Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 764
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Ecotoxicol Environ Saf ; 192: 110254, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32007746

RESUMO

The direct interactions of bacterial membranes and polycyclic aromatic hydrocarbons (PAHs) strongly influence the biological processes, such as metabolic activity and uptake of substrates due to changes in membrane lipids. However, the elucidation of adaptation mechanisms as well as membrane phospholipid alterations in the presence of phenanthrene (PHE) from α-proteobacteria has not been fully explored. This study was conducted to define the degradation efficiency of PHE by Sphingopyxis soli strain KIT-001 in a newly isolated from Jeonju river sediments and to characterize lipid profiles in the presence of PHE in comparison to cells grown on glucose using quantitative lipidomic analysis. This strain was able to respectively utilize 1-hydroxy-2-naphthoic acid and salicylic acid as sole carbon source and approximately 90% of PHE (50 mg/L) was rapidly degraded via naphthalene route within 1 day incubation. In the cells grown on PHE, strain KIT-001 appeared to dynamically change profiles of metabolite and lipid in comparison to cells grown on glucose. The levels of primary metabolites, phosphatidylethanolamines (PE), and phosphatidic acids (PA) were significantly decreased, whereas the levels of phosphatidylcholines (PC) and phosphatidylglycerols (PG) were significantly increased. The adaptation mechanism of Sphingopyxis sp. regarded mainly the accumulation of bilayer forming lipids and anionic lipids to adapt more quickly under restricted nutrition and toxicity condition. Hence, these findings are conceivable that strain KIT-001 has a good adaptive ability and biodegradation for PHE through the alteration of phospholipids, and will be helpful for applications for effective bioremediation of PAHs-contaminated sites.


Assuntos
Fenantrenos/metabolismo , Fosfolipídeos/metabolismo , Sphingomonadaceae/metabolismo , Biodegradação Ambiental , Sedimentos Geológicos/microbiologia , Lipidômica , Metabolômica , Naftalenos/metabolismo , Naftóis/metabolismo , Fosfolipídeos/química , Ácido Salicílico/metabolismo , Sphingomonadaceae/isolamento & purificação
2.
Proc Natl Acad Sci U S A ; 117(2): 872-876, 2020 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-31888984

RESUMO

Virtually all proton-pumping terminal respiratory oxygen reductases are members of the heme-copper oxidoreductase superfamily. Most of these enzymes use reduced cytochrome c as a source of electrons, but a group of enzymes have evolved to directly oxidize membrane-bound quinols, usually menaquinol or ubiquinol. All of the quinol oxidases have an additional transmembrane helix (TM0) in subunit I that is not present in the related cytochrome c oxidases. The current work reports the 3.6-Å-resolution X-ray structure of the cytochrome aa 3 -600 menaquinol oxidase from Bacillus subtilis containing 1 equivalent of menaquinone. The structure shows that TM0 forms part of a cleft to accommodate the menaquinol-7 substrate. Crystals which have been soaked with the quinol-analog inhibitor HQNO (N-oxo-2-heptyl-4-hydroxyquinoline) or 3-iodo-HQNO reveal a single binding site where the inhibitor forms hydrogen bonds to amino acid residues shown previously by spectroscopic methods to interact with the semiquinone state of menaquinone, a catalytic intermediate.


Assuntos
Bacillus subtilis/metabolismo , Cobre/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Heme/química , Hidroquinonas/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Grupo dos Citocromos b/química , Transporte de Elétrons , Ligação de Hidrogênio , Modelos Moleculares , Naftóis/metabolismo , Oxirredutases , Conformação Proteica , Subunidades Proteicas/química , Bombas de Próton/química , Bombas de Próton/metabolismo , Terpenos/metabolismo , Vitamina K 2/análogos & derivados , Vitamina K 2/química
3.
Sci Total Environ ; 704: 135331, 2020 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-31831232

RESUMO

Rhodococcus sp. WB9, a strain isolated from polycyclic aromatic hydrocarbons contaminated soil, degraded phenanthrene (PHE, 100 mg L-1) completely within 4 days. 18 metabolites were identified during PHE degradation, including 5 different hydroxyphenanthrene compounds resulted from multiple routes of initial monooxygenase attack. Initial dioxygenation dominantly occurred on 3,4-C positions, followed by meta-cleavage to form 1-hydroxy-2-naphthoic acid (1H2N). More than 95.2% of 1H2N was transported to and kept in extracellular solution without further degradation. However, intracellular 1H2N was converted to 1,2-naphthalenediol that was branched to produce salicylate and phthalate. Furthermore, 131 genes in strain WB9 genome were related to aromatic hydrocarbons catabolism, including the gene coding for salicylate 1-monooxygenase that catalyzed the oxidation of 1H2N to 1,2-naphthalenediol, and complete gene sets for the transformation of salicylate and phthalate toward tricarboxylic acid (TCA) cycle. Metabolic and genomic analyses reveal that strain WB9 has the ability to metabolize intracellular 1H2N to TCA cycle intermediates, but the extracellular 1H2N can't enter the cells, restricting 1H2N bioavailability and PHE mineralization.


