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1.
Genes (Basel) ; 10(7)2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31323901

RESUMO

2-Methylketones are involved in plant defense and fragrance and have industrial applications as flavor additives and for biofuel production. We isolated three genes from the crop plant Solanum melongena (eggplant) and investigated these as candidates for methylketone production. The wild tomato methylketone synthase 2 (ShMKS2), which hydrolyzes ß-ketoacyl-acyl carrier proteins (ACP) to release ß-ketoacids in the penultimate step of methylketone synthesis, was used as a query to identify three homologs from S. melongena: SmMKS2-1, SmMKS2-2, and SmMKS2-3. Expression and functional characterization of SmMKS2s in E. coli showed that SmMKS2-1 and SmMKS2-2 exhibited the thioesterase activity against different ß-ketoacyl-ACP substrates to generate the corresponding saturated and unsaturated ß-ketoacids, which can undergo decarboxylation to form their respective 2-methylketone products, whereas SmMKS2-3 showed no activity. SmMKS2-1 was expressed at high level in leaves, stems, roots, flowers, and fruits, whereas expression of SmMKS2-2 and SmMKS2-3 was mainly in flowers and fruits, respectively. Expression of SmMKS2-1 was induced in leaves by mechanical wounding, and by methyl jasmonate or methyl salicylate, but SmMKS2-2 and SmMKS2-3 genes were not induced. SmMKS2-1 is a candidate for methylketone-based defense in eggplant, and both SmMKS2-1 and SmMKS2-2 are novel MKS2 enzymes for biosynthesis of methylketones as feedstocks to biofuel production.


Assuntos
Hexanonas/metabolismo , Solanum lycopersicum/enzimologia , Solanum melongena/metabolismo , Tioléster Hidrolases/metabolismo , Sequência de Aminoácidos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Hexanonas/química , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Solanum melongena/classificação , Solanum melongena/genética , Tioléster Hidrolases/química , Tioléster Hidrolases/genética
2.
Chem Biol Interact ; 305: 156-162, 2019 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-30849340

RESUMO

In terms of drug disposal and metabolism SDR21C1 (carbonyl reductase 1; CBR1) exerts an assorted substrate spectrum among a large variety of clinically relevant substances. Additionally, this short-chain dehydrogenase/reductase is extensively expressed in most tissues of the human body, thus underpinning its role in xenobiotic metabolism. Reduction of the chemotherapeutic daunorubicin (DAUN) to daunorubicinol (DAUNol) is a prominent example of its metabolic properties in terms of chemoresistance and cardiotoxicity. The hop-derived prenylated chalcone xanthohumol (XN) and its physiological metabolites isoxanthohumol (IX) and 8-prenylnaringenin (8-PN) have previously been reported to inhibit other DAUN reducing reductases and dehydrogenases including AKR1B1 and AKR1B10. Also with regard to their effects by means of interacting with cancer-related molecular pathways, XN and related prenylated flavonoids in particular have been in the focus of recent studies. In this study, inhibitory properties of these substances were examined with CBR1-mediated 2,3-hexanedione and DAUN reduction. All substances tested in this study turned out to efficiently inhibit recombinant human CBR1 within a low micromolar to submicromolar range. Among the substances tested, 8-PN turned out to be the most effective inhibitor when using 2,3-hexanedione as a substrate (Ki(app) = 180 ±â€¯20 nM). Inhibition rates of recombinant CBR1-mediated DAUN reduction were somewhat weaker with IC50-values ranging from 11 to 20 µM. XN, IX and 8-PN also efficiently inhibited DAUN reduction by SW480 colon adenocarcinoma cytosol (IC50 = 3.71 ±â€¯0.26 µM with 8-PN as inhibitor). This study identifies prenylated inhibitors, which might potentially interact with endogenous CBR1-driven (de-)toxication systems.


