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Jorge C Escalantesemerena - One of the best experts on this subject based on the ideXlab platform.
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cobb a new member of the sir2 family of eucaryotic regulatory proteins is required to compensate for the lack of nicotinate mononucleotide 5 6 dimethylbenzimidazole phosphoribosyltransferase activity in cobt mutants during cobalamin biosynthesis in salmonella typhimurium lt2
Journal of Biological Chemistry, 1998Co-Authors: Allen W Tsang, Jorge C EscalantesemerenaAbstract:The cobB gene of Salmonella typhimurium LT2 has been isolated and genetically and biochemically characterized. cobB was located by genetic means to the 27-centisome region of the chromosome. Genetic crosses established the gene order to be cobB pepT phoQ, and the direction of cobB transcription was shown to be clockwise. The nucleotide sequence of cobB (711 base pairs) predicted a protein of 237 amino acids length with a molecular mass of 26.3 kDa, a mass consistent with the experimentally determined one of approximately 28 kDa. The cobB gene was defined genetically by deletions (10), insertions (5), and point mutations (15). The precise location of a Tn10d(Tc) element within cobB was established by sequencing. DNA sequence analysis of the region flanking cobB located it 81 base pairs 3' of the potABCD operon, with the potABCD operon and cobB being divergently transcribed. cobB was overexpressed to approximately 30% of the total soluble protein using a T7 overexpression system. In vitro activity assays showed that cell-free extracts enriched for CobB catalyzed the synthesis of the cobalamin biosynthetic intermediate N1-(5-phospho-alpha-D-ribosyl)-5, 6-dimethylbenzimidazole (also known as alpha-ribazole-5'-phosphate) from nicotinate mononucleotide and 5,6-dimethylbenzimidazole, the reaction known to be catalyzed by the CobT phosphoribosyltransferase enzyme (EC 2.4.2.21) (Trzebiatowski, J. R. and Escalante-Semerena, J. C. (1997) J. Biol. Chem. 272, 17662-17667). Computer analysis of the primary amino acid sequence of the CobB protein identified the sequences GAGISAESGIRTFR and YTQNID which are diagnostic of members of the SIR2 family of eucaryotic transcriptional regulators. Possible roles of CobB as a regulator are discussed within the context of the catabolism of propionate, a pathway known to require cobB function (Tsang, A. W. and Escalante-Semerena, J. C. (1996) J. Bacteriol. 178, 7016-7019).
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cobb a new member of the sir2 family of eucaryotic regulatory proteins is required to compensate for the lack of nicotinate mononucleotide 5 6 dimethylbenzimidazole phosphoribosyltransferase activity in cobt mutants during cobalamin biosynthesis in salmonella typhimurium lt2
Journal of Biological Chemistry, 1998Co-Authors: Allen W Tsang, Jorge C EscalantesemerenaAbstract:Abstract The cobB gene ofSalmonella typhimurium LT2 has been isolated and genetically and biochemically characterized. cobB was located by genetic means to the 27-centisome region of the chromosome. Genetic crosses established the gene order to be cobB pepT phoQ, and the direction of cobB transcription was shown to be clockwise. The nucleotide sequence of cobB (711 base pairs) predicted a protein of 237 amino acids length with a molecular mass of 26.3 kDa, a mass consistent with the experimentally determined one of ∼28 kDa. The cobB gene was defined genetically by deletions (10), insertions (5), and point mutations (15). The precise location of a Tn10d(Tc) element withincobB was established by sequencing. DNA sequence analysis of the region flanking cobB located it 81 base pairs 3′ of the potABCD operon, with the potABCD operon andcobB being divergently transcribed. cobB was overexpressed to ∼30% of the total soluble protein using a T7 overexpression system. In vitro activity assays showed that cell-free extracts enriched for CobB catalyzed the synthesis of the cobalamin biosynthetic intermediateN 1-(5-phospho-α-d-ribosyl)-5,6-dimethylbenzimidazole (also known as α-ribazole-5′-phosphate) from nicotinate mononucleotide and 5,6-dimethylbenzimidazole, the reaction known to be catalyzed by the CobT phosphoribosyltransferase enzyme (EC 2.4.2.21) (Trzebiatowski, J. R. and Escalante-Semerena, J. C. (1997)J. Biol. Chem. 272, 17662–17667). Computer analysis of the primary amino acid sequence of the CobB protein identified the sequences GAGISAESGIRTFR andYTQNID which are diagnostic of members of the SIR2 family of eucaryotic transcriptional regulators. Possible roles of CobB as a regulator are discussed within the context of the catabolism of propionate, a pathway known to require cobB function (Tsang, A. W. and Escalante-Semerena, J. C. (1996)J. Bacteriol. 178, 7016–7019).
