Alkylation resistance of E. coli cells expressing different isoforms of human alkyladenine DNA glycosylase (hAAG)

The alkyladenine DNA glycosylase (AAG) has been cloned from mouse and humans. AAG knock out mouse cells are sensitized to a variety of alkylating and cross-linking agents suggesting AAG is active on a variety of substrates. In humans, two isoforms have been characterized that are generated by alternative splicing and contain either exon 1a or 1b (hAAG1 or hAAG2). In this study, we examine the ability of the both known isoforms of human AAG (hAAG) to contribute to survival of Escherichia coli from treatments with simple alkylating agents and cross-linking alkylating agents. Our results show that hAAG is effective at repairing methyl lesions when expressed in E. coli, but is unable to afford increased resistance to alkylating agents producing larger alkyl lesions such as ethyl lesions or lesions produced by the cross-linking alkylating agents N,N'-bis-chloroethyl-N-nitrosourea (BCNU), N-(2-chloroethyl)-N-nitrosourea (CNU) or mitomycin C. In the case of CNU, expression of hAAG causes increased sensitivity rather than resistance, suggesting deleterious effects of hAAG activity. We also demonstrate that there are no apparent differences between the two isoforms of hAAG when recovery from damage produced by all alkylating agents is tested.     

Effect of surfactant concentration, compression ratio and compression rate on the surface activity and dynamic properties of a lung surfactant

This paper reports dynamic surface tension experiments of a lung surfactant preparation, BLES, for a wide range of concentrations, compression ratios and compression rates. These experiments were performed using Axisymmetric Drop Shape Analysis-Constrained Sessile Drop (ADSA-CSD). The main purpose of the paper is to interpret the results in terms of physical parameters using the recently developed Compression-Relaxation Model (CRM). In the past, only the minimum surface tension was used generally for the characterization of lung surfactant films; however, this minimum value is not a physical parameter and depends on the compression protocol. CRM is based on the assumption that the dynamic surface tension response is governed by surface elasticities, adsorption and desorption of components of the lung surfactant. The ability of CRM to fit the surface tension response closely for a wide variety of parameters (compression ratio, compression rate and surfactant concentration) and produce sensible values for the elastic and kinetic parameters supports the validity of CRM.     

ERK1/2 map kinase metabolic pathway is responsible for phosphorylation of translation initiation factor eIF4E during in vitro maturation of pig oocytes

Eukaryotic initiation factor 4E (eIF4E) plays an important role in mRNA translation by binding the 5'-cap structure of the mRNA and facilitating the recruitment to the mRNA of other translation factors and the 40S ribosomal subunit. eIF4E undergoes regulated phosphorylation on Ser-209 and this phosphorylation is believed to be important for its binding to mRNA and to other initiation factors. The findings showing that the translation initiation factor eIF4E becomes gradually phosphorylated during in vitro maturation (IVM) of pig oocytes with a maximum in metaphase II (M II) stage oocytes have been documented by us recently (Ellederova et al., 2006). The aim of this work was to study in details the metabolic pathways involved in this process. Using inhibitors of cyclin-dependent kinases, Butyrolactone I (BL I) and protein phosphatases, okadaic acid (OA) we show that ERK1/2 MAP kinase pathway is involved in this phosphorylation. We also demonstrate that activation and phosphorylation of ERK1/2 MAP kinase and eIF4E is associated with the activating phosphorylation of Mnk1 kinase, one of the two main kinases phosphorylating eIF4E in somatic cells.     

Enzymatic synthesis of three pNP-α-galactobiopyranosides: application of the library of fungal α-galactosidases

The regioselectivity of the transglycosylation reaction catalyzed by extracellular α-galactosidases from filamentous fungi was studied using p-nitrophenyl α- -galactopyranoside. Regioisomers of p-nitrophenyl α- -galactobiopyranoside α(1→2), α(1→3) and α(1→6) were isolated and characterized. α-Galactosidases with pronounced regioselectivity towards α-Gal-O-R acceptor were identified.     

Stress-induced expression of cucumber chitinase and <Emphasis Type="Italic">Nicotiana plumbaginifolia</Emphasis><Emphasis Type="Italic">β</Emphasis>-1,3-glucanase genes in transgenic potato plants

The genes encoding for a cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase were co-introduced into Slovak potato (Solanum tuberosum L.) cultivar ETA using Agrobacterium tumefaciens. Expression of both genes was driven by wound-inducible polyubiquitin promoter isolated from Slovak potato breeding line 116/86. Analyses showed inducible, peel-specific expression of both transgenes under stress conditions. The effect of transgene expression on fungal susceptibility of transformants was evaluated in vitro and in vivo. Experiments with crude protein extracts isolated from transgenic microtubers showed growth inhibition of Rhizoctonia solani hyphae in the range from 7.3 to 14.2%. In contrast, experiments performed in growth chamber conditions revealed that the polyubiquitin promoter driven transgene expression did not ensure any obvious increase of transgenic potato resistance against Rhizoctonia solani.     

Geographic variation in seed dormancy among populations of Echinochloa crus-galli

In 1991–1993, we investigated the incidence of seed dormancy in 25 local populations of barnyard grass, Echinochloa crus-galli (L.) P.Beauv., in the western Czech Republic. The percentage of germination after 4 months afterripening of dry seeds at 25°C varied between 0.0 and 83.6%. Although there were significant annual differences in the percentage of germination at some localities, typical proportions of dormant seeds persisted over 3 years at field sites where the seed bank was not disturbed. One-way ANOVA (using data from 14 cultivated or abandoned fields) revealed that 73.0% of variance in seed dormancy incidence could be attributed to the effect of locality (P<0.001). Incidence of dormancy was not correlated with mother plant stature (dry above-ground biomass, number of tillers, maximal stem height) nor seed mass. There was a significant correlation (r ²=0.403, P<0.005) between dormancy incidence at natural localities in 1991 and in F₁ offspring sown at experimental grounds at Praha-Ruzyn in 1992. The results indicate that heredity is important in maintaining local variation in seed dormancy, probably favoured by the self-pollinating reproduction of barnyard grass.     

Ochratoxinogenicity of Aspergillus ochraceus strains from nephropathic and non-nephropathic areas in Yugoslavia

Mycological analyses of 855 samples of stored grains and dried meat collected in period 1980–1987 from individual households in the nephropathic and wider non-nephropathic area in SR Croatia in Yugoslavia showed 10% of samples to be contaminated with Aspergillus ochraceus. Ability to produce ochratoxin A (OA) was tested in 70 samples (27 from nephropathic areas and 43 from non-nephropathic areas). The detection was carried out under UV-light (365 nm) (light blue fluorescence) and 6 OA-producers were found.A biosynthetic procedure on liquid nutritional substrate with sacharose and yeast extract as well as a method using wet crushed wheat revealed that 37% of the samples from a nephropathic area, and 35% of the samples from a non-nephropathic area produce OA.In the nephropathic area 1/10 strains was a strong producer of OA (concentration crushed wheat 135 mg/kg, and 240 mg/l on YES liquid substrate), 1/10 strains was a moderate producer (concentration 16.6 mg/l and 0.07–7.0 mg/l and 0.1–10.4 mg/kg).Among the strains isolated from a wider non-nephropathic area no strong producers of OA were found, but 2/15 strains were moderate producers of OA (concentration of OA 20.4–27.0 mg/l and 15.0–33.7 mg/kg). The other strains, 8/10 on the crushed wheat and 13/15 on the liquid substrate, were weak producers of OA with concentrations of OA between 0.2–9.0 mg/l and 0.2–10.0 mg/kg with the two methods respectively.