First record of picophytoplankton diversity in Central European hypersaline lakes

Our survey has revealed that the phytoplankton in the anthropo-hypersaline lakes of the Transylvanian Basin (Romania) was often dominated by photoautotrophic picoplankton (PPP, cells with a diameter <2?μm). Therefore, the aim of this study was to identify PPP members both in the summer and the winter communities using molecular biological techniques, denaturing gradient gel electrophoresis (DGGE) and sequence analysis. The applied PCR-DGGE methods were highly specific to cyanobacteria and green algae. A total of 11 different plankton taxa were identified that were related to several distant taxonomic groups. PPP were represented by a simple community and consisted of two major genotypes, one from the green algal species Picochlorum oklahomense and the other related to marine Synechococcus isolates (Cyanobacteria). These marine PPP species were recorded for the first time in inland saline lakes from Europe. Besides picoplankton, several additional marine taxa (e.g. cryptophytes and haptophytes) were detected among the nanoplankton species. The presence of the identified marine and hypersaline species could be explained by wind, precipitation or waterfowl transfer; however, this latter could have smaller importance.     

Membrane cholesterol selectively modulates the activity of the human ABCG2 multidrug transporter

The human ABCG2 multidrug transporter provides protection against numerous toxic compounds and causes multidrug resistance in cancer. Here we examined the effects of changes in membrane cholesterol on the function of this protein. Human ABCG2 was expressed in mammalian and in Sf9 insect cells, and membrane cholesterol depletion or enrichment was achieved by preincubation with beta cyclodextrin or its cholesterol-loaded form. We found that mild cholesterol depletion of intact mammalian cells inhibited ABCG2-dependent dye and drug extrusion in a reversible fashion, while the membrane localization of the transporter protein was unchanged. Cholesterol enrichment of cholesterol-poor Sf9 cell membrane vesicles greatly increased ABCG2-driven substrate uptake, substrate-stimulated ATPase activity, as well as the formation of a catalytic cycle intermediate (nucleotide trapping). Interestingly, modulation of membrane cholesterol did not significantly affect the function of the R482G or R482T substrate mutant ABCG2 variants, or that of the MDR1 transporter. The selective, major effect of membrane cholesterol on the wild-type ABCG2 suggests a regulation of the activity of this multidrug transporter during processing or in membrane micro-domain interactions. The experimental recognition of physiological and pharmacological substrates of ABCG2, as well as the fight against cancer multidrug resistance may be facilitated by demonstrating the key role of membrane cholesterol in this transport activity.     

Synthesis and characterization of PAMAM dendrimer-based multifunctional nanodevices for targeting alphavbeta3 integrins

We have synthesized a stable and clinically relevant nanodevice (cRGD-BT-ND; ND for short) that exhibits superior binding to the biologic target alphavbeta3 integrins, when either compared to the same free cRGD peptide or to the biotinylated nanodevice without covalently attached peptides (BT-ND). Selective targeting of alphavbeta3 integrins was achieved by coupling cyclic cRGD peptides to the nanodevice (ND) surface, while biotin groups (BT) were used for amplified detection of bound cRGD-BT-ND by anti-biotin antibody or avidin linked to horseradish peroxidase after binding. The synthesis involved the following steps: the amino-terminated ethylenediamine core generation 5 poly(amidoamine) (PAMAM_E5.NH2) dendrimer was first partially acetylated and then biotinylated, and residual primary amine termini were converted to succinamic acid groups (SAH), some of which finally were conjugated with cRGD peptide residues through the amino group of the lysine side chain. The starting material and all derivatives were extensively characterized by polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography (SEC), potentiometric acid-base titration, MALDI-TOF, and NMR. Cytotoxicity of all dendrimer derivatives was examined in B16F10 melanoma cell cultures using the XTT colorimetric assay for cellular viability. Binding of nanodevices to the biological target was determined using plates coated with human alphavbeta3 integrin and alphavbeta3 receptor expressing human dermal microvascular endothelial cells (HDMECs). The PAMAM_E5.(NHAc)72(NHBT)8(NHSAH)35(NHSA-cR GD)4 nanodevice is nontoxic within physiologic concentration ranges and specifically binds to the alphavbeta3 integrins, apparently much stronger than the cyclic cRGD peptide itself.     

Intranasal PUVA phototherapy in nasal polyposis--a pilot study

Nasal polyposis (NP) affects 4% of the general population, representing a major health problem. In spite of complex (surgical and medical) treatment, the relapse rate is high and it has a negative impact on the quality of life. Recently we found that intranasal photochemotherapy with ultraviolet A light (PUVA) is effective in allergic rhinitis. In the present study PUVA was administered for 6 weeks in 7 patients with NP. Nasal lavages were performed in all patients before and at the end of the treatment; from four patients a biopsy specimen was also collected. Eosinophils significantly decreased in patients with NP and slightly in a patient who had associated aspirin sensitivity. IL-5 and eosinophil cationic protein (ECP) levels showed a decreasing trend in patients with NP and an increasing trend in patients with associated aspirin sensitivity. Our results suggest that intranasal PUVA might represent a future therapeutic method in a subset of patients with NP.     

