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1.
Food Chem ; 293: 529-536, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31151644

RESUMO

The effect of glucose oxidase (GOx) catalytic oxidation on the efficacy of gallic acid (GA) to modify the chemical structure and gelling behavior of myofibrillar protein (MP) was investigated. In contrast to non-oxidized MP samples where GA induced very little changes, GA (0, 6, 30, and 60 µmol/g MP) under GOx treatment promoted sulfhydryl and amine loss (up to 58% and 49%, respectively). The attenuation of intrinsic tryptophan fluorescence in the GA/GOx-treated MP corroborated the finding. The gelling capacity of MP, corresponding to disulfide and non-disulfide bond formation in protein aggregates, was markedly enhanced by 60 µmol GA under GOx, up to 86% in gel storage modulus G' and 53% in gel strength. The GOx-aided GA modification of MP could be a potential ingredient strategy in meat processing to promote textural attributes of cooked products.


Assuntos
Ácido Gálico/química , Géis/química , Glucose Oxidase/metabolismo , Proteínas Musculares/química , Aminas/química , Aminas/metabolismo , Animais , Ácido Gálico/metabolismo , Carne/análise , Microscopia Eletrônica de Varredura , Proteínas Musculares/metabolismo , Oxirredução , Reologia , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo
2.
J Enzyme Inhib Med Chem ; 34(1): 1193-1198, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31237157

RESUMO

A series of histamine bis-Schiff bases and bis-spinaceamine derivatives were synthesised and investigated as activators of four human (h) carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic hCA I, II and VII, and the membrane-associated hCA IV. All isoforms were effectively activated by the new derivatives, with activation constants in the range of 4.73-10.2 µM for hCA I, 6.15-42.1 µM for hCA II, 2.37-32.7 µM for hCA IV and 32 nM-18.7 µM for hCA VII, respectively. The nature of the spacer between the two histamine/spinaceamine units of these molecules was the main contributor to the diverse activating efficacy, with a very different fine tuning for the diverse isoforms. As CA activators recently emerged as interesting agents for enhancing cognition, in the management of CA deficiencies, or for therapy memory and artificial tissues engineering, our compounds may be considered as candidates for such applications.


Assuntos
Aminas/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Histamina/metabolismo , Isoenzimas/antagonistas & inibidores , Bases de Schiff/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Humanos , Espectroscopia de Prótons por Ressonância Magnética , Espectroscopia de Infravermelho com Transformada de Fourier
3.
Methods Mol Biol ; 1965: 297-311, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31069683

RESUMO

BACKGROUND: After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde-fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, both of which correlate to apoptotic activity. Thus, LT is a good indicator of apoptosis visualized by confocal microscopy. Results of LT staining of apoptotic cell death correlate well with other whole mount apoptosis vital dyes such as Nile blue sulfate and neutral red, with the added benefit of being fixable in situ. Nile blue sulfate can also be used as a non-vital, nonspecific dye to visualize general morphology. Stains such as acridine orange can be used for surface staining of fixed embryos to yield confocal images that are similar to scanning electron micrographs. METHODS: Mouse embryos were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Embryos are mounted on depression slides, and serial sections are imaged by confocal microscopy, followed by 3-D reconstruction. RESULTS: Embryos or tissues as thick as 500 microns (µm) can be visualized after clearing with BABB. LysoTracker staining reveals apoptotic regions in organogenesis-stage mouse embryos. Morphological observation of tissue was facilitated by combining autofluorescence with Nile blue sulfate staining of fixed embryos or opaque surface staining with acridine orange staining. CONCLUSIONS: The use of BABB for clearing LT vital-stained and fixed embryos matches the refractive index of the tissue to the suspending medium, allowing increased penetration of laser light in a confocal microscope. Nile blue sulfate used as a non-vital dye provides a nonspecific staining of fixed embryos that can then be cleared with methyl salicylate for confocal observation. Sample preparation and staining procedures described here, with optimization of confocal laser scanning microscopy, allow for the detection and visualization of morphological structure and apoptosis in embryos up to 500 µm thick, and stained specimens can be fixed and mounted on depression slides.