Assuntos
Oxirredutases do Álcool/metabolismo , Flavanonas/química , Flavonoides/química , Propiofenonas/química , Xantonas/química , Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/genética , Linhagem Celular Tumoral , Chalconas/química , Daunorrubicina/química , Daunorrubicina/metabolismo , Flavanonas/metabolismo , Flavonoides/metabolismo , Hexanonas/química , Hexanonas/metabolismo , Humanos , Concentração Inibidora 50 , Cinética , Oxirredução , Propiofenonas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Xantonas/metabolismo
3.
J Chem Ecol ; 45(3): 217-226, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30706280

RESUMO

Many species of longhorn beetles (Coleoptera: Cerambycidae) utilize male-produced aggregation-sex pheromones that attract both sexes. However, the reasons why and the details of how this type of pheromone is used by cerambycids and other coleopteran species that utilize analogous male-produced pheromones remain unclear. Thus, our goals were to test the hypotheses that 1) cerambycids respond to pheromones in a dose-dependent (= release rate-dependent) manner and 2) pheromone emission is density-dependent. If true, these characteristics of pheromone use could suggest that cerambycids utilize an optimal density strategy to limit competition for scarce and ephemeral hosts, i.e., the stressed or dying trees that typically constitute their larval hosts. Attraction of beetles to a range of release rates of two common pheromone components - 2-methylbutanol and 3-hydroxyhexan-2-one - was tested in field trials. Responses, as measured by the number of beetles caught in pheromone-baited traps, increased with release rates for five endemic species, even at the highest rates tested (~1450 µg/h for 2-methylbutanol and ~720 µg/h for 3-hydroxyhexan-2-one). The effect of density of conspecific males on per capita pheromone production was tested by collecting the volatiles produced by individuals, pairs, or groups of three or four male Phymatodes grandis Casey. Frequency of pheromone production was significantly different among the treatment densities, and emission rates of the pheromone (R)-2-methylbutanol decreased with increasing density. These results are discussed in the context of a possible optimal density strategy used by cerambycids, and more broadly, in relation to the use of male-produced aggregation-sex pheromones by other coleopterans. In addition, we report the identification of the pheromones of four of our five test species, specifically, Phymatodes obliquus Casey, Brothylus conspersus LeConte, Brothylus gemmulatus LeConte, and Xylotrechus albonotatus Casey.


Assuntos
Besouros/fisiologia , Atrativos Sexuais/metabolismo , Animais , Besouros/classificação , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Hexanonas/metabolismo , Masculino , Pentanóis/metabolismo , Especificidade da Espécie , Compostos Orgânicos Voláteis/metabolismo
4.
J Chem Ecol ; 45(1): 28-36, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30413986

RESUMO

The number of longhorn beetles with confirmed aggregation-sex pheromones has increased rapidly in recent years. However, the species that have been studied most intensively are pests, whereas much less is known about the pheromones of longhorn beetles that are rare or threatened. We studied the cerambycid beetle Plagionotus detritus ssp. detritus with the goal of confirming the presence and composition of an aggregation-sex pheromone. This species has suffered widespread population decline due to habitat loss in Western Europe, and it is now considered threatened and near extinction in several countries. Beetles from a captive breeding program in Sweden were used for headspace sampling. Gas chromatography-mass spectrometry revealed that collections from males contained large quantities of two compounds, identified as (R)-3-hydroxy-2-hexanone (major component) and (S)-2-hydroxy-3-octanone (minor component), in addition to smaller quantities of 2,3-hexanedione and 2,3-octanedione. None of the compounds was present in collections from females. When tested singly in a field bioassay, racemic 3-hydroxy-2-hexanone and 2-hydroxy-3-octanone were not attractive to P. detritus, whereas a 5:1 blend elicited significant attraction. Both compounds are known as components of the pheromones of conspecific beetles, but, to our knowledge, this is the first cerambycid shown to use two compounds with different chain lengths, in which the positions of the hydroxyl and carbonyl functions are interchanged between the two. The pheromone has potential as an efficient tool to detect and monitor populations of P. detritus, and may also be useful in more complex studies on the ecology and conservation requirements of this species.


