RESUMO
OBJECTIVES: The aims of this study were to assess aetiology and clinical characteristics in childhood meningitis, and develop clinical decision rules to distinguish bacterial meningitis from other similar clinical syndromes. METHODS: Children aged <16 years hospitalised with suspected meningitis/encephalitis were included, and prospectively recruited at 31 UK hospitals. Meningitis was defined as identification of bacteria/viruses from cerebrospinal fluid (CSF) and/or a raised CSF white blood cell count. New clinical decision rules were developed to distinguish bacterial from viral meningitis and those of alternative aetiology. RESULTS: The cohort included 3002 children (median age 2·4 months); 1101/3002 (36·7%) had meningitis, including 180 bacterial, 423 viral and 280 with no pathogen identified. Enterovirus was the most common pathogen in those aged <6 months and 10-16 years, with Neisseria meningitidis and/or Streptococcus pneumoniae commonest at age 6 months to 9 years. The Bacterial Meningitis Score had a negative predictive value of 95·3%. We developed two clinical decision rules, that could be used either before (sensitivity 82%, specificity 71%) or after lumbar puncture (sensitivity 84%, specificity 93%), to determine risk of bacterial meningitis. CONCLUSIONS: Bacterial meningitis comprised 6% of children with suspected meningitis/encephalitis. Our clinical decision rules provide potential novel approaches to assist with identifying children with bacterial meningitis. FUNDING: This study was funded by the Meningitis Research Foundation, Pfizer and the NIHR Programme Grants for Applied Research.
Assuntos
Meningites Bacterianas , Meningite Viral , Vacinas Conjugadas , Humanos , Criança , Lactente , Meningites Bacterianas/diagnóstico , Meningites Bacterianas/líquido cefalorraquidiano , Meningites Bacterianas/microbiologia , Pré-Escolar , Adolescente , Feminino , Masculino , Estudos Prospectivos , Meningite Viral/diagnóstico , Meningite Viral/líquido cefalorraquidiano , Regras de Decisão Clínica , Reino Unido/epidemiologia , Neisseria meningitidis/isolamento & purificação , Streptococcus pneumoniae/isolamento & purificação , Técnicas de Apoio para a DecisãoRESUMO
This article focuses on implementing multivariate analysis to evaluate biochar-derived carbonaceous nanomaterials (BCN) from three different feedstocks for the detection and differentiation of heavy metal ions in aqueous systems. The BCN were produced from dairy manure, rice straw and sorghum straw biochar using our NanoRefinery process. The NanoRefinery process transforms biochar into advanced nanomaterials using depolymerisation/chemical oxidation and purification of nanomaterials using solvent extraction. Dairy manure biochar-derived carbonaceous nanomaterials (DMB-CN), rice straw biochar-derived carbonaceous nanomaterials (RSB-CN) and sorghum straw biochar-derived carbonaceous nanomaterials (SSB-CN) were utilised as probes for the evaluation of their fluorescent properties and the detection of heavy metal ions. The BCN fluorescence quenching and fluorescence recovery was tested with lead (Pb2+), nickel (Ni2+), copper (Cu2+) and mercury (Hg2+). Principal component analysis (PCA) and discriminant analysis were used to differentiate among heavy metal ions in water samples. The BCN from different feedstocks had different characteristics and produced different interactions with heavy metal ions. DMB-CN had the highest quenching for Hg2+ and Ni2+ while SSB-CN and RSB-CN responded best to Cu2+ and Pb2+, respectively. The fluorescence quenching was modelled using linear and empirical functions. PCA and discriminant analysis used the quenching measurements to differentiate heavy metal ions in aqueous system. A key result was that the discriminant analysis had a 100% accuracy to detect Pb2+, 66% for Ni2+ and Cu2+, and 33% for Hg2+. This study has shown that biochar-derived carbonaceous nanomaterials could be used in heavy metal ions sensing applications. This is the first step in the development of a fast and accurate method for the detection of heavy metal ions in waters using environmentally friendly BCN.
