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1.
Chem Pharm Bull (Tokyo) ; 67(9): 945-952, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31474734

RESUMO

Salt and cocrystal formulations are widely used as techniques to improve physicochemical properties of compounds. Some spectrometric techniques to distinguish cocrystals from salts have been reported; however, it has not been possible to adapt these formulations with many compounds, because of limitations, high difficulty, and exceptions. Therefore, we focused on the possibility of UV spectrometry, which had not been reported as a classification technique for salts and cocrystals. The integration values of solid-state UV/visible (Vis) spectra of indomethacin salts were larger than those of physical mixtures of indomethacin and counter molecules, while that of indomethacin cocrystal was not large compared with that of the physical mixture. From these results, differences between a salt and a cocrystal were observed in their solid-state UV/Vis absorption spectra for indomethacin complexes. Therefore, it is suggested that solid-state UV/Vis absorption spectra can be used as a new technique to classify salts and cocrystals.


Assuntos
Indometacina/química , Espectrofotometria Ultravioleta/métodos , Arginina/química , Cristalização , Espectroscopia de Ressonância Magnética , Meglumina/química , Sacarina/química , Sais/química , Difração de Raios X
2.
Phys Chem Chem Phys ; 21(32): 17893-17900, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31380529

RESUMO

The dispersion interaction was reported to play a critical role in the stabilization of model dipeptide Z-Arg-OH, even greater than the conventional hydrogen bond (HB), which is opposite to the traditional opinion. Here the conformation of Z-Arg-OH has been systematically searched by the effective fragment based step-by-step strategy. All the newly-found low-energy conformers determined at the advanced DSD-PBEP86-D3(BJ)/aug-cc-pVTZ level are clearly in the stretched form with strong conventional HBs, rather than the reported folded structures with emphasis on the dispersion interactions. The simulated IR spectra of the stretched conformers fit better than those of the folded ones compared with the previous experimental observations. Near-edge X-ray absorption fine-structure (NEXAFS) spectra and X-ray photoelectron spectra (XPS) at C, N and O K-edges have also been simulated to unambiguously identify different isomers. This work thus provides valuable insight into the competitions between the conventional HB and the dispersion interactions and demonstrates that the conventional hydrogen bonding is still more important for such small peptides.


Assuntos
Arginina/análogos & derivados , Arginina/química , Dipeptídeos/química , Modelos Moleculares , Ligações de Hidrogênio , Isomerismo , Fenômenos Físicos , Conformação Proteica , Estabilidade Proteica , Solventes/química , Termodinâmica
3.
Nat Commun ; 10(1): 2917, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266949

RESUMO

Novel antibacterial agents are needed to address the emergence of global antibiotic resistance. MraY is a promising candidate for antibiotic development because it is the target of five classes of naturally occurring nucleoside inhibitors with potent antibacterial activity. Although these natural products share a common uridine moiety, their core structures vary substantially and they exhibit different activity profiles. An incomplete understanding of the structural and mechanistic basis of MraY inhibition has hindered the translation of these compounds to the clinic. Here we present crystal structures of MraY in complex with representative members of the liposidomycin/caprazamycin, capuramycin, and mureidomycin classes of nucleoside inhibitors. Our structures reveal cryptic druggable hot spots in the shallow inhibitor binding site of MraY that were not previously appreciated. Structural analyses of nucleoside inhibitor binding provide insights into the chemical logic of MraY inhibition, which can guide novel approaches to MraY-targeted antibiotic design.


Assuntos
Antibacterianos/química , Bactérias/enzimologia , Proteínas de Bactérias/química , Produtos Biológicos/química , Inibidores Enzimáticos/química , Nucleosídeos/antagonistas & inibidores , Transferases/química , Aminoglicosídeos/química , Arginina/análogos & derivados , Arginina/química , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Transferases/antagonistas & inibidores , Transferases/genética , Transferases/metabolismo
4.
Food Chem ; 295: 320-326, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174764

RESUMO

This research focused on the effects of l-arginine (l-Arg) and l-histidine (l-His) on the heat-induced aggregation of fish myosin. l-Arg/l-His increased the pH of the myosin solution from 6.82 to 8.74 and 7.24, respectively, and decreased the turbidity, aggregate size, shear modulus, and breaking force. The incorporation of l-Arg/l-His decreased the surface hydrophobicity during setting, but increased it during the two-step heating. The heat-induced aggregation of myosin was suppressed by both amino acids, with the inhibitory effect being greater for l-Arg than l-His. On one hand, the change in the pH played a critical role in suppressing the heat-induced aggregation of myosin. On the other hand, the characteristics of l-Arg/l-His themselves, such as net charges and particular R-groups, were another main contributor to aggregation suppression. Particularly, l-Arg/l-His could interact with exposed aromatic residues of myosin, and the interactions may dominate and overwhelm the burial of aromatic residues during two-step heating.