Assuntos
Embrião de Mamíferos/ultraestrutura , Lisossomos/metabolismo , Organogênese , Aminas/metabolismo , Animais , Apoptose , Embrião de Mamíferos/metabolismo , Imagem Tridimensional , Camundongos , Microscopia Confocal , Fagocitose
4.
Appl Microbiol Biotechnol ; 103(14): 5727-5737, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31123770

RESUMO

Transaminase enzymes (TAms) are becoming increasingly valuable in the chemist's toolbox as a biocatalytic route to chiral amines. Despite high profile successes, the lack of (R)-selective TAms and robustness under harsh industrial conditions continue to prove problematic. Herein, we report the isolation of the first haloarchaeal TAm (BC61-TAm) to be characterised for the purposes of pharmaceutical biocatalysis. BC61-TAm is an (R)-selective enzyme, cloned from an extremely halophilic archaeon, isolated from a Triassic period salt mine. Produced using a Haloferax volcanii-based expression model, the resulting protein displays a classic halophilic activity profile, as well as thermotolerance (optimum 50 °C) and organic solvent tolerance. Molecular modelling predicts the putative active site residues of haloarchaeal TAms, with molecular dynamics simulations providing insights on the basis of BC61-TAm's organic solvent tolerance. These results represent an exciting advance in the study of transaminases from extremophiles, providing a possible scaffold for future discovery of biocatalytic enzymes with robust properties.


Assuntos
Archaea/enzimologia , Proteínas Arqueais/metabolismo , Mineração , Cloreto de Sódio , Transaminases/metabolismo , Aminas/metabolismo , Archaea/genética , Proteínas Arqueais/genética , Biocatálise , Haloferax volcanii/enzimologia , Haloferax volcanii/genética , Simulação de Dinâmica Molecular , Solventes/metabolismo , Especificidade por Substrato , Termotolerância , Transaminases/genética
5.
Eur J Med Chem ; 176: 11-20, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31091477

RESUMO

A novel series of dihydroquinazolin-2-amine derivatives were synthesized and evaluated for their anti-HIV-1 activity in MT-4 cell cultures. All of the molecules were active against wild-type HIV-1 with EC50 values ranging from 0.61 µM to 0.84 nM. The most potent inhibitor, compound 4b, had an EC50 value of 0.84 nM against HIV-1 strain IIIB, and thus was more active than the reference drugs efavirenz and etravirine. Moreover, most of the compounds maintained high activity (low-micromolar EC50 values) against strains bearing the reverse transcriptase (RT) E138K mutation. Compound 4b had EC50 values of 3.5 nM and 66 nM against non-nucleoside reverse transcriptase inhibitor-resistant strains bearing the RT E138K and RES056 mutations. In enzyme activity assays, compound 4b exhibited an IC50 value of 10 nM against HIV-1 RT. Preliminary SARs and molecular docking studies provide valuable insights for further optimization.


Assuntos
Aminas/farmacologia , Fármacos Anti-HIV/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/enzimologia , Quinazolinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Aminas/síntese química , Aminas/metabolismo , Aminas/toxicidade , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/toxicidade , Sítios de Ligação , Linhagem Celular Tumoral , Transcriptase Reversa do HIV/química , Transcriptase Reversa do HIV/metabolismo , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Quinazolinas/síntese química , Quinazolinas/metabolismo , Quinazolinas/toxicidade , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/metabolismo , Inibidores da Transcriptase Reversa/toxicidade , Relação Estrutura-Atividade
6.
Molecules ; 24(7)2019 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-30934681

RESUMO

Enhancing the thermostability of (R)-selective amine transaminases (AT-ATA) will expand its application in the asymmetric synthesis of chiral amines. In this study, mutual information and coevolution networks of ATAs were analyzed by the Mutual Information Server to Infer Coevolution (MISTIC). Subsequently, the amino acids most likely to influence the stability and function of the protein were investigated by alanine scanning and saturation mutagenesis. Four stabilized mutants (L118T, L118A, L118I, and L118V) were successfully obtained. The best mutant, L118T, exhibited an improved thermal stability with a 3.7-fold enhancement in its half-life (t1/2) at 40 °C and a 5.3 °C increase in T5010 compared to the values for the wild-type protein. By the differential scanning fluorimetry (DSF) analysis, the best mutant, L118T, showed a melting temperature (Tm) of 46.4 °C, which corresponded to a 5.0 °C increase relative to the wild-type AT-ATA (41.4 °C). Furthermore, the most stable mutant L118T displayed the highest catalytic efficiency among the four stabilized mutants.


