Identification of mitogen-activated protein/extracellular signal-responsive kinase kinase 2 as a novel partner of the scaffolding protein human homolog of disc-large

Human disc-large homolog (hDlg), also known as synapse-associated protein 97, is a scaffold protein, a member of the membrane-associated guanylate kinase family, implicated in neuronal synapses and epithelial-epithelial cell junctions whose expression and function remains poorly characterized in most tissues, particularly in the vasculature. In human vascular tissues, hDlg is highly expressed in smooth muscle cells (VSMCs). Using the yeast two-hybrid system to screen a human aorta cDNA library, we identified mitogen-activated protein/extracellular signal-responsive kinase (ERK) kinase (MEK)2, a member of the ERK cascade, as an hDlg binding partner. Site-directed mutagenesis showed a major involvement of the PSD-95, disc-large, ZO-1 domain-2 of hDlg and the C-terminal sequence RTAV of MEK2 in this interaction. Coimmunoprecipitation assays in both human VSMCs and human embryonic kidney 293 cells, demonstrated that endogenous hDlg physically interacts with MEK2 but not with MEK1. Confocal microscopy suggested a colocalization of the two proteins at the inner layer of the plasma membrane of confluent human embryonic kidney 293 cells, and in a perinuclear area in human VSMCs. Additionally, hDlg also associates with the endoplasmic reticulum and microtubules in these latter cells. Taken together, these findings allow us to hypothesize that hDlg acts as a MEK2-specific scaffold protein for the ERK signaling pathway, and may improve our understanding of how scaffold proteins, such as hDlg, differentially tune MEK1/MEK2 signaling and cell responses.     

Drosophila p24 homologues eclair and baiser are necessary for the activity of the maternally expressed Tkv receptor during early embryogenesis

p24 proteins are assumed to play an important role in the transport of secreted and transmembrane proteins into membranes. However, only few cargo proteins are known that partially, but in no case completely require p24 proteins for membrane transport. Here, we show that two p24 proteins are essential for dorsoventral patterning of Drosophila melanogaster embryo. Mutations in the genes, eclair (eca) and baiser (bai), encoding two p24 proteins reduce signalling by the TGF-beta homologue, Dpp, in early embryos. This effect is strictly maternal and specific to early embryogenesis, as Dpp signalling in other contexts is not notably affected. We provide genetic evidence that in the absence of eca or bai function in the oocyte, the maternally expressed type I TGF-beta receptor Tkv is not active. We propose that during early embryogenesis eca and bai are specifically required for the activity of the maternal Tkv, while the zygotic Tkv is not affected in the mutant embryos. Mutations in either eca or bai are sufficient for the depletion of Tkv activity and no enhancement of the phenotypes was observed in embryos derived from oocytes mutant for both genes. The dependence of maternal Tkv protein on the products of p24 genes may serve as an in vivo model for studying p24 proteins.     

ABA may affect the dormancy in triticale caryopses by inhibition of embryo class I RNases,enzymes involved in P<Subscript>i</Subscript> release

The main objective of this study was to analyze the differences in profiles of RNase activities from triticale embryos (Triticosecale, cv. Ugo) between dormant and non-dormant caryopses and to determine the influence of exogenous abscisic acid (ABA) on the activities of these enzymes. The major RNase from the examined tissue was detected following SDS-PAGE, with substrate-based gel assay, described by Yen and Green (Plant Physiol 97:1487–1493, 1991). The activities of enzymes were characterized according to their pH optima, ion dependence, EDTA sensitivity and DNase activity. In embryos with arrested growth (in a natural way by dormancy or artificially by ABA treatment), the activity of two enzymes—24 and 27 kDa—belonging to class I RNases was completely inhibited, whereas that of two other RNases of this family—23 and 25 kDa—was detectable. However, the activity of the class I ribonucleases (enzymes responsible for cellular P_i release) was very low. Moreover, in contrast with non-dormant caryopses, imbibing embryos of dormant or ABA-treated seeds contained 13- and 14-kDa enzymes. These enzymes have not been classified so far, and their specific properties are different from the generally accepted properties of ribonucleolytic enzymes. In addition to the above results, the P_i content in the analyzed samples was determined by the Ames (Methods Enzymol 8:115–118, 1966) method. The results suggest a very low and constant level of inorganic phosphate in dormant samples as well as an evidently decreasing P_i content in embryos under the influence of ABA treatment. The inhibition of the class I RNases activity induced by abscisic acid implies that one of the roles of ABA in seed dormancy may consist in arresting the catabolic release of P_i, which results in retarding the embryo’s growth.     