Harry L Malech - One of the best experts on this subject based on the ideXlab platform.
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crispr mediated knockout of CYBB in nsg mice establishes a model of chronic granulomatous disease for human stem cell gene therapy transplants
Human Gene Therapy, 2017Co-Authors: Colin L Sweeney, Uimook Choi, Sherry Koontz, Chengyu Liu, Harry L MalechAbstract:Chronic granulomatous disease (CGD) is characterized by defects in the production of microbicidal reactive oxygen species (ROS) by phagocytes. Testing of gene and cell therapies for the treatment of CGD in human hematopoietic cells requires preclinical transplant models. The use of the lymphocyte-deficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse strain for human hematopoietic cell xenografts to test CGD therapies is complicated by the presence of functional mouse granulocytes capable of producing ROS for subsequent bacterial and fungal killing. To establish a phagocyte-defective mouse model of X-linked CGD (X-CGD) in NSG mice, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 was utilized for targeted knockout of mouse CYBB on the X-chromosome by microinjection of NSG mouse zygotes with Cas9 mRNA and CRISPR single-guide RNA targeting CYBB exon 1 or exon 3. This resulted in a high incidence of indel formation at the CRISPR target site, with all mice exhibiting deletions in at lea...
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targeted repair of CYBB in x cgd ipscs requires retention of intronic sequences for expression and functional correction
Molecular Therapy, 2017Co-Authors: Colin L Sweeney, Uimook Choi, Jizhong Zou, Randall K Merling, Suk See De Ravin, Alexander Liu, Aaron Bodansky, Sandra Burkett, Jungwoong Kim, Harry L MalechAbstract:X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the CYBB gene, resulting in absent or defective gp91 phox protein expression. To correct CYBB exon 5 mutations while retaining normal gene regulation, we utilized TALEN or Cas9 for exon 5 replacement in induced pluripotent stem cells (iPSCs) from patients, which restored gp91 phox expression and ROS production in iPSC-derived granulocytes. Alternate approaches for correcting the majority of X-CGD mutations were assessed, involving TALEN- or Cas9-mediated insertion of CYBB minigenes at exon 1 or 2 of the CYBB locus. Targeted insertion of an exon 1–13 minigene into CYBB exon 1 resulted in no detectable gp91 phox expression or ROS activity in iPSC-derived granulocytes. In contrast, targeted insertion of an exon 2–13 minigene into exon 2 restored both gp91 phox and ROS activity. This demonstrates the efficacy of two correction strategies: seamless repair of specific CYBB mutations by exon replacement or targeted insertion of an exon 2–13 minigene to CYBB exon 2 while retaining exon/intron 1. Furthermore, it highlights a key issue for targeted insertion strategies for expression from an endogenous promoter: retention of intronic elements can be necessary for expression.
