RESUMO
We have proposed a rapid phylogenetic classification at the strain level by MALDI-TOF MS using ribosomal protein matching profiling. In this study, the S10-spc-alpha operon, encoding half of the ribosomal subunit proteins and highly conserved in eubacterial genomes, was selected for construction of the ribosomal protein database as biomarkers for bacterial identification by MALDI-TOF MS analysis to establish a more reliable phylogenetic classification. Our method revealed that the 14 reliable and reproducible ribosomal subunit proteins with less than m/z 15,000, except for L14, coded in the S10-spc-alpha operon were significantly useful biomarkers for bacterial classification at species and strain levels by MALDI-TOF MS analysis of genus Pseudomonas strains. The obtained phylogenetic tree was consisted with that based on genetic sequence (gyrB). Since S10-spc-alpha operons of genus Pseudomonas strains were sequenced using specific primers designed based on nucleotide sequences of genome-sequenced strains, the ribosomal subunit proteins encoded in S10-spc-alpha operon were suitable biomarkers for construction and correction of the database. MALDI-TOF MS analysis using these 14 selected ribosomal proteins is a rapid, efficient, and versatile bacterial identification method with the validation procedure for the obtained results.
Assuntos
Proteínas de Bactérias/análise , Pseudomonas/metabolismo , Proteínas Ribossômicas/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Proteínas de Bactérias/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Bases de Dados de Proteínas , Dados de Sequência Molecular , Óperon , Filogenia , Subunidades Proteicas/análise , Subunidades Proteicas/genética , Pseudomonas/classificação , Pseudomonas/genética , Proteínas Ribossômicas/genética , Análise de Sequência de DNA , Especificidade da EspécieRESUMO
The effect of 32 flavonoids on androgen (AR) and glucocorticoid receptors (GR) was investigated using an MDA-kb2 human breast cancer cell line to predict potential AR and GR activities. Among them, 5-hydroxyflavone (7) had the highest AR antagonistic activity with an IC(50) value of 0.3 microM, whereas 6-methoxyflavone (11) had the highest induced luciferase activity with an EC(150) value of 0.7 microM. Genistein (2) and daizein (1) showed a sufficient increase of luciferase activities as their concentrations increased with EC(150) values of 4.4 and 10.1 microM, respectively. These findings provide evidence of a fundamental property of their structure-activity relationship with AR and/or GR.
Assuntos
Antagonistas de Receptores de Andrógenos , Androgênios , Flavonoides/farmacologia , Genes Reporter/genética , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/antagonistas & inibidores , Linhagem Celular Tumoral , Flavonoides/química , Humanos , Receptores Androgênicos/metabolismo , Receptores de Glucocorticoides/metabolismo , Relação Estrutura-AtividadeRESUMO
A search of streptomycin-resistant bacteria was carried out using ten creek water samples collected in Saga prefecture by spreading the sample water on an R2A medium containing 10 microg/ml of streptomycin. It was clarified that such streptomycin-resistant bacteria as Bacillus, Novosphigobium, Sphingopyxis and Oceanobacillus were distributed in the creek water. Further, 60% of the isolates didn't form an inhibitory zone by the application of 700 microg/ml streptomycin solution in the cup method assay. Further, the effectiveness of the MALDI-TOF MS analysis for the grouping of the isolates was examined. The discriminating ability of MALDI-TOF MS analysis was higher than that of RFLP analysis and it was almost equal to that of sequence analysis using 16S rDNA. Considering the high-throughput ability of the MALDI-TOF MS instrument, MALDI-TOF mass spectral identification of bacteria will be a powerful method in the construction of a MALDI-TOF mass spectra database.
Assuntos
Agroquímicos/farmacologia , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/isolamento & purificação , Rios/microbiologia , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Estreptomicina/farmacologia , Bactérias/classificação , Farmacorresistência Bacteriana , Polimorfismo de Fragmento de Restrição , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Identification of several environmental chemicals capable of binding to the androgen receptor (AR) and interfering with its normal function has heightened concern about adverse effects across a broad spectrum of environmental chemicals. We previously demonstrated AR antagonist activity of the organophosphate (OP) pesticide fenitrothion. In this study, we characterized AR activity of analogues of fenitrothion to probe the structural requirements for AR activity among related chemicals. AR activity was measured using HepG2 human hepatoma cells transfected with human AR plus an androgen-responsive luciferase reporter gene, MMTV-luc. AR antagonist activity decreased as alkyl chain length of the phosphoester increased, whereas electron-donating properties of phenyl substituents of the tested compounds did not influence AR activity. Oxon derivatives of fenitrothion, which are more likely to undergo hydrolytic degradation, had no detectable AR antagonist activity. Molecular modeling results suggest that hydrogen-bond energies and the maximum achievable interatomic distance between two terminal H-bond capable sites may influence both the potential to interact with the AR and the nature of the interaction (agonist vs. antagonist) within this series of chemicals. This hypothesis is supported by the results of recent AR homology modeling and crystallographic studies relative to agonist- and antagonist-bound AR complexes. The present results are placed in the context of structure-activity knowledge derived from previous modeling studies as well as studies aimed toward designing nonsteroidal antiandrogen pharmaceuticals. Present results extend understanding of the structural requirements for AR activity to a new class of nonsteroidal, environmental, OP-related chemicals.
