Embryonic poly(A)-binding protein is differently expressed and interacts with the messenger RNAs in the mouse oocytes and early embryos

The embryonic poly(A)-binding protein (EPAB) functions in the translational regulation of the maternal messenger RNAs (mRNAs) required during oocyte maturation, fertilization, and early embryo development. Since there is no antibody specific to mammalian EPAB protein, all studies related to the Epab gene could be performed at the mRNA levels except for the investigations in the Xenopus. In this study, we have produced an EPAB-specific antibody. When we examined its expressional distribution in the mouse gonadal and somatic tissues, the EPAB protein was found to be expressed only in the mouse ovary and testis tissues, but it is undetectable level in the somatic tissues including stomach, liver, heart, small intestine, and kidney. Additionally, the spatial and temporal expression patterns of the EPAB and poly(A)-binding protein cytoplasmic 1 (PABPC1) proteins were analyzed in the mouse germinal vesicle (GV) and metaphase II (MII) oocytes, one-cell, and two-cell embryos. While EPAB expression gradually decreased from GV oocytes to two-cell embryos, the PABPC1 protein level progressively increased from GV oocytes to one-cell embryos and remarkably declined in the two-cell embryos ( P < 0.05). We have also described herein that the EPAB protein interacted with Epab, Pabpc1, Ccnb1, Gdf9, and Bmp15 mRNAs dependent upon the developmental stages of the mouse oocytes and early embryos. As a result, we have first produced an EPAB-specific antibody and characterized its expression patterns and interacting mRNAs in the mouse oocytes and early embryos. The findings suggest that EPAB in cooperation with PABPC1 implicate in the translational control of maternal mRNAs during oogenesis and early embryo development.     

DNA Methylation Mediates Persistent Epileptiform Activity In Vitro and In Vivo

Epilepsy is a chronic brain disorder involving recurring seizures often precipitated by an earlier neuronal insult. The mechanisms that link the transient neuronal insult to the lasting state of epilepsy are unknown. Here we tested the possible role of DNA methylation in mediating long-term induction of epileptiform activity by transient kainic acid exposure using in vitro and in vivo rodent models. We analyzed changes in the gria2 gene, which encodes for the GluA2 subunit of the ionotropic glutamate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid receptor and is well documented to play a role in epilepsy. We show that kainic acid exposure for two hours to mouse hippocampal slices triggers methylation of a 5’ regulatory region of the gria2 gene. Increase in methylation persists one week after removal of the drug, with concurrent suppression of gria2 mRNA expression levels. The degree of kainic acid-induced hypermethylation of gria2 5’ region varies between individual slices and correlates with the changes in excitability induced by kainic acid. In a rat in vivo model of post kainic acid-induced epilepsy, we show similar hypermethylation of the 5’ region of gria2. Inter-individual variations in gria2 methylation, correlate with the frequency and intensity of seizures among epileptic rats. Luciferase reporter assays support a regulatory role for methylation of gria2 5’ region. Inhibition of DNA methylation by RG108 blocked kainic acid-induced hypermethylation of gria2 5’ region in hippocampal slice cultures and bursting activity. Our results suggest that DNA methylation of such genes as gria2 mediates persistent epileptiform activity and inter-individual differences in the epileptic response to neuronal insult and that pharmacological agents that block DNA methylation inhibit epileptiform activity raising the prospect of DNA methylation inhibitors in epilepsy therapeutics.     

Combined point mutations in codon 12 and 13 of KRAS oncogene in prostate carcinomas

Prostate cancer is a common malignancy that develops by structural mutation(s) and/or other genetic alterations in specific genes.The G to T transversions in codon 12 and C to T transitions in codon 13 of KRAS proto-oncogene are predominant point mutations that occur in about 20% of different cancers in human. In the current study it was aimed to investigate the prevalence and predictive significance of KRAS mutations in patients with prostate carcinomas. In a total of 30 fresh tumoural tissue specimens were investigated in patients with prostate carcinoma. All tumoural specimens were histo-pathologically diagnosed and genotyped for codon 12, 13 KRAS point mutations by reverse hybridisation and direct sequencing methods. KRAS mutations were found in 12 (40%) samples with 29 samples deriving from adenocarcinomas and 1 sample was small cell prostate carcinoma. In 1 (3.44%) sample codon 12 was found to be mutated and in 2 (6.8%) samples codon 13 and in 9 (31%) samples combined codon 12 and 13 were found to be mutated particularly in higher grade of tumoural tissues. Our study, based on representative collection of human prostate tumours, indicates that combined mutations in codons 12 and 13 KRAS are relatively infrequent and most commonly occur in prostate carcinomas.     

Production and partial characterization of the exopolysaccharide from Pleurotus sajor caju

Microbial exopolysaccharides (EPSs) are very important because they are used in biotechnological applications in different industrial areas. The aim of the study was to determine the best EPS producer Pleurotus sp., to optimize EPS production and to perform partial purification and characterization of the produced EPS. After the production conditions were optimized, the EPS was isolated and partially purified. EPS was characterized by HP-TLC, 1H-NMR, FT-IR, and TGA. Hydroxyl, superoxide, and DPPH radical scavenging activities of the EPS were also investigated spectrophotometrically. The best EPS producer and its incubation period in submerged fermentation were determined as Pleurotus sajor caju and on 5 days, respectively. Culture conditions to increase EPS production were optimized as follows (in per liter): 90 g of glucose, 10 g of yeast extract, 10 g of peptone, and 100 mM of Mg2+. The optimal initial pH, temperature, and an agitation rate of culture were determined as 5.0, 25 °C, and 150 rate min−1, respectively. The highest EPS production was determined as 33.32 ± 1.6 g L−1. After isolation of EPS, one active fraction was obtained by gel filtration chromatography. EPS is composed mainly of glucose according to HP-TLC analysis. To the results, EPS had a complex structure by having carbohydrate and protein contents. The produced EPS had high degradation temperature as well as high antioxidant activity.     

IL-4Rα-Dependent Alternative Activation of Macrophages Is Not Decisive for Mycobacterium tuberculosis Pathology and Bacterial Burden in Mice

Classical activation of macrophages (caMph or M1) is crucial for host protection against Mycobacterium tuberculosis (Mtb) infection. Evidence suggests that IL-4/IL-13 alternatively activated macrophages (aaMph or M2) are exploited by Mtb to divert microbicidal functions of caMph. To define the functions of M2 macrophages during tuberculosis (TB), we infected mice deficient for IL-4 receptor α on macrophages (LysM^creIL-4Rα^-/lox) with Mtb. We show that absence of IL-4Rα on macrophages does not play a major role during infection with Mtb H37Rv, or the clinical Beijing strain HN878. This was demonstrated by similar mortality, bacterial burden, histopathology and T cell proliferation between infected wild-type (WT) and LysM^creIL-4Rα^-/lox mice. Interestingly, we observed no differences in the lung expression of inducible nitric oxide synthase (iNOS) and Arginase 1 (Arg1), well-established markers for M1/M2 macrophages among the Mtb-infected groups. Kinetic expression studies of IL-4/IL-13 activated bone marrow-derived macrophages (BMDM) infected with HN878, followed by gene set enrichment analysis, revealed that the MyD88 and IL-6, IL-10, G-CSF pathways are significantly enriched, but not the IL-4Rα driven pathway. Together, these results suggest that IL-4Rα-macrophages do not play a central role in TB disease progression.     

Population genetic structure of turbot (Scophthalmus maximus L., 1758) in the Black Sea

Turbot, Scophthalmus maximus, is a commercially important demersal flatfish species distributed throughout the Black Sea. Several studies performed locally with a limited number of specimens using both mitochondrial DNA (mtDNA) and microsatellite markers evidenced notable genetic variation among populations. However, comprehensive population genetic studies are required to help management of the species in the Black Sea. In the present study eight microsatellite loci were used to resolve the population structure of 414 turbot samples collected from 12 sites across the Black Sea. Moreover, two mtDNA genes, COI and Cyt-b, were used for taxonomic identification. Microsatellite markers of Smax-04 and B12-I GT14 were excluded from analysis due to scoring issues. Data analysis was performed with the remaining six loci. Loci were highly polymorphic (average of 17.8 alleles per locus), indicating high genetic variability. Locus 3/20CA17, with high null allele frequency (>30%), significantly deviated from HW equilibrium. Pairwise comparison of the F_ST index showed significant differences between most of the surveyed sampling sites (P < 0.01). Cluster analysis evidenced the presence of three genetic groups among sampling sites. Significant genetic differentiation between Northern (Sea of Azov and Crimea) and Southern (Turkish Black Sea Coast) Black Sea sampling sites were detected. The Mantel test supported an isolation by distance model of population structure. These findings are vital for long-term sustainable management of the species and development of conservation programs. Moreover, generated mtDNA sequences would be useful for the establishment of a database for S. maximus.     

Thymoquinone attenuates doxorubicin‐cardiotoxicity in rats

Contrary to the fact that doxorubicin is a powerful chemotherapeutic agent for the treatment of neoplastic diseases, cardiotoxicity is too important to be ignored. Thymoquinone serves as a powerful free radical scavenger. In the study, the effects of thymoquinone against doxorubicin‐cardiotoxicity will be evaluated. Forty rats were divided into five groups. Group I: control group (n = 8); group II: olive oil group (n = 8); group III: thymoquinone group (n = 8); given 10 mg/kg thymoquinone intraperitoneally per day throughout the experiment; group IV: doxorubicin group (n = 8); injected with a single dose of 15 mg/kg ip doxorubicin on the 7th day of the experiment; group V: doxorubicin + thymoquinone group (n = 8); administered with 10 mg/kg thymoquinone per day during the experiment and 15 mg/kg doxorubicin ip on the 7th day. The experiment was planned for 14 days. Immunohistochemically, heat shock protein (HSP) 70 and HSP90, glucose‐regulated protein 78 (GRP78), caspase‐3 were stained. We made terminal deoxynucleotidyl transferase dUTP nick end labeling for apoptotic evaluation. Total oxidant status (TOS) levels and total antioxidant status (TAS) were measured in the heart tissue. Atrial natriuretic peptide (ANP) and pro‐B type natriuretic peptide (proBNP) were evaluated. In the study, HSP70, HSP90, GRP78, and caspase‐3 levels increased in group IV. TOS and TAS levels were significant compared to group I. Doxorubicin significantly increased ANP and NT‐proBNP levels. Thymoquinone revealed significant differences in these values. Thymoquinone can be an important cardioprotective agent against doxorubicin‐cardiotoxicity.     

Quantitative trait loci conferring grain mineral nutrient concentrations in durum wheat × wild emmer wheat RIL population

Mineral nutrient malnutrition, and particularly deficiency in zinc and iron, afflicts over 3 billion people worldwide. Wild emmer wheat, Triticum turgidum ssp. dicoccoides, genepool harbors a rich allelic repertoire for mineral nutrients in the grain. The genetic and physiological basis of grain protein, micronutrients (zinc, iron, copper and manganese) and macronutrients (calcium, magnesium, potassium, phosphorus and sulfur) concentration was studied in tetraploid wheat population of 152 recombinant inbred lines (RILs), derived from a cross between durum wheat (cv. Langdon) and wild emmer (accession G18-16). Wide genetic variation was found among the RILs for all grain minerals, with considerable transgressive effect. A total of 82 QTLs were mapped for 10 minerals with LOD score range of 3.2–16.7. Most QTLs were in favor of the wild allele (50 QTLs). Fourteen pairs of QTLs for the same trait were mapped to seemingly homoeologous positions, reflecting synteny between the A and B genomes. Significant positive correlation was found between grain protein concentration (GPC), Zn, Fe and Cu, which was supported by significant overlap between the respective QTLs, suggesting common physiological and/or genetic factors controlling the concentrations of these mineral nutrients. Few genomic regions (chromosomes 2A, 5A, 6B and 7A) were found to harbor clusters of QTLs for GPC and other nutrients. These identified QTLs may facilitate the use of wild alleles for improving grain nutritional quality of elite wheat cultivars, especially in terms of protein, Zn and Fe.     

Genetic differentiation of Mediterranean horse mackerel (Trachurus mediterraneus) populations as revealed by mtDNA PCR-RFLP analysis

The genetic population structure of Mediterranean horse mackerel, Trachurus mediterraneus , from seven locations throughout the Black, Marmara, Aegean and eastern Mediterranean seas was investigated using restriction fragment length polymorphism (RFLP) analysis of the mtDNA 16S rDNA region. An approximately 2000-bp segment was screened in 280 individuals using six restriction enzymes, resulting in 10 composite haplotypes. The most common haplotype was present in 56.42% individuals; the next most frequent haplotype was present in 22.85% individuals. Average haplotype diversity within samples was moderate (0.38), and nucleotide diversity was low (0.00435). Mean nucleotide divergence for the seven sampling sites was 0.0028. Nucleotide divergence among samples was moderate, with the highest value detected between the Aegean Sea (Izmir) and the eastern Black Sea (Trabzon) populations (0.007055), and the lowest (−0.000043) between the Marmara Sea (Adalar) and the western Black Sea (Sile) populations. In Monte Carlo pairwise comparisons of haplotype frequencies, the Sinop from the middle Black Sea, Trabzon from the eastern Black Sea, and Iskenderun Bay from the north-eastern Mediterranean Sea exhibited highly significant (P   <   0.001) geographical differentiation from each other and from all other populations. Mantel's test indicated that the nucleotide divergence among populations of T. mediterraneus was not significantly associated with their geographical isolation ( r  = −0.2963; P   >   0.05). Consequently, the mtDNA 16S rDNA region provided evidence for the existence of three distinct T. mediterraneus populations (Sinop, Trabzon and Iskenderun Bay) in the Black and north-eastern Mediterranean seas.