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1.
Food Chem ; 337: 127759, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32777568

RESUMO

High-resolution ultrasonic spectroscopy (HR-US) was applied for real-time monitoring of ß-casein hydrolysis by trypsin at various conditions for the first time. The technique is based on the precision measurement of hydration changes proportional to the number of peptide bond hydrolyzed. As HR-US exhibits ultrasonic transparency for most solution, the analysis did not require optical transparency like for 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay. Appropriate enzymatic models were fitted with degree of hydrolysis (dh) profiles to provide kinetic and mechanistic description of proteolysis in terms of initial hydrolysis rate, r0, and rate constant of hydrolysis, kh, and enzyme inactivation, kd. Maximal r0 and dh were obtained at 45 °C and pH 8. The exponential dependence of kinetic parameters allowed determination of the activation (EA = 50.3 ± 7 kJ/mol) and deactivation (ED = 62.23 ± 3 kJ/mol) energies of hydrolysis. The ultrasonic assay provided rapid detection of trypsin activity even at sub-nanomolar concentration.

2.
Sensors (Basel) ; 20(19)2020 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-33003538

RESUMO

This review is focused on the application of surface and volume-sensitive acoustic methods for the detection of milk proteases such as trypsin and plasmin. While trypsin is an important protein of human milk, plasmin is a protease that plays an important role in the quality of bovine, sheep and goat milks. The increased activity of plasmin can cause an extensive cleavage of ß-casein and, thus, affect the milk gelation and taste. The basic principles of surface-sensitive acoustic methods, as well as high-resolution ultrasonic spectroscopy (HR-US), are presented. The current state-of-the-art examples of the application of acoustic sensors for protease detection in real time are discussed. The application of the HR-US method for studying the kinetics of the enzyme reaction is demonstrated. The sensitivity of the acoustics biosensors and HR-US methods for protease detection are compared.

3.
Sensors (Basel) ; 20(17)2020 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-32839389

RESUMO

The determination of antibiotics in food is important due to their negative effect on human health related to antimicrobial resistance problem, renal toxicity, and allergic effects. We propose an impedimetric aptasensor for the determination of kanamycin A (KANA), which was assembled on the glassy carbon electrode by the deposition of carbon black in a chitosan matrix followed by carbodiimide binding of aminated aptamer mixed with oligolactide derivative of thiacalix[4]arene in a cone configuration. The assembling was monitored by cyclic voltammetry, electrochemical impedance spectroscopy, and scanning electron microscopy. In the presence of the KANA, the charge transfer resistance of the inner interface surprisingly decreased with the analyte concentration within 0.7 and 50 nM (limit of detection 0.3 nM). This was attributed to the partial shielding of the negative charge of the aptamer and of its support, a highly porous 3D structure of the surface layer caused by a macrocyclic core of the carrier. The use of electrostatic assembling in the presence of cationic polyelectrolyte decreased tenfold the detectable concentration of KANA. The aptasensor was successfully tested in the determination of KANA in spiked milk and yogurt with recoveries within 95% and 115%.

4.
Biosensors (Basel) ; 10(3)2020 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-32138172

RESUMO

This paper describes the anti-fouling capability of the novel monolayer-forming surface linker 3-(3-(trichlorosilylpropyloxy) propanoyl chloride (MEG-Cl). This compound was successfully attached to quartz crystal surfaces which are employed in an electromagnetic piezoelectric acoustic sensor (EMPAS) configuration. The MEG-Cl coated surface was both employed with Ni-NTA for the binding of recombinant proteins and for the tandem property of the avoidance of fouling from serum and milk. The MEG-Cl coated surfaces were found to provide a large degree of anti-fouling on the EMPAS device, and were comparable to previously studied MEG-OH surfaces. Importantly, the monolayer continued to provide anti-fouling capability to the biosensor following extension with Ni-NTA in place. Accordingly, this surface linker provides an attractive system for use in biosensor technology in terms of both its anti-fouling and linking properties.