Assuntos
Monitoramento Ambiental/métodos , Metais Pesados/análise , Nanoestruturas/química , Poluentes Químicos da Água/análise , Carvão Vegetal/química , Análise MultivariadaRESUMO
This article describes an efficient method, combining chemical oxidation and acetone extraction, to produce carbonaceous nanomaterials from dairy manure biochar. The optical and mechanical properties are similar to methods previously reported carbonaceous nanomaterials from biomass. Our novel process cuts the processing time in half and drastically reduces the energy input required. The acetone extraction produced 10 fractions with dairy manure biochar-derived carbonaceous nanomaterials (DMB-CNs). The fraction with the carbonaceous nanomaterials, DMB-CN-E1, with highest fluorescence was selected for in-depth characterisation and for initial testing across a range of applications. DMB-CN-E1 was characterised using atomic force microscope, electrophoresis, and spectrophotometric methods. DMB-CN-E1 exhibited a lateral dimension between 11 and 28â¯nm, a negative charge, and excitation/emission maxima at 337/410â¯nm, respectively. The bioimaging potential of DMB-CN-E1 evidenced different locations and different interactions with the cellular models evaluated. DMB-CN-E1 was quenched by several heavy metal ions showing a future application of these materials in heavy metal ion detection and/or removal. The demonstrated capabilities in bioimaging and environmental sensing create the opportunity for generating added-value nanomaterials (NanoRefinery) from dairy manure biochar gasification and, thus, increasing the economic viability of gasification plants.
Assuntos
Carvão Vegetal/química , Monitoramento Ambiental/instrumentação , Esterco/análise , Metais Pesados/análise , Nanotubos de Carbono/análise , Acetona/química , Animais , Bovinos , Indústria de Laticínios , Nanotubos de Carbono/química , Oxirredução , PolimerizaçãoRESUMO
This research seeks a coupled solution for managing the large amounts of biochar produced by microalgae biofuel production, and the necessity for novel, economic and accurate heavy metal sensing methods. Therefore, this study evaluated the transformation of microalgae biochar (MAB) into carbon dots (Cdots) and their subsequent application as heavy metal ion sensors in aqueous systems. The experimental phase included the transformation of MAB into microalgae biochar-derived carbon dots (MAB-Cdots), MAB-Cdot characterisation and the evaluation of the MAB-Cdots as transducers for the detection of four heavy metal ions (Pb2+, Cu2+, Cd2+, and Ni2+). MAB-Cdot fluorescence was stable over a wide range of pH and resistant to photo-bleaching, making them suitable as fluorescence probes. The MAB-Cdot fluorescence was quenched by all of the metal ions and displayed different quenching levels. Depending upon the ions involved, MAB-Cdots were used to detect the presence of heavy metal ions from concentrations of 0.012⯵M up to 2â¯mM by measuring the reduction in fluorescence intensity. Neutral and slightly alkaline pHs were optimal for Cu2+ Ni2+ and Pb2+ heavy metal quenching. To quantify the concentration of the heavy metal ions, linear and logarithmic functions were used to model the MAB-Cdot fluorescence quenching. The sensing mechanism was determined to be reversible and purely collisional with some fluorophores less accessible than the others. This work demonstrated the ability to produce Cdots from microalgae biochar, examined their application as a transducer for detecting heavy metal ions in aqueous systems and paves the way for novel sensing systems using MAB-Cdots.
