RESUMO
Selenium is an essential inorganic compound in human and animal nutrition, involved in the proper functioning of the body. As a micronutrient, it actively contributes to the regulation of various metabolic activities, i.e., thyroid hormone, and protection against oxidative stress. However, Se exhibits a narrow concentration window between having a positive effect and exerting a toxic effect. In higher doses, it negatively affects living organisms and causes DNA damage through the formation of free radicals. Increased reactivity of Se anions can also disrupt the integrity and function of DNA-repairing proteins. As the permissible concentration of Se in drinking water is 10 µg/L, it is vital to develop sensitive and robust methods of Se detection in aqueous samples. In this study, for the first time, we proposed a selective aptamer for selenate ion detection, chosen following the SELEX process, and its application in the construction of an electrochemical aptasensor towards SeO42- ions. Measurement conditions such as the used redox marker and pH value of the measurement solution were chosen. The proposed aptasensor is characterized by good selectivity and an LOD of 1 nM. Conditions for biosensor regeneration and storage were also investigated in this research.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Técnicas Eletroquímicas , Ácido Selênico , Aptâmeros de Nucleotídeos/química , Ácido Selênico/química , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Íons , Técnica de Seleção de Aptâmeros/métodos , Humanos , Limite de DetecçãoRESUMO
Currently, tuberculosis immunoprophylaxis is based solely on Bacillus Calmette-Guérin (BCG) vaccination, and some of the new potential tuberculosis vaccines are based on the BCG genome. Therefore, it is reasonable to analyze the genomes of individual BCG substrains. The aim of this study was the genetic characterization of the BCG-Moreau Polish (PL) strain used for the production of the BCG vaccine in Poland since 1955. Sequencing of different BCG lots showed that the strain was stable over a period of 59 years. As a result of comparison, BCG-Moreau PL with BCG-Moreau Rio de Janeiro (RDJ) 143 single nucleotide polymorphisms (SNPs) and 32 insertion/deletion mutations (INDELs) were identified. However, the verification of these mutations showed that the most significant were accumulated in the BCG-Moreau RDJ genome. The mutations unique to the Polish strain genome are 1 SNP and 2 INDEL. The strategy of combining short-read sequencing with long-read sequencing is currently the most optimal approach for sequencing bacterial genomes. With this approach, the only available genomic sequence of BCG-Moreau PL was obtained. This sequence will primarily be a reference point in the genetic control of the stability of the vaccine strain in the future. The results enrich knowledge about the microevolution and attenuation of the BCG vaccine substrains. IMPORTANCE: The whole genome sequence obtained is the only genomic sequence of the strain that has been used for vaccine production in Poland since 1955. Sequencing of different BCG lots showed that the strain was stable over a period of 59 years. The comprehensive genomic analysis performed not only enriches knowledge about the microevolution and attenuation of the BCG vaccine substrains but also enables the utilization of identified markers as a reference point in the genetic control and identity tests of the stability of the vaccine strain in the future.