Suppression of neuronal network excitability and seizure-like events by 2-methyl-4-oxo-3H-quinazoline-3-acetyl piperidine in juvenile rat hippocampus: involvement of a metabotropic glutamate receptor

We present data on the antiepileptic potency of 2-methyl-4-oxo-3H-quinazoline-3-acetyl piperidine (Q5) in juvenile (P9-13) rat hippocampal slices and in particular Q5's action mechanism and target. Q5 (200-500 microM), but not alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/Kainate receptor antagonists blocked low-[Mg2+]-induced seizure-like events (SLE) in the CA3 region. Q5 (100 microM) decreased Glu-induced [35S]guanosine 5'-O-(3-thiotriphosphate) binding enhancement in brain homogenates, without interaction with ionotropic Glu receptor sites and Glu transport. In voltage-clamped CA3 pyramidal cells, Q5 (500 microM) depressed activities of spontaneous excitatory and inhibitory postsynaptic currents without affecting miniature inhibitory currents. Metabotropic Glu receptor (mGluR) subtype antagonists affected network excitability dissimilarly. Intracellular Ca2+ ion transients induced by the mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) were suppressed by Q5. Agreeing predictions obtained by modelling Q5 binding to different experimental conformations of mGlu1, Q5 was bound partially to an mGluR binding site in the presence of 1mM ACPD. Findings suggest the apparent involvement of a novel phenotype of action or a new mGluR subtype in the specific suppression of epileptiform activity by Q5 through the depression of network excitability.     

Human HLTF mediates postreplication repair by its HIRAN domain-dependent replication fork remodelling

Defects in the ability to respond properly to an unrepaired DNA lesion blocking replication promote genomic instability and cancer. Human HLTF, implicated in error-free replication of damaged DNA and tumour suppression, exhibits a HIRAN domain, a RING domain, and a SWI/SNF domain facilitating DNA-binding, PCNA-polyubiquitin-ligase, and dsDNA-translocase activities, respectively. Here, we investigate the mechanism of HLTF action with emphasis on its HIRAN domain. We found that in cells HLTF promotes the filling-in of gaps left opposite damaged DNA during replication, and this postreplication repair function depends on its HIRAN domain. Our biochemical assays show that HIRAN domain mutant HLTF proteins retain their ubiquitin ligase, ATPase and dsDNA translocase activities but are impaired in binding to a model replication fork. These data and our structural study indicate that the HIRAN domain recruits HLTF to a stalled replication fork, and it also provides the direction for the movement of the dsDNA translocase motor domain for fork reversal. In more general terms, we suggest functional similarities between the HIRAN, the OB, the HARP2, and other domains found in certain motor proteins, which may explain why only a subset of DNA translocases can carry out fork reversal.     

The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis

Successful and accurate completion of the replication of damage-containing DNA requires mainly recombination and RAD18-dependent DNA damage tolerance pathways. RAD18 governs at least two distinct mechanisms: translesion synthesis (TLS) and template switching (TS)-dependent pathways. Whereas TS is mainly error-free, TLS can work in an error-prone manner and, as such, the regulation of these pathways requires tight control to prevent DNA errors and potentially oncogenic transformation and tumorigenesis. In humans, the PCNA-associated recombination inhibitor (PARI) protein has recently been shown to inhibit homologous recombination (HR) events. Here, we describe a biochemical mechanism in which PARI functions as an HR regulator after replication fork stalling and during double-strand break repair. In our reconstituted biochemical system, we show that PARI inhibits DNA repair synthesis during recombination events in a PCNA interaction-dependent way but independently of its UvrD-like helicase domain. In accordance, we demonstrate that PARI inhibits HR in vivo, and its knockdown suppresses the UV sensitivity of RAD18-depleted cells. Our data reveal a novel human regulatory mechanism that limits the extent of HR and represents a new potential target for anticancer therapy.     

Rapid Brain Uptake of Manganese(II) Across the Blood-Brain Barrier

Abstract: ⁵⁴Mn²⁺ uptake into brain and choroid plexus from the circulation was studied using the in situ rat brain perfusion technique. Initial uptake from blood was linear with time (30 s to 6 min) and extrapolated to zero with an average transfer coefficient of ∼6 × 10^-5 ml/s/g for brain and ∼7 × 10^-3 ml/s/g for choroid plexus. Influx from physiologic saline was three- to fourfold more rapid and exceeded that predicted for passive diffusion by more than one order of magnitude. The lower uptake rate from blood could be explained by plasma protein binding as the free fraction of ⁵⁴Mn²⁺ in rat plasma was ≤30%. Purified albumin, transferrin, and α₂-macroglobulin were each found to bind ⁵⁴Mn²⁺ significantly and to restrict brain ⁵⁴Mn²⁺ influx. The results demonstrate that ⁵⁴Mn²⁺ is readily taken up into the CNS, most likely as the free ion, and that transport is critically affected by plasma protein binding. The results support the hypothesis that Mn²⁺ transport across the blood-brain barrier is facilitated by either an active or a passive mechanism.