Polyamidoamine dendrimer impairs mitochondrial oxidation in brain tissue

Background: The potential nanocarrier polyamidoamine (PAMAM) generation 5 (G5-NH2) dendrimer has been shown to evoke lasting neuronal depolarization and cell death in a concentration-dependent manner. In this study we explored the early progression of G5-NH2 action in brain tissue on neuronal and astroglial cells.Results: In order to describe early mechanisms of G5-NH2 dendrimer action in brain tissue we assessed G5-NH2 trafficking, free intracellular Ca2+ and mitochondrial membrane potential (ΨMITO) changes in the rat hippocampal slice by microfluorimetry. With the help of fluorescent dye conjugated G5-NH2, we observed predominant appearance of the dendrimer in the plasma membrane of pyramidal neurons and glial cells within 30 min. Under this condition, G5-NH2 evoked robust intracellular Ca2+ enhancements and ΨMITO depolarization both in pyramidal neurons and astroglial cells. Intracellular Ca2+ enhancements clearly preceded ΨMITO depolarization in astroglial cells. Comparing activation dynamics, neurons and glia showed prevalence of lasting and transient ΨMITO depolarization, respectively. Transient as opposed to lasting ΨMITO changes to short-term G5-NH2 application suggested better survival of astroglia, as observed in the CA3 stratum radiatum area. We also showed that direct effect of G5-NH2 on astroglial ΨMITO was significantly enhanced by neuron-astroglia interaction, subsequent to G5-NH2 evoked neuronal activation.Conclusion: These findings indicate that the interaction of the PAMAM dendrimer with the plasma membrane leads to robust activation of neurons and astroglial cells, leading to mitochondrial depolarization. Distinguishable dynamics of mitochondrial depolarization in neurons and astroglia suggest that the enhanced mitochondrial depolarization followed by impaired oxidative metabolism of neurons may be the primary basis of neurotoxicity. © 2013 Nyitrai et al.; licensee BioMed Central Ltd

Influence of acid-induced conformational variability on protein separation in reversed phase high performance liquid chromatography

Influence of acid concentration in the mobile phase on protein separation was studied in a wide concentration range of using trifluoroacetic acid (TFA) and formic acid (FA). At low, 0.001-0.01 v/v% TFA concentration and appropriate solvent strength proteins elute before the column's dead time. This is explained by the proteins having a structured, but relatively extended conformation in the eluent; and are excluded from the pores of the stationary phase. Above ca. 0.01-0.05 v/v% TFA concentration proteins undergo further conformational change, leading to a compact, molten globule-like structure, likely stabilized by ion pairing. Proteins in this conformation enter the pores and are retained on the column. The results suggest a pore exclusion induced separation related to protein conformation. This effect is influenced by the pH and type of acid used, and is likely to involve ion-pair formation. The TFA concentration needed to result in protein folding (and therefore to observe retention on the column) depends on the protein; and therefore can be utilized to improve chromatographic performance. Conformation change was monitored by circular dichroism spectroscopy and mass spectrometry; and it was shown that not only TFA, but FA can also induce molten globule formation

Synthesis and serum protein binding of novel ring-substituted harmine derivatives

A series of new derivatives of the natural β-carboline alkaloid harmine, introducing hydrophobic substituents into positions 7 and 9 were synthetized as potential anticancer agents. Their binding affinities for human serum albumin (HSA) and α1-acid glycoprotein (AAG) were investigated by affinity chromatography combined with fluorescence, circular dichroism (CD) and UV absorption spectroscopy. The weak binding of harmine to both proteins (Ka ~ 3 × 104 M-1) was highly increased by aromatic substitutions (Ka ~ 105-106 M-1). Derivatives having a substituted benzyl group in the N9-position of the β-carboline nucleus showed about tenfold and hundredfold affinity enhancement for HSA and AAG, respectively. Such a strong plasma protein interaction would be of pharmacokinetic relevance for these potential drug candidates. Induced CD spectra indicated the variant selective, dimeric binding of the 7-pyridylethoxy derivative to AAG. Absorbance and fluorescence spectra refer to the binding preference of the neutral form of the studied β-carbolines for both proteins

The Janus facet of nanomaterials

Application of nanoscale materials (NMs) displays a rapidly increasing trend in electronics, optics, chemical catalysis, biotechnology, and medicine due to versatile nature of NMs and easily adjustable physical, physicochemical, and chemical properties. However, the increasing abundance of NMs also poses significant new and emerging health and environmental risks. Despite growing efforts, understanding toxicity of NMs does not seem to cope with the demand, because NMs usually act entirely different from those of conventional small molecule drugs. Currently, large-scale application of available safety assessment protocols, as well as their furthering through case-by-case practice, is advisable. We define a standard work-scheme for nanotoxicity evaluation of NMs, comprising thorough characterization of structural, physical, physicochemical, and chemical traits, followed by measuring biodistribution in live tissue and blood combined with investigation of organ-specific effects especially regarding the function of the brain and the liver. We propose a range of biochemical, cellular, and immunological processes to be explored in order to provide information on the early effects of NMs on some basic physiological functions and chemical defense mechanisms. Together, these contributions give an overview with important implications for the understanding of many aspects of nanotoxicity