Assuntos
Técnicas Biossensoriais/métodos , Sondas Moleculares/uso terapêutico , Humanos
5.
Sensors (Basel) ; 19(22)2019 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-31739501

RESUMO

DNA sensors were assembled by consecutive deposition of thiacalix[4]arenes bearing oligolactic fragments, poly(ethylene imine), and DNA onto the glassy carbon electrode. The assembling of the layers was monitored with scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. The configuration of the thiacalix[4]arene core determined self-assembling of the polymeric species to the nano/micro particles with a size of 70-350 nm. Depending on the granulation, the coatings show the accumulation of a variety of DNA quantities, charges, and internal pore volumes. These parameters were used to optimize the DNA sensors based on these coatings. Thus, doxorubicin was determined to have limits of detection of 0.01 nM (cone configuration), 0.05 nM (partial cone configuration), and 0.10 nM (1,3-alternate configuration of the macrocycle core). Substitution of native DNA with aptamer specific to aflatoxin M1 resulted in the detection of the toxin in the range of 20 to 200 ng/L (limit of detection 5 ng/L). The aptasensor was tested in spiked milk samples and showed a recovery of 80 and 85% for 20 and 50 ng/L of the aflatoxin M1, respectively.


Assuntos
Aflatoxina M1/isolamento & purificação , Técnicas Biossensoriais , DNA/química , Poliésteres/química , Aflatoxina M1/química , Animais , Aptâmeros de Nucleotídeos/química , Carbono/química , Bovinos , Espectroscopia Dielétrica , Ouro/química , Humanos , Microscopia Eletrônica de Varredura , Leite/química , Leite/microbiologia
6.
Chemphyschem ; 20(23): 3177-3180, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31639248

RESUMO

Herein, we report ultrasound-propelled graphene-oxide coated gold nanowire motors, functionalized with fluorescein-labeled DNA aptamers (FAM-AIB1-apt), for qualitative detection of overexpressed AIB1 oncoproteins in MCF-7 breast cancer cells. The movement of nanomotors under the ultrasound field facilitated intracellular uptake and resulted in a faster aptamer binding with the target protein and thus faster fluorescence recovery. The propulsion behavior of the aptamer functionalized nanomotors greatly enhanced the fluorescence intensity compared to static conditions. The new aptamer@nanomotor-based strategy offers considerable potential for further development of sensing methodologies towards diagnosis of breast cancer.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Fluoresceína/química , Aptâmeros de Nucleotídeos/química , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Ouro/química , Humanos , Células MCF-7 , Microscopia Eletrônica de Varredura , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Nanofios/química , Sonicação
7.
Biosensors (Basel) ; 9(2)2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31137893

RESUMO

Detection of the breast cancer cells is important for early diagnosis of the cancer. We applied thickness shear mode acoustics method (TSM) for detection of SK-BR-3 breast cancer cells using DNA aptamers specific to HER2 positive membrane receptors. The biotinylated aptamers were immobilized at the neutravidin layer chemisorbed at gold surface of TSM transducer. Addition of the cells resulted in decrease of resonant frequency, fs, and in increase of motional resistance, Rm. Using gold nanoparticles (AuNPs), modified by aptamers it was possible improving the limit of detection (LOD) that reached 550 cells/mL, while without amplification the sensitivity of the detection of SK-BR-3 cells was 1574 cells/mL. HER2 negative cell line MDA-MB-231 did not resulted in significant changes of fs. The viability studies demonstrated that cells are stable at experimental conditions used during at least 8 h. AuNPs were not toxic on the cells up to concentration of 1 µg/mL.