Molecular mechanisms of <Emphasis Type="Italic">Bombyx batryticatus</Emphasis> ethanol extract inducing gastric cancer SGC-7901 cells apoptosis

Bombyx batryticatus is a traditional Chinese medicine. To understand apoptotic effect of B. batryticatus ethanol extract (BBE), we investigated the role of BBE in inducing apoptosis of human gastric cancer cells SGC-7901. Cells treated with BBE and apoptosis was assessed by methyl thiazolyl tetrazolium (MTT) assay, morphological changes, DNA fragmentation and flow cytometry assays. The expression of Bcl-2, Bax and P21 were evaluated by western blot analysis and real time polymerase chain reaction. MTT assay showed that the cytotoxicity of BBE extract on SGC-7901 cells was correlated with treatment time and concentration. After treatment with 6 mg/mL of BBE the microscopy showed that, the majority of SGC-7901 cells were obviously reduced, distorted and grew slowly. Annexin-V/propidium iodide double-staining assay emerge the early apoptosis and the late apoptosis after treatment with different times by laser confocal fluorescence microscopy and flow cytometer. Cell cycle analysis of SGC 79 cells showed that BBE induced cell cycle arrest in the G1 and G2 phases. DNA fragmentation indicated the trend of BBE inducing apoptosis on SGC-7901 cells. The qRT-PCR and western blot analysis indicated that the mRNA and protein expressions of Bax and P21 were significantly up-regulated whereas that of Bc1-2 was down-regulated after treatment with BBE for 24 h. Our results revealed a correlation between gene regulation and BBE-induced apoptosis, which might indicate the potential of BBE in cancer therapy.     

The influence of spatial pulsed magnetic field application on neuropathic pain after tibial nerve transection in rat

The purpose of the study was to examine the influence of the spatial variable magnetic field (induction: 150–300?µT, 80–150?µT, 20–80?µT; frequency 40?Hz) on neuropathic pain after tibial nerve transection. The experiments were carried out on 64 male Wistar C rats. The exposure of animals to magnetic field was performed 1?d/20?min., 5?d/week, for 28?d. Behavioural tests assessing the intensity of allodynia and sensitivity to mechanical and thermal stimuli were conducted 1?d prior to surgery and 3, 7, 14, 21 and 28?d after the surgery. The extent of autotomy was examined. Histological and immunohistochemical analysis was performed. The use of extremely low-frequency magnetic fields of minimal induction values (20–80?µT/40?Hz) decreased pain in rats after nerve transection. The nociceptive sensitivity of healthy rats was not changed following the exposition to the spatial magnetic field of the low frequency. The results of histological and immunohistochemical investigations confirm those findings. Our results indicate that extremely low-frequency magnetic field may be useful in the neuropathic pain therapy.     

Phylogeographic patterns of steppe species in Eastern Central Europe: a review and the implications for conservation

The phylogeography of species associated with European steppes and extrazonal xeric grasslands is poorly understood. This paper summarizes the results of recent studies on the phylogeography and conservation genetics of animals (20 taxa of beetles, butterflies, reptiles and rodents) and flowering plants (18 taxa) of such, "steppic" habitats in Eastern Central Europe. Most species show a similar phylogeographic pattern: relatively high genetic similarity within regional groups of populations and moderate-to-high genetic distinctiveness of populations from currently isolated regions located in the studied area. This distinctiveness of populations suggests a survival here during glacial maxima, including areas north of the Bohemian Massif-Carpathians arc. Steppic species generally do not follow the paradigmatic patterns known for temperate biota (south-north “contraction–expansion”), but to some extent are similar to those of arctic-alpine taxa. There are three main groups of taxa within Eastern Central Europe that differ in their contemporary distribution pattern, which may reflect historical origin and expansion routes. Present diversity patterns of the studied steppic species suggest that they share a unique genetic signature and distinct assemblages exist in each of the now isolated areas rich in steppic habitats. At least some of these areas probably act as present “interglacial refugia” for steppic species. This study strongly supports the need to protect steppic species throughout their entire ranges in the region, as the continuous destruction of steppic habitats in some areas may lead not only to the disappearance of local populations, but also to the extinction of unique evolutionary units.     