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557 targeted CYBB minigene insertion into the CYBB locus for correction of x cgd ipscs requires intronic elements for expression
Molecular Therapy, 2016Co-Authors: Colin L Sweeney, Uimook Choi, Jizhong Zou, Randall K Merling, Suk See De Ravin, Harry L MalechAbstract:X-linked chronic granulomatous disease (X-CGD) is an immune deficiency characterized by defective phagocyte production of microbicidal reactive oxygen species (ROS), resulting in recurring, life-threatening infections and hyper-inflammation. Mutations causing X-CGD span the entire 13 exons or intronic splice sites of the >30-kb CYBB gene encoding gp91phox, resulting in a loss of gp91phox protein expression. We previously tested a TALEN-mediated targeted gene therapy approach to insert a codon-optimized CYBB minigene into the start site of endogenous CYBB. Although targeted insertion into the endogenous start site was achieved in X-CGD patient iPSCs, little or no gp91phox expression or ROS activity was observed upon granulocyte differentiation, suggesting that downstream intronic or regulatory elements may be necessary for efficient gene expression from the CYBB promoter. To test this hypothesis, we tested CRISPR-mediated targeted insertion of a codon-optimized CYBB cDNA consisting of exons 2 through 13 (CYBB2-13) together with a puromycin-resistance gene cassette into exon 2 of the CYBB locus. In iPSCs from X-CGD patients with a CYBB mutation in exon 5, exon 7, or intron 10, the efficiency of targeted insertion of the CYBB2-13 plasmid donor without random inserts in puromycin-selected clones was 50-66%. Upon granulocyte differentiation of CYBB2-13 corrected X-CGD iPSCs, gp91phox expression and ROS production were restored to levels 64-100% (gp91phox) and 68-76% (DHR) of normal healthy donor controls. As expected for expression from the endogenous CYBB promoter, expression of gp91phox was specific to CD13+ granulocytes, and was undetected in undifferentiated iPSCs. This targeted gene therapy approach should allow correction of ~90% of X-CGD patient mutations (those involving mutations in exons 2 through 13), to restore ROS activity while maintaining normal regulation of CYBB expression. Further, these findings demonstrate a key issue for the design of targeted gene insertion to capture expression from an endogenous promoter: for some endogenous promoters, the inclusion of intronic elements is necessary for efficient expression of the insert.
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57 seamless targeted correction of CYBB exon 5 mutations restores granulocyte function in x linked chronic granulomatous disease ipscs
Molecular Therapy, 2015Co-Authors: Colin L Sweeney, Uimook Choi, Jizhong Zou, Randall K Merling, Suk See Deravin, Harry L MalechAbstract:X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from lack of production of microbicidal reactive oxygen species (ROS) by phagocytic cells. Mutations causing X-CGD can occur throughout the >30-kb CYBB gene encoding gp91phox, with the majority of patients exhibiting a single causative mutation within one of the 13 exons or adjoining intronic splice sites, resulting in a loss of gp91phox protein expression. We previously demonstrated targeted “safe-harbor” gene therapy of X-CGD in iPSCs through insertion of a codon-optimized CYBB minigene into the AAVS1 locus, resulting in constitutive gp91phox expression from a CAG promoter. Another approach targeting insertion of the codon-optimized minigene or normal CYBB cDNA to the start site of endogenous CYBB resulted in little or no gp91phox expression or ROS activity in iPSC-derived granulocytes, suggesting that regulatory elements downstream of the CYBB promoter may be required for efficient expression at this locus. In order to maintain normal regulation of gp91phox expression with minimal alterations in corrected cells, we now demonstrate a strategy for seamless targeted repair of CYBB exon 5 mutations using an exon 5-specific TALEN pair or CRISPR, along with a donor plasmid containing the corrected 146-bp exon 5 interrupted by a piggyBac transposon cassette containing a PGK promoter and puroDtk gene for positive/negative selection, and flanked by ~900-bp upstream and downstream intron sequences for homologous recombination. iPSCs from X-CGD patients with exon 5 458T>G mutation or 461A deletion were nucleofected with donor and TALEN or CRISPR expression plasmids. Both TALENs and CRISPR enabled efficient targeted insertion in 82-98% of puromycin-resistant clones, 25-65% of which contained off-target inserts, for an overall efficiency of 29-75% of selected clones containing only the targeted insert. Targeted iPSC clones lacking off-target inserts were nucleofected with “excision-only” piggyBac transposase expression plasmid and selected with ganciclovir for removal of the PGK-puroDtk cassette, leaving only a silent codon change to accommodate the TTAA sequence left behind after piggyBac excision, without additional alterations detected beyond correction of the CYBB mutation. Corrected iPSCs maintained pluripotency and upon in vitro granulocyte differentiation exhibited restoration of gp91phox expression and ROS production comparable to normal blood neutrophils (3100% of normal gp91phox expression and 85-99% of normal ROS activity by mean fluorescence intensity in DHR assay). Our findings demonstrate efficient and seamless targeted repair of exon 5 mutations in X-CGD iPSCs with an exon replacement strategy, resulting in normal regulation of gp91phox expression and functional correction of the disease defect in iPSC-derived granulocytes. The development of similar approaches for the other exons of CYBB will enable seamless repair of the majority of causative mutations found in X-CGD patients.