Assuntos
Fenitrotion/efeitos adversos , Inseticidas/efeitos adversos , Receptores Androgênicos/efeitos dos fármacos , Receptores Androgênicos/fisiologia , Carcinoma Hepatocelular/patologia , Interações Medicamentosas , Humanos , Ligação de Hidrogênio , Neoplasias Hepáticas/patologia , Modelos Moleculares , Relação Estrutura-Atividade , Transfecção , Células Tumorais CultivadasRESUMO
Joyce et al. proposed the hypothesis that the sugar moiety of nucleic acid evolved from some achiral, stable and acyclic sugar into the ribose or the deoxyribose. According to their hypothesis, we designed and synthesized new pseudonucleotides having pentaerythritol moiety as a sugar moiety of nucleic acid and estimated their biological activities. Although all chemicals were not toxic to Lepidium sativum, Rhodotolula rubra and Cercospora kikuchi in the eucaryotes, three compounds having adenine, benzimidazole or 6-chloropurine residue as the base component of nucleotide exhibited the growth inhibiting activity to a prokaryote Spirulina platensis at 100 ppm. In the plaque formation test with Vero cells, a chemical with 6-chloropurine moiety inhibited 65.7% of plaque formation by Herpes simplex virus(HSV-1) at 500 ppm. Three chemicals with 6-chloropurine, 2-mercaptomethylbenzimidazole or guanine as the nucleic base moiety inhibited 62.3, 63.1 and 52.5% of plaque formation by Parainfluenza virus(PIFV) at 500 ppm, respectively. The prepared chemicals exhibited no effect on the Vero cells at the same concentration.
Assuntos
Carboidratos/química , Evolução Molecular , Ácidos Nucleicos/química , Nucleotídeos/química , Nucleotídeos/síntese química , Animais , Antivirais/síntese química , Antivirais/química , Antivirais/farmacologia , Células Cultivadas , Chlorocebus aethiops , Cianobactérias/crescimento & desenvolvimento , Depressão Química , Herpesvirus Humano 1/crescimento & desenvolvimento , Nucleotídeos/farmacologia , Paramyxoviridae/crescimento & desenvolvimento , Relação Estrutura-Atividade , Células Vero/virologiaRESUMO
The bacteria Sphingomonas sp. strain BSN22, isolated from bean fields, degraded octylphenol polyethoxylates (OPEO(n)) to octylphenol (OP) under aerobic conditions. This biodegradation mechanism proceeded by the following two-step degradation process: (1) degradation of OPEO(n) to octylphenol triethoxylate (OPEO(3)), (2) degradation from OPEO(3) to OP via octylphenoxy acetic acid (OPEC(1)). The chemical structure of OPEC(1) was confirmed by analysis using (18)O-labeled water. Quantitative studies revealed that magnesium (Mg(2+)) and calcium (Ca(2+)) ions were essential for the biodegradation of OPEO(n). Furthermore, the rate of biodegradation was especially accelerated by ferric ions (Fe(3+)), and the accumulated amounts of endocrine active chemicals, such as OP, OPEO(1), and OPEC(1), significantly increased to the concentration of 22.8, 221.7, and 961.1 microM in the presence of 37.0 microM Fe(3+), respectively. This suggests that environmental elements significantly influence the resultant ecotoxicity as well as the rate of their biodegradation in the environment. This study on the mechanism of OPEO(n) biodegradation may play an important role in understanding and managing environmental safety, including drinking water safety.
