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1.
PLoS Genet ; 15(2): e1007957, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30742617

RESUMO

Mucormycosis-an emergent, deadly fungal infection-is difficult to treat, in part because the causative species demonstrate broad clinical antifungal resistance. However, the mechanisms underlying drug resistance in these infections remain poorly understood. Our previous work demonstrated that one major agent of mucormycosis, Mucor circinelloides, can develop resistance to the antifungal agents FK506 and rapamycin through a novel, transient RNA interference-dependent mechanism known as epimutation. Epimutations silence the drug target gene and are selected by drug exposure; the target gene is re-expressed and sensitivity is restored following passage without drug. This silencing process involves generation of small RNA (sRNA) against the target gene via core RNAi pathway proteins. To further elucidate the role of epimutation in the broad antifungal resistance of Mucor, epimutants were isolated that confer resistance to another antifungal agent, 5-fluoroorotic acid (5-FOA). We identified epimutant strains that exhibit resistance to 5-FOA without mutations in PyrF or PyrG, enzymes which convert 5-FOA into the active toxic form. Using sRNA hybridization as well as sRNA library analysis, we demonstrate that these epimutants harbor sRNA against either pyrF or pyrG, and further show that this sRNA is lost after reversion to drug sensitivity. We conclude that epimutation is a mechanism capable of targeting multiple genes, enabling Mucor to develop resistance to a variety of antifungal agents. Elucidation of the role of RNAi in epimutation affords a fuller understanding of mucormycosis. Furthermore, it improves our understanding of fungal pathogenesis and adaptation to stresses, including the evolution of drug resistance.


Assuntos
Farmacorresistência Fúngica Múltipla/genética , Mucor/efeitos dos fármacos , Mucor/patogenicidade , Antifúngicos/farmacologia , Epigênese Genética , Genes Fúngicos , Humanos , Mucor/genética , Mucormicose/tratamento farmacológico , Mucormicose/microbiologia , Mutação , Orotato Fosforribosiltransferase/genética , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , Orotidina-5'-Fosfato Descarboxilase/genética , Interferência de RNA , RNA Fúngico/genética , Sirolimo/farmacologia , Tacrolimo/farmacologia
2.
Nat Commun ; 10(1): 201, 2019 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-30643150

RESUMO

Under hypoxia, most of glucose is converted to secretory lactate, which leads to the overuse of glutamine-carbon. However, under such a condition how glutamine nitrogen is disposed to avoid over-accumulating ammonia remains to be determined. Here we identify a metabolic flux of glutamine to secretory dihydroorotate, which is indispensable to glutamine-carbon metabolism under hypoxia. We found that glutamine nitrogen is necessary to nucleotide biosynthesis, but enriched in dihyroorotate and orotate rather than processing to its downstream uridine monophosphate under hypoxia. Dihyroorotate, not orotate, is then secreted out of cells. Furthermore, we found that the specific metabolic pathway occurs in vivo and is required for tumor growth. The identified metabolic pathway renders glutamine mainly to acetyl coenzyme A for lipogenesis, with the rest carbon and nitrogen being safely removed. Therefore, our results reveal how glutamine carbon and nitrogen are coordinatively metabolized under hypoxia, and provide a comprehensive understanding on glutamine metabolism.


Assuntos
Glutamina/metabolismo , Redes e Vias Metabólicas , Metaboloma , Neoplasias/metabolismo , Ácido Orótico/análogos & derivados , Acetilcoenzima A/metabolismo , Amônia/metabolismo , Amônia/toxicidade , Animais , Carbono/química , Carbono/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Glucose/metabolismo , Glutamina/química , Células HEK293 , Humanos , Ácido Láctico/metabolismo , Lipogênese , Metabolômica , Camundongos , Camundongos Nus , Neoplasias/sangue , Neoplasias/mortalidade , Neoplasias/patologia , Nitrogênio/química , Nitrogênio/metabolismo , Nucleotídeos/biossíntese , Ácido Orótico/metabolismo , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Fungal Genet Biol ; 124: 1-7, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30562583

