Identification of 19-epi-okadaic Acid,a New Diarrhetic Shellfish Poisoning Toxin,by Liquid Chromatography with Mass Spectrometry Detection

Okadaic acid (1) (OA) and its congeners are mainly responsible for diarrhetic shellfish poisoning (DSP) syndrome. The presence of several OA derivatives have already been confirmed in Prorocentrum and Dinophysis spp. In this paper, we report on the detection and identification of a new DSP toxin, the OA isomer 19-epi-okadaic acid (2) (19-epi-OA), isolated from cultures of Prorocentrum belizeanum, by determining its retention time (RT) and fragmentation pattern using liquid chromatography coupled with mass spectrometry (LC–MS/MS).     

Kaiware Daikon (Raphanus sativus L.) extract: a naturally multipotent chemopreventive agent

Brassica vegetables are attracting major attention as healthy foods because of their content of glucosinolates (GLs) that release the corresponding isothiocyanates (ITCs) upon myrosinase hydrolysis. A number of studies have so far documented the chemopreventive properties of some ITCs. On the other hand, single nutrients detached from the food itself risk being somewhat "reductive", since plants contain several classes of compounds endowed with a polyhedral mechanism of action. Our recent finding that 4-methylthio-3-butenyl isothiocyanate (GRH-ITC) and 4-methylsulfinyl-3-butenyl isothiocyanate (GRE-ITC), released by the GLs purified from Japanese (Kaiware) Daikon (Raphanus sativus L.) seeds and sprouts, had selective cytotoxic/apoptotic activity on three human colon carcinoma cell lines prompted further research on the potential chemopreventive role of a standardized Kaiware Daikon extract (KDE), containing 10.5% w/w GRH and 3.8% w/w GRE, compared to its isolated components. KDE administered in combination with myrosinase at doses corresponding to 50 microM GRH-ITC plus 15 microM GRE-ITC (50 microM KDE-ITC) to three human cancer cell lines (LoVo, HCT-116 and HT-29) significantly reduced cell growth by 94-96% of control in six days (p < 0.05), outperforming pure GRH-ITC or GRE-ITC at the same dose. On the other hand, the same treatment had no significant toxicity on normal human T-lymphocytes. A 50 microM concentration of KDE-ITC had relevant apoptosis induction in all tested cancer cell lines, as confirmed by annexin V assay (e.g., 33% induction in LoVo compared to control, p < 0.05), Bax protein induction (e.g., +20% in HT-29, p < 0.05), and Bcl2 downregulation (e.g.-20% in HT-29, p < 0.05), and induced caspase-1 and PARP-1 activation in all cancer cells as shown by Western blot analysis. Unlike pure GRH or GRH-ITC, KDE also had significant chain-breaking antioxidant activity, retarding the AAPH-initiated autoxidation of methyl linoleate in SDS micelles at concentrations as low as 4.4 ppm (-50% in oxygen consumption rate), as monitored by Clark-type microelectrode oxygen-uptake kinetics, and induced very fast quenching of DPPH. radical in methanol with t(1/2) (s) = (1.47 +/- 0.25) x 10(-2)/[KDE; (g/L)], measured by stopped-flow UV-vis kinetics at 298 K. The potential chemopreventive role of KDE is discussed.     

Oxidative Stress, the Paradigm of Ozone Toxicity in Plants and Animals

Tropospheric ozone represents a relevant atmospheric pollutant, because of its strong oxidizing potential. The risk for animal (human) and plant health, at molecular and cellular level, arises from the oxidative damage to lipids, proteins and nucleic acids, depending on the dose. Therefore, ozone concentration and exposure time determine the chronic or acute toxicity and, consequently, the severity of injury at biochemical and physiological level. In living organisms, reactive oxygen species (ROS), directly or indirectly derived from ozone exposure, are scavenged by enzymatic and non-enzymatic antioxidant defensive mechanisms, overall deputed to preserve cell structures and biomacromolecules from the oxidative damage. These defences are essentially those also involved in detoxifying the ROS inevitably produced by the metabolism of organisms living in oxygenic atmosphere.     

Quality Evaluation of Sediments from 24 Tributaries of the Po River, Italy

Sediment samples from 24 tributaries of the Po River (Italy) were screened for selected trace elements (Cd, Cu, Hg, Pb, and Zn) and extractable organic compounds; a proxy for contamination by organic microcontaminants. The toxicity of sediment extracts was evaluated using a battery of biotests (Dugesia gonocephala, Paracentrotus lividus, and Tamnocephalus platyurus). Contamination by trace elements (including very high Hg pollution – 4 to 16ppm total Hg – in one sub-basin) reached potentially harmful levels only in the sediments of four tributaries; while contamination by organic microcontaminants was present in most sub-basins. Sediments from most study sites did actually show signs of anthropogenic stress and were able to elicit a toxic response. A more detailed evaluation of sediment quality in the Po River tributaries seems to be urgently needed for developing the necessary remediation strategies. Research priorities should include more thorough testing of sediment toxicity, determination of metal background levels in the various sub-basins and a more detailed identification of the organic micropollutants of possible concern.     

Characterization and propagation of some medicinal plants in the central-south region of Chile

The increase of land use for crop cultivation and forest in South Central Chile, and the increasing wildcrafting of medicinal plants has resulted in a significant reduction of the plant population density of many native and endemic medicinal plants. Their cultivation and domestication is very limited, and there are no regulations or legislation for wildcrafting in Chile. The objectives of this study were to collect genetic material from five native medicinal plants (Adesmia emarginata, Buddleja globosa, Fabiana imbricata, Linum chamissonis, and Sophora macrocarpa), characterize the environmental conditions in which these grow in the Bio-Bio Region, Chile, and to determine the content of specific bioactive molecules. A maximum of 10 accessions of each species were collected in 2003, 2004, and 2005 in a longitudinal and transversal transect of the Bio-Bio Region (36°00′-38°30′S; 71°W). Data of altitude, light conditions, soil chemical and physical characteristics and associated flora of the site collection were recorded. Seed germination requirements and the content of specific bioactive molecules (flavonoids or rutin) were also determined. In general, all these species are adapted to grow in poor soils with different pH values, P and K levels, low organic matter and N content. Flavonoids were determined in both A. emarginata (0.6-1%) and B. globosa (9.7-13.9%) leaves, while rutin concentration was determined in F. imbricata leaves and stems (1.3-5.3%). Results showed great variations for the content of active principles with medicinal activity, which indicates a corresponding variation in the quality of raw materials for the pharmaceutical industry. Due to the fact that information on the domestication, propagation, cultivation and agronomic practices can ensure good pharmaceutical quality, this study provides a basis for further research on Chilean medicinal plants.     

On-farm evaluation of a low-input rice production system in Panama

On-farm trials were conducted to evaluate the potential of the System of Rice Intensification (SRI), a low-input crop management system, to increase rice yields and reduce water consumption on subsistence farms in several regions of Panama and to determine how inherent soil fertility might affect SRI yields and the yield response to SRI management in the first season of SRI management. SRI practices increased yield by 47% on average and showed potential to increase yield by over 