Assuntos
Biodegradação Ambiental , Naftóis/metabolismo , Fenantrenos/metabolismo , Rhodococcus/metabolismo
4.
Infect Immun ; 88(1)2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31685546

RESUMO

Listeria monocytogenes, a Gram-positive, facultative intracellular pathogen, survives and replicates in the cytosol of host cells. Synthesis of 1,4-dihydroxy-2-naphthoate (DHNA), an intermediate of menaquinone biosynthesis, is essential for cytosolic survival of L. monocytogenes independent from its role in respiration. Here, we demonstrate that DHNA is essential for virulence in a murine model of listeriosis due to both respiration-dependent and -independent functions. In addition, DHNA can be both secreted and utilized as an extracellular shared metabolite to promote cytosolic survival inside host macrophages. To understand the role(s) of DHNA in L. monocytogenes intracellular survival and virulence, we isolated DHNA-deficient (ΔmenD strain) suppressor mutants that formed plaques in monolayers of fibroblasts. Five ΔmenD suppressor (mds) mutants additionally rescued at least 50% of the cytosolic survival defect of the parent ΔmenD mutant. Whole-genome sequencing revealed that four of the five suppressor mutants had independent missense mutations in a putative transcriptional regulator, ytoI (lmo1576). Clean deletion and complementation in trans confirmed that loss of ytoI could restore plaquing and cytosolic survival of DHNA-deficient L. monocytogenes RNA-seq transcriptome analysis revealed five genes (lmo0944, lmo1575, lmo1577, lmo2005, and lmo2006) expressed at a higher level in the ΔytoI strain than in the wild-type strain, whereas two genes (lmo1917 and lmo2103) demonstrated lower expression in the ΔytoI mutant. Intriguingly, the majority of these genes are involved in controlling pyruvate flux. Metabolic analysis confirmed that acetoin, acetate, and lactate flux were altered in a ΔytoI mutant, suggesting a critical role for regulating these metabolic programs. In conclusion, we have demonstrated that, similar to findings in select other bacteria, DHNA can act as a shared resource, and it is essential for cytosolic survival and virulence of L. monocytogenes Furthermore, we have identified a novel transcriptional regulator in L. monocytogenes and determined that its metabolic regulation is implicated in cytosolic survival of L. monocytogenes.


Assuntos
Listeria monocytogenes/metabolismo , Listeriose/microbiologia , Proteínas Mutantes/metabolismo , Naftóis/metabolismo , Supressão Genética , Fatores de Transcrição/metabolismo , Animais , Citosol/química , Modelos Animais de Doenças , Listeria monocytogenes/genética , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/patogenicidade , Camundongos , Viabilidade Microbiana , Proteínas Mutantes/genética , Fatores de Transcrição/genética , Virulência , Vitamina K 2/análise , Sequenciamento Completo do Genoma
5.
Sci Total Environ ; 689: 789-796, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31280161

RESUMO

The sesquiterpene geosmin, mainly originating from cyanobacteria, is considered one of the problematic odor compounds responsible for unpleasant-tasting and -smelling water episodes in freshwater supplies. The biochemistry and genetics of geosmin synthesis in cyanobacteria is well-elucidated and the geosmin synthase gene (geo) has been cloned and characterized in recent years. However, understanding the diversity, origin, and evolution of geo has been hindered by the limited availability of geo sequences to date. On the basis of the cloned geo sequences from16 filamentous geosmin-producing cyanobacterial species, representing 11 genera in Nostocales and Oscillatoriales, the diversity and evolution of geo in cyanobacteria was systematically analyzed in this study. Homologous alignment revealed that geo is highly conserved among the examined cyanobacterial species, with DNA sequence identities >0.72. Phylogenetic reconstruction and codon bias analysis based on geo suggest that cyanobacterial geo form a monophyletic branch with a common origin and ancestor for cyanobacteria, actinomycetes, and myxobacteria. The global ratio of nonsynonymous/synonymous nucleotide substitutions (dN/dS) was 0.125, which is substantially <1 and indicates strong purifying selection in the evolution of cyanobacterial geo. To add to further interest, horizontal gene transfer of cyanobacterial geo in evolutionary history was confirmed by the discovery of an incongruent coevolutionary relationship between geo and housekeeping genes 16S rDNA and rpoC. The present study enhances the fundamental understanding of cyanobacterial geo in diversity and evolution, and sheds light on the development of molecular assays for detection and molecular ecology research of geosmin-producing cyanobacteria.


