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1.
J Zoo Wildl Med ; 52(1): 253-258, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33827183

RESUMO

While electrophoresis is considered the standard method for evaluation of protein concentrations as a result of its direct measurement, albumin is often quantified with biochemical assays. Many laboratory-based chemistry analyzers and clinic-based point-of-care analyzers use the dye bromocresol green (BCG) for the quantitation of albumin. Several studies have shown that albumin concentrations obtained by the standard (BCG) dye-binding method are significantly different from those obtained by protein electrophoresis in avian species and chelonia. The goal of this study was to compare plasma albumin concentrations obtained by the BCG method with those derived from electrophoresis in bearded dragons (Pogona vitticeps). Thirty-six heparinized plasma samples were obtained from 13 clinically healthy male bearded dragons. Albumin was quantified by protein electrophoresis and by the BCG dye-binding method. The two methods were significantly different (P < 0.0001, paired t-test; P < 0.0001, Wilcoxon signed-rank test), with the BCG measurement always equal to or higher than the electrophoretic result. The measurements from both methods were significantly correlated (r = 0.8634, P < 0.0001), but concordance between the two techniques was poor. The Bland-Altman plot appeared to show a greater difference between the two measurements with lower albumin values and lesser difference with higher values. These results indicate that bearded dragon plasma albumin concentration measurements obtained by the BCG dye-binding method are unreliable when compared to those obtained with electrophoresis, suggesting that albumin should be measured by protein electrophoresis for health assessment in bearded dragons.


Assuntos
Eletroforese das Proteínas Sanguíneas/veterinária , Lagartos/sangue , Albumina Sérica/análise , Animais , Masculino , Albumina Sérica/química
2.
Nat Commun ; 12(1): 1543, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33750839

RESUMO

Protein engineering has great potential for devising multifunctional recombinant proteins to serve as next-generation protein therapeutics, but it often requires drastic modifications of the parental protein scaffolds e.g., additional domains at the N/C-terminus or replacement of a domain by another. A discovery platform system, called RaPID (Random non-standard Peptides Integrated Discovery) system, has enabled rapid discovery of small de novo macrocyclic peptides that bind a target protein with high binding specificity and affinity. Capitalizing on the optimized binding properties of the RaPID-derived peptides, here we show that RaPID-derived pharmacophore sequences can be readily implanted into surface-exposed loops on recombinant proteins and maintain both the parental peptide binding function(s) and the host protein function. We refer to this protein engineering method as lasso-grafting and demonstrate that it can endow specific binding capacity toward various receptors into a diverse set of scaffolds that includes IgG, serum albumin, and even capsid proteins of adeno-associated virus, enabling us to rapidly formulate and produce bi-, tri-, and even tetra-specific binder molecules.


Assuntos
Peptídeos/química , Peptídeos/farmacologia , Engenharia de Proteínas/métodos , Proteínas do Capsídeo/química , Proteínas de Transporte/química , Linhagem Celular , Dependovirus , Humanos , Imunoglobulina G/química , Modelos Moleculares , Albumina Sérica/química , Bibliotecas de Moléculas Pequenas
3.
Molecules ; 26(5)2021 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-33670879

RESUMO

Four flavanone Schiff bases (E)-1-(2-phenylchroman-4-ylidene)thiosemicarbazide (FTSC) (1), N',2-bis((E)-2-phenylchroman-4-ylidene)hydrazine-1-carbothiohydrazide (FTCH) (2), (E)-N'-(2-phenylchroman-4-ylidene)benzohydrazide (FHSB) (3) and (E)-N'-(2-phenylchroman-4-ylidene)isonicotinohydrazide (FIN) (4) were synthesized and evaluated for their electronic and physicochemical properties using experimental and theoretical methods. One of them, (2), consists of two flavanone moieties and one substituent, the rest of the compounds (1, 3, 4) comprises of a flavanone-substituent system in relation to 1:1. To uncover the structural and electronic properties of flavanone Schiff bases, computational simulations and absorption spectroscopy were applied. Additionally, binding efficiencies of the studied compounds to serum albumins were evaluated using fluorescence spectroscopy. Spectral profiles of flavanone Schiff bases showed differences related to the presence of substituent groups in system B of the Schiff base molecules. Based on the theoretically predicted chemical descriptors, FTSC is the most chemically reactive among the studied compounds. Binding regions within human and bovine serum albumins of the ligands studied are in the vicinity of the Trp residue and a static mechanism dominates in fluorescence quenching.


