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Longstaff C.,UK National Institute for Biological Standards and Control | Varju I.,Semmelweis University | Sotonyi P.,Semmelweis University | Szabo L.,Institute of Materials and Environmental Chemistry | And 6 more authors.
Journal of Biological Chemistry | Year: 2013

Background: Neutrophil extracellular traps (NETs) composed of DNA and proteins form a scaffold in thrombi, supplementing the fibrin matrix. Results: DNA and histones modify the structure of fibrin and render it resistant to mechanical and enzymatic destruction. Conclusion: NET components are essential factors in thrombus stability. Significance: Therapeutic strategies could be optimized to enhance fibrinolysis in clots containing DNA and histones. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.


Hoell A.,Helmholtz Center Berlin | Varga Z.,Institute of Molecular Pharmacology | Raghuwanshi V.S.,Humboldt University of Berlin | Krumrey M.,Physikalisch - Technische Bundesanstalt | And 2 more authors.
Journal of Applied Crystallography | Year: 2014

The formation and growth of nanosized CaF2 crystallites by heat treatment of an oxyfluoride glass of composition 7.65Na2O-7.69K2O-10.58CaO-12.5CaF2-5. 77Al2O3-55.8SiO2 (wt%) was investigated using anomalous small-angle X-ray scattering (ASAXS). A recently developed vacuum version of the hybrid pixel detector Pilatus 1M was used for the ASAXS measurements below the Ca K-edge of 4038 eV down to 3800 eV. ASAXS investigation allows the determination of structural parameters such as size and size distribution of nanoparticles and characterizes the spatial distribution of the resonant element, Ca. The method reveals quantitatively that the growing CaF2 crystallites are surrounded by a shell of lower electron density. This depletion shell of growing thickness hinders and finally limits the growth of CaF2 crystallites. Moreover, in samples that were annealed for 10 h and more, additional very small heterogeneities (1.6 nm diameter) were found. © 2014 International Union of Crystallography.


Szabo M.,Institute of Molecular Pharmacology | Veres Z.,Institute of Molecular Pharmacology | Batai-Konczos A.,Institute of Molecular Pharmacology | Kekesi O.,Institute of Molecular Pharmacology | And 3 more authors.
Toxicology in Vitro | Year: 2014

Several studies have reported that statins occasionally cause impairment of liver functions characterized by elevated serum bilirubin levels, which might be due to altered function of the multidrug resistance-associated proteins (Mrp2/3). We aimed to study the modulation of the hepatobiliary transport of bilirubin by four statin derivatives, atorvastatin, fluvastatin, pravastatin, and rosuvastatin in sandwich-cultured rat hepatocytes. All statins except pravastatin significantly inhibited the uptake of bilirubin. The biliary efflux of bilirubin conjugates was increased by pravastatin and rosuvastatin concentration dependently. Rosuvastatin stimulated not only the Mrp2 mediated biliary, but the Mrp3 mediated sinusoidal elimination, resulting in decreased intracellular bilirubin accumulation. The significantly induced Mrp2/3 protein levels (ranging from 1.5 to 1.8-fold) accounted for the elevated efflux. Cell polarization, the formation of biliary network was also significantly increased by fluvastatin, pravastatin and rosuvastatin (151%, 216% and 275% of the control, respectively). The simultaneous inhibition of the uptake and the stimulation of the sinusoidal and canalicular elimination may explain, at least in part, the clinical observation of elevated serum bilirubin levels. In conclusion, our results suggest that in spite of the elevated serum bilirubin levels, the altered Mrp2 and Mrp3 functions by statins is probably not associated with hepatotoxic effects. © 2014.


Du L.,CAS Institute of Chemistry | Huang S.,CAS Institute of Chemistry | Zhuang Q.,CAS Institute of Chemistry | Jia H.,CAS Institute of Chemistry | And 4 more authors.
Nanoscale | Year: 2014

