New activities of a catalytic antibody with a peroxidase activity: formation of Fe(II)-RNO complexes and stereoselective oxidation of sulfides.

In order to estimate the size of the cavity remaining around the heme of the 3A3-microperoxidase 8 (MP8) hemoabzyme, the formation of 3A3-MP8-Fe(II)-nitrosoalkane complexes upon oxidation of N-monosubstituted hydroxylamines was examined. This constituted a new reaction for hemoabzymes and is the first example of fully characterized Fe(II)-metabolite complexes of antibody-porphyrin. Also, via a comparison of the reactions with N-substituted hydroxylamines of various size and hydrophobicity, antibody 3A3 was confirmed to bring about a partial steric hindrance on the distal face of MP8. Subsequently, the influence of the antibody on the stereoselectivity of the S-oxidation of sulfides was examined. Our results showed that MP8 alone and the antibody-MP8 complex catalyze the oxidation of thioanisole by H(2)O(2) and tert-butyl hydroperoxide, following a peroxidase-like two-step oxygen-transfer mechanism involving a radical-cation intermediate. The best system, associating H(2)O(2) as oxidant and 3A3-MP8 as a catalyst, in the presence of 5% tert-butyl alcohol, led to the stereoselective S-oxidation of thioanisole with a 45% enantiomeric excess in favour of the R isomer. This constitutes the highest enantiomeric excess reported to date for the oxidation of sulfides catalyzed by hemoabzymes.     

Comparative effect of different dietary inulin sources and probiotics on growth performance and blood characteristics in growing–finishing pigs

The study was focused on assessment of the effect of an extract of long-chain inulin (LCI) and dried tubers of Jerusalem artichoke (JA) and a multispecies probiotic preparation as well as a combination thereof on growth performance and blood parameters of fattening pigs. In total, 144 pigs (initial body weight 30.0 ± 0.5 kg) were used in a 98-d experiment. The six dietary treatments consisted of the control diet (Con), diet Con supplemented with probiotics (ConP) and four diets supplemented with LCI or JA alone or with probiotics (diets LCIP and JAP). Throughout the fattening period, there was a beneficial effect of the probiotic supplementation to the inulin-containing diets and the average daily gain (ADG) was increased by supplementation of probiotics in combination with inulin sources (p < 0.05). At the end of the fattening period, ADG and feed conversion ratio (FCR) were higher after supplementation of LCI only (p < 0.05). Compared with group ConP, in groups LCI and JA, the ADG and FCR were improved (p < 0.05). Only in the first fattening stage, the addition of the prebiotics and/or probiotics had an impact on the level of white blood cells and some biochemical indices in pigs. In younger animals, probiotic or LCI supplementation increased the IgG level (p < 0.05). There was also an interaction between the probiotics and JA resulting in increased IgG and IgA concentrations (p < 0.05). In the finishing period, LCI addition increased the IgM level (p < 0.05), whereas JA addition increased IgG and IgM levels as well (p < 0.05). In conclusion, both dietary sources of inulin and probiotic supplementation can improve the fattening performance and health status of growing pigs.     

Microbial transformations of flavanone and 6-hydroxyflavanone by Aspergillus niger strains

Flavanone (1) and 6-hydroxyflavanone (2) were subjected to transformation by means of Aspergillus niger strains (one wild and three UV mutants). For both substrates the biotransformation resulted in reduction of the carbonyl group (products 5 and 7) and dehydrogenation at C-2 and C-3 (3 and 8). Additionally, for flavanone (1) reduction of C-4 together with hydroxylation at C-7 (6) and dehydrogenation at C-2, C-3 along with hydroxylation at C-3 (4) were observed.     

A different methylation profile of circadian genes promoter in breast cancer patients according to clinicopathological features

ABSTRACT

One of the supposed mechanisms that may lead to breast cancer (BC) is an alteration of circadian gene expression and DNA methylation. We undertook an integrated approach to identify methylation pattern of core circadian promoter regions in BC patients with regard to clinical features. We performed a quantitative methylation-specific real-time PCR analysis of a promoter methylation profile in 107 breast tumor and matched non-tumor tissues. A panel of circadian genes CLOCK, BMAL1, PERIOD (PER1, 2, 3), CRYPTOCHROME (CRY1, 2) and TIMELESS as well as their association with clinicopathological characteristics were included in the analysis. Three out of the eight analyzed genes exhibited marked hypermethylation (PER1, 2, 3), whereas CLOCK, BMAL1, CRY2 showed significantly lower promoter CpG methylation in the BC tissues when compared to the non-tumor tissues. Among variously methylated genes we found an association between the elevated methylation level of PERs promoter region and molecular subtypes, histological subtypes and tumor grading of BC. Methylation status may be associated with a gene expression level of circadian genes in BC patients. An aberrant methylation pattern in circadian genes in BC may provide information that could be used as novel biomarkers in clinics and molecular epidemiology as well as play an important role in BC etiology.     