RESUMO

Rhizopus delemar causes devastating mucormycosis in immunodeficient individuals. Despite its medical importance, R. delemar remains understudied largely due to the lack of available genetic markers, the presence of multiple gene copies due to genome duplication, and mitotically unstable transformants resulting from conventional and limited genetic approaches. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) system induces efficient homologous and non-homologous break points and generates individual and multiple mutant alleles without requiring selective marker genes in a wide variety of organisms including fungi. Here, we have successfully adapted this technology for inducing gene-specific single nucleotide (nt) deletions in two clinical strains of R. delemar: FGSC-9543 and CDC-8219. For comparative reasons, we first screened for spontaneous uracil auxotrophic mutants resistant to 5-fluoroorotic acid (5-FOA) and obtained one substitution (f1) mutationin the FGSC-9543 strain and one deletion (f2) mutation in the CDC-8219 strain. The f2 mutant was then successfully complemented with a pyrF-dpl200 marker gene. We then introduced a vector pmCas9:tRNA-gRNA that expresses both Cas9 endonuclease and pyrF-specific gRNA into FGSC-9543 and CDC-8219 strains and obtained 34 and 42 5-FOA resistant isolates, respectively. Candidate transformants were successively transferred eight times by propagating hyphal tips prior to genotype characterization. Sequencing of the amplified pyrF allele in all transformants tested revealed a single nucleotide (nt) deletion at the 4th nucleotide before the protospacer adjacent motif (PAM) sequence, which is consistent with CRISPR-Cas9 induced gene mutation through non-homologous end joining (NHEJ). Our study provides a new research tool for investigating molecular pathogenesis mechanisms of R. delemar while also highlighting the utilization of CRISPR-Cas9 technology for generating specific mutants of Mucorales fungi.


Assuntos
Mutação Puntual , Rhizopus/genética , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Genes Fúngicos , Vetores Genéticos , Orotato Fosforribosiltransferase/genética , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , Rhizopus/efeitos dos fármacos , Rhizopus/enzimologia , Uracila
4.
Appl Environ Microbiol ; 85(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30504216

RESUMO

The discovery of hyperthermophiles has dramatically changed our understanding of the habitats in which life can thrive. However, the extreme high temperatures in which these organisms live have severely restricted the development of genetic tools. The archaeon Pyrococcus yayanosii A1 is a strictly anaerobic and piezophilic hyperthermophile that is an ideal model for studies of extreme environmental adaptation. In the present study, we identified a high hydrostatic pressure (HHP)-inducible promoter (P hhp ) that controls target gene expression under HHP. We developed an HHP-inducible toxin-antitoxin cassette (HHP-TAC) containing (i) a counterselectable marker in which a gene encoding a putative toxin (virulence-associated protein C [PF0776 {VapC}]) controlled by the HHP-inducible promoter was used in conjunction with the gene encoding antitoxin PF0775 (VapB), which was fused to a constitutive promoter (P hmtB ), and (ii) a positive marker with the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase-encoding gene from P. furiosus controlled by the constitutive promoter P gdh The HHP-TAC was constructed to realize markerless gene disruption directly in P. yayanosii A1 in rich medium. The pop-out recombination step was performed using an HHP-inducible method. As proof, the PYCH_13690 gene, which encodes a 4-α-glucanotransferase, was successfully deleted from the strain P. yayanosii A1. The results showed that the capacity for starch hydrolysis in the Δ1369 mutant decreased dramatically compared to that in the wild-type strain. The inducible toxin-antitoxin system developed in this study greatly increases the genetic tools available for use in hyperthermophiles.IMPORTANCE Genetic manipulations in hyperthermophiles have been studied for over 20 years. However, the extremely high temperatures under which these organisms grow have limited the development of genetic tools. In this study, an HHP-inducible promoter was used to control the expression of a toxin. Compared to sugar-inducible and cold-shock-inducible promoters, the HHP-inducible promoter rarely has negative effects on the overall physiology and central metabolism of microorganisms, especially piezophilic hyperthermophiles. Previous studies have used auxotrophic strains as hosts, which may interfere with studies of adaptation and metabolism. Using an inducible toxin-antitoxin (TA) system as a counterselectable marker enables the generation of a markerless gene disruption strain without the use of auxotrophic mutants and counterselection with 5-fluoroorotic acid. TA systems are widely distributed in bacteria and archaea and can be used to overcome the limitations of high growth temperatures and dramatically extend the selectivity of genetic tools in hyperthermophiles.


Assuntos
Adaptação Fisiológica/genética , Antitoxinas/genética , Archaea/genética , Proteínas Arqueais/metabolismo , Pressão Hidrostática , Pyrococcus/genética , Toxinas Biológicas/genética , Archaea/fisiologia , Proteínas Arqueais/genética , Proteínas de Bactérias , Sequência de Bases , DNA Arqueal , Proteínas de Ligação a DNA , Regulação da Expressão Gênica em Archaea , Genes Arqueais/genética , Temperatura Alta , Fontes Hidrotermais , Hidroximetilglutaril-CoA Redutases/genética , Glicoproteínas de Membrana , Ácido Orótico/análogos & derivados , Regiões Promotoras Genéticas , Pyrococcus/fisiologia , Toxinas Biológicas/metabolismo , Transformação Genética
5.
Appl Environ Microbiol ; 84(3)2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29150512