Assuntos
Flavanonas/química , Bases de Schiff/química , Albumina Sérica/química , Sequência de Aminoácidos , Animais , Bovinos , Teoria da Densidade Funcional , Humanos , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Espectrometria de Fluorescência , Espectrofotometria , Relação Estrutura-Atividade
4.
Artigo em Inglês | MEDLINE | ID: mdl-33388526

RESUMO

The present study developed an analytical technique to investigate the possible covalent adduct formation of albumin with the herbicide atrazine, and to characterize the protein modifications in vitro using liquid chromatography separation coupled with high resolution time-of-flight mass spectrometry (LC-TOF-MS). Tandem mass spectrum analysis (MS/MS) with collision induced dissociation (CID) revealed the specific sites of rat, human and bovine serum albumin adduct with atrazine. The formation of b-ion, y-ion series in MS/MS showed a covalent adduct with an addition mass of 179.1 Da located on Cys-34 of serum albumin from rats, human and bovine. This clearly indicated that the chemical group C8H13N5 forms an adduct with Cys-34 despite the sequences differences between of rat, human and bovine serum albumin. To confirm the method reliability, concentration-dependent and time-dependent formation of adducts between serum albumins and atrazine were also investigated. Our results confirmed that atrazine can directly react with Cys-34 of serum albumin and form covalent adducts without prior metabolism.


Assuntos
Atrazina , Cromatografia Líquida de Alta Pressão/métodos , Albumina Sérica , Espectrometria de Massas em Tandem/métodos , Animais , Atrazina/análise , Atrazina/química , Atrazina/metabolismo , Bovinos , Cisteína/química , Herbicidas/análise , Herbicidas/química , Herbicidas/metabolismo , Humanos , Ratos , Albumina Sérica/análise , Albumina Sérica/química , Albumina Sérica/metabolismo
5.
Food Funct ; 12(3): 1271-1290, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33434253

RESUMO

The aim of the study was to broadly determine the biological activities of purple potato ethanolic extract of the Blue Congo variety (BCE). The antioxidant activity of BCE was determined in relation to liposome membranes, and peroxidation was induced by UVB and AAPH. To clarify the antioxidant activity of BCE, we investigated its interactions with hydrophilic and hydrophobic regions of a membrane using fluorimetric and FTIR methods. Next, we investigated the cytotoxicity and pro-apoptotic activities of BCE in two human colon cancer cell lines (HT-29 and Caco-2) and in normal cells (IPEC-J2). In addition, the ability to inhibit enzymes that are involved in pro-inflammatory reactions was examined. Furthermore, BCE interactions with serum albumin and plasmid DNA were investigated using steady state fluorescence spectroscopy and a single molecule fluorescence technique (TCSPC-FCS). We proved that BCE effectively protects lipid membranes against the process of peroxidation and successfully inhibits the cyclooxygenase and lipoxygenase enzymes. Furthermore, it interacts with the hydrophilic and hydrophobic parts of lipid membranes as well as with albumin and plasmid DNA. It was observed that BCE is more cytotoxic against colon cancer cell lines than normal IPEC-J2 cells; it also induces apoptosis in cancer cell lines, but does not induce cell death in normal cells.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Antioxidantes/farmacologia , Extratos Vegetais/farmacologia , Solanum tuberosum/química , Albuminas , Antineoplásicos Fitogênicos/química , Antioxidantes/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/química , Inibidores de Ciclo-Oxigenase/farmacologia , Humanos , Lipídeos/química , Lipossomos , Inibidores de Lipoxigenase/química , Inibidores de Lipoxigenase/farmacologia , Extratos Vegetais/química , Plasmídeos , Ligação Proteica , Espécies Reativas de Oxigênio , Albumina Sérica/química , Albumina Sérica/metabolismo
6.
Food Chem ; 342: 128378, 2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33508903

