Myocardial regeneration by transplantation of modified endothelial progenitor cells expressing SDF-1 in a rat model

Cell based therapy has been shown to attenuate myocardial dysfunction after myocardial infarction (MI) in different acute and chronic animal models. It has been further shown that stromal‐cell derived factor‐1α (SDF‐1α) facilitates proliferation and migration of endogenous progenitor cells into injured tissue. The aim of the present study was to investigate the role of exogenously applied and endogenously mobilized cells in a regenerative strategy for MI therapy. Lentivirally SDF‐1α‐infected endothelial progenitor cells (EPCs) were injected after 90 min. of ligation and reperfusion of the left anterior descending artery (LAD) intramyocardial and intracoronary using a new rodent catheter system. Eight weeks after transplantation, echocardiography and isolated heart studies revealed a significant improvement of LV function after intramyocardial application of lentiviral with SDF‐1 infected EPCs compared to medium control. Intracoronary application of cells did not lead to significant differences compared to medium injected control hearts. Histology showed a significantly elevated rate of apoptotic cells and augmented proliferation after transplantation of EPCs and EPCs + SDF‐1α in infarcted myocardium. In addition, a significant increased density of CD31⁺ vessel structures, a lower collagen content and higher numbers of inflammatory cells after transplantation of SDF‐1 transgenic cells were detectable. Intramyocardial application of lentiviral‐infected EPCs is associated with a significant improvement of myocardial function after infarction, in contrast to an intracoronary application. Histological results revealed a significant augmentation of neovascularization, lower collagen content, higher numbers of inflammatory cells and remarkable alterations of apoptotic/proliferative processes in infarcted areas after cell transplantation.     

Gene expression profiles of vitrified in vitro- and in vivo-derived bovine blastocysts

Vitrification is becoming a preferred method for pre‐implantation embryo cryopreservation. The objective of this study was to determine the differentially expressed genes of in vivo‐ and in vitro‐produced bovine embryos after vitrification. In vitro‐ (IVF) and in vivo‐derived (IVV) bovine blastocysts were identified as follows: in vitro‐produced fresh (IVF‐F), in vitro‐produced vitrified (IVF‐V), in vivo‐derived fresh (IVV‐F), in vivo‐derived vitrified (IVV‐V). The microarray results showed that 53 genes were differentially regulated between IVF and IVV, and 121 genes were differentially regulated between fresh and vitrified blastocysts (P < 0.05). There were 6, 268, 962, and 17 differentially regulated genes between IVF‐F × IVV‐F, IVF‐V × IVV‐V, IVF‐F × IVF‐V, and IVV‐F × IVV‐V, respectively (P < 0.05). While gene expression was significantly different between fresh and vitrified IVF blastocysts (P < 0.05), it was similar between fresh and vitrified IVV blastocysts. Significantly up‐regulated KEGG pathways included ribosome, oxidative phosphorylation, spliceosome, and oocyte meiosis in the fresh IVF blastocyst samples, while sphingolipid and purine metabolisms were up‐regulated in the vitrified IVF blastocyst. The results showed that in vitro bovine blastocyst production protocols used in this study caused no major gene expression differences compared to those of in vivo‐produced blastocysts. After vitrification, however, in vitro‐produced blastocysts showed major gene expression differences compared to in vivo blastocysts. This study suggests that in vitro‐produced embryos are of comparable quality to their in vivo counterparts. Vitrification of in vitro blastocysts, on the other hand, causes significant up‐regulation of genes that are involved in stress responses. Mol. Reprod. Dev. 79: 613–625, 2012. © 2012 Wiley Periodicals, Inc.     

Effectors of alcohol-induced cell killing in Drosophila

Heavy alcohol consumption provokes an array of degenerative pathologies but the signals that couple alcohol exposure to regulated forms of cell death are poorly understood. Using Drosophila as a model, we genetically establish that the severity of ethanol challenge dictates the type of death that occurs. In contrast to responses seen under acute exposure, cytotoxic responses to milder challenges required gene encoding components of the apoptosome, Dronc and Dark. We conducted a genome-wide RNAi screen to capture targets that specifically mediate ethanol-induced cell death. One effector, Drat, encodes a novel protein that contains an ADH domain but lacks essential residues in the catalytic site. In cultured cells and neurons in vivo, depletion of Drat conferred protection from alcohol-induced apoptosis. Adults mutated for Drat showed both improved survival and enhanced propensities toward sedation after alcohol challenge. Together, these findings highlight novel effectors that support regulated cell death incited by alcohol stress in vitro and in vivo.     

