Double-Stranded RNA Attenuates the Barrier Function of Human Pulmonary Artery Endothelial Cells

Circulating RNA may result from excessive cell damage or acute viral infection and can interact with vascular endothelial cells. Despite the obvious clinical implications associated with the presence of circulating RNA, its pathological effects on endothelial cells and the governing molecular mechanisms are still not fully elucidated. We analyzed the effects of double stranded RNA on primary human pulmonary artery endothelial cells (hPAECs). The effect of natural and synthetic double-stranded RNA (dsRNA) on hPAECs was investigated using trans-endothelial electric resistance, molecule trafficking, calcium (Ca²⁺) homeostasis, gene expression and proliferation studies. Furthermore, the morphology and mechanical changes of the cells caused by synthetic dsRNA was followed by in-situ atomic force microscopy, by vascular-endothelial cadherin and F-actin staining. Our results indicated that exposure of hPAECs to synthetic dsRNA led to functional deficits. This was reflected by morphological and mechanical changes and an increase in the permeability of the endothelial monolayer. hPAECs treated with synthetic dsRNA accumulated in the G1 phase of the cell cycle. Additionally, the proliferation rate of the cells in the presence of synthetic dsRNA was significantly decreased. Furthermore, we found that natural and synthetic dsRNA modulated Ca²⁺ signaling in hPAECs by inhibiting the sarco-endoplasmic Ca²⁺-ATPase (SERCA) which is involved in the regulation of the intracellular Ca²⁺ homeostasis and thus cell growth. Even upon synthetic dsRNA stimulation silencing of SERCA3 preserved the endothelial monolayer integrity. Our data identify novel mechanisms by which dsRNA can disrupt endothelial barrier function and these may be relevant in inflammatory processes.     

Orexinergic Input to Dopaminergic Neurons of the Human Ventral Tegmental Area

The mesolimbic reward pathway arising from dopaminergic (DA) neurons of the ventral tegmental area (VTA) has been strongly implicated in reward processing and drug abuse. In rodents, behaviors associated with this projection are profoundly influenced by an orexinergic input from the lateral hypothalamus to the VTA. Because the existence and significance of an analogous orexigenic regulatory mechanism acting in the human VTA have been elusive, here we addressed the possibility that orexinergic neurons provide direct input to DA neurons of the human VTA. Dual-label immunohistochemistry was used and orexinergic projections to the VTA and to DA neurons of the neighboring substantia nigra (SN) were analyzed comparatively in adult male humans and rats. Orexin B-immunoreactive (IR) axons apposed to tyrosine hydroxylase (TH)-IR DA and to non-DA neurons were scarce in the VTA and SN of both species. In the VTA, 15.0±2.8% of TH-IR perikarya in humans and 3.2±0.3% in rats received orexin B-IR afferent contacts. On average, 0.24±0.05 and 0.05±0.005 orexinergic appositions per TH-IR perikaryon were detected in humans and rats, respectively. The majority (86–88%) of randomly encountered orexinergic contacts targeted the dendritic compartment of DA neurons. Finally, DA neurons of the SN also received orexinergic innervation in both species. Based on the observation of five times heavier orexinergic input to TH-IR neurons of the human, compared with the rat, VTA, we propose that orexinergic mechanism acting in the VTA may play just as important roles in reward processing and drug abuse in humans, as already established well in rodents.     

Analysis of biofilm bacterial communities responsible for carbon removal through a reactor cascade treating wastewater

In this study molecular microbiological and multivariate statistical analyses were carried out to determine the structure and dynamics of bacterial communities through a biofilm based, pilot-scale wastewater treatment cascade system comprised of eight reactors. Results indicated a vertical as well as horizontal differentiation of biofilm bacterial communities within individual reactors and through the reactor series, respectively. The richness of biofilm samples taken from dissolved oxygen rich sections of reactors were relatively lower than of samples taken from less oxygenized sections (one-way ANOVA P = 0.07). The Euclidean distance based one-way ANOSIM pointed out that in bacteriological point of view: (1) no statistically significant difference could be observed among the first five reactors (P ≥ 0.1); (2) the first seven reactors differed significantly from the last reactor, (P ≤ 0.03); (3) reactors 1 and 2 differed significantly from reactors 6 and 7 (P ≈ 0.02) and (4) reactor 3 from reactor 7 (P ≈ 0.03). 16S rRNA gene cloning revealed that through the cascade system the initially dominant heterotrophic bacteria (Acinetobacter, Acidovorax, Parabacteroides, Thauera, Desulfobacterium and Desulfomicrobium) were gradually replaced or supplemented by autotrophic nitrifying bacteria (Nitrosomonas, ‘Candidatus Nitrotoga’ and Nitrospira). Our results indicate that the vertical alteration of bacterial community structure within a particular reactor was driven by the alteration of dissolved oxygen concentration, while the horizontal alteration of bacterial community structure through the cascade system was driven mainly by the gradually decreasing dissolved organic matter content and increasing dissolved oxygen concentration.     