RESUMO

Rheum ribes L. (Rhubarb) is one of the most important edible medicinal plants in the Eastern Anatolia region and is called "Iskin" by local people. Resveratrol and 6-O-methylalaternin were isolated from the Rhubarb for the first time in addition to well-known secondary metabolites including emodin, aloe-emodin, ß-sitosterol and rutin. The new semi-synthetic anthraquinone derivatives with the NαFmoc-l-Lys and ethynyl group were synthesized from the isolated anthraquinones emodin and aloe-emodin of Rhubarb to increase the bioactivities. Aloe-emodin derivative with NαFmoc-l-Lys shows the highest inhibition values by 94.11 ± 0.12 and 82.38 ± 0.00% against HT-29 and HeLa cell lines, respectively, at 25 µg/mL. Further, modification of the aloe-emodin with both the ethynyl and the NαFmoc-l-Lys groups showed an antioxidant activity-enhancing effect. From molecular docking studies, the relative binding energies of the emodin and aloe-emodin derivatives to human serum albumin ranged from -7.30 and -10.62 kcal/mol.


Assuntos
Antraquinonas/química , Antineoplásicos/síntese química , Resveratrol/química , Rheum/química , Antraquinonas/síntese química , Antraquinonas/isolamento & purificação , Antraquinonas/metabolismo , Antraquinonas/farmacologia , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Emodina/química , Emodina/isolamento & purificação , Emodina/metabolismo , Emodina/farmacologia , Humanos , Simulação de Acoplamento Molecular , Componentes Aéreos da Planta/química , Componentes Aéreos da Planta/metabolismo , Resveratrol/isolamento & purificação , Resveratrol/farmacologia , Rheum/metabolismo , Albumina Sérica/química , Albumina Sérica/metabolismo
7.
Anal Chem ; 93(4): 1944-1950, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33399445

RESUMO

Carboxyl-group specific chemical cross-linking is gaining an increased interest as a structural mass spectrometry/structural proteomics technique that is complementary to the more commonly used amine-specific chemistry using succinimide esters. One of these protocols uses a combination of dihydrazide linkers and the coupling reagent DMTMM [4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium] chloride, which allows performing the reaction at neutral pH. The reaction yields two types of products, carboxyl-carboxyl cross-links that incorporate the dihydrazide linker and zero-length carboxyl-amine cross-links induced by DMTMM alone. Until now, it has not been systematically investigated how the balance between the two products is affected by experimental conditions. Here, we studied the role of the ratios of the two reagents (using pimelic dihydrazide and DMTMM) and demonstrate that the concentration of the two reagents can be systematically adjusted to favor one reaction product over the other. Using a set of five model proteins, we observed that the number of identified cross-linked peptides could be more than doubled by a combination of three different reaction conditions. We also applied this strategy to the bovine 20S proteasome and the Escherichia coli 70S ribosome, again demonstrating complementarity and increased cross-link coverage.


Assuntos
Reagentes para Ligações Cruzadas/química , Proteínas/química , Proteômica , Animais , Catalase/química , Catalase/metabolismo , Conalbumina/química , Conalbumina/metabolismo , Creatina Quinase/química , Creatina Quinase/metabolismo , Espectrometria de Massas/métodos , Proteínas/metabolismo , Albumina Sérica/química , Albumina Sérica/metabolismo , Transferrina/química , Transferrina/metabolismo
8.
Int J Biol Macromol ; 169: 143-152, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33338529

RESUMO

Advanced glycation endproducts (AGEs) are the final product of glycation, highly reactive in nature and contribute directly or indirectly to numerous complications related to diabetes. In this study, the antiglycation activity of glyburide was investigated using HSA as model protein, both against glucose and methylglyoxal mediated glycation. The possible mechanism of action was also deciphered using biophysical and computational tools. Approximately 70% inhibition of both early and advanced glycation end products were recorded in the presence of glyburide. Free lysine modification was reduced by glyburide treatment and improvement in biochemical markers such as free thiol groups and carbonyl content was observed. Interaction studies revealed that glyburide showed moderate to strong binding affinity towards HSA with binding constant in the order of 106 M-1. The interaction of glyburide with HSA was entropically favourable and spontaneous in nature. Molecular dynamics simulation deciphered that glyburide-HSA complex was quite stable where RMSD, RMSF, Rg, SASA, and secondary structure of HSA remained approximately same over the entire simulation period. The average binding energy of the MD simulation for glyburide-HSA complex was found to be -15.386 kJ mol-1. The findings demonstrate the antiglycation potential of glyburide and its possible mechanism of action.