Assuntos
Carbono/química , Carvão Vegetal/química , Monitoramento Ambiental/métodos , Metais Pesados/análise , Nanoestruturas/química , Poluentes Químicos da Água/análise , Fluorescência , Microalgas/químicaRESUMO
The binding and cytochrome P45051 (CYP51) inhibition properties of a novel antifungal compound, VT-1161, against purified recombinant Candida albicans CYP51 (ERG11) and Homo sapiens CYP51 were compared with those of clotrimazole, fluconazole, itraconazole, and voriconazole. VT-1161 produced a type II binding spectrum with Candida albicans CYP51, characteristic of heme iron coordination. The binding affinity of VT-1161 for Candida albicans CYP51 was high (dissociation constant [Kd], ≤ 39 nM) and similar to that of the pharmaceutical azole antifungals (Kd, ≤ 50 nM). In stark contrast, VT-1161 at concentrations up to 86 µM did not perturb the spectrum of recombinant human CYP51, whereas all the pharmaceutical azoles bound to human CYP51. In reconstitution assays, VT-1161 inhibited Candida albicans CYP51 activity in a tight-binding fashion with a potency similar to that of the pharmaceutical azoles but failed to inhibit the human enzyme at the highest concentration tested (50 µM). In addition, VT-1161 (MIC = 0.002 µg ml(-1)) had a more pronounced fungal sterol disruption profile (increased levels of methylated sterols and decreased levels of ergosterol) than the known CYP51 inhibitor voriconazole (MIC = 0.004 µg ml(-1)). Furthermore, VT-1161 weakly inhibited human CYP2C9, CYP2C19, and CYP3A4, suggesting a low drug-drug interaction potential. In summary, VT-1161 potently inhibited Candida albicans CYP51 and culture growth but did not inhibit human CYP51, demonstrating a >2,000-fold selectivity. This degree of potency and selectivity strongly supports the potential utility of VT-1161 in the treatment of Candida infections.
Assuntos
Inibidores de 14-alfa Desmetilase/química , Antifúngicos/química , Candida albicans/química , Proteínas Fúngicas/antagonistas & inibidores , Piridinas/química , Esterol 14-Desmetilase/química , Tetrazóis/química , Inibidores de 14-alfa Desmetilase/síntese química , Antifúngicos/síntese química , Sítios de Ligação , Candida albicans/enzimologia , Citocromo P-450 CYP2C19/química , Citocromo P-450 CYP2C9/química , Citocromo P-450 CYP3A/química , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Expressão Gênica , Humanos , Ligação Proteica , Piridinas/síntese química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade da Espécie , Esterol 14-Desmetilase/genética , Tetrazóis/síntese química , Voriconazol/químicaRESUMO
This study demonstrates use of recombinant yeast to simultaneously saccharify and ferment grass juice (GJ) to bioethanol. A modified Bacillus subtilis levanase gene (sacC) in which the native bacterial signal sequence was replaced with a yeast α-factor domain, was synthesised with yeast codon preferences and transformed into Saccharomyces cerevisiae (strain AH22) using the expression vector pMA91. AH22:psacC transformants secreted sacCp as an active, hyper-glycosylated (>180 kDa) protein allowing them to utilise inulin (ß[2-1] linked fructose) and levan (ß[2-6] linkages) as growth substrates. The control (AH22:pMA91) strain, transformed with empty plasmid DNA was not able to utilise inulin or levan. When cultured on untreated GJ levels of growth and bioethanol production were significantly higher in experiments with AH22:psacC than with AH22:pMA91. Bioethanol yields from AH22:psacC grown on GJ (32.7[±4] mg mL(-1)) compared closely to those recently achieved (Martel et al., 2010) using enzymatically pre-hydrolysed GJ (36.8[±4] mg mL(-1)).