RESUMO

Thermoanaerobacter kivui is one of the very few thermophilic acetogenic microorganisms. It grows optimally at 66°C on sugars but also lithotrophically with H2 + CO2 or with CO, producing acetate as the major product. While a genome-derived model of acetogenesis has been developed, only a few physiological or biochemical experiments regarding the function of important enzymes in carbon and energy metabolism have been carried out. To address this issue, we developed a method for targeted markerless gene deletions and for integration of genes into the genome of T. kivui The strain naturally took up plasmid DNA in the exponential growth phase, with a transformation frequency of up to 3.9 × 10-6 A nonreplicating plasmid and selection with 5-fluoroorotate was used to delete the gene encoding the orotate phosphoribosyltransferase (pyrE), resulting in a ΔpyrE uracil-auxotrophic strain, TKV002. Reintroduction of pyrE on a plasmid or insertion of pyrE into different loci within the genome restored growth without uracil. We subsequently studied fructose metabolism in T. kivui The gene fruK (TKV_c23150) encoding 1-phosphofructosekinase (1-PFK) was deleted, using pyrE as a selective marker via two single homologous recombination events. The resulting ΔfruK strain, TKV003, did not grow on fructose; however, growth on glucose (or on mannose) was unaffected. The combination of pyrE as a selective marker and the natural competence of the strain for DNA uptake will be the basis for future studies on CO2 reduction and energy conservation and their regulation in this thermophilic acetogenic bacterium.IMPORTANCE Acetogenic bacteria are currently the focus of research toward biotechnological applications due to their potential for de novo synthesis of carbon compounds such as acetate, butyrate, or ethanol from H2 + CO2 or from synthesis gas. Based on available genome sequences and on biochemical experiments, acetogens differ in their energy metabolism. Thus, there is an urgent need to understand the carbon and electron flows through the Wood-Ljungdahl pathway and their links to energy conservation, which requires genetic manipulations such as deletion or overexpression of genes encoding putative key enzymes. Unfortunately, genetic systems have been reported for only a few acetogenic bacteria. Here, we demonstrate proof of concept for the genetic modification of the thermophilic acetogenic species Thermoanaerobacter kivui The genetic system will be used to study genes involved in biosynthesis and energy metabolism, and may further be applied to metabolically engineer T. kivui to produce fuels and chemicals.


Assuntos
Frutose/metabolismo , Genoma Bacteriano , Thermoanaerobacter/genética , Ciclo do Carbono , Metabolismo Energético/genética , Frutose/farmacologia , Deleção de Genes , Glucose/farmacologia , Recombinação Homóloga , Manose/farmacologia , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , Fosfofrutoquinases/deficiência , Fosfofrutoquinases/genética , Thermoanaerobacter/efeitos dos fármacos , Thermoanaerobacter/enzimologia , Thermoanaerobacter/crescimento & desenvolvimento
6.
J Am Chem Soc ; 139(45): 16048-16051, 2017 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-29058891

RESUMO

Orotidine 5'-monophosphate decarboxylase (OMPDC) catalyzes the decarboxylation of 5-fluoroorotate (FO) with kcat/Km = 1.4 × 10-7 M-1 s-1. Combining this and related kinetic parameters shows that the 31 kcal/mol stabilization of the transition state for decarboxylation of OMP provided by OMPDC represents the sum of 11.8 and 10.6 kcal/mol stabilization by the substrate phosphodianion and the ribosyl ring, respectively, and an 8.6 kcal/mol stabilization from the orotate ring. The transition state for OMPDC-catalyzed decarboxylation of FO is stabilized by 5.2, 7.2, and 9.0 kcal/mol, respectively, by 1.0 M phosphite dianion, d-glycerol 3-phosphate and d-erythritol 4-phosphate. The stabilization is due to the utilization of binding interactions of the substrate fragments to drive an enzyme conformational change, which locks the orotate ring of the whole substrate, or the substrate pieces in a caged complex. We propose that enzyme-activation is a possible, and perhaps probable, consequence of any substrate-induced enzyme conformational change.


Assuntos
Ácido Orótico/análogos & derivados , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Biocatálise , Cinética , Modelos Moleculares , Conformação Molecular , Ácido Orótico/química , Ácido Orótico/metabolismo , Orotidina-5'-Fosfato Descarboxilase/química , Especificidade por Substrato
7.
Yeast ; 34(12): 483-494, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28810289

RESUMO

The basidiomycetous yeast Pseudozyma antarctica is a remarkable producer of industrially valuable enzymes and extracellular glycolipids. In this study, we developed a method for targeted gene replacement in P. antarctica. In addition, transformation conditions were optimized using lithium acetate, single-stranded carrier DNA and polyethylene glycol (lithium acetate treatment), generally used for ascomycetous yeast transformation. In the rice-derived P. antarctica strain GB-4(0), PaURA3, a homologue of the Saccharomyces cerevisiae orotidine-5'-phosphate decarboxylase gene (URA3), was selected as the target locus. A disruption cassette was constructed by linking the nouseothricine resistance gene (natMX4) to homologous DNA fragments of PaURA3, then electroporated into the strain GB-4(0). We obtained strain PGB015 as one of the PaURA3 disruptants (Paura3Δ::natMX4). Then the PCR-amplified PaURA3 fragment was introduced into PGB015, and growth of transformant colonies but not background colonies was observed on selective media lacking uracil. The complementation of uracil-auxotrophy in PGB015 by introduction of PaURA3 was also performed using lithium acetate treatment, which resulted in a transformation efficiency of 985 CFU/6.8 µg DNA and a gene-targeting ratio of two among 30 transformants. Copyright © 2017 John Wiley & Sons, Ltd.