Assuntos
Glibureto/química , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/metabolismo , Complicações do Diabetes/metabolismo , Diabetes Mellitus/metabolismo , Glucose/química , Glibureto/farmacologia , Glicosilação , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Albumina Sérica/química , Albumina Sérica/metabolismo
9.
Int J Nanomedicine ; 15: 10331-10347, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33376324

RESUMO

Background: Lung cancer is the leading cause of cancer patient death in the world. There are many treatment options for lung cancer, including surgery, radiation therapy, chemotherapy, targeted therapy, and combined therapy. Despite significant progress has been made in the diagnosis and treatment of lung cancer during the past few decades, the prognosis is still unsatisfactory. Purpose: To resolve the problem of chemotherapy failure, we developed a magnetite-based nanomedicine for chemotherapy acting synergistically with loco-regional hyperthermia. Methods: The targeting carrier consisted of a complex of superparamagnetic iron oxide (SPIO) and poly(sodium styrene sulfonate) (PSS) at the core and a layer-by-layer shell with cisplatin (CDDP), together with methotrexate - human serum albumin conjugate (MTX-HSA conjugate) for lung cancer-specific targeting, referred to hereafter as SPIO@PSS/CDDP/HSA-MTX nanoparticles (NPs). Results: SPIO@PSS/CDDP/HSA-MTX NPs had good biocompatibility and stability in physiological solutions. Furthermore, SPIO@PSS/CDDP/HSA-MTX NPs exhibited a higher temperature increase rate than SPIO nanoparticles under irradiation by a radiofrequency (RF) generator. Therefore, SPIO@PSS/CDDP/HSA-MTX NPs could be used as a hyperthermia inducer under RF exposure after nanoparticles preferentially targeted and then accumulated at tumor sites. In addition, SPIO@PSS/CDDP/HSA-MTX NPs were developed to be used during combined chemotherapy and hyperthermia therapy, exhibiting a synergistic anticancer effect better than the effect of monotherapy. Conclusion: Both in vitro and in vivo results suggest that the designed SPIO@PSS/CDDP/HSA-MTX NPs are a powerful candidate nanoplatform for future antitumor treatment strategies.


Assuntos
Óxido Ferroso-Férrico/química , Hipertermia Induzida , Neoplasias Pulmonares/terapia , Nanomedicina/métodos , Animais , Linhagem Celular Tumoral , Cisplatino/química , Cisplatino/uso terapêutico , Terapia Combinada , Portadores de Fármacos/química , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Metotrexato/química , Nanopartículas/química , Albumina Sérica/química
10.
PLoS One ; 15(10): e0239282, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33095778

RESUMO

OBJECTIVES: To determine if the URO-MCP-1 mouse model for bladder IC/BPS is associated with in vivo bladder hyper-permeability, as measured by contrast-enhanced MRI (CE-MRI), and assess whether molecular-targeted MRI (mt-MRI) can visualize in vivo claudin-2 expression as a result of bladder hyper-permeability. Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic, painful condition of the bladder that affects primarily women. It is known that permeability plays a substantial role in IC/BPS. Claudins are tight junction membrane proteins that are expressed in epithelia and endothelia and form paracellular barriers and pores that determine tight junction permeability. Claudin-2 is a molecular marker that is associated with increased hyperpermeability in the urothelium. MATERIALS AND METHODS: CE-MRI was used to measure bladder hyper-permeability in the URO-MCP-1 mice. A claudin-2-specific mt-MRI probe was used to assess in vivo levels of claudin-2. The mt-MRI probe consists of an antibody against claudin-2 conjugated to albumin that had Gd-DTPA (gadolinium diethylenetriamine pentaacetate) and biotin attached. Verification of the presence of the mt-MRI probe was done by targeting the biotin moiety for the probe with streptavidin-horse radish peroxidase (SA-HRP). Trans-epithelial electrical resistance (TEER) was also used to assess bladder permeability. RESULTS: The URO-MCP-1 mouse model for IC/BPS was found to have a significant increase in bladder permeability, following liposaccharide (LPS) exposure, compared to saline-treated controls. mt-MRI- and histologically-detectable levels of the claudin-2 probe were found to increase with LPS -induced bladder urothelial hyper-permeability in the URO-MCP-1 IC mouse model. Levels of protein expression for claudin-2 were confirmed with immunohistochemistry and immunofluorescence imaging. Claudin-2 was also found to highly co-localize with zonula occlidens-1 (ZO-1), a tight junction protein. CONCLUSION: The combination of CE-MRI and TEER approaches were able to demonstrate hyper-permeability, a known feature associated with some IC/BPS patients, in the LPS-exposed URO-MCP-1 mouse model. This MRI approach could be clinically translated to establish which IC/BPS patients have bladder hyper-permeability and help determine therapeutic options. In addition, the in vivo molecular-targeted imaging approach can provide invaluable information to enhance our understanding associated with bladder urothelium hyper-permeability in IC/BPS patients, and perhaps be used to assist in developing further therapeutic strategies.