The detection of free radicals and related species has attracted significant attention in recent years because of their critical roles in physiological and pathological processes. Among the methods for the detection of free radicals, electron spin resonance (ESR) coupled with the use of the spin trapping technique has been an effective approach for characterization and quantification of these species due to its high specificity. However, its application in biological systems, especially in in vivo systems, has been greatly limited partially due to the low reaction rate between the currently available spin traps with biological radicals. To overcome this drawback, we herein report the first example of nitrone functionalized gold nanoparticles (Au@EMPO) as highly efficient spin traps in which the thiolated EMPO (2-(ethoxycarbonyl)-2-methyl-3,4-dihydro-2H-pyrrole 1-oxide) derivative was self-assembled on gold nanoparticles. Kinetic studies showed that Au@EMPO has a 137-fold higher reaction rate constant with OH than PBN (N-tert-butyl-α- phenylnitrone). Owing to the high rate of trapping OH by Au@EMPO as well as the high stability of the resulting spin adduct (t1/2 ∼ 56 min), Au@EMPO affords 124-fold higher sensitivity for OH than EMPO. Thus, this new nanospin trap shows great potential in trapping the important radicals such as OH in various biological systems and provides a novel strategy to design spin traps with much improved properties. © 2013 The Royal Society of Chemistry.


Telbisz A.,Institute of Molecular Pharmacology | Ozvegy-Laczka C.,National Blood Center | Hegedus T.,National Blood Center | Hegedus T.,Hungarian Academy of Sciences | And 5 more authors.
Biochemical Journal | Year: 2013

The human ABCG2 multidrug transporter actively extrudes a wide range of hydrophobic drugs and xenobiotics recognized by the transporter in the membrane phase. In order to examine the molecular nature of the transporter and its effects on the lipid environment, we have established an efficient protocol for the purification and reconstitution of the functional protein.We found that the drug-stimulated ATPase and the transport activity of ABCG2 are fully preserved by applying excess lipids and mild detergents during solubilization, whereas a detergent-induced dissociation of the ABCG2 dimer causes an irreversible inactivation. By using the purified and reconstituted protein we demonstrate that cholesterol is an essential activator, whereas bile acids are important modulators ofABCG2 activity. Bothwild-type ABCG2 and its R482G mutant variant require cholesterol for full activity, although they exhibit different cholesterol sensitivities. Bile acids strongly decrease the basal ABCG2-ATPase activity both in the wild-type ABCG2 and in the mutant variant. These data reinforce the results for the modulatory effects of cholesterol and bile acids of ABCG2 investigated in a complex cellmembrane environment. Moreover, these experiments open the possibility to perform functional and structural studies with a purified, reconstituted and highly active ABCG2 multidrug transporter. © 2013 Biochemical Society.


Arus D.,University of Szeged | Dancs A.,University of Szeged | Nagy N.V.,Institute of Molecular Pharmacology | Gajda T.,University of Szeged
Dalton Transactions | Year: 2013

The brain specific zinc transporter protein ZnT3 can be related to the amyloid neuropathology of Alzheimer's disease. In order to analyze the metal binding ability of human ZnT3 protein, here we report a potentiometric and solution structural (UV-Vis, CD, EPR, NMR) study of nickel(ii), copper(ii) and zinc(ii) complexes of three peptides mimicking the possible metal binding sequences of this protein. The peptide L1 (Ac-RHQAGPPHSHR-NH 2) is a minimalist, the cyclic peptide L2 (cyclo(Ac-CKLHQAGPPHSHGSRGAEYAPLEEGPEEKC-NH2) is a more complete model of the intracellular His-rich loop, which is widely accepted as a putative metal binding site. The peptide L3 (Ac-PFHHCHRD-NH2) is the model of the conserved cytoplasmic N-terminal -HHCH- sequence. In the physiological pH-range, the ZnL1, ZnH3L2 and ZnL3 complexes are the major species in the corresponding binary systems, with {3Nim}, {3Nim,2/3Oamide} and {3Nim,S-} coordination environments, respectively. The species ZnL3 has 3-4 orders of magnitude higher stability than the other two complexes, indicating the presence of a high-affinity zinc-binding site at the N-terminal tail of the human ZnT3 transporter. Moreover, L 3 shows preferred zinc binding as compared to nickel (log β(ZnL3) - log β(NiL3) = 2.3), probably due to the higher preference of zinc(ii) for tetrahedral geometry. These facts suggest that zinc binding to the N-terminal -HHCH- sequence of human ZnT3 may be involved in the biological activity of this zinc transporter protein in zinc sensing, binding or translocation processes. This journal is © The Royal Society of Chemistry.


