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1.
Bioresour Technol ; 304: 123014, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32088628

RESUMO

In this study, a lab-scale multiple draft tubes airlift loop membrane bioreactor (Mt-ALMBR) was used for treating acidic 7-amino cephalosporanic acid (7-ACA) wastewater under different pHs (3.54-6.20) and hydraulic retention time (HRT) (48 h, 36 h, 24 h and 16 h). During about 200 days operation, under HRT of 48 h and pH condition about 6.0, the optimum average COD and BOD5 removal rates were reach to 84.4 ± 2.1% and 94.9 ± 0.8%, and the highest 7-ACA removal rate also observed as 77.6%. Biodegradation, membrane rejection, hydrolysis and sludge adsorption were the four main pathways of 7-ACA removal. With the increase of pH, biodegradation, membrane rejection and hydrolysis had significant positive impacts on 7-ACA removal, while adsorption had a negative impact. Moreover, mathematical models for 7-ACA removal rate and pH were calculated to guide the operation of Mt-ALMBR. Biodegradation was the main pathway to remove 7-ACA when pH was >4.17.


Assuntos
Eliminação de Resíduos Líquidos , Águas Residuárias , Aminoácidos Acídicos , Reatores Biológicos , Esgotos
2.
Chem Commun (Camb) ; 54(95): 13443-13446, 2018 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-30430176

RESUMO

Surface charge engineering of nanosized CuS via acidic amino acid modification is proved to be an efficient strategy to realize high peroxidase-mimicking catalytic activity at neutral pH. As a proof-of-concept application, one-pot high-performance colorimetric sensing of glucose by coupling the engineered nanozyme with glucose oxidase is realized.


Assuntos
Aminoácidos Acídicos/química , Técnicas Biossensoriais , Cobre/química , Glucose/análise , Nanopartículas/química , Peroxidase/química , Engenharia de Proteínas , Concentração de Íons de Hidrogênio , Tamanho da Partícula , Peroxidase/metabolismo , Propriedades de Superfície
4.
Spectrochim Acta A Mol Biomol Spectrosc ; 201: 367-375, 2018 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-29772516

RESUMO

Nanomaterials have triggered tremendous interest to mimick peroxidase but rarely attention has been paid to small molecules. Herein we first found that acidic amino acids including l-glutamic acid (L-Glu) and l-aspartic acid (L-Asp) exhibited an intrinsic peroxidase-like activity, endowing acidic amino acids with the capability of catalysing the oxidation of the peroxidase substrates 3,3',5,5'-tetramethylbenzidine (TMB) to produce color reaction in the presence of H2O2. Reaction mechanism was further investigated by means of electron spin resonance spectroscopy (ESR), enzyme kinetics assay and quantum theoretical calculations, to verify and provide a good deal of insight into the catalytic process. Based on the above discovery, a colorimetric platform was successfully developed for sensing glucose in the range of 0.10 µM to 10 µM with a detection limit of 40 nM, as well as evaluating the inhibitory effect of antioxidants on reactive oxygen species. This extraordinary finding not only extends the new biological function of acidic amino acids, but also opens new opportunities to deepen the knowledge of the new class of small molecule enzymes.


Assuntos
Aminoácidos Acídicos/metabolismo , Antioxidantes/metabolismo , Técnicas Biossensoriais/métodos , Aminoácidos Acídicos/análise , Antioxidantes/análise , Benzidinas , Glicemia , Colorimetria/métodos , Humanos , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/metabolismo , Limite de Detecção , Modelos Moleculares , Peroxidase/análise , Peroxidase/metabolismo
5.
Comput Biol Chem ; 72: 96-104, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29414100

RESUMO

The electrostatic (ES) energy of each residue was for the first time quantitatively evaluated in a flavin mononucleotide binding protein (FBP). A residue electrostatic energy (RES) was obtained as the sum of the ES energies between atoms in each residue and all other atoms in the FBP dimer using atomic coordinates obtained by a molecular dynamics (MD) simulation. ES is one of the most important energies among the interaction energies in a protein. It is determined from the RES, the residues which mainly contribute to stabilize the structure of each subunit, and the binding energy between two subunits can be estimated. The RES of all residues in subunit A (Sub A) and subunit B (Sub B) were attractive forces, even though the residues contain net negative or positive charges. This reveals that the ES energies of any of the residues can contribute to stabilize the protein structure. The total binding ES energy over all residues among the subunits was distributed between -0.2 to -1.2 eV (mean = -0.67 eV) from the MD simulation time.