Assuntos
Claudina-2/metabolismo , Cistite Intersticial/patologia , Imageamento por Ressonância Magnética/métodos , Sondas Moleculares/química , Bexiga Urinária/fisiopatologia , Animais , Anticorpos/química , Anticorpos/imunologia , Claudina-2/imunologia , Cistite Intersticial/metabolismo , Modelos Animais de Doenças , Gadolínio DTPA/química , Imuno-Histoquímica , Lipopolissacarídeos/toxicidade , Camundongos , Permeabilidade/efeitos dos fármacos , Albumina Sérica/química
11.
J Med Chem ; 63(11): 6057-6065, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32372648

RESUMO

Toxicity concerns related to Gd(III)-based magnetic resonance imaging (MRI) agents prompted an intensive research toward their replacement by complexes of essential metal ions, like Mn(II). Here, we report a macrocyclic chelate, [Mn(PC2A-BP)], which possesses high thermodynamic stability (log KMnL = 14.86 and pMn=8.35) and kinetic inertness (t1/2pH=7.4 = 286.2 h) as well as as remarkable relaxivity (r1p = 23.5 mM-1 s-1, 0.49 T, 37 °C) in the presence of human serum albumin, allowing a significant MRI signal intensity increase in the vasculature even at low dose (25 µmol/kg) of the complex.


Assuntos
Meios de Contraste/química , Imageamento por Ressonância Magnética/métodos , Manganês/química , Complexos de Coordenação/química , Estabilidade de Medicamentos , Humanos , Cinética , Ligantes , Albumina Sérica/química , Termodinâmica
12.
J Med Chem ; 63(13): 6847-6862, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32469516

RESUMO

Every day, hundreds of millions of people worldwide take nonsteroidal anti-inflammatory drugs (NSAIDs), often in conjunction with multiple other medications. In the bloodstream, NSAIDs are mostly bound to serum albumin (SA). We report the crystal structures of equine serum albumin complexed with four NSAIDs (ibuprofen, ketoprofen, etodolac, and nabumetone) and the active metabolite of nabumetone (6-methoxy-2-naphthylacetic acid, 6-MNA). These compounds bind to seven drug-binding sites on SA. These sites are generally well-conserved between equine and human SAs, but ibuprofen binds to both SAs in two drug-binding sites, only one of which is common. We also compare the binding of ketoprofen by equine SA to binding of it by bovine and leporine SAs. Our comparative analysis of known SA complexes with FDA-approved drugs clearly shows that multiple medications compete for the same binding sites, indicating possibilities for undesirable physiological effects caused by drug-drug displacement or competition with common metabolites. We discuss the consequences of NSAID binding to SA in a broader scientific and medical context, particularly regarding achieving desired therapeutic effects based on an individual's drug regimen.


Assuntos
Anti-Inflamatórios não Esteroides/metabolismo , Albumina Sérica/metabolismo , Animais , Anti-Inflamatórios não Esteroides/sangue , Sítios de Ligação , Transporte Biológico , Modelos Moleculares , Conformação Proteica , Albumina Sérica/química
13.
Chem Commun (Camb) ; 56(35): 4797-4800, 2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32227051

RESUMO

The measurement of exchangeable Cu2+ levels in biological samples is gaining interest in the context of copper-related pathologies. Here, we report a Tb3+ luminescent turn-off sensor for Cu2+ based on the specific and suitable-affinity Xxx-Zzz-His (ATCUN) peptide motif, enabling Cu2+ detection in the presence of a biological fluorescent background.