Assuntos
Arginina/química , Proteínas de Peixes da Dieta/química , Histidina/química , Miosinas/química , Animais , Cyprinidae , Proteínas de Peixes da Dieta/metabolismo , Géis/química , Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Miosinas/metabolismo
5.
Chem Commun (Camb) ; 55(52): 7482-7485, 2019 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-31184653

RESUMO

Protein arginine (Arg) phosphorylation regulates stress responses and virulence in bacteria. With fluorescent activity probes, we show that McsB, a protein Arg kinase, can dephosphorylate phosphoarginine (pArg) residues to produce ATP from ADP, implicating the dynamic control of protein pArg levels by the kinase even without a phosphatase.


Assuntos
Arginina Quinase/metabolismo , Corantes Fluorescentes/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Arginina/análogos & derivados , Arginina/análise , Arginina/química , Arginina/metabolismo , Cromatografia Líquida de Alta Pressão , Compostos Organofosforados/análise , Fosforilação
6.
J Agric Food Chem ; 67(20): 5754-5763, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31045365

RESUMO

Recently, although ginseng ( Panax ginseng C. A. Meyer) and its main component saponins (ginsenosides) have been reported to exert protective effects on cisplatin (CDDP)-induced acute kidney injury (AKI), the beneficial activities of non-saponin on CDDP-induced AKI is little known. This research was designed to explore the protective effect and underlying mechanism of arginyl-fructosyl-glucose (AFG), a major and representative non-saponin component generated during the process of red ginseng, on CDDP-caused AKI. AFG at doses of 40 and 80 mg/kg remarkably reversed CDDP-induced renal dysfunction, accompanied by the decreased levels of serum creatinine and blood urea nitrogen. Interestingly, all of oxidative stress indices were ameliorated after pretreatment with AFG continuously for 10 days. Importantly, AFG relieved CDDP-induced inflammation and apoptosis in part by mitigating the cascade initiation steps of nuclear factor κB signals and regulating the participation of the phosphatidylinositol 3-kinase/protein kinase B signal pathway. In conclusion, these results clearly provide strong rationale for the development of AFG to prevent CDDP-induced AKI.


Assuntos
Lesão Renal Aguda/tratamento farmacológico , Arginina/análogos & derivados , Cisplatino/efeitos adversos , Medicamentos de Ervas Chinesas/administração & dosagem , Glucose/administração & dosagem , Glicina/análogos & derivados , NF-kappa B/metabolismo , Panax/química , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Lesão Renal Aguda/induzido quimicamente , Lesão Renal Aguda/metabolismo , Lesão Renal Aguda/fisiopatologia , Animais , Apoptose/efeitos dos fármacos , Arginina/administração & dosagem , Arginina/química , Creatinina/metabolismo , Medicamentos de Ervas Chinesas/química , Glucose/química , Glicina/administração & dosagem , Glicina/química , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Reação de Maillard , Masculino , Camundongos Endogâmicos ICR , NF-kappa B/genética , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/efeitos dos fármacos
7.
Chem Commun (Camb) ; 55(46): 6610-6613, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31119219

RESUMO

The X-ray crystal structure of F43Y/T67R myoglobin revealed unique Tyr-heme double cross-links between Tyr43 and the heme 4-vinyl group, which represents a novel post-translational modification of heme proteins. Moreover, with the feature of a distal His-Arg pair, the designed artificial enzyme exhibited a peroxidase activity comparable to that of native peroxidases, such as the most efficient horseradish peroxidase.


