DNA methylation-associated colonic mucosal immune and defense responses in treatment-na?ve pediatric ulcerative colitis

Inflammatory bowel diseases (IBD) are emerging globally, indicating that environmental factors may be important in their pathogenesis. Colonic mucosal epigenetic changes, such as DNA methylation, can occur in response to the environment and have been implicated in IBD pathology. However, mucosal DNA methylation has not been examined in treatment-naïve patients. We studied DNA methylation in untreated, left sided colonic biopsy specimens using the Infinium HumanMethylation450 BeadChip array. We analyzed 22 control (C) patients, 15 untreated Crohn’s disease (CD) patients, and 9 untreated ulcerative colitis (UC) patients from two cohorts. Samples obtained at the time of clinical remission from two of the treatment-naïve UC patients were also included into the analysis. UC-specific gene expression was interrogated in a subset of adjacent samples (5 C and 5 UC) using the Affymetrix GeneChip PrimeView Human Gene Expression Arrays. Only treatment-naïve UC separated from control. One-hundred-and-twenty genes with significant expression change in UC (> 2-fold, P < 0.05) were associated with differentially methylated regions (DMRs). Epigenetically associated gene expression changes (including gene expression changes in the IFITM1, ITGB2, S100A9, SLPI, SAA1, and STAT3 genes) were linked to colonic mucosal immune and defense responses. These findings underscore the relationship between epigenetic changes and inflammation in pediatric treatment-naïve UC and may have potential etiologic, diagnostic, and therapeutic relevance for IBD.     

Production of DON-related trichothecenes byFusarium graminearum Schw from Krasnodarski krai of the USSR

34Fusarium graminearum Schw isolates produced 4-deoxynivalenol to form significant amounts of 4, 7 — dideoxynivalenol and lesser amounts of 4 — deoxynivalenol monoacetates on grain substratesin vitro. This is the first report on the capability a large group of naturally occurring isolates to produce 4,7-dideoxynivalenol. The average levels of 4,7-dideoxynivalenol on rice, corn, barley, and wheat as a substrate were respectively 26.8, 14.0, 12.8, and 10.5% of the level of 4-deoxynivalenol. 4, 7 — dideoxynivalenol was present in all examined naturally contaminated wheat kernel samples at levels of 1.7 to 7.9% of the level of 4-deoxynivalenol. These findings suggest that more attention should be given to the occurrence of 4,7-dideoxynivalenol in cereals.     

Plant virus infection development as affected by heavy metal stress

The work was focused on the investigation of possible dependencies between the development of viral infection in plants and the presence of high heavy metal concentrations in soil. Field experiments have been conducted in order to study the development of systemic tobacco mosaic virus (TMV) infection in Lycopersicon esculentum L. cv. Miliana plants under effect of separate salts of heavy metals Cu, Zn and Pb deposited in soil. As it is shown, simultaneous effect of viral infection and heavy metals in tenfold maximum permissible concentration leads to decrease of total chlorophyll content in experiment plants mainly due to the degradation of chlorophyll a. The reduction of chlorophyll concentration under the combined influence of both stress factors was more serious comparing to the separate effect of every single factor. Plants' treatment with toxic concentrations of lead and zinc leaded to slight delay in the development of systemic TMV infection together with more than twofold increase of virus content in plants that may be an evidence of synergism between these heavy metal's and virus' effects. Contrary, copper although decreased total chlorophyll content but showed protective properties and significantly reduced amount of virus in plants.     

Production of antibodies to roridin and their applications

Polyclonal rabbit antibodies against a conjugate synthesized through condensing BSA and disubstituted roridin A hemisuccinate allowed roridin A to be determined in solutions at a sensitivity of 0.2 ng/ml. The cross-reactivity of structural analogues—roridin A, verrucarin A, and verrucarol—amounted to 100, 2.5, and 0.03%, respectively. The data showed that these antibodies determine roridin A in an indirect heterogeneous enzyme immunoassay in cereal straw samples at a sensitivity of 20 μg/kg.     

Caffeine-induced oscillations of the membrane potential inAplysia neurons

It has been found in culturedAplysia neurons, including L7 and L2–L6 neurons, that bath application of 40 mM caffeine evokes oscillations of the membrane potential (MP) with the amplitude of about 40 mV. The frequency of oscillations, on the crest of which action potentials (AP) arise, varied from 0.2 to 0.5 sec¹. The effect of caffeine was completely reversible. The MP waves demonstrated high sensitivity to membrane polarization: artificial depolarization increased the frequency of oscillations, while even subtle hyperpolarization resulted in a decrease in the frequency up to their complete disappearance. External application of CdCl₂ (1 mM), a nonspecific blocker of calcium channels, or ryanodine (50 μM, 20 min), release of Ca²⁻ from the intracellular stores, replacement of Ca²⁺ in the external medium by Mg²⁻, or Na⁺ by Li⁺, did not exert visible effect on the parameters of MP waves. It was concluded that Ca ions (changing of intracellular concentration of which is due to such processes as inward calcium current, ryanodine-sensitive caffeine-induced calcium release from the intracellular, stores, sodium-calcium exchange through the plasma membrane) do not play any significant part in generation of the MP waves. The most probable mechanism of caffeine-induced oscillations in the studied nerve cells is inhibition of voltage-activated outward potassium current and, as could be seen from our mathematical modeling, slowdown of inactivation of inward sodium current. It seems likely that these oscillations have a purely membrane origin. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 102–111, March–April, 2000.     

Termination of translation in eukaryotes is mediated by the quaternary eRF1*eRF3*GTP*Mg2+ complex. The biological roles of eRF3 and prokaryotic RF3 are profoundly distinct

GTP hydrolysis catalyzed in the ribosome by a complex of two polypeptide release factors, eRF1 and eRF3, is required for fast and efficient termination of translation in eukaryotes. Here, isothermal titration calorimetry is used for the quantitative thermodynamic characterization of eRF3 interactions with guanine nucleotides, eRF1 and Mg²⁺. We show that (i) eRF3 binds GDP (K_d = 1.9 μM) and this interaction depends only minimally on the Mg²⁺ concentration; (ii) GTP binds to eRF3 (K_d = 0.5 μM) only in the presence of eRF1 and this interaction depends on the Mg²⁺ concentration; (iii) GTP displaces GDP from the eRF1•eRF3•GDP complex, and vice versa; (iv) eRF3 in the GDP-bound form improves its ability to bind eRF1; (v) the eRF1•eRF3 complex binds GDP as efficiently as free eRF3; (vi) the eRF1•eRF3 complex is efficiently formed in the absence of GDP/GTP but requires the presence of the C-terminus of eRF1 for complex formation. Our results show that eRF1 mediates GDP/GTP displacement on eRF3. We suggest that after formation of eRF1•eRF3•GTP•Mg²⁺, this quaternary complex binds to the ribosomal pretermination complex containing P-site-bound peptidyl-tRNA and the A-site-bound stop codon. The guanine nucleotide binding properties of eRF3 and of the eRF3•eRF1 complex profoundly differ from those of prokaryotic RF3.     

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.