Effect of UV irradiation on free radicals in different types of melanins and melanin complexes with diamagnetic Cd(II) and paramagnetic Cu(II) was examined by the use of electron paramagnetic resonance (EPR) spectroscopy. The aim of this studies was to compare o-semiquinone free radicals formation in two model eumelanins synthesized from 3,4-dihydroxyphenylalanine (DOPA) and tyrosine in the presence of tyrosinase, and in synthetic pheomelanin, under exposition on ultraviolet, because of the important role of free radicals and melanins in human organism. UV may change free radical concentrations in melanin. Changes in EPR spectra of DOPA-melanin-Cd(II) and DOPA-melanin-Cu(II) complexes after UV irradiation were determined. Diamagnetic Cd(II) strongly increased free radical concentrations in DOPA-melanin. UV irradiation during 30 and 60 min slightly increased and decreased free radical concentrations in DOPA-melanin-Cd(II) complexes, respectively. Paramagnetic Cu(II) quenched free radical lines of DOPA-melanin, and only the Cu(II) signals were detected for both UV-irradiated and nonirradiated samples. Free radical concentration in both eumelanins increased after UV irradiation, but it decreased in irradiated pheomelanin. EPR spectra of free radicals in the studied samples were homogeneously broadened. Slow spin-lattice relaxation processes exist in all the examined melanins and DOPA-melanin-Cd(II) complexes. Fast spin-lattice relaxation processes characterized Cu(II) in DOPA-melanin-Cu(II) complexes.

• MeSH
• dihydroxyfenylalanin účinky záření   MeSH
• elektronová paramagnetická rezonance * metody   využití   MeSH
• epidermis imunologie   účinky záření   MeSH
• lidé MeSH
• melaniny * izolace a purifikace   účinky záření   MeSH
• melanocyty imunologie   účinky záření   MeSH
• statistika jako téma MeSH
• tyrosinasa biosyntéza   účinky záření   MeSH
• ultrafialové záření * diagnostické užití   škodlivé účinky   MeSH
• volné radikály izolace a purifikace   škodlivé účinky   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

A combined modeling and spectroscopic approach is used to describe Cd(II), Cr(VI), and Pb(II) adsorption onto nanomaghemite and nanomaghemite coated quartz. A pseudo-second order kinetic model fitted the adsorption data well. The sorption capacity of nanomaghemite was evaluated using a Langmuir isotherm model, and a diffuse double layer surface complexation model (DLM) was developed to describe metal adsorption. Adsorption mechanisms were assessed using X-ray photoelectron spectroscopy and X-ray absorption spectroscopy. Pb(II) adsorption occurs mainly via formation of inner-sphere complexes, whereas Cr(VI) likely adsorbs mainly as outer-sphere complexes and Cd(II) as a mixture of inner- and outer-sphere complexes. The simple DLM describes well the pH-dependence of single adsorption edges. However, it fails to adequately capture metal adsorption behavior over broad ranges of ionic strength or metal-loading on the sorbents. For systems with equimolar concentrations of Pb(II), Cd(II), and Cr(VI). Pb(II) adsorption was reasonably well predicted by the DLM, but predictions were poorer for Cr(VI) and Cd(II). This study demonstrates that a simple DLM can describe well the adsorption of the studied metals in mixed sorbate-sorbent systems, but only under narrow ranges of ionic strength or metal loading. The results also highlight the sorption potential of nanomaghemite for metals in complex systems.

