Hedge Mustard (Sisymbrium officinale): Chemical Diversity of Volatiles and Their Antimicrobial Activity

Volatile compounds of hedge mustard (Sysimbrium officinale) have been investigated for the first time. Forthy‐two compounds were identified after hydrodistillation (without or upon autolysis) after gas chromatography and gas chromatography/mass spectrometry analyses. In addition, after decoction and hydrolysis of O‐glycosides, 18 volatile O‐aglycones were identified. In general, the main volatiles found in hydrodistillates were: isopropyl isothiocyanate (27.6–48.9%), 2‐methylpropanenitrile (0.5–18.8%), (Z)‐hex‐3‐en‐1‐ol (0.5–18.0%), sec‐butyl isothiocyanate (4.9–9.4%), (E)‐hex‐2‐enal (3.5–8.6%), (Z)‐hex‐2‐en‐1‐ol (0.3–8.4%), octanoic (0.5–8.6%) and dodecanoic acid (0–5.0%), 2‐methylbutanenitrile (0–4.6%), dibutyl phthalate (0–4.5%), and ethyl linolenate (0–3.6%). The main volatile O‐aglycones were: 2‐phenylethyl alcohol (21.5%), 6,7‐dehydro‐7,8‐dihydro‐3‐oxo‐α‐ionol (9.3%), eugenol (8.3%), benzyl alcohol (7.0%), ethyl vanillate (5.2%), 6‐(tert‐butyl)‐5‐methylphenol (5.1%), vanillin acetone (4.7%), ethyl 4‐hydroxybenzoate (4.3%), and 2‐hydroxy‐β‐ionone (3.8%). All hydrodistillates exhibited great potential of antibacterial activity against five Gram‐positive bacteria, nine ampicillin‐resistant Gram‐negative bacteria, and four fungi at a concentration of 500 μg/ml using the disc diffusion method.     

The phylogenetic position of <Emphasis Type="Italic">Obolarina dryophila</Emphasis> (Xylariales)

The inconspicuous inner-bark parasite Obolarina dryophila is reported from wood of Quercus petraea and as an endophyte of Salix alba. In addition, viable ascospores of O. dryophila have been found in the gut of the oak bark weevil Gasterocercus depressirostris, suggesting a possible dissemination mechanism for the fungus. A phylogenetic analysis based on three genes (nrDNA, actin, β-tubulin) placed Obolarina inside the genus Biscogniauxia as a close relative of the oak pathogens B. atropunctata and B. mediterranea.     

Elastic,not plastic species: Frozen plasticity theory and the origin of adaptive evolution in sexually reproducing organisms

Background  

Darwin's evolutionary theory could easily explain the evolution of adaptive traits (organs and behavioral patterns) in asexual but not in sexual organisms. Two models, the selfish gene theory and frozen plasticity theory were suggested to explain evolution of adaptive traits in sexual organisms in past 30 years.     

Ammodytoxin content of Vipera ammodytes ammodytes venom as a prognostic factor for venom immunogenicity

Venoms are complex mixtures of proteins, peptides and other compounds whose biochemical and biological variability has been clearly demonstrated. These molecules have been used as antigens for immunization of anti-venom-producing animals (horses or sheep). Ammodytoxins (Atx) are potently neurotoxic compounds, and the most toxic compounds isolated so far from the Vipera ammodytes ammodytes (Vaa) venom. Recently we have shown that the level of antibodies specific to Vaa venom's most toxic component, ammodytoxin A (AtxA), (anti-AtxA IgG) in Vaa venom immunized rabbit sera highly correlated to the venom toxicity–neutralization potential of these sera. Here we investigated whether Atx content of Vaa venom could influence the outcome of immunization procedure. The novel ELISA was developed for precise determination of Atx content and Atx was quantified in venom samples used for immunization of rabbits. We clearly showed that animals immunized with the venom containing lower amount of Atx produced sera with significantly lower venom toxicity–neutralizing power and, vice versa, animals immunized with venoms containing higher amount of Atx produced sera with higher venom toxicity–neutralizing ability. Thus, the content of Atx in Vaa venom is a relevant parameter of its suitability in the production of highly protective Vaa anti-venom.     

