdysone inactivation in the silkworm provides hints for the artificial regulation of the silkworm development and biological control of pests. PMID:22215981

  11. Sex chromosome-specific regulation in the Drosophila male germline but little evidence for chromosomal dosage compensation or meiotic inactivation.

    Directory of Open Access Journals (Sweden)

    Colin D Meiklejohn

    2011-08-01

    Full Text Available The evolution of heteromorphic sex chromosomes (e.g., XY in males or ZW in females has repeatedly elicited the evolution of two kinds of chromosome-specific regulation: dosage compensation--the equalization of X chromosome gene expression in males and females--and meiotic sex chromosome inactivation (MSCI--the transcriptional silencing and heterochromatinization of the X during meiosis in the male (or Z in the female germline. How the X chromosome is regulated in the Drosophila melanogaster male germline is unclear. Here we report three new findings concerning gene expression from the X in Drosophila testes. First, X chromosome-wide dosage compensation appears to be absent from most of the Drosophila male germline. Second, microarray analysis provides no evidence for X chromosome-specific inactivation during meiosis. Third, we confirm the previous discovery that the expression of transgene reporters driven by autosomal spermatogenesis-specific promoters is strongly reduced when inserted on the X chromosome versus the autosomes; but we show that this chromosomal difference in expression is established in premeiotic cells and persists in meiotic cells. The magnitude of the X-autosome difference in transgene expression cannot be explained by the absence of dosage compensation, suggesting that a previously unrecognized mechanism limits expression from the X during spermatogenesis in Drosophila. These findings help to resolve several previously conflicting reports and have implications for patterns of genome evolution and speciation in Drosophila.

  12. CFTR inactivation by lentiviral vector-mediated RNA interference and CRISPR-Cas9 genome editing in human airway epithelial cells.

    Science.gov (United States)

    Bellec, Jessica; Bacchetta, Marc; Losa, Davide; Anegon, Ignacio; Chanson, Marc; Nguyen, Tuan Huy

    2015-01-01

    Polarized airway epithelial cell cultures modelling Cystic Fibrosis Transmembrane conductance Regulator (CFTR) defect are crucial for CF and biomedical research. RNA interference has proven its value to generate knockdown models for various pathologies. More recently, genome editing using CRISPR-Cas9 artificial endonuclease was a valuable addition to the toolbox of gene inactivation. Calu-3 cells and primary HAECs were transduced with HIV-1-derived lentiviral vectors (LVV) encoding small hairpin RNA (shRNA) sequence or CRISPR-Cas9 components targeting CFTR alongside GFP. After sorting of GFP-positive cells, CFTR expression was measured by RT-qPCR and Western blot in polarized or differentiated cells. CFTR channel function was assessed in Ussing chambers. Il-8 secretion, proliferation and cell migration were also studied in transduced cells. shRNA interference and CRISPRCas9 strategies efficiently decreased CFTR expression in Calu-3 cells. Strong CFTR knockdown was confirmed at the functional level in CRISPR-Cas9-modified cells. CFTR-specific shRNA sequences did not reduce gene expression in primary HAECs, whereas CRISPR-Cas9-mediated gene modification activity was correlated with a reduction of transepithelial secretion and response to a CFTR inhibitor. CFTR inactivation in the CRISPR-Cas9-modified Calu-3 cells did not affect migration and proliferation but slightly increased basal interleukin-8 secretion. We generated CFTR inactivated cell lines and demonstrated that CRISPR-Cas9 vectorised in a single LVV efficiently promotes CFTR inactivation in primary HAECs. These results provide a new protocol to engineer CF primary epithelia with their isogenic controls and pave the way for manipulation of CFTR expression in these cultures.

  13. Mitotic recombination and compound-heterozygous mutations are predominant NF1-inactivating mechanisms in children with juvenile myelomonocytic leukemia and neurofibromatosis type 1

    OpenAIRE

    Steinemann, Doris; Arning, Larissa; Praulich, Inka; Stuhrmann, Manfred; Hasle, Henrik; Starý, Jan; Schlegelberger, Brigitte; Niemeyer, Charlotte M.; Flotho, Christian

    2009-01-01

    Children with neurofibromatosis type 1 (NF-1), being constitutionally deficient for one allele of the NF1 gene, are at greatly increased risk of juvenile myelomonocytic leukemia (JMML). NF1 is a negative regulator of RAS pathway activity, which has a central role in JMML. To further clarify the role of biallelic NF1 gene inactivation in the pathogenesis of JMML, we investigated the somatic NF1 lesion in 10 samples from children with JMML/NF-1. We report that two-thirds of somatic events invol...

  14. The carbapenem inactivation method (CIM, a simple and low-cost alternative for the Carba NP test to assess phenotypic carbapenemase activity in gram-negative rods.