Impact of osmotic stress on physiological and biochemical characteristics in drought-susceptible and drought-resistant wheat genotypes

In this study, the seedlings of two wheat cultivars were used: drought-resistant Chinese Spring (CS) and drought-susceptible (SQ1). Seedlings were subjected to osmotic stress in order to assess the differences in response to drought stress between resistant and susceptible genotype. The aim of the experiment was to evaluate the changes in physiological and biochemical characteristics and to establish the optimum osmotic stress level in which differences in drought resistance between the genotypes could be revealed. Plants were subjected to osmotic stress by supplementing the root medium with three concentrations of PEG 6000. Seedlings were grown for 21 days in control conditions and then the plants were subjected to osmotic stress for 7 days by supplementing the root medium with three concentrations of PEG 6000 (D1, D2, D3) applied in two steps: during the first 3 days of treatment ?0.50, ?0.75 and ?1.00 and next ?0.75, ?1.25 and ?1.5 MPa, respectively. Measurements of gas exchange parameters, chlorophyll content, height of seedlings, length of root, leaf and root water content, leaf osmotic potential, lipid peroxidation, and contents of soluble carbohydrates and proline were taken. The results highlighted statistically significant differences in most traits for treatment D2 and emphasized that these conditions were optimum for expressing differences in the responses to osmotic stress between SQ1 and CS wheat genotypes. The level of osmotic stress defined in this study as most suitable for differentiating drought resistance of wheat genotypes will be used in further research for genetic characterization of this trait in wheat through QTL analysis of mapping population of doubled haploid lines derived from CS and SQ1.     

The chemical synthesis and cytotoxicity of new sulfur analogues of 2-methoxy-lysophosphatidylcholine

The chemical synthesis of phosphorothioate/phosphorodithioate analogues of 2-methoxy-lysophosphatidylcholine has been described. For the preparation of new sulfur derivatives of lysophosphatidylcholine both oxathiaphospholane and dithiaphospholane approaches have been employed. Each lysophospholipid analogue was synthesized as a series of five compounds, bearing different fatty acid residues both saturated (12:0, 14:0, 16:0, 18:0) and unsaturated (18:1). The methylation of glycerol 2-hydroxyl function was applied in order to increase the stability of prepared analogues by preventing 1→2 acyl migration. The cellular toxicity of newly synthesized 2-methoxy-lysophosphatidylcholine derivatives was measured using MTT viability assay and lactate dehydrogenase release method.     

High Content Screening as High Quality Assay for Biological Evaluation of Photosensitizers In Vitro

A novel single step assay approach to screen a library of photdynamic therapy (PDT) compounds was developed. Utilizing high content analysis (HCA) technologies several robust cellular parameters were identified, which can be used to determine the phototoxic effects of porphyrin compounds which have been developed as potential anticancer agents directed against esophageal carcinoma. To demonstrate the proof of principle of this approach a small detailed study on five porphyrin based compounds was performed utilizing two relevant esophageal cancer cell lines (OE21 and SKGT-4). The measurable outputs from these early studies were then evaluated by performing a pilot screen using a set of 22 compounds. These data were evaluated and validated by performing comparative studies using a traditional colorimetric assay (MTT). The studies demonstrated that the HCS assay offers significant advantages over and above the currently used methods (directly related to the intracellular presence of the compounds by analysis of their integrated intensity and area within the cells). A high correlation was found between the high content screening (HCS) and MTT data. However, the HCS approach provides additional information that allows a better understanding of the behavior of these compounds when interacting at the cellular level. This is the first step towards an automated high-throughput screening of photosensitizer drug candidates and the beginnings of an integrated and comprehensive quantitative structure action relationship (QSAR) study for photosensitizer libraries.     