Assuntos
Materiais Biomiméticos/química , Heme/química , Mioglobina/química , Tirosina/química , Animais , Arginina/química , Benzotiazóis/química , Guaiacol/química , Histidina/química , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Cinética , Estrutura Molecular , Mutação , Mioglobina/genética , Oxirredução , Peroxidases/química , Processamento de Proteína Pós-Traducional , Cachalote , Ácidos Sulfônicos/química
8.
Phys Chem Chem Phys ; 21(20): 10621-10634, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31080981

RESUMO

The solvation of lidocaine in three newly designed deep eutectic solvents is studied using combined experimental and theoretical methods that include density functional theory and molecular dynamics methods. The intermolecular forces between lidocaine and the hydrogen bond acceptors and hydrogen bond donors of the deep eutectic solvents were analysed regarding the type and the strength of inter- and intra-molecular bonding. The structure, composition and properties of the lidocaine solvation shells are analysed together with the possible lidocaine-clustering around the studied deep eutectic solvents and their constituent molecules. Furthermore, the changes in the solvent structures upon lidocaine solubilization are also studied. Natural product-based eutectic solvents showed considerably high solvation of lidocaine in all three deep eutectics based on the strong solute-solvent intermolecular interactions accompanied by a slight volume expansion and minor solvent structural changes. These non-toxic and almost null-volatile therapeutic deep eutectic solvents can be considered as suitable solubilization media for developing pharmaceutical applications and they can be considered as effective drug delivery vehicles for active pharmaceutical ingredients.


Assuntos
Arginina/química , Sistemas de Liberação de Medicamentos , Lidocaína/química , Solventes/química , Lidocaína/administração & dosagem , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/química , Solubilidade
9.
Phys Chem Chem Phys ; 21(19): 9957-9968, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31041955

RESUMO

The ethylene-forming enzyme (EFE) is a unique member of the Fe(ii)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenases. It converts 2OG into ethylene plus three CO2 molecules (ethylene-forming reaction) and also catalyzes the C5 hydroxylation of l-arginine coupled to the oxidative decarboxylation of 2OG (l-Arg hydroxylation reaction). To uncover the mechanisms of the dual transformations by EFE, quantum mechanical/molecular mechanical (QM/MM) calculations were carried out. Based on the results, a branched mechanism was proposed. An FeII-peroxysuccinate complex with a dissociated CO2 generated through the nucleophilic attack of the superoxo moiety of the Fe-O2 species on the keto carbon of 2OG is the key common intermediate in both reactions. A competition between the subsequent CO2 insertion (a key step in the ethylene-forming pathway) and the O-O bond cleavage (leading to the formation of succinate) governs the product selectivity. The calculated reaction barriers suggested that the CO2 insertion is favored over the O-O bond cleavage. This is consistent with the product preference observed in experiments. By comparison with the results of AsqJ (an Fe/2OG oxygenase that leads to substrate oxidation exclusively), the protein environment was found to be crucial for the selectivity. Further calculations demonstrated that the local electric field of the protein environment in EFE promotes ethylene formation by acting as a charge template, exemplifying the importance of the electrostatic interaction in enzyme catalysis. These findings offer mechanistic insights into the EFE catalysis and provide important clues for better understanding the unique ethylene-forming capability of EFE compared with other Fe/2OG oxygenases.


Assuntos
Arginina/metabolismo , Etilenos/biossíntese , Ácidos Cetoglutáricos/metabolismo , Liases/metabolismo , Arginina/química , Biocatálise , Teoria da Densidade Funcional , Etilenos/química , Hidroxilação , Ácidos Cetoglutáricos/química , Liases/química , Estrutura Molecular , Oxirredução
10.
J Agric Food Chem ; 67(20): 5874-5881, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31050431

RESUMO

Dicarbonyl compounds such as methylglyoxal (MGO) and 3-deoxyglucosone (3-DG) are formed via caramelization and the Maillard reaction in food during heating or in vivo as byproducts of glycolysis. Recently, it was shown that creatine, an amino compound linked to the energy metabolism in vertebrate muscle, reacts rapidly with methylglyoxal under physiological conditions to form N-(4-methyl-5-oxo-1-imidazolin-2-yl)sarcosine (MG-HCr), a methylglyoxal-derived hydroimidazolone of creatine. Based on the observation that heated meat contains only small amounts of MGO and 3-DG when compared to many other foodstuffs, the aim of this study was to investigate a possible reaction of creatine with 3-DG and MGO in meat. From incubation mixtures consisting of 3-DG and creatine, a new hydroimidazolone of creatine, namely N-(4-butyl-1,2,3-triol-5-oxo-1-imidazolin-2-yl)sarcosine (3-DG-HCr), was isolated and characterized via spectroscopic means. To quantitate 3-DG-HCr and MG-HCr, meat and fish products were analyzed via HPLC-MS/MS using isotopically labeled standard material. Whereas samples of raw fish and meat contained only trace amounts of the hydroimidazolones (below 5 µg/kg), up to 28.3 mg/kg MG-HCr and up to 15.3 mg/kg 3-DG-HCr were found in meat and fish products. The concentrations were dependent on the heat treatment and presumably on the smoking process. In comparison to the lysine and arginine derivatives CEL, pyrraline, and MG-H1, the derivatization rate of creatine as MG-HCr and 3-DG-HCr was higher than of lysine and arginine, which clearly demonstrates the 1,2-dicarbonyl scavenging properties of creatine in meat.


