Free energy simulations of ligand binding to the aspartate transporter Glt(Ph)

Glutamate/Aspartate transporters cotransport three Na⁺ and one H⁺ ions with the substrate and countertransport one K⁺ ion. The binding sites for the substrate and two Na⁺ ions have been observed in the crystal structure of the archeal homolog Glt_Ph, while the binding site for the third Na⁺ ion has been proposed from computational studies and confirmed by experiments. Here we perform detailed free energy simulations of Glt_Ph, giving a comprehensive characterization of the substrate and ion binding sites, and calculating their binding free energies in various configurations. Our results show unequivocally that the substrate binds after the binding of two Na⁺ ions. They also shed light into Asp/Glu selectivity of Glt_Ph, which is not observed in eukaryotic glutamate transporters.     

TRPM2 Channel Membrane Currents in Primary Rat Megakaryocytes Were Activated by the Agonist ADP-Ribose but Not Oxidative Stress

Melastatin-like transient receptor potential 2 (TRPM2) channel activation/inhibition mechanisms in response to ADP-ribose (ADPR), oxidative stress, flufenamic acid (FFA) and 2-aminoethoxydiphenyl borate (2-APB) are not clear. We tested the effects of FFA and 2-APB on ADPR-induced TRPM2 cation channel currents in rat native bone marrow megakaryocytes. Megakaryocyte cells were freshly isolated from rat bone marrow and studied with the conventional whole-cell patch-clamp technique. Extracellular H₂O₂, FFA and 2-APB were added through the patch chamber, while intracellular ADPR was applied through the pipette. Nonselective cation currents were consistently induced by ADPR but not H₂O₂. Current density of ADPR in the cells was significantly (P < 0.001) higher than in control. The time courses of ADPR effects in the megakaryocytes were characterized by a delay of 2.24 ± 0.73. The ADPR-induced Ca²⁺ gate was not blocked by either the IP₃ receptor inhibitor 2-APB or the PLC inhibitor FFA. In conclusion, TRPM2 channels were constitutively activated by intracellular ADPR, although the channel currents in rat native megakaryocytes were not affected by extracellular H₂O₂, 2-APB or FFA. Activation of TRPM2 channels in megakaryocytes seems to be intracellular and ADPR-dependent.     

Effects of Nutrient Level on Maternal Choice and Siring Success in <Emphasis Type="Italic">Cucumis sativus</Emphasis> (Cucurbitaceae)

Plants have evolved many mechanisms to increase the chance of gene dispersal mainly through pollen and environmental factors play an important role. Understanding the mechanism behind gene dispersal is therefore crucial in the correct evaluation of the use of genetically modified crops for cultivation. In this paper we address the question of weather nutrient availability for the female affects the outcome of pollen competition between two pollen donor cultivars of Cucumis sativus. We do this by carrying out controlled crosses of female plants grown at three different nutrient levels. We separated the effect of a specific donor from the effect of pollen tube growth rate by using reversed crosses of fast and slow pollen. Our results show that female effects on siring ability vary with nutrient level. Pollen with a high pollen tube growth rate was more successful when nutrient availability for the female was high. This could be the result of selection on the female to adjust preference according to environmental circumstances. Pollen tube growth rate was measured under nutrient rich circumstances, thus high performers possessed traits adapted to a nutrient rich situation. Due to trade-off effects, these traits might not be advantageous in poor environments. Instead, individuals adapted to low nutrient circumstances will have a higher pollen tube growth rate. If siring ability varies with the environment of the recipient plant, this means that assessments of gene flow must account for this variation and include both pollen donors and recipient plants subjected to a range of environmental circumstances. In risk assessments of transgenic plants, plants are often kept under experimental, homogenous conditions. If our results also apply to other species, estimates of gene flow under constant conditions may be misleading. Selection on siring ability and female preference have fundamental effects on gene flow and need to be considered in risk assessments of transgenic plants.Co-ordinating editor: I. Olivieri     

Lymphoma development of simultaneously combined exposure to two radiofrequency signals in AKR/J mice

There are public concerns regarding possible carcinogenic or cancer-promoting effects of radiofrequency electromagnetic fields (RF-EMFs) because of the extensive use of wireless mobile phones and other telecommunication devices in daily life. However, so far it is unclear if non-thermal exposure of single EMF exposure in animal studies has a direct influence on carcinogenesis. Here, carcinogenic effects of combined signal RF-EMFs on AKR/J mice, which were used for the lymphoma animal model, were investigated. Six-week-old AKR/J mice were simultaneously exposed to two types of RF signals: single code division multiple access (CDMA) and wideband code division multiple access (WCDMA). AKR/J mice were exposed to combined RF-EMFs for 45 min/day, 5 days/week, for a total of 42 weeks. The whole-body average specific absorption rate (SAR) of CDMA and WCDMA fields was 2.0 W/kg each, 4.0 W/kg in total. When we examined final survival, lymphoma incidence, and splenomegaly incidence, no differences were found between sham- and RF-exposed mice. However, occurrence of metastasis infiltration to the brain in lymphoma-bearing mice was significantly different in RF-exposed mice when compared to sham-exposed mice, even though no consistent correlation (increase or decrease) was observed between male and female mice. However, infiltration occurrence to liver, lung, and spleen was not different between the groups. From the results, we suggested that simultaneous exposure to CDMA and WCDMA RF-EMFs did not affect lymphoma development in AKR/J mice.     

Local kallikrein�Ckinin system is involved in podocyte apoptosis under diabetic conditions

The kallikrein-kinin system (KKS) serves as the physiologic counterbalance to the renin-angiotensin system. This study was conducted to examine the changes in the expression of KKS components in podocytes under diabetic conditions and to elucidate the functional role of bradykinin (BK) in diabetes-associated podocyte apoptosis. Thirty-two rats were injected with either diluent (n = 16, C) or with streptozotocin intraperitoneally (n = 16, DM), and 8 rats from each group were treated with BK infusion for 6 weeks. Immortalized mouse podocytes were cultured in media containing 5.6 mmol/l glucose (NG), NG + 10(-7) mol/l AII (AII), or 30 mmol/l glucose (HG) with or without 10(-8) mol/l BK. Urinary albumin excretion was significantly higher in DM rats, and this increase was ameliorated by BK. Not only kininogen, kallikrein, and BK B1- and B2-receptor expression but also BK levels were significantly decreased in DM glomeruli and in cultured podocytes exposed to HG. The changes in the expressions of apoptosis-related molecules and the increase in the number of apoptotic cells in DM glomeruli as well as in HG- and AII-stimulated podocytes were significantly abrogated by BK. The suppressed KSS within podocytes under diabetic condition was associated with podocyte apoptosis, suggesting that BK may be beneficial in preventing podocyte loss in diabetic nephropathy.     

Association between Epstein-Barr virus infection and chemoresistance to docetaxel in gastric carcinoma

Epstein-Barr virus (EBV) is associated with human cancers such as nasopharyngeal carcinoma, Burkitt’s lymphoma, Hodgkin’s disease, and gastric carcinoma (GC). EBV is associated with about 10% of all GC cases globally. EBV-associated GC has distinct features from EBV-negative GC. However, it is still unclear if EBV infection has any effect on GC chemoresistance. Cell proliferation assay, cell cycle analysis, and active caspase Western blot revealed that the EBV-positive GC cell line (AGS-EBV) showed chemoresistance to docetaxel compared to the EBV-negative GC cell line (AGS). Docetaxel treatment increased expression of Bax similarly in AGS and AGS-EBV cell lines. However, Bcl-2 induction was markedly higher in AGS-EBV cells, after docetaxel treatment. Although docetaxel increased the expression of p53 to a similar extent in both cell lines, induction of p21 in AGS-EBV cells was lower than in AGS cells. Furthermore, expression of survivin was higher in AGS-EBV cells than in AGS cells following docetaxel treatment as well as at basal state. EBVlytic gene expression was induced by docetaxel treatment in AGS-EBV cells. The results suggest that EBV infection and lytic induction confers chemoresistance to GC, possibly by regulating cellular and EBV latent and lytic gene expression.     

Inflammatory markers associated with abdominal aortic aneurysm

Purpose

Inflammation with leukocytic infiltration, degradation of extracellular matrix (ECM), and depletion of vascular smooth muscle cells (VSMC) are pathological hallmarks of abdominal aortic aneurysm (AAA). The aim of this study was to further evaluate relationships betweenAAAand inflammatory biomarkers, interleukin- 6 (IL-6), tumour necrosis factor-α (TNF-α), endothelin-1 (ET-1) and soluble urokinase-type plasminogen activator receptor (suPAR), by comparing levels in 65-year-old men with and without AAA at ultrasound screening.We also evaluated whether any biomarker can independently predict AAA at screening, and clarified potential correlations between aortic diameter and blood levels of these biomarkers.

Results

There were significant (p ? 0.05) differences between subjects with and without AAA for the following variables: p-leukocyte count (TLC) (p<0.001), p-homocysteine (p<0.001), p-TNF-α (p = 0.023), p-IL-6 (p<0.001), p-ET-1 (p = 0.002), p-suPAR (p<0.001), ankle brachial index (ABI) (p<0.001), plasma (p)-creatinine (p = 0.049), p-total cholesterol (p<0.001), p-high density lipoprotein (HDL) (p<0.001) and low density lipoprotein (LDL) cholesterol (p = 0.001), smoking habits (p<0.001), and use of antihypertensive (p<0.001) and lipid-lowering (p = 0.001) drugs. When the above variables were stepwise excluded in a logistic regression model, only p-IL-6 (p = 0.002), p-homocysteine (p = 0.015), p-HDL (p = 0.004), ABI in the right (p = 0.005) and left (p = 0.094) leg, smoking habits (p = 0.003), and antihypertensive drug use (p = 0.045), differed between groups. Significant correlations with aortic diameter existed for p-TNF-α (p = 0.028), p-IL-6 (p<0.001), p-ET-1 (p = 0.002) and p-suPAR (p<0.001) in the entire study population, and for p-TNF-α (p = 0.023), p-ET-1 (p = 0.009) and p-suPAR (p = 0.001) among men with AAA.

Conclusions

Several inflammatory biomarkers were significantly elevated and correlated with aortic diameter among 65-year old men with AAA at ultrasound screening. IL-6, homocysteine and use of antihypertensive medication remained elevated in the logistic regression model, together with known risk markers for AAA such as smoking and signs of atherosclerosis.