Assuntos
Proteínas de Bactérias/genética , Cianobactérias/genética , Evolução Molecular , Variação Genética , Naftóis/metabolismo , Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , DNA Bacteriano/análise , Filogenia , Análise de Sequência de DNA , Especificidade da Espécie
6.
Elife ; 82019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31232690

RESUMO

Some anaerobic bacteria use insoluble minerals as terminal electron acceptors and discovering the ways in which electrons move through the membrane barrier to the exterior acceptor forms an active field of research with implications for both bacterial physiology and bioenergy. A previous study suggested that Shewanella oneidensis MR-1 utilizes a small, polar, redox active molecule that serves as an electron shuttle between the bacteria and insoluble acceptors, but the shuttle itself has never been identified. Through isolation and synthesis, we identify it as ACNQ (2-amino-3-carboxy-1,4-naphthoquinone), a soluble analog of menaquinone. ACNQ is derived from DHNA (1,4-dihydroxy-2-naphthoic acid) in a non-enzymatic process that frustrated genetic approaches to identify the shuttle. Both ACNQ and DHNA restore reduction of AQDS under anaerobic growth in menaquinone-deficient mutants. Bioelectrochemistry analyses reveal that ACNQ (-0.32 VAg/AgCl) contributes to the extracellular electron transfer (EET) as an electron shuttle, without altering menaquinone generation or EET related cytochrome c expression.


Assuntos
Transporte de Elétrons , Naftoquinonas/metabolismo , Shewanella/metabolismo , Anaerobiose , Naftóis/metabolismo , Oxirredução
7.
J Agric Food Chem ; 67(23): 6523-6531, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31117507

RESUMO

Identification of diterpene synthase-encoding genes together with synthetic biology technology offers an opportunity for the biosynthesis of cis-abienol. The methylerythritol phosphate (MEP) and the mevalonate (MVA) pathways were both engineered for cis-abienol production in Escherichia coli, which improved the cis-abienol yield by approximately 7-fold and 31-fold, respectively, compared to the yield obtained by overexpression of the MEP pathway alone or the original MEP pathway. Furthermore, systematic optimization of cis-abienol biosynthesis was performed, such as diterpene synthase screening and two-phase cultivation. The combination of bifunctional class I/II cis-abienol synthase from Abies balsamea ( AbCAS) and class II abienol synthase from Salvia sclarea ( SsTPS2) was found to be the most effective. By using isopropyl myristate as a solvent in two-phase cultivation, cis-abienol production reached 634.7 mg/L in a fed-batch bioreactor. This work shows the possibility of E. coli utilizing glucose as a carbon source for cis-abienol biosynthesis through a modified pathway.


Assuntos
Diterpenos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Metabólica , Ácido Mevalônico/metabolismo , Naftóis/metabolismo , Abies/enzimologia , Vias Biossintéticas , Diterpenos/química , Fermentação , Glucose/metabolismo , Naftóis/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Salvia/enzimologia
8.
ACS Chem Biol ; 14(6): 1227-1234, 2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31141338

RESUMO

Heterologous expression of secondary metabolite genes and gene clusters has been proven to be a successful strategy for identification of new natural products of cryptic or silent genes hidden in the genome sequences. It is also a useful tool to produce designed compounds by synthetic biology approaches. In this study, we demonstrate the potential usage of the gene locus pcr4401 in the fast-growing filamentous fungus Penicillium crustosum as an integration site for heterologous gene expression. The deduced polyketide synthase (PKS) Pcr4401 is involved in the dihydroxynaphthalene (DHN)-melanin pigment formation, and its deletion in P. crustosum PRB-2 led to an albino phenotype. Heterologous expression of pcr4401 in Aspergillus nidulans proved its function as the melanin precursor YWA1 synthase. To ensure gene expression after genomic integration and to easily identify the potential transformants by visualization, the gene locus of pcr4401 was chosen as an integration site. For heterologous expression in P. crustosum, the expression constructs were created by ligation-independent homologous recombination in Escherichia coli or Saccharomyces cerevisiae. A pyrG deficient strain was also created, so that both the pyrG and hph resistance gene can be used as selection markers. Successful expression in P. crustosum was demonstrated by using one uncharacterized PKS gene from Aspergillus and two from Penicillium strains. All three genes were successfully introduced, heterologously expressed, and their biosynthetic products elucidated. The results presented in this study demonstrated that P. crustosum can be used as a suitable host for heterologous expression of secondary metabolite genes.