Assuntos
Vacina BCG , Genoma Bacteriano , Mycobacterium bovis , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Genoma , Vacina BCG/genética , Vacina BCG/imunologia , Mycobacterium bovis/genética , Mycobacterium bovis/classificação , Polônia , Humanos , Tuberculose/prevenção & controle , Tuberculose/microbiologia , Mutação INDEL , MutaçãoRESUMO
Heavy metals are one of the major pollutants found in drinking water and their abnormal level may pose a threat to human's health and life. Manganese also belongs to heavy metals group, and it is generally used in production of batteries, fertilizers, and ceramics. Even though, Mn is necessary for proper development of central nervous system, its elevated concentration might lead to certain diseases such as epilepsies, cell death in focal cerebral ischemia as well as neurodegenerative diseases such as Huntington and Alzheimer. Hence, it is crucial to elaborate novel methods for manganese ions detection that could be applied for in situ analysis of water samples. Herein, we present the studies on the electrochemical detection of manganese ions using aptamer-modified electrodes. This is the first attempt of application of aptamer strands as receptor layers for electrochemical analysis of manganese ions and for that purpose gold disk electrodes served as transducers, which were further modified with disulfide - based aptamers and 6-mercapto-1-hexanol blocking agent. The electrochemical measurements concerned the choice of the conditions for formation of aptamer receptor layer as well as the type of redox indicator that served as the source of current signal. The studies referred to the definition of aptasensor working parameters including the verification of the possibility of manganese ion detection in cell culture media. It was shown that it was possible to detect Mn2+ ions within 25 nM-1 µM concentration and the proposed aptasensor exhibited high selectivity towards target analyte for which at least 2 - times higher response was recorded than for interfering ions. Moreover, the possibility of Mn2+ detection in real samples was depicted followed by stability and regeneration studies.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Humanos , Manganês , Oxirredução , Ouro , Eletrodos , Íons , Técnicas Eletroquímicas/métodos , Técnicas Biossensoriais/métodos , Limite de DetecçãoRESUMO
The importance of understanding the mercury (II) ion interactions with thymine-rich DNA sequences is the reason for multiple comparative investigations carried out with the use of optical detection techniques directly in the depth of solution. However, the results of such investigations have limited applicability in the interpretation of the Hg2+ binding phenomenon by DNA sequences in thin, interfacial (electrode/solution), self-organized monolayers immobilized on polarizable surfaces, often used for sensing purposes in electrochemical biosensors. Overlooking the careful optimization of the measurement conditions is the source of discrepancies in the interpretation of the registered electrochemical signal. In this study, the chosen effects accompanying the efficiency of surface related recognition of Hg2+ by polyThymine DNA sequences labelled with methylene blue were investigated by voltammetry, QCM and spectro-electrochemical techniques. As was shown, the composition of the biosensing layer and buffers or the analytical procedures have a significant impact on the registered electrochemical readout which translates into signal stability, the biosensor's working parameters or even the mechanism of detection. After elucidation of the above factors, the complete and ready-to-use biosensor-based analytical solution was proposed offering subpicomolar mercury ion determination with high selectivity (also in aqueous real samples), reusability, and high signal stability even after long-term storage. The developed procedures were successfully used during the miniaturization process with self-prepared (PVD) elastic transducers. The obtained sensor, together with the simplicity of its use, low manufacturing cost, and attractive analytical parameters (i.e., LOD < < Hg2+ WHO limit) can present an interesting alternative for on-site mercury ion detection in environmental samples.
Assuntos
Técnicas Biossensoriais , Mercúrio , Mercúrio/química , Ouro/química , Água/química , Azul de Metileno/química , Técnicas Biossensoriais/métodosRESUMO
Lead poses severe effects on living organisms, and since Pb2+ ions tend to accumulate in different organs, it is crucial to monitor Pb2+ concentration in samples such as water and soil. One of the approaches is the utilization of biosensors combined with aptamer-based layers for the electrochemical detection of lead ions. Herein, we present the studies of applying miniaturized screen-printed transducers as solid surfaces to fabricate aptamer layers. As the research is the direct continuation of our previous studies regarding the use of gold disk electrodes, the working parameters of elaborated aptasensors were defined, including the range of linear response (10-100 nM), selectivity as well as stability, regeneration, and feasibility of application for the analysis of real samples. This was achieved using voltammetric techniques including cyclic and square-wave voltammetry in the presence of methylene blue redox indicator.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Técnicas Eletroquímicas/métodos , Chumbo , Aptâmeros de Nucleotídeos/química , Oxirredução , Técnicas Biossensoriais/métodos , Ouro/química , Eletrodos , Limite de DetecçãoRESUMO
Recently nucleic acids gained considerable attention as selective receptors of metal ions. This is because of the possibility of adjusting their sequences in new aptamers selection, as well as the convenience of elaborating new detection mechanisms. Such a flexibility allows for easy utilization of newly emerging nanomaterials for the development of detection devices. This, in turn, can significantly increase, e.g., analytical signal intensity, both optical and electrochemical, and the same can allow for obtaining exceptionally low detection limits and fast biosensor responses. All these properties, together with low power consumption, make nucleic acids biosensors perfect candidates as detection elements of fully automatic portable microfluidic devices. This review provides current progress in nucleic acids application in monitoring environmentally and clinically important metal ions in the electrochemical or optical manner. In addition, several examples of such biosensor applications in portable microfluidic devices are shown.