Assuntos
Bacillus subtilis/enzimologia , Biocombustíveis/análise , Etanol/síntese química , Fermentação , Glicosídeo Hidrolases/metabolismo , Poaceae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Biotecnologia , Metabolismo dos Carboidratos , Dados de Sequência Molecular , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transformação GenéticaRESUMO
The recent decrease in the sensitivity of the Western European population of the wheat pathogen Mycosphaerella graminicola to azole fungicides has been associated with the emergence and subsequent spread of mutations in the CYP51 gene, encoding the azole target sterol 14alpha-demethylase. In this study, we have expressed wild-type and mutated M. graminicola CYP51 (MgCYP51) variants in a Saccharomyces cerevisiae mutant carrying a doxycycline-regulatable tetO(7)-CYC promoter controlling native CYP51 expression. We have shown that the wild-type MgCYP51 protein complements the function of the orthologous protein in S. cerevisiae. Mutant MgCYP51 proteins containing amino acid alterations L50S, Y459D, and Y461H and the two-amino-acid deletion DeltaY459/G460, commonly identified in modern M. graminicola populations, have no effect on the capacity of the M. graminicola protein to function in S. cerevisiae. We have also shown that the azole fungicide sensitivities of transformants expressing MgCYP51 variants with these alterations are substantially reduced. Furthermore, we have demonstrated that the I381V substitution, correlated with the recent decline in the effectiveness of azoles, destroys the capacity of MgCYP51 to complement the S. cerevisiae mutant when introduced alone. However, when I381V is combined with changes between residues Y459 and Y461, the function of the M. graminicola protein is partially restored. These findings demonstrate, for the first time for a plant pathogenic fungus, the impacts that naturally occurring CYP51 alterations have on both azole sensitivity and intrinsic protein function. In addition, we also provide functional evidence underlying the order in which CYP51 alterations in the Western European M. graminicola population emerged.
Assuntos
Ascomicetos/enzimologia , Azóis/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Fungicidas Industriais/farmacologia , Ascomicetos/genética , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Mutação , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esterol 14-Desmetilase , Esteróis/biossíntese , Triticum/microbiologiaRESUMO
Microbial inulinases find application in food, pharmaceutical and biofuel industries. Here, a novel Lactobacillus paracasei beta-fructosidase was overexpressed as truncated cytosolic protein ((t)fosEp) in Escherichia coli. Purified (t)fosEp was thermostable (10-50 degrees C) with a pH optimum of 5; it showed highest affinity for bacterial levan (beta[2-6] linked fructose) followed by nystose, chicory inulin, 1-kestose (beta[2-1] linkages) and sucrose (K(m) values of 0.5, 15, 15.6, 49 and 398 mM, respectively). Hydrolysis of polyfructose moieties in agriculturally-sourced grass juice (GJ) with (t)fosEp resulted in the release of >13 mg/ml more bioavailable fructose than was measured in untreated GJ. Bioethanol yields from fermentation experiments with Brewer's yeast and GJ+(t)fosEp were >25% higher than those achieved using untreated GJ feedstock (36.5[+/-4.3] and 28.2[+/-2.7]mg ethanol/ml, respectively). This constitutes the first specific study of the potential to ferment ethanol from grass juice and the utility of a novel core domain of beta-fructosidase from L. paracasei.
Assuntos
Biocombustíveis , Etanol/metabolismo , Frutanos/metabolismo , Lactobacillus/enzimologia , Poaceae/metabolismo , Proteínas Recombinantes/isolamento & purificação , beta-Frutofuranosidase/química , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Fermentação , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Especificidade por Substrato , Leveduras/crescimento & desenvolvimentoRESUMO
Inhibition of sterol-14 alpha-demethylase, a cytochrome P450 (CYP51, Erg11p), is the mode of action of azole antifungal drugs, and with high frequencies of fungal infections new agents are required. New drugs that target fungal CYP51 should not inhibit human CYP51, although selective inhibitors of the human target are also of interest as anticholesterol agents. A strain of Saccharomyces cerevisiae that was humanized with respect to the amino acids encoded at the CYP51 (ERG11) yeast locus (BY4741:huCYP51) was produced. The strain was validated with respect to gene expression, protein localization, growth characteristics, and sterol content. The MIC was determined and compared to that for the wild-type parental strain (BY4741), using clotrimazole, econazole, fluconazole, itraconazole, ketoconazole, miconazole, and voriconazole. The humanized strain showed up to >1,000-fold-reduced susceptibility to the orally active azole drugs, while the topical agents showed no difference. Data from growth kinetic measurements substantiated this finding but also revealed reduced effectiveness against the humanized strain for the topical drugs. Cellular sterol profiles reflected the decreased susceptibility of BY4741:huCYP51 and showed a smaller depletion of ergosterol and accumulation of 14 alpha-methyl-ergosta-8, 24(28)-dien-3beta-6 alpha-diol than the parental strain under the same treatment conditions. This strain provides a useful tool for initial specificity testing for new drugs targeting CYP51 and clearly differentiates azole antifungals in a side-by-side comparison.
Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Sequência de Bases , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/genética , DNA Fúngico/genética , Farmacorresistência Fúngica/genética , Farmacorresistência Fúngica/fisiologia , Genes Fúngicos , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Esterol 14-DesmetilaseRESUMO
The complete DNA sequence of Candida albicans DIT2, encoding cytochrome P450 family 56 (CYP56), was obtained, and heterologous expression was achieved in Escherichia coli, where CYP56 was targeted to the membrane fraction. In reconstituted assays with the purified enzyme, CYP56 was shown to catalyze the conversion of N-formyl tyrosine into N,N'-bisformyl dityrosine, a reaction that was dependent on cytochrome P450 reductase, NADPH, and oxygen, yielding a turnover of 21.6 min(-1) and a k(s) of 26 microM. The Hill number was calculated as 1.6, indicating that two molecules of the substrate could bind to the protein. Azole antifungals could bind to the heme of CYP56 as a sixth ligand with high affinity. Both chromosomal alleles of CYP56 were disrupted using the SAT1 flipper technique, and CYP56 was found to be nonessential for cell viability under the culture conditions investigated. Susceptibility to azole drugs that bind to cytochromes P450 was tested, and the mutant showed unaltered susceptibility. However, the mutant showed increased susceptibility to the echinocandin drug caspofungin, suggesting an alteration in 1,3-glucan synthase and/or cell wall structure mediated by the presence of dityrosine. Phenotypically, the wild-type and mutant strains were morphologically similar when cultured in rich yeast extract-peptone-dextrose medium. However in minimal medium, the cyp56Delta mutant strain exhibited hyphal growth, in contrast to the wild-type strain, which grew solely in the yeast form. Furthermore, CYP56 was essential for chlamydospore formation.
Assuntos
Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Sequência de Bases , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Parede Celular/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Primers do DNA/genética , DNA Fúngico/genética , Farmacorresistência Fúngica/genética , Farmacorresistência Fúngica/fisiologia , Deleção de Genes , Expressão Gênica , Genes Fúngicos , Mutação , Fenótipo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tirosina/análogos & derivados , Tirosina/biossínteseRESUMO
CYP (cytochrome P450) biodiversity and biotechnology is of importance given the industrial applications and potential for the huge array of genes and proteins that can constitute up to 1% of a coding genome. Historical biotechnological roles for CYPs in mutant fungi diverting the flux of metabolites towards penicillin production, in biotransformations allowing the production of corticosteroids and CYPs as drug targets contribute to interest in the roles of orphan CYPs in the emerging genomes. This area includes studies related to biotransformations and bioremediation, natural product synthesis and its manipulation, tools for exploiting CYPs and using CYPs as biomarkers and drug targets. Fundamental studies on diverse structure and function, on the ecological and evolution of CYPs through geological time and in drug/pesticide resistance also contribute distinctively to this field of CYP research.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Variação Genética , Animais , Biotecnologia/tendências , TrypanosomaRESUMO
The ergosterol pathway in fungal pathogens is an attractive antimicrobial target because it is unique from the major sterol (cholesterol) producing pathway in humans. Lanosterol 14alpha-demethylase is the target for a major class of antifungals, the azoles. In this study we have isolated the gene for this enzyme from Cryptococcus neoformans. The gene, ERG11, was recovered using degenerate PCR with primers designed with a novel algorithm called CODEHOP. Sequence analysis of Erg11p identified a highly conserved region typical of the cytochrome P450 class of mono-oxygenases. The gene was present in single copy in the genome and mapped to one end of the largest chromosome. Comparison of the protein sequence to a number of major human fungal pathogen Erg11p homologs revealed that the C. neoformans protein was highly conserved, and most closely related to the Erg11p homologs from other basidiomycetes. Functional studies demonstrated that the gene could complement a Saccharomyces cerevisiae erg11 mutant, which confirmed the identity of the C. neoformans gene.