Assuntos
Acetatos/farmacologia , Proteínas Fúngicas/genética , Reparo Gênico Alvo-Dirigido/métodos , Transformação Genética , Ustilaginales/genética , Sequência de Aminoácidos , DNA Fúngico/genética , Farmacorresistência Fúngica/genética , Eletroporação , Temperatura Alta , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , Orotidina-5'-Fosfato Descarboxilase/química , Orotidina-5'-Fosfato Descarboxilase/genética , Plasmídeos/genética , Estreptotricinas/farmacologia , Árvores/microbiologia , Ustilaginales/efeitos dos fármacos , Ustilaginales/crescimento & desenvolvimento
8.
Biosci Biotechnol Biochem ; 81(6): 1227-1234, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28290772

RESUMO

We screened for factors involved in the cellulose-responsive induction of cellulose biomass-degrading enzyme genes from approximately 12,000 Aspergillus aculeatus T-DNA insertion mutants harboring a transcriptional fusion between the FIII-avicelase gene (cbhI) promoter and the orotidine 5'-monophosphate decarboxylase gene. Analysis of 5-fluoroorodic acid (5-FOA) sensitivity, cellulose utilization, and cbhI expression of the mutants revealed that a mutant harboring T-DNA at the dipeptidyl peptidase IV (dppIV) locus had acquired 5-FOA resistance and was deficient in cellulose utilization and cbhI expression. The deletion of dppIV resulted in a significant reduction in the cellulose-responsive expression of both cbhI as well as genes controlled by XlnR-independent and XlnR-dependent signaling pathways at an early phase in A. aculeatus. In contrast, the dppIV deletion did not affect the xylose-responsive expression of genes under the control of XlnR. These results demonstrate that DppIV participates in cellulose-responsive induction in A. aculeatus.


Assuntos
Aspergillus/genética , Celulases/genética , Celulose/metabolismo , Dipeptidil Peptidase 4/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Orotidina-5'-Fosfato Descarboxilase/genética , Aspergillus/efeitos dos fármacos , Aspergillus/enzimologia , Celulases/metabolismo , Celulose/farmacologia , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Dipeptidil Peptidase 4/agonistas , Dipeptidil Peptidase 4/metabolismo , Proteínas Fúngicas/metabolismo , Deleção de Genes , Mutagênese Insercional , Ácido Orótico/análogos & derivados , Ácido Orótico/metabolismo , Ácido Orótico/farmacologia , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Xilose/metabolismo , Xilose/farmacologia
9.
Inflammopharmacology ; 25(2): 271-274, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28155119

RESUMO

Approximately, one-third of those who develop major depression will have a poor response to treatment and over time can become treatment resistant. Intestinal dysbiosis has been implicated in depression with systemic inflammation and vagal and enteric nerve impairment. We report on a sequel pilot study (n = 12) with a combination probiotics/magnesium orotate formulation adjuvant administered with SSRIs for treatment resistant depression. At the end of an 8-week intervention mean changes for depression scores and quality of life in the group was clinically significantly improved (p < 0.001) with all but 4 participants experiencing a benefit. An intestinal anti-inflammatory response was suggested. At 16-weeks follow-up while still on SSRI medications, the group had relapsed after cessation of the test intervention.


Assuntos
Anti-Inflamatórios/administração & dosagem , Transtorno Depressivo Resistente a Tratamento/diagnóstico , Transtorno Depressivo Resistente a Tratamento/tratamento farmacológico , Ácido Orótico/análogos & derivados , Probióticos/administração & dosagem , Inibidores de Captação de Serotonina/administração & dosagem , Adulto , Antidepressivos/administração & dosagem , Estudos de Coortes , Quimioterapia Combinada , Feminino , Seguimentos , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Ácido Orótico/administração & dosagem , Projetos Piloto
10.
J Vis Exp ; (119)2017 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-28190067

RESUMO

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.