Assuntos
Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Melaninas/genética , Penicillium/genética , Pigmentos Biológicos/genética , Aspergillus nidulans/genética , Escherichia coli/genética , Melaninas/metabolismo , Naftóis/metabolismo , Penicillium/metabolismo , Pigmentos Biológicos/metabolismo , Policetídeo Sintases/metabolismo , Saccharomyces cerevisiae/genética
9.
PLoS One ; 14(4): e0214958, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30978223

RESUMO

The menaquinone biosynthetic pathway presents a promising drug target against Mycobacterium tuberculosis and potentially other Gram-positive pathogens. In the present study, the essentiality, steady state kinetics of MenA from M. tuberculosis and the mechanism of MenA inhibition by Ro 48-8071 were characterized. MenA [isoprenyl diphosphate:1,4-dihydroxy-2-naphthoate (DHNA) isoprenyltransferase] catalyzes a critical reaction in menaquinone biosynthesis that involves the conversion of cytosolic DHNA, to membrane bound demethylmenaquinone by transferring a hydrophobic 45-carbon isoprenoid chain (in the case of mycobacteria) to the ring nucleus of DHNA. Rv0534c previously identified as the gene encoding MenA in M. tuberculosis complemented a menA deletion in E. coli and an E. coli host expressing Rv0534c exhibited an eight-fold increase in MenA specific activity over the control strain harboring empty vector under similar assay conditions. Expression of Rv0534c is essential for mycobacterial survival and the native enzyme from M. tuberculosis H37Rv was characterized using membrane preparations as it was not possible to solubilize and purify the recombinant enzyme. The enzyme is absolutely dependent on the presence of a divalent cation for optimal activity with Mg+2 being the most effective and is active over a wide pH range, with pH 8.5 being optimal. The apparent Km values for DHNA and farnesyl diphosphate were found to be 8.2 and 4.3 µM, respectively. Ro 48-8071, a compound previously reported to inhibit mycobacterial MenA activity, is non-competitive with regard to DHNA and competitive with regard to the isoprenyldiphosphate substrate.


Assuntos
Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Viabilidade Microbiana , Mycobacterium tuberculosis/enzimologia , Alquil e Aril Transferases/genética , Proteínas de Bactérias/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Deleção de Genes , Teste de Complementação Genética , Mycobacterium tuberculosis/genética , Naftóis/química , Naftóis/metabolismo , Especificidade por Substrato
10.
Anal Chim Acta ; 1066: 121-130, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31027526

RESUMO

A visual, rapid, and sensitive method for the detection of two algal metabolites, geosmin (GSM) and 2-methylisoborneol (2-MIB) using a competitive displacement technique based on molecular imprinted polymers (MIPs) and fluorescent tags was developed. In this method, fluorescent tags that bind to synthetic receptor sites of MIPs were designed and synthesised. In the presence of target analytes (geosmin and 2-methylisoborneol respectively), the tags are displaced leading to fluorescence signals. The MIPs were derived from the polymerisation of functional monomers and crosslinkers in the presence of suitable templates. Good to high binding capacities and selectivities were obtained with the MIPs. The displacement of fluorescent-tagged substrates from the respective MIPs by the target analytes enabled the quantitative detection of geosmin at concentrations as low as 0.38 µM (69 µg L-1), while the LOD for 2-methylisoborneol is 0.29 µM (48 µg L-1) without any cross-reactivity, non-specific (false-positive) binding, and matrix complications. Qualitative detection of geosmin and 2-methylisoborneol is also possible via visualisation of fluorescence using a hand held UV lamp, with LOD for geosmin and 2-methylisoborneol at 0.44 µM (80 µg L-1) and 0.35 µM (60 µg L-1), respectively. The sensitivity of the system can be improved with a pre-concentration step using the respective MIPs as a sorbent.


