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.     

Simulation of leaf photosynthesis of C3 plants under fluctuating light and different temperatures

An induction-dependent empirical model was developed to simulate the C₃ leaf photosynthesis under fluctuating light and different temperatures. The model also takes into account the stomatal conductance when the light intensity just exceeds the compensation point after a prolonged period of darkness (initial stomatal conductance, $ g_{{{\text{S}}_{\text{ini}} }} $ ). The model was parameterized for both Chrysanthemum morifolium and Spinacia oleracea by artificially changing the induction states of the leaves in the climate chamber. The model was tested under natural conditions that were including frequent light flecks due to partial cloud cover and varying temperatures. The temporal course of observed photosynthesis rate and the carbon gain was compared to the simulation. The ability of the current model to predict the carbon assimilation rate was assessed using different statistical indexes. The model predictions were accurate but the model slightly underestimated the actual overall carbon gain. The accuracy of the simulation was largely dependent on the parameters that were calculated for the particular plant species, of which the simulation is intended for. In particular, the rate of change of induction and the initial stomatal conductance were found to be highly important and these were species-specific parameters for the predictions. The model is suitable for estimating instantaneous leaf CO₂ assimilation for different herbaceous plant species under dynamic environmental conditions. It can be simply calibrated for other crops, by estimating the individual parameters.     

Grain zinc, iron and protein concentrations and zinc-efficiency in wild emmer wheat under contrasting irrigation regimes

Micronutrient malnutrition, and particularly deficiency in zinc (Zn) and iron (Fe), afflicts over three billion people worldwide, and nearly half of the world’s cereal-growing area is affected by soil Zn deficiency. Wild emmer wheat [Triticum turgidum ssp. dicoccoides (Körn.) Thell.], the progenitor of domesticated durum wheat and bread wheat, offers a valuable source of economically important genetic diversity including grain mineral concentrations. Twenty two wild emmer wheat accessions, representing a wide range of drought resistance capacity, as well as two durum wheat cultivars were examined under two contrasting irrigation regimes (well-watered control and water-limited), for grain yield, total biomass production and grain Zn, Fe and protein concentrations. The wild emmer accessions exhibited high genetic diversity for yield and grain Zn, Fe and protein concentrations under both irrigation regimes, with a considerable potential for improvement of the cultivated wheat. Grain Zn, Fe and protein concentrations were positively correlated with one another. Although irrigation regime significantly affected ranking of genotypes, a few wild emmer accessions were identified for their advantage over durum wheat, having consistently higher grain Zn (e.g., 125 mg kg^?1), Fe (85 mg kg^?1) and protein (250 g kg^?1) concentrations and high yield capacity. Plants grown from seeds originated from both irrigation regimes were also examined for Zn efficiency (Zn deficiency tolerance) on a Zn-deficient calcareous soil. Zinc efficiency, expressed as the ratio of shoot dry matter production under Zn deficiency to Zn fertilization, showed large genetic variation among the genotypes tested. The source of seeds from maternal plants grown under both irrigation regimes had very little effect on Zn efficiency. Several wild emmer accessions revealed combination of high Zn efficiency and drought stress resistance. The results indicate high genetic potential of wild emmer wheat to improve grain Zn, Fe and protein concentrations, Zn deficiency tolerance and drought resistance in cultivated wheat.     

Additive Antiatherogenic Effects of CETP rs708272 on Serum LDL Subfraction Levels in Patients with CHD Under Statin Therapy

Recently, subfraction analysis of serum low density lipoprotein (LDL) is considered to be a better predictor of the risk of coronary heart disease (CHD) compared to the other lipid parameters. The aim of this study was to examine the effects of the HDL-associated Taq1B (rs708272) SNP of cholesterol ester transfer protein (CETP) gene on serum LDL subfractions in patients with CHD. Serum lipid levels were measured enzymatically and LDL subfraction analysis was carried out by the Lipoprint System (Quantimetrix, CA, USA). The CETP rs708272 SNP was studied in 66 healthy controls and 79 patients with CHD receiving statin therapy by the PCR–RFLP technique. The CHD patients had elevated antiatherogenic LDL-1 subfraction (p = 0.042), decreased atherogenic IDL-C subfraction (p = 0.023), and total IDL (p = 0.030) levels compared to the healthy controls. The CETP rs708272 Taq1B minor B2 allele was associated with increased levels of antiatherogenic LDL-1 (B2: 0.40 ± 0.20 vs. B1B1: 0.25 ± 0.08, p = 0.004) and large-LDL (LDL 1–2) subfractions in the CHD group (B2 allele: 0.68 ± 0.41 vs. B1B1: 0.42 ± 0.20; p < 0.05), while it was associated with reduced levels of the large-LDL subfraction in healthy subjects (B2 allele: 0.29 ± 0.14 vs. B1B1: 0.54 ± 0.24; p = 0.017). However, there was no statistically significant association between the CETP rs708272 SNP and small dense LDL subfraction (LDL 3–7) and lipoprotein levels (p > 0.05). Our findings have indicated that the CETP rs708272 SNP together with statin therapy may show a favorable effect on antiatherogenic LDL-1 and large-LDL subfractions in CHD patients with an atherogenic effect on large-LDL subfraction in healthy subjects. Based on these results, it can be concluded that the effects of the CETP variation on LDL subfraction could change in cardiometabolic events such as CHD and statin therapy.     

Site-directed mutagenesis of five conserved residues of subunit i of the cytochrome cbb3 oxidase in Rhodobacter capsulatus

Cytochrome cbb(3) oxidase is a member of the heme-copper oxidase superfamily that catalyses the reduction of molecular oxygen to the water and conserves the liberated energy in the form of a proton gradient. Comparison of the amino acid sequences of subunit I from different classes of heme-copper oxidases showed that transmembrane helix VIII and the loop between transmembrane helices IX and X contain five highly conserved polar residues; Ser333, Ser340, Thr350, Asn390 and Thr394. To determine the relationship between these conserved amino acids and the activity and assembly of the cbb(3) oxidase in Rhodobacter capsulatus, each of these five conserved amino acids was substituted for alanine by site-directed mutagenesis. The effects of these mutations on catalytic activity were determined using a NADI plate assay and by measurements of the rate of oxygen consumption. The consequence of these mutations for the structural integrity of the cbb(3) oxidase was determined by SDS-PAGE analysis of chromatophore membranes followed by TMBZ staining. The results indicate that the Asn390Ala mutation led to a complete loss of enzyme activity and that the Ser333Ala mutation decreased the activity significantly. The remaining mutants cause a partial loss of catalytic activity. All of the mutant enzymes, except Asn390Ala, were apparently correctly assembled and stable in the membrane of the R. capsulatus.     

Seroprevalence of Borrelia burgdorferi infection among forestry workers and farmers in Duzce, north-western Turkey

Borrelia burgdorferi infection is the most frequent tick-transmitted disease worldwide. Our aim was to assess the seroprevalence of B. burgdorferi infection among forestry workers and farmers in Duzce, in the north-west region of Turkey. Blood samples from 349 forestry workers and farmers and 193 healthy blood donors were obtained to determine the presence of antibodies to B. burgdorferi. A two-step testing strategy was used; the sera were initially tested by ELISA and then by Western blot (WB) IgG. Demographic data regarding residence, age, gender, profession, tick bite history, contact with animals, and symptoms involving the skin, nervous system, and osteoarticular system were collected by questionnaire. All results were evaluated statistically using the chi2 test. The seroprevalence of B. burgdorferi was 10.9% (n=38) in forestry workers and farmers and 2.6% (n=5) in blood donors by ELISA, with a statistically significant difference (p<0.001). Seropositivity rates were related to age, gender, and common risk factors for the disease. IgG seropositivity was confirmed in four (1.1%) sera by WB. In this first seroepidemiological report from the northwest region of Turkey, tick bite exposure was found to be high, whereas B. burgdorferi infection was not common. Preventive measures against tick exposure and further studies to determine the distribution of Lyme disease in Turkey are proposed.     

Paraoxonase and Arylesterase Levels in Behcet’s Disease and Their Relations with the Disease Activity

The aim of this study was to determine the paraoxonase (PON) and arylesterase (ARE) enzyme activity levels in Behcet’s disease (BD) and to investigate whether they are associated with the disease activity. Twenty-six patients (study group) with active BD and 28 healthy controls (control group) were included in this study. While the patients who had at least one of the symptoms related to genital ulcer, skin lesions, active uveitis, arthritis, thrombophlebitis, or central nervous system involvement in addition to oral ulcers were considered as the active group, the patients who did not show clinical symptoms in the last one month due to the medical treatment were considered as the inactive group in the clinical evaluation of patients with BD. The PON and ARE levels were found to be significantly lower in the study group than the control group (p < 0.05). The PON levels of the active and inactive groups were 96.23 ± 57.84 and 112.2 ± 65.14, respectively. The ARE levels of the active and inactive groups were 30.49 ± 5.81 and 30.85 ± 6.40, respectively. No significant correlations were found between clinical findings and the activity levels of PON and ARE in the active patient group (p > 0.05). The activities of the antioxidant PON and ARE enzymes are reduced in BD. Therefore, it may be useful to add antioxidant therapy to the conventional treatment of the disease.     

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.     

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.