Assuntos
Besouros/fisiologia , Hexanonas/metabolismo , Atrativos Sexuais/metabolismo , Animais , Ecossistema , Espécies em Perigo de Extinção , Feminino , Hexanonas/análise , Masculino , Atrativos Sexuais/análise , Comportamento Sexual Animal , Suécia
5.
J Biosci ; 43(1): 49-58, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29485114

RESUMO

In Dictyostelium discoideum, cells that become part of the stalk or basal disc display behaviour that can be interpreted as altruistic. Atzmony et al. (Curr Sci 72:142-145, 1997) had hypothesised that this behaviour could be the outcome of an adaptive strategy based on differing intrinsic quality as reflected by phenotypes that indicate differences in potential for survival and reproduction, followed by intercellular competition among amoebae of differing qualities. Low-quality amoebae would have a poor chance of succeeding in the competition to form spores; they could enhance their chances of survival by adopting a presumptive stalk strategy. Here we extend the hypothesis by making use of recent findings. Our approach is based on the view that an evolutionary explanation for the apparent altruism of stalk cells in D. discoideum must apply broadly to other cellular slime moulds (CSMs) that exhibit stalk cell death. Further, it must be capable of being modified to cover social behaviour in CSMs with an extracellular stalk, as well as in sorocarpic amoebae whose stalk cells are viable. With regard to D. discoideum, we suggest that (a) differentiation-inducing factor, thought of as a signal that inhibits amoebae from forming spores and induces them to differentiate into basal disc cells, is better viewed as a mediator of competition among post-aggregation amoebae and (b) the products of the 'recognition genes', tgrB and tgrC, allow an amoeba to assess its quality relative to that of its neighbours and move to a position within the aggregate that optimises its reproductive fitness. From this perspective, all cells behave in a manner that is 'selfish' rather than 'altruistic', albeit with different expectations of success.


Assuntos
Dictyostelium/genética , Hexanonas/metabolismo , Morfogênese/genética , Proteínas de Protozoários/genética , Esporos de Protozoários/genética , Adaptação Fisiológica , Evolução Biológica , Dictyostelium/citologia , Dictyostelium/metabolismo , Expressão Gênica , Variação Genética , Proteínas de Protozoários/metabolismo , Esporos de Protozoários/metabolismo
6.
J Nat Prod ; 80(10): 2716-2722, 2017 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-28921976

RESUMO

Eight chlorinated alkylresorcinols, monochasiol A-H (1-8), were isolated from the fruiting bodies of Dictyostelium monochasioides. Compounds 1-8 were synthesized to confirm their structures and to obtain sufficient material for performing biological tests. Monochasiol A (1) selectively inhibited the concanavalin A-induced interleukin-2 production in Jurkat cells, a human T lymphocyte cell line. Monochasiols were biogenetically synthesized by the combination of biosynthetic enzymes relating to the principal polyketides, MPBD and DIF-1, produced by Dictyostelium discoideum.


Assuntos
Dictyostelium/química , Hidrocarbonetos Clorados , Resorcinóis , Sobrevivência Celular/efeitos dos fármacos , Concanavalina A/farmacologia , Dictyosteliida/química , Células HeLa , Hexanonas/metabolismo , Humanos , Hidrocarbonetos Clorados/química , Hidrocarbonetos Clorados/isolamento & purificação , Hidrocarbonetos Clorados/farmacologia , Interleucina-2/biossíntese , Células Jurkat , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Policetídeos/metabolismo , Resorcinóis/química , Resorcinóis/isolamento & purificação , Resorcinóis/farmacologia
7.
Cell Chem Biol ; 24(8): 935-943.e7, 2017 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-28820963

RESUMO

The α-oxoaldehyde methylglyoxal is a ubiquitous and highly reactive metabolite known to be involved in aging- and diabetes-related diseases. If not detoxified by the endogenous glyoxalase system, it exerts its detrimental effects primarily by reacting with biopolymers such as DNA and proteins. We now demonstrate that during ketosis, another metabolic route is operative via direct non-enzymatic aldol reaction between methylglyoxal and the ketone body acetoacetate, leading to 3-hydroxyhexane-2,5-dione. This novel metabolite is present at a concentration of 10%-20% of the methylglyoxal level in the blood of insulin-starved patients. By employing a metabolite-alkyne-tagging strategy it is clarified that 3-hydroxyhexane-2,5-dione is further metabolized to non-glycating species in human blood. The discovery represents a new direction within non-enzymatic metabolism and within the use of alkyne-tagging for metabolism studies and it revitalizes acetoacetate as a competent endogenous carbon nucleophile.


Assuntos
Acetoacetatos/química , Corpos Cetônicos/química , Aldeído Pirúvico/sangue , Acetoacetatos/metabolismo , Alcinos/química , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Hexanonas/análise , Hexanonas/sangue , Hexanonas/metabolismo , Humanos , Corpos Cetônicos/metabolismo , Espectrometria de Massas , Aldeído Pirúvico/análise , Aldeído Pirúvico/metabolismo , Albumina Sérica/química , Albumina Sérica/metabolismo
8.
Insect Mol Biol ; 26(5): 642-653, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28621451

RESUMO

Insect sex pheromones (SPs) are central to mate-finding behaviour, and play an essential role in the survival and reproduction of organisms. Understanding the roles, biosynthetic pathways and evolution of insect chemical communication systems has been an exciting challenge for biologists. Compared with Lepidoptera, little is known about the mechanisms underlying pheromone biosynthesis in Hemiptera. In this study, we isolated and characterized two new desaturase-like genes, termed Asutdes1 and Asutdes2, from Adelphocoris suturalis, an important agricultural pest in China. Although the two genes encode an identical protein, Southern blot analysis revealed that they are duplicated genes. The Asutdes2 transcript is more abundant than Asutdes1 in the tissues tested, in particular the metathoracic scent gland and fat body. Silencing Asutdes expression in females by injecting double-stranded RNA (dsAsutdes) against a portion of the coding sequence shared by the two genes enhanced the production of (E)-4-oxo-2-hexenal, a component of the A. suturalis SP blend, and dramatically suppressed the sexual attractiveness of A. suturalis females. We conclude that dsAsutdes is associated with the SP biosynthetic pathway in A. suturalis.


Assuntos
Aldeídos/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Hemípteros/metabolismo , Hexanonas/metabolismo , Proteínas de Insetos/metabolismo , Atrativos Sexuais/metabolismo , Comportamento Sexual Animal/fisiologia , Sequência de Aminoácidos , Animais , Ácidos Graxos Dessaturases/genética , Feminino , Hemípteros/genética , Proteínas de Insetos/genética , Masculino , Dados de Sequência Molecular , Interferência de RNA , Glândulas Odoríferas/metabolismo , Análise de Sequência de DNA
9.
Mar Drugs ; 15(5)2017 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-28505073

RESUMO

Bioactivity-guided isolation of a crude extract from a culture broth of Bacillus sp. has led to the isolation of (-)-4-hydroxysattabacin (1). The inhibitory effect of (-)-4-hydroxysattabacin (1) was investigated on melanogenesis in the murine melanoma cell line, B16F10, and human melanoma cell line, MNT-1, as well as a pigmented 3D-human skin model. (-)-4-Hydroxysattabacin treatment decreased melanin contents in a dose-dependent manner in α-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 cells. Quantitative real time PCR (qRT-PCR) demonstrated that treatment with (-)-4-hydroxysattabacin down-regulated several melanogenic genes, including tyrosinase, tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2) while their enzymatic activities were unaffected. The anti-melanogenic effects of (-)-4-hydroxysattabacin were further demonstrated in a pigmented 3D human epidermal skin model, MelanodermTM, and manifested as whitening and regression of melanocyte activation in the tissue.


Assuntos
Organismos Aquáticos , Bacillus/metabolismo , Hexanonas/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Relação Dose-Resposta a Droga , Epiderme/efeitos dos fármacos , Hexanonas/química , Hexanonas/metabolismo , Humanos , Levodopa/administração & dosagem , Levodopa/farmacologia , Melaninas/metabolismo , Melanoma/metabolismo , Camundongos , Estrutura Molecular , Pigmentação/efeitos dos fármacos
10.
J Microbiol Biotechnol ; 27(6): 1065-1070, 2017 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-28297749

RESUMO

This study aimed to examine the anti-candidal efficacy of a novel ketone derivative isolated from Diaporthe sp. ED2, an endophytic fungus residing in medicinal herb Orthosiphon stamieus Benth. The ethyl acetate extract of the fungal culture was separated by open column and reverse phase high-performance liquid chromatography (HPLC). The eluent at retention time 5.64 min in the HPLC system was the only compound that exhibited anti-candidal activity on Kirby-Bauer assay. The structure of the compound was also elucidated by nuclear magnetic resonance and spectroscopy techniques. The purified anti-candidal compound was obtainedas a colorless solid and characterized as 3-hydroxy-5-methoxyhex-5-ene-2,4-dione. On broth microdilution assay, the compound also exhibited fungicidal activity on a clinical strain of Candida albicans at a minimal inhibitory concentration of 3.1 µg/ml. The killing kinetic analysis also revealed that the compound was fungicidal against C. albicans in a concentration- and time-dependent manner. The compound was heat-stable up to 70°C, but its anti-candidal activity was affected at pH 2.