Assuntos
/análise , Clorófitas/química , Fluorescência , Impressão Molecular , Naftóis/análise , Polímeros/química , /metabolismo , Clorófitas/metabolismo , Cinética , Estrutura Molecular , Naftóis/metabolismo , Tamanho da Partícula , Polímeros/síntese química , Propriedades de Superfície
11.
mBio ; 10(2)2019 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-30914505

RESUMO

The pathogenic fungus Aspergillus fumigatus is able to adapt to extremely variable environmental conditions. The A. fumigatus genome contains four genes coding for mitogen-activated protein kinases (MAPKs), which are important regulatory knots involved in diverse cellular responses. From a clinical perspective, MAPK activity has been connected to salvage pathways, which can determine the failure of effective treatment of invasive mycoses using antifungal drugs. Here, we report the characterization of the Saccharomyces cerevisiae Fus3 ortholog in A. fumigatus, designated MpkB. We demonstrate that MpkB is important for conidiation and that its deletion induces a copious increase of dihydroxynaphthalene (DHN)-melanin production. Simultaneous deletion of mpkB and mpkA, the latter related to maintenance of the cell wall integrity, normalized DHN-melanin production. Localization studies revealed that MpkB translocates into the nuclei when A. fumigatus germlings are exposed to caspofungin stress, and this is dependent on the cross-talk interaction with MpkA. Additionally, DHN-melanin formation was also increased after deletion of genes coding for the Gα protein GpaA and for the G protein-coupled receptor GprM. Yeast two-hybrid and coimmunoprecipitation assays confirmed that GpaA and GprM interact, suggesting their role in the MpkB signaling cascade.IMPORTANCE Aspergillus fumigatus is the most important airborne human pathogenic fungus, causing thousands of deaths per year. Its lethality is due to late and often inaccurate diagnosis and the lack of efficient therapeutics. The failure of efficient prophylaxis and therapy is based on the ability of this pathogen to activate numerous salvage pathways that are capable of overcoming the different drug-derived stresses. A major role in the protection of A. fumigatus is played by melanins. Melanins are cell wall-associated macromolecules classified as virulence determinants. The understanding of the various signaling pathways acting in this organism can be used to elucidate the mechanism beyond melanin production and help to identify ideal drug targets.


Assuntos
Aspergillus fumigatus/crescimento & desenvolvimento , Aspergillus fumigatus/metabolismo , Melaninas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Naftóis/metabolismo , Mapas de Interação de Proteínas , Esporos Fúngicos/crescimento & desenvolvimento , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno/genética
12.
Int Immunopharmacol ; 69: 225-234, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30738992

RESUMO

Heme oxygenase-1 (HO-1) has an anti-inflammatory action in acute pancreatitis (AP). However, its mechanism of action and natural compounds/drugs to induce HO-1 in pancreas are not well understood. In this study, we investigated the regulatory mechanisms of HO-1 during AP using desoxo-narchinol-A (DN), the natural compound inducing HO-1 in the pancreas. Female C57/BL6 Mice were intraperitoneally injected with supramaximal concentrations of cerulein (50 µg/kg) hourly for 6 h to induce AP. DMSO or DN was administered intraperitoneally, then mice were sacrificed 6 h after the final cerulein injection. Administration of DN increased pancreatic HO-1 expression through activation of activating protein-1, mediated by mitogen-activated protein kinases. Furthermore, DN treatment reduced the pancreatic weight-to-body weight ratio as well as production of digestive enzymes and pro-inflammatory cytokines. Inhibition of HO-1 by tin protoporphyrin IX abolished the protective effects of DN on pancreatic damage. Additionally, DN treatment inhibited neutrophil infiltration into the pancreas via regulation of chemokine (C-X-C motif) ligand 2 (CXCL2) by HO-1. Our results suggest that DN is an effective inducer of HO-1 in the pancreas, and that HO-1 regulates neutrophil infiltration in AP via CXCL2 inhibition.


Assuntos
Quimiocina CXCL2/metabolismo , Heme Oxigenase-1/metabolismo , Neutrófilos/fisiologia , Pâncreas/metabolismo , Pancreatite/metabolismo , Doença Aguda , Amilases/sangue , Animais , Ceruletídeo/administração & dosagem , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Naftóis/metabolismo , Infiltração de Neutrófilos , Pâncreas/patologia , Pancreatite/patologia
13.
Bioorg Chem ; 85: 420-430, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30665036

RESUMO

An air- and moisture-stable molybdenum Schiff base complexes immobilized on magnetic iron oxide nanoparticles with a core-shell structure was developed for utilization as a new heterogeneous catalyst. The surface, structural and magnetic characteristics of the nanomaterials were characterized by using Fourier transform infrared (FT-IR) spectroscopy, X-ray powder diffraction (XRD), vibrating sample magnetometry (VSM), scanning electron microscopy (SEM), energy dispersive X-ray (EDX) and inductively coupled plasma atomic emission spectroscopy (ICP-AES). In application point of view, an ultrasonic assisted one-pot multicomponent protocol for the synthesis of 1-(α-aminoalkyl)-2-naphthol derivatives have been demonstrated under mild condition with short reaction times, high yields and TON values up to 570. To survey the generality of the procedure, we studied the synthesis of α-aminonitrile derivatives with different aldehydes, trimethylsilyl cyanide (TMSCN) and aniline under the same conditions. Additionally, binding interaction of 1-(phenyl(pyridin-2-ylamino)methyl)naphthalen-2-ol (AMAN-1) with various types of rigid DNA and HSA has been investigated by molecular modeling study. In vitro studies under physiological conditions showed that the desired derivative interacts with calf-thymus DNA (ct-DNA) via an intercalative binding mode.