Zsila F.,Institute of Molecular Pharmacology
Journal of Physical Chemistry B | Year: 2013

This work demonstrates for the first time that binding of various compounds within subdomain IB of human serum albumin (HSA) provokes characteristic changes in the near-UV circular dichroism (CD) spectrum of the protein. It can be inferred from the spectroscopic features of difference ellipticity signals and from CD displacement experiments that tyrosine residues located in subdomain IB are the source of the observed spectral alterations. It is proposed that inclusion of some ligand molecules (bile acids, dehydroepiandrosterone sulfate, steroidal terpenes, fatty acids, ibuprofen, and gemfibrozil) into the pocket of subdomain IB disrupts the Tyr138-Tyr161 interhelical π-π stacking interaction, which is reflected in the CD spectrum. This phenomenon can be utilized for the CD detection of subdomain IB specific binding of endo- as well as exogenous agents and to study the drug binding associated local conformational adaptation of the HSA molecule. © 2013 American Chemical Society.


Zsila F.,Institute of Molecular Pharmacology
Molecular Pharmaceutics | Year: 2013

According to the conventional view, noncovalent association of small molecules with human serum albumin (HSA) occurs principally at the so-called Sudlow's sites located in subdomain IIA and IIIA. By employing a circular dichroism (CD) spectroscopic approach, it is shown that biliverdin is the specific CD label of an additional drug binding area in subdomain IB. CD competition experiments disclosed the entrapment of a diverse assortment of acidic, neutral, and basic molecules within subdomain IB including anticancer agents (camptothecin, doxorubicin, daunorubicin, teniposide, suramin, tyrosine kinase inhibitors), anticoagulants (dicoumarol), various steroids (bile acids, carbenoxolone), nonsteroidal antiinflammatory drugs, natural substances (aristolochic acid, glycyrrhetinic acid), and synthetic dyes (methyl orange, azocarmine B). These finding imply that subdomain IB can be considered as the third major drug binding region of HSA featured with promiscuous ligand recognition ability. Additionally, subdomain IB is allosterically coupled with the Sudlow's sites, the ligand binding of which is shown to alter the HSA binding mode and affinity of biliverdin and hemin. Brief case studies are presented to illustrate how the evaluation of spectral changes of tetrapyrrole CD probes gains new insight into the HSA binding properties of endogenous as well as pharmaceutical compounds. © 2013 American Chemical Society.


Hegyi B.,Institute of Molecular Pharmacology | Kornyei Z.,Institute of Experimental Medicine | Ferenczi S.,Institute of Experimental Medicine | Fekete R.,Institute of Experimental Medicine | And 5 more authors.
Stem Cells and Development | Year: 2014

Mesenchymal stems or stromal cells (MSCs) are rare multipotent cells with potent regenerative and immunomodulatory properties. Microglial cells (MGs) are specialized tissue macrophages of the central nervous system (CNS) that continuously survey their environment with highly motile extensions. Recently, several studies have shown that MSCs are capable of reprogramming microglia into an "M2-like" phenotype characterized by increased phagocytic activity and upregulated expression of anti-inflammatory mediators in vitro. However, the precise polarization states of microglia in the presence of MSCs under physiological or under inflammatory conditions remain largely unknown. In this study, we found that MSCs induce a mixed microglia phenotype defined as Arg1-high, CD86-high, CD206-high, IL-10-high, PGE2-high, MCP-1/CCL2-high, IL-1β-moderate, NALP-3-low, and TNF-α-low cells. These MSC-elicited MGs have high phagocytic activity and antigen-presenting ability. Lipopolysaccharide is able to shape this microglia phenotype quantitatively, but not qualitatively in the presence of MSCs. This unique polarization state resembles a novel regulatory microglia phenotype, which might contribute to the resolution of inflammation and to tissue repair in the CNS. © Mary Ann Liebert, Inc. 2014.


Heja L.,Institute of Molecular Pharmacology
Current Medicinal Chemistry | Year: 2014

Although glial proliferation of the epileptic loci is recognized for more than a century in certain focal epilepsies, the role of astrocytes in epileptic conditions is receiving significant attention only in recent years. The present review will highlight current knowledge about the various ways astrocytes control neuronal excitability and contribute to genesis, maintenance and suppression of seizures. Besides the widely recognized astrocytic tasks like glutamate clearance, the role of gliotransmission, glutamate, GABA and ATP release as well as gap junctional communication will also be discussed along with the contribution of blood-brain barrier dysfunction, inflammatory pathways and alterations in mircoRNA expression profile to epilepsy. The mechanisms described will help to understand the astrocytic mechanisms contributing to the antiepileptic effect of existing anti-epileptic drugs (AEDs) and current therapeutic strategies and also signifies the potential of specific astrocyte-based AED development. © 2014 Bentham Science Publishers.

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