A New Zearalenone Biodegradation Strategy Using Non-Pathogenic Rhodococcus pyridinivorans K408 Strain

Zearalenone (hereafter referred to as ZEA) is a nonsteroidal estrogenic mycotoxin produced by several Fusarium spp. on cereal grains. ZEA is one of the most hazardous natural endocrine disrupting chemicals (EDC) which induces hyper estrogenic responses in mammals. This can result in reproductive disorders in farm animals as well as in humans. Consequently, detoxification strategies for contaminated crops are crucial for food safety. In this study we have developed a bacterial based detoxification system using a non-pathogen Rhodococcus pyridinivorans K408 strain. Following 5 days treatment of ZEA with R. pyridinivorans K408 strain HPLC analyses showed an 87.21% ZEA-degradation efficiency of the bacterial enzyme systems. In another approach, the strain biotransformation ability has also been confirmed by a bioluminescent version of the yeast estrogen screening system (BLYES), which detected an 81.75% of biodegradability of ZEA, in a good agreement with the chemical analyses. Furthermore, the capacity of R. pyridinivorans to eliminate the estrogenic effects of ZEA was tested by using an immature uterotrophic assay. Prepubertal female rats were treated with vehicle (olive oil), 17β-estradiol, ZEA (0.1-1-5-10 mg/kg body weight) and LB broth containing 500 mg/l ZEA that has already been incubated with or without Rhodococcus pyridinivorans K408 strain. Uterine weights were measured and the mRNA level changes relating to apelin, aquaporin 5, complement component 2, and calbindin-3 genes were measured by qRT-PCR. These genes represent the major pathways that are affected by estromimetic compounds. Zearalenone feeding significantly increased the uterus weight in a dose dependent manner and at the same time upregulated complement component 2 and calbindin-3 expression as well as decreased apelin and aquaporin 5 mRNA levels comparable to that seen in 17β-estradiol exposed rats. In contrast, LB broth in which ZEA was incubated with Rhodococcus pyridinivorans K408 prior to the feeding did not display any estrogenic effect neither on uterine weight nor on the expression of estrogen-regulated genes. Consequently, the identification of Rhodococcus pyridinivorans K408 strain in ZEA biodegradation proved to be a very efficient biological tool that is able to eliminate the complete estrogenic effects of ZEA. It is also remarkable that this biotransformation pathway of ZEA did not result in any residual estrogenic effects.     

Coherence of phytoplankton and attached diatom-based ecological status assessment in Lake Balaton

Coherence between ecological status assessment by phytoplankton and attached diatoms was analyzed in the littoral zone of Lake Balaton. Sampling of periphytic diatoms, phytoplankton, and water were carried out at ten different littoral sites in the northern and southern shores of the lake for a year. Phytoplankton species were sorted into functional groups and ecological status was assessed by means of the phytoplankton assemblage Q index. The index TDIL was calculated using quantitative attached diatom data. Significant differences were found between the ecological assessments based on phytoplankton and phytobenthos metrics, both seasonally and spatially. The Q index indicated ecological states varying from bad to good, while the average of diatom indices varied from moderate to high conditions. The Q index provided more realistic ecological status of Lake Balaton, compared with trophic status based on TP values, especially in the summer period. Differences in the response-time indication of phytoplankton and attached diatoms suggest that lack of coherence should also be expected between the responses of other BQEs.     

Adhesion and stress relaxation forces between melanoma and cerebral endothelial cells

Mechanical parameters play a crucial role in proper cellular functions. This article examines the process of the appearance and breaking of adhesion forces during contact between the confluent cerebral endothelial cell layer and a melanoma cell attached to a tipless cantilever. This adhesion is the initial phase of melanoma transmigration through the endothelial cell layer. Taking the force measurement, if the contact was prolonged for several seconds, a decrease in the load force was observed, which corresponds to stress relaxation of the cells. The dependence of adhesion force and stress relaxation on dwell time showed a saturation-like behavior. These stress relaxation curves could be fitted with the sum of two exponentials, suggesting that two independent processes take place simultaneously. The breakup of the adhesion during the retraction of the cantilever with the attached melanoma cell is not continuous but shows jumps. Between living endothelial and melanoma cells, a minimum jump size of about 20 pN could be determined. The minimum jump is independent of the dwell time and load force. It seems to be the elementary binding force between these two cell types. In case of fixed endothelial cells, the adhesion force was strongly decreased and the jumps disappeared, whereas the stress relaxation did not show considerable change upon fixation.     