Assuntos
Complexos de Coordenação/química , DNA/metabolismo , Nanopartículas de Magnetita/química , Naftóis/metabolismo , Nitrilos/metabolismo , Bases de Schiff/química , Animais , Catálise , Bovinos , DNA/química , Humanos , Simulação de Acoplamento Molecular , Molibdênio/química , Naftóis/síntese química , Nitrilos/síntese química , Albumina Sérica Humana/metabolismo , Dióxido de Silício/química , Viscosidade
14.
Ecotoxicol Environ Saf ; 172: 1-10, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30665150

RESUMO

Benzo[a]pyrene (BaP) is a model compound of polycyclic aromatic hydrocarbons. The relationship between its toxicity and some target biomolecules has been investigated. To reveal the interactions of BaP biodegradation and metabolic network, BaP intermediates, proteome, carbon metabolism and ion transport were analyzed. The results show that 76% BaP was degraded by Brevibacillus brevis within 7 d through the cleavage of aromatic rings with the production of 1-naphthol and 2-naphthol. During this process, the expression of xylose isomerase was induced for xylose metabolism, whereas, α-cyclodextrin could no longer be metabolized. Lactic acid, acetic acid and oxalic acid at 0.1-1.2 mg dm-3 were released stemming from their enhanced biosynthesis in the pathways of pyruvate metabolism and citrate cycle, while 5-7 mg dm-3 of PO43- were transported for energy metabolism. The relative abundance of 43 proteins was significantly increased for pyruvate metabolism, citrate cycle, amino acid metabolism, purine metabolism, ribosome metabolism and protein synthesis.


Assuntos
Benzo(a)pireno/metabolismo , Brevibacillus/metabolismo , Bacillus/metabolismo , Benzo(a)pireno/química , Biodegradação Ambiental , Carbono/metabolismo , Metabolismo Energético , Naftóis/química , Naftóis/metabolismo , Proteoma/metabolismo , Proteômica
15.
Plant Physiol ; 179(3): 1001-1012, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30643014

RESUMO

Transcription activator-like effectors (TALEs) are bacterial Type-III effector proteins from phytopathogenic Xanthomonas species that act as transcription factors in plants. The modular DNA-binding domain of TALEs can be reprogrammed to target nearly any DNA sequence. Here, we designed and optimized a two-component AND-gate system for synthetic circuits in plants based on TALEs. In this system, named split-TALE (sTALE), the TALE DNA binding domain and the transcription activation domain are separated and each fused to protein interacting domains. Physical interaction of interacting domains leads to TALE-reconstitution and can be monitored by reporter gene induction. This setup was used for optimization of the sTALE scaffolds, which result in an AND-gate system with an improved signal-to-noise ratio. We also provide a toolkit of ready-to-use vectors and single modules compatible with Golden Gate cloning and MoClo syntax. In addition to its implementation in synthetic regulatory circuits, the sTALE system allows the analysis of protein-protein interactions in planta.


Assuntos
Plantas/genética , Biologia Sintética/métodos , Efetores Semelhantes a Ativadores de Transcrição/fisiologia , Xanthomonas/genética , Diterpenos/metabolismo , Engenharia Genética/métodos , Naftóis/metabolismo , Mapeamento de Interação de Proteínas , Efetores Semelhantes a Ativadores de Transcrição/genética , Efetores Semelhantes a Ativadores de Transcrição/metabolismo
16.
ACS Sens ; 4(3): 660-669, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30698007