Assuntos
Técnicas Biossensoriais , Nanoestruturas , Ácidos Nucleicos , Ácidos Nucleicos/química , Metais/química , Íons , Nanoestruturas/química , Técnicas EletroquímicasRESUMO
In this study, we established a dynamic micromodel of urinary tract infection to analyze the impact of UT-segment-specific urinary outflow on the persistence of E. coli colonization. We found that the adherence of Dr+ E. coli to bladder T24 transitional cells and type IV collagen is maximal at lowest shear stress and is reduced by any increase in flow velocity. The analyzed adherence was effective in the whole spectrum of physiological shear stress and was almost irreversible over the entire range of generated shear force. Once Dr+ E. coli bound to host cells or collagen, they did not detach even in the presence of elevated shear stress or of chloramphenicol, a competitive inhibitor of binding. Investigating the role of epithelial surface architecture, we showed that the presence of budding cells-a model microarchitectural obstacle-promotes colonization of the urinary tract by E. coli. We report a previously undescribed phenomenon of epithelial cell "rolling-shedding" colonization, in which the detached epithelial cells reattach to the underlying cell line through a layer of adherent Dr+ E. coli. This rolling-shedding colonization progressed continuously due to "refilling" induced by the flow-perturbing obstacle. The shear stress of fluid containing free-floating bacteria fueled the rolling, while providing an uninterrupted supply of new bacteria to be trapped by the rolling cell. The progressive rolling allows for transfer of briefly attached bacteria onto the underlying monolayer in a repeating cascading event.
Assuntos
Infecções por Escherichia coli/microbiologia , Escherichia coli/química , Escherichia coli/fisiologia , Infecções Urinárias/microbiologia , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/metabolismo , Aderência Bacteriana , Escherichia coli/genética , Humanos , Estresse MecânicoRESUMO
The impact of the differently hydrated non-electrolytes (protein structure destabilizers) on the fibrillation of hen egg white lysozyme (HEWL) was investigated. Two isomeric urea derivatives i.e. butylurea (BU) and N,N,N',N'-tetramethylurea (TMU) were chosen as a tested compounds. The obtained results show that butylurea exerts greater impact on HEWL and its fibrillation than tetramethylurea. Both substances decrease the time of induction of the fibrillation (lag time) but only BU increases the efficiency of amyloidogenesis. For the systems with equivalent reduction of the HEWL stability (250mM BU and 500mM TMU) the not-equivalent increase of the protein fibrillation was recorded (higher for BU). This fact suggests that specific interactions with protein, possibly water mediated, are responsible for the action of the tested substances.
Assuntos
Amiloide/química , Muramidase/química , Água/química , Animais , Galinhas , Dicroísmo Circular , Microscopia de Força Atômica , Muramidase/metabolismo , Estabilidade Proteica , Soluções/química , Ureia/químicaRESUMO
Nucleases are an important group of hydrolases that degrade nucleic acids, with broad spectrum of applications in science and industry. In this paper, we report the identification and characterization of the nuclease from extremely psychrophilic bacterium Psychromonas ingrahamii that grows exponentially at 5 °C, but may also grow at even lower temperatures (down to - 12 °C). The putative endonuclease I gene, identified in the genome of P. ingrahamii, was cloned and expressed in Pichia pastoris. The recombinant protein was purified and its nucleolytic features were studied. The new enzyme, named by us as PinNuc, displays the features characteristic for the nonselective endonucleases, and has the ability to degrade different forms of nucleic acids. It is very active at room temperature in low ion-strength buffer and in the presence of low concentrations of magnesium ions. The enzyme, which possesses six cysteine residues, the most likely all engaged in disulphide bridges, is active only in oxidized form, and can be efficiently inactivated by the addition of low amounts of a reducing agent. According to our knowledge, it is the first nuclease, belonging to endonuclease I family, isolated from such extremely psychrophilic organism.