• MeSH
• adsorpce MeSH
• chrom chemie   MeSH
• fotoelektronová spektroskopie MeSH
• kadmium chemie   MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• křemen chemie   MeSH
• nanostruktury chemie   MeSH
• olovo chemie   MeSH
• osmolární koncentrace MeSH
• rentgenová absorpční spektroskopie MeSH
• teoretické modely * MeSH
• železité sloučeniny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: The majority of eukaryotic promoters utilize multiple transcription start sites (TSSs). How multiple TSSs are specified at individual promoters across eukaryotes is not understood for most species. In Saccharomyces cerevisiae, a pre-initiation complex (PIC) comprised of Pol II and conserved general transcription factors (GTFs) assembles and opens DNA upstream of TSSs. Evidence from model promoters indicates that the PIC scans from upstream to downstream to identify TSSs. Prior results suggest that TSS distributions at promoters where scanning occurs shift in a polar fashion upon alteration in Pol II catalytic activity or GTF function. RESULTS: To determine the extent of promoter scanning across promoter classes in S. cerevisiae, we perturb Pol II catalytic activity and GTF function and analyze their effects on TSS usage genome-wide. We find that alterations to Pol II, TFIIB, or TFIIF function widely alter the initiation landscape consistent with promoter scanning operating at all yeast promoters, regardless of promoter class. Promoter architecture, however, can determine the extent of promoter sensitivity to altered Pol II activity in ways that are predicted by a scanning model. CONCLUSIONS: Our observations coupled with previous data validate key predictions of the scanning model for Pol II initiation in yeast, which we term the shooting gallery. In this model, Pol II catalytic activity and the rate and processivity of Pol II scanning together with promoter sequence determine the distribution of TSSs and their usage.

• MeSH
• DNA-polymerasa II metabolismus   MeSH
• iniciace genetické transkripce * MeSH
• modely genetické MeSH
• počátek transkripce * MeSH
• promotorové oblasti (genetika) MeSH
• Saccharomyces cerevisiae enzymologie   genetika   MeSH
• transkripční faktory hlavní metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Mitochondria-targeting peptides represent an emergent tool for cancer inhibition. Here supramolecular assemblies of novel amphiphilic cell-penetrating peptides for targeting cancer cell mitochondria are reported. The employed strategy aims at amplifying the apoptotic stimuli by weakening the mitochondrial VDAC1 (voltage-dependent anion channel-1)-hexokinase-II (HK-II) interaction. Peptide engineering is performed with the N-terminus of the HK-II protein, which binds to VDAC1. First, a designed positively charged segment (pKV) is anchored to the specific 15 amino acid sequence (MIASHLLAYFFTELN) to yield a cell-penetrating peptide (pHK-pKV). Second, a lipid chain (Pal) is conjugated to the N-terminus of pHK-pKV in order to enhance the intracellular delivery of the HK-II scaffold. The self-assembly properties of these two synthetic peptides are investigated by synchrotron small-angle X-ray scattering (BioSAXS) and cryogenic transmission electron (cryo-TEM) imaging, which evidence the formation of nanoassemblies of ellipsoid-like shapes. Circular dichroism (CD) spectroscopy demonstrates the induction of partial α-helical structures in the amphiphilic peptides. Confocal microscopy reveals the specific mitochondrial location of Pal-pHK-pKV assemblies in human non-small cell lung cancer (NSCLC) A549 cells. The cytotoxicity and apoptotic studies indicate the enhanced bioactivity of Pal-pHK-pKV self-assembled reservoirs, which cause massive A549 cell death with regard to pHK-pKV. Of significance, Pal-pHK-pKV treatment of non-cancerous NCM460 cells resulted in substantially lower cytotoxicity. The results demonstrate the potential of self-assembled lipo-peptide (HK-II-derived) conjugates as a promising strategy in cancer therapy.

• MeSH
• buněčná smrt účinky léků   MeSH
• buňky A549 MeSH
• hexokinasa metabolismus   MeSH
• lékové transportní systémy metody   MeSH
• lidé MeSH
• lipidy chemie   MeSH
• lipopeptidy chemická syntéza   terapeutické užití   MeSH
• mitochondrie metabolismus   MeSH
• nádory plic farmakoterapie   patologie   MeSH
• napětím ovládaný aniontový kanál 1 metabolismus   MeSH
• penetrační peptidy chemická syntéza   metabolismus   MeSH
• povrchově aktivní látky metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH

A mononuclear cadmium(II) complex of formula [Cd(5,5'-dmbipy)2(OAc)2]·2H2O (5,5'-dmbipy = 5,5'-dimethyl-2,2'-bipyridine and OAc = acetato ligand) has been synthesized and characterized by FT-IR, UV-Vis, 1H-NMR, elemental analysis and single-crystal X-ray structure analysis. The molecular structure of the complex shows a distorted tetragonal antiprism CdN4O4 coordination geometry around the cadmium atom, resulting in coordination by four nitrogen atoms from two 5,5'-dmbipy ligands and four oxygen atoms from two acetate anions. The interaction of this complex to FS-DNA (fish sperm DNA) has also been studied by electronic absorption, fluorescence and gel electrophoresis techniques. Binding constant (Kb), Stern-Volmer constant (Ksv), number of binding sites (n) and bimolecular quenching rate constant (kq) have been calculated from these spectroscopic data. These results have revealed that the metal complex can bind effectively to FS-DNA via groove binding. The calculated thermodynamic parameters (ΔH°, ΔS° and ΔG°) show that hydrogen bonding and van der Waals forces have an important function in the Cd(II) complex-DNA interaction. The antibacterial effects of the synthesized cadmium complex have also been examined in vitro against standard bacterial strains: one Gram-positive (Staphylococcus aureus, ATCC 25923) and one Gram-negative (Escherichia coli, ATCC 25922) bacteria, using disk diffusion and macro-dilution broth methods. The obtained results show that the Cd(II) complex exhibits a marked antibacterial activity which is significantly better than those observed for its free ligand and metal salt for both Gram-positive and Gram-negative bacteria. However, this metal complex is a more potent antibacterial agent against the Gram-positive than that of the Gram-negative bacteria.Communicated by Ramaswamy H. Sarma.

• MeSH
• algoritmy MeSH
• antibakteriální látky chemie   farmakologie   MeSH
• Bacteria účinky léků   MeSH
• DNA chemie   MeSH
• kadmium chemie   MeSH
• krystalografie rentgenová MeSH
• ligandy MeSH
• magnetická rezonanční spektroskopie MeSH
• mikrobiální testy citlivosti MeSH
• molekulární modely * MeSH
• molekulární struktura MeSH
• pyridiny chemická syntéza   chemie   farmakologie   MeSH
• spektroskopie infračervená s Fourierovou transformací MeSH
• techniky syntetické chemie MeSH
• termodynamika MeSH
• vodíková vazba MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Comparing a parasitic lineage to its free-living relatives is a powerful way to understand how that evolutionary transition to parasitism occurred. Giardia intestinalis (Fornicata) is a leading cause of gastrointestinal disease world-wide and is famous for its unusual complement of cellular compartments, such as having peripheral vacuoles instead of typical endosomal compartments. Endocytosis plays an important role in Giardia's pathogenesis. Endosomal sorting complexes required for transport (ESCRT) are membrane-deforming proteins associated with the late endosome/multivesicular body (MVB). MVBs are ill-defined in G. intestinalis, and roles for identified ESCRT-related proteins are not fully understood in the context of its unique endocytic system. Furthermore, components thought to be required for full ESCRT functionality have not yet been documented in this species. RESULTS: We used genomic and transcriptomic data from several Fornicata species to clarify the evolutionary genome streamlining observed in Giardia, as well as to detect any divergent orthologs of the Fornicata ESCRT subunits. We observed differences in the ESCRT machinery complement between Giardia strains. Microscopy-based investigations of key components of ESCRT machinery such as GiVPS36 and GiVPS25 link them to peripheral vacuoles, highlighting these organelles as simplified MVB equivalents. Unexpectedly, we show ESCRT components associated with the endoplasmic reticulum and, for the first time, mitosomes. Finally, we identified the rare ESCRT component CHMP7 in several fornicate representatives, including Giardia and show that contrary to current understanding, CHMP7 evolved from a gene fusion of VPS25 and SNF7 domains, prior to the last eukaryotic common ancestor, over 1.5 billion years ago. CONCLUSIONS: Our findings show that ESCRT machinery in G. intestinalis is far more varied and complete than previously thought, associates to multiple cellular locations, and presents changes in ESCRT complement which pre-date adoption of a parasitic lifestyle.