Assuntos
Proteínas de Bactérias/química , Flavoproteínas/química , Aminoácidos Acídicos/química , Aminoácidos Básicos/química , Desulfovibrio vulgaris , Simulação de Dinâmica Molecular , Multimerização Proteica , Eletricidade Estática
6.
Food Chem ; 242: 22-28, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29037682

RESUMO

The objective of this paper is to investigate the potential affecting mechanisms of l-lysine (Lys)/l-arginine (Arg) on myosin solubility. The results showed that both Lys and Arg increased the solubility of myosin at the examined pH values. Additionally, both Lys and Arg decreased the hydrodynamic size of myosin but increased the hydration capacity (HC), the surface aromatic hydrophobicity of myosin, the surface tension of the myosin solution and the absolute transfer free energy (TFE) of the major amino acids that constitute myosin. The results indicate that the properties of Lys or Arg that result in an inhibition of myosin aggregation and an interaction with hydrophobic amino acid residues may play important roles in increasing the myosin solubility. The results are attractive to the meat industry.


Assuntos
Aminoácidos Acídicos/química , Arginina/farmacologia , Lisina/farmacologia , Miosinas/efeitos dos fármacos , Arginina/química , Interações Hidrofóbicas e Hidrofílicas , Lisina/química , Miosinas/química , Agregados Proteicos/efeitos dos fármacos , Solubilidade
7.
J Cell Physiol ; 233(4): 2681-2692, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-28833090

RESUMO

CRSBP-1 (mammalian LYVE-1) is a membrane glycoprotein highly expressed in lymphatic endothelial cells (LECs). It has multiple ligands, including hyaluronic acid (HA) and growth factors/cytokines (e.g., PDGF-BB and VEGF-A) containing CRS motifs (clusters of basic amino-acid residues). The ligand binding activities are mediated by Link module and acidic-amino-acid-rich (AAAR) domains, respectively. These CRSBP-1/LYVE-1 ligands have been shown to induce opening of lymphatic intercellular junctions in LEC monolayers and in lymphatic vessels in wild-type mice. We hypothesize that CRSBP-1/LYVE-1 ligands, particularly CRS-containing growth factors/cytokines, are secreted by immune and cancer cells for lymphatic entry during adaptive immune responses and lymphatic metastasis. We have looked into the origin of the Link module and AAAR domain of LYVE-1 in evolution and its association with the development of lymph nodes and efficient adaptive immunity. Lymph nodes represent the only major recent innovation of the adaptive immune systems in evolution particularly to mammals and bird. Here we demonstrate that the development of the LYVE-1 gene with the AAAR domain in evolution is associated with acquisition of lymph nodes and adaptive immunity. LYVE-1 from other species, which have no lymph nodes, lack the AAAR domain and efficient adaptive immunity. Synthetic CRSBP-1 ligands PDGF and VEGF peptides, which contain the CRS motifs of PDGF-BB and VEGF-A, respectively, specifically bind to CRSBP-1 but do not interact with either PDGFßR or VEGFR2. These peptides function as adjuvants by enhancing adaptive immunity of pseudorabies virus (PRV) vaccine in pigs. These results support the notion that LYVE-1 is involved in adaptive immunity in mammals.