Assessment of rpoB and 16S rRNA genes as targets for PCR-based identification of Pasteurella pneumotropica

Diagnosis of Pasteurella pneumotropica in laboratory animals relies on isolation of the organism, biochemical characterization, and, more recently, DNA-based diagnostic methods. 16S rRNA and rpoB gene sequences were examined for development of a real-time PCR assay. Partial sequencing of rpoB (456 bp) and 16S rRNA (1368 bp) of Pasteurella pneumotropica isolates identified by microbiologic and biochemical assays indicated that either gene sequence can be used to distinguish P. pneumotropica from other members of the Pasteurellaceae family. However, alignment of rpoB sequences from the Pasteurella pneumotropica Heyl (15 sequences) and Jawetz (16 sequences) biotypes with other Pasteurellaceae sequences from GenBank indicated that although rpoB DNA sequencing could be used for diagnosis, development of diagnostic primers and probes would be difficult, because the sequence variability between Heyl and Jawetz biotypes is not clustered in any particular region of the rpoB sequence. In contrast, alignment of 16S rRNA sequences revealed a region with unique and stable nucleotide motifs sufficient to permit development of a specific fluorogenic real-time PCR assay to confirm P. pneumotropica isolated by culture and to differentiate Heyl and Jawetz biotypes.     

Diagnostic role of p16/INK4A protein in Human Papillomavirus (HPV) induced cervical dysplasia

The p16/INK4A protein is a cellular regulatory polypeptide over-expressed in the presence of high levels of the Human Papillomavirus (HPV) coded E7 protein. This review outlines the use of p16 antigen staining in cervical biopsies as well as in PAP smears summarizing the corresponding literature and commenting the authors’ own experience. The p16 antigen is a reliable marker for dysplastic cells in CINII/CINIII (HSIL) lesions as viewed in cervical biopsies. When PAP smears were examined at large scale screening for p16 antigenreactive and atypical cells, considerable variations could be found especially in ASCUS graded lesions. Therefore, the presence of p16-reactive atypical cells in PAP smears should be interpreted together with the cytological signs of dysplasia, such as the altered N/C ratio. In addition, women revealing p16-positive ASCUS/LSIL specimens should be examined for the presence of HPV DNA. Detection of HPV DNA alone, i.e. in the absence of cytological screening has a low predictive value, since the clearance of HPV may occur even in the absence of morphological alterations. Combined cytological as well as molecular follow up contributes to the efficiency of diagnostic and increases the probability of correct interpretation of the pre-cancerous lesions by non-invasive techniques.     

The contents of risk elements,arsenic speciation,and possible interactions of elements and betalains in beetroot (<Emphasis Type="Italic">Beta vulgaris</Emphasis>, L.) growing in contaminated soil

The effect of enhanced soil risk element contents on the uptake of As, Cd, Pb, and Zn was determined in two pot experiments. Simultaneously, transformation of arsenic and its compounds in beetroot (Beta vulgaris L.) plants was investigated. The mobile fractions of elements were determined in 0.05 mol L⁻¹ (NH₄)₂SO₄ extracts and did not exceed 2% of total soil arsenic, 9% of total cadmium, 3% of total lead, and 8% of total zinc, respectively. Although the soils were extremely contaminated the mobile portions of the elements represented only a small fragment of the total element content. Arsenic contents in beet plants reached up to 25 mg As kg⁻¹ in roots and 48 mg As kg⁻¹ in leaves in the soil characterized by the highest mobile arsenic portion. Arsenic portions extractable with water and phosphate buffer from the beetroot samples did not show significant differences between the extraction agents but the extractability was affected by the arsenic concentration. Arsenic was almost quantitatively extractable from the samples with the lowest total arsenic concentration, whereas in the samples with the highest total arsenic concentration less than 25% was extractable. Arsenate was the dominant arsenic compound in the extracts (70% in phosphate buffer, 50% in water extracts). A small portion of dimethylarsinic acid, not exceeding 0.5%, was detected only in the sample growing in the soil with the highest arsenic concentration. The role of betalains (betanin, isobetanin, vulgaxanthin I and vulgaxanthin II) in transformation/detoxification of arsenic in plants was not confirmed in this experiment because the plants were able to grow in the contaminated soil without any symptoms of arsenic toxicity.     