Assuntos
Proteínas de Bactérias/fisiologia , Desoxirribonuclease I/fisiologia , Extremófilos/enzimologia , Gammaproteobacteria/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Desoxirribonuclease I/química , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Concentração de Íons de Hidrogênio , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , TemperaturaRESUMO
The study investigates the role of the electrostatic interactions in the fibrillation of the hen egg white lysozyme (HEWL). In order to achieve this aim the influence of the cations Na+, Mg2+ and Al3+ on the amyloid fibril formation and amorphous aggregation was tested. The amyloids are formed in the solution without added salt but the Thioflavin T fluorescence gives the false-negative result. In these conditions, the HEWL fibrils are long and curvy. If the ionic strength of the solution is sufficiently high, the formed amyloids are shorter and fragmented. Our study shows that the addition of the aluminium salt promotes protein fibrillation. The amorphous aggregation dominates in the high concentration of electrolyte. The in vitro amyloid fibril formation seems to be regulated by universal mechanisms. The theories implemented in the polymer science or for colloidal solutions give the qualitative description of the aggregation phenomena. However, the specific interactions and the additional effects (e.g. fibril fragmentation) modulate the amyloidogenesis.
Assuntos
Amiloide/química , Muramidase/química , Agregados Proteicos , Animais , Concentração OsmolarRESUMO
The DNA coding sequence of TaqStoffel polymerase was fused with the DNA-binding domain of Pyrococcus furiosus ligase. The resulting novel recombinant gene was cloned and expressed in E. coli. The recombinant enzyme was purified and its enzymatic features were studied. The fusion protein (PfuDBDlig-TaqS) was found to have enhanced processivity as a result of the conversion of the TaqDNA polymerase from a relatively low processive to a highly processive enzyme. The abovementioned processivity enhancement was about threefold as compared to the recombinant TaqStoffel DNA polymerase (TaqS), and the recombinant fusion protein was more thermostable. It had a half-life of 23 min at 99 °C as compared to 10 min for TaqS. The fusion protein also showed a significantly higher resistance to PCR inhibitors such as heparin or lactoferrin and the fusion polymerase-amplified GC-rich templates much more efficiently and was efficient even with 78% GC pairs.
Assuntos
Proteínas de Ligação a DNA/química , DNA Polimerase Dirigida por DNA/química , Ligases/química , Pyrococcus furiosus/enzimologia , Clonagem Molecular , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Ligases/genética , Reação em Cadeia da Polimerase , Domínios Proteicos , Pyrococcus furiosus/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
DNA polymerases are present in all organisms and are important enzymes that synthesise DNA molecules. They are used in various fields of science, predominantly as essential components for in vitro DNA syntheses, known as PCR. Modern diagnostics, molecular biology and genetic engineering need DNA polymerases which demonstrate improved performance. This study was aimed at obtaining a new NeqSSB-TaqS fusion DNA polymerase from the Taq DNA Stoffel domain and a single-stranded DNA binding-like protein of Nanoarchaeum equitans in order to significantly improve the properties of DNA polymerase. The DNA coding sequence of Taq Stoffel DNA polymerase and the nonspecific DNA-binding protein of Nanoarchaeum equitans (NeqSSB-like protein) were fused. A novel recombinant gene was obtained which was cloned into the pET-30 Ek/LIC vector and introduced into E. coli for expression. The recombinant enzyme was purified and its enzymatic properties including DNA polymerase activity, PCR amplification rate, thermostability, processivity and resistance to inhibitors, were tested. The yield of the target protein reached approximately 18 mg/l after 24 h of the IPTG induction. The specific activity of the polymerase was 2200 U/mg. The recombinant NeqSSB-TaqS exhibited a much higher extension rate (1000 bp template in 20 s), processivity (19 nt), thermostability (half-life 35 min at 95°C) and higher tolerance to PCR inhibitors (0.3-1.25% of whole blood, 0.84-13.5 µg of lactoferrin and 4.7-150 ng of heparin) than Taq Stoffel DNA polymerase. Furthermore, our studies show that NeqSSB-TaqS DNA polymerase has a high level of flexibility in relation to Mg2+ ions (from 1 to 5 mM) and KCl or (NH4)2SO4 salts (more than 60 mM and 40 mM, respectively). Using NeqSSB-TaqS DNA polymerase instead of the Taq DNA polymerase could be a better choice in many PCR applications.
