Unusual evolutionary conservation of 5S rRNA pseudogenes inAspergillus nidulans: Similarity of the DNA sequence associated with the pseudogenes with the mouse immunoglobulin switch region

Summary AllAspergillus nidulans 5S rRNA pseudogenes known so far are the result of integration of an approx. 0.2-kbp-long DNA sequence into the 5S rRNA genes. This sequence, called block C, is present in at least five copies in theA. nidulans genome and seems to be associated either with 5S rRNA genes or pseudogenes. In contrast to the 78% sequence conservation of the C-block in pseudogenes, the truncated 5 halves of the pseudogenes are very highly conserved (96.9–100%). We postulate that the 5S rRNA pseudogenes are still a subject of concerted evolution. The C-block sequence shows similarity to the switch region of the mouse heavy chain immunoglobulin gene. A characteristic motif GGGTGAG is repeated several times in both sequences; the sequence conservation is 63%.     

Enhanced production of the pharmaceutically important polyphenolic compounds in Vitex agnus castus L. shoot cultures by precursor feeding strategy

Agitated Vitex agnus castus L. shoot cultures were established to analyse the content of selected pharmaceutically important flavonoids and phenolic acids. Two variants (selected from nine ones) of MS medium were prepared: A (BAP 1 mg/L; NAA 0.5 mg/L; GA₃ 0.25 mg/L) and B (BAP 2 mg/L; NAA 0.5 mg/L). The biomass was harvested after 1, 2, 3,4, 5 and 6 weeks. Four‐week cultures (variant A) were selected to perform the precursor feeding experiment. The L‐phenylalanine dose of 1.6 g/L appears to be the most advantageous. Compared to the control cultures, the content of the individual compounds increased in a range from 1.4 to 17.3‐fold (e.g. p‐coumaric acid – 17.3 fold; casticin – 4.8‐fold). The biomass from in vitro cultures is richer in neochlorogenic acid (16‐fold), p‐coumaric acid (5.3‐fold), rutin (2.8‐fold), caffeic acid (1.5‐fold) and cinaroside (1.5‐fold) than the leaves of its parent greenhouse‐cultivated plants. Extracts contained 30 mg/100 g DW of casticin, but after the hydrolysis its amount increased up to 200 mg/100 g DW and twice exceeded the content in the greenhouse leaves. The results indicate that V. agnus castus agitated shoot cultures might be considered as a potential biotechnological source of some pharmaceutically important compounds, especially casticin, rutin, neochlorogenic and p‐coumaric acids.     

Induction of apoptosis in human glioma cell lines of various grades through the ROS-mediated mitochondrial pathway and caspase activation by <Emphasis Type="Italic">Rhaponticum carthamoides</Emphasis> transformed root extract

The placenta plays a major role in embryo-fetal defects and intrauterine growth retardation after maternal alcohol consumption. Our aims were to determine the oxidative status and cellular and molecular oxidative stress effects on uterine myometrium and trophoblast-decidual tissue following perigestational alcohol intake at early organogenesis. CF-1 female mice were administered with 10% alcohol in drinking water for 17 days prior to and up to day 10 of gestation. Control females received ethanol-free water. Treated mice had smaller implantation sites compared to controls (p < 0.05), diminished maternal vascular lumen, and irregular/discontinuous endothelium of decidual vessels. The trophoblast giant cell layer was disorganized and presented increased abnormal nuclear frequency. The myometrium of treated females had reduced nitrite content, increased superoxide dismutase activity, and reduced glutathione (GSH) content (p < 0.05). However, the trophoblast-decidual tissue of treated females had increased nitrite content (p < 0.05), increased GSH level (p < 0.001), increased thiobarbituric acid-reactive substance concentration (p < 0.001), higher 3-nitrotyrosine immunoreaction, and increased apoptotic index (p < 0.05) compared to controls. In summary, perigestational alcohol ingestion at organogenesis induced oxidative stress in the myometrium and trophoblast-decidual tissue, mainly affecting cells and macromolecules of trophoblast and decidual tissues around early organogenesis, in CF-1 mouse, and suggests that oxidative-induced abnormal early placental formation probably leads to risk of prematurity and fetal growth impairment at term.     

Behavior of sperm nuclei in intact and bisected metaphase II mouse oocytes fertilized in the presence of colcemid

Our objective was to examine the developmental fate of sperm nuclei in oocytes fertilized under conditions of meiotic arrest. Therefore zona-free metaphase II oocytes and oocyte fragments (nucleate and anucleate) were fertilized in the presence of colcemid. In anucleate oocyte fragments, normal male pronuclei develop. In contrast, in intact oocytes and nucleate fragments sperm nuclei after initial decondensation undergo secondary condensation. This state is maintained as long as the oocytes are treated with colcemid. When the drug is removed 3 h after insemination, the meiotic spindle(s) is reconstructed, the second polar body(ies) is extruded, and a female pronucleus (or micronuclei) forms. At the same time the sperm nucleus decondenses again and transforms into a male pronucleus. In addition oocytes fertilized in the presence of colcemid could not be refertilized. These observations suggest that oocytes and oocyte fragments fertilized in the presence of colcemid undergo activation despite the failure of pronucleus formation. The inhibitory effect of colcemid on the formation of pronuclei is expressed only in the presence of oocyte chromosomes. We suggest that colcemid stabilizes factors responsible for chromosome condensation that are associated with oocyte chromosomes but not factors (whether the same or different) present in the cytoplasm.     

Comparison of glyoxalase I purified from yeast (Saccharomyces cerevisiae) with the enzyme from mammalian sources

Glyoxalase I from yeast (Saccharomyces cerevisiae) purified by affinity chromatography on S-hexylglutathione-Sepharose 6B was characterized and compared with the enzyme from rat liver, pig erythrocytes and human erythrocytes. The molecular weight of glyoxalase I from yeast was, like the enzyme from Rhodospirillum rubrum and Escherichia coli, significantly less (approx. 32000) than that of the enzyme from mammals (approx. 46000). The yeast enzyme is a monomer, whereas the mammalian enzymes are composed of two very similar or identical subunits. The enzymes contain 1Zn atom per subunit. The isoelectric points (at 4 degrees C) for the yeast and mammalian enzymes are at pH7.0 and 4.8 respectively; tryptic-peptide ;maps' display corresponding dissimilarities in structure. These and some additional data indicate that the microbial and the mammalian enzymes may have separate evolutionary origins. The similarities demonstrated in mechanistic and kinetic properties, on the other hand, indicate convergent evolution. The k(cat.) and K(m) values for the yeast enzyme were both higher than those for the enzyme from the mammalian sources with the hemimercaptal adduct of methylglyoxal or phenylglyoxal as the varied substrate and free glutathione at a constant and physiological concentration (2mm). Glyoxalase I from all sources investigated had a k(cat.)/K(m) value near 10(7)s(-1).m(-1), which is close to the theoretical diffusion-controlled rate of enzyme-substrate association. The initial-velocity data show non-Michaelian rate saturation and apparent non-linear inhibition by free glutathione for both yeast and mammalian enzyme. This rate behaviour may have physiological importance, since it counteracts the effects of fluctuations in total glutathione concentrations on the glyoxalase I-dependent metabolism of 2-oxoaldehydes.     

The effect of experimental exposure to low doses of zearalenone on uterine histology and morphometry in prepubertal bitches

The experiment involved 30 clinically healthy prepubertal bitches aged approximately 70 days with an estimated initial body weight (BW) of 8 kg. The animals were randomly divided into two experimental groups (EI and EII) and a control group of 10 animals each. Group EI was administered 50 μg zearalenone (ZEN)/kg BW per os for 42 days, group EII received 75 μg zearalenone/kg BW per os for 42 days, and the control group was administered placebo per os for 42 days. The bitches were hysterectomized at the end of treatment, and samples of uterine tissue were collected for histological and morphometric analyses. The results of the study indicate that exposure to very low doses of ZEN (100% and 150% of the NOAEL) causes simple glandular hyperplasia of the endometrium accompanied by adenogenesis, angiogenesis, and vasodilatation with the related consequences. The noted changes were more pronounced in group EI and less visible in group EII in comparison with group C, which could be indicative of a hormetic dose response.     

Multiple cysteine residues are necessary for sorting and transport activity of the arsenite permease Acr3p from Saccharomyces cerevisiae

The yeast transporter Acr3p is a low affinity As(III)/H⁺ and Sb(III)/H⁺ antiporter located in the plasma membrane. It has been shown for bacterial Acr3 proteins that just a single cysteine residue, which is located in the middle of the fourth transmembrane region and conserved in all members of the Acr3 family, is essential for As(III) transport activity. Here, we report a systematic mutational analysis of all nine cysteine residues present in the Saccharomyces cerevisiae Acr3p. We found that mutagenesis of highly conserved Cys151 resulted in a complete loss of metalloid transport function. In addition, lack of Cys90 and Cys169, which are conserved in eukaryotic members of Acr3 family, impaired Acr3p trafficking to the plasma membrane and greatly reduced As(III) efflux, respectively. Mutagenesis of five other cysteines in Acr3p resulted in moderate reduction of As(III) transport capacities and sorting perturbations. Our data suggest that interaction of As(III) with multiple thiol groups in the yeast Acr3p may facilitate As(III) translocation across the plasma membrane.     

cis-Prenyltransferase AtCPT6 produces a family of very short-chain polyisoprenoids in planta

cis-Prenyltransferases (CPTs) comprise numerous enzymes synthesizing isoprenoid hydrocarbon skeleton with isoprenoid units in the cis (Z) configuration. The chain-length specificity of a particular plant CPT is in most cases unknown despite the composition of the accumulated isoprenoids in the tissue of interest being well established. In this report AtCPT6, one of the nine Arabidopsis thaliana CPTs, is shown to catalyze the synthesis of a family of very short-chain polyisoprenoid alcohols of six, seven, and eight isoprenoid units, those of seven units dominating. The product specificity of AtCPT6 was established in vivo following its expression in the heterologous system of the yeast Saccharomyces cerevisiae and was confirmed by the absence of specific products in AtCPT6 T-DNA insertion mutants and their overaccumulation in AtCPT6-overexpressing plants. These observations are additionally validated in silico using an AtCPT6 model obtained by homology modeling. AtCPT6 only partially complements the function of the yeast homologue of CPT-Rer2 since it restores the growth but not protein glycosylation in rer2Δ yeast. This is the first in planta characterization of specific products of a plant CPT producing polyisoprenoids. Their distribution suggests that a joint activity of several CPTs is required to produce the complex mixture of polyisoprenoid alcohols found in Arabidopsis roots.     

Diversity of Selected Lupinus angustifolius L. Genotypes at the Phenotypic and DNA Level with Respect to Microscopic Seed Coat Structure and Thickness

The paper investigates seed coat characteristics (as a percentage of overall seed diameter) in Lupinus angustifolius L., a potential forage crop. In the study ten L. angustifolius genotypes, including three Polish cultivars, two Australian cultivars, three mutants originated from cv. ‘Emir’, and one Belarusian and one Australian breeding line were evaluated. The highest seed coat percentage was recorded in cultivars ‘Sonet’ and ‘Emir’. The lowest seed coat thickness percentage (below 20%) was noted for breeding lines 11257-19, LAG24 and cultivar ‘Zeus’ (17.87%, 18.91% 19.60%, respectively). Despite having low seed weight, the Australian line no. 11257-19 was characterized by a desirable proportion of seed coat to the weight of seeds. In general, estimation of the correlation coefficient indicated a tendency that larger seeds had thinner coats. Scanning Electron Microscopy images showed low variation of seed coat sculpture and the top of seeds covered with a cuticle. Most of the studied genotypes were characterized by a cristatepapillate seed coat surface, formed by elongated polygonal cells. Only breeding line no. 11267-19 had a different shape of the cells building the surface layer of the coat. In order to illustrate genetic diversity among the genotypes tested, 24 ISSR primers were used. They generated a total of 161 polymorphic amplification products in 10 evaluated narrow-leaved lupin genotypes.