Assuntos
Aspergillus/fisiologia , Mutação , Transaminases/metabolismo , Aminas/química , Aminas/metabolismo , Estabilidade Enzimática , Cinética , Conformação Molecular , Mutagênese Sítio-Dirigida , Relação Estrutura-Atividade , Termodinâmica , Transaminases/química
7.
Carbohydr Polym ; 212: 215-221, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30832850

RESUMO

DrzBC and DrzBS (10-23 DNAzyme) could block the expression of HBV e- and s- gene respectively. But the application of 10-23 DNAzyme was limited owing to the lack of appropriate delivery vehicles. Chitosan oligosaccharide-SS-Octadecylamine (CSSO), a redox-responsive nano-sized polymeric carrier, could self-aggregate and bind with DNA by electrostatic interaction at proper mass ratio. Compared with the traditional commercial carrier Lipo2000, CSSO exhibited lower cytotoxicity, efficient cellular uptake by targeting cells, and rapidly DNA released in cytoplasm after escaping from endosomes. Including the same DNA concentration, Lipo2000/(DrzBC or DrzBS) showed maximum inhibitory rate on HBeAg (47.29 ±â€¯1.86%) and HBsAg (33.58 ±â€¯0.72%) secretion after 48 h incubation, and then both decreased. In contrast, HBeAg secretion inhibition by CSSO/DrzBC and HBsAg secretion inhibition by CSSO/DrzBS were up to 73.86 ±â€¯1.77% and 67.80 ±â€¯2.51% at 48 h, and further increased to 83.83 ±â€¯2.34% and 76.79 ±â€¯2.18% at 72 h, respectively. CSSO is a promising redox-responsive polymeric carrier for efficient anti-Hepatitis B Virus gene therapy.


Assuntos
Aminas/administração & dosagem , Quitosana/administração & dosagem , Terapia Genética/métodos , Vírus da Hepatite B/efeitos dos fármacos , Oligossacarídeos/administração & dosagem , Polímeros/administração & dosagem , Aminas/metabolismo , Quitosana/metabolismo , DNA Viral/efeitos dos fármacos , DNA Viral/genética , DNA Viral/metabolismo , Relação Dose-Resposta a Droga , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/metabolismo , Células Hep G2 , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Humanos , Oligossacarídeos/metabolismo , Oxirredução/efeitos dos fármacos , Polímeros/metabolismo
8.
Nat Commun ; 10(1): 795, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30770837

RESUMO

Herein, we design and prepare cellulose-based ratiometric fluorescent materials with superior amine-response, which offers the real-time and visual detection of seafood freshness. Through utilizing the reactive hydroxyl groups along cellulose chains, we covalently immobilize the fluorescein isothiocyanate (FITC) as indicator and protoporphyrin IX (PpIX) as internal reference onto cellulose acetate (CA), respectively. Subsequently, a series of dual-emission solid fluorescent materials are achieved by simply blending green emitting CA-FITC with red-emitting CA-PpIX with varying ratios. They exhibit a sensitive, color-responsive, rapid and linear response to ammonia in a wide range of 5.0 ppm to 2.5 × 104 ppm. Benefiting from the excellent solubility and processibility of cellulose derivatives, the as-prepared materials are readily processed into different material forms, including printing ink, coating, flexible film, and nanofibrous membrane. The electrospun nanofibrous membrane is successfully employed as a low-cost, high-contrasting, quick-responsive fluorescent trademark for visual monitoring the freshness of shrimp and crab.