Assuntos
Histonas/genética , Mutagênese Sítio-Dirigida/métodos , Reação em Cadeia da Polimerase/métodos , Saccharomycetales/genética , Alelos , Recombinação Homóloga , Fases de Leitura Aberta , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , Plasmídeos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomycetales/efeitos dos fármacos
11.
Curr Genet ; 63(4): 697-707, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28011993

RESUMO

It is well known that 5-fluoroorotic acid (5-FOA)-resistant mutants isolated from wild-type Cryptococcus neoformans are exclusively either ura3 or ura5 mutants. Unexpectedly, many of the 5-FOA-resistant mutants isolated in our selective regime were Ura+. We identified CNM00460 as the gene responsible for these mutations. Cnm00460 belongs to the nucleobase cation symporter 1/purine-related transporter (NCS1/PRT) super family of fungal transporters, representative members of which are uracil transporter, uridine transporter and allantoin transporter of Saccharomyces cerevisiae. Since the CNM00460 gene turned out to be involved in utilization of orotic acid, most probably as transporter, we designated this gene Orotic Acid Transporter 1 (OAT1). This is the first report of orotic acid transporter in this family. C. neoformans has four members of the NCS1/PRT family, including Cnm00460, Cnm02550, Cnj00690, and Cnn02280. Since the cnm02550∆ strain showed resistance to 5-fluorouridine, we concluded that CNM02550 encodes uridine permease and designated it URidine Permease 1 (URP1). We found that oat1 mutants were sensitive to 5-FOA in the medium containing proline as nitrogen source. A mutation in the GAT1 gene, a positive transcriptional regulator of genes under the control of nitrogen metabolite repression, in the genetic background of oat1 conferred the phenotype of weak resistance to 5-FOA even in the medium using proline as nitrogen source. Thus, we proposed the existence of another orotic acid utilization system (tentatively designated OAT2) whose expression is under the control of nitrogen metabolite repression at least in part. We found that the OAT1 gene is necessary for full pathogenic activity of C. neoformans var. neoformans.


Assuntos
Transporte Biológico/genética , Cryptococcus neoformans/genética , Proteínas de Membrana Transportadoras/genética , Cryptococcus neoformans/efeitos dos fármacos , Cryptococcus neoformans/patogenicidade , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Mutação , Nitrogênio/metabolismo , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , Uracila/metabolismo
12.
Biochimie ; 131: 45-53, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27650727

RESUMO

De novo pyrimidine biosynthesis pathway is well developed and functional in protozoan parasite Leishmania donovani. The dihydroorotase (LdDHOase) is third enzyme of the pathway. The enzyme was cloned, expressed in E. coli BL21 (DE3), purified to homogeneity and biochemically characterized. The estimated kcat for the forward reaction and reverse reactions were 2.1 ± 0.1 s-1 and 1.1 ± 0.15 s-1, respectively. Homology modeling and docking studies were done to find out potential inhibitors for LdDHOase. Biotin sulfone and Kaempferol were found to be potential inhibitors of LdDHOase based on docking studies. These inhibitors were verified using recombinant LdDHOase and their anti-leishmanial effect was evaluated. Moreover, alterations in expressions of de novo as well as salvage pathways enzymes, after treatment of L. donovani with dihydroorotase inhibitor(s) were evaluated and discussed as survival mechanism of the pathogen. Further, effect of inhibition of cytidine deaminase, a key enzyme of salvage pathway of pyrimidine biosynthesis, was also evaluated on parasitic survival and alteration in gene expression of enzymes of both pathways. Further, effect of both pathways inhibition was also evaluated. The data suggests that the inhibition of single pathway can be overcome by increased expression of enzyme(s) of alternate pathway and both pathways seem to be equally important in the pathogen. When both pathways are simultaneously inhibited, parasite shows significant DNA damage and parasitic death.


Assuntos
Di-Hidro-Orotase/metabolismo , Leishmania donovani/metabolismo , Proteínas de Protozoários/metabolismo , Pirimidinas/metabolismo , Biotina/análogos & derivados , Biotina/química , Biotina/farmacologia , Citidina Desaminase/antagonistas & inibidores , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Di-Hidro-Orotase/antagonistas & inibidores , Di-Hidro-Orotase/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica , Quempferóis/química , Quempferóis/farmacologia , Cinética , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/genética , Simulação de Acoplamento Molecular , Estrutura Molecular , Ácido Orótico/análogos & derivados , Ácido Orótico/química , Ácido Orótico/metabolismo , Domínios Proteicos , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade por Substrato , Sulfonas/química , Sulfonas/farmacologia
13.
Fungal Biol ; 120(9): 1146-55, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27567720

RESUMO

Of all of the natural polymers, lignin, an aromatic heteropolymer in plant secondary cell walls, is the most resistant to biological degradation. White-rot fungi are the only known organisms that can depolymerize or modify wood lignin. Investigating the mechanisms underlying lignin biodegradation by white-rot fungi would contribute to the ecofriendly utilization of woody biomass as renewable resources in the future. Efficient gene disruption, which is generally very challenging in the white-rot fungi, was established in Pleurotus ostreatus (the oyster mushroom). Some of the genes encoding manganese peroxidases, enzymes that are considered to be involved in lignin biodegradation, were disrupted separately, and the phenotype of each single-gene disruptant was analysed. However, it remains difficult to generate multi-gene disruptants in this fungus. Here we developed a new genetic transformation marker in P. ostreatus and demonstrated two marker recycling methods that use counter-selection to generate a multigene disruptant. This study will enable future genetic studies of white-rot fungi, and it will increase our understanding of the complicated mechanisms, which involve various enzymes, including lignin-degrading enzymes, underlying lignin biodegradation by these fungi.