Plant Rho-type (Rop) GTPase-dependent activation of receptor-like cytoplasmic kinases in vitro

Plants have evolved distinct mechanisms to link Rho-type (Rop) GTPases to downstream signaling pathways as compared to other eukaryotes. Here, experimental data are provided that members of the Medicago, as well as Arabidopsis, receptor-like cytoplasmic kinase family (RLCK Class VI) were strongly and specifically activated by GTP-bound Rop GTPases in vitro. Deletion analysis indicated that the residues implicated in the interaction might be distributed on various parts of the kinases. Using a chimaeric Rop GTPase protein, the importance of the Rho-insert region in kinase activation could also be verified. These data strengthen the possibility that RLCKs may serve as Rop GTPase effectors in planta.     

Soil tillage systems to reduce the harmful effect of extreme weather and hydrological situations

Soil as the largest potential natural water reservoir in the Carpathian Basin has increasing importance under conditions of predicted climate change resulting in increase of probability of extreme hydrological events. Soil management changes soil structure and has a major effect on soil water, heat and nutrition regimes. In this study the effect of four tillage treatments in combination with catch crop management was studied on soil hydraulic properties and water regime under semi-arid conditions. Investigations were carried out in a long-term soil tillage experiment established on Calcic Chernozem soil in Hungary. Tillage variants comprised mouldboard ploughing, disking, loosening combined with disking and direct drilling. The crop sequence between September 2003 and September 2004 comprised maize (main crop), rye (catch crop) and pea (forage). In May 2004, disturbed samples and undisturbed soil cores were collected from each tillage treatment/catch crop combination. The main soil physical and hydrophysical properties were determined in laboratory. In each treatment, capacitive soil moisture probes were installed up to 80 cm depth to ensure continuous measurement of soil water content. Total soil water amounts of chosen soil layers and soil water content dynamics as a function of depth were evaluated for selected periods in order to quantify the effect of the studied management systems on soil water regime. The main conclusion from the experiment is that under such (or similar) ecological conditions, the uniform, „over-standardized“ adaptation of tillage methods for soil moisture conservation is rather risky, their application needs special care and the future is for site-specific precision technologies. These are, in combination with catch crop application can be efficient measures of environmental protection and soil structure and water conservation.     

Role of protein phosphatase 2A in the regulation of endothelial cell cytoskeleton structure

Our recently published data suggested the involvement of protein phosphatase 2A (PP2A) in endothelial cell (EC) barrier regulation (Tar et al. [2004] J Cell Biochem 92:534-546). In order to further elucidate the role of PP2A in the regulation of EC cytoskeleton and permeability, PP2A catalytic (PP2Ac) and A regulatory (PP2Aa) subunits were cloned and human pulmonary arterial EC (HPAEC) were transfected with PP2A mammalian expression constructs or infected with PP2A recombinant adenoviruses. Immunostaining of PP2Ac or of PP2Aa + c overexpressing HPAEC indicated actin cytoskeleton rearrangement. PP2A overexpression hindered or at least dramatically reduced thrombin- or nocodazole-induced F-actin stress fiber formation and microtubule (MT) dissolution. Accordingly, it also attenuated thrombin- or nocodazole-induced decrease in transendothelial electrical resistance indicative of barrier protection. Inhibition of PP2A by okadaic acid abolished its effect on agonist-induced changes in EC cytoskeleton; this indicates a critical role of PP2A activity in EC cytoskeletal maintenance. The overexpression of PP2A significantly attenuated thrombin- or nocodazole-induced phosphorylation of HSP27 and tau, two cytoskeletal proteins, which potentially could be involved in agonist-induced cytoskeletal rearrangement and in the increase of permeability. PP2A-mediated dephosphorylation of HSP27 and tau correlated with PP2A-induced preservation of EC cytoskeleton and barrier maintenance. Collectively, our observations clearly demonstrate the crucial role of PP2A in EC barrier protection.     

Single amino acid (482) variants of the ABCG2 multidrug transporter: major differences in transport capacity and substrate recognition

The human ABCG2 protein is an ATP binding cassette half-transporter, which protects our cells and tissues against various xenobiotics, while overexpression of ABCG2 in tumor cells confers multidrug resistance. It has been documented that single amino acid changes at position 482 resulted in altered drug resistance and transport capacity. In this study, we have generated nine Arg-482 mutants (G, I, M, S, T, D, N, K, Y) of ABCG2, and expressed them in insect cells. All ABCG2 variants showed cell surface expression and, in isolated membranes, an ABCG2-specific ATPase activity. When methotrexate accumulation was measured in inside-out membrane vesicles, this transport was supported only by the wild-type ABCG2. In intact cells, mitoxantrone was transported by all ABCG2 variants, except by R482K. Rhodamine 123 was extruded by most of the mutants, except by R482K, Y and by wild-type ABCG2. Hoechst 33342 was pumped out from cells expressing the wild-type and all Arg-482 variants, but not from those expressing R482K and Y. Our study demonstrates that the substrate specificity of the Arg (wild-type) form is unique and that amino acid replacements at position 482 induce major alterations in both the transport activity and substrate specificity of this protein.