Assuntos
Creatina/química , Desoxiglucose/análogos & derivados , Imidazóis/química , Carne/análise , Aldeído Pirúvico/química , Animais , Arginina/química , Bovinos , Galinhas , Cromatografia Líquida de Alta Pressão , Culinária , Desoxiglucose/química , Temperatura Alta , Lisina/química , Reação de Maillard , Suínos , Espectrometria de Massas em Tandem
11.
Nat Chem Biol ; 15(5): 510-518, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962626

RESUMO

Protein phosphorylation regulates key processes in all organisms. In Gram-positive bacteria, protein arginine phosphorylation plays a central role in protein quality control by regulating transcription factors and marking aberrant proteins for degradation. Here, we report structural, biochemical, and in vivo data of the responsible kinase, McsB, the founding member of an arginine-specific class of protein kinases. McsB differs in structure and mechanism from protein kinases that act on serine, threonine, and tyrosine residues and instead has a catalytic domain related to that of phosphagen kinases (PhKs), metabolic enzymes that phosphorylate small guanidino compounds. In McsB, the PhK-like phosphotransferase domain is structurally adapted to target protein substrates and is accompanied by a novel phosphoarginine (pArg)-binding domain that allosterically controls protein kinase activity. The identification of distinct pArg reader domains in this study points to a remarkably complex signaling system, thus challenging simplistic views of bacterial protein phosphorylation.


Assuntos
Arginina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Arginina/química , Modelos Moleculares , Fosforilação
12.
Mol Cell ; 74(5): 922-935.e6, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-30979585

RESUMO

Enteropathogenic E. coli NleB and related type III effectors catalyze arginine GlcNAcylation of death domain (DD) proteins to block host defense, but the underlying mechanism is unknown. Here we solve crystal structures of NleB alone and in complex with FADD-DD, UDP, and Mn2+ as well as NleB-GlcNAcylated DDs of TRADD and RIPK1. NleB adopts a GT-A fold with a unique helix-pair insertion to hold FADD-DD; the interface contacts explain the selectivity of NleB for certain DDs. The acceptor arginine is fixed into a cleft, in which Glu253 serves as a base to activate the guanidinium. Analyses of the enzyme-substrate complex and the product structures reveal an inverting sugar-transfer reaction and a detailed catalytic mechanism. These structural insights are validated by mutagenesis analyses of NleB-mediated GlcNAcylation in vitro and its function in mouse infection. Our study builds a structural framework for understanding of NleB-catalyzed arginine GlcNAcylation of host death domain.


Assuntos
Escherichia coli Enteropatogênica/genética , Proteínas de Escherichia coli/química , Interações Hospedeiro-Patógeno/genética , Conformação Proteica , Fatores de Virulência/química , Animais , Apoptose/genética , Arginina/química , Arginina/genética , Coenzima A Ligases/química , Coenzima A Ligases/genética , Cristalografia por Raios X , Domínio de Morte/genética , Escherichia coli Enteropatogênica/patogenicidade , Proteínas de Escherichia coli/genética , Guanidina/química , Humanos , Manganês/química , Camundongos , Mutagênese , Proteína de Domínio de Morte Associada a Receptor de TNF/química , Proteína de Domínio de Morte Associada a Receptor de TNF/genética , Fatores de Virulência/genética
13.
Plant Mol Biol ; 100(6): 571-578, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30953277

