Uptake of microperoxidase by segmenting rat ova in vitro

Summary Uptake and incidence of microperoxidase (Sigma) in the rat ova during cleavage at the 1-, 2-, 8-cell and blastocyst stages were studied after 30 min incubation with the enzyme (molecular weight of 1,900 and size of molecule of 2 nm).Evidence was furnished of the presence of a small amount of reaction product of microperoxidase in the zona pellucida and its local concentration in some parts of the perivitelline space. The larger part of surface of the ovum, however, was free of microperoxidase. In the cytoplasm microperoxidase was found in pinocytotic vesicles, less frequently in large vacuoles and starting with the eight-cell stage in secondary lysosomes. The largest amount of microperoxidase was ascertained at the stage of blastocyst, chiefly in the cells of the trophoblast. In the cells of the embryoblast, on the contrary, microperoxidase was found but occasionally. The reaction product of microperoxidase was also present in the intercellular space of ova in negligible amount, and likewise on the side of blastocysts' cavity. In comparison with the ingestion of horseradish peroxidase the incidence of microperoxidase in the segmenting rat ova was less frequent.     

Corticotropin-Releasing Hormone Affects Short Immobilization Stress-Induced Changes in Lung Cytosolic and Membrane Glucocorticoid Binding Sites

Glucocorticoids act via glucocorticoid receptors (GR), typically localized in the cytosol (cGR). Rapid action is probably mediated via membrane receptors (mGR). In corticotropin-releasing hormone knockouts (CRH-KO), basal plasma glucocorticoid levels do differ from wild type levels (WT), but are approximately ten times lower during exposure to immobilization stress (IMMO) in comparison to WT. We tested the following hypotheses: (1) the mice lung tissue GR basal numbers would not be changed in CRH-KO (because of similar glucocorticoid levels), (2) the number of GR would be changed in WT but not in KO during short (30, 90, and 120 min) IMMO (because of higher increase of glucocorticoid levels in WT). The basal levels of cGR were not changed in CRH-KO (compared to WT), while mGR were significantly lower (62 %) in CRH-KO. In WT, there was the only decrease (to 32 %) in cGR after 120 min when we also found an increase in mGR in WT (to 201 %). In CRH-KO, IMMO caused gradual decrease in cGR (to 52 % after 30 min, to 46 % after 90 min, and to 32 % after 120 min). In CRH-KO, the only increase in mGR appeared already at 30 min of IMMO. These data suggest, on the contrary to our hypotheses, that CRH-KO are more susceptible to GR changes in early phases of stress.     

Phenotypic profiling of mGlu7 knockout mice reveals new implications for neurodevelopmental disorders

Neurodevelopmental disorders are characterized by deficits in communication, cognition, attention, social behavior and/or motor control. Previous studies have pointed to the involvement of genes that regulate synaptic structure and function in the pathogenesis of these disorders. One such gene, GRM7, encodes the metabotropic glutamate receptor 7 (mGlu₇), a G protein‐coupled receptor that regulates presynaptic neurotransmitter release. Mutations and polymorphisms in GRM7 have been associated with neurodevelopmental disorders in clinical populations; however, limited preclinical studies have evaluated mGlu₇ in the context of this specific disease class. Here, we show that the absence of mGlu₇ in mice is sufficient to alter phenotypes within the domains of social behavior, associative learning, motor function, epilepsy and sleep. Moreover, Grm7 knockout mice exhibit an attenuated response to amphetamine. These findings provide rationale for further investigation of mGlu₇ as a potential therapeutic target for neurodevelopmental disorders such as idiopathic autism, attention deficit hyperactivity disorder and Rett syndrome.     