Assuntos
Resinas Acrílicas/química , Sphingomonas/metabolismo , Oligoelementos/metabolismo , Resinas Acrílicas/metabolismo , Biodegradação Ambiental , Cálcio/metabolismo , Magnésio/metabolismo , Microbiologia do Solo , Sphingomonas/isolamento & purificaçãoRESUMO
Because limes have been used as important fertilizers to neutralize acidified farmland in Japan, our interest in this study was focused on the effect of calcium ion on the biodegradation of octylphenol polyethoxylates (OPEOn) by a pure culture of Pseudomonas putida S5 isolated from a rice paddy field in Japan. In the presence of calcium ion, P. putida S5 accelerated the formation of octylphenol oligoethoxy carboxylates (OPECn) rather than that of octylphenol oligoethoxylates under an aerobic condition, indicating that more soluble biodegradates with terminal carboxyl group may liquate out easily to surface and ground water rather than more hydrophobic biodegradates with shorter ethylene oxide residues. Therefore, the androgen receptor (AR) activity of their degradation products was characterized using an in vitro reporter gene assay. As ethylene oxide chain length decreased, the biodegradates, OPEOn (n < 3), increased their AR antagonist activity. However, OPECn (n < 3) were unable to determine their AR activity because of their cytotoxicity in our reporter gene assay system.
Assuntos
Antagonistas de Androgênios/metabolismo , Cálcio/farmacologia , Fenóis/metabolismo , Pseudomonas putida/metabolismo , Antagonistas de Androgênios/química , Antagonistas de Androgênios/farmacologia , Antagonistas de Receptores de Andrógenos , Biodegradação Ambiental/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Humanos , Japão , Estrutura Molecular , Fenóis/química , Pseudomonas putida/efeitos dos fármacosRESUMO
A new method for phylogenetic classification of bacterial strains using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is proposed. This method was developed using a bioinformatics-based approach to the rapid identification of bacteria as previously proposed by Demirev and co-workers, which uses ribosomal proteins composed of approximately 50 subunit proteins as biomarkers. Although the amino acid sequences of ribosomal proteins are highly conserved, slight sequence variations can occur at the strain level. Since ribosomal subunit proteins are a complex of housekeeping proteins that have different phylogenetic evolution rates, sequence variation detected as mass differences by MALDI-MS may be useful for the phylogenetic classification of bacteria at strain level. In our proposed method, the first step is the selection of reliable biomarkers through characterization of the expressed ribosomal subunit proteins of a reference strain (usually a genome-sequenced strain) by MALDI-MS. The observed masses in the MALDI mass spectra of cell lysates of sample strains are then compared with the biomarker masses of the reference strain. The biomarkers for each sample strain were designated as present or absent at the reference masses, indicated by 1 or 0, respectively, which were summarized in a table. This table is processed by cluster analysis, generating a phylogenetic tree. In this study, the success of this approach was confirmed by classification of Pseudomonas putida strains because its classification is much more complicated than that of other bacterial strains. Forty-three reliable biomarkers were selected from ribosomal sub-unit proteins of a genome-sequenced strain, P. putida KT2440. The numbers and kinds of biomarkers observed for 16 strains of P. putida, including different biovars, were markedly different, reflecting the variety of the strains. The classification results by the proposed method were highly comparable to those based on the DNA gyrase subunit B gene (gyrB) sequence analysis, suggesting our proposed method would be a useful high-throughput method for phylogenetic classification of newly isolated bacteria.
Assuntos
Filogenia , Pseudomonas putida/classificação , Pseudomonas putida/genética , Proteínas Ribossômicas/análise , Proteínas Ribossômicas/genética , Subunidades Ribossômicas/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Biomarcadores , Pseudomonas putida/química , Proteínas Ribossômicas/química , Proteínas Ribossômicas/classificação , Subunidades Ribossômicas/química , Subunidades Ribossômicas/classificaçãoRESUMO
Octylphenol polyethoxylate (OPEO(n)) biodegradation by Pseudomonas putida S-5 under aerobic conditions is initiated by the oxidation of its terminal alcohol group by alcohol dehydrogenase. A DNA fragment, containing an alcohol dehydrogenase gene (adh1), was isolated using a combination of degenerate PCR and inverse PCR. The predicted translation product of adh1 showed significant sequence similarity to bacterial alcohol dehydrogenases. Furthermore, a flavin-binding motif and signature patterns conserved in type III FAD-dependent alcohol oxidases were detected. Two open reading frames (ORFs) were found upstream of adh1, encoding a putative acyl-CoA synthetase and a putative esterase. Downstream of adh1 and located on the opposite strand was an ORF encoding a putative aldehyde dehydrogenase. Transcription analysis using RT-PCR showed that adh1 is cotranscribed with the putative acyl-CoA synthetase and esterase genes during growth on OPEO(n). ADH1 overproduced in Escherichia coli exhibited activity not only toward various alcohols, including OPEO(n)s, but also toward primary aliphatic and aromatic aldehydes.