Assuntos
Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/genética , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/genética , Oxirredutases/química , Oxirredutases/genética , Algoritmos , Sequência de Aminoácidos , Antifúngicos/farmacologia , Clonagem Molecular , Primers do DNA/química , DNA Complementar/metabolismo , Genes Fúngicos , Teste de Complementação Genética , Íntrons , Dados de Sequência Molecular , Mutação , Filogenia , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Esterol 14-DesmetilaseRESUMO
1. Cardiovascular responses to the stress of orthostasis, forearm (FA) ischaemia (reactive hyperaemia) and FA exercise (postexercise hyperaemia) are well described. Although sex differences in responses to orthostatic stress have been reported, few studies have examined the impact of sex on reactive hyperaemia and none has commented with regard to postexercise hyperaemia. 2. We investigated 11 men (mean (+/-SEM) age 18.5 +/- 0.3 years) and 10 women (18.8 +/- 0.8 years), all of whom were sedentary, with women being studied in the mid-follicular phase of their menstrual cycle. We measured blood pressure (BP), heart rate (HR) and forearm blood flow (FBF) in response to a fixed sequence of orthostatic, ischaemic and exercise stressors. 3. Orthostatic stress (10 min at -50 mmHg lower body negative pressure; LBNP) induced presyncopal signs in one man and three women. In all other subjects, BP was well maintained, with FBF decreasing and HR increasing similarly in both sexes. The tachycardia was earlier in onset in men and reached significantly higher absolute levels in women during the final 5 min of LBNP, but the percentage changes and integrated responses of both HR and FBF were not different between sexes. 4. The increases in FBF following either 10 min FA ischaemia or 10 min FA exercise were similar in men and women in terms of peak blood flow, percentage change, rate of recovery and total blood flow response. 5. In conclusion, although women were less tolerant of orthostatic stress than men, the cardiovascular responses to this and the other stressors appeared essentially independent of sex.
Assuntos
Hemodinâmica/fisiologia , Hipotensão Ortostática/fisiopatologia , Postura/fisiologia , Estresse Fisiológico/fisiopatologia , Adolescente , Adulto , Pressão Sanguínea/fisiologia , Exercício Físico/fisiologia , Feminino , Antebraço/irrigação sanguínea , Frequência Cardíaca/fisiologia , Humanos , Isquemia/fisiopatologia , Masculino , Contração Muscular/fisiologia , Fluxo Sanguíneo Regional/fisiologia , Caracteres Sexuais , Dobras CutâneasRESUMO
BACKGROUND/AIMS: SEN virus (SENV) was discovered in 1999 as a DNA virus with hepatotropic properties. Nine genotypes (A-I) have been identified with genotypes D and H being more prevalent in cases of chronic hepatitis. Attempts to determine whether SENV causes liver disease have been hampered by limited diagnostic testing. METHODS: In the present study, we developed two PCR based assays; a general SENV screening and genotype-specific assay. RESULTS: By screening PCR, the specificity for all SENV genotypes and SENV-related sequences was 20/20 (100%) with confirmation of the results being provided by genomic sequencing. With the genotype-specific PCR, specificities for SENV-D and SENV-H were 7/7 (100%) and 7/11 (64%), respectively. All screening PCR products were cloned and sequenced. The results of sequencing showed high genetic diversity in representative SENV genotypes. Five of twenty patients (25%) had mixed infections with several SENV genotypes. CONCLUSIONS: The screening PCR was useful for identifying cases of SENV infection. However, because of high genetic divergence and mixed co-infection, it was difficult to establish a specific method for genotype distinction. Hence, sequencing is still required for further investigations of SENV as a potential cause of liver disease.