Development of novel chiral capillary electrophoresis methods for the serotonin receptor (5-HT2A) antagonist MDL 100,907 (volinanserin) and for its key intermediate compound

Enantioselective capillary electrophoretic methods were elaborated for the determination of the enantiomeric purity of (R)-MDL 100,907 and its preparatively resolved key intermediate compound during the synthesis route. The pKa values of the intermediate compound and the end product determined by CE were 10.5±0.1 and 9.0±0.1, respectively. The enantiopurity of the intermediate compound can be monitored in fully protonated state by applying 15mM sulfobutylether-β-cyclodextrin at pH 5 when the peak belonging to the impurity migrates before the main component. The fact that the consecutive steps of the synthesis do not affect the enantiomeric purity was verified by the other, newly developed CE method. The enantiomers of rac-MDL 100,907 were resolved by 15mM carboxymethyl-γ-cyclodextrin at pH 3. The applicability (selectivity, LOD, LOQ, repeatability, precision and accuracy) of the methods was studied as well

Biológiailag jelentős nem-kovalens kölcsönhatások tanulmányozása: fehérje-kötődés, nukleinsav-kötődés, önszerveződés = Investigations on non-covalent interactions of biological importance: protein binding, nucleic acid binding, self-assembly

Nem-kovalens kölcsönhatások (fehérjekötődés, aggregáció) kisérleti vizsgálatára UV-Vis, fluoreszcencia és kiroptikai spektroszkópiai módszereket alkalmaztak, a vizsgálati minták tisztítását kromatográfiás (HPLC, kapilláris elektroforézis) technikákkal végezték. A humán vérplazma minor fehérje komponensének (alfa-1 savas glikoprotein, AGP) részletes vizsgálata kimutatta, hogy az AGP genetikai variánsai eltérő, a kötődési erősségtől függő módon csökkentik a myeloid leukémia gyógyszerének (Imatinib, Glivec', Novartis) hatásosságát. Daganatos betegek klinikai vérmintáiból kidolgozták nagy tisztaságú AGP kinyerését és kapilláris elektroforézissel igazolták, hogy az AGP cukorláncának változatossága (glycoform heterogeneity) különféle daganatos megbetegedések érzékeny markere. Enantiomerek elválasztására egy ciklodextrin származékot tartalmazó új királis állófázist fejlesztettek ki. Megfigyelték, hogy AGP kötődésben királis inverzió következhet be. A kiroptikai spektrumokban megjelenő exciton sávpár intenzitását nem-kovalens kölcsönhatások esetére elméleti alapon indokolták. | UV-Vis, fluorescence and chiroptical spectroscopic methods have been applied for experimental studies of non-covalent interactions, the purification of the samples were done by chromatographic (HPLC, capillary electrophoresis) techniques. Detailed studies of the minor protein component of human blood plasma, alpha-1 acid glycoprotein (AGP) revealed that the decrease in the efficiency of the medication against chronic myelogenous leukaemia, Imatinib (Glivec', Novartis) brought about by the genetic variants of AGP was in correlation with their binding strength. Highly purified AGP samples were prepared from clinical blood samples of cancer patients and their capillary electrophoretic analysis proved the variability of sugar-chain of AGP (glycoform heterogeneity) to be a sensitive marker of several malignant diseases. New chiral stationary phase containing a cyclodextrin derivative has been developed for enantiomer separation. Chiral inversion has been demonstrated in AGP binding. The intensity of exciton couplets appearing in chiroptical spectra has been explained on theoretical grounds for non-covalent interactions

Synthesis and pharmacological investigation of new N-hydroxyalkyl-2-aminophenothiazines exhibiting marked MDR inhibitory effect

Novel N-hydroxyalkyl-2-aminophenothiazines implying a tetrazole moiety at the alkyl chain have been synthesized by hydroboration-oxidation of dienes followed by Buchwald-Hartwig cross-coupling reaction. Also, some sulfoxide and sulfone derivatives have been prepared by selective oxidations. MDR inhibition studies on rat hepatocyte cell culture revealed that some derivatives exhibit marked biological efficacy exceeding that of the standard verapamil (e.g. 3h, 4h, 16). Selected derivatives were subjected to chemical resolution to provide both enantiomers which were shown of similar activity on P-gp interaction measurements. The new compounds exhibited no toxicit

Cryptocapsinepoxide-type Carotenoids from Red Mamey, Pouteria sapota

Three new carotenoids, cryptocapsin-5,6-epoxide, 3ʹ-deoxycapsanthin-5,6-epoxide, and cryptocapsin-5,8-epoxides, have been isolated from the ripe fruits of red mamey (Pouteria sapota). Cryptocapsin-5,6-epoxide was prepared by partial synthesis via epoxidation of cryptocapsin and the (5R,6S)- and (5S,6R)-stereoisomers were identified by HPLC-ECD analysis. Spectroscopic data of the natural (anti) and semisynthetic (syn) derivatives obtained by acid-catalyzed rearrangement of cryptocapsin-5,8-epoxide stereoisomers were compared for structural elucidation. Chiral HPLC separation of natural and semisynthetic samples of cryptocapsin-5,8-epoxides was performed and HPLC-ECD analysis allowed configurational assignment of the separated stereoisomers