Assuntos
Antifúngicos/metabolismo , Flucitosina/metabolismo , Ácido Orótico/análogos & derivados , Pleurotus/genética , Pleurotus/metabolismo , Seleção Genética , Proteínas Fúngicas/genética , Técnicas de Inativação de Genes/métodos , Marcadores Genéticos , Genética Microbiana/métodos , Ácido Orótico/metabolismo , Peroxidases/genética
14.
Mol Genet Metab ; 119(1-2): 83-90, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27370710

RESUMO

BACKGROUND: Miller syndrome (post-axial acrofacial dysostosis) arises from gene mutations for the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH). Nonetheless, despite demonstrated loss of enzyme activity dihydroorotate (DHO) has not been shown to accumulate, but paradoxically urine orotate has been reported to be raised, confusing the metabolic diagnosis. METHODS: We analysed plasma and urine from a 4-year-old male Miller syndrome patient. DHODH mutations were determined by PCR and Sanger sequencing. Analysis of DHO and orotic acid (OA) in urine, plasma and blood-spot cards was performed using liquid chromatography-tandem mass spectrometry. In vitro stability of DHO in distilled water and control urine was assessed for up to 60h. The patient received a 3-month trial of oral uridine for behavioural problems. RESULTS: The patient had early liver complications that are atypical of Miller syndrome. DHODH genotyping demonstrated compound-heterozygosity for frameshift and missense mutations. DHO was grossly raised in urine and plasma, and was detectable in dried spots of blood and plasma. OA was raised in urine but undetectable in plasma. DHO did not spontaneously degrade to OA. Uridine therapy did not appear to resolve behavioural problems during treatment, but it lowered plasma DHO. CONCLUSION: This case with grossly raised plasma DHO represents the first biochemical confirmation of functional DHODH deficiency. DHO was also easily detectable in dried plasma and blood spots. We concluded that DHO oxidation to OA must occur enzymatically during renal secretion. This case resolved the biochemical conundrum in previous reports of Miller syndrome patients, and opened the possibility of rapid biochemical screening.


Assuntos
Anormalidades Múltiplas/genética , Deformidades Congênitas dos Membros/genética , Disostose Mandibulofacial/genética , Micrognatismo/genética , Ácido Orótico/análogos & derivados , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Anormalidades Múltiplas/sangue , Anormalidades Múltiplas/fisiopatologia , Anormalidades Múltiplas/urina , Pré-Escolar , Genótipo , Humanos , Deformidades Congênitas dos Membros/sangue , Deformidades Congênitas dos Membros/fisiopatologia , Deformidades Congênitas dos Membros/urina , Masculino , Disostose Mandibulofacial/sangue , Disostose Mandibulofacial/fisiopatologia , Disostose Mandibulofacial/urina , Micrognatismo/sangue , Micrognatismo/fisiopatologia , Micrognatismo/urina , Mutação , Ácido Orótico/sangue , Ácido Orótico/urina , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/sangue , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/urina , Uridina/sangue , Uridina/urina
15.
J Biosci Bioeng ; 122(6): 645-651, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27401770

RESUMO

Shewanella livingstonensis Ac10, a psychrotrophic bacterium isolated from Antarctic seawater, grows well at low temperatures close to 0°C. The bacterium is useful as a host in a low-temperature protein expression system. It is also useful as a model microorganism to investigate the mechanisms of microbial cold-adaptation. Versatile genetic manipulation techniques would be useful to investigate the biology of this bacterium and to develop its applications. In this study, we developed a method for targeted gene deletion and insertion by using the gene coding for orotidine-5'-phosphate decarboxylase (pyrF), which is involved in pyrimidine synthesis. We found that S. livingstonensis Ac10 is sensitive to 5-fluoroorotic acid (5-FOA), which is converted to a highly toxic compound by the product of pyrF. A uracil-auxotrophic strain resistant to 5-FOA was constructed by deleting pyrF, thus allowing the use of a plasmid-borne copy of pyrF for selection of recombinants. We constructed the pyrF complementation suicide plasmid pKKP, which contains pyrF, the R6K replication origin, the mob site of RP4, an antibiotic marker gene, and a multiple cloning site. To demonstrate pyrF-based gene replacement, we deleted the internal region of orf5, the gene coding for an eicosapentaenoic acid (EPA) synthesis enzyme. We also successfully inserted a His6-tag-coding sequence into orf8, the gene coding for another EPA synthesis enzyme. This system allows the markerless deletion and insertion of desired sequences at specific sites in the genome, which remarkably facilitates genetic manipulation of this bacterium.


