Applications of the ETS-NOCV method in descriptions of chemical reactions

The present study characterizes changes in the electronic structure of reactants during chemical reactions based on the combined charge and energy decomposition scheme, ETS-NOCV (extended transition state–natural orbitals for chemical valence). Decomposition of the activation barrier, ΔE ^#, into stabilizing (orbital interaction, ΔE _orb, and electrostatic, ΔE _elstat) and destabilizing (Pauli repulsion, ΔE _Pauli, and geometry distortion energy, ΔE _dist) factors is discussed in detail for the following reactions: (I) hydrogen cyanide to hydrogen isocyanide, HCN → CNH isomerization; (II) Diels-Alder cycloaddition of ethene to 1,3-butadiene; and two catalytic processes, i.e., (III) insertion of ethylene into the metal-alkyl bond using half-titanocene with phenyl-phenoxy ligand catalyst; and (IV) B–H bond activation catalyzed by an Ir-containing catalyst. Various reference states for fragments were applied in ETS-NOCV analysis. We found that NOCV-based deformation densities (Δρ _i) and the corresponding energies ΔE _orb(i) obtained from the ETS-NOCV scheme provide a very useful picture, both qualitatively and quantitatively, of electronic density reorganization along the considered reaction pathways. Decomposition of the barrier ΔE^# into stabilizing and destabilizing contributions allowed us to conclude that the main factor responsible for the existence of positive values of ΔE ^# for all processes (I, II, III and IV) is Pauli interaction, which is the origin of steric repulsion. In addition, in the case of reactions II, III and IV, a significant degree of structural deformation of the reactants, as measured by the geometry distortion energy, plays an important role. Depending on the reaction type, stabilization of the transition state (relatively to the reactants) originating either from the orbital interaction term or from electrostatic attraction can be of vital importance. Finally, use of the ETS-NOCV method to describe catalytic reactions allows extraction of information on the role of catalysts in determination of ΔE ^#.     

Interaction between cadmium, zinc and silver-substituted plastocyanin and cytochrome b 6 f complex — heavy metals toxicity towards photosynthetic apparatus

This paper reports the results of research on the interaction between the cytochrome f of the active cytochrome b ₆ f complex (incubated with Cd-, Zn-, and Ag-substituted plastocyanins) and Cu-plastocyanin. The presented studies show, that the metal derivatives of plastocyanin can have an influence on the photosynthetic electron transfer path: cytochrome b ₆ f complex — photosystem I. The metal-substituted plastocyanins occupy the plastocyanin electron transfer site of the cytochrome f. The stopped-flow measurements show, that although the metal derivatives of plastocyanin do not influence the rate of cyt f- Pc electron transfer, creation of the non-electron-transfer complexes characterised by a strong binding between the cyt f and substituted plastocyanins and their slow release, dependent on the redox state of the substituted metal, results in the decrease of a turnover of the cytochrome complex. The research was done in the Department of Plant Biochemistry, Freiburg University, Sch?nzlestrasse 1, 79 104 Freiburg, Germany     

Predictive Factors of Late Venous Aortocoronary Graft Failure: Ultrastructural Studies

Background

Venous aortocoronary graft arterialization may precede a preterm occlusion in some coronary artery bypass grafting (CABG) patients. The aim of the present study was to identify ultrastructural variations in the saphenous vein wall that may have an impact on the development of venous graft disease in CABG patients.

Methods

The study involved 365 consecutive patients with a mean age of 62.9±9.4 years who underwent isolated CABG. The thickness and area of the whole venous wall, the tunica intima, the tunica media and the adventitia and the number and shape (length, thickness and length/thickness ratio) of the nuclei in the medial smooth muscle cells nuclei in the distal saphenous vein segments were evaluated by ultrastructural studies. Patients were followed up for 41 to 50 months (mean 45.1±5.1). Saphenous vein graft patency was assessed by follow-up coronary angiography. Logistic regression models were used to identify independent risk factors for late graft failure.

