Metallothionein (MT) plays the important role in the detoxification of heavy metals, protection against oxidative compounds and as a prognostic marker in the development of tumors. It is important to find selective, stable and sensitive tools and probes to evaluate the presence of MT in biological fluids or tissues. QDs linked with ligands such as peptides or small molecules are a promising tool for selective, fast, and sensitive tagging and imaging in medicine. In previous findings, the authors proved the possibility of interaction with QDs (particularly with CdTe) and analyzed the stability of the formed complexes between CdTe and MT during incubation over time. Following that, an initial analysis of the interactions between CdTe quantum dots (QDs) and human metallothionein (MT) was performed. Complexes of mercaptosuccinic acid-covered CdTe QDs + MT were investigated using fluorescence intensity changes along a timeline, quenching analysis, stability interpretation based on zeta potential, and quenching intensity. Based on the preliminary results, it appears as though the possible interactions depend on the size of the CdTe QDs. Additionally, the formation of complexes between CdTe and human MT likely depends mostly on structural changes and conformational reorganization rather than on electrostatic interactions. Both types of interactions are responsible for complex creation and stabilization.
- MeSH
- fluorescence MeSH
- fluorescenční spektrometrie MeSH
- kvantové tečky chemie MeSH
- lidé MeSH
- metalothionein chemie MeSH
- povrchové vlastnosti MeSH
- sloučeniny kadmia chemie MeSH
- telur chemie MeSH
- velikost částic MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Thanks to quantum dots' (QDs) properties, they can be used as selective and sensitive biomarkers in molecular imaging. In a previous paper, we confirmed the possibility of interaction between mercaptosuccinic acid-capped cadmium telluride QDs (MSA-CdTe) and human metallothionein (MT). The aim of this study was to expand on our previous research with an evaluation of the stability of the formed complexes between human MT and four CdTe compounds of the following sizes: 3.4nm (blue QDs), 3.8nm (green QDs), 4.5nm (yellow QDs), and 5.2nm (red QDs). Complexes were evaluated over time using fluorescence intensity and differential pulse voltammetry. Differences between the voltammograms obtained for standard solutions and for CdTe+MT show that complexes were formed. An increase in fluorescence intensity was observed for blue (Δ%≈40 for t=1→120min) and red (Δ%≈30 for t=1→120min) CdTe-MT complexes than CdTe alone, whereas green and yellow CdTe-MT complexes had a lower fluorescence intensity than CdTe alone. A stronger time dependence of the mercaptosuccinic acid (MSA) peak height on the timeline and differences in the MSA peak shape (in CdTe, and CdTe+MT complexes) were also observed by voltammetry. Authors noticed a decrease in the Cat2 signal of the red and green CdTe+MT complexes at the time of conjugation. Our results reveal that the size of QDs has an impact on the interaction between CdTe and human MT, as well as on the stability of complexes formed during these interactions. The bioconjugates' stability was also found to depend on the time of interaction.
- MeSH
- časové faktory MeSH
- elektrochemické techniky metody MeSH
- fluorescence MeSH
- fluorescenční spektrometrie metody MeSH
- kinetika MeSH
- kvantové tečky * MeSH
- lidé MeSH
- metalothionein chemie metabolismus MeSH
- sloučeniny kadmia chemie metabolismus MeSH
- telur chemie metabolismus MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The translation of metallothioneins (MTs) is one of the defense strategies by which organisms protect themselves from metal-induced toxicity. MTs belong to a family of proteins comprising MT-1, MT-2, MT-3, and MT-4 classes, with multiple isoforms within each class. The main aim of this study was to determine the behavior of MT in dependence on various externally modelled environments, using electrochemistry. In our study, the mass distribution of MTs was characterized using MALDI-TOF. After that, adsorptive transfer stripping technique with differential pulse voltammetry was selected for optimization of electrochemical detection of MTs with regard to accumulation time and pH effects. Our results show that utilization of 0.5 M NaCl, pH 6.4, as the supporting electrolyte provides a highly complicated fingerprint, showing a number of non-resolved voltammograms. Hence, we further resolved the voltammograms exhibiting the broad and overlapping signals using curve fitting. The separated signals were assigned to the electrochemical responses of several MT complexes with zinc(II), cadmium(II), and copper(II), respectively. Our results show that electrochemistry could serve as a great tool for metalloproteomic applications to determine the ratio of metal ion bonds within the target protein structure, however, it provides highly complicated signals, which require further resolution using a proper statistical method, such as curve fitting.