• MeSH
• biologická evoluce MeSH
• endozomální třídící komplexy pro transport * genetika   metabolismus   MeSH
• endozomy metabolismus   MeSH
• Giardia lamblia * genetika   metabolismus   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The PsbO protein is an essential extrinsic subunit of photosystem II, the pigment-protein complex responsible for light-driven water splitting. Water oxidation in photosystem II supplies electrons to the photosynthetic electron transfer chain and is accompanied by proton release and oxygen evolution. While the electron transfer steps in this process are well defined and characterized, the driving forces acting on the liberated protons, their dynamics and their destiny are all largely unknown. It was suggested that PsbO undergoes proton-induced conformational changes and forms hydrogen bond networks that ensure prompt proton removal from the catalytic site of water oxidation, i.e. the Mn4 CaO5 cluster. This work reports the purification and characterization of heterologously expressed PsbO from green algae Chlamydomonas reinhardtii and two isoforms from the higher plant Solanum tuberosum (PsbO1 and PsbO2). A comparison to the spinach PsbO reveals striking similarities in intrinsic protein fluorescence and CD spectra, reflecting the near-identical secondary structure of the proteins from algae and higher plants. Titration experiments using the hydrophobic fluorescence probe ANS revealed that eukaryotic PsbO proteins exhibit acid-base hysteresis. This hysteresis is a dynamic effect accompanied by changes in the accessibility of the protein's hydrophobic core and is not due to reversible oligomerization or unfolding of the PsbO protein. These results confirm the hypothesis that pH-dependent dynamic behavior at physiological pH ranges is a common feature of PsbO proteins and causes reversible opening and closing of their β-barrel domain in response to the fluctuating acidity of the thylakoid lumen.

• MeSH
• Chlamydomonas reinhardtii metabolismus   MeSH
• fotosystém II (proteinový komplex) metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• Spinacia oleracea metabolismus   MeSH
• tylakoidy metabolismus   MeSH
• vodíková vazba MeSH
• Publikační typ
• časopisecké články MeSH

Silicon was shown to alleviate the negative effects of various biotic and abiotic stresses on plant growth. Although the positive role of Si on toxic and heavy metal Cd has been already described, the mechanisms have been explained only partially and still remain unclear. In the present study we investigated the effect of Si on photosynthetic-related processes in maize exposed to two different levels of Cd via measurements of net photosynthetic rate (AN), chlorophyll a fluorescence and pigment analysis, as well as studies of leaf tissue anatomy and cell ultrastructure using bright-field and transmission electron microscopy. We found that Si actively alleviated the toxic syndromes of Cd by increasing AN, effective photochemical quantum yield of photosystem II (ϕPSII) and content of assimilation pigments, although did not decrease the concentration of Cd in leaf tissues. Cadmium did not affect the leaf anatomy and ultrastructure of leaf mesophyll's cell chloroplasts; however, Cd negatively affected thylakoid formation in chloroplasts of bundle sheath cells, and this was alleviated by Si. Improved thylakoid formation in bundle sheath's cell chloroplasts may contribute to Si-induced enhancement of photosynthesis and related increase in biomass production in C4 plant maize.