RESUMO

The rise of three-dimensional cell culture systems that provide in vivo-like environments for pharmaco-toxicological models has prompted the need for simple and robust viability assays suitable for complex cell architectural structures. This study addresses that challenge with the development of an in vitro enzyme based electrochemical sensor for viability/cytotoxicity assessment of two-dimensional (2D) monolayer and three-dimensional (3D) spheroid culture formats. The biosensor measures the cell viability/toxicity via electrochemical monitoring of the enzymatic activity of nonspecific esterases of viable cells, through the hydrolysis of 1-naphthyl acetate to 1-naphthol. The proposed sensor demonstrated strong correlation ( r = 0.979) with viable cell numbers. Furthermore, the model intestinal toxicants diclofenac (DFC, pharmaceutical), okadaic acid (OA, food-safety), and mancozeb (MZB, environmental) were used for the functional evaluation of the proposed sensor using 2D and 3D culture formats. Sensor performance showed high consistency with conventional cell viability/cytotoxicity assays (MTT/CFDA-AM) for all toxicants, with the sensor IC50 values matching the relevant viability LC50 values at the 95% confidence interval range for 2D (DCF: 1.19-1.26 mM, MZB: 10.28-14.18 µM, OA: 40.91-77.13 nM) and 3D culture formats (DCF: 1.02-4.78 mM, MZB: 11.26-15.16 µM, OA: 162.09-179.67 nM). The presented results demonstrate the feasibility of the proposed sensor as a robust endpoint screening tool for both 2D and 3D cytotoxicity assessment.


Assuntos
Técnicas Biossensoriais/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Poluentes Ambientais/toxicidade , Inocuidade dos Alimentos , Intestinos/citologia , Esferoides Celulares/efeitos dos fármacos , Testes de Toxicidade/métodos , Sobrevivência Celular/efeitos dos fármacos , Eletroquímica , Esterases/metabolismo , Humanos , Concentração Inibidora 50 , Naftóis/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo
17.
Bioorg Med Chem ; 27(5): 708-720, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30679134

RESUMO

A series of new pyrazole linked benzothiazole-ß-naphthol derivatives were designed and synthesized using a simple, efficient and ecofriendly route under catalyst-free conditions in good to excellent yields. These derivatives were evaluated for their cytotoxicity on selected human cancer cell lines. Among those, the derivatives 4j, 4k and 4l exhibited considerable cytotoxicity with IC50 values ranging between 4.63 and 5.54 µM against human cervical cancer cells (HeLa). Structure activity relationship was elucidated by varying different substituents on benzothiazoles and pyrazoles. Further, flow cytometric analysis revealed that these derivatives induced cell cycle arrest in G2/M phase and spectroscopic studies such as UV-visible, fluorescence and circular dichroism studies showed that these derivatives exhibited good DNA binding affinity. Additionally, these derivatives can effectively inhibit the topoisomerase I activity. Viscosity studies and molecular docking studies demonstrated that the derivatives bind with the minor groove of the DNA.


Assuntos
Benzotiazóis/farmacologia , Naftóis/farmacologia , Pirazóis/farmacologia , Inibidores da Topoisomerase I/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Benzotiazóis/síntese química , Benzotiazóis/metabolismo , Bisbenzimidazol/farmacologia , Linhagem Celular Tumoral , DNA/química , DNA/metabolismo , Doxorrubicina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Substâncias Intercalantes/síntese química , Substâncias Intercalantes/metabolismo , Substâncias Intercalantes/farmacologia , Simulação de Acoplamento Molecular , Naftóis/síntese química , Naftóis/metabolismo , Pirazóis/síntese química , Pirazóis/metabolismo , Inibidores da Topoisomerase I/síntese química , Inibidores da Topoisomerase I/metabolismo , Viscosidade
18.
Chembiochem ; 20(7): 949-954, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30537293

RESUMO

Phenylpropanoids and phenylpropanoid-derived plant polyphenols find numerous applications in the food and pharmaceutical industries. In recent years, several microbial platform organisms have been engineered towards producing such compounds. However, for the most part, microbial (poly)phenol production is inspired by nature, so naturally occurring compounds have predominantly been produced to date. Here we have taken advantage of the promiscuity of the enzymes involved in phenylpropanoid synthesis and exploited the versatility of an engineered Escherichia coli strain harboring a synthetic monolignol pathway to convert supplemented natural and unnatural phenylpropenoic acids into their corresponding monolignols. The performed biotransformations showed that this strain is able to catalyze the stepwise reduction of chemically interesting unnatural phenylpropenoic acids such as 3,4,5-trimethoxycinnamic acid, 5-bromoferulic acid, 2-nitroferulic acid, and a "bicyclic" p-coumaric acid derivative, in addition to six naturally occurring phenylpropenoic acids.