Screening for free radical scavenging and cell aggregation inhibitory activities by secondary metabolites from Turkish Verbascum species

Free radical scavenging and cell aggregation inhibitory activities of 36 secondary metabolites isolated from the methanolic extracts of Verbascum cilicicum Boiss., V. lasianthum Boiss. ex Bentham, V pterocalycinum var. mutense Hub.-Mor., and V. salviifolium Boiss. (Scrophulariaceae) were investigated. The isolated compounds, 6-O-vaniloyl ajugol (1), ilwensisaponin A (2), ilwensisaponin C (3), verbascoside (4), beta-hydroxyacteoside (5), martynoside (6), poliumoside (7), forsythoside B (8), angoroside A (9), dehydrodiconiferyl alcohol-9-O-beta-D-glucopyranoside (10), dehydrodiconiferyl alcohol-9'-O-beta-D-glucopyranoside (11), apigenin 7-O-beta-glucopyranoside (12), luteolin 7-O-beta-glucopyranoside (13), luteolin 3'-O-beta-glucopyranoside (14) and chrysoeriol 7-O-beta-glucopyranoside (15), exhibited a dose-dependent inhibition of bioautographic and spectrophotometric DPPH activities. Verbascoside (4) was the most active (IC50 4.0 microg/ml) comparing it to vitamin C (IC50 4.4 microg/ml) to inhibit phorbol 12-myristate 13-acetate (PMA)-induced peroxide-catalyzed oxidation of 2',7'-dichlorofluorescein (DCFH) by reactive oxygen species (ROS) within human promyelocytic HL-60 cells. Ilwensisaponin A (2) (MIC 6.9 microg/ml) showed moderate in vitro activity on lymphocyte-associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1)-mediated aggregation using the HL-60 cell line [positive control was cytochalasin B (MIC 2.3 microg/ml)]. None of the other compounds showed free radical scavenging and cell aggregation inhibitory activities.     

Idebenone Ameliorates Rotenone-Induced Parkinson’s Disease in Rats Through Decreasing Lipid Peroxidation

Neurochemical Research - Oxidative stress is considered one of the mechanisms responsible for neurodegenerative diseases, especially for Parkinson’s disease. Since oxidative stress causes...     

The therapeutic effects of anti-oxidant and anti-inflammatory drug quercetin on aspiration-induced lung injury in rats

Aspiration pneumonitis refers to acute chemical lung injury caused by aspiration of sterile gastric contents. The aim of this study was to evaluate the role of quercetin (QC) in acid aspiration-induced lung injury in rats. Twenty-eight female Sprague–Dawley rats were used and divided into the following groups (n = 7): sham (aspirated normal saline, S), hydrochloric acid (aspirated HCl), S plus treatment with QC (S + QC), and HCl plus treatment with QC (HCl + QC). After aspiration, the treatment groups received QC 60 mg/kg/day intraperitoneally once a day for 7 days. As a result of acid aspiration, an increase was observed in the levels of serum clara cell protein-16 (CC-16) and advanced oxidation protein products, whereas there was a decrease in serum thiobarbituric acid-reactive substances, superoxide dismutase (SOD), and catalase levels. There was a significant decrease in peribronchial inflammatory cell infiltration, alveolar septal infiltration, alveolar edema, and alveolar exudate scores, except in the alveolar histiocytes in the HCl + QC group. The expression of nitric oxide synthase, which increased after aspiration in the HCl group, showed a statistically significant decrease after the QC treatment. After the treatment with QC, an increase in the serum SOD level was observed, whereas a significant decrease was determined in the serum CC-16 level relative to that of the aspiration group (HCl). The antioxidant QC is effective in the treatment of lung injury following acid aspiration and can be used as a serum CC-16 biomarker in predicting the severity of oxidative lung injury.     