Assuntos
Imunidade Adaptativa , Aminoácidos Acídicos/metabolismo , Evolução Molecular , Linfonodos/imunologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Imunidade Adaptativa/efeitos dos fármacos , Adjuvantes Imunológicos/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Ligantes , Linfonodos/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Peptídeos/farmacologia , Filogenia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Domínios Proteicos , Vacinas contra Pseudorraiva/imunologia , Alinhamento de Sequência , Tubarões , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Sus scrofa , Fator A de Crescimento do Endotélio Vascular/farmacologia , Peixe-Zebra
8.
Sci Rep ; 7(1): 16838, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29203783

RESUMO

The current study aimed to investigate the association between dietary amino acid patterns and incidence of hypertension, using principal components factor analyses. This study was conducted within the framework of Tehran Lipid and Glucose Study on 4288 adults, who were free of hypertension at baseline (2008-2011) and were followed for three years (2011-2014). Principal component factor analyses were conducted based on eight amino acid groups and three amino acid patterns were extracted. The first pattern was characterized by branched chain, aromatic, and alcoholic amino acids, and proline. Acidic amino acids and proline were highly loaded in the second pattern and the third was characterized by sulphuric and small amino acids. Adjusted odds ratio of the highest quartile of the first pattern was 1.83 (95%CI: 1.21-2.77, P for trend = 0.002) compared to the lowest one. The first pattern had high positive correlation with dietary intakes of animal protein and dairy, but was negatively correlated with plant protein, fruit, and vegetable. There was no significant association for the second and third patterns. Findings indicate that the dietary amino acid pattern, rich in branched chain, aromatic, and alcoholic amino acids, and proline could increase the risk of hypertension.


Assuntos
Aminoácidos/administração & dosagem , Dieta , Adulto , Aminoácidos Acídicos/administração & dosagem , Aminoácidos de Cadeia Ramificada/administração & dosagem , Índice de Massa Corporal , Exercício Físico , Feminino , Humanos , Hipertensão/epidemiologia , Incidência , Irã (Geográfico)/epidemiologia , Masculino , Pessoa de Meia-Idade , Razão de Chances , Análise de Componente Principal , Prolina/administração & dosagem
9.
Methods Mol Biol ; 1608: 71-77, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28695504

RESUMO

ADP-ribosylation, a posttranslational modification catalyzed by a family of enzymes known as poly(ADP-ribose) polymerases (PARPs, 17 in humans), regulates diverse cellular processes. To aid in understanding the functions of ADP-ribosylation in cells, we developed a clickable aminooxy probe, AO-alkyne, which detects ADP-ribosylation of acidic amino acids. AO-alkyne can be used to detect auto-ADP-ribosylation of PARP10 in cells following Cu-catalyzed click conjugation to an azide reporter. This method can be extended to other PARP family members that catalyze ADP-ribosylation on acidic amino acids, providing a convenient and direct readout of PARP activity in cells.


Assuntos
ADP-Ribosilação/fisiologia , Química Click/métodos , Poli(ADP-Ribose) Polimerases/metabolismo , Aminoácidos Acídicos/química , Aminoácidos Acídicos/metabolismo , Animais , Humanos , Processamento de Proteína Pós-Traducional/fisiologia
10.
Nat Commun ; 8: 15066, 2017 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-28406143

RESUMO

Chirality is ubiquitous in biology, including in biomineralization, where it is found in many hardened structures of invertebrate marine and terrestrial organisms (for example, spiralling gastropod shells). Here we show that chiral, hierarchically organized architectures for calcium carbonate (vaterite) can be controlled simply by adding chiral acidic amino acids (Asp and Glu). Chiral, vaterite toroidal suprastructure having a 'right-handed' (counterclockwise) spiralling morphology is induced by L-enantiomers of Asp and Glu, whereas 'left-handed' (clockwise) morphology is induced by D-enantiomers, and sequentially switching between amino-acid enantiomers causes a switch in chirality. Nanoparticle tilting after binding of chiral amino acids is proposed as a chiral growth mechanism, where a 'mother' subunit nanoparticle spawns a slightly tilted, consequential 'daughter' nanoparticle, which by amplification over various length scales creates oriented mineral platelets and chiral vaterite suprastructures. These findings suggest a molecular mechanism for how biomineralization-related enantiomers might exert hierarchical control to form extended chiral suprastructures.