    Directory of Open Access Journals (Sweden)

    Kim van der Zwaluw

    Full Text Available A new phenotypic test, called the Carbapenem Inactivation Method (CIM, was developed to detect carbapenemase activity in Gram-negative rods within eight hours. This method showed high concordance with results obtained by PCR to detect genes coding for the carbapenemases KPC, NDM, OXA-48, VIM, IMP and OXA-23. It allows reliable detection of carbapenemase activity encoded by various genes in species of Enterobacteriaceae (e.g., Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae, but also in non-fermenters Pseudomonas aeruginosa and Acinetobacter baumannii. The CIM was shown to be a cost-effective and highly robust phenotypic screening method that can reliably detect carbapenemase activity.

  15. Molecular analyses of novel ASAH1 mutations causing Farber lipogranulomatosis: analyses of exonic splicing enhancer inactivating mutation.

    Science.gov (United States)

    Bashyam, M D; Chaudhary, A K; Kiran, M; Reddy, V; Nagarajaram, H A; Dalal, A; Bashyam, L; Suri, D; Gupta, A; Gupta, N; Kabra, M; Puri, R D; RamaDevi, R; Kapoor, S; Danda, S

    2014-12-01

    Farber lipogranulomatosis is a rare autosomal recessive lysosomal storage disorder caused by mutations in the ASAH1 gene. In the largest ever study, we identified and characterized ASAH1 mutations from 11 independent Farber disease (FD) families. A total of 13 different mutations were identified including 1 splice, 1 polypyrimidine tract (PPT) deletion and 11 missense mutations. Eleven mutations were exclusive to the Indian population. The IVS6+4A>G splice and IVS5-16delTTTTC PPT deletion mutations resulted in skipping of exon 6 precluding thereby the region responsible for cleavage of enzyme precursor. A missense mutation (p.V198A) resulted in skipping of exon 8 due to inactivation of an exonic splicing enhancer (ESE) element. This is the first report of mutations affecting PPT and ESE in the ASAH1 gene resulting in FD. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. The dynamic changes of X chromosome inactivation during early culture of human embryonic stem cells.

    Science.gov (United States)

    Xie, Pingyuan; Ouyang, Qi; Leng, Lizhi; Hu, Liang; Cheng, Dehua; Tan, Yueqiu; Lu, Guangxiu; Lin, Ge

    2016-07-01

    X chromosome inactivation (XCI) is required for dosage compensation of X-linked genes in human female cells. Several previous reports have described the promiscuous XCI status in long-term cultured female human embryonic stem cells (hESCs), and the majority of them exhibit non-random XCI. However, when and how such female hESCs acquire the aberrant XCI states during culture is unknown. Herein, through comparing the XCI states in 18 paired hES cell lines throughout early culture, we revealed a uniform dynamic change during this culture period under a widely used culture condition. The female initial hESCs (ihESCs, P4-P9) expressed XIST RNA, H3K27me3 punctate enrichment and displayed random XCI pattern. By further culturing, the female early hESCs (ehESCs, P20-P30) lost the expression of XIST RNA, H3K27me3 punctate enrichment and exhibited a completely skewed XCI pattern. Importantly, a subset of X-linked genes was up-regulated in ehESCs, including some cancer-related genes. At last, we found 5% physiological oxygen was beneficial for the expression of XIST and H3K27me3 punctate enrichment, but not for the XCI pattern. We conclude that the XCI dynamic change is a frequent epigenetic instability event during early culture, which is accompanied by the up-regulation of some X-linked genes. Furthermore, we emphasize that physiological oxygen is beneficial for XCI fidelity. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  17. Biallelic inactivation of protoporphyrinogen oxidase and hydroxymethylbilane synthase is associated with liver cancer in acute porphyrias.

    Science.gov (United States)

    Schneider-Yin, Xiaoye; van Tuyll van Serooskerken, Anne-Moon; Siegesmund, Marko; Went, Philip; Barman-Aksözen, Jasmin; Bladergroen, Reno S; Komminoth, Paul; Cloots, Roy H E; Winnepenninckx, Véronique J; zur Hausen, Axel; Weber, Markus; Driessen, Ann; Poblete-Gutiérrez, Pamela; Bauer, Peter; Schroeder, Christopher; van Geel, Michel; Minder, Elisabeth I; Frank, Jorge

    2015-03-01

    Variegate porphyria (VP) and acute intermittent porphyria (AIP), the two most common types of acute porphyrias (AHPs), result from a partial deficiency of protoporphyrinogen oxidase (PPOX) and hydroxymethylbilane synthase (HMBS), respectively. A rare but serious complication in the AHPs is hepatocellular carcinoma (HCC). However, the underlying pathomechanisms are yet unknown. We performed DNA sequence analysis in cancerous and non-cancerous liver tissue of a VP and an AIP patient, both with HCC. In samples of both cancerous and non-cancerous liver tissues from the patients, we identified the underlying PPOX and HMBS germline mutations, c.1082dupC and p.G111R, respectively. Additionally, we detected a second somatic mutation, only in the cancer tissue i.e., p.L416X in the PPOX gene of the VP patient and p.L220X in the HMBS gene of the AIP patient, both located in trans to the respective germline mutations. Both somatic mutations were not detected in 10 non-porphyria-associated HCCs. Our data demonstrate that in the hepatic cancer tissue of AHP patients, somatic second-hit mutations result in nearly complete inactivation of the enzymes catalyzing major steps in the heme biosynthetic pathway. Both PPOX and HMBS, which might act as tumor suppressors, play a crucial role in the development of HCC in these individuals. Copyright © 2014 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