The Plant Membrane-Associated REMORIN1.3 Accumulates in Discrete Perihaustorial Domains and Enhances Susceptibility to Phytophthora infestans

Filamentous pathogens such as the oomycete Phytophthora infestans infect plants by developing specialized structures termed haustoria inside the host cells. Haustoria are thought to enable the secretion of effector proteins into the plant cells. Haustorium biogenesis, therefore, is critical for pathogen accommodation in the host tissue. Haustoria are enveloped by a specialized host-derived membrane, the extrahaustorial membrane (EHM), which is distinct from the plant plasma membrane. The mechanisms underlying the biogenesis of the EHM are unknown. Remarkably, several plasma membrane-localized proteins are excluded from the EHM, but the remorin REM1.3 accumulates around P. infestans haustoria. Here, we used overexpression, colocalization with reporter proteins, and superresolution microscopy in cells infected by P. infestans to reveal discrete EHM domains labeled by REM1.3 and the P. infestans effector AVRblb2. Moreover, SYNAPTOTAGMIN1, another previously identified perihaustorial protein, localized to subdomains that are mainly not labeled by REM1.3 and AVRblb2. Functional characterization of REM1.3 revealed that it is a susceptibility factor that promotes infection by P. infestans. This activity, and REM1.3 recruitment to the EHM, require the REM1.3 membrane-binding domain. Our results implicate REM1.3 membrane microdomains in plant susceptibility to an oomycete pathogen.Filamentous plant pathogens, including oomycetes of the genus Phytophthora, downy mildews and white rusts, as well as powdery mildews and rust fungi, are among the most devastating plant pathogens. These biotrophic parasites associate closely with plant cells through specialized infection structures called haustoria. Haustoria are specialized pathogen hyphal structures formed within host cells and enveloped by a perimicrobial membrane called the extrahaustorial membrane (EHM), a key interface between plant pathogens and the host cell. Haustoria are critical for successful parasitic infection by many filamentous plant pathogens and are a signature of the biotrophic lifestyle. In fungi, haustoria function as feeding structures (Voegele et al., 2001). In addition, haustoria are thought to enable the delivery of host-translocated virulence proteins, known as effectors, by both fungal and oomycete pathogens (Catanzariti et al., 2006; Whisson et al., 2007). However, little is known about the molecular mechanisms underlying the biogenesis and function of haustoria and EHM (Kemen and Jones, 2012; Lu et al., 2012).The EHM is thought to be continuous with the host plasma membrane (PM), yet it is a highly specialized membrane compartment that develops only in plant cells that accommodate haustoria (haustoriated cells; Coffey and Wilson, 1983). On the plant side, all eight PM proteins tested by Koh et al. (2005) were excluded from the EHM in Arabidopsis (Arabidopsis thaliana) cells infected with the powdery mildew fungus Golovinomyces cichoracearum. Conversely, the atypical Arabidopsis resistance protein Resistance to Powdery Mildew8.2 (RPW8.2) exclusively localizes to the EHM in this interaction (Wang et al., 2009). Ultrastructure analyses of the Golovinomyces orontii powdery mildew pathosystem revealed that the EHM is asymmetric, thicker and more electron opaque than the PM, and can be highly convoluted around mature haustoria (Micali et al., 2011). More recently, a survey of Arabidopsis and Nicotiana benthamiana plants infected by the oomycete pathogens Hyaloperonospora arabidopsidis and Phytophthora infestans, respectively, revealed that several integral host PM proteins are excluded from the EHM (Lu et al., 2012). Nevertheless, the remorin REM1.3 and the SYNAPTOTAGMIN1 (SYT1) peripheral membrane proteins localized to undetermined subcellular compartments around haustoria in P. infestans-plant interactions (Lu et al., 2012). Whether the differential accumulation of membrane proteins at the EHM is due to interference with the lateral diffusion of proteins from the PM or targeted secretion of specialized vesicles remains unclear (Lu et al., 2012).The subcellular distribution of effectors inside plant cells provides valuable clues about the host cell compartments they modify to promote disease, and effectors have emerged as useful molecular probes for plant cell biology (Whisson et al., 2007; Bozkurt et al., 2012). Heterologous expression of fluorescently tagged effectors in plant cells has been used to determine their subcellular localization in uninfected and infected tissue. This approach has been successful with the RXLR and CRINKLER (CRN) effectors, the