Assuntos
Acústica/instrumentação , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Neoplasias da Mama/patologia , Receptor ErbB-2/análise , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Ouro/química , Humanos , Nanopartículas Metálicas/química , Técnicas de Diagnóstico Molecular/métodos , Receptor ErbB-2/metabolismo
8.
Sensors (Basel) ; 19(3)2019 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-30678376

RESUMO

A DNA sensor has been proposed on the platform of glassy carbon electrode modified with native DNA implemented between two electropolymerized layers of polyaniline. The surface layer was assembled by consecutive stages of potentiodynamic electrolysis, DNA drop casting, and second electrolysis, which was required for capsulation of the DNA molecules and prevented their leaching into the solution. Surface layer assembling was controlled by cyclic voltammetry, electrochemical impedance spectroscopy, atomic force, and scanning electron microscopy. For doxorubicin measurement, the DNA sensor was first incubated in the Methylene blue solution that amplified signal due to DNA intercalation and competition with the doxorubicin molecules for the DNA binding sites. The charge transfer resistance of the inner layer interface decreased with the doxorubicin concentration in the range from 1.0 pM to 0.1 µM (LOD 0.6 pM). The DNA sensor was tested for the analysis of spiked artificial urine samples and showed satisfactory recovery in concentration range of 0.05⁻10 µM. The DNA sensor developed can find application in testing of antitumor drugs and some other DNA damaging factors.


Assuntos
DNA/química , Doxorrubicina/química , Técnicas Eletroquímicas/métodos , Compostos de Anilina/química , Técnicas Biossensoriais/métodos , Espectroscopia Dielétrica/métodos
9.
Chemphyschem ; 20(4): 545-554, 2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30552789

RESUMO

By using the thickness shear mode acoustics method (TSM) and single-molecule force spectroscopy (SMFS) we studied the interactions between DNA aptamers (sgc8c) specific to the protein tyrosine kinase 7 (PTK7), which is localized in the membranes of leukemia lymphoblastics (MOLT-4), and lymphocyte (Jurkat) cell lines, as well with PTK7-negative U266 myeloid leukemia cells. The TSM method allowed the development of a highly sensitive, label-free biosensor for the detection leukemia cells with a limit of detection of (195±20) cells/mL. SMFS approved the high selectivity of the sgc8c aptamers to the PTK7 receptors at the cell surface and allowed determining the binding probability of the aptamers to the PTK7 receptors at different cell lines.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , DNA/química , Leucemia/diagnóstico , Acústica , Moléculas de Adesão Celular/química , Linhagem Celular Tumoral , Humanos , Leucemia/sangue , Limite de Detecção , Receptores Proteína Tirosina Quinases/química
10.
Sensors (Basel) ; 18(12)2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30513767

RESUMO

We performed a comparative analysis of the sensitivity of aptamer-based biosensors for detection mycotoxin aflatoxin M1 (AFM1) depending on the method of immobilization of DNA aptamers and method of the detection. Label-free electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV) for ferrocene labeled neutravidin layers were used for this purpose. Amino-modified DNA aptamers have been immobilized at the surface of polyamidoamine dendrimers (PAMAM) of fourth generation (G4) or biotin-modified aptamers were immobilized at the neutravidin layer chemisorbed at gold surface. In the first case the limit of detection (LOD) has been determined as 8.47 ng/L. In the second approach the LOD was similar 8.62 ng/L, which is below of allowable limits of AFM1 in milk and milk products. The aptasensors were validated in a spiked milk samples with good recovery better than 78%. Comparative analysis of the sensitivity of immuno- and aptasensors was also performed and showed comparable sensitivity.


