In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent epigenetic mechanism. Here, the effects of Cd treatment on the DNA methylation patten are examined together with its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in actively growing organs, under short- (6 h) and long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd, through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach, respectively. The expression of one member of the CHROMOMETHYLASE (CMT) family, a DNA methyltransferase, was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd perturbs the DNA methylation status through the involvement of a specific methyltransferase. Such changes are linked to nuclear chromatin reconfiguration likely to establish a new balance of expressed/repressed chromatin. Overall, the data show an epigenetic basis to the mechanism underlying Cd toxicity in plants.

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In Posidonia oceanica cadmium induces changes in DNA methylation and chromatin patterning

Antioxidant activity of Citrus fruits

Characterization of the solid-state: spectroscopic techniques.

The 2,2-diphenylpicrylhydrazyl (DPPH) assay is widely used in plant biochemistry to evaluate the properties of plant constituents for scavenging free radicals. The method is based on the spectrophotometric measurement of the DPPH concentration change resulting from the reaction with an antioxidant. Several protocols have been followed for this assay using different conditions such as different reaction times, solvents, pH and different compounds used as antioxidant standards. This review shows to what extent the mentioned parameters have the influence on the presented results.

Application of free radical diphenylpicrylhydrazyl (DPPH) to estimate the antioxidant capacity of food samples

Objectives To catalog what is known about the safety of interventions containing Lactobacillus, Bifidobacterium, Saccharomyces, Streptococcus, Enterococcus, and/or Bacillus strains used as probiotic agents in research to reduce the risk of, prevent, or treat disease. Data sources We searched 12 electronic databases, references of included studies, and pertinent reviews for studies addressing the safety of probiotics from database inception to August 2010 without language restriction. Review methods We identified intervention studies on probiotics that reported the presence or absence of adverse health outcomes in human participants, without restriction by study design, participant type, or clinical field. We investigated the quantity, quality, and nature of adverse events. Results The search identified 11,977 publications, of which 622 studies were included in the review. In 235 studies, only nonspecific safety statements were made ("well tolerated"); the remaining 387 studies reported the presence or absence of specific adverse events. Interventions and adverse events were poorly documented. A number of case studies described fungemia and some bacteremia potentially associated with administered probiotic organisms. Controlled trials did not monitor routinely for such infections and primarily reported on gastrointestinal adverse events. Based on reported adverse events, randomized controlled trials (RCTs) showed no statistically significantly increased relative risk (RR) of the overall number of experienced adverse events (RR 1.00; 95% confidence interval [CI]: 0.93, 1.07, p=0.999); gastrointestinal; infections; or other adverse events, including serious adverse events (RR 1.06; 95% CI: 0.97, 1.16; p=0.201), associated with short-term probiotic use compared to control group participants; long-term effects are largely unknown. Existing studies primarily examined Lactobacillus alone or in combination with other genera, often Bifidobacterium. Few studies directly compared the safety among different intervention or participant characteristics. Indirect comparisons indicated that effects of delivery vehicles (e.g., yogurt, dairy) should be investigated further. Case studies suggested that participants with compromised health are most likely to experience adverse events associated with probiotics. However, RCTs in medium-risk and critically ill participants did not report a statistically significantly increased risk of adverse events compared to control group participants. Conclusions There is a lack of assessment and systematic reporting of adverse events in probiotic intervention studies, and interventions are poorly documented. The available evidence in RCTs does not indicate an increased risk; however, rare adverse events are difficult to assess, and despite the substantial number of publications, the current literature is not well equipped to answer questions on the safety of probiotic interventions with confidence.

Safety of probiotics used to reduce risk and prevent or treat disease.

Several species of the Veratrum genus are associated with toxicity in humans and animals. The principal toxins are steroid alkaloids; some have a modified steroid template, whereas others differ in their esterified acid moieties. These alkaloids act by increasing the permeability of the sodium channels of nerve cells, causing them to fire continuously. Increased stimulation, associated with the vagal nerve results in a reflex that causes the triad of responses known as the Bezold-Jarisch reflex: hypotension, bradycardia and apnoea. Clinically, various Veratrum extracts were marketed for clinical use as antihypertensive drugs, but because of their narrow therapeutic index were withdrawn from the market. Following the ingestion of Veratrum alkaloids, expected signs and symptoms include vomiting and abdominal pain, followed by cardiovascular effects such as bradycardia, hypotension and cardiac conduction abnormalities and death. Similar symptoms arise in other mammalian species ingesting these alkaloids; teratogenic effects may occur to the fetuses of animals that have grazed on Veratrum californicum. Treatment consists of supportive care, with an emphasis on haemodynamic stability with fluid replacement, atropine and vasopressors. The onset of symptoms occurs between 30 minutes and 4 hours, and the duration of the illness can range from 1 to 10 days; however, with prompt supportive care, patients typically make a full recovery within 24 hours.