Assuntos
Aminoácidos Acídicos/química , Ácido Aspártico/química , Carbonato de Cálcio/química , Ácido Glutâmico/química , Microscopia Eletrônica , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Estereoisomerismo , Difração de Raios X
11.
Nucleus ; 8(4): 360-369, 2017 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-28406743

RESUMO

Acetylation of non-histone proteins plays important roles in regulating protein functions but the mechanisms of action are poorly understood. Our recent study uncovered a previously unknown mechanism by which C-terminal domain (CTD) acetylation of p53 serves as a "switch" to determine the interaction between a unique group of acidic domain-containing proteins and p53, as well as revealed that acidic domains may act as a novel class of "readers" for unacetylated p53. However, the properties of acidic domain "readers" are not well elucidated yet. Here, we identified that the charge effect between acidic domain "readers" and the p53 CTD is necessary for their interaction. Both the length and the amino acid composition of a given acidic domain contributed to its ability to recognize the p53 CTD. Finally, we summarized the characteristic features of our identified acidic domains, which would distinguish this kind of "readers" from other types of acidic amino acid-containing domains.


Assuntos
Biologia Computacional , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Sequência de Aminoácidos , Aminoácidos Acídicos , Western Blotting , Humanos , Domínios Proteicos , Proteína Supressora de Tumor p53/química
12.
Plant Biotechnol J ; 15(2): 237-248, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27500592

RESUMO

Heat stress transcription factors (HSFs) compose a large gene family, and different members play differential roles in regulating plant responses to abiotic stress. The objectives of this study were to identify and characterize an A2-type HSF, FaHsfA2c, in a cool-season perennial grass tall fescue (Festuca arundinacea Schreb.) for its association with heat tolerance and to determine the underlying physiological functions and regulatory mechanisms of FaHsfA2c imparting plant tolerance to heat stress. FaHsfA2c was localized in nucleus and exhibited a rapid transcriptional increase in leaves and roots during early phase of heat stress. Ectopic expression of FaHsfA2c improved basal and acquired thermotolerance in wild-type Arabidopsis and also restored heat-sensitive deficiency of hsfa2 mutant. Overexpression of FaHsfA2c in tall fescue enhanced plant tolerance to heat by triggering transcriptional regulation of heat-protective gene expression, improving photosynthetic capacity and maintaining plant growth under heat stress. Our results indicated that FaHsfA2c acted as a positive regulator conferring thermotolerance improvement in Arabidopsis and tall fescue, and it could be potentially used as a candidate gene for genetic modification and molecular breeding to develop heat-tolerant cool-season grass species.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Festuca/fisiologia , Proteínas de Choque Térmico/fisiologia , Proteínas de Plantas/fisiologia , Termotolerância/genética , Fatores de Transcrição/fisiologia , Aminoácidos Acídicos , Arabidopsis/genética , Proteínas de Arabidopsis , Clorofila/metabolismo , Embaralhamento de DNA , Proteínas de Ligação a DNA/genética , Festuca/genética , Festuca/crescimento & desenvolvimento , Genes de Plantas , Fatores de Transcrição de Choque Térmico , Proteínas de Choque Térmico/genética , Temperatura Alta , Mutação , Fenótipo , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Estações do Ano , Alinhamento de Sequência , Estresse Fisiológico/genética , Taxa de Sobrevida , Fatores de Transcrição/genética
13.
Mater Sci Eng C Mater Biol Appl ; 54: 150-7, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26046278

RESUMO

In situ fabrication of carbonated hydroxyapatite (CHA) remineralization layer on an enamel slice was completed in a novel, biomimetic two-step method. First, a CaCO3 layer was synthesized on the surface of demineralized enamel using an acidic amino acid (aspartic acid or glutamate acid) as a soft template. Second, at the same concentration of the acidic amino acid, rod-like carbonated hydroxyapatite was produced with the CaCO3 layer as a sacrificial template and a reactant. The morphology, crystallinity and other physicochemical properties of the crystals were characterized using field emission scanning electron microscopy (FESEM), Fourier transform infrared spectrometry (FTIR), X-ray diffraction (XRD) and energy-dispersive X-ray analysis (EDAX), respectively. Acidic amino acid could promote the uniform deposition of hydroxyapatite with rod-like crystals via absorption of phosphate and carbonate ions from the reaction solution. Moreover, compared with hydroxyapatite crystals coated on the enamel when synthesized by a one-step method, the CaCO3 coating that was synthesized in the first step acted as an active bridge layer and sacrificial template. It played a vital role in orienting the artificial coating layer through the template effect. The results show that the rod-like carbonated hydroxyapatite crystals grow into bundles, which are similar in size and appearance to prisms in human enamel, when using the two-step method with either aspartic acid or acidic glutamate (20.00 mmol/L).