Assuntos
Cobre/análise , Peptídeos/química , Térbio/química , Animais , Cobre/química , Luminescência , Albumina Sérica/química , Suínos
14.
Exp Oncol ; 42(1): 40-45, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32231185

RESUMO

AIM: To assess oxidative stress and structural changes of the serum albumin in rats with transplanted Walker-256 carcinosarcoma (W256) strains with varying sensitivity to doxorubicin (Dox). MATERIALS AND METHODS: The study was performed on female Wistar rats with transplanted W256. On the 9th day after tumor cell transplantation an analysis of peripheral blood, oxidative stress parameters, and structural changes of serum albumin of experimental animals was performed. RESULTS: On the 9th day after W256 transplantation a significant increase in the leukocyte counts was observed in the groups of animals with the Dox-resistant and parental (Dox-sensitive) W256 tumors compared with the group of the intact animals: up to 14.24 ± 1.92 â€¢ 103/µl and 9.78 ± 1.03 â€¢ 103/µl, vs 8.92 ± 1.04 â€¢ 103/µl, respectively, due to the increase of granulocyte and monocyte counts. The number of lymphocytes was within the normal range. The level of hemoglobin and the erythrocyte counts were also within normal limits, but hematocrit in both groups of animals with tumors somewhat increased against the background of 1.2-fold elevation of the mean erythrocyte volume. In the group of rats with Dox-resistant W256, there was observed a decrease in the plateletcrit by almost 22% and thrombocyte counts - by 28%. Analysis of oxidative stress indices revealed a significant increase in the level of reactive oxygen species, 2-fold increase of malonic dialdehyde level and the degree of oxidative damage of blood plasma proteins, as well as a decrease in the activity of catalase in hemolysates (by 12-15%) in both groups of tumor-bearing rats. With the use of differential scanning calorimetry, UV and fluorescence spectroscopy we have revealed anomalous conformational changes of albumin caused by tumor development: structural rearrangements in the region of its first drug binding site located in the IIA domain, separation of globular parts of albumin molecule, and partial "opening" in a protein molecular three-domain structure resulting a loss of its thermal resistance. CONCLUSION: The development of transplanted Walker-256 carcinosarcoma, especially its Dox-resistant variant, results in severe metabolic intoxication reflected in alteration of hematological parameters, and indices of oxidative stress, as well as architectonic changes of serum albumin.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Carcinoma 256 de Walker/sangue , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Albumina Sérica/química , Animais , Carcinoma 256 de Walker/metabolismo , Carcinoma 256 de Walker/patologia , Feminino , Transplante de Neoplasias , Estresse Oxidativo/efeitos dos fármacos , Conformação Proteica , Ratos Wistar
15.
Sci Rep ; 10(1): 4166, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32139799

RESUMO

A nitrobenzoxadiazole-based fluoroprobe (NBD-Bu) is designed to probe cellular metabolic activity in cancer and normal cells. NBD-Bu shows a significant fluorescence enhancement upon selective binding to the transport protein serum albumin in PBS buffer at ambient conditions. Encouraged by this finding, the site- specificity of NBD-Bu has been explored through a competitive displacement assay in the presence of site-specific markers such as warfarin and ibuprofen. Notably, even at micromolar concentrations, the probe possesses the ability to displace the site marker drug ibuprofen, efficiently. Subsequently, high-resolution fluorescence imaging results consolidated the potential of NBD-Bu for detection of abnormal cellular metabolic activity.