two major classes of cytoplasmic (host-translocated) oomycete effectors (Bozkurt et al., 2012). The 49 H. arabidopsidis RXLR effectors studied by Caillaud et al. (2012) localized to the nucleus, the cytoplasm, or various plant membrane compartments. In contrast, CRN effectors from several oomycete species exclusively accumulate in the plant cell nucleus (Schornack et al., 2010; Stam et al., 2013). The P. infestans effectors AVRblb2 and AVR2 accumulate around haustoria when expressed in infected N. benthamiana cells, highlighting the PM and the EHM as important sites for effector activity (Bozkurt et al., 2011; Saunders et al., 2012). These effectors, therefore, can serve as useful probes for plant cell biology to dissect vesicular trafficking and focal immunity, processes that have proved difficult to study using standard genetic approaches (Bozkurt et al., 2011; Win et al., 2012).REM1.3 is one of two plant membrane-associated proteins detected around haustoria during the interaction between P. infestans and the model plant N. benthamiana (Lu et al., 2012). Therefore, we hypothesized that studying REM1.3 should prove useful for understanding the mechanisms governing the function and formation of perihaustorial membranes. REM1.3 belongs to a diverse family of plant-specific proteins containing a Remorin_C domain (PF03763) and has known orthologs in potato (Solanum tuberosum; StREM1.3), tomato (Solanum lycopersicum; SlREM1.2), tobacco (Nicotiana tabacum; NtREM1.2), and Arabidopsis (AtREM1.1–AtREM1.4; Raffaele et al., 2007). Several proteins from the remorin family, including REM1.3, are preferentially associated with membrane rafts, nanometric sterol- and sphingolipid-rich domains in PMs (Pike, 2006; Simons and Gerl, 2010). Indeed, StREM1.3 and NtREM1.2 are highly enriched in detergent-insoluble membranes (DIMs) and form sterol-dependent domains of approximately 75 nm in purified PMs (Mongrand et al., 2004; Shahollari et al., 2004; Raffaele et al., 2009). StREM1.3 directly binds to the cytoplasmic leaflet of the PM through a C-terminal anchor domain (RemCA) that folds into a hairpin of aliphatic α-helices in polar environments (Raffaele et al., 2009; Perraki et al., 2012). StREM1.3 is differentially phosphorylated upon the perception of polygalacturonic acid (Reymond et al., 1996). AtREM1.3 is differentially recruited to DIMs and differentially phosphorylated upon flg22 (for flagellin) peptide perception (Benschop et al., 2007; Keinath et al., 2010; Marín et al., 2012), suggesting a role in plant defense signaling. StREM1.3 and SlREM1.2 prevent Potato virus X spreading by interacting with the Triple Gene Block protein1 (TGBp1) viral movement protein, presumably in plasmodesmata or at the PM (Raffaele et al., 2009; Perraki et al., 2012). AtREM1.2 belongs to protein complexes formed by a negative regulator of immune responses, Resistance to Pseudomonas syringae pv maculicola1 (RPM1)-INTERACTING PROTEIN4, at the PM (Liu et al., 2009). Furthermore, Medicago truncatula MtSYMREM1 is enriched in root cell DIMs (Lefebvre et al., 2007) and localizes to patches at the peribacteroid membrane during symbiosis with Sinorhizobium meliloti (Lefebvre et al., 2010). MtSYMREM1 is important for nodule formation and interacts with the Lysin motif domain–containing receptor-like kinase3 (LYK3) symbiotic receptor (Lefebvre et al., 2010). Multiple lines of evidence, therefore, implicate several remorins in cell surface signaling and the accommodation of microbes during plant-microbe interactions (Raffaele et al., 2007; Jarsch and Ott, 2011; Urbanus and Ott, 2012). Nevertheless, little is known about REM1.3’s molecular function, and its role in immunity against filamentous plant pathogens has not been reported to date.In this study, we analyzed in detail the localization and function of REM1.3 during host colonization by P. infestans. We found that REM1.3 localizes exclusively to the vicinity of the PM and the EHM around noncallosic haustoria. Furthermore, our results suggest that the EHM is likely formed by multiple microdomains. REM1.3 silencing and overexpression experiments demonstrated that it promotes susceptibility to P. infestans in N. benthamiana and tomato. We also show that the REM1.3 membrane anchor domain is required for its localization at the EHM and for the promotion of susceptibility to P. infestans. This work demonstrates the importance of the dynamic reorganization of the PM in response to haustoria-forming pathogens. Our study also revealed that the effector AVRblb2 localizes to remorin-containing host membrane domains at the host-pathogen interface, possibly as a pathogen strategy to facilitate the accommodation of infection structures inside plant cells.     