RESUMO

KEY MESSAGE: A mutation in the nuclear localization signal of squamosa promoter binding like-protein 9 (SPL9) delays vegetative phase change by disrupting its nuclear localization. The juvenile-to-adult phase transition is a critical developmental process in plant development, and it is regulated by a decrease in miR156/157 and a corresponding increase in their targets, squamosa promoter binding protein-like (SPL) genes. SPL proteins contain a conserved SBP domain with putative nuclear localization signals (NLSs) at their C-terminals. Some SPLs promote vegetative phase change by promoting miR172 expression, but the function of nuclear localization signals in those SPLs remains unknown. Here, we identified a loss-of-function mutant, which we named del6, with delayed vegetative phase change phenotypes in a forward genetic screen. Map-based cloning, the whole genome resequencing, and allelic complementation test demonstrate that a G-to-A substitution in the SPL9 gene is responsible for the delayed vegetative phase change phenotypes. In del6, the mutation causes a substitution of the glutamine (Gln) for the conserved basic amino acid arginine (Arg) in the NLS of the SBP domain, and disrupts the normal nuclear localization and function of SPL9. Therefore, our work demonstrates that the NLSs in the SBP domain of SPL9 are indispensable for its nuclear localization and normal function in Arabidopsis.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Arabidopsis/genética , Sinais de Localização Nuclear , Regiões Promotoras Genéticas , Transativadores/genética , Transativadores/fisiologia , Alelos , Arginina/química , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Vetores Genéticos , Genoma de Planta , Glutamina/química , MicroRNAs/genética , Mutação , Fenótipo , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas
14.
Eur J Mass Spectrom (Chichester) ; 25(2): 212-218, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31018697

RESUMO

Electrostatic interactions are one of the main factors influencing biomolecular conformation. The formation of noncovalent complexes by electrostatic interactions is governed by certain amino acid residues and post-translational modifications. It has been demonstrated that adjacent arginine forms noncovalent complex with phosphate; however, histidine noncovalent complexes have rarely been investigated. In the present work, we compare the interaction between basic epitopes (NLRRITRVN, SHHGLHSTPD) and diverse acidic and aromatic-rich peptides using both MALDI and ESI Mass spectrometry. We show that adjacent histidines can also form stable noncovalent bonds and that those bonds are probably formed by a salt bridge between the phosphate or the acid residues and the histidines. However, noncovalent complexes with the arginine epitopes form more readily and are stronger than those with histidine-containing epitopes.


Assuntos
Arginina/química , Histidina/química , Conformação Molecular , Peptídeos/química , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Eletricidade Estática
15.
Food Microbiol ; 82: 46-52, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31027806

RESUMO

Although Staphylococcus aureus is a major cause of food poisoning, little is known about its response to growth on food. Utilizing a transcriptional profiling and metabolomics approach, we compared S. aureus grown on autoclaved chicken breast (ACB) to Luria broth agar. ACB cultures demonstrated increased expression of genes associated with protein synthesis, cofactors, secondary metabolites, nitrogen and nucleotide metabolism, amino acid transport, and reduced expression of general stress, lipid metabolism, and virulence genes. The ACB culture also displayed characteristics of catabolite de-repression and anaerobic growth, and increased expression of arginine biosynthesis genes (argFGH) and an arginine/ornithine antiporter gene (arcD). S. aureus synthesizes arginine from proline and the ACB culture exhibited increased expression of proline transport genes (opuBA, opuBB and putP) and increased proline accumulation. Amino acid and sugar content in the ACB grown culture increased, and this was attributed to the consumption of ACB, transport of amino acids, and gluconeogenesis. Genes involved with biotin biosynthesis and uptake were upregulated and biotin is required for amino acid catabolism. Genes encoding urease and urease activity were upregulated in ACB cultures, while urea levels were reduced. This research provides fundamental information on the response of S. aureus growing on chicken meat that could find application in future attempts to reduce the growth of S. aureus in food.


Assuntos
Proteínas de Bactérias/genética , Perfilação da Expressão Gênica , Temperatura Alta , Metabolômica , Produtos Avícolas/microbiologia , Staphylococcus aureus/genética , Aminoácidos/química , Anaerobiose , Animais , Arginina/química , Biotina/biossíntese , Repressão Catabólica , Galinhas , Gluconeogênese/genética , Staphylococcus aureus/crescimento & desenvolvimento , Urease/metabolismo , Virulência
16.
Biosens Bioelectron ; 133: 86-93, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30909017