Assuntos
Aflatoxina M1/isolamento & purificação , Técnicas Biossensoriais/métodos , Espectroscopia Dielétrica , Leite/química , Aflatoxina M1/química , Animais , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Avidina/química , Bovinos , Ouro/química , Humanos , Limite de Detecção , Oxirredução
11.
Sensors (Basel) ; 18(10)2018 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-30332841

RESUMO

Voltammetric DNA sensor has been proposed on the platform of glassy carbon electrode covered with carbon black with adsorbed pillar[5]arene molecules. Electropolymerization of Neutral Red performed in the presence of native or oxidatively damaged DNA resulted in formation of hybrid material which activity depended on the DNA conditions. The assembling of the surface layer was confirmed by scanning electron microscopy and electrochemical impedance spectroscopy. The influence of DNA and pillar[5]arene on redox activity of polymeric dye was investigated and a significant increase of the peak currents was found for DNA damaged by reactive oxygen species generated by Cu2+/H2O2 mixture. Pillar[5]arene improves the electron exchange conditions and increases the response and its reproducibility. The applicability of the DNA sensor developed was shown on the example of ascorbic acid as antioxidant. It decreases the current in the concentration range from 1.0 µM to 1.0 mM. The possibility to detect antioxidant activity was qualitatively confirmed by testing tera infusion. The DNA sensor developed can find application in testing of carcinogenic species and searching for new antitumor drugs.


Assuntos
Dano ao DNA , DNA/análise , Espectroscopia Dielétrica/métodos , Compostos de Amônio Quaternário/química , Animais , Calixarenos , Cobre/química , Espectroscopia Dielétrica/instrumentação , Eletrodos , Peróxido de Hidrogênio/química , Ácidos Nucleicos Imobilizados/análise , Ácidos Nucleicos Imobilizados/química , Vermelho Neutro/química , Oxirredução , Polímeros/química , Compostos de Amônio Quaternário/análise
12.
Gen Physiol Biophys ; 37(5): 597-601, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30307406

RESUMO

The commercial solution of fourth generation (G4) of poly-amidoamine (PAMAM) dendrimers contains methanol, which is toxic for human body. Our differential scanning calorimetry (DSC) study of dendrimers confirmed the existence of this threat. The recommendation is done on how to prepare dendrimer solutions for practical and safely use in gene delivery. DSC have been also used to study the thermodynamic properties of DNA/dendrimer complexes (dendriplexes). We showed that up to DNA/dendrimer ratio 43 ± 3 (w/w) the solution was homogeneous, but stable aggregates were formed at higher PAMAM content. DSC experiments performed with homogeneous solution of dendriplexes revealed existence of the pH-dependent melting curves that contain several endothermic peaks associated with melting of GC-rich regions.


Assuntos
DNA/metabolismo , Dendrímeros/metabolismo , DNA/química , Dendrímeros/toxicidade , Quadruplex G/efeitos dos fármacos , Modelos Moleculares , Termodinâmica
13.
Analyst ; 143(15): 3686-3692, 2018 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-29978167

RESUMO

Graphene oxide (GO), a partially oxidized two-dimensional allotrope of carbon, is an attractive nanocarrier for cancer diagnostics and therapy. The nanometer-sized GO is known to permeate cell membranes. Herein we studied the cellular uptake pathways of GO nanoflakes by cancer and non-cancerous cell lines. By employing confocal Raman imaging, we were able to track the GO cellular uptake in living cells (C33 and MDCK) without any additional fluorescent or plasmonic labels. This specific progress in label-free Raman imaging of GO facilitates the monitoring of nanoflakes at the cellular level.


Assuntos
Grafite/metabolismo , Microscopia Confocal , Nanopartículas/metabolismo , Análise Espectral Raman , Animais , Linhagem Celular Tumoral , Cães , Humanos , Células Madin Darby de Rim Canino , Oxirredução , Óxidos
14.
Adv Food Nutr Res ; 85: 263-310, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29860976

RESUMO

This chapter reviews recent achievements in methods of detection of mycotoxins in food. Special focus is on the biosensor technology that utilizes antibodies and nucleic acid aptamers as receptors. Development of biosensors is based on the immobilization of antibodies or aptamers onto various conventional supports like gold layer, but also on nanomaterials such as graphene oxide, carbon nanotubes, and quantum dots that provide an effective platform for achieving high sensitivity of detection using various physical methods, including electrochemical, mass sensitive, and optical. The biosensors developed so far demonstrate high sensitivity typically in subnanomolar limit of detection. Several biosensors have been validated in real samples. The sensitivity of biosensors is similar and, in some cases, even better than traditional analytical methods such as ELISA or chromatography. We believe that future trends will be focused on improving biosensor properties toward practical application in food industry.