Assuntos
Antifúngicos/farmacologia , Ascomicetos/química , Candida/efeitos dos fármacos , Endófitos/química , Hexanonas/farmacologia , Cetonas/metabolismo , Cetonas/farmacologia , Antifúngicos/química , Antifúngicos/metabolismo , Ascomicetos/metabolismo , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Cromatografia Líquida de Alta Pressão , Endófitos/metabolismo , Hexanonas/química , Hexanonas/isolamento & purificação , Hexanonas/metabolismo , Concentração de Íons de Hidrogênio , Cetonas/química , Cetonas/isolamento & purificação , Lamiaceae/microbiologia , Testes de Sensibilidade Microbiana , Temperatura
11.
FEBS Lett ; 590(6): 760-8, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26919666

RESUMO

Differentiation-inducing factor 1(DIF-1) and DIF-2 are signaling molecules that control chemotaxis in Dictyostelium discoideum. Whereas DIF-1 suppresses chemotaxis in shallow cAMP gradients, DIF-2 enhances chemotaxis under the same conditions via a phosphodiesterase, response regulator A (RegA), which is a part of the DhkC-RdeA-RegA two-component signaling system. In this study, to investigate the mechanism of the chemotaxis regulation by DIF-2, we examined the effects of DIF-2 (and DIF-1) on chemotaxis in rdeA(-) and dhkC(-) mutant strains. In the parental wild-type strains, chemotactic cell movement was suppressed with DIF-1 and enhanced with DIF-2 in shallow cAMP gradients. In contrast, in both rdeA(-) and dhkC(-) strains, chemotaxis was suppressed with DIF-1 but unaffected by DIF-2. The results suggest that DIF-2 modulates chemotaxis via the DhkC-RdeA-RegA signaling system.


Assuntos
Dictyostelium/fisiologia , Pentanonas/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Protozoários/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Quimiotaxia/fisiologia , AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dictyostelium/genética , Dictyostelium/crescimento & desenvolvimento , Deleção de Genes , Técnicas de Silenciamento de Genes , Genes de Protozoários , Hexanonas/metabolismo , Histidina Quinase , Modelos Biológicos , Mutação , Proteínas Quinases/genética , Proteínas de Protozoários/genética , Transdução de Sinais , Regulação para Cima
12.
J Mol Biol ; 427(23): 3722-33, 2015 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-26284972

RESUMO

Aggregative multicellularity, resulting in formation of a spore-bearing fruiting body, evolved at least six times independently amongst both eukaryotes and prokaryotes. Amongst eukaryotes, this form of multicellularity is mainly studied in the social amoeba Dictyostelium discoideum. In this review, we summarise trends in the evolution of cell-type specialisation and behavioural complexity in the four major groups of Dictyostelia. We describe the cell-cell communication systems that control the developmental programme of D. discoideum, highlighting the central role of cAMP in the regulation of cell movement and cell differentiation. Comparative genomic studies showed that the proteins involved in cAMP signalling are deeply conserved across Dictyostelia and their unicellular amoebozoan ancestors. Comparative functional analysis revealed that cAMP signalling in D. discoideum originated from a second messenger role in amoebozoan encystation. We highlight some molecular changes in cAMP signalling genes that were responsible for the novel roles of cAMP in multicellular development.


Assuntos
Evolução Biológica , AMP Cíclico/metabolismo , Dictyostelium/fisiologia , Proteínas de Protozoários/metabolismo , AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Dictyostelium/metabolismo , Genoma de Protozoário , Genômica/métodos , Hexanonas/metabolismo , Histidina Quinase , Proteínas Quinases/metabolismo , Proteínas de Protozoários/genética , Percepção de Quorum , Transdução de Sinais
13.
J Theor Biol ; 370: 135-50, 2015 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-25665718