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CYBB mutation analysis in x linked chronic granulomatous disease
Clinical Immunology, 2002Co-Authors: Orathai Jirapongsananuruk, Harry L Malech, Julie E Niemela, Thomas A FleisherAbstract:Abstract Chronic granulomatous disease (CGD) results from mutations of phagocyte NADPH oxidase. Seventy percent are X-linked (X-)CGD with absent or defective gp91 phox protein encoded by the CYBB gene. A subset of X-CGD patients demonstrates partial oxidase activity and/or varied levels of the gp91 phox protein. Definitive genotypic diagnosis in these unusual patients requires mutation analysis. Typically, CYBB mutation analysis has relied on initial screening of cDNA by single-stranded conformation polymorphism analysis, followed by selective sequencing. We report a fluorescent, automated method for CYBB mutation analysis using genomic DNA that provides more rapid and reliable results. Moreover, the use of genomic DNA in this approach allows mutation detection in the mRNA coding region, promoter/enhancer region, and intronic sequences flanking splice junctions and does not require mRNA preparation. The PCR conditions were optimized for each exon, including those with A+T-rich regions. We analyzed DNA from two unusual X-CGD patients and established the genetic basis for their phenotype. We also sequenced 100 normal X chromosomes to establish wild-type consensus sequences and identify polymorphisms.
Dirk Roos - One of the best experts on this subject based on the ideXlab platform.
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primary immunodeficiency caused by an exonized retroposed gene copy inserted in the CYBB gene
Human Mutation, 2014Co-Authors: Martin De Boer, Karin Van Leeuwen, Judy Geissler, C M R Weemaes, Timo K Van Den Berg, Taco W Kuijpers, Adilia Warris, Dirk RoosAbstract:Retrotransposon-mediated insertion of a long interspersed nuclear element (LINE)-1 or an Alu element into a human gene is a well-known pathogenic mechanism. We report a novel LINE-1-mediated insertion of a transcript from the TMF1 gene on chromosome 3 into the CYBB gene on the X-chromosome. In a Dutch male patient with chronic granulomatous disease, a 5.8-kb, incomplete and partly exonized TMF1 transcript was identified in intron 1 of CYBB, in opposite orientation to the host gene. The sequence of the insertion showed the hallmarks of a retrotransposition event, with an antisense poly(A) tail, target site duplication, and a consensus LINE-1 endonuclease cleavage site. This insertion induced aberrant CYBB mRNA splicing, with inclusion of an extra 117-bp exon between exons 1 and 2 of CYBB. This extra exon contained a premature stop codon. The retrotransposition took place in an early stage of fetal development in the mother of the patient, because she showed a somatic mosaicism for the mutation that was not present in the DNA of her parents. However, the mutated allele was not expressed in the patient's mother because the insertion was found only in the methylated fraction of her DNA.
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Chronic Granulomatous Disease: A 25-Year Patient Registry Based on a Multistep Diagnostic Procedure, from the Referral Center for Primary Immunodeficiencies in Greece
Journal of Clinical Immunology, 2013Co-Authors: Maria Raptaki, Ioanna Varela, Kleopatra Spanou, Marianna Tzanoudaki, Sofia Tantou, Manolis Liatsis, Nikki Constantinidou, Chryssa Bakoula, Dirk Roos, Maria KanariouAbstract:Chronic Granulomatous Disease (CGD) is an uncommon primary immunodeficiency caused by the absence or dysfunction of one of NADPH oxidase subunits, with heterogeneous genetic aetiologies. The aim of this study was the CGD patient registry in Greece, the identification of the responsible genotype and the potential correlation with the patient’s clinical phenotype. Medical charts of 24 CGD patients, investigated by NBT test or DHR for NADPH oxidase activity, Western blot analysis for NADPH oxidase component expression and DNA sequencing (pyro- and cycle sequencing) for mutation analysis, were reviewed. All patients, but one, were classified into the different types of CGD. Sixteen from 14 unrelated families had X-linked CGD (66.7 %), four patients had mutations in the NCF1 gene (16.7 %), and three, from two unrelated families, had mutations in NCF2 (12.5 %). Fifteen mutations were detected in the CYBB gene, including nonsense (53.8 %), splice site (30.8 %) and missense mutations (7.7 %), and deletions (7.7 %). Two novel mutations were identified; one in CYBB and one in NCF1 . Carrier detection for X-CGD revealed that the de novo mutation rate was about 7 %. Prenatal diagnosis identified one affected male in three male fetuses tested. In both the X-linked and the autosomal recessive (AR-CGD) group, the gastrointestinal and respiratory manifestations were more common, followed by lympadenopathy in X-CGD and skin infections in the AR-CGD group. The patients with a mutation in CYBB had a wider variability of clinical manifestations and earlier diagnosis (4.6 years) compared to the AR-CGD group (12.9 years). The incidence of CGD in Greece is estimated at 0.90 (95 % CI 0.89–0.91) per 100,000 live births for the last decade.