Positive selection and convergent evolution shape molecular phenotypic traits of innate immunity receptors in tits (Paridae)

Despite widespread variability and redundancy abounding animal immunity, little is currently known about the rate of evolutionary convergence (functionally analogous traits not inherited from a common ancestor) in host molecular adaptations to parasite selective pressures. Toll‐like receptors (TLRs) provide the molecular interface allowing hosts to recognize pathogenic structures and trigger early danger signals initiating an immune response. Using a novel combination of bioinformatic approaches, here we explore genetic variation in ligand‐binding regions of bacteria‐sensing TLR4 and TLR5 in 29 species belonging to the tit family of passerine birds (Aves: Paridae). Three out of the four consensual positively selected sites in TLR4 and six out of 14 positively selected positions in TLR5 were located on the receptor surface near the functionally important sites, and based on the phylogenetic pattern evolved in a convergent (parallel) manner. This type of evolution was also seen at one N‐glycosylation site and two positively selected phosphorylation sites, providing the first evidence of convergence in post‐translational modifications in evolutionary immunology. Finally, the overall mismatch between phylogeny and the clustering of surface charge distribution demonstrates that convergence is common in overall TLR4 and TLR5 molecular phenotypes involved in ligand binding. Our analysis did not reveal any broad ecological traits explaining the convergence observed in electrostatic potentials, suggesting that information on microbial symbionts may be needed to explain TLR evolution. Adopting state‐of‐the‐art predictive structural bionformatics, we have outlined a new broadly applicable methodological approach to estimate the functional significance of positively selected variation and test for the adaptive molecular convergence in protein‐coding polymorphisms.     

Partial silencing of fucosyltransferase 8 gene expression inhibits proliferation of Ishikawa cells,a cell line of endometrial cancer

Endometrial cancer is the most common gynecologic malignancy and is associated with increased morbidity each year, including young people. However, its mechanisms of proliferation and progression are not fully elucidated. It is well known that abnormal glycosylation is involved in oncogenesis, and fucosylation is one of the most important types of glycosylation. In particular, fucosyltransferase 8 (FUT8) is the only FUT responsible for α1, 6-linked fucosylation (core fucosylation), and it is involved in various physiological as well as pathophysiological processes, including cancer biology. Therefore, we aimed to identify the expression of FUT8 in endometrial endometrioid carcinoma and investigate the effect of the partial silencing of the FUT8 gene on the cell proliferation of Ishikawa cells, an epithelial-like endometrial cancer cell line. Quantitative real-time PCR analysis showed that FUT8 gene expression was significantly elevated in the endometrial endometrioid carcinoma, compared to the normal endometrium. The immunostaining of FUT8 and Ulex europaeus Agglutinin 1 (UEA-1), a kind of lectin family specifically binding to fucose, was detected endometrial endometrioid carcinoma. The proliferation assay showed FUT8 partial knockdown by transfection of siRNA significantly suppressed the proliferation of Ishikawa cells, concomitant with the upregulation in the gene expressions associated with the interesting pathways associated with de-ubiquitination, aspirin trigger, mesenchymal-epithelial transition (MET) et al. It was suggested that the core fucosylation brought about by FUT8 might be involved in the proliferation of endometrial endometrioid carcinoma cells.     

Predictive markers of safety and immunogenicity of adjuvanted vaccines

Vaccination represents one of the greatest public health triumphs; in part due to the effect of adjuvants that have been included in vaccine preparations to boost the immune responses through different mechanisms. Although a variety of novel adjuvants have been under development, only a limited number have been approved by regulatory authorities for human vaccines. This report reflects the conclusions of a group of scientists from academia, regulatory agencies and industry who attended a conference on the current state of the art in the adjuvant field. Held at the U.S. Pharmacopeial Convention (USP) in Rockville, Maryland, USA, from 18 to 19 April 2013 and organized by the International Association for Biologicals (IABS), the conference focused particularly on the future development of effective adjuvants and adjuvanted vaccines and on overcoming major hurdles, such as safety and immunogenicity assessment, as well as regulatory scrutiny. More information on the conference output can be found on the IABS website, http://www.iabs.org/.     

Adenosine modulates LPS-induced cytokine production in porcine monocytes

Adenosine plays an important role during inflammation, particularly through modulation of monocyte function. The objective of the present study was to evaluate the effect of synthetic adenosine analogs on cytokine production by porcine monocytes. The LPS-stimulated cytokine production was measured by flow cytometry and quantitative real-time PCR. Adenosine receptor expression was measured by quantitative real-time PCR. The present study demonstrates that adenosine analog N-ethylcarboxyamidoadenosine (NECA) down-regulates TNF-α production and up-regulates IL-8 production by LPS-stimulated porcine monocytes. The effect was more pronounced in CD163^? subset of monocytes compared to the CD163⁺ subset. Although both monocyte subsets express mRNA for A1, A2A, A2B and A3 adenosine receptors, the treatment of monocytes with various adenosine receptor agonists and antagonists proved that the effect of adenosine is mediated preferentially via A2A adenosine receptor. Moreover, the study suggests that the effect of NECA on porcine monocytes alters the levels of the cytokines which could play a role in the differentiation of naive T cells into Th17 cells. The results suggest that adenosine plays an important role in modulation of cytokine production by porcine monocytes.     

Oxidative stress in small-for-gestational age (SGA) term newborns and their mothers

This study used malondialdehyde (MDA) determination by HPLC and enzymatic assays for total serum peroxides and antioxidant capacity to evaluate oxidative stress in 47 healthy full-term small-for-gestational age (SGA) newborns vs 67 appropriate-for-gestational age (AGA) newborns. Blood samples were collected at delivery from umbilical cord artery and vein and from peripheral blood of the babies on the third day after birth. Blood samples of mothers were also collected and compared with blood of 29 normal non-pregnant women (NPW). Serum peroxide values were significantly higher in both groups of mothers than in NPW, decreasing towards the third day in AGA mothers, while persisting in SGA mothers. Antioxidant capacity of sera of both groups of mothers was lower than NPW. Both SGA mothers and babies had increased MDA at delivery, unlike AGA counterparts. MDA levels in umbilical vein were higher than in umbilical arteries, while immunohistochemistry revealed abundant presence of 4-hydroxynonenal (HNE)-protein adducts only in stroma of the SGA placenta. These results show that both mothers and babies are exposed to oxidative stress during and after delivery, which is more pronounced and persistent in the perinatal period of the SGA group, while lipid peroxidation in placenta could play a role in SGA pathophysiology.     

Synthesis and Evaluation of Acyclic Nucleotide Analogs

Abstract

Acyclic nucleotide analogs derived from antiviral 9-(2-phosphonylmethoxyethyl)adenine by modification at the side chain or by alternation of the heterocyclic base were synthesized and investigated for their antiviral activity.     

A high density physical map of chromosome 1BL supports evolutionary studies,map-based cloning and sequencing in wheat

Background

As for other major crops, achieving a complete wheat genome sequence is essential for the application of genomics to breeding new and improved varieties. To overcome the complexities of the large, highly repetitive and hexaploid wheat genome, the International Wheat Genome Sequencing Consortium established a chromosome-based strategy that was validated by the construction of the physical map of chromosome 3B. Here, we present improved strategies for the construction of highly integrated and ordered wheat physical maps, using chromosome 1BL as a template, and illustrate their potential for evolutionary studies and map-based cloning.

Results

Using a combination of novel high throughput marker assays and an assembly program, we developed a high quality physical map representing 93% of wheat chromosome 1BL, anchored and ordered with 5,489 markers including 1,161 genes. Analysis of the gene space organization and evolution revealed that gene distribution and conservation along the chromosome results from the superimposition of the ancestral grass and recent wheat evolutionary patterns, leading to a peak of synteny in the central part of the chromosome arm and an increased density of non-collinear genes towards the telomere. With a density of about 11 markers per Mb, the 1BL physical map provides 916 markers, including 193 genes, for fine mapping the 40 QTLs mapped on this chromosome.

Conclusions

Here, we demonstrate that high marker density physical maps can be developed in complex genomes such as wheat to accelerate map-based cloning, gain new insights into genome evolution, and provide a foundation for reference sequencing.