RESUMO

Pediatric adrenocortical carcinoma (pACC) is a rare and aggressive malignancy of high occurrence in Southern Brazil. pACC is characterized by the usual overproduction of dehydroepiandrosterone sulfate (DHEAS), whose detection in serum or plasma can be effective to the early diagnosis of the disease. Therefore, the present paper reports, for the first time, the construction and application of a label-free impedimetric immunosensor to detect DHEAS, which was based on the modification of an oxidized glassy carbon electrode with arginine-functionalized gold nanoparticles (AuNPs-ARG) and anti-DHEA IgM antibodies (ox-GCE/AuNPs-ARG/IgM). AuNPs-ARG was synthesized by a green route, and characterized by UV-VIS spectroscopy, FTIR, TEM, DLS, and XRD. The construction of ox-GCE/AuNPs-ARG/IgM was optimized through factorial design and response surface methodology. Cyclic voltammetry and electrochemical impedance spectroscopy measurements were employed to characterize the optimized immunosensor. The DHEAS detection principle was based on the variation of charge transfer resistance (∆Rct) relative to the Fe(CN)64-/3- electrochemical probe after immunoassays in the presence of the biomarker. A linear relationship between ∆Rct and DHEAS concentration was verified in the range from 10.0 to 110.0 µg dL-1, with a LOD of 7.4 µg dL-1. Besides the good sensitivity, the immunosensor displayed accuracy, stability, and specificity to detect DHEAS. The promising analytical performance of ox-GCE/AuNPs-ARG/IgM was confirmed by quantifying DHEAS in real patient plasma samples, with results that were comparable to the reference chemiluminescence assay. Our results suggest that the presented immunosensor can find clinical applications in the early diagnosis of pACC and to monitor DHEAS levels in other adrenal pathologies.


Assuntos
Carcinoma Adrenocortical/diagnóstico , Biomarcadores Tumorais/isolamento & purificação , Técnicas Biossensoriais , Nanopartículas Metálicas/química , Carcinoma Adrenocortical/genética , Arginina/química , Biomarcadores Tumorais/química , Carbono/química , Técnicas Eletroquímicas , Ouro/química , Humanos , Limite de Detecção
17.
Virus Res ; 265: 132-137, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30926385

RESUMO

In our previous study, we produced a monoclonal antibody EB2 that recognized an epitope in the HA1 domain on the hemagglutinin (HA) of H6N1 influenza virus (A/chicken/Taiwan/2838 V/00). The residue Arg-201 (R201) on this epitope was protected by the glycan at Asn-167 (N167) from tryptic digestion; therefore, the infectivity of the virus was retained. R201 was extremely conserved in various subtypes of the influenza virus. To explore the role of R201 and the protecting glycan, we developed a bi-cistronic baculovirus expression system for the production of H6HA1 and H6HA0 (nearly full-length HA), which were glycosylated in insect cells. The expressed H6HA1 was mostly found in the trimeric form, and the H6HA0 protein was only found in the monomeric form. The trimeric H6HA1 was resistant to tryptic digestion; however, it could not bind to fetuin, a glycoprotein containing sialylated N-linked and O-linked glycans. By contrast, the monomeric H6HA0 could bind to fetuin but was sensitive to tryptic digestion. We found that the positive charge on R201 was critical for binding HA to the negatively charged surface of host cells because the mutant R201A of H6HA0 lost its binding capacity substantially. Moreover, this binding capacity was dependent on the pH value and inhibited by free electrically charged amino acids. We propose a two-step model for binding the influenza virus with a host cell. The first step involved the specific recognition of the receptor binding site on HA to the sialylated glycan on the host cell. After the virus is engulfed by the acidic endosome, R201 could bind to the cell surface with stronger interactions and trigger the fusion process.


Assuntos
Arginina/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Interações entre Hospedeiro e Microrganismos , Vírus da Influenza A/fisiologia , Internalização do Vírus , Animais , Anticorpos Monoclonais/imunologia , Baculoviridae/genética , Sítios de Ligação , Galinhas , Epitopos/imunologia , Glicosilação , Influenza Aviária/virologia , Polissacarídeos/imunologia
18.
J Chromatogr A ; 1595: 97-107, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-30833023