Assuntos
Deleção de Genes , Marcação de Genes/métodos , Genes Bacterianos , Mutagênese Insercional/métodos , Mutagênese Sítio-Dirigida/métodos , Orotidina-5'-Fosfato Descarboxilase/genética , Shewanella/genética , Adaptação Biológica/genética , Sequência de Bases , Temperatura Baixa , Organismos Geneticamente Modificados , Ácido Orótico/análogos & derivados , Ácido Orótico/metabolismo , Orotidina-5'-Fosfato Descarboxilase/metabolismo , Shewanella/enzimologia , Shewanella/metabolismo
16.
ACS Synth Biol ; 5(7): 632-8, 2016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27138234

RESUMO

Predictable integration of foreign biological signals and parts remains a key challenge in the systematic engineering of synthetic cellular actuations, and general methods to improve signal transduction and sensitivity are needed. To address this problem we modeled and built a molecular signal buffer network in Saccharomyces cerevisiae inspired by chemical pH buffer systems. The molecular buffer system context-insulates a riboswitch enabling synthetic control of colony formation and modular signal manipulations. The riboswitch signal is relayed to a transcriptional activation domain of a split transcription factor, while interacting DNA-binding domains mediate the transduction of signal and form an interacting molecular buffer. The molecular buffer system enables modular signal inversion through integration with repressor modules. Further, tuning of input sensitivity was achieved through perturbation of the buffer pair ratio guided by a mathematical model. Such buffered signal tuning networks will be useful for domestication of RNA-based sensors enabling tunable outputs and library-wide selections for drug discovery and metabolic engineering.


Assuntos
Engenharia Genética/métodos , Riboswitch , Transdução de Sinais/genética , DNA/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Modelos Teóricos , Ácido Orótico/análogos & derivados , Ácido Orótico/farmacologia , Domínios Proteicos , Riboswitch/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
17.
Redox Rep ; 21(2): 84-9, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26193444

RESUMO

OBJECTIVES: This study is an extension to our finding of direct anti-oxidant activities of lanthanide(III) complexes with the heterocyclic compound, 5-aminoorotic acid (AOA). In this experiment, we used AOA and coumarin-3-carboxylic acid as the two heterocyclic compounds with anti-oxidant potential, to produce the complexes with different lanthanides. METHODS: Lanthanide(III) complexes were tested on the iron-driven Fenton reaction. The product of this reaction, the hydroxyl radical, was detected by HPLC. RESULTS: All complexes as well as their ligands had positive or neutral effect on the Fenton reaction but their behavior was different. Both pure ligands in low concentration ratio to iron were inefficient in contrast to some of their complexes. Complexes of neodymium, samarium, gadolinium, and partly of cerium blocked the Fenton reaction at very low ratios (in relation to iron) but the effect disappeared at higher ratios. In contrast, lanthanum complexes appeared to be the most promising. Both blocked the Fenton reaction in a dose-dependent manner. CONCLUSION: Lanthanide(III) complexes were proven to block the iron-driven production of the hydroxyl radical. Second, the lanthanide(III) element appears to be crucial for the anti-oxidant effect. Overall, lanthanum complexes may be promising direct anti-oxidants for future testing.


Assuntos
Elementos da Série dos Lantanídeos/química , Antioxidantes , Cumarínicos/química , Peróxido de Hidrogênio/química , Ferro/química , Ligantes , Ácido Orótico/análogos & derivados , Ácido Orótico/química
18.
Appl Environ Microbiol ; 82(4): 1346-52, 2016 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-26682856

RESUMO

The pathophysiology of Treponema denticola, an oral pathogen associated with both periodontal and endodontic infections, is poorly understood due to its fastidious growth and recalcitrance to genetic manipulations. Counterselectable markers are instrumental in constructing clean and unmarked mutations in bacteria. Here, we demonstrate that pyrF, a gene encoding orotidine-5'-monophosphate decarboxylase, can be used as a counterselectable marker in T. denticola to construct marker-free mutants. T. denticola is susceptible to 5-fluoroorotic acid (5-FOA). To establish a pyrF-based counterselectable knockout system in T. denticola, the pyrF gene was deleted. The deletion conferred resistance to 5-FOA in T. denticola. Next, a single-crossover mutant was constructed by reintroducing pyrF along with a gentamicin resistance gene (aacC1) back into the chromosome of the pyrF mutant at the locus of choice. In this study, we chose flgE, a flagellar hook gene that is located within a large polycistronic motility gene operon, as our target gene. The obtained single-crossover mutant (named FlgE(in)) regained the susceptibility to 5-FOA. Finally, FlgE(in) was plated on solid agar containing 5-FOA. Numerous colonies of the 5-FOA-resistant mutant (named FlgE(out)) were obtained and characterized by PCR and Southern blotting analyses. The results showed that the flgE gene was deleted and FlgE(out) was free of selection markers (i.e., pyrF and aacC1). Compared to previously constructed flgE mutants that contain an antibiotic selection marker, the deletion of flgE in FlgE(out) has no polar effect on its downstream gene expression. The system developed here will provide us with a new tool for investigating the genetics and pathogenicity of T. denticola.