Assuntos
Escherichia coli/metabolismo , Fenilpropionatos/metabolismo , Propanóis/metabolismo , Oxirredutases do Álcool/genética , Aldeído Oxirredutases/genética , Amônia-Liases/genética , Coenzima A Ligases/genética , Escherichia coli/genética , Engenharia Metabólica/métodos , Naftóis/metabolismo , Petroselinum/enzimologia , Fenóis/metabolismo , Rhodobacter sphaeroides/enzimologia , Zea mays/enzimologia
19.
Ecotoxicol Environ Saf ; 167: 331-337, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30359899

RESUMO

Although ascomycetes occupy a vaster niche in soil than the well-studied basidiomycetes, they have received limited attention in studies related to bioremediation. In this study, the degradation of carbaryl by Xylaria sp. was studied in different culture conditions and its possible metabolic pathway was elucidated. In liquid culture, 99% of the added carbaryl was eliminated when cytochrome P450 (CYP450) was active, which was similar to the degradation rate of Pleurotus ostreatus, a fungus with strong bioremediation ability. Mn2+ is beneficial to the degradation of carbaryl. Compared to the 72.17% degradation rate in sterile soil, 59.0% carbaryl was eliminated in non-sterile soil, which suggested that Xylaria sp. BNL1 can resist microorganismal infection. Furthermore, the intracellular fractions containing laccase, CYP450, and carbaryl esterase efficiently degraded carbaryl. The presence of carbaryl metabolites suggested that Xylaria sp. BNL1 initiated its attack on carbaryl via carbaryl esterase to release α-naphthol, which was further degraded to 1,4-naphthoquinone and benzoic acid by CYP450 and laccase. Thus, our study highlights the potential of using Xylaria sp. for bioremediation.


Assuntos
Biodegradação Ambiental , Carbaril/metabolismo , Xylariales/metabolismo , Ácido Benzoico/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Lacase/metabolismo , Manganês/metabolismo , Redes e Vias Metabólicas , Naftóis/metabolismo , Naftoquinonas/metabolismo , Pleurotus/metabolismo , Microbiologia do Solo
20.
Appl Environ Microbiol ; 84(24)2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30315077

RESUMO

Carbaryl is the most widely used carbamate family pesticide, and its persistent nature causes it to pollute both soil and water ecosystems. Microbes maintain the Earth's biogeochemical cycles by metabolizing various compounds present in the matter, including xenobiotics, as a sole source of carbon, nitrogen, and energy. Soil isolate Pseudomonas sp. strain C5pp metabolizes carbaryl efficiently as the carbon source. Periplasmic carbaryl hydrolase catalyzes the conversion of carbaryl to 1-naphthol and methylamine. 1-Naphthol was further used as a carbon source via gentisate, whereas the metabolic fate of methylamine is not known. Here, we demonstrate that strain C5pp showed efficient growth on carbaryl when supplied as a carbon and nitrogen source, suggesting that the methylamine generated was used as the nitrogen source. Genes involved in the methylamine metabolism were annotated and characterized at the biochemical and molecular level. Transcriptional and enzyme activity studies corroborate that the γ-glutamylmethylamide/N-methylglutamate (GMA/NMG) pathway is involved in the metabolism of carbaryl and methylamine as a nitrogen source. Compared to carbaryl, methylamine was found to be an effective inducer for the metabolic and transporter genes. Strain C5pp also harbored genes involved in sarcosine metabolism that were cotranscribed and induced by sarcosine. The presence of inducible pathways for metabolism of carbaryl as a nitrogen and carbon source helps in complete and efficient mineralization of carbaryl by strain C5pp, thereby maintaining the biogeochemical cycles.IMPORTANCE The degradation of xenobiotics plays a significant role in the environment to maintain ecological systems as well as to prevent the imbalance of biogeochemical cycles via carbon-nitrogen cycling. Carbaryl is the most widely used pesticide from the carbamate family. Pseudomonas sp. strain C5pp, capable of utilizing carbaryl as a carbon and nitrogen source for its growth, subsequently helps in complete remediation of carbaryl. Thus, it maintains the ecosystem by balancing the biogeochemical cycles. The metabolic versatility and genetic diversity of strain C5pp for the transformation of contaminants like carbaryl and 1-naphthol into less harmful products make it a suitable candidate from the perspective of bioremediation.


Assuntos
Carbaril/metabolismo , Carbono/metabolismo , Redes e Vias Metabólicas , Metilaminas/metabolismo , Nitrogênio/metabolismo , Pseudomonas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Biodegradação Ambiental , Carbamatos , Ciclo do Carbono/genética , Clonagem Molecular , Ecossistema , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Gentisatos/metabolismo , Glutamato-Amônia Ligase/genética , Hidrolases , Cinética , Redes e Vias Metabólicas/genética , Família Multigênica , Naftóis/metabolismo , Ciclo do Nitrogênio/genética , Periplasma/metabolismo , Pseudomonas/genética , Pseudomonas/crescimento & desenvolvimento , Sarcosina/metabolismo , Alinhamento de Sequência , Microbiologia do Solo , Xenobióticos/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA