Autotaxin protects microglial cells against oxidative stress

Oxidative stress occurs when antioxidant defenses are overwhelmed by oxygen-reactive species and can lead to cellular damage, as seen in several neurodegenerative disorders. Microglia are specialized cells in the central nervous system that act as the first and main form of active immune defense in the response to pathological events. Autotaxin (ATX) plays an important role in the modulation of critical cellular functions, through its enzymatic production of lysophosphatidic acid (LPA). In this study, we investigated the potential role of ATX in the response of microglial cells to oxidative stress. We show that treatment of a microglial BV2 cell line with hydrogen peroxide (H(2)O(2)) stimulates ATX expression and LPA production. Stable overexpression of ATX inhibits microglial activation (CD11b expression) and protects against H(2)O(2)-treatment-induced cellular damage. This protective effect of ATX was partially reduced in the presence of the LPA-receptor antagonist Ki16425. ATX overexpression was also associated with a reduction in intracellular ROS formation, carbonylated protein accumulation, proteasomal activity, and catalase expression. Our results suggest that up-regulation of ATX expression in microglia could be a mechanism for protection against oxidative stress, thereby reducing inflammation in the nervous system.     

Relaxant effects of different fractions from Tymus vulgaris on guinea-pig tracheal chains

In previous studies, the relaxant effect of Tymus vulgaris has been demonstrated on guinea pig tracheal chains. Therefore, in the present study, the relaxant effects of n-hexane, dichloromethane, methanol and aqueous fractions of Tymus vulgaris on tracheal chains of guinea pigs were examined. The relaxant effects of four cumulative concentrations of each fraction (0.4, 0.8, 1.2 and 1.6 g%) in comparison to saline as negative control and four cumulative concentrations of theophylline (0.2, 0.4, 0.6 and 0.8 mM) were examined for their relaxant effects on precontracted tracheal chains of guinea pig by 60 mM KCl (group 1) and 10 ìì methacholine (group 2, n = 7 for each group). In group 1, all concentrations of the n-hexane fraction and theophylline and three last concentrations (0.8, 1.2 and 1.6 g%) of dichloromethane and two higher concentrations (1.2 and 1.6 g%) of methanol fractions showed significant relaxant effects compared to that of saline (p<0.05 to p<0.001). In group 2, all concentrations of theophylline, n-hexane and dichloromethane fractions and three concentrations (0.8, 1.2 and 1.6 g%) of methanol and two higher concentrations (1.2 and 1.6 g%) of aqueous fractions showed significant relaxant effects compared to that of saline (p<0.05 to p<0.001). In addition, with group 1, the relaxant effect of all concentrations of all fractions except the n-hexane fraction, were significantly less than those of theophylline (p<0.05 to p<0.001). The n-hexane fraction showed higher relaxant effect than theophylline. The relaxant effect of all concentrations of the n-hexane fraction and the three last concentrations (0.8, 1.2 and 1.6 g%) of dichloromethane and aqueous fractions were significantly greater in group 2 than in group 1 (p<0.05 to p<0.001). There were significant positive correlations between the relaxant effects and concentrations for theophylline and all fractions (except aqueous fraction in group 1) in both groups, but a negative correlation for the aqueous fraction in group 1 (p<0.05 to p<0.001). These results showed a potent relaxant effect for n-hexane and weaker relaxant effect for other fractions from Tymus vulgaris on tracheal chains of guinea pigs.     

Prevalence of coagulation factor II G20210A and factor V G1691A Leiden polymorphisms in Chechans, a genetically isolated population in Jordan

Background Coagulation factor II G20210A and coagulation factor V (Leiden) G1691A single nucleotide polymorphisms (SNPs) are major inherited risk factors of venous thromboembolism. In view of the heterogeneity in their world distribution and lack of sufficient information about their distribution among Chechans, we addressed the prevalence of these SNPs in the Chechan population in Jordan, a genetically isolated population. Methods and Results factor II G20210A and factor V Leiden SNPs were analysed by polymerase chain reaction and restriction fragment length polymorphism (PCR?CRFLP) method and Amplification refractory mutation detection system (ARMS) respectively in 120 random unrelated subjects from the Chechan population in Jordan. Among the subjects studied for factor II G20210A mutation there were three individuals carrying this mutation as heterozygous (one female and two male), giving a prevalence of 2.5?% and an allele frequency of 1.25?%. No homozygous factor II allele was found. Factor V Leiden G1691A mutation was detected as heterozygous in 22 of 120 of individuals (17 female and five male) indicating a prevalence of 18.3?% and allele frequency of 9.2?%. No homozygous allele was found. Conclusion Our results indicated that prevalence of factor II G20210A mutation in the Chechan population is similar to prevalence in Jordan and Caucasian populations (1?C6?%) while the prevalence of factor V Leiden was higher in the Chechan population compared to Jordan and Caucasian populations (2?C15?%).     