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unusual late presentation of x linked chronic granulomatous disease in an adult female with a somatic mosaic for a novel mutation in CYBB
Blood, 2005Co-Authors: Baruch Wolach, Yitshak Scharf, Ronit Gavrieli, Martin De Boer, Dirk RoosAbstract:Most patients with chronic granulomatous disease (CGD) have mutations in the X-linked CYBB gene that encodes gp91 phox , a component of the phagocyte NADPH oxidase. The resulting X-linked form of CGD is usually manifested in boys. Rarely, X-CGD is encountered in female carriers with extreme expression of the mutated gene. Here, we report on a woman with a novel mutation in CYBB (CCG[90-92] 3 GGT), predicting Tyr30Arg31 3 stop, Val in gp91 phox , who presented with clinical symptoms at the age of 66. The mutation was present in heterozygous form in genomic DNA from her leukocytes but was fully expressed in mRNA from these cells, indicating that in her leukocytes the X chromosome carrying the nonmutated CYBB allele had been inactivated. Indeed, only 0.4% to 2% of her neutrophils showed NADPH oxidase activity. This extreme skewing of her Xchromosome inactivation was not found in her cheek mucosal cells and is thus not due to a general defect in gene methylation on one X chromosome. Moreover, the CYBB mutation was not present in the DNA from her cheek cells and was barely detectable in the DNA from her memory T lymphocytes. Thus, this patient shows a somatic mosaic for the CYBB mutation, which probably originated during her lifetime in her bone marrow. (Blood. 2005; 105:61-66)
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point mutations in the promoter region of the CYBB gene leading to mild chronic granulomatous disease
Clinical and Experimental Immunology, 2000Co-Authors: R S Weening, Taco W Kuijpers, M De Boer, V M E Neefjes, W W M Hack, Dirk RoosAbstract:Chronic granulomatous disease (CGD) is a clinical syndrome of recurrent bacterial and fungal infections caused by a rare disorder of phagocytic cells. In CGD, the phagocytes are unable to generate oxygen radicals after stimulation of these cells, due to a defect in the NADPH oxidase system. This NADPH oxidase is a multicomponent enzyme of at least four subunits, of which the β-subunit of cytochrome b558, gp91-phox, is encoded by an X-linked gene (called CYBB). We report here five patients from two families; in each family we found a different mutation in the promoter region of CYBB. Both mutations prevented the expression of gp91-phox in the patients' neutrophils and thus caused inability of these cells to generate oxygen radicals. However, the mutations left the gp91-phox expression and the function of the NADPH oxidase in the patients' eosinophils intact. The relatively mild course of the CGD in these patients can probably be attributed to the fact that the eosinophils have retained their oxidative capacity. Furthermore, our results indicate that neutrophils and eosinophils differ in their regulation of gp91-phox expression.
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a novel polymorphism in the coding region of CYBB the human gp91 phox gene
Human Genetics, 1996Co-Authors: Futoshi Kuribayashi, Martin De Boer, Jeanette H W Leusen, Arthur J Verhoeven, Dirk RoosAbstract:We have identified a rare polymorphism (G to C at nucleotide 1102) in CYBB, which codes for gp91-phox, a component of NADPH oxidase. Polymorphonuclear leukocytes with this enzyme produced normal amounts of superoxide anion.
Maria Lucia Correagiannella - One of the best experts on this subject based on the ideXlab platform.