Assuntos
Técnicas Biossensoriais , Cromatografia/métodos , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Imunoensaio/métodos , Micotoxinas/química , Animais , Análise de Alimentos/instrumentação , Humanos
15.
Anal Bioanal Chem ; 409(11): 2767-2776, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28229174

RESUMO

We studied the interaction of the specific DNA aptamer sgc8c immobilized at the AFM tip with its corresponding receptor, the protein tyrosine kinase-7 (PTK7) embedded in the membrane of acute lymphoblastic leukemia (ALL) cells (Jurkat T-cells). Performing single molecule force spectroscopy (SMFS) experiments, we showed that the aptamer sgc8c bound with high probability (38.3 ± 7.48%) and high specificity to PTK7, as demonstrated by receptor blocking experiments and through comparison with the binding behavior of a nonspecific aptamer. The determined kinetic off-rate (koff = 5.16 s-1) indicates low dissociation of the sgc8c-PTK7 complex. In addition to the pulling force experiments, simultaneous topography and recognition imaging (TREC) experiments using AFM tips functionalized with sgc8c aptamers were realized on the outer regions surface of surface-immobilized Jurkat cells for the first time. This allowed determination of the distribution of PTK7 without any labeling and at near physiological conditions. As a result, we could show a homogeneous distribution of PTK7 molecules on the outer regions of ALL cells with a surface density of 325 ± 12 PTK7 receptors (or small receptor clusters) per µm2. Graphical Abstract The specific interaction of the DNA aptamer sgc8c and protein tyrosine kinase-7 (PTK7) on acute lymphoblastic leukemia (ALL) cells was characterized. AFM based single molecule force spectroscopy (SMFS) yielded a kinetic off-rate of 5.16 s-1 of the complex. Simultaneous topography and recognition imaging (TREC) revealed a PTK7 density of 325 ± 12 molecules or clusters per µm2 in the cell membrane.


Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Moléculas de Adesão Celular/metabolismo , Microscopia de Força Atômica/métodos , Imagem Molecular/métodos , Mapeamento de Interação de Proteínas/métodos , Receptores Proteína Tirosina Quinases/metabolismo , Linfócitos T/metabolismo , Sítios de Ligação , Técnicas Biossensoriais/métodos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Humanos , Células Jurkat , Ligação Proteica , Linfócitos T/ultraestrutura
16.
Colloids Surf B Biointerfaces ; 150: 373-383, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-27842930

RESUMO

The interaction of the promising drug carriers poly(alkyl cyanoacrylate) nanoparticles (PACA NPs) with lipid monolayers modeling the cell membrane and with RBE4 immortalized rat brain endothelial cells was compared to assess the relevance of lipid monolayer-based cell membrane models for PACA NP cellular uptake. NP properties such as size and charge of NPs and density of poly(ethylene glycol) coating (PEG) were kept in a narrow range to assess whether the type of PEG coating and the PACA monomer affected NP-monolayer and NP-cell interactions. The interaction with lipid monolayers was evaluated using surface pressure measurements and Brewster angle microscopy. NP association with and uptake by cells were assessed using flow cytometry and confocal laser scanning microscopy. The interaction between NPs and both lipid monolayers and the plasma membrane depended on the type of PEG. PEG density affected cellular uptake but not interaction with lipid monolayers. NP monomer, NPs size and charge had no effect on the interaction. This might be due to the fact that the size and charge distribution was kept rather narrow to study the effect of PACA monomer and PEG type. In conclusion, while modeling solely the passive aspect of NP-cell interactions, lipid monolayers nevertheless proved a valuable cell membrane model whose interaction with PACA NPs correlated well with NP-cell interaction. In addition, both NP-monolayer and NP-cell interactions were dependent on PEGylation type, which could be used in the design of NPs to either facilitate or hinder cellular uptake, depending on the intended purpose.