Assuntos
Álcool Desidrogenase/genética , Octoxinol/metabolismo , Pseudomonas putida/enzimologia , Álcool Desidrogenase/biossíntese , Álcool Desidrogenase/química , Sequência de Aminoácidos , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Octoxinol/química , Pseudomonas putida/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Análise de Sequência de DNA , Especificidade por SubstratoRESUMO
The androgen receptor (AR) activity of listed chemicals, so called SPEED 98, by the Ministry of the Environment, Japan, and structurally related chemicals was characterized using MDA-kb2 human breast cancer cells stably expressing an androgen-responsive luciferase reporter gene, MMTV-luc. Since our results suggested that chemicals with diverse chemical structures were capable of disrupting the endocrine systems mediated by AR, a comparative molecular field analysis (CoMFA) model was developed to analyze the structural requirements necessary to disrupt AR function. A significant CoMFA model with r(2)=0.825 and q(2)=0.332 was developed for AR antagonist activity of 35 pure antagonists excluding procymidone. On the other hand, a good CoMFA model with r(2)=0.983 and q(2)=0.555 was obtained for antagonist activity of 13 chemicals with both agonist and antagonist activities. The steric and electrostatic properties were sufficient to describe the structural requirements for AR antagonist activity. In addition, the structural difference of AR agonists and antagonists was explained based on CoMFA results and the AR-LBD crystal structure. As several ERalpha agonists such as diethylstilbestrol (DES) acted as AR antagonists, the surface area of the AR ligand-binding domain (LBD) was compared with that of the ERalpha-LBD based on their reported crystal structures to analyze how those ligands interact with LBDs. The surface area of AR-LBD was shown to be smaller than that of ERalpha-LBD and therefore compounds with both estrogenic and antiandrogenic activities can fit well into the ERalpha-LBD but may protrude from the AR-LBD. It is likely that this subtle difference of the surface areas of the LBDs determines whether an ERalpha agonist acts as an AR antagonist or an agonist.
Assuntos
Antagonistas de Androgênios/química , Receptores Androgênicos/efeitos dos fármacos , Antagonistas de Androgênios/farmacologia , Antagonistas de Receptores de Andrógenos , Androgênios , Linhagem Celular Tumoral , Genes Reporter , Humanos , Modelos Moleculares , Relação Quantitativa Estrutura-AtividadeRESUMO
Topsoil samples were collected from eight golf courses in Yamaguchi Prefecture, Japan, and enrichment cultures were carried out with a basal-salt medium containing 0.2% 4-tert-octylphenol polyethoxylate (OPPEO) as sole carbon source. OPPEO-degrading activity was detected in one of the samples, from which a strain of OPPEO-degrading bacterium was isolated. The isolated bacterium grew on a nutritionally enriched medium (NE medium) containing 0.2% OPPEO as sole carbon source, and accumulated 4-tert-octylphenol diethoxylate (OP2EO) (63%), 4-tert-octylphenol triethoxylate (OP3EO) (14%), and 4-tert-octylphenol monoethoxylate (OP1EO) (2%) after 7 d cultivation under aerobic conditions. The addition of clay mineral (vermiculite) to the medium accelerated the degradation of OP2EO (40%) and OP3EO (4%) to OP1EO (23%). This is the first report about bacteria that can degrade OPPEO to OP1EO under aerobic conditions. The strain was identified as Sphingomonas macrogoltabidus, based on the homology of a 16S rDNA sequence.
Assuntos
Etil-Éteres/metabolismo , Fenóis/metabolismo , Sphingomonas/isolamento & purificação , Aerobiose , Silicatos de Alumínio/farmacologia , Biodegradação Ambiental/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , DNA Bacteriano/genética , Etil-Éteres/química , Cromatografia Gasosa-Espectrometria de Massas , Estrutura Molecular , Fenóis/química , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 16S/genética , Microbiologia do Solo , Poluentes do Solo/análise , Poluentes do Solo/metabolismo , Sphingomonas/genética , Sphingomonas/metabolismoRESUMO
The effect of the surfactant, alpha-[4-(1,1,3,3-tetramethylbutyl) phenyl]-omega-hydroxypolyoxy-1,2-ethanediyl, on the adsolubilization of cholesterol and/or dotriacontane as model compounds of the epicuticular wax of mature tomato (Lycopersicon esculentum Mill.) fruit was investigated. Cholesterol as a model compound of such triterpenols as alpha- and beta-amyrins was solubilized in a concentration-dependent manner above the critical micelle concentration (cmc), while non-detectable quantities of the saturated hydrocarbon, dotriacontane, was solubilized at any concentration used. However, the surfactants solubilized more cholesterol from mixed than single membranes. The surfactants with a shorter polyoxyethylene (POE) chain length solubilized greater quantities than those with longer POE chains, suggesting that the microenvironment of micelles related to the polyoxyethylene moiety had an important effect on surfactant solubilization and that the octylphenol moiety must be capable of adsorbing to a specific region of the reconstituted membrane like dotriacontane.