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sex specific associations of variants in regulatory regions of nadph oxidase 2 CYBB and glutathione peroxidase 4 gpx4 genes with kidney disease in type 1 diabetes
Free Radical Research, 2013Co-Authors: Maria Beatriz Monteiro, Thiago A Patente, Kamel Mohammedi, Marcia Silva Queiroz, Mirela Jobim De Azevedo, Luis Henrique Santos Canani, Maria Candida Ribeiro Parisi, Michel Marre, Gilberto Velho, Maria Lucia CorreagiannellaAbstract:AbstractOxidative stress is involved in the pathophysiology of diabetic nephropathy. The superoxide-generating nicotinamide adenine dinucleotide phosphate-oxidase 2 (NOX2, encoded by the CYBB gene) and the antioxidant enzyme glutathione peroxidase 4 (GPX4) play opposing roles in the balance of cellular redox status. In the present study, we investigated associations of single nucleotide polymorphisms (SNPs) in the regulatory regions of CYBB and GPX4 with kidney disease in patients with type 1 diabetes. Two functional SNPs, rs6610650 (CYBB promoter region, chromosome X) and rs713041 (GPX4 3'untranslated region, chromosome 19), were genotyped in 451 patients with type 1 diabetes from a Brazilian cohort (diabetic nephropathy: 44.6%) and in 945 French/Belgian patients with type 1 diabetes from Genesis and GENEDIAB cohorts (diabetic nephropathy: 62.3%). The minor A-allele of CYBB rs6610650 was associated with lower estimated glomerular filtration rate (eGFR) in Brazilian women, and with the prevalence of estab...
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sex specific associations of variants in regulatory regions of nadph oxidase 2 CYBB and glutathione peroxidase 4 gpx4 genes with kidney disease in type 1 diabetes
Free Radical Research, 2013Co-Authors: Maria Beatriz Monteiro, Thiago A Patente, Kamel Mohammedi, Marcia Silva Queiroz, Mirela Jobim De Azevedo, Luis Henrique Santos Canani, Maria Candida Ribeiro Parisi, Michel Marre, Gilberto Velho, Maria Lucia CorreagiannellaAbstract:Oxidative stress is involved in the pathophysiology of diabetic nephropathy. The superoxide-generating nicotinamide adenine dinucleotide phosphate-oxidase 2 (NOX2, encoded by the CYBB gene) and the antioxidant enzyme glutathione peroxidase 4 (GPX4) play opposing roles in the balance of cellular redox status. In the present study, we investigated associations of single nucleotide polymorphisms (SNPs) in the regulatory regions of CYBB and GPX4 with kidney disease in patients with type 1 diabetes. Two functional SNPs, rs6610650 (CYBB promoter region, chromosome X) and rs713041 (GPX4 3'untranslated region, chromosome 19), were genotyped in 451 patients with type 1 diabetes from a Brazilian cohort (diabetic nephropathy: 44.6%) and in 945 French/Belgian patients with type 1 diabetes from Genesis and GENEDIAB cohorts (diabetic nephropathy: 62.3%). The minor A-allele of CYBB rs6610650 was associated with lower estimated glomerular filtration rate (eGFR) in Brazilian women, and with the prevalence of established/advanced nephropathy in French/Belgian women (odds ratio 1.75, 95% CI 1.11-2.78, p = 0.016). The minor T-allele of GPX4 rs713041 was inversely associated with the prevalence of established/advanced nephropathy in Brazilian men (odds ratio 0.30, 95% CI 0.13-0.68, p = 0.004), and associated with higher eGFR in French/Belgian men. In conclusion, these heterogeneous results suggest that neither CYBB nor GPX4 are major genetic determinants of diabetic nephropathy, but nevertheless, they could modulate in a gender-specific manner the risk for renal disease in patients with type 1 diabetes.
Allen W Tsang - One of the best experts on this subject based on the ideXlab platform.