Assuntos
Aminas/metabolismo , Braquiúros , Celulose/metabolismo , Isotiocianatos/metabolismo , Penaeidae , Alimentos Marinhos/análise , Aminas/química , Animais , Celulose/química , Cor , Fluoresceína/química , Fluoresceína/metabolismo , Fluorescência , Análise de Alimentos/métodos , Qualidade dos Alimentos , Isotiocianatos/química , Protoporfirinas/química , Protoporfirinas/metabolismo , Reprodutibilidade dos Testes , Fatores de Tempo
9.
Phytochemistry ; 159: 75-89, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30597374

RESUMO

Solanum tuberosum, commonly known as the potato, is a worldwide food staple. During harvest, storage, and distribution the crop is at risk of mechanical damage. Wounding of the tuber skin can also become a point of entry for bacterial and fungal pathogens, resulting in substantial agricultural losses. Building on the proposal that potato tubers produce metabolites to defend against microbial infection during early stages of wound healing before protective suberized periderm tissues have developed, we assessed extracts of wound tissues from four potato cultivars with differing skin morphologies (Norkotah Russet, Atlantic, Chipeta, and Yukon Gold). These assays were conducted at 0, 1, 2, 3 and 7 days post wounding against the plant pathogen Erwinia carotovora and a non-pathogenic Escherichia coli strain that served as a control. For each of the potato cultivars, only polar wound tissue extracts demonstrated antibacterial activity. The polar extracts from earlier wound-healing time points (days 0, 1 and 2) displayed notably higher antibacterial activity against both strains than the later wound-healing stages (days 3 and 7). These results support a burst of antibacterial activity at early time points. Parallel metabolite profiling of the extracts revealed differences in chemical composition at different wound-healing time points and allowed for identification of potential marker compounds according to healing stage for each of the cultivars. It was possible to monitor the transformations in the metabolite profiles that could account for the phenomenon of temporal resistance by looking at the relative quantities of various metabolite classes as a function of time.


Assuntos
Antibacterianos/farmacologia , Pectobacterium carotovorum/efeitos dos fármacos , Extratos Vegetais/farmacologia , Tubérculos/metabolismo , Solanum tuberosum/metabolismo , Cicatrização/efeitos dos fármacos , Alcaloides/metabolismo , Aminas/metabolismo , Biomarcadores/metabolismo , Escherichia coli/fisiologia , Testes de Sensibilidade Microbiana , Pectobacterium carotovorum/patogenicidade , Fenóis/metabolismo , Tubérculos/microbiologia , Solanum tuberosum/classificação , Solanum tuberosum/microbiologia , Especificidade da Espécie
10.
J Med Chem ; 62(2): 762-773, 2019 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-30525583

RESUMO

Dynamic combinatorial chemistry (DCC) has emerged as a promising strategy for template-driven selection of high-affinity ligands for biological targets from equilibrating combinatorial libraries. However, only a few examples using disulfide-exchange-based DCC are reported for nucleic acid targets. Herein, we have demonstrated that gold-coated magnetic nanoparticle-conjugated DNA targets can be used as templates for dynamic selection of ligands from an imine-based combinatorial library. The implementation of DCC using DNA nanotemplates enables efficient identification of the lead compounds, from the dynamic combinatorial library via magnetic decantation. It further allows quick separation of DNA nanotemplates for reuse in DCC reactions. The identified lead compound exhibits significant quadruplex versus duplex DNA selectivity and suppresses promoter activity of c-MYC gene that contains G-quadruplex DNA forming sequence in the upstream promoter region. Further cellular experiments indicated that the lead compound is able to permeate into cell nuclei and trigger a DNA damage response in cancer cells.