  18. Genetically dependent modulation of serotonergic inactivation in the human prefrontal cortex.

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    Passamonti, Luca; Cerasa, Antonio; Gioia, Maria Cecilia; Magariello, Angela; Muglia, Maria; Quattrone, Aldo; Fera, Francesco

    2008-04-15

    Previous research suggests that genetic variations regulating serotonergic neurotransmission mediate individual differences in the neural network underlying impulsive and aggressive behaviour. Although with conflicting findings, the monoamine oxidase-A (MAOA) and the serotonin transporter (5HTT) gene polymorphisms have been associated with an increased risk to develop impulsive and aggressive behaviour. Double knock-out mice studies have also demonstrated that MAOA and 5HTT genes strongly interact in the metabolic pathway leading to the serotonergic inactivation; however, their potential interactive effect in human brain remains uninvestigated. We used blood oxygenation level-dependent (BOLD) functional magnetic resonance imaging (fMRI) to assess the independent and interactive effects of both MAOA and 5HTT polymorphisms on the brain activity elicited by a response inhibition task in healthy volunteers. Multivariate analysis demonstrated an individual effect of both MAOA and 5HTT polymorphisms and a strong allele-allele interaction in the anterior cingulate cortex (ACC), a key region implicated in cognitive control and in the pathophysiology of impulsive and aggressive behaviour. These findings suggest that the MAOAx5HTT allelic interaction exerts a significant modulation on the BOLD response associated with response inhibition and contribute to validate haplotype models as useful tools for a better understanding of the neurobiology underlying complex cognitive functions.

  19. Influence of activation and germination on high pressure inactivation of ascospores of the mould Eurotium repens.

    Science.gov (United States)

    Eicher, R; Ludwig, H

    2002-03-01

    We investigated heat activation and germination of Eurotium repens ascospores to follow high pressure inactivation. Activation energy and entropy values strengthen the idea of protein denaturation as the underlying mechanism of heat activation. Preceding activation, germination or a combination of both affected high pressure inactivation in different ways. Activation followed immediately by high pressure treatment led to the most efficient improvement in inactivation. However, a pause after activation caused a partial re-establishment of the spores' stability and less efficient high pressure inactivation. Germination stabilized the spores against high pressure. A combined treatment of activation and germination led to an initially fast inactivation, but compared to high pressure treatment of only activated spores the time course of inactivation was slowed down.

  20. Arabidopsis JASMONATE-INDUCED OXYGENASES down-regulate plant immunity by hydroxylation and inactivation of the hormone jasmonic acid.

    Science.gov (United States)

    Caarls, Lotte; Elberse, Joyce; Awwanah, Mo; Ludwig, Nora R; de Vries, Michel; Zeilmaker, Tieme; Van Wees, Saskia C M; Schuurink, Robert C; Van den Ackerveken, Guido

    2017-06-13

    The phytohormone jasmonic acid (JA) is vital in plant defense and development. Although biosynthesis of JA and activation of JA-responsive gene expression by the bioactive form JA-isoleucine have been well-studied, knowledge on JA metabolism is incomplete. In particular, the enzyme that hydroxylates JA to 12-OH-JA, an inactive form of JA that accumulates after wounding and pathogen attack, is unknown. Here, we report the identification of four paralogous 2-oxoglutarate/Fe(II)-dependent oxygenases in Arabidopsis thaliana as JA hydroxylases and show that they down-regulate JA-dependent responses. Because they are induced by JA we named them JASMONATE-INDUCED OXYGENASES (JOXs). Concurrent mutation of the four genes in a quadruple Arabidopsis mutant resulted in increased defense gene expression and increased resistance to the necrotrophic fungus Botrytis cinerea and the caterpillar Mamestra brassicae In addition, root and shoot growth of the plants was inhibited. Metabolite analysis of leaves showed that loss of function of the four JOX enzymes resulted in overaccumulation of JA and in reduced turnover of JA into 12-OH-JA. Transformation of the quadruple mutant with each JOX gene strongly reduced JA levels, demonstrating that all four JOXs inactivate JA in plants. The in vitro catalysis of 12-OH-JA from JA by recombinant enzyme could be confirmed for three JOXs. The identification of the enzymes responsible for hydroxylation of JA reveals a missing step in JA metabolism, which is important for the inactivation of the hormone and subsequent down-regulation of JA-dependent defenses.