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cobb a new member of the sir2 family of eucaryotic regulatory proteins is required to compensate for the lack of nicotinate mononucleotide 5 6 dimethylbenzimidazole phosphoribosyltransferase activity in cobt mutants during cobalamin biosynthesis in salmonella typhimurium lt2
Journal of Biological Chemistry, 1998Co-Authors: Allen W Tsang, Jorge C EscalantesemerenaAbstract:The cobB gene of Salmonella typhimurium LT2 has been isolated and genetically and biochemically characterized. cobB was located by genetic means to the 27-centisome region of the chromosome. Genetic crosses established the gene order to be cobB pepT phoQ, and the direction of cobB transcription was shown to be clockwise. The nucleotide sequence of cobB (711 base pairs) predicted a protein of 237 amino acids length with a molecular mass of 26.3 kDa, a mass consistent with the experimentally determined one of approximately 28 kDa. The cobB gene was defined genetically by deletions (10), insertions (5), and point mutations (15). The precise location of a Tn10d(Tc) element within cobB was established by sequencing. DNA sequence analysis of the region flanking cobB located it 81 base pairs 3' of the potABCD operon, with the potABCD operon and cobB being divergently transcribed. cobB was overexpressed to approximately 30% of the total soluble protein using a T7 overexpression system. In vitro activity assays showed that cell-free extracts enriched for CobB catalyzed the synthesis of the cobalamin biosynthetic intermediate N1-(5-phospho-alpha-D-ribosyl)-5, 6-dimethylbenzimidazole (also known as alpha-ribazole-5'-phosphate) from nicotinate mononucleotide and 5,6-dimethylbenzimidazole, the reaction known to be catalyzed by the CobT phosphoribosyltransferase enzyme (EC 2.4.2.21) (Trzebiatowski, J. R. and Escalante-Semerena, J. C. (1997) J. Biol. Chem. 272, 17662-17667). Computer analysis of the primary amino acid sequence of the CobB protein identified the sequences GAGISAESGIRTFR and YTQNID which are diagnostic of members of the SIR2 family of eucaryotic transcriptional regulators. Possible roles of CobB as a regulator are discussed within the context of the catabolism of propionate, a pathway known to require cobB function (Tsang, A. W. and Escalante-Semerena, J. C. (1996) J. Bacteriol. 178, 7016-7019).
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cobb a new member of the sir2 family of eucaryotic regulatory proteins is required to compensate for the lack of nicotinate mononucleotide 5 6 dimethylbenzimidazole phosphoribosyltransferase activity in cobt mutants during cobalamin biosynthesis in salmonella typhimurium lt2
Journal of Biological Chemistry, 1998Co-Authors: Allen W Tsang, Jorge C EscalantesemerenaAbstract:Abstract The cobB gene ofSalmonella typhimurium LT2 has been isolated and genetically and biochemically characterized. cobB was located by genetic means to the 27-centisome region of the chromosome. Genetic crosses established the gene order to be cobB pepT phoQ, and the direction of cobB transcription was shown to be clockwise. The nucleotide sequence of cobB (711 base pairs) predicted a protein of 237 amino acids length with a molecular mass of 26.3 kDa, a mass consistent with the experimentally determined one of ∼28 kDa. The cobB gene was defined genetically by deletions (10), insertions (5), and point mutations (15). The precise location of a Tn10d(Tc) element withincobB was established by sequencing. DNA sequence analysis of the region flanking cobB located it 81 base pairs 3′ of the potABCD operon, with the potABCD operon andcobB being divergently transcribed. cobB was overexpressed to ∼30% of the total soluble protein using a T7 overexpression system. In vitro activity assays showed that cell-free extracts enriched for CobB catalyzed the synthesis of the cobalamin biosynthetic intermediateN 1-(5-phospho-α-d-ribosyl)-5,6-dimethylbenzimidazole (also known as α-ribazole-5′-phosphate) from nicotinate mononucleotide and 5,6-dimethylbenzimidazole, the reaction known to be catalyzed by the CobT phosphoribosyltransferase enzyme (EC 2.4.2.21) (Trzebiatowski, J. R. and Escalante-Semerena, J. C. (1997)J. Biol. Chem. 272, 17662–17667). Computer analysis of the primary amino acid sequence of the CobB protein identified the sequences GAGISAESGIRTFR andYTQNID which are diagnostic of members of the SIR2 family of eucaryotic transcriptional regulators. Possible roles of CobB as a regulator are discussed within the context of the catabolism of propionate, a pathway known to require cobB function (Tsang, A. W. and Escalante-Semerena, J. C. (1996)J. Bacteriol. 178, 7016–7019).