Heat processing of soybean kernel and its effect on lysine availability and protein solubility

Soybean kernels of cultivars Bosa and ZPS 015 were used in the experiment. The contents of available lysine as well as water and salt soluble proteins, were analysed in fresh soybean kernels, soybean products made after the processes of dry extrusion, micronisation, microwave toasting and autoclaving. Utilizing a technological procedure of processing, kernels were exposed to temperatures from 57 to 150°C. The duration of exposure of the soybean kernels to the increased temperatures, ranged from 25-30 seconds in dry extrusion to 30 minutes in autoclaving. All treatments were subjected to different sources of heat, causing different thermodynamic processes to take place in kernels and change their chemical composition; i.e. nutritive quality. The content of water and salt soluble proteins decreased under the influence of higher temperatures in the course of all treatments of processing. The drop of solubility already was drastically effected by temperatures of 100°C in dry extrusion, while there was a gradual decrease in other treatments. The content of available lysine was determined by the modified Carpenter methods with DNFB. The processes of micronisation and microwave toasting showed the greatest effect on the reduction of lysine availability. Dry extrusion and autoclaving, performed within closed systems — in which the increased moisture content has a special effect — resulted in significantly smaller changes of the available lysine content.     

Multiplexed flow cytometric analyses of pro- and anti-inflammatory cytokines in the culture media of oxysterol-treated human monocytic cells and in the sera of atherosclerotic patients.

BACKGROUND: Some oxysterols are identified in atheromatous plaques and in plasma of atherosclerotic patients. We asked whether they might modulate cytokine secretion on human monocytic cells. In healthy and atherosclerotic subjects, we also investigated the relationships between circulating levels of C-reactive protein (CRP), conventional markers of hyperlipidemia, some oxysterols (7beta-hydroxycholesterol, 7-ketocholesterol, and 25-hydroxycholesterol), and various cytokines. METHODS: Different flow cytometric bead-based assays were used to quantify some cytokines (IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, G-CSF, GM-CSF, IFN-gamma, MCP-1, MIP-1beta, or TNF-alpha) in the culture media of oxysterol-treated U937 and THP-1 cells, and in the sera of healthy and atherosclerotic subjects. CRP and markers of hyperlipidemia were determined with routine analytical methods. Oxysterols were quantified by gas chromatography/mass spectrometry. Flow cytometric and biochemical methods were used to measure IL-8 mRNA levels, intracellular IL-8 content, and protein phosphorylation in the mitogenic extracellular kinase/extracellular signal-regulated kinase1/2 (MEK/ERK1/2) signaling pathway. RESULTS: All oxysterols investigated are potent in vitro inducers of MCP-1, MIP-1beta, TNF-alpha, and/or IL-8 secretion, the latter involving the MEK/ERK1/2 cell signaling pathway. In healthy and atherosclerotic subjects, no relationships were found between cytokines (IL-8, IL-1beta, IL-6, IL-10, TNF-alpha, IL-12, and MCP-1), CRP, conventional markers of hyperlipidemia, and oxysterols. However, in patients with arterial disorders of the lower limbs, small but statistically significant differences in the circulating levels of CRP, TNF-alpha, and IL-10 were observed comparatively to healthy subjects and according to the atherosclerotic stage considered. CONCLUSIONS: Flow cytometric bead-based assays are well adapted to measure variations of cytokine secretion in the culture media of oxysterol-treated cells and in the sera of healthy and atherosclerotic subjects. They underline the in vitro proinflammatory properties of oxysterols and may permit to distinguish healthy and atherosclerotic subjects, as well as various atherosclerotic stages.     

The bacterial artificial chromosome (BAC) library of the narrow-leafed lupin (Lupinus angustifolius L.)

The narrow-leafed lupin possesses valuable traits for environment-friendly agriculture and for the production of unconventional agricultural products. Despite various genetic and environmental studies, the breeding of improved cultivars has been slow due to the limited knowledge of its genomic structure. Further advances in genomics require, among other things, the availability of a genomic DNA library with large inserts. We report here on the construction of the first DNA library cloned in a BAC (bacterial artificial chromosome) vector from diploid Lupinus angustifolius L. cv. Sonet. The high molecular weight DNA used for its preparation was isolated from interphase nuclei that were purified by flow cytometry. The library comprises 55,296 clones and is ordered in 144×384-well microtitre plates. With an average insert size of 100 kb, the library represents six haploid genome equivalents. Thanks to the purification of the nuclei by flow cytometry, contamination with chloroplast DNA and mitochondrial DNA was negligible. The availability of a BAC library opens avenues for the development of a physical contig map and positional gene cloning, as well as for the analysis of the plant’s genome structure and evolution.