RBM10 inhibits cell proliferation of lung adenocarcinoma via RAP1/AKT/CREB signalling pathway

Initial functional studies have demonstrated that RNA‐binding motif protein 10 (RBM10) can promote apoptosis and suppress cell proliferation; however, the results of several studies suggest a tumour‐promoting role for RBM10. Herein, we assessed the involvement of RBM10 in lung adenocarcinoma cell proliferation and explored the potential molecular mechanism. We found that, both in vitro and in vivo, RBM10 overexpression suppresses lung adenocarcinoma cell proliferation, while its knockdown enhances cell proliferation. Using complementary DNA microarray analysis, we previously found that RBM10 overexpression induces significant down‐regulation of RAP1A expression. In this study, we have confirmed that RBM10 decreases the activation of RAP1 and found that EPAC stimulation and inhibition can abolish the effects of RBM10 knockdown and overexpression, respectively, and regulate cell growth. This effect of RBM10 on proliferation was independent of the MAPK/ERK and P38/MAPK signalling pathways. We found that RBM10 reduces the phosphorylation of CREB via the AKT signalling pathway, suggesting that RBM10 exhibits its effect on lung adenocarcinoma cell proliferation via the RAP1/AKT/CREB signalling pathway.     

Biogenic and Risk Elements in Wines from the Slovak Market with the Estimation of Consumer Exposure

Wine consumption delivers macroelements and microelements necessary for the proper metabolism. On the other hand, wine can be an important source of toxic metals. The aim of this study was to estimate the concentrations of Ca, Cd, Cu, Fe, Hg, Mg, Ni, Pb, and Zn in the Slovak and non-Slovak wines. The concentration of metals was evaluated with respect to the type, the alcohol content, and the age of Slovak wine. The general scheme of concentrations found was as follows Ca > Mg > Fe > Zn > Pb > Cd > Ni > Cu > Hg. The type of wine and the alcohol content do not have a significant impact on metal concentrations. Also, the age of wine has no influence on the mean concentration of metals, except for Zn. Metal concentrations in Slovak and non-Slovak wines indicate similar contents of metals, except for Ni. The contribution to both dietary reference values (DRVs) and provisional tolerable weekly intake (PTWI) evaluations in the Slovak wine suggested low dietary exposure to Ca, Cu, Fe, Mg, Ni, Zn, Cd, Hg, and Pb, respectively. However, we do not suggest that the consumption of all Slovak wines is healthy. The maximum Pb concentrations in Slovak wines exceed the maximum permitted level proposed by the European Commission. This might be proved by the results of the margin of the exposure (MOE) value evaluation in the samples containing the maximum Pb concentrations, showing a high risk of CKD and SBP in high and extreme consumption groups.     

The regulation of necroptosis by ubiquitylation

Necroptosis is a programmed necrosis that is mediated by receptor-interacting protein kinases RIPK1, RIPK3 and the mixed lineage kinase domain-like protein, MLKL. Necroptosis must be strictly regulated to maintain normal tissue homeostasis, and dysregulation of necroptosis leads to the development of various inflammatory, infectious, and degenerative diseases. Ubiquitylation is a widespread post-translational modification that is essential for balancing numerous physiological processes. Over the past decade, considerable progress has been made in the understanding of the role of ubiquitylation in regulating necroptosis. Here, we will discuss the regulatory functions of ubiquitylation in necroptosis signaling pathway. An enhanced understanding of the ubiquitylation enzymes and regulatory proteins in necroptotic signaling pathway will be exploited for the development of new therapeutic strategies for necroptosis-related diseases.