RESUMO

Understanding the mechanisms that control tissue morphogenesis and homeostasis is a central goal not only in developmental biology but also has great relevance for our understanding of various diseases, including cancer. A model organism that is widely used to study the control of tissue morphogenesis and proportioning is the Dictyostelium discoideum. While there are mathematical models describing the role of chemotactic cell motility in the Dictyostelium assembly and morphogenesis of multicellular tissues, as well as models addressing possible mechanisms of proportion regulation, there are no models incorporating both these key aspects of development. In this paper, we introduce a 1D hyperbolic model to investigate the role of two morphogens, DIF and cAMP, on cell movement, cell sorting, cell-type differentiation and proportioning in Dictyostelium discoideum. First, we use the non-spatial version of the model to study cell-type transdifferentiation. We perform a steady-state analysis of it and show that, depending on the shape of the differentiation rate functions, multiple steady-state solutions may occur. Then we incorporate spatial dynamics into the model, and investigate the transdifferentiation and spatial positioning of cells inside the newly formed structures, following the removal of prestalk or prespore regions of a Dictyostelium slug. We show that in isolated prespore fragments, a tipped mound-like aggregate can be formed after a transdifferentiation from prespore to prestalk cells and following the sorting of prestalk cells to the centre of the aggregate. For isolated prestalk fragments, we show the formation of a slug-like structure containing the usual anterior-posterior pattern of prestalk and prespore cells.


Assuntos
Agregação Celular , Diferenciação Celular , Movimento Celular , Dictyostelium/citologia , Comunicação Celular , Simulação por Computador , AMP Cíclico/metabolismo , Dictyostelium/metabolismo , Hexanonas/metabolismo , Modelos Biológicos , Análise Numérica Assistida por Computador , Transdução de Sinais
14.
Mol Biol Cell ; 26(4): 805-20, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25518940

RESUMO

Differentiation-inducing factor-1 (DIF-1) is a polyketide that induces Dictyostelium amoebae to differentiate as prestalk cells. We performed a global quantitative screen for phosphorylation changes that occur within the first minutes after addition of DIF-1, using a triple-label SILAC approach. This revealed a new world of DIF-1-controlled signaling, with changes in components of the MAPK and protein kinase B signaling pathways, components of the actinomyosin cytoskeletal signaling networks, and a broad range of small GTPases and their regulators. The results also provide evidence that the Ca(2+)/calmodulin-dependent phosphatase calcineurin plays a role in DIF-1 signaling to the DimB prestalk transcription factor. At the global level, DIF-1 causes a major shift in the phosphorylation/dephosphorylation equilibrium toward net dephosphorylation. Of interest, many of the sites that are dephosphorylated in response to DIF-1 are phosphorylated in response to extracellular cAMP signaling. This accords with studies that suggest an antagonism between the two inducers and also with the rapid dephosphorylation of the cAMP receptor that we observe in response to DIF-1 and with the known inhibitory effect of DIF-1 on chemotaxis to cAMP. All MS data are available via ProteomeXchange with identifier PXD001555.


Assuntos
Dictyostelium/metabolismo , Hexanonas/metabolismo , Hidrocarbonetos Clorados/metabolismo , Policetídeos/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Calcineurina/química , Quimiotaxia , AMP Cíclico/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Fosforilação , Transdução de Sinais
15.
Metab Eng ; 26: 67-76, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25241399

RESUMO

We previously reported development of a metabolic pathway in Escherichia coli for overproduction of medium-chain methyl ketones (MK), which are relevant to the biofuel and flavor-and-fragrance industries. This MK pathway was a re-engineered version of ß-oxidation designed to overproduce ß-ketoacyl-CoAs and involved overexpression of the fadM thioesterase gene. Here, we document metabolic engineering modifications that have led to a MK titer of 3.4 g/L after ~45 h of fed-batch glucose fermentation and attainment of 40% of the maximum theoretical yield (the best values reported to date for MK). Modifications included balancing overexpression of fadR and fadD to increase fatty acid flux into the pathway, consolidation of the pathway from two plasmids into one, codon optimization, and knocking out key acetate production pathways. In vitro studies confirmed that a decarboxylase is not required to convert ß-keto acids into MK and that FadM is promiscuous and can hydrolyze several CoA-thioester pathway intermediates.


Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/fisiologia , Melhoramento Genético/métodos , Hexanonas/metabolismo , Engenharia Metabólica/métodos , Transdução de Sinais/fisiologia
16.
Chembiochem ; 15(4): 527-32, 2014 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-24474719

RESUMO

Two new acyloin compounds were isolated from the thermophilic bacterium Thermosporothrix hazakensis SK20-1(T) . Genome sequencing of the bacterium and biochemical studies identified the thiamine diphosphate (TPP)-dependent enzyme Thzk0150, which is involved in the formation of acyloin. Through extensive analysis of the Thzk0150-catalyzed reaction products, we propose a putative reaction mechanism involving two substrates: 4-methyl-2-oxovalerate as an acyl donor and phenyl pyruvate as an acyl acceptor.


Assuntos
Chloroflexi/química , Álcoois Graxos/metabolismo , Proteínas de Bactérias/metabolismo , Biocatálise , Candida albicans/efeitos dos fármacos , Chloroflexi/metabolismo , Álcoois Graxos/química , Álcoois Graxos/farmacologia , Hexanonas/química , Hexanonas/metabolismo , Hexanonas/farmacologia , Cetoácidos/química , Cetoácidos/metabolismo , Ácidos Fenilpirúvicos/química , Ácidos Fenilpirúvicos/metabolismo
17.
Int Arch Occup Environ Health ; 87(6): 655-62, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24078145

RESUMO

PURPOSE: The formation of pyrrole adducts might be responsible for peripheral nerve injury caused by n-hexane, but there is not an effective biomarker for monitoring occupational exposure of n-hexane. The current study was designed to investigate the changes of pyrrole adducts in serum and urine of rats exposed to 2,5-hexanedione (2,5-HD) and analyze the correlation between pyrrole adducts and 2,5-HD. METHODS: Two groups of male Wistar rats (n = 8) were administered a single dose of 200 and 400 mg/kg 2,5-HD (i.p.), and another two groups (n = 8) were given daily dose of 200 and 400 mg/kg 2,5-HD (i.p.) for 5 days. Pyrrole adducts and 2,5-HD in serum and urine were determined, at different time points after dosing, using Ehrlich's reagent and gas chromatography, respectively. RESULTS: The levels of pyrrole adducts in serum accumulated in a time-dependant manner after repeated exposure to 2,5-HD, while pyrrole adducts in urine, and 2,5-HD in serum and urine were kept stable. The half-life times (t1/2) of 2,5-HD and pyrrole adducts in serum were 2.27 ± 0.28 and 25.3 ± 3.34 h, respectively. Furthermore, the levels of pyrrole adducts in urine were significantly correlated with the levels of 2,5-HD in serum (r = 0.736, P < 0.001) and urine (r = 0.730, P < 0.001), and the levels of pyrrole adducts in serum were correlated with the cumulative dosage of 2,5-HD (r = 0.965, P < 0.001). CONCLUSION: The results suggested that pyrrole adducts in serum and urine might be markers of chronic exposure to n-hexane or 2,5-HD.


Assuntos
Monitoramento Ambiental , Hexanonas/metabolismo , Neurotoxinas/metabolismo , Pirróis/metabolismo , Animais , Área Sob a Curva , Biomarcadores/sangue , Biomarcadores/urina , Hexanos/metabolismo , Hexanos/toxicidade , Hexanonas/toxicidade , Masculino , Neurotoxinas/toxicidade , Pirróis/toxicidade , Ratos , Ratos Wistar , Espectrofotometria , Toxicocinética
18.
PLoS One ; 8(9): e76011, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24098756

RESUMO

BACKGROUND: The formation of pyrrole adducts might be responsible for peripheral nerve injury caused by n-hexane. The internal dose of pyrrole adducts would supply more information for the neurotoxicity of n-hexane. The current study was designed to investigate the tissue distributions of 2, 5-hexanedione (2,5-HD) and pyrrole adducts in rats exposed to n-hexane, and analyze the correlation between pyrrole adducts and 2,5-HD in tissues. METHODS: Male Wistar rats were given daily dose of 500,1000, 2000, 4000 mg/kg bw n-hexane by gavage for 5 days. The rats were sacrificed 24 hours after the last administration. The levels of 2, 5-hexanedione and pyrrole adducts in tissues were measured by gas chromatography and Ehrlich's reagent, respectively. The correlations between 2, 5-hexanedione and pyrrole adducts were analyzed by linear regression. RESULTS: Dose-dependent effects were observed between the dosage of n-hexane and 2, 5-hexanedione, and pyrrole adducts in tissues. The highest level of 2, 5-hexanedione was found in urine and the lowest in sciatic nerve, while the highest level of pyrrole adducts was seen in liver and the lowest in serum. There were significant correlations among the free 2, 5-hexanedione, total 2, 5-hexanedione and pyrrole adducts within the same tissues. Pyrrole adducts in serum showed the most significant correlation with free 2, 5-hexanedione or pyrrole adducts in tissues. CONCLUSION: The findings suggested that pyrrole adducts in serum might be a better indicator for the internal dose of free 2, 5-hexanedione and pyrrole adducts in tissues.