Assuntos
Modelos Químicos , Polietilenoglicóis/química , Tensoativos/química , SolubilidadeRESUMO
Lunularic acid (LA) inhibited not only the germination and the growth of cress and lettuce at 1 mM but also the gibberellic acid (GA3)-induced alpha-amylase induction in embryoless barley seeds at 120 microM, which was recognized as a specific activity of abscisic acid (ABA). Moreover LA and ABA equally inhibited the growth of Lunularia cruciata A18 strain callus at 40 and 120 microM. A computational analysis revealed that the stable conformers of LA could be superimposed on the stable ABA conformers. In addition, the antibody raised against the conjugate of C1-ABA-bovine serum albumin (ABA-BSA) reacted with LA-horse-radish peroxidase (LA-HRP) conjugate as well as ABA-HRP conjugate, apparently. These results can explain why LA has ABA-like activity in higher plants. Moreover the results suggest that LA and ABA bind to the same receptor in higher plants.
Assuntos
Ácido Abscísico/química , Ácido Abscísico/farmacologia , Lactuca/efeitos dos fármacos , Lepidium/efeitos dos fármacos , Salicilatos/química , Salicilatos/farmacologia , Sementes/efeitos dos fármacos , Estilbenos , Indução Enzimática/efeitos dos fármacos , Hordeum/enzimologia , Cinética , Modelos Moleculares , Conformação Molecular , Sementes/enzimologia , alfa-Amilases/biossínteseRESUMO
Topsoil samples were collected from 36 different paddy fields in West Japan. Each soil sample was incubated with a basal salt-medium containing 0.2% OPPEO. Twelve samples possessed OPPEO-degrading activity, from which twelve cultures of OPPEO-degrading bacteria were isolated. The isolated bacteria grew on a medium containing 0.2% OPPEO as the sole carbon source, and OP2EO and OP3EO were accumulated in the medium under aerobic conditions. OP1EO and octylphenol, which have often been identified in surface water together with OP2EO, were not observed in this experiment. The bacterial isolates were gram negative and tentatively identified as Pseudomonas putida (10 isolates) and Burkholderia cepacia (one isolate) by BIOLOG and 16S rDNA RFLP analyses.
Assuntos
Burkholderia cepacia/isolamento & purificação , Burkholderia cepacia/metabolismo , Octoxinol/química , Octoxinol/metabolismo , Pseudomonas/isolamento & purificação , Pseudomonas/metabolismo , Microbiologia do Solo , Agricultura , Biodegradação Ambiental , Burkholderia cepacia/genética , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Cromatografia Gasosa-Espectrometria de Massas , Estrutura Molecular , Oryza , Polimorfismo de Fragmento de Restrição , Pseudomonas/genética , RNA Ribossômico 16S/genética , Fatores de TempoRESUMO
This paper reports the characterization of the biodegradation intermediates of octylphenol octaethoxylate (OP(8)EO) by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The biodegradation test study was carried out in a pure culture (Pseudomonas putida S-5) under aerobic conditions using OP(8)EO as the sole carbon source and (18)O-labeled water as an incubation medium. In the MALDI-MS spectra of biodegraded samples, a series of OP(n)EO molecules with n = 2-8 EO units and their corresponding carboxylic acid products (OP(n)EC) were observed. The use of purified OP(8)EO enabled one to distinguish the shortened OPEO molecules as biodegradation intermediates. Furthermore, the formation of OP(8)EC (the oxidized product of OP(8)EO) supported the notion that terminal oxidation is a step in the biodegradation process. When biodegradation study was carried out in (18)O-labeled water, incorporation of (18)O atoms into the carboxyl group was observed for OPEC, while no incorporation was observed for the shortened OPEO products. These results could provide some rationale to the biodegradation mechanism of alkylphenol polyethoxylates.