Assuntos
Técnicas de Inativação de Genes/métodos , Genética Microbiana/métodos , Orotidina-5'-Fosfato Descarboxilase/genética , Seleção Genética , Treponema denticola/genética , Farmacorresistência Bacteriana , Ácido Orótico/análogos & derivados , Ácido Orótico/toxicidade , Treponema denticola/efeitos dos fármacos , Treponema denticola/crescimento & desenvolvimento
19.
Ter Arkh ; 87(6): 88-97, 2015.
Artigo em Russo | MEDLINE | ID: mdl-26281202

RESUMO

AIM: To make a meta-analysis of clinical trials of magnerot (magnesium orotate) used in cardiac patients. SUBJECTS AND METHODS: The meta-analysis covered the data of 19 randomized trials including a total of 603 patients treated with magnerot (a case group) and 587 receiving placebo (a control group). The patients' mean age was 36 ± 19 years. On the average, the patients took magnerot 1878 ± 823 mg/day for 4.2 ± 29 months. RESULTS: Associations between the intake of magnerot and the risk of 50 pathological conditions were analyzed. Significant associations were established between the drug's administration and the reduced risk of conditions, such as hypomagnesemia (relative risk (RR) = 0.06; 95% confidence intervals (C): 0.04 to 0.09; p = 2 · 10(-46)), exercise intolerance (RR = 0.41; 95% CI: 0.27 to 0.62; p = 0.0004), dysautonomia (RR = 0.08; 95% CI: 0.04 to 0.14; p = 2 · 10(-21)), morning headache (RR = 0.16; 95% CI: 0.09 to 0.29; p = 1.5-10(-6)), tension headache (RR = 0.16; 95% Cl: 0.09 to 0.27; p = 5 · 10(-10)), dizziness (RR = 0.28; 95% CI: 0.15 to 0.50; p = 0.0004), first-degree mitral valve prolapse (MVP) (RR = 0.05; 95% CI: 0.03 to 0.09; p = 1.2 · 10(-25)), grade 1 regurgitation (RR = 0.29; 95 CI: 0.14 to 0.60; p = 0.0075), supraventricular (RR = 0.30; 95% CI: 0.21 to 0.44; p = 1 · 10(-8)) and ventricular (RR = 0.48; 95% CI: 0.30 to 0.76; p = 0.019) premature contraction, paroxysmal supraventricular tachycardia (RR = 0.28; 95% CI: 0.15 to 0.50; p = 0.0002), and hypertension (RR = 0.32; 95% CI: 0.17 to 0.58; p = 0.0027). CONCLUSION: The use of magnesium orotate is promising not only in treating MVP and compensating for hypomagnesemia, but also in preventing and treating cardiac arrhythmias, regulating blood pressure, and improving the function of the autonomic nervous system.


Assuntos
Sistema Cardiovascular/efeitos dos fármacos , Deficiência de Magnésio/tratamento farmacológico , Ácido Orótico/análogos & derivados , Ensaios Clínicos Controlados Aleatórios como Assunto , Medicina Baseada em Evidências/métodos , Humanos , Ácido Orótico/uso terapêutico
20.
Phys Chem Chem Phys ; 17(27): 17790-6, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26087682

RESUMO

The dihydroorotate dehydrogenase (DHOD) enzyme catalyzes the unique redox reaction in the de novo pyrimidine biosynthesis pathway. In this reaction, the oxidation of dihydroorotate (DHO) to orotate (OA) and reduction of the flavin mononucleotide (FMN) cofactor is catalysed by DHOD. The class 2 DHOD, to which the human enzyme belongs, was experimentally shown to follow a stepwise mechanism but the data did not allow the determination of the order of bond-breaking in a stepwise oxidation of DHO. The goal of this study is to understand the reaction mechanism at the molecular level of class 2 DHOD, which may aid in the design of inhibitors that selectively impact the activity of only certain members of the enzyme family. In this paper, the catalytic mechanism of oxidation of DHO to OA in human DHOD was studied using a hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) approach and Molecular Dynamics (MD) simulations. The free energy barriers calculated reveal that the mechanism in human DHOD occurs via a stepwise reaction pathway. In the first step, a proton is abstracted from the C5 of DHO to the deprotonated Ser215 side chain. Whereas, in the second step, the transfer of the hydride or hydride equivalent from the C6 of DHO to the N5 of FMN, where free energy barrier calculated by the DFT/MM level is 10.84 kcal mol(-1). Finally, a residual decomposition analysis was carried out in order to elucidate the influence of the catalytic region residues during DHO oxidation.


Assuntos
Simulação de Dinâmica Molecular , Ácido Orótico/análogos & derivados , Ácido Orótico/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Teoria Quântica , Sítios de Ligação , Biocatálise , Mononucleotídeo de Flavina/química , Humanos , Ácido Orótico/química , Oxirredução , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Estrutura Terciária de Proteína , Termodinâmica
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