Assuntos
Aminoácidos Acídicos/química , Durapatita/síntese química , Remineralização Dentária/métodos , Ácido Aspártico/química , Biomimética , Carbonato de Cálcio/síntese química , Cristalização , Esmalte Dentário/química , Humanos , Microscopia Eletrônica de Varredura , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios X
14.
Sci Rep ; 5: 9881, 2015 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-25955787

RESUMO

During the long-term evolution of animal toxins acting on potassium channels, the acidic residues can orientate the toxin binding interfaces by adjusting the molecular polarity. Based on the evolutionary function of toxin acidic residues, de novo peptide drugs with distinct binding interfaces were designed for the immunotherapeutic target, the Kv1.3 channel. Using a natural basic toxin, BmKTX, as a template, which contains 2 acidic residues (Asp19 and Asp33), we engineered two new peptides BmKTX-19 with 1 acidic residue (Asp33), and BmKTX-196 with 2 acidic residues (Asp6 and Asp33) through only adjusting acidic residue distribution for reorientation of BmKTX binding interface. Pharmacological experiments indicated that BmKTX-19 and BmKTX-196 peptides were specific inhibitors of the Kv1.3 channel and effectively suppressed cytokine secretion. In addition to the structural similarity between the designed and native peptides, both experimental alanine-scanning mutagenesis and computational simulation further indicated that the binding interface of wild-type BmKTX was successfully reoriented in BmKTX-19 and BmKTX-196, which adopted distinct toxin surfaces as binding interfaces. Together, these findings indicate not only the promising prospect of BmKTX-19 and BmKTX-196 as drug candidates but also the desirable feasibility of the evolution-guided peptide drug design for discovering numerous peptide drugs for the Kv1.3 channel.


Assuntos
Aminoácidos Acídicos/toxicidade , Desenho de Fármacos , Evolução Molecular , Imunoterapia , Peptídeos/química , Sequência de Aminoácidos , Citocinas/metabolismo , Células HEK293 , Humanos , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/química , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Ligação Proteica/efeitos dos fármacos , Venenos de Escorpião/química , Soluções , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo
15.
Artigo em Inglês | MEDLINE | ID: mdl-25589253

RESUMO

Glutamic acid (Glu) and aspartic acid (Asp), as two important neurotransmitters, have been the focus of increasingly intense research over the past several years. Glu and Asp are present in biological fluids such as serum at trace levels, but complex components in biological matrices make it difficult to determine them in biological samples. In this paper, a sensitive and simple method coupled with indirect UV detection, using benzoic acid (BA) as the UV-absorbing probe, was developed and validated for the quantitative determination of Glu and Asp in human serum and Compound Amino Acid Injection-18 AA. The method combines a dynamic pH junction with a sweeping technique using ß-cyclodextrin (ß-CD) as the complexing agent for sweeping. Employing this proposed method, low detection limits of 0.061µg/mL for Glu and 0.032µg/mL for Asp were obtained. The sensitivity was improved 30- and 55-fold for Glu and Asp compared to conventional CE method. Standard curves were linear (r>0.999) over the concentration range of 0.1-8.0µg/mL. To further improve the resolution of Asp from interfering substances in human serum, 6% (v/v) methanol was added to the sample matrix, and resulted in the detection limits of 0.125µg/mL for Glu and 0.057µg/mL for Asp. With a simple precipitation of protein, the method has been successfully applied to the analysis of human serum, and the recoveries (82% for Glu and 87% for Asp) were achieved with relative standard deviations of 1.9% and 2.0%, respectively.