Sensitization of Children to Storage Mites in Kutahya, Turkey

Specific IgE against Acarus siro, Glycphagus domesticus, Tyrophagus putrescentiae, and Lepidoglyphus destructor have been investigated by ELISA in sera of 92 children. Of them, 41 were found to be specific IgE positive (≥ 0.35 IU/ml) against at least one of house dust mite species, Dermatophagoides pteronyssinus and Dermatophagoides farinae, by an immunoblot. In 65.9% of the dust mite-sensitized children, specific IgE against at least one of these mite species was found. Sensitization levels, including co-sensitization cases were found to be 35.7% against A. siro, 24.4% against T. putrescentiae, 31.7% against L. destructor, and 26.8% against G. domesticus. In non-sensitized children, dust mite sensitization level was found to be 25.5%. Breakdown of sensitization by individual species in this group was; against A. siro and T. putrescentiae at 7.8%, against L. destructor at 13.7%, and against G. domesticus at 9.8%. When all children were reckoned, 43.5% was found to be sensitized against at least one storage mite species, with sensitizations against A. siro at 18.5%, T. putrescentiae at 26.1%, L. destructor at 21.7%, and G. domesticus at 17.4%. In dust samples collected from the dwellings of children, distribution of species was found to be A. siro (17%), G. domesticus (23%), T. putrescentiae (29%), L. destructor (25%), and unidentified (6%). In Fisher''s chi-square test on SPSS program, there was a relationship between dust mite sensitization and storage mite sensitization (P < 0.05), but no meaningful relationship was found on the basis of individual mite species.     

Autoantibodies against the replication protein A complex in systemic lupus erythematosus and other autoimmune diseases

Replication protein A (RPA), a heterotrimer with subunits of molecular masses 70, 32, and 14 kDa, is a single-stranded-DNA-binding factor involved in DNA replication, repair, and recombination. There have been only three reported cases of anti-RPA in systemic lupus erythematosus (SLE) and Sjögren syndrome (SjS). This study sought to clarify the clinical significance of autoantibodies against RPA. Sera from 1,119 patients enrolled during the period 2000 to 2005 were screened by immunoprecipitation (IP) of ³⁵S-labeled K562 cell extract. Antigen-capture ELISA with anti-RPA32 mAb, immunofluorescent antinuclear antibodies (ANA) and western blot analysis with purified RPA were also performed. Our results show that nine sera immunoprecipitated the RPA70–RPA32–RPA14 complex and all were strongly positive by ELISA (titers 1:62,500 to 1:312,500). No additional sera were positive by ELISA and subsequently confirmed by IP or western blotting. All sera showed fine speckled/homogeneous nuclear staining. Anti-RPA was found in 1.4% (4/276) of SLE and 2.5% (1/40) of SjS sera, but not in rheumatoid arthritis (0/35), systemic sclerosis (0/47), or polymyositis/dermatomyositis (0/43). Eight of nine patients were female and there was no racial predilection. Other positive patients had interstitial lung disease, autoimmune thyroiditis/hepatitis C virus/pernicious anemia, or an unknown diagnosis. Autoantibody specificities found in up to 40% of SLE and other diseases, such as anti-nRNP, anti-Sm, anti-Ro, and anti-La, were unusual in anti-RPA-positive sera. Only one of nine had anti-Ro, and zero of nine had anti-nRNP, anti-Sm, anti-La, or anti-ribosomal P antibodies. In summary, high titers of anti-RPA antibodies were found in nine patients (1.4% of SLE and other diseases). Other autoantibodies found in SLE were rare in this subset, suggesting that patients with anti-RPA may form a unique clinical and immunological subset.     

Cholinesterase inhibitory and antioxidant properties of Verbascum mucronatum Lam. and its secondary metabolites

The aqueous extract of Verbascum mucronatum Lam. along with its fractions and secondary metabolites were assessed for their antioxidant, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) inhibitory activities. The antioxidant activity was evaluated by three methods: as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, ferrous ion-chelating effect, and ferric-reducing antioxidant power (FRAP) tests. The AChE activity was determined by the Ellman method using an ELISA microplate reader. Phytochemical investigations revealed the presence of four iridoid glucosides, ajugol (1), aucubin (2), lasianthoside I (3) and catalpol (4), two saponins, ilwensisaponin A (5) and C (6), and a phenylethanoid glycoside, verbascoside (7), in Verbascum mucronatum. Their structures were elucidated by spectral techniques. The aqueous extract and fractions including the phenylethanoid glycoside 7 showed DPPH scavenger effect and had the best FRAP. Besides these results, one of the phenylethanoid fractions displayed the highest ferrous ion-chelating effect. While only 7 was found to possess moderate AChE inhibition, the extract, fractions, and all other tested compounds did not inhibit AChE and BChE.