Molecular identification and characterization of a new type of bovine enterovirus

Bovine enteroviruses belong to the family Picornaviridae. Little is known about their pathogenic potential; however, they cause asymptomatic infections in cattle and are excreted in feces. In the present study, viruses isolated from environmental samples were sequenced. According to phylogenetic analyses and standard picornavirus nomenclature, these isolates constitute a new type of bovine enterovirus serogroup A.     

Toward tailored exosomes: the exosomal tetraspanin web contributes to target cell selection

Exosomes are discussed as potent therapeutics due to efficient transfer of proteins, mRNA and miRNA in selective targets. However, therapeutic exosome application requires knowledge on target structures to avoid undue delivery. Previous work suggesting exosomal tetraspanin-integrin complexes to be involved in target cell binding, we aimed to control this hypothesis and to define target cell ligands. Exosomes are rich in tetraspanins that associate besides other molecules with integrins. Co-immunoprecipitation of exosome lysates from rat tumor lines that differ only with respect to Tspan8 and beta4 revealed promiscuity of tetraspanin-integrin associations, but also few preferential interactions like that of Tspan8 with alpha4 and beta4 integrin chains. These minor differences in exosomal tetraspanin-complexes strongly influence target cell selection in vitro and in vivo, efficient exosome-uptake being seen in hematopoietic cells and solid organs. Exosomes expressing the Tspan8-alpha4 complex are most readily taken up by endothelial and pancreas cells, CD54 serving as a major ligand. Selectivity of uptake was confirmed with exosomes from an alpha4 cDNA transfected Tspan8(+) lymph node stroma line. Distinct from exosomes from the parental line, the latter preferentially targeted endothelial cells and in vivo the pancreas. Importantly, pulldown experiments provided strong evidence that exosome-uptake occurs in internalization-prone membrane domains. This is the first report on the exosomal tetraspanin web contributing to target cell selection such that predictions can be made on potential targets, which will facilitate tailoring exosomes for drug delivery.     

Epinephrine deficiency results in intact glucose counter-regulation, severe hepatic steatosis and possible defective autophagy in fasting mice

Epinephrine is one of the major hormones involved in glucose counter-regulation and gluconeogenesis. However, little is known about its importance in energy homeostasis during fasting. Our objective is to study the specific role of epinephrine in glucose and lipid metabolism during starvation. In our experiment, we subject regular mice and epinephrine-deficient mice to a 48-h fast then we evaluate the different metabolic responses to fasting. Our results show that epinephrine is not required for glucose counter-regulation: epinephrine-deficient mice maintain their blood glucose at normal fasting levels via glycogenolysis and gluconeogenesis, with normal fasting-induced changes in the peroxisomal activators: peroxisome proliferator activated receptor γ coactivator α (PGC-1α), fibroblast growth factor 21 (FGF-21), peroxisome proliferator activated receptor α (PPAR-α), and sterol regulatory element binding protein (SREBP-1c). However, fasted epinephrine-deficient mice develop severe ketosis and hepatic steatosis, with evidence for inhibition of hepatic autophagy, a process that normally provides essential energy via degradation of hepatic triglycerides during starvation. We conclude that, during fasting, epinephrine is not required for glucose homeostasis, lipolysis or ketogenesis. Epinephrine may have an essential role in lipid handling, possibly via an autophagy-dependent mechanism.     

Zfp423 expression identifies committed preadipocytes and localizes to adipose endothelial and perivascular cells

Progress has been made in elucidating the cell-surface phenotype of primary adipose progenitors; however, specific functional markers and distinct molecular signatures of fat depot-specific preadipocytes have remained elusive. In this study, we label committed murine adipose progenitors through expression of GFP from the genetic locus for Zfp423, a gene controlling preadipocyte determination. Selection of GFP-expressing fibroblasts from either subcutaneous or visceral adipose-derived stromal vascular cultures isolates stably committed preadipocytes that undergo robust adipogenesis. Immunohistochemistry for Zfp423-driven GFP expression in?vivo confirms a perivascular origin of preadipocytes within both white and brown adipose tissues. Interestingly, a small subset of capillary endothelial cells within white and brown fat also express this marker, suggesting a contribution of specialized endothelial cells to the adipose lineage. Zfp423(GFP) mice represent a simple tool for the specific localization and isolation of molecularly defined preadipocytes from distinct adipose tissue depots.     

O‐12BLAND DYSKARYOSIS: A NEW PITFALL IN THINPREP® LIQUID BASED CYTOLOGY

Liquid based cytology (LBC) has improved cell visualization and preservation in cervical cytology. There has been a reduction in inadequate rate and some data to suggest an increase in sensitivity for dyskaryosis. Training for LBC has focused on differences in distribution of abnormal cells, but in most cases the morphological appearance of the dyskaryotic cells themselves is similar to that seen in conventional cytology. We are describing a new presentation of dyskaryosis which may be a cause of false negative cytology. We have referred to this as ‘Bland dyskaryosis’ because cells appear deceptively bland on low power examination, and can be misinterpreted as metaplastic or endocervical cells. Bland dyskaryosis cells are seen in groups. The architecture of the group is very disorganized, and adjacent cells show variation in size. Cells have a high nuclear/cytoplasmic ratio and smooth nuclear membranes. Chromatin is finely granular and evenly distributed. This is an unusual presentation of high‐grade dyskaryosis and we feel that there is a learning curve in laboratories converting to liquid based cytology. The spectrum of appearances of squamous dyskaryosis needs to be delineated to allow further increases in sensitivity for dyskaryosis.     

Sustained Action of Ceramide on the Insulin Signaling Pathway in Muscle Cells: IMPLICATION OF THE DOUBLE-STRANDED RNA-ACTIVATED PROTEIN KINASE*

In vivo, ectopic accumulation of fatty acids in muscles leads to alterations in insulin signaling at both the IRS1 and Akt steps. However, in vitro treatments with saturated fatty acids or their derivative ceramide demonstrate an effect only at the Akt step. In this study, we adapted our experimental procedures to mimic the in vivo situation and show that the double-stranded RNA-dependent protein kinase (PKR) is involved in the long-term effects of saturated fatty acids on IRS1. C2C12 or human muscle cells were incubated with palmitate or directly with ceramide for short or long periods, and insulin signaling pathway activity was evaluated. PKR involvement was assessed through pharmacological and genetic studies. Short-term treatments of myotubes with palmitate, a ceramide precursor, or directly with ceramide induce an inhibition of Akt, whereas prolonged periods of treatment show an additive inhibition of insulin signaling through increased IRS1 serine 307 phosphorylation. PKR mRNA, protein, and phosphorylation are increased in insulin-resistant muscles. When PKR activity is reduced (siRNA or a pharmacological inhibitor), serine phosphorylation of IRS1 is reduced, and insulin-induced phosphorylation of Akt is improved. Finally, we show that JNK mediates ceramide-activated PKR inhibitory action on IRS1. Together, in the long term, our results show that ceramide acts at two distinct levels of the insulin signaling pathway (IRS1 and Akt). PKR, which is induced by both inflammation signals and ceramide, could play a major role in the development of insulin resistance in muscle cells.     

Identification of extremely halophilic archaea associated with adult <Emphasis Type="Italic">Artemia urmiana</Emphasis>

The brine shrimp, Artemia is the dominant macrozooplankton present in many hypersaline environments. Artemia urmiana is the only macroscopic organism in Urmia Salt Lake (Iran), and the high salinity of the lake makes it a suitable environment for halophilic archaea too. Because of common environment for Artemia and extreme halophiles; this investigation is concentrated on studying the relationship between Artemia and halophilic archaea in Urmia Lake. In this study first the procedure of arhaea isolation was done. Then, isolated strains were sub-cultured and DNA was extracted and amplified by PCR using specific primers for amplifying archaeal 16S rRNA. The amplified archeal DNA fragments were purified, and sequenced. 16S rRNA sequences were compared to known sequences using the NCBI BLAST program. Sequences relating to Halorubrum, Haloarcula and Halobacterium species were identified in Urmia Salt Lake water and Artemia adults and the phylogenetic tree of different species was constructed. Only Halorubrum species were present in association with Artemia. They belong to Halobacteriaceae family of archeae which are isolated from different salt lakes in different parts of world and we could show their existence in adult Artemia, another organism living in hypersaline enviroments.