Assuntos
Técnicas de Química Combinatória/métodos , DNA/química , Quadruplex G , Ligantes , Nanopartículas Metálicas/química , Aldeídos/química , Aminas/química , Aminas/metabolismo , Aminas/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , DNA/metabolismo , Ouro/química , Humanos , Microscopia Confocal , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética
11.
J Biosci Bioeng ; 127(4): 520-527, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30563742

RESUMO

Metabolomics has been an evolving science with a wide range of applications in various fields. However, previous studies have rarely focused on metabolite chirality. In this study, to achieve metabolic profiling of chiral amino acids and related metabolites, we developed a high-throughput method using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The combination of two types of chiral columns (with binaphthyl-based crown ether and cinchona alkaloid-derived zwitterionic stationary phases) enabled the analysis of 115 chiral and non-chiral metabolites. By finely optimizing MS/MS parameters, the method allowed the highly sensitive (0.001-50 nmol/mL) and wide dynamic range detection of targeted analytes in a standard solution without derivatization. We applied the method to food samples (cheese), and successfully quantified trace levels of metabolites such as d-amino acids in samples. Additionally, we performed principal component analysis on the metabolome data and obtained unique profiles that reflected metabolite chirality. These results demonstrated the applicability and feasibility of the LC-MS/MS method as an effective tool for wide-targeted chiral metabolome analysis.


Assuntos
Aminoácidos/análise , Metaboloma , Metabolômica/métodos , Espectrometria de Massas em Tandem/métodos , Aminas/análise , Aminas/metabolismo , Aminoácidos/metabolismo , Aminoácidos Básicos/análise , Aminoácidos Básicos/metabolismo , Diamino Aminoácidos/análise , Diamino Aminoácidos/metabolismo , Animais , Queijo/análise , Cromatografia Líquida/instrumentação , Cromatografia Líquida/métodos , Estudos de Viabilidade , Análise de Alimentos/instrumentação , Análise de Alimentos/métodos , Metabolômica/instrumentação , Estereoisomerismo , Espectrometria de Massas em Tandem/instrumentação
12.
Sci Rep ; 8(1): 17774, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30538262

RESUMO

The development of Bretschneider's histidine-tryptophan-ketoglutarate (HTK) cardioplegia solution represented a major advancement in cardiac surgery, offering significant myocardial protection. However, metabolic changes induced by this additive in the whole body have not been systematically investigated. Using an untargeted mass spectrometry-based method to deeply explore the urine metabolome, we sought to provide a holistic and systematic view of metabolic perturbations occurred in patients receiving HTK. Prospective urine samples were collected from 100 patients who had undergone cardiac surgery, and metabolomic changes were profiled using a high-performance chemical isotope labeling liquid chromatography-mass spectrometry (LC-MS) method. A total of 14,642 peak pairs or metabolites were quantified using differential 13C-/12C-dansyl labeling LC-MS, which targets the amine/phenol submetabolome from urine specimens. We identified 223 metabolites that showed significant concentration change (fold change > 5) and assembled several potential metabolic pathway maps derived from these dysregulated metabolites. Our data indicated upregulated histidine metabolism with subsequently increased glutamine/glutamate metabolism, altered purine and pyrimidine metabolism, and enhanced vitamin B6 metabolism in patients receiving HTK. Our findings provide solid evidence that HTK solution causes significant perturbations in several metabolic pathways and establish a basis for further study of key mechanisms underlying its organ-protective or potential harmful effects.


Assuntos
Metaboloma/efeitos dos fármacos , Urina/química , Adulto , Idoso , Aminas/metabolismo , Líquidos Corporais/química , Líquidos Corporais/metabolismo , Isótopos de Carbono/química , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Cromatografia Líquida/métodos , Feminino , Glucose/efeitos adversos , Glucose/farmacologia , Humanos , Marcação por Isótopo , Masculino , Manitol/efeitos adversos , Manitol/farmacologia , Espectrometria de Massas/métodos , Metabolômica/métodos , Pessoa de Meia-Idade , Fenóis/metabolismo , Cloreto de Potássio/efeitos adversos , Cloreto de Potássio/farmacologia , Procaína/efeitos adversos , Procaína/farmacologia , Estudos Prospectivos , Espectrometria de Massas em Tandem
13.
Inorg Chem ; 57(20): 12521-12535, 2018 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-30281299