Results

In 71 patients significant lesions in the saphenous vein grafts were observed. The whole venous wall thickness (437.5 µm vs. 405.5 µm), tunica media thickness (257.2 µm vs. 211.5 µm), whole venous wall area (2.23 mm² vs. 2.02 mm²) and tunica media area (1.09 mm² vs. 0.93 mm²) were significantly larger for this group of patients than for those without graft disease. In the latter group more elongated smooth muscle cell nuclei (higher length/thickness ratio) were found in the tunica media of the saphenous vein segments. Thickening of the saphenous vein tunica media and chunky smooth muscle cell nuclei were identified as independent risk factors for graft disease development.

Conclusions

Saphenous vein tunica media hypertrophy (resulting in wall thickening) and chunky smooth muscle cell nuclei might predict the development of venous graft disease.     

An alginate-layer technique for culture of Brassica oleracea L. protoplasts

Ten accessions belonging to the Brassica oleracea subspecies alba and rubra, and to B. oleracea var. sabauda were used in this study. Protoplasts were isolated from leaves and hypocotyls of in vitro grown plants. The influence of selected factors on the yield, viability, and mitotic activity of protoplasts immobilized in calcium alginate layers was investigated. The efficiency of protoplast isolation from hypocotyls was lower (0.7 ± 0.1 × 10⁶ ml⁻¹) than for protoplasts isolated from leaf mesophyll tissue (2 ± 0.1 × 10⁶ ml⁻¹). High (70–90%) viabilities of immobilized protoplasts were recorded, independent of the explant sources. The highest proportion of protoplasts undergoing divisions was noted for cv. Reball F1, both from mesophyll (29.8 ± 2.2%) and hypocotyl (17.5 ± 0.3%) tissues. Developed colonies of callus tissue were subjected to regeneration and as a result plants from six accessions were obtained.     

Pooled sample-based GWAS: a cost-effective alternative for identifying colorectal and prostate cancer risk variants in the Polish population

Background

Prostate cancer (PCa) and colorectal cancer (CRC) are the most commonly diagnosed cancers and cancer-related causes of death in Poland. To date, numerous single nucleotide polymorphisms (SNPs) associated with susceptibility to both cancer types have been identified, but their effect on disease risk may differ among populations.

Methods

To identify new SNPs associated with PCa and CRC in the Polish population, a genome-wide association study (GWAS) was performed using DNA sample pools on Affymetrix Genome-Wide Human SNP 6.0 arrays. A total of 135 PCa patients and 270 healthy men (PCa sub-study) and 525 patients with adenoma (AD), 630 patients with CRC and 690 controls (AD/CRC sub-study) were included in the analysis. Allele frequency distributions were compared with t-tests and χ²-tests. Only those significantly associated SNPs with a proxy SNP (p<0.001; distance of 100 kb; r²>0.7) were selected. GWAS marker selection was conducted using PLINK. The study was replicated using extended cohorts of patients and controls. The association with previously reported PCa and CRC susceptibility variants was also examined. Individual patients were genotyped using TaqMan SNP Genotyping Assays.

Results

The GWAS selected six and 24 new candidate SNPs associated with PCa and CRC susceptibility, respectively. In the replication study, 17 of these associations were confirmed as significant in additive model of inheritance. Seven of them remained significant after correction for multiple hypothesis testing. Additionally, 17 previously reported risk variants have been identified, five of which remained significant after correction.

Conclusion

Pooled-DNA GWAS enabled the identification of new susceptibility loci for CRC in the Polish population. Previously reported CRC and PCa predisposition variants were also identified, validating the global nature of their associations. Further independent replication studies are required to confirm significance of the newly uncovered candidate susceptibility loci.     

Selected airborne allergenic fungal spores and meteorological factors in Szczecin,Poland, 2004–2006

Airborne fungal spore concentrations in Szczecin, Poland, were studied between 2004 and 2006 with the objective of determining a seasonal variation in the concentrations of selected fungal spore types in relation to meteorological parameters. The presence of spores of five taxa, namely, Cladosporium, Ganoderma, Alternaria, Leptosphaeria and Didymella, was recorded using a volumetric method (Hirst type). Fungal spores were present in the air in large numbers during the summer, with the highest concentrations recorded mainly in June, July and August. The peak concentrations of two of the studied spore types, Ganoderma and Alternaria, occurred in August, while the concentrations of Cladosporium, Leptosphaeria and Didymella spores were the highest in July. Multiple regression analysis was performed for three fungal seasons—2004, 2005 and 2006. Spore concentration was found to be positively correlated with the minimum temperature. For some spore types, there was also a significant correlation between concentrations, relative humidity and rain.     

SUMOylation regulates nuclear localization of Krüppel-like factor 5

SUMOylation is a form of post-translational modification shown to control nuclear transport. Krüppel-like factor 5 (KLF5) is an important mediator of cell proliferation and is primarily localized to the nucleus. Here we show that mouse KLF5 is SUMOylated at lysine residues 151 and 202. Mutation of these two lysines or two conserved nearby glutamates results in the loss of SUMOylation and increased cytoplasmic distribution of KLF5, suggesting that SUMOylation enhances nuclear localization of KLF5. Lysine 151 is adjacent to a nuclear export signal (NES) that resembles a consensus NES. The NES in KLF5 directs a fused green fluorescence protein to the cytoplasm, binds the nuclear export receptor CRM1, and is inhibited by leptomycin and site-directed mutagenesis. SUMOylation facilitates nuclear localization of KLF5 by inhibiting this NES activity, and enhances the ability of KLF5 to stimulate anchorage-independent growth of HCT116 colon cancer cells. A survey of proteins whose nuclear localization is regulated by SUMOylation reveals that SUMOylation sites are frequently located in close proximity to NESs. A relatively common mechanism for SUMOylation to regulate nucleocytoplasmic transport may lie in the interplay between neighboring NES and SUMOylation motifs.     

Discovery of thieno[2,3-c]pyridines as potent COT inhibitors

Evaluation of hit chemotypes from high throughput screening identified a novel series of 2,4-disubstituted thieno[2,3-c]pyridines as COT kinase inhibitors. Structural modifications exploring SAR at the 2- and 4-positions resulting in inhibitors with improved enzyme potency and cellular activity are disclosed.     

Chemically-induced affinity star restriction specificity: a novel TspGWI/sinefungin endonuclease with theoretical 3-bp cleavage frequency

The type IIS/IIC restriction endonuclease TspGWI recognizes the sequence 5'-ACGGA-3', cleaving DNA 11/9 nucleotides downstream. Here we show that sinefungin, a cofactor analog of S-adenosyl methionine, induces a unique type of relaxation in DNA recognition specificity. In the presence of sinefungin, TspGWI recognizes and cleaves at least 12 degenerate variants of the original recognition sequence that vary by single base pair changes from the original 5-bp restriction site with only a single degeneracy per variant appearing to be allowed. In addition, sinefungin was found to have a stimulatory effect on cleavage at these nondegenerate TspGWI recognition sites, irrespective of their number or the DNA topology. Interestingly, no fixed "core" could be identified among the new recognition sequences. Theoretically, TspGWI cleaves DNA every 1024 bp, while sinefungin-induced activity cleaves every 78.8 bp, corresponding to a putative 3-bp long recognition site. Thus, the combination of sinefungin and TspGWI represents a novel frequent cutter, next only to CviJI/CviJI*, that should prove useful in DNA cloning methodologies.     

Bio-oxidation of tripropylene glycol under aerobic conditions

Aerobic biodegradation of tripropylene glycol (PG3) was investigated under the conditions of the OECD screening test 301E and the Continuous Flow Activated Sludge Simulation test (CFAS). A modified two-chamber facility with a denitrification stage was used for the CFAS test. Primary PG3 biodegradation was measured by the HPLC with fluorimetric detection and analyte derivatisation. Metabolites were identified by LC-MS with electrospray ionisation and GC-MS with electron impact ionisation, as well as semiquantitatively determined by the LC-MS technique. PG3 was found to be inherently biodegradable and it exhibits a strong poisonous effect on activated sludge after exceeding the threshold concentration (10 mg l⁻¹). Metabolite accumulation onto the activated sludge is probably responsible for this poisonous effect. Probable biotransformation products of tripropylene glycol under the aerobic conditions include metabolites with a single terminal aldehyde or a ketone group and metabolites with two terminal aldehyde or ketone groups. Their concentration rises at the end of the OECD screening test.