Assuntos
Aminoácidos Acídicos/sangue , Eletroforese Capilar/métodos , Humanos , Concentração de Íons de Hidrogênio
16.
Mol Microbiol ; 96(1): 14-27, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25588312

RESUMO

Hydrophobins are amphipathic proteins secreted by filamentous fungi. When the industrial fungus Aspergillus oryzae is grown in a liquid medium containing the polyester polybutylene succinate co-adipate (PBSA), it produces RolA, a hydrophobin, and CutL1, a PBSA-degrading cutinase. Secreted RolA attaches to the surface of the PBSA particles and recruits CutL1, which then condenses on the particles and stimulates the hydrolysis of PBSA. Here, we identified amino acid residues that are required for the RolA-CutL1 interaction by using site-directed mutagenesis. We quantitatively analyzed kinetic profiles of the interactions between RolA variants and CutL1 variants by using a quartz crystal microbalance (QCM). The QCM analyses revealed that Asp142, Asp171 and Glu31, located on the hydrophilic molecular surface of CutL1, and His32 and Lys34, located in the N-terminus of RolA, play crucial roles in the RolA-CutL1 interaction via ionic interactions. RolA immobilized on a QCM electrode strongly interacted with CutL1 (K(D) = 6.5 nM); however, RolA with CutL1 variants, or RolA variants with CutL1, showed markedly larger KD values, particularly in the interaction between the double variant RolA-H32S/K34S and the triple variant CutL1-E31S/D142S/D171S (K(D) = 78.0 nM). We discuss a molecular prototype model of hydrophobin-based enzyme recruitment at the solid-water interface.


Assuntos
Aminoácidos Acídicos/metabolismo , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Sequência de Aminoácidos , Hidrolases de Éster Carboxílico/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Interações Hidrofóbicas e Hidrofílicas , Íons , Modelos Moleculares , Mutagênese Sítio-Dirigida , Poliésteres/metabolismo , Polímeros/metabolismo , Técnicas de Microbalança de Cristal de Quartzo
17.
Biosens Bioelectron ; 65: 204-10, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25461159

RESUMO

A simple mix-and-detect photoluminescence method was developed for the turn-on detection of acidic amino acids. To achieve this, graphene quantum dots (GQDs), which emit both down-conversion and up-conversion photoluminescence were prepared by solvothermal synthesis. The carboxylic acid-rich surface not only increases the water solubility of the prepared GQDs, but also makes Eu(3+)-triggered GQDs aggregation possible, thus causing the photoluminescence quenching of GQDs. The quenched photoluminescence can be recovered by the competition between acidic amino acids and GQDs for Eu(3+). Under optimized conditions, sensitive and specific acidic amino acids quantitation can be achieved by utilizing the changes in either down-conversion or up-conversion photoluminescence. Up-conversion mode gives a little lower detection limit than the down-conversion one. Nearly overlapped calibration curves were obtained for the two acidic amino acids, glutamic acid (Glu) and aspartic acid (Asp), thus suggesting that the proposed method can be used not only for the quantitation of individual acidic amino acids, but also for the detection of total amount of them.


Assuntos
Aminoácidos Acídicos/sangue , Európio/química , Grafite/química , Medições Luminescentes/métodos , Pontos Quânticos/química , Aminoácidos Acídicos/análise , Animais , Cátions/química , Bovinos , Limite de Detecção , Pontos Quânticos/ultraestrutura
18.
J Mol Biol ; 426(24): 3946-3959, 2014 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-25315822

RESUMO

Processing of Holliday junctions is essential in recombination. We have identified the gene for the junction-resolving enzyme GEN1 from the thermophilic fungus Chaetomium thermophilum and expressed the N-terminal 487-amino-acid section. The protein is a nuclease that is highly selective for four-way DNA junctions, cleaving 1nt 3' to the point of strand exchange on two strands symmetrically disposed about a diagonal axis. CtGEN1 binds to DNA junctions as a discrete homodimer with nanomolar affinity. Analysis of the kinetics of cruciform cleavage shows that cleavage of the second strand occurs an order of magnitude faster than the first cleavage so as to generate a productive resolution event. All these properties are closely similar to those described for bacterial, phage and mitochondrial junction-resolving enzymes. CtGEN1 is also similar in properties to the human enzyme but lacks the problems with aggregation that currently prevent detailed analysis of the latter protein. CtGEN1 is thus an excellent enzyme with which to engage in biophysical and structural analysis of eukaryotic GEN1.