RESUMO

Superoxide dismutases (SODs) utilize a ping-pong mechanism in which a redox-active metal cycles between oxidized and reduced forms that differ by one electron to catalyze the disproportionation of superoxide to dioxygen and hydrogen peroxide. Nickel-dependent SOD (NiSOD) is a unique biological solution for controlling superoxide levels. This enzyme relies on the use of cysteinate ligands to bring the Ni(III/II) redox couple into the range required for catalysis (∼300 mV vs. NHE). The use of cysteine thiolates, which are not found in any other SOD, is a curious choice because of their well-known oxidation by peroxide and dioxygen. The NiSOD active site cysteinate ligands are resistant to oxidation, and prior studies of synthetic and computational models point to the backbone N-donors in the active site (the N-terminal amine and the amide N atom of Cys2) as being involved in stabilizing the cysteines to oxidation. To test the role of the backbone N-donors, we have constructed a variant of NiSOD wherein an alanine residue was added to the N-terminus (Ala0-NiSOD), effectively altering the amine ligand to an amide. X-ray absorption, electronic absorption, and magnetic circular dichroism (MCD) spectroscopic analyses of as-isolated Ala0-NiSOD coupled with density functional theory (DFT) geometry optimized models that were evaluated on the basis of the spectroscopic data within the framework of DFT and time-dependent DFT computations are consistent with a diamagnetic Ni(II) site with two cysteinate, one His1 amide, and one Cys2 amidate ligands. The variant protein is catalytically inactive, has an altered electronic absorption spectrum associated with the nickel site, and is sensitive to oxidation. Mass spectrometric analysis of the protein exposed to air shows the presence of a mixture of oxidation products, the principal ones being a disulfide, a bis-sulfenate, and a bis-sulfinate derived from the active site cysteine ligands. Details of the electronic structure of the Ni(III) site available from the DFT calculations point to subtle changes in the unpaired spin density on the S-donors as being responsible for the altered sensitivity of Ala0-NiSOD to O2.


Assuntos
Amidas/metabolismo , Aminas/metabolismo , Níquel/química , Superóxido Dismutase/metabolismo , Amidas/química , Aminas/química , Escherichia coli/metabolismo , Regulação Enzimológica da Expressão Gênica , Modelos Moleculares , Conformação Proteica , Superóxido Dismutase/química
14.
Carbohydr Res ; 467: 33-44, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30075363

RESUMO

Azide- and amine-substituted sugars can be useful tools in the probing of biological systems as well as in the assembly of libraries of derivatives using click chemistry or simple amine coupling approaches. A collection of methylumbelliferyl glycosides of various azido- and amino-deoxy sugar derivatives of glucose, galactose and xylose was synthesised via azide displacement of the corresponding triflate derivatives and subsequent modification. These compounds will be used as substrates in a high-throughput screen to identify glycosidases that can process such modified sugars. The α-glycosyl fluoride derivatives of each modified sugar were also synthesised to serve as substrates for glycosynthases derived from the enzymes identified in the screen.


Assuntos
Aminas/química , Azidas/química , Fluoretos/síntese química , Glicosídeo Hidrolases/química , Glicosídeos/síntese química , Aminas/metabolismo , Azidas/metabolismo , Fluoretos/química , Fluoretos/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosídeos/química , Glicosídeos/metabolismo , Ensaios de Triagem em Larga Escala , Estrutura Molecular , Biblioteca de Peptídeos , Especificidade por Substrato
16.
J Chromatogr A ; 1567: 219-225, 2018 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-30005940

RESUMO

Analysis of metabolites is often performed using separations coupled to mass spectrometry which is challenging due to their vast structural heterogeneity and variable charge states. Metabolites are often separated based on their class/functional group which in large part determine their acidity or basicity. This charge state dictates the ionization mode and efficiency of the molecule. To improve the sensitivity and expand the coverage of the mammalian metabolome, multifunctional derivatization with sheathless CE-ESI-MS was undertaken. In this work, amines, hydroxyls and carboxylates were labeled with tertiary amines tags. This derivatization was performed in under 100 min and resulted in high positive charge states for all analytes investigated. Amino acids and organic acids showed average limits of detection of 76 nM with good linearity of 0.96 and 10% RSD for peak area. Applying this metabolomic profiling system to bovine aortic endothelial cells showed changes in 15 metabolites after treatment with high glucose. The sample injection volume on-capillary was <300 cells for quantitative analyses. Targeted metabolites were found in human tissue, which indicates possible application of the system complex metabolome quantitation.