Synthesis and in vitro evaluation of antiviral and cytostatic properties of novel 8-triazolyl acyclovir derivatives

Abstract

As a part of the research aimed on identification of new nucleobase derivatives with improved biological properties, a series of novel 8-substituted acyclovir derivatives were synthesized. The 8-azidoguanosine 4 and novel 8-azidoacyclovir 9 were synthesized from commercially available guanosine 1 and acyclovir 6 which were transformed into 8-bromopurine derivatives 2 and 7 and hydrazine derivatives 3 and 8, respectively. 8-Triazolylguanosine 5 and 8-triazolylacyclovir analogs 10–12 were successfully synthesized via the Cu(I) catalyzed 1,3-dipolar cycloaddition reaction of azides 4 and 9 with propargyl alcohol, 4-pentyn-1-ol and 5-hexyn-1-ol. The novel 1,4-disubstituted 1,2,3-triazolyl compounds 5, 10–12 were evaluated for antiviral activity against selected DNA and RNA viruses and cytostatic activity against normal Madine Darby canine kidney (MDCK I) cells, and seven tumor cell lines (HeLa, CaCo-2, NCI-H358, Jurkat, K562, Raji and HuT78). While tested compounds exerted no antiviral activity at nontoxic concentrations, the 8-triazolyl acyclovir derivative 10, with the shortest alkyl substituent at the C-4 of triazole ring, was found to be the most active against the CaCo-2 cell line.     

TLR9 2848 GA Heterozygotic Status Possibly Predisposes Fetuses and Newborns to Congenital Infection with Human Cytomegalovirus

Background

Some single nucleotide polymorphisms (SNP), located in Toll-like receptor (TLR) genes, were reported to be associated with human cytomegalovirus (HCMV) infections. The study was aimed to assess the correlation of SNPs at TLR4 and TLR9 genes with the occurrence of congenital cytomegaly, based on available samples.

Methods

Reported case-control study included both HCMV infected and non-infected fetuses and newborns. The specimens were classified to the molecular analyses, based on serological features of the recent infection and HCMV DNAemia in body fluids. TLR SNPs were studied, using multiplex nested PCR-RFLP assay, and determined genotypes were confirmed by sequencing. Hardy-Weinberg equilibrium was assessed for the identified genotypes. The linkage disequilibrium was also estimated for TLR4 SNPs. A relationship between the status of TLR genotypes and congenital cytomegaly development was estimated, using a logistic regression model.

Results

Hardy Weinberg equilibrium was observed for almost all SNPs, both infected and non-infected patients, with exception of TLR4 896 A>G polymorphism in the control group (P≤0.050). TLR4 896 A>G and 1196 C>T SNPs were found in linkage disequilibrium in both study groups (P≤0.050). The CC genotype at TLR4 1196 SNP and the GA variant at TLR9 2848 G>A SNP were significantly associated with HCMV infection (P≤0.050). The risk of congenital cytomegaly was higher in heterozygotes at TLR9 SNP than in the carriers of other genotypic variants at the reported locus (OR 4.81; P≤0.050). The GC haplotype at TLR4 SNPs and GCA variants at TLR4 and TLR9 SNPs were significantly associated with HCMV infection (P≤0.0001). The ACA variants were more frequent among fetuses and neonates with symptomatic, rather than asymptomatic cytomegaly (P≤0.0001).

Conclusions

TLR4 and TLR9 polymorphisms may contribute to the development of congenital infection with HCMV in fetuses and neonates. The TLR9 2848 GA heterozygotic status possibly predisposes to HCMV infection, increasing the risk of congenital cytomegaly development.     

Refugial debate: on small sites according to their function and capacity

The occurrence and location of long-term refugia determine the current patterns of biodiversity on Earth. The importance of the refugial debate is certain to increase in response to observed and expected species extinctions caused by climate change. Small areas where species survive outside their core range are important, as unique natural phenomena and model systems for observing the response of species to climate change. They can play a crucial role as potential sources for species recovery in the future or can act as progenitors of a new species. While most authors believe that sites connected with only long-term isolation should be included into the refugium concept, this approach can result in the loss of linkage between the ecological and evolutionary processes taking place during different phases of the species range dynamics. Moreover, the papers often interpret the nature of described phenomena in different ways. In response, the conceptual scheme given in our letter summarises the patterns which occur during species range shift. It proposes an equivalent scheme for small refugial sites according to their function and capacity, based on the relict species concept. This approach and proposed terminology is tested on the example of two plant species with different pattern of the long term range dynamics. Our paper highlights the importance of sites harboring ‘trailing-edge’ young relicts for the future long-term persistence of the species (as old relicts) under unfavorable regional conditions. By considering the age gradients of small refugial sites it is possible to reveal community interactions, species traits or genes that drive the responses of biota to climate changes.