- MeSH
- chlorid sodný chemie MeSH
- elektrochemie MeSH
- elektrolyty MeSH
- komplexní sloučeniny chemie metabolismus MeSH
- kovy chemie metabolismus MeSH
- metalothionein chemie metabolismus MeSH
- protein - isoformy MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
The present study suggests and describes the application of a delivery system for antisense oligonucleotides against mRNA encoding estrogen receptor proteins α and β. The delivery system is composed of a cationic liposome envelope containing 17β-estradiol (E2) in its structure. Cationic liposomes protect cargo against the extracellular matrix, and E2 can increase its shuttling efficiency into cells. Using MCF-7 cells derived from estrogen receptor-positive ductal carcinoma, treatment with liposomes against ERα was found to decrease MCF-7 proliferation, and importantly the application of both the antisense against ERα and β exhibited an antiproliferative effect expressed as cell viability. Using qRT-PCR, it was shown that MT1A, NF-κB1 and K-ras genes, but not TFF1, were downregulated using E2-based liposomes (evaluated at P=0.05). Further indicators of oxidative stress were employed to assess the effect on treatment efficiency. Glutathione (GSH/GSSG redox ratio), metallothionein (MT) and malondialdehyde (MDA) confirmed a positive effect of antisense therapy resulting in their decreased levels in the MCF-7 cells. Based on these data, we suggest that E2-based liposomes offer sufficient transfer efficiency and moreover, due to the effect on NF-κB1, MT and GSH, tumor cells can be chemosensitized to increase treatment effectiveness.
- MeSH
- antitumorózní látky chemie MeSH
- estradiol metabolismus MeSH
- genetická terapie metody MeSH
- glutathion chemie MeSH
- koncentrace vodíkových iontů MeSH
- kyslík chemie MeSH
- lidé MeSH
- liposomy chemie MeSH
- malondialdehyd chemie MeSH
- metalothionein chemie MeSH
- MFC-7 buňky MeSH
- mikroskopie fázově kontrastní MeSH
- nádorové buněčné linie MeSH
- nádory prsu metabolismus MeSH
- oxidace-redukce MeSH
- oxidační stres MeSH
- proliferace buněk MeSH
- receptory pro estrogeny metabolismus MeSH
- regulace genové exprese MeSH
- systémy cílené aplikace léků * MeSH
- viabilita buněk MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Arsenic compounds belong to the most controversial agents concerning human health. Arsenic (As) is considered as a top environmental element influencing human health due to its adverse effects including cancer, diabetes, cardiovascular disease, and reproductive or developmental problems. Despite the proven mutagenic, teratogenic and carcinogenic effects, the arsenic compounds are used for centuries to treat infectious diseases. In our work, we focused on studying of interactions of As(III) and/or As(V) with DNA. Interactions between arsenic ions and DNA were monitored by UV/vis spectrophotometry by measuring absorption and fluorescence spectra, atomic absorption spectrometry, electrochemical measurements (square wave voltammetry) and agarose gel electrophoresis. Using these methods, we observed a stable structure of DNA with As(III) within the concentration range 0.4-6.25 μg mL(-1). Higher As(III) concentration caused degradation of DNA. However, similar effects were not observed for As(V).
- MeSH
- antitumorózní látky chemie terapeutické užití MeSH
- arsen škodlivé účinky terapeutické užití MeSH
- DNA chemie účinky léků genetika MeSH
- fragmentace DNA účinky léků MeSH
- infekční nemoci farmakoterapie patologie MeSH
- ionty chemie MeSH
- lidé MeSH
- metalothionein chemie genetika MeSH
- spektrofotometrie atomová MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this study, we focused on the effect of heavy metal ions in resistant strains of gram-positive bacteria Staphylococcus aureus using biochemical methods and mass spectrometry. Five nitrate solutions of heavy metals (Ag(+), Cu(2+), Cd(2+), Zn(2+) and Pb(2+)) were used to create S. aureus resistant strains. Biochemical changes of resistant strains in comparison with the non-resistant control strain of S. aureus were observed by microbiological (measuring - growth curves and inhibition zones) and spectrophotometric methods (antioxidant activity and alaninaminotransferase, aspartateaminotransferase, alkaline phosphatase, γ-glutamyltransferase activities). Mass spectrometry was employed for the qualitative analysis of the samples (changes in S. aureus protein composition) and for the identification of the strains database MALDI Biotyper was employed. Alterations, in terms of biochemical properties and protein composition, were observed in resistant strains compared to non-resistant control strain. Our results describe the possible option for the analysis of S. aureus resistant strains and may thus serve as a support for monitoring of changes in genetic information caused by the forming of resistance to heavy metals.
- MeSH
- hmotnostní spektrometrie MeSH
- ionty metabolismus farmakologie MeSH
- kovy metabolismus farmakologie MeSH
- metabolom MeSH
- metabolomika metody MeSH
- metalothionein chemie metabolismus MeSH
- mikrobiální viabilita účinky léků MeSH
- RNA ribozomální genetika MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- Staphylococcus aureus účinky léků genetika růst a vývoj metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Interaction between semiconductor nanocrystals, cadmium telluride quantum dots (CdTe QDs) capped with mercaptosuccinic acid (MSA) and metallothionein (MT) was investigated. MSA-capped CdTe QDs were synthesized in aqueous solution. Mixture of MT and MSA-capped CdTe QDs has been investigated by various analytical methods as follows: tris-tricine gel electrophoresis, fluorescence evaluation and electrochemical detection of catalysed hydrogen evolution. The obtained results demonstrate that MSA-capped CdTe QDs and MT do not create firmly bound stabile complex. However, weak electrostatic interactions contribute to the interaction of MT with MSA-capped CdTeQDs. It can be concluded that QDs size influences the QDs and MT interaction. The smallest QDs had the highest affinity to MT and vice versa.