Assuntos
Chaetomium/enzimologia , DNA Cruciforme/metabolismo , Proteínas Fúngicas/metabolismo , Resolvases de Junção Holliday/metabolismo , Algoritmos , Sequência de Aminoácidos , Aminoácidos Acídicos/genética , Aminoácidos Acídicos/metabolismo , Sequência de Bases , Ligação Competitiva , Chaetomium/genética , DNA Cruciforme/química , DNA Cruciforme/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Resolvases de Junção Holliday/classificação , Resolvases de Junção Holliday/genética , Hidrólise , Cinética , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Filogenia , Ligação Proteica , Multimerização Proteica , Homologia de Sequência de Aminoácidos
19.
PLoS One ; 9(6): e99376, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24901998

RESUMO

Anoctamin1 (Ano1, or TMEM16A) is a Ca2+-activated chloride channel that is gated by both voltage and Ca2+. We have previously identified that the first intracellular loop that contains a high density of acidic residues mediates voltage- and calcium-dependent gating of Ano1. Mutation of the four consecutive glutamates (444EEEE447) inhibits the voltage-dependent activation of Ano1, whereas deletion of these residues decreases apparent Ca2+ sensitivity. In the present study, we further found that deletion of 444EEEEEAVKD452 produced a more than 40-fold decrease in the apparent Ca2+ sensitivity with altered activation kinetics. We then systematically mutated each acidic residue into alanine, and analyzed the voltage- and calcium dependent activation of each mutation. Activation kinetics of wild type Ano1 consisted of a fast component (τfast) that represented voltage-dependent mode, and a slow component (τslow) that reflected the Ca2+-dependent modal gating. E444A, E445A, E446A, E447A, E448A, and E457A mutations showed a decrease in the τfast, significantly inhibited voltage-dependent activation of Ano1 in the absence of Ca2+, and greatly shifted the G-V curve to the right, suggesting that these glutamates are involved in voltage-gating of Ano1. Furthermore, D452A, E464A, E470A, and E475A mutations that did not alter voltage-dependent activation of the channel, significantly decreased Ca2+ dependence of G-V curve, exhibited an increase in the τslow, and produced a 2-3 fold decrease in the apparent Ca2+ sensitivity, suggesting that these acidic residues are involved in Ca2+-dependent gating of the channel. Our data show that acidic residues in the first intracellular loop are the important structural determinant that couples the voltage and calcium dependent gating of Ano1.


Assuntos
Aminoácidos Acídicos/metabolismo , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Proteínas de Neoplasias/metabolismo , Potenciais de Ação/fisiologia , Sequência de Aminoácidos , Anoctamina-1 , Canais de Cloreto/química , Canais de Cloreto/genética , Células HEK293 , Humanos , Cinética , Mutação , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Técnicas de Patch-Clamp
20.
Molecules ; 19(5): 6349-67, 2014 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-24840903

RESUMO

Aryl-keto-containing α-amino acids are of great importance in organic chemistry and biochemistry. They are valuable intermediates for the construction of hydroxyl α-amino acids, nonproteinogenic α-amino acids, as well as other biofunctional components. Friedel-Crafts acylation is an effective method to prepare aryl-keto derivatives. In this review, we summarize the preparation of aryl-keto containing α-amino acids by Friedel-Crafts acylation using acidic α-amino acids as acyl-donors and Lewis acids or Brönsted acids as catalysts.


Assuntos
Aminoácidos Acídicos/química , Ácidos de Lewis/química , Mesilatos/química , Acilação , Aminoácidos Acídicos/metabolismo , Catálise , Ácidos de Lewis/metabolismo , Mesilatos/metabolismo
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