Assuntos
Eletroforese Capilar/métodos , Mamíferos/metabolismo , Metabolômica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Aminas/metabolismo , Aminoácidos/metabolismo , Animais , Tampões (Química) , Ácidos Carboxílicos/metabolismo , Bovinos , Linhagem Celular , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Glucose/farmacologia , Humanos , Metaboloma/efeitos dos fármacos , Padrões de Referência
17.
Drug Deliv ; 25(1): 1161-1174, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29792353

RESUMO

Gabapentin (GBP), an antiepileptic and anti-neuropathic agent, suffers from short half-life (5-7 h), has narrow absorption window, and is absorbed via carrier-mediated mechanism resulting in frequent dosing, poor compliance, and poor bioavailability (<60%). Moreover, GBP is a freely water-soluble drug, thus it is considered a challenging candidate to be formulated as extended release dosage form. In this study, raft forming systems were investigated as a potential drug delivery system for prolonging gastric residence time of GBP. A 23 full factorial design was adopted to study the effect of formulation variables (% gellan gum, % GMO, and % LM-pectin 101), on the percent of GBP released at different time intervals (1, 5, and 8 h) as well as the gel strength, and thus was achieved an optimized formula with zero-order release profile suitable for once-daily administration. In vivo assessment was performed in rats to evaluate gastric residence of the gel formed. In addition, the oral bioavailability of GBP relative to commercially available Neurontin® immediate release oral solution was also investigated. Significant increase was observed for Cmax, AUC(0-t), and AUC(0-∞). The increase in relative bioavailability of GBP from the optimized formula was 1.7 folds.


Assuntos
Aminas/química , Aminas/metabolismo , Ácidos Cicloexanocarboxílicos/química , Ácidos Cicloexanocarboxílicos/metabolismo , Preparações de Ação Retardada/química , Preparações de Ação Retardada/metabolismo , Portadores de Fármacos/química , Mucosa Gástrica/metabolismo , Ácido gama-Aminobutírico/química , Ácido gama-Aminobutírico/metabolismo , Administração Oral , Animais , Disponibilidade Biológica , Química Farmacêutica/métodos , Sistemas de Liberação de Medicamentos/métodos , Excipientes/química , Gabapentina , Géis/química , Géis/farmacologia , Meia-Vida , Masculino , Pectinas/química , Polissacarídeos Bacterianos/química , Ratos
18.
N Biotechnol ; 47: 18-24, 2018 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-29758351

RESUMO

The industrial importance of optically pure compounds has thrown a spotlight on ω-transaminases that have shown a high potential for the synthesis of bioactive compounds with a chiral amine moiety. The implementation of biocatalysts in industrial processes relies strongly on fast and cost effective process development, including selection of a biocatalyst form and the strategy for its immobilization. Here, microscale reactors with selected surface-immobilized amine-transaminase (ATA) in various forms are described as platforms for high-throughput process development. Wild type ATA (ATA-wt) from a crude cell extract, as well as Escherichia coli cells intracellularly overexpressing the enzyme, were immobilized on the surfaces of meander microchannels of disposable plastics by means of reactor surface silanization and glutaraldehyde bonding. In addition, a silicon/glass microchannel reactor was used for immobilization of an ATA-wt, genetically engineered to contain a silica-binding module (SBM) at the N-terminus (N-SBM-ATA-wt), leading to immobilization on the non-modified inner microchannel surface. Microreactors with surface-immobilized biocatalysts were coupled with a quenching system and at-line HPLC analytics and evaluated based on continuous biotransformation, yielding acetophenone and l-alanine. E. coli cells and N-SBM-ATA-wt were efficiently immobilized and yielded a volumetric productivity of up to 14.42 g L-1 h-1, while ATA-wt small load resulted in two orders of magnitude lower productivity. The miniaturized reactors further enabled in-operando characterization of biocatalyst stability, crucial for successful transfer to a production scale.


Assuntos
Biocatálise , Reatores Biológicos , Enzimas Imobilizadas/metabolismo , Microtecnologia/instrumentação , Aminação , Aminas/metabolismo , Biotransformação , Células Imobilizadas/metabolismo , Escherichia coli/metabolismo , Vidro/química , Polímeros/química , Dióxido de Silício/química , Propriedades de Superfície , Transaminases/metabolismo
19.
J Biosci Bioeng ; 126(3): 293-300, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29628267

RESUMO

d-Stereospecific amidohydrolase from Streptomyces sp. 82F2 (DAH) recognizes d-amino acyl ester derivatives as substrates and catalyzes hydrolysis and aminolysis to yield d-amino acids and d-amino acyl peptides or amide derivatives, respectively. Crystallographic analysis has revealed that DAH possesses a large cavity with a small pocket at the bottom. Because the pocket is close to the catalytic center and is thought to interact with substrates, we examined the function of the eight residues that form the pocket in terms of substrate recognition and aminolysis via mutational analysis. Formation of the acyl-enzyme intermediate and catalysis of aminolysis by DAH were changed by substitutions of selected residues with Ala. In particular, I338A DAH exhibited a significant increase in the condensation product of Ac-d-Phe methyl ester and 1,8-diaminooctane (Ac-d-Phe-1,8-diaminooctane) compared with the wild-type DAH. A similar effect was observed by the mutation of Ile338 to Gly and Ser. The pocket shapes and local flexibility of the mutants I338G, I338A, and I338S are thought to resemble each other. Thus, changes in the shape and local flexibility of the pocket of DAH by mutation presumably alter substrate recognition for aminolysis.


Assuntos
Amidoidrolases/química , Amidoidrolases/metabolismo , Aminas/metabolismo , Domínio Catalítico , Streptomyces/enzimologia , Aminas/química , Sítios de Ligação , Catálise , Domínio Catalítico/fisiologia , Hidrólise , Cinética , Estereoisomerismo , Streptomyces/metabolismo , Especificidade por Substrato
20.
Water Res ; 137: 290-300, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29554533

RESUMO

Laboratory-scale incubation experiments in water/sediment systems were conducted to test the transformation behavior of the anticonvulsant gabapentin (GBP) under different environmental conditions (aerobic, anaerobic, with abiotic controls). GBP was transformed by biological processes as it was eliminated quickly under aerobic conditions (dissipation time 50% of initial concentration (DT50): 2-7 days) whereas no decrease was observed under anaerobic conditions. Measurements via high resolution mass spectrometry (LC-Orbitrap-MS) revealed eight biological transformation products (TPs). Three of them were identified with reference standards (GBP-Lactam, TP186, TP213), while for the other five TPs tentative structures were proposed from information by MS2/MS3 experiments. Furthermore, the quantitatively most relevant TP GBP-Lactam was formed via intramolecular amidation (up to 18% of initial GBP concentration). Incubation experiments with GBP-Lactam revealed a higher stability against biotic degradation (DT50: 12 days) in contrast to GBP, while it was stable under anaerobic and abiotic conditions. Besides GBP, GBP-Lactam was detected in surface water in the µg L-1 range. Finally, GBP and GBP-Lactam were found in potable water with concentrations up to 0.64 and 0.07 µg L-1, respectively. According to the elevated environmental persistence of GBP-Lactam compared to GBP and its presumed enhanced toxicity, we recommend to involve GBP-Lactam into monitoring programs.


Assuntos
Aminas/metabolismo , Anticonvulsivantes/metabolismo , Ácidos Cicloexanocarboxílicos/metabolismo , Poluentes Químicos da Água/metabolismo , Ácido gama-Aminobutírico/metabolismo , Aerobiose , Aminas/análise , Anticonvulsivantes/análise , Compostos Aza/análise , Biotransformação , Cromatografia Líquida , Ácidos Cicloexanocarboxílicos/análise , Água Potável/análise , Gabapentina , Água Subterrânea/análise , Oxirredução , Rios/química , Compostos de Espiro/análise , Espectrometria de Massas em Tandem , Poluentes Químicos da Água/análise , Ácido gama-Aminobutírico/análise
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