RESUMO

Salt solutions are widely used as eluents for ion-exchange chromatography. In general, salts reduce the retention of applied solutes on ion-exchange columns via electrostatic screening effects. The reverse phenomenon, namely, salt-enhanced retention, has not been reported. Here, we report that cations, including arginine, guanidine and sodium ions, enhance the retention of uncharged aromatic solutes on a cation-exchange resin, i.e., a negatively charged resin, with carboxyl groups, where we used alkyl gallates as model uncharged aromatic solutes and a carboxymethyl agarose gel (CM Sepharose) as a model negatively charged resin. Enhancement of retention was observed at concentrations of tens of millimolar of the salts, in which arginine hydrochloride was more effective than guanidinium salts and NaCl. Similar trends were observed for other phenolic compounds, including phenol and 4-hydroxybenzyl alcohol. Molecular dynamics simulations showed that the binding free energy between the alkyl gallate molecule and the CM Sepharose resin ligand molecule increased with increasing salt concentration. The increase in binding free energy caused by the salts was accounted for by the binding of the salt cations to the aromatic moiety of the alkyl gallate via cation-π interactions, leading to attenuation of intrinsic repulsive interactions between the ligand carboxyl group and the alkyl gallate aromatic moiety. Therefore, the salt-enhanced retention of the uncharged aromatic solutes on the negatively charged resins was ascribable to the increase in binding free energy induced by the cation-π interactions. This unique reverse phenomenon of the effect of salts on solute retention indicates the importance of cation-π interactions in ion-exchange chromatography. This phenomenon can be used for selective chromatographic separation of aromatic solutes, including organic solutes, proteins and nucleic acids.


Assuntos
Arginina/química , Resinas de Troca de Cátion/química , Cátions/química , Cromatografia por Troca Iônica/métodos , Sódio/química , Guanidina/química , Ligantes , Simulação de Dinâmica Molecular , Proteínas/química , Cloreto de Sódio/química , Soluções
19.
J Pharm Biomed Anal ; 169: 82-89, 2019 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-30844626

RESUMO

Pediatric chronic kidney disease (CKD) is currently assessed using glomerular filtration rate (GFR), which is calculated from different equations based on serum creatinine concentration. However, serum creatinine is affected by several factors and is not reliable enough for the diagnosis of CKD, especially at early stages. Recent targeted and untargeted metabolomics studies found 7 new potential biomarkers that could be useful for early pediatric chronic kidney disease diagnosis. Thus, a new LC-QQQ-MS analytical method has been developed and validated aimed at routine analysis of these 7 potential biomarkers: NG,NG'-dimethyl-l-arginine di(p-hydroxyazobenzene-p'-sulfonate) (SDMA), S-adenosyl-l-methionine (SAM), n-butyryl-l-carnitine (nC4), iso-butyryl-l-carnitine (iC4), citrulline (CIT), creatinine (CNN) and d-erytro-sphingosine-1-phosphate (S1P), in addition to creatinine, the classical biomarker for CKD diagnosis. Then, this analytical method has been used for the quantification of plasma samples from a heterogeneous group of CKD pediatric patients and a control pediatric population. Data analysis of these results showed that it is possible to differentiate between CKD and control populations based on the metabolite concentration in plasma.


Assuntos
Biomarcadores/sangue , Biomarcadores/química , Plasma/química , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/diagnóstico , Arginina/análogos & derivados , Arginina/sangue , Arginina/química , Criança , Pré-Escolar , Cromatografia Líquida/métodos , Citrulina/química , Creatinina/sangue , Diagnóstico Precoce , Feminino , Taxa de Filtração Glomerular/fisiologia , Humanos , Masculino , Metabolômica/métodos , S-Adenosilmetionina/sangue , S-Adenosilmetionina/química , Espectrometria de Massas em Tandem/métodos
20.
Chem Commun (Camb) ; 55(38): 5431-5434, 2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-30916680

RESUMO

Downstream processing to obtain enantiopure compounds from a racemic mixture relies mainly on crystallization. Natural transporters can specifically translocate enantiomers through membranes. Here a ß-barrel transmembrane protein FhuA is re-engineered into a chiral channel protein (FhuAF4) to resolve racemic mixtures of d-/l-arginine. The engineered FhuAF4 variant exhibits an enantioselectivity (E-value) of 1.92 and an enantiomeric excess percentage (ee%) of 23.91 at 52.39% conversion. OmniChange mutant libraries at the computationally identified "filter-regions" likely help to identify FhuA variants for enantiomeric separation of other compounds.


Assuntos
Arginina/química , Arginina/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Nanoestruturas , Engenharia de Proteínas , Estereoisomerismo
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