Assuntos
DNA de Cadeia Simples , Proteínas de Ligação a DNA/metabolismo , Nanoarchaeota/enzimologia , Taq Polimerase/metabolismo , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética , Nanoarchaeota/genética , Plasmídeos/metabolismo , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Dr fimbriae are homopolymeric adhesive organelles of uropathogenic Escherichia coli composed of DraE subunits, responsible for the attachment to host cells. These structures are characterized by enormously high stability resulting from the structural properties of an Ig-like fold of DraE. One feature of DraE and other fimbrial subunits that makes them peculiar among Ig-like domain-containing proteins is a conserved disulfide bond that joins their A and B strands. Here, we investigated how this disulfide bond affects the stability and folding/unfolding pathway of DraE. We found that the disulfide bond stabilizes self-complemented DraE (DraE-sc) by â¼50 kJ mol-1 in an exclusively thermodynamic manner, i.e. by lowering the free energy of the native state and with almost no effect on the free energy of the transition state. This finding was confirmed by experimentally determined folding and unfolding rate constants of DraE-sc and a disulfide bond-lacking DraE-sc variant. Although the folding of both proteins exhibited similar kinetics, the unfolding rate constant changed upon deletion of the disulfide bond by 10 orders of magnitude, from â¼10-17 s-1 to 10-7 s-1 Molecular simulations revealed that unfolding of the disulfide bond-lacking variant is initiated by strands A or G and that disulfide bond-mediated joining of strand A to the core strand B cooperatively stabilizes the whole protein. We also show that the disulfide bond in DraE is recognized by the DraB chaperone, indicating a mechanism that precludes the incorporation of less stable, non-oxidized DraE forms into the fimbriae.
Assuntos
Adesinas Bacterianas/metabolismo , Cistina/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Modelos Moleculares , Escherichia coli Uropatogênica/fisiologia , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Aderência Bacteriana , Linhagem Celular Tumoral , Sequência Conservada , Cisteína/química , Transferência de Energia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Fímbrias/química , Proteínas de Fímbrias/genética , Humanos , Cinética , Simulação de Dinâmica Molecular , Mutação , Oxirredução , Conformação Proteica , Dobramento de Proteína , Redobramento de Proteína , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
PCR has become an essential tool in biological science. However, researchers often encounter problems with difficult targets, inhibitors accompanying the samples, or PCR trouble related to DNA polymerase. Therefore, PCR optimization is necessary to obtain better results. One solution is using modified DNA polymerases with desirable properties for the experiments. In this article, PCR troubleshooting, depending on the DNA polymerase used, is shown. In addition, the reasons that might justify the need for modification of DNA polymerases, type of modifications, and links between modified DNA polymerases and PCR efficiency are described.