- MeSH
- elektrochemické techniky MeSH
- fluorescenční spektrometrie MeSH
- králíci MeSH
- kvantové tečky * MeSH
- metalothionein chemie MeSH
- molekulární struktura MeSH
- nanočástice * MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Metallomics is an area that studies the interactions of metal ions with proteins and other biomolecules and their function in living organisms. Metallothioneins (MTs) belong to a large family of metalloproteins in this field. As it is known, the presence of Cd2+ ions causes higher expression of MTs. Therefore we focused on determination of MT and Cd content in liver from chicken embryos (model organism) by electrochemistry and atomic absorption spectroscopy (AAS) after the exposure of chicken embryos to different concentrations of Cd(NO3)2. We also determined the spatial distribution of MT in chicken liver tissue slices by matrix assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF MSI) and evaluated the toxicity of Cd(NO3)2 to chicken embryos. Finally, we attempted to find the correlation between 2D MSI maps of MT and concentration of MT in chicken liver.
- MeSH
- elektrochemické techniky MeSH
- játra * chirurgie metabolismus účinky léků MeSH
- kadmium * metabolismus toxicita MeSH
- kuřecí embryo MeSH
- metalothionein * chemie metabolismus MeSH
- organokovové sloučeniny toxicita MeSH
- spektrofotometrie atomová MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- zvířata MeSH
- Check Tag
- kuřecí embryo MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Homeostatic mechanisms preventing the toxicity of free Zn ions in cells involve, among others, cytosolic Zn-binding ligands, particularly the cysteine-rich metallothioneins (MTs). Here we examined the Zn-binding peptides of Russula atropurpurea, an ectomycorrhizal fungus known for its ability to accumulate high amounts of Zn in its sporocarps. The Zn complexes and their peptide ligands were characterized using chromatography, electrophoresis after fluorescent labeling of cysteine residues, and tandem mass spectrometry. Functional complementation assays in Saccharomyces cerevisiae were used to obtain and characterize cDNA sequences. Zn-speciation analysis showed that nearly 80% of the Zn extracted from the sporocarps was associated with cysteine-containing peptides in a 5 kDa complex. Screening of an R. atropurpurea cDNA library for sequences encoding peptides capable of sequestering divalent heavy metals was conducted in the Cd-hypersensitive ycf1Δ yeast. This allowed identification of two cDNAs, RaZBP1 and RaZBP2, which protected the metal-sensitive yeast mutants against Cd and Zn, but not Co, Mn or Cu, toxicity. The corresponding RaZBP1 and RaZBP2 peptides consisting of 53 amino acid (AA) residues and sharing 77% identity showed only a limited sequence similarity to known MTs, particularly due to the absence of multiple Cys-AA-Cys motifs. Both RaZBPs were detected in a native Zn-complex of R. atropurpurea and the recombinant RaZBP1 was found associated with Zn and Cd in yeasts. Altogether, the results point to an important role of RaZBPs in the handling of a substantial portion of the Zn pool in R. atropurpurea.
- MeSH
- Basidiomycota metabolismus MeSH
- intracelulární prostor metabolismus MeSH
- kadmium metabolismus MeSH
- metalothionein chemie izolace a purifikace metabolismus MeSH
- molekulární sekvence - údaje MeSH
- mutace genetika MeSH
- mykorhiza metabolismus MeSH
- peptidy chemie izolace a purifikace metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- sekvence aminokyselin MeSH
- sekvenční analýza proteinů MeSH
- sekvenční seřazení MeSH
- zinek chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Metallothioneins (MTs) were discovered in 1957 and identified as low-molecular weight sulfhydryl-rich proteins. MTs belong to a superfamily of intracellular metal-binding proteins, present in virtually all living organisms, with features common to the archetypal. MT was first isolated from horse kidney and characterized by Margoshes and Vallee [1]. In this work, we wish to briefly summarize the current knowledge regarding the MT forms. All vertebrates examined contain two or more distinct MT isoforms designated MT-1 through MT-4. The three-dimensional structures of MTs from mammalian that have been determined so far show a monomeric protein composed of two globular domains, each encompassing a metal–thiolate cluster. The metallothionein isoform A (MTA) is a 64-residue metalloprotein, which contains essentially the same number of metal-chelating Cys–Cys and Cys–Xxx–Cys motifs (where Xxx stands for any amino acid, other than Cys) and metal ions [2, 3]. These cysteine-rich proteins are localized in cytoplasm and some organelles, predominantly in mitochondria, where their presence is sensitively and strictly regulated by the oxidative state induced by mitochondrial respiration. Depending on the cell state, but especially presence of oxidative stress, MTs are rapidly translocated to the nucleus through nuclear pore complexes. MT localized in the nuclei is oxidized there and it is transported to cytosol; this system is balanced [3].
- MeSH
- kovy MeSH
- lidé MeSH
- metalothionein * analýza fyziologie chemie MeSH
- nádorové biomarkery * analýza metabolismus MeSH
- nádory diagnóza metabolismus MeSH
- protein - isoformy MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