Veratrum Poisoning

Objectives To use an online assay to identify key antioxidants in Sonchus oleraceus leaf extracts and to investigate the effect of leaf position and extraction conditions on antioxidant concentration and activity. Methods Separation of phytochemicals and simultaneous assessment of antioxidant activity were performed online using HPLC and post-column reaction with a free-radical reagent (2, 2-diphenylpicrylhydrazyl, DPPH). Active compounds were identified using nuclear magnetic resonance spectroscopy and mass spectrometry. We applied the online HPLC-DPPH radical assay to evaluate antioxidants in leaves from different positions on the plant and to assess the effect of pre-treatment of leaves with liquid N(2) before grinding, extraction time, extraction temperature and method of concentrating extracts. Key findings Key antioxidants identified in S. oleraceus leaf extracts were caftaric acid, chlorogenic acid and chicoric acid. Middle leaves contained the highest total amount of the three key antioxidant compounds, consisting mainly of chicoric acid. Pre-treatment with liquid N(2), increasing the extraction temperature and time and freeze-drying the extract did not enhance the yield of the key antioxidants. Conclusion The online HPLC-DPPH radical assay was validated as a useful screening tool for investigating individual antioxidants in leaf extracts. Optimized extraction conditions were middle leaves pre-treated with liquid N(2), extraction at 25°C for 0.5 h and solvent removal by rotary evaporation.

Application of an online post-column derivatization HPLC-DPPH assay to detect compounds responsible for antioxidant activity in Sonchus oleraceus L. leaf extracts.

Aim: To carry out a pilot study to investigate the effect of short-term oral probiotic administration on the metabolism of sulfasalazine (SSZ) in patients with rheumatoid arthritis (RA) stabilized on SSZ.



Methods:  Twelve subjects with RA taking stable doses of SSZ for a minimum of 3 months prior to the study, received a probiotic preparation contained three strains of bacteria (1.8 × 109 CFU/day) twice daily for 1 week. Single point blood and 12-h urine samples were taken before and after probiotic treatment and 3 weeks following discontinuation of probiotics, for determination of SSZ and its metabolites. The presence of the probiotic bacteria in the feces of patients was investigated by denaturing gradient gel electrophoresis (DGGE).



Results:  Adverse events recorded were three instances of gastrointestinal disturbance and one flare of RA. Plasma and urinary levels of SSZ and its metabolites showed no statistically significant changes after probiotic administration and the incidence of gastrointestinal disturbance did not appear to be ascribed to higher sulfapyridine plasma levels. Probiotic-specific DGGE bands were detected in the feces of some patients after the treatment period.



Conclusions:  Short-term treatment of RA patients with a multi-strain probiotic did not significantly influence SSZ metabolism as has been demonstrated in animal models.

The effects of an orally administered probiotic on sulfasalazine metabolism in individuals with rheumatoid arthritis: a preliminary study.

The cytokinin content in fruit tissue of the kiwifruit (Actinidia deliciosa [A. Chev.] C. E Liang et A. R. Ferguson var. deliciosa cv. Hayward) was monitored during fruit development to identify which cytokinins were present and if they were linked with specific stages of fruit growth. Cytokinins were isolated and purified by column chromatography and high-performance liquid chromatography and quantified by radioimmunoassay. A novel HPLC step utilising an amine column was successfully introduced as a preparative step in the separation of the O- and 9-glucosides from the free bases and ribosides. The radioimmunoassay results were validated, and the different cytokinins identified, by gas chromatography-mass spectrometry. Cytokinins detected in fruit included the cytokinin free bases, zeatin and isopentenyladenine, their ribosides, nucleotides and both O- and 9-glucosides. Both qualitative and quantitative changes of the cytokinins occurred during fruit development. A decrease in cytokinin concentration occurred after anthesis (from 342 pmol g -1 fresh weight at anthesis to 41 pmol g -1 fresh weight 27 days after anthesis). A large increase in cytokinin concentration and content per fruit occurred as the fruit reached commercial maturity (to 1 900 pmol g -1 fresh weight). Individual cytokinins showed quite different patterns. Zeatin, in particular, showed a peak in concentration (13 pmol g -1 fresh weight) 11 days after anthesis that correlated with the beginning of the cell division phase of fruit growth. The accumulation of cytokinin (mostly zeatin riboside or zeatin nucleotide) in mature fruit may be of significance for the postharvest storage of kiwifruit fruit.

Cytokinins and fruit development in the kiwifruit (Actinidia deliciosa). I. Changes during fruit development

Raman spectroscopy was used to classify a group of seized counterfeit medications associated with erectile dysfunction. Using appropriate data preprocessing, principal component analysis and the classification method soft independent modelling of class analogy (SIMCA), it was possible to classify genuine from unregistered generic and counterfeit Cialis® batches. However, SIMCA did not effectively classify samples on the basis of their active pharmaceutical ingredient (API). Partial least squares discriminant analysis, principal component regression and support vector machines effectively distinguished between the API of the samples but were unable to correctly distinguish all samples as genuine or generic/counterfeit. This study highlights the importance of choosing the correct preprocessing procedures and classification method for the data set.



Despite diverse tablet contents in addition to the drug, it was possible to quantify the levels of drug in the medicines as high, medium or low (within ±20 mg g−1 tablet concentration).



Overall, the potential for Raman spectroscopy combined with multivariate analysis for qualitative and semiquantitative analysis of counterfeit medicines was demonstrated, and the approach may be used to determine the potential level of harm in counterfeit medicines on the basis of API identity and amount. Copyright © 2013 John Wiley & Sons, Ltd.

David M. Schmierer

Papers

Veratrum Poisoning

Application of an online post-column derivatization HPLC-DPPH assay to detect compounds responsible for antioxidant activity in Sonchus oleraceus L. leaf extracts.

The effects of an orally administered probiotic on sulfasalazine metabolism in individuals with rheumatoid arthritis: a preliminary study.

Cytokinins and fruit development in the kiwifruit (Actinidia deliciosa). I. Changes during fruit development

Simultaneous qualitative and quantitative analysis of counterfeit and unregistered medicines using Raman spectroscopy