Assuntos
4-Cloro-7-nitrobenzofurazano/química , Albumina Sérica/química , Animais , Células CHO , Linhagem Celular , Cricetulus , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Microscopia Confocal , Soroalbumina Bovina/química , Albumina Sérica Humana/química
16.
Subcell Biochem ; 94: 383-397, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32189308

RESUMO

Albumin is widely conserved from vertebrates to invertebrates, and nature of mammalian albumins permit them to bind various endogenous ligands and drugs in the blood. It is known that at least two major ligand binding sites are present on the albumin molecule, which are referred to as Site I and Site II. These binding sites are thought to be almost completely conserved among mammals, even though the degree of binding to these sites are different depending on the physical and chemical properties of drugs and differences in the microenvironment in the binding pockets. In addition, the binding sites for medium and long-chain fatty acids are also well conserved among mammals, and it is considered that there are at least seven binding sites, including Site I and Site II. These bindings properties of albumin in the blood are also widely known to be important for transporting drugs and fatty acids to various tissues. It can therefore be concluded that albumin is one of the most important serum proteins for various ligands, and information on human albumin can be very useful in predicting the ligand binding properties of the albumin of other vertebrates.


Assuntos
Ácidos Graxos/metabolismo , Preparações Farmacêuticas/metabolismo , Albumina Sérica/metabolismo , Animais , Sítios de Ligação , Ácidos Graxos/química , Humanos , Preparações Farmacêuticas/química , Ligação Proteica , Albumina Sérica/química
17.
Mutat Res ; 849: 503127, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32087848

RESUMO

The environmental and food contaminant, benzo[a]pyrene {B[a]P, a polycyclic aromatic hydrocarbon (PAH)}, is classified as a human carcinogen by the International Agency for Research on Cancer. The carcinogenicity of B[a]P is linked to the formation of electrophilic metabolites, namely B[a]P-diol epoxides (BPDEs) occurring as stereoisomers. In this work, we quantified the metabolic formation of BPDE isomers and the genotoxic effect in B[a]P-exposed mice, with an aim to estimate the genotoxic potency of B[a]P per in vivo dose of its most potent metabolite [i.e. (+)-anti-BPDE]. The increase in frequency of micronuclei (fMN) in erythrocytes was measured as a biomarker for genotoxic effect. Covalent adducts to serum albumin (SA) and those to DNA from the BPDEs were analysed using liquid chromatography tandem mass spectrometry (LC-MS/MS), as adducts to histidine (BPDE-His-Pro) and deoxyguanosine (BPDE-dG), respectively. For the first time in animal experiments it was possible to resolve adducts to SA from (+)-anti-, (-)-anti- and (±)-syn-BPDE isomers by LC-MS/MS. The adduct levels in the protein were about 16 fmol/mg SA, which was orders of magnitude lower than that in the nucleic acid, 28 pmol/mg DNA, in mice exposed to 100 mg B[a]P per kg body weight (bw). Using SA adduct levels, the in vivo dose of (+)-anti-BPDE was calculated to be approximately 50 nM·h per mg B[a]P per kg bw. This allowed to make a preliminary estimate of the genotoxic potency as 2‰ fMN per µM·h of (+)-anti-BPDE. This estimate was compared to that from another food toxicant, glycidol, studied with similar methods, which indicated that the BPDE has several orders of magnitude higher genotoxic potency. The demonstrated approach on integrating biomarkers of internal dose of a causative agent and that of genotoxic effect for assessing genotoxic potency, using B[a]P as a model, has a potential for improving cancer risk assessment procedures for PAHs.


Assuntos
Benzo(a)pireno/toxicidade , Carcinógenos/toxicidade , Adutos de DNA/química , Micronúcleos com Defeito Cromossômico/estatística & dados numéricos , Albumina Sérica/química , Animais , Biotransformação , Compostos de Epóxi/química , Compostos de Epóxi/toxicidade , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes para Micronúcleos , Propanóis/toxicidade
18.
J Oleo Sci ; 69(1): 65-72, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31902896