Assuntos
Cianoacrilatos/química , Lipídeos/química , Nanopartículas/química , Polietilenoglicóis/química , Animais , Encéfalo/metabolismo , Membrana Celular/metabolismo , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Células Endoteliais/metabolismo , Citometria de Fluxo , Microscopia Confocal , Tamanho da Partícula , Fosfolipídeos/química , Polímeros/farmacologia , Ratos , Propriedades de Superfície
17.
Chem Phys Lipids ; 202: 13-20, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27884594

RESUMO

We studied the surface properties of monolayers composed of polyunsaturated conjugated ethylene glycol phospholipids (carotenoid lipids), compared the data with monolayers of dipalmitoylphosphatidylcholine (DPPC) to which carotenoids were added and evaluated the impact of the unsaturated glycol lipids on monolayers with the glycerolipid DPPC. The carotenoid based glycol lipids formed monolayers at the air/water interface. Using the Langmuir method we obtained series of pressure-area (π-A) isotherms and determined the limiting area A per molecule of three glycol lipids, C30:9-C0A=42.6±1.4Å2, C30:9-C2A=76.1±2.5Ǻ2 and C30:9-C12A=354.0±12.0Å2 and their mixtures with DPPC at various mole fraction X. C30:9-C0 and C30:9-C2 did not affect significantly the shape of the isotherm, but caused their slight shift toward a lower and larger molecular area, respectively. C30:9-C12 at mole fractions X>0.02 affected the shape of isotherm. The compressibility modulus Cs-1 of monolayers depended on the surface pressure. Cs-1 value was substantially higher for DPPC monolayers in comparison with those of pure glycol lipids. At low surface pressure π=5-10mN/m and low mole fractions X<0.02 the glycol lipids formed complexes with DPPC; at higher surface pressure the separation of pure components took place. The dipole potential of the monolayers composed of cationic glycol lipids C30:9-C2 and C30:9-C12 was higher in comparison with those of zwitterionic DPPC and C30:9-C0. This may be connected with various contributions of dipole moments of the molecules and their orientation in the monolayer.


Assuntos
Glicóis/química , Fosfolipídeos/química , Polienos/química , Estrutura Molecular , Propriedades de Superfície
18.
Biochim Biophys Acta ; 1858(12): 3005-3016, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27620331

RESUMO

In this study, dendrimers have been purposed as an alternative approach for delivery of HIV peptides to dendritic cells. We have investigated the interaction of dendriplexes formed from polyanionic HIV peptide Nef and cationic carbosilane dendrimer (CBD) with model lipid membranes - large unilamellar vesicles (LUVs), Langmuir monolayers and supported lipid membranes (sBLMs) containing various molar ratio of zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG2000). In our experiments, the lipid membranes represented the model of the plasma membrane of the cell. PEGylated lipids were used in order to model glycocalyx which constitutes the outer leaflet of cellular membranes. The presence of PEGylated lipids resulted in an increase of the phase transition temperature of the lipid bilayer of LUVs, in a decrease of specific volume and adiabatic compressibility. Fluorescence anisotropy study suggests that PEGylated LUVs possessed higher lipid order and decreased fluidity when compared to zwitterionic DMPC vesicles. The interaction of dendriplexes with monolayers was accompanied by the formation of the aggregates as revealed by BAM experiments. This conclusion has been confirmed also by AFM imaging of sBLMs. We have demonstrated that dendriplexes interact with lipid membranes for all types of lipid composition. Moreover, the stronger interaction of cationic dendrimer/peptide complexes with lipid monolayers, vesicles and sBLMs was observed for membranes composed of zwitterionic lipids than for PEGylated lipid membranes. Increased concentration of PEGylated lipids made this interaction weaker.


Assuntos
Dendrímeros/química , Bicamadas Lipídicas/química , Polietilenoglicóis/farmacologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/química , Polarização de Fluorescência , Microscopia de Força Atômica , Espalhamento de Radiação , Termodinâmica
19.
Biochim Biophys Acta ; 1864(9): 1072-1082, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27216152

RESUMO

BACKGROUND: Nicking endonucleases are enzymes that recognize specific sites in double-stranded DNA and cleave only one strand at a predetermined position. These enzymes are involved in DNA replication and repair; they can also function as subunits of bacterial heterodimeric restriction endonucleases. One example of such a proteins is the restriction endonuclease BspD6I (R.BspD6I) from Bacillus species strain D6, which consists of the large subunit - nicking endonuclease BspD6I (Nt.BspD6I), and the small subunit (ss.BspD6I). Nt.BspD6I can function independently. Similar enzymes are now widely used in numerous biotechnological applications. The aim of this study was to investigate the fundamental properties of two subunits of R.BspD6I and their interdependence in the course of R.BspD6I activity. METHODS: The binding and hydrolysis of DNA duplexes by R.BspD6I are primary analyzed by gel electrophoresis. To elucidate the difference between Nt.BspD6I interaction with the substrate and product of hydrolysis, the thickness shear mode acoustic method is used. RESULTS AND CONCLUSIONS: The thermodynamic and kinetic parameters of the Nt.BspD6I interaction with DNA are determined. For the first time we demonstrated that Nt.BspD6I bends the DNA during complex formation. Nt.BspD6I is able to form complexes with the product nicked in the top strand and ss.BspD6I cleaves the bottom strand of the DNA consecutively. Furthermore, the influence of dA methylation in the R.BspD6I recognition site on ss.BspD6I activity is analyzed. GENERAL SIGNIFICANCE: The obtained results provide evidence that Nt.BspD6I coordinates the activity of R.BspD6I by strictly coupling of the bottom strand cleavage by ss.BspD6I to the top strand cleavage.


Assuntos
DNA/química , Desoxirribonuclease I/química , Subunidades Proteicas/química , Bacillus/química , Sítios de Ligação , Clonagem Molecular , DNA/metabolismo , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Hidrólise , Cinética , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica
20.
Biochim Biophys Acta ; 1848(4): 907-15, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25576765

RESUMO

AIMS: We have investigated the effect of surface charge of model lipid membranes on their interactions with dendriplexes formed by HIV-derived peptides and 2 types of positively charged carbosilane dendrimers (CBD). METHODS: Interaction of dendriplexes with lipid membranes was measured by fluorescence anisotropy, dynamic light scattering and Langmuir-Blodgett techniques. The morphology of the complexes was examined by transmission electron microscopy. RESULTS: All dendriplexes independent of the type of peptide interacted with model lipid membranes. Negatively charged vesicles composed of a mixture of DMPC/DPPG interacted more strongly, and it was accompanied by an increase in anisotropy of the fluorescent probe localized in polar domain of lipid bilayers. There was also an increase in surface pressure of the lipid monolayers. Mixing negatively charged liposomes with dendriplexes increased liposome size and made their surface charges more positive. CONCLUSIONS: HIV-peptide/dendrimer complexes interact with model lipid membranes depending on their surface charge. Carbosilane dendrimers can be useful as non-viral carriers for delivering HIV-peptides into cells.


Assuntos
Dendrímeros/metabolismo , HIV-1/química , Bicamadas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Fragmentos de Peptídeos/metabolismo , Silanos/metabolismo , Dendrímeros/química , Polarização de Fluorescência , Humanos , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Lipossomos , Fluidez de Membrana , Lipídeos de Membrana/química , Microscopia Eletrônica de Transmissão , Fragmentos de Peptídeos/química , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Silanos/química
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