Assuntos
Biomarcadores/sangue , Hexanos/toxicidade , Hexanonas/metabolismo , Pirróis/metabolismo , Animais , Benzaldeídos , Cromatografia Gasosa , Relação Dose-Resposta a Droga , Hexanos/administração & dosagem , Hexanonas/farmacocinética , Hexanonas/urina , Modelos Lineares , Fígado/metabolismo , Masculino , Pirróis/sangue , Pirróis/farmacocinética , Ratos , Ratos Wistar
19.
PLoS One ; 8(8): e72118, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23977224

RESUMO

Differentiation-inducing factor-3 (DIF-3), found in the cellular slime mold Dictyostelium discoideum, and its derivatives such as butoxy-DIF-3 (Bu-DIF-3) are potent anti-tumor agents. However, the precise mechanisms underlying the actions of DIF-3 remain to be elucidated. In this study, we synthesized a green fluorescent derivative of DIF-3, BODIPY-DIF-3, and a control fluorescent compound, Bu-BODIPY (butyl-BODIPY), and investigated how DIF-like molecules behave in human cervical cancer HeLa cells by using both fluorescence and electron microscopy. BODIPY-DIF-3 at 5-20 µ M suppressed cell growth in a dose-dependent manner, whereas Bu-BODIPY had minimal effect on cell growth. When cells were incubated with BODIPY-DIF-3 at 20 µM, it penetrated cell membranes within 0.5 h and localized mainly in mitochondria, while Bu-BODIPY did not stain the cells. Exposure of cells for 1-3 days to DIF-3, Bu-DIF-3, BODIPY-DIF-3, or CCCP (a mitochondrial uncoupler) induced substantial mitochondrial swelling, suppressing cell growth. When added to isolated mitochondria, DIF-3, Bu-DIF-3, and BOIDPY-DIF-3, like CCCP, dose-dependently promoted the rate of oxygen consumption, but Bu-BODIPY did not. Our results suggest that these bioactive DIF-like molecules suppress cell growth, at least in part, by disturbing mitochondrial activity. This is the first report showing the cellular localization and behavior of DIF-like molecules in mammalian tumor cells.


Assuntos
Antineoplásicos/metabolismo , Dictyostelium/química , Hexanonas/metabolismo , Mitocôndrias/metabolismo , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Compostos de Boro/química , Compostos de Boro/metabolismo , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Permeabilidade da Membrana Celular , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Ciclina D/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Células HeLa , Hexanonas/química , Hexanonas/isolamento & purificação , Hexanonas/farmacologia , Humanos , Mitocôndrias/efeitos dos fármacos , Consumo de Oxigênio , Ionóforos de Próton/farmacologia
20.
Sci Rep ; 3: 2272, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23893301

RESUMO

Solitons have been observed in various physical phenomena. Here, we show that the distinct characteristics of solitons are present in the mass cell movement of non-chemotactic mutants of the cellular slime mould Dictyostelium discoideum. During starvation, D. discoideum forms multicellular structures that differentiate into spore or stalk cells and, eventually, a fruiting body. Non-chemotactic mutant cells do not form multicellular structures; however, they do undergo mass cell movement in the form of a pulsatile soliton-like structure (SLS). We also found that SLS induction is mediated by adhesive cell-cell interactions. These observations provide novel insights into the mechanisms of biological solitons in multicellular movement.


Assuntos
Movimento Celular/fisiologia , Dictyostelium/fisiologia , Cafeína/farmacologia , Adesão Celular , Movimento Celular/efeitos dos fármacos , Quimiotaxia/fisiologia , AMP Cíclico/metabolismo , Genes de Protozoários/genética , Hexanonas/metabolismo , Mutação , Transdução de Sinais
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