Assuntos
Infecções por Circoviridae/diagnóstico , Circoviridae/genética , Circoviridae/isolamento & purificação , Hepatite Crônica/virologia , Reação em Cadeia da Polimerase/métodos , Sequência de Aminoácidos , Circoviridae/classificação , Infecções por Circoviridae/virologia , DNA Viral/sangue , DNA Viral/química , DNA Viral/isolamento & purificação , Variação Genética , Genótipo , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Sensibilidade e Especificidade , Proteínas Virais/químicaRESUMO
BACKGROUND/AIMS: Transgenic mice that express HBV X protein (HBx) have increased sensitivity to hepatocarcinogens. In the present study, we hypothesized that HBx interferes with the DNA protective increases in telomerase activity that occur in proliferating hepatocytes. METHODS: Male CD-1 mice (4-6/grp) were killed and hepatic telomerase activity measured at 0, 6, 12, 24, 36, 48 h post partial hepatectomy (PHx). Four HBx transgenic mice were killed at 12 h post-PHx when maximum telomerase activity was observed in CD-1 non-transgenic mice. mRNA of the telomerase catalytic subunit; murine telomerase reverse transcriptase (mTERT), was measured by reverse transcription-polymerase chain reaction. Telomerase activity and human TERT (hTERT) were also measured in Chang and PLC/PRF/5 cells following transient transfection with HBx cDNA. RESULTS: Telomerase activity peaked at 12 h post-PHx in normal mice, however, in HBx transgenic mice, telomerase activity was significantly lower, both at baseline (P<0.05) and 12 h post-PHx (P<0.01). Following PHx, mTERT mRNA expression remained constant in normal mice but decreased significantly (P<0.01) in HBx transgenic mice. Transfection of HBx in Chang and PLC/PRF/5 cells had no effect on telomerase activity or hTERT mRNA expression. CONCLUSIONS: The results of this study suggest that HBx expression may play a role in hepatocellular carcinogenesis by interfering with telomerase activity during hepatocyte proliferation.
Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Regeneração Hepática/fisiologia , Telomerase/metabolismo , Transativadores/genética , Animais , Carcinoma Hepatocelular/fisiopatologia , Proteínas de Ligação a DNA , Hepatectomia , Neoplasias Hepáticas/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Telômero/metabolismo , Transfecção , Regulação para Cima , Proteínas Virais Reguladoras e AcessóriasRESUMO
The raison d'ítre for the drug discovery and development process is to provide safe and effective treatments for diseases. Bringing a new drug to market, however, is a time-consuming and expensive process and it remains an imperative for drug companies that they identify ways in which they can accelerate the identification of potential targets and their screening and development in order to maintain a competitive edge. Successful drug discovery efforts include biochemical, biophysical, genetic and immunological approaches, targeting such processes as signal transduction, cell cycle control, apoptosis, gene regulation and metastasis. As the number of these biological targets increases, reliance on bioinformatics and chemoinformatics to improve decision making, by identifying characteristics of successful drugs and sharing knowledge gained within the scientific community, has become a burgeoning area in the post-genomic era of drug discovery.
Assuntos
Desenho de Fármacos , Genômica/métodos , Humanos , Estrutura Molecular , Ligação ProteicaRESUMO
Human sterol 14alpha-demethylase (P45051; CYP51) catalyzes the oxidative removal of the C32 methyl group of dihydrolanosterol, an essential step in the cholesterol biosynthetic pathway. The reaction is dependent upon NADPH cytochrome P450 reductase (CPR) that donates the electrons for the catalytic cycle. Here we used a recombinant yeast CPR to investigate the abilities of four different forms of cytochrome b(5) to support sterol demethylation activity of CYP51. The cytochrome b(5) derivatives were genetically engineered forms of the native rat cytochrome b(5) core-tail: the soluble globular b(5) core (core), the core linked at its N-terminus with the secretory signal sequence of alkaline phosphatase (signal-core), and the signal sequence linked to the native b(5) (signal-core-tail). The rat core-tail enzyme greatly stimulated sterol demethylation, whereas the signal-core-tail was only marginally active. In contrast, the core and signal-core constructs were completely inactive in stimulating the demethylation reaction. Additionally, cytochrome b(5) enhanced sterol demethylation by more than threefold by accepting electrons from soluble yeast CPR and in its ability to reduce P450. We show that the nature of transient linkage between the hemoproteins and the redox partners is most likely brought about electrostatically, although productive interaction between cytochrome b(5) and CYP51 is governed by the membrane-insertable hydrophobic region in the cytochrome b(5) which in turn determines the correct spatial orientation of the core. This is the first report showing the stimulation of CYP51 by cytochrome b(5).