Assuntos
DNA Polimerase Dirigida por DNA/química , Reação em Cadeia da Polimerase/métodos , Proteínas de Ligação a DNA/química , Mutação , Proteínas Recombinantes de Fusão/químicaRESUMO
Previous studies have suggested that NK cells may limit T cell responses by their ability to eradicate dendritic cells, as demonstrated by NK cell-mediated killing of dendritic cells generated from mouse bone marrow cells or human monocytes with GM-CSF. In the present study, we demonstrated that conventional dendritic cells, generated in vitro with Flt3 ligand or from spleens, were resistant to NK cell-mediated lysis. However, upon stimulation with GM-CSF, NK cells could mediate lysis of these dendritic cells. GM-CSF-stimulated Flt3 ligand dendritic cells or splenic dendritic cells increased surface expression of costimulatory molecules and known NK cell ligands. Likewise, NK cells could target dendritic cells in vivo, which could be inhibited, in part, by anti-GM-CSF antibodies. The blocking of CD54 or CD226 inhibited NK cell-mediated cytotoxicity of the GM-CSF-stimulated Flt3 ligand conventional dendritic cells. Furthermore, the CD226+NKG2A- subset of NK cells was selectively better at targeting GM-CSF-stimulated Flt3 ligand conventional dendritic cells. However, CD155, a known ligand for CD226, could also act as an inhibitor of NK cell-mediated lysis, as dendritic cells lacking CD155 were more sensitive to NK cell-mediated lysis than wild-type dendritic cells. We hypothesize that by only permitting a subset of NK cells to target activated dendritic cells during inflammation, this would allow the immune system to balance between dendritic cells able to drive adaptive immune responses and dendritic cells targeted for elimination by NK cells to hinder, e.g., spread of infection.
Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Citotoxicidade Imunológica/imunologia , Células Dendríticas/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Células Matadoras Naturais/imunologia , Animais , Células Cultivadas , Células Dendríticas/transplante , Genes RAG-1 , Rejeição de Enxerto/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Inflamação , Interleucina-18/farmacologia , Proteínas de Membrana/farmacologia , Camundongos , Camundongos Knockout , Receptor de Morte Celular Programada 1/deficiência , Receptores Virais/deficiência , Proteínas Recombinantes/farmacologia , Baço/imunologiaRESUMO
We report on investigation of detonation nanodiamond annealed at 800C°in chlorine atmosphere by means of 1H, 13C and 35Cl nuclear magnetic resonance and X-ray photoelectron spectroscopy. The results of these methods are found to be consistent with each other and evidence formation of chlorine-carbon groups and sp2 carbon shell on the nanodiamond surface. The data obtained provide detailed information about the structure and bonding in this diamond nanoparticle. Interaction of nuclear spins with unpaired electron spins of dangling bonds results in fast 13C nuclear spin-lattice relaxation.
Assuntos
Cloro/química , Nanocompostos/química , Nanocompostos/ultraestrutura , Nanodiamantes/química , Nanodiamantes/ultraestrutura , Absorção Fisico-Química , Adsorção , Sítios de Ligação , Espectroscopia de Ressonância Magnética , Teste de Materiais , Tamanho da Partícula , Espectroscopia Fotoeletrônica , Propriedades de SuperfícieRESUMO
BACKGROUND: SSB (single-stranded DNA-binding) proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis. RESULTS: This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein). This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity). The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7 ± 1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100 °C and melting temperature (T(m)) is 100.2 °C as shown by differential scanning calorimetry (DSC) analysis. CONCLUSION: NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids.
Assuntos
Proteínas Arqueais/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nanoarchaeota/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas de Ligação a DNA/química , Dados de Sequência Molecular , Nanoarchaeota/química , Estabilidade Proteica , Alinhamento de Sequência , Especificidade por Substrato , TemperaturaRESUMO
Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are amongst the most common bacterial infectious diseases in the developed world. The urovirulence of UPEC is mainly associated with the surface-exposed fimbrial adhesins and adhesins of the autotransporter (AT) family. The best studied of these proteins is antigen Ag43 mediating cell aggregation, adhesion and biofilm development as the causes of chronic UTIs. The E. coli IH11128 Dr(+) (dra (+)) strain of the Dr/Afa(+) family of adhesins possesses two major surface-exposed virulence factors: Dr fimbrial polyadhesin and DraD protein (fimbrial tip subunit or protein component of the adhesive sheath). Here, we identified for the first time, to our knowledge, the agn43 gene encoding Ag43 in the WT clinical isolate of UPEC Dr(+) as a new virulence factor not yet tested. We also found that Dr fimbrial expression, which like Ag43 is under the control of a phase-variable mechanism, did not exclude Ag43 surface presentation. However, the presence of Dr fimbriae supported by other structures on the cell surface caused a physical neutralization of Ag43-mediated autoaggregation during in vitro growth. The fimbrial bundling further increased the distance between the adjacent Ag43(+) cells, thus preventing head-to-tail association between surface-exposed Ag43 subunits and their interactions with the host cells. The investigations showed that Ag43 did not act as a specific adhesin and invasin, conversely to the major virulence factors of E. coli Dr(+), but played significant roles in the viability and metabolic activity of bacterial cells forming biofilm, and in the survival of bacteria within invaded epithelial cells.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Escherichia coli/imunologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/fisiologia , Escherichia coli Uropatogênica/patogenicidade , Adesinas de Escherichia coli/genética , Adesinas de Escherichia coli/imunologia , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Aderência Bacteriana/genética , Aderência Bacteriana/imunologia , Biofilmes , Linhagem Celular , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/imunologia , Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Viabilidade Microbiana/genética , Viabilidade Microbiana/imunologia , Fenótipo , Domínios e Motivos de Interação entre Proteínas , Fatores de Virulência/genética , Fatores de Virulência/imunologiaRESUMO
In vitro inhibition of the formation of fibrous aggregates of proteins (amyloids) has gained increasing attention due to the number of diseases associated with protein misfolding and fibrillation. An interesting group of compounds for which pronounced activity against this phenomenon can be expected consists of low molecular weight substances (osmolytes) which have the ability to change protein stability. Here we investigate the influence of trimethylamine N-oxide (TMAO) in acidic solution (pH=2) on the fibrillation of hen egg white lysozyme (HEWL). The process was monitored by five techniques: circular dichroism in the UV region, atomic force microscopy, dynamic light scattering, densimetry and gel electrophoresis. The obtained results show that protonated TMAO in a concentration of 400 mM inhibits amyloidogenesis. In the conditions of the experiment the HEWL molecules form clusters about 30 nm in diameter containing a relatively high fraction of covalent-bonded dimers.
Assuntos
Amiloide/química , Clara de Ovo/química , Metilaminas/química , Muramidase/química , Amiloide/ultraestrutura , Animais , Feminino , Concentração de Íons de HidrogênioRESUMO
BACKGROUND: Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination and repair in Bacteria, Archaea and Eukarya. In recent years, there has been an increasing interest in SSBs, since they find numerous applications in diverse molecular biology and analytical methods. RESULTS: We report the characterization of single-stranded DNA-binding proteins from the psychrophilic bacteria Desulfotalea psychrophila (DpsSSB), Flavobacterium psychrophilum (FpsSSB), Psychrobacter arcticus (ParSSB), Psychrobacter cryohalolentis (PcrSSB), Psychromonas ingrahamii (PinSSB), Photobacterium profundum (PprSSB), and Psychroflexus torquis (PtoSSB). The proteins show a high differential within the molecular mass of their monomers and the length of their amino acid sequences. The high level of identity and similarity in respect to the EcoSSB is related to the OB-fold and some of the last amino acid residues. They are functional as homotetramers, with each monomer encoding one single stranded DNA binding domain (OB-fold). The fluorescence titrations indicated that the length of the ssDNA-binding site size is approximately 30 ± 2 nucleotides for the PinSSB, 31 ± 2 nucleotides for the DpsSSB, and 32 ± 2 nucleotides for the ParSSB, PcrSSB, PprSSB and PtoSSB. They also demonstrated that it is salt independent. However, when the ionic strength was changed from low salt to high, binding-mode transition was observed for the FpsSSB, at 31 ± 2 nucleotides and 45 ± 2 nucleotides, respectively. As expected, the SSB proteins under study cause duplex DNA destabilization. The greatest decrease in duplex DNA melting temperature was observed in the presence of the PtoSSB 17 °C. The SSBs in question possess relatively high thermostability for proteins derived from cold-adapted bacteria. CONCLUSION: The results showed that SSB proteins from psychrophilic microorganisms are typical bacterial SSBs and possess relatively high thermostability, offering an attractive alternative to other thermostable SSBs in molecular biology applications.