RESUMO

The secondary structures of human serum albumin (HSA) and bovine serum albumin (BSA) were disrupted in the solution of sodium dodecyl sulfate (SDS), while being hardly damaged in the solution of the bile salt, sodium cholate (NaCho). In the present work, the removal of dodecyl sulfate (DS) ions bound to these proteins was attempted by adding various amounts of NaCho. The extent of removal was estimated by the restoration of α-helical structure of each protein disrupted by SDS. Increases and decreases in α-helical structure were examined using the mean residue ellipticity at 222 nm, [θ]222, which was frequently used as a measure of α-helical structure content. The magnitudes of [θ]222 of HSA and BSA, weakened by SDS, were restrengthened upon the addition of NaCho. This indicated that the α-helical structures of HSA and BSA that were disrupted by the binding of DS ions were nearly reformed by the addition of NaCho. The NaCho concentration at which the maximum restoration of [θ]222 of each protein was attained increased nearly linearly with SDS concentration. These results indicated that most of the bound DS ions were removed from the proteins but the removal was incomplete. The removal of DS ions, examined by means of the equilibrium dialysis, was also incomplete. The α-helical structure restoration and the DS ion removal by NaCho were considered to be due to the ability of cholate anions to strip the surfactant ions bound to HSA and BSA. These stripped DS ions appeared to be more likely to form SDS-NaCho mixed micelles in bulk rather than SDS-NaCho mixed aggregates on the proteins.


Assuntos
Soroalbumina Bovina/química , Albumina Sérica/química , Colato de Sódio/química , Dodecilsulfato de Sódio/isolamento & purificação , Tensoativos/isolamento & purificação , Animais , Bovinos , Humanos , Ligação Proteica , Psicoterapia Breve , Dodecilsulfato de Sódio/química , Tensoativos/química
19.
J Phys Chem Lett ; 11(3): 1170-1177, 2020 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-31967479

RESUMO

Understanding nanoscale protein conformational changes at solid-liquid interfaces is critical for predicting how proteins will impact the performance of biomaterials in vivo. Crowding is an important contributor to conformational stability. Here we apply single-molecule high resolution imaging with photobleaching to directly measure dye-conjugated fibronectin's unfolding in varying conditions of crowding with human serum albumin on aminosilanized glass. Using this approach, we identify serum albumin's crowding mechanism. We find that fibronectin achieves larger degrees of unfolding when not crowded by coadsorbed serum albumin. Serum albumin does not as effectively constrict fibronectin's conformation if it is sequentially, rather than simultaneously, introduced, suggesting that serum albumin's crowding mechanism is dependent on its ability to sterically block fibronectin's unfolding during the process of adsorption. Because fibronectin's conformation is dependent on interfacial macromolecular crowding under in vitro conditions, it is important to consider the role of in vivo crowding on protein activity.


Assuntos
Fibronectinas/química , Albumina Sérica/química , Fibronectinas/metabolismo , Vidro/química , Humanos , Nanotecnologia/métodos , Estabilidade Proteica , Desdobramento de Proteína , Albumina Sérica/metabolismo , Propriedades de Superfície
20.
Int J Biol Macromol ; 148: 533-542, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31954794

RESUMO

The present study was aimed at investigating the binding between an important drug of Alzheimer's therapy, Rivastigmine tartrate (RT), with Bovine serum albumin (BSA). BSA is a model protein that is increasingly being used for studies related to drug-protein interaction owing to its structural similarity with human serum albumin (HSA) which is extremely abundant in the circulatory system comprising around 60% of the total plasma protein. Fluorescence spectroscopy implied that complex formation is taking place between BSA and RT; binding constant calculated was of the order of 104 M-1 implicative of the strength of this interaction. Fluorescence spectroscopy was carried out at three different temperatures in a bid to find out the operative mode of quenching; static quenching was taking place for RT-BSA interaction with a binding constant of 2.5 × 104 M-1 at 298 K. Further, changes in Far UV CD spectra clearly implied that RT induces structural transition in BSA suggestive of RT-BSA complex formation. The negative value of ∆G0 as obtained from fluorescence spectroscopy and isothermal titration calorimetry (ITC) suggests the reaction to be spontaneous and thermodynamically favorable. Additionally, molecular docking was employed to investigate different forces and critical residues involved in RT-BSA interaction. Furthermore, all-atom molecular dynamics simulation for 50 ns was performed on the BSA-RT complex to investigate its conformational behavior, stability and dynamics.


Assuntos
Rivastigmina/química , Rivastigmina/farmacologia , Albumina Sérica , Algoritmos , Doença de Alzheimer/tratamento farmacológico , Sítios de Ligação , Humanos , Ligação de Hidrogênio , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Albumina Sérica/química , Albumina Sérica/metabolismo , Análise Espectral , Relação Estrutura-Atividade , Termodinâmica
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