Assuntos
Membrana Celular/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Citocromos b5/metabolismo , Oxirredutases/metabolismo , Sequência de Aminoácidos , Animais , Sistema Enzimático do Citocromo P-450/genética , Citocromos b5/genética , Citocromos b5/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Humanos , Hidroxilação/efeitos dos fármacos , Dados de Sequência Molecular , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oxirredução , Oxirredutases/genética , Ligação Proteica/fisiologia , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência/genética , Esterol 14-Desmetilase , Relação Estrutura-AtividadeRESUMO
Native yeast NADPH-cytochrome P450 oxidoreductase (CPR; EC 1.6.2.4) and a soluble derivative lacking 33 amino acids of the NH(2)-terminus have been overexpressed as recombinant proteins in Escherichia coli. The presence of a hexahistidine sequence at the N-terminus allowed protein purification in a single step using nickel-chelating affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the predicted molecular weights of the proteins and indicated a purity of >95%. Protein functionality was demonstrated by cytochrome c reduction and reconstitution of CYP61-mediated sterol Delta(22)-desaturation. Steady-state kinetics of cytochrome c reductase activity revealed a random Bi-Bi mechanism with NADPH donating electrons directly to CPR to produce a reduced intermediary form of the enzyme. The kinetic mechanism studies showed no difference between the two yeast CPRs in mechanism or after reconstitution with CYP61-mediated 22-desaturation, confirming that the retention of the NH(2)-terminable membrane anchor is functionally dispensable.
Assuntos
NADPH-Ferri-Hemoproteína Redutase/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Eletroforese em Gel de Poliacrilamida , Cinética , NADP/metabolismo , NADPH-Ferri-Hemoproteína Redutase/isolamento & purificação , Oxirredutases/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiaeRESUMO
Azole fungicides were thought to have much greater affinity for the fungal cytochrome P450 enzyme, sterol 14 alpha-demthylase (CYP51) than the plant orthologue. Using purified CYP51 from the plant Sorghum bicolor L Moenech, a direct comparison of the sensitivity to the fungicides triadimenol and tebuconazole has been carried out. S. bicolor CYP51 was purified to homogenity as determined by SDS--PAGE and specific heme content. Addition of the azole fungicides triadimenol and tebuconazole induced type II spectral changes, with saturation occurring at equimolar azole/P450 concentrations. Inhibition of reconstituted activities revealed only a threefold insensitivity of the plant CYP51 compared to a fungal CYP51, from the phytopathogen Ustilago maydis, as judged by IC(50) values. The implications for fungicide mode of action and application are discussed.
Assuntos
Inibidores das Enzimas do Citocromo P-450 , Fungicidas Industriais/farmacologia , Oxirredutases/antagonistas & inibidores , Proteínas de Plantas/antagonistas & inibidores , Triazóis/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Escherichia coli/genética , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/metabolismo , Fungicidas Industriais/metabolismo , Concentração Inibidora 50 , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Espectrofotometria , Esterol 14-Desmetilase , Triazóis/metabolismoRESUMO
Cytochrome P450 51 (CYP51) is sterol 14alpha-demethylase, known also as Erg11p in yeast. First studied in yeast, where it is one of three CYPs in the genome, it has subsequently gained attention as the only CYP found so far in different kingdoms of life. As such it is central to considerations of CYP evolution. Recent use of CYP51-inhibiting antifungal drugs, such as fluconazole, has also been associated with dramatic CYP51 evolution to numerous resistant forms in fungal pathogens. CYP51 has also been discovered in mycobacteria where antifungal azoles have effect and might be of value against tuberculosis. Evolutionary and therapeutic aspects of CYP51 studies are discussed.