• MeSH
• chlorofyl analogy a deriváty   metabolismus   MeSH
• chloroplasty účinky léků   metabolismus   ultrastruktura   MeSH
• fluorescence MeSH
• fotosyntéza účinky léků   MeSH
• fotosystém II (proteinový komplex) metabolismus   MeSH
• kadmium toxicita   MeSH
• křemík farmakologie   MeSH
• kukuřice setá účinky léků   metabolismus   ultrastruktura   MeSH
• listy rostlin účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cadmium (Cd) is an important environmental pollutant and is poisonous to most organisms. We aimed to unravel the mechanisms of Cd toxicity in the model water plant Ceratophyllum demersum exposed to low (nM) concentrations of Cd as are present in nature. Experiments were conducted under environmentally relevant conditions, including nature-like light and temperature cycles, and a low biomass to water ratio. We measured chlorophyll (Chl) fluorescence kinetics, oxygen exchange, the concentrations of reactive oxygen species and pigments, metal binding to proteins, and the accumulation of starch and metals. The inhibition threshold concentration for most parameters was 20 nM. Below this concentration, hardly any stress symptoms were observed. The first site of inhibition was photosynthetic light reactions (the maximal quantum yield of photosystem II (PSII) reaction centre measured as Fv /Fm , light-acclimated PSII activity ΦPSII , and total Chl). Trimers of the PSII light-harvesting complexes (LHCIIs) decreased more than LHC monomers and detection of Cd in the monomers suggested replacement of magnesium (Mg) by Cd in the Chl molecules. As a consequence of dysfunctional photosynthesis and energy dissipation, reactive oxygen species (superoxide and hydrogen peroxide) appeared. Cadmium had negative effects on macrophytes at much lower concentrations than reported previously, emphasizing the importance of studies applying environmentally relevant conditions. A chain of inhibition events could be established.

• MeSH
• fotosyntéza * MeSH
• fotosystém II (proteinový komplex) metabolismus   MeSH
• kadmium toxicita   MeSH
• Magnoliopsida účinky léků   fyziologie   účinky záření   MeSH
• peroxid vodíku metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• superoxidy metabolismus   MeSH
• světlo MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: The diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) relies on flow cytometric demonstration of loss of glycosyl-phosphatidyl inositol (GPI)-anchored proteins from red blood cells (RBC) and white blood cells (WBC). High-sensitivity multiparameter assays have been developed to detect loss of GPI-linked structures on PNH neutrophils and monocytes. High-sensitivity assays to detect PNH phenotypes in RBCs have also been developed that rely on the loss of GPI-linked CD59 on CD235a-gated mature RBCs. The latter is used to delineate PNH Type III (total loss of CD59) and PNH Type II RBCs (partial loss of CD59) from normal (Type I) RBCs. However, it is often very difficult to delineate these subsets, especially in patients with large PNH clones who continue to receive RBC transfusions, even while on eculizumab therapy. METHODS: We have added allophycocyanin (APC)-conjugated CD71 to the existing CD235aFITC/CD59PE RBC assay allowing simultaneous delineation and quantification of PNH Type III and Type II immature RBCs (iRBCs). RESULTS: We analyzed 24 medium to large-clone PNH samples (>10% PNH WBC clone size) for PNH Neutrophil, PNH Monocyte, Type III and Type II PNH iRBCs, and where possible, Type III and Type II PNH RBCs. The ability to delineate PNH Type III, Type II, and Type I iRBCs was more objective compared to that in mature RBCs. Additionally, total PNH iRBC clone sizes were very similar to PNH WBC clone sizes. CONCLUSIONS: Addition of CD71 significantly improves the ability to analyze PNH clone sizes in the RBC lineage, regardless of patient hemolytic and/or transfusion status.

• MeSH
• antigeny CD59 metabolismus   MeSH
• buněčná diferenciace MeSH
• CD antigeny krev   fyziologie   MeSH
• diferenciální diagnóza MeSH
• erytrocyty metabolismus   patologie   MeSH
• glykoforin metabolismus   MeSH
• imunofenotypizace přístrojové vybavení   metody   normy   MeSH
• kohortové studie MeSH
• leukocyty patologie   MeSH
• lidé MeSH
• monocyty metabolismus   patologie   MeSH
• neutrofily metabolismus   patologie   MeSH
• paroxysmální hemoglobinurie krev   klasifikace   diagnóza   patologie   MeSH
• počet leukocytů přístrojové vybavení   metody   MeSH
• průtoková cytometrie přístrojové vybavení   metody   normy   MeSH
• receptory transferinu krev   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH