Interaction of dipeptydil peptidase IV with amyloid peptides

The aggregates of amyloid beta peptides (Aβs) are regarded as one of the main pathological hallmarks of Alzheimer’s disease (AD). An imbalance between the rates of synthesis and clearance of Aβs is considered to be a possible cause for the onset of AD. Dipeptidyl peptidases II and IV (DPPII and DPPIV) are serine proteases removing N-terminal dipeptides from polypeptides and proteins with proline or alanine on the penultimate position. Alanine is an N-terminal penultimate residue in Аβs, and we presumed that DPPII and DPPIV could cleave them. The results of present in vitro research demonstrate for the first time the ability of DPPIV to truncate the commercial Aβ40 and Aβ42 peptides, to hinder the fibril formation by them and to participate in the disaggregation of preformed fibrils of these peptides. The increase of absorbance at 334 nm due to complex formation between primary amines with o-phtalaldehyde was used to show cleaving of Aβ40 and Aβ42. The time-dependent increase of the quantity of primary amines during incubation of peptides in the presence of DPPIV suggested their truncation by DPPIV, but not by DPPII. The parameters of the enzymatic breakdown by DPPIV were determined for Aβ40 (K_m = 37.5 μM, k_cat/K_m = 1.7 × 10³ M⁻¹sec⁻¹) and Aβ42 (K_m = 138.4 μM, k_cat/K_m = 1.90 × 10² M⁻¹sec⁻¹). The aggregation-disaggregation of peptides was controlled by visualization on transmission electron microscope and by Thioflavin-T fluorescence on spectrofluorimeter and fluorescent microscope. DPPIV hindered the peptide aggregation/fibrillation during 3-4 days incubation in 20 mM phosphate buffer, pH 7.4, 37 °C by 50–80%. Ovalbumin, BSA and DPPII did not show this effect. In the presence of DPPIV, the preformed fibrils were disaggregated by 30–40%. Conclusion: for the first time it was shown that the Aβ40 and Aβ42 are substrates of DPPIV. DPPIV prohibits the fibrillation of peptides and promotes disaggregation of their preformed aggregates.     

The density-dependency of dark- and low-background radiation effects on water and water solution properties

The effects of dark -(Ev=0 lux) and low-background radiation (BGR), where R<1μRongen/h, on physicochemical properties (specific electrical conductivity, heat fusion, hydrogen peroxide (H2O2), and oxygen contents) of distilled water (DW) and physiological solution (PS) at 4°C and 18°C were studied. The incubation of DW and PS samples in dark and in low BGR (under dark) medium at 4°C and 18°C brings to changes of their physicochemical properties compared with DW and PS samples incubated in light and normal BGR condition (Ev=500-550 lux and R=17 μRoentgen/h). The observed changes of DW and PS properties depended on their initial temperature, density and ionic composition. It is suggested that water molecules dissociation and ions hydration are sensitive to illumination and BGR. Therefore, the cell-bathing medium can be considered as a messenger through which direct and non direct (by modulating of others factors-induced effects) influences of illumination and BGR on cell metabolism are realized.     

P element-mediated duplications of genomic regions in Drosophila melanogaster

Previously we have described highly unstable yellow mutations induced by chimeric elements that consist of genomic sequences originating from different regions of the X chromosome flanked by identical copies of an internally deleted 1.2 kb P element. To study further the origin and the mechanism of formation of chimeric mobile elements, we analyzed complex y-sc mutations, induced by inversions between P elements located in the neighboring yellow and scute loci. The breakpoints of the inversions are flanked by two P elements in head-to-head orientation on one side and by one P element on the other side. Such an arrangement of P elements leads to frequent duplication into the site between the two P element copies located in head-to-head orientation of the yellow sequences adjacent to the single P element. The duplicated yellow sequences either partly replace the sequence of one of the P elements or are inserted between the conserved head-to-head oriented P elements. In some cases two copies of the yellow sequence are duplicated between the P elements in inverted tail-to-tail orientation. The structure of the P elements at the place of duplication and of the P element- yellow junction suggests that the described duplications, which form chimeric mobile elements, are generated through the previously proposed synthesis-dependent strand annealing mechanism.     

Glucose isomerase production on a xylan-based medium by Bacillus thermoantarcticus

Effects of medium components on intracellular glucose isomerase (GI) production were investigated by Bacillus thermoantarcticus. The highest GI activity was obtained as 1630 U dm^?3 in the medium containing (g dm^?3): 10.6, birchwood-xylan; 5.6, yeast extract; 5.9 (NH₄)₂SO₄ at T = 55 °C in 33 cm^?3 shake-flasks. When birchwood-xylan was replaced with oat spelt- or beechwood-xylan, GI activity decreased to 1372 and 1308 U dm^?3, respectively. Effects of pH at uncontrolled-pH (pH_UC = 6.0) and controlled-pH (pH_C = 6.0) operations, and oxygen transfer at the air inlet rate of 0.5 vvm and agitation rates of 300, 500 and 700 min^?1, were investigated in 3.0 dm³ bioreactor system with 1.65 dm³ working volume in the designed medium. The highest GI activity was attained at 500 min^?1, 0.5 vvm, pH_UC = 6 as 1840 U dm^?3 where cell concentration was 2.3 g dm^?3. The use of agricultural waste xylan, as the carbon source resulted in concomitant production of xylanase and GI. The highest xylanase activity was attained as 9300 U dm^?3 at 500 min^?1 and 0.5 vvm. K_La varied between 0.008–0.033 s^?1 whereas the highest oxygen uptake rate was 0.002 mmol dm^?3 s^?1. Initially biochemical reaction limitations were effective; thereafter, mass transfer resistances became more effective.     

High-throughput sequencing of mGluR signaling pathway genes reveals enrichment of rare variants in autism

Identification of common molecular pathways affected by genetic variation in autism is important for understanding disease pathogenesis and devising effective therapies. Here, we test the hypothesis that rare genetic variation in the metabotropic glutamate-receptor (mGluR) signaling pathway contributes to autism susceptibility. Single-nucleotide variants in genes encoding components of the mGluR signaling pathway were identified by high-throughput multiplex sequencing of pooled samples from 290 non-syndromic autism cases and 300 ethnically matched controls on two independent next-generation platforms. This analysis revealed significant enrichment of rare functional variants in the mGluR pathway in autism cases. Higher burdens of rare, potentially deleterious variants were identified in autism cases for three pathway genes previously implicated in syndromic autism spectrum disorder, TSC1, TSC2, and SHANK3, suggesting that genetic variation in these genes also contributes to risk for non-syndromic autism. In addition, our analysis identified HOMER1, which encodes a postsynaptic density-localized scaffolding protein that interacts with Shank3 to regulate mGluR activity, as a novel autism-risk gene. Rare, potentially deleterious HOMER1 variants identified uniquely in the autism population affected functionally important protein regions or regulatory sequences and co-segregated closely with autism among children of affected families. We also identified rare ASD-associated coding variants predicted to have damaging effects on components of the Ras/MAPK cascade. Collectively, these findings suggest that altered signaling downstream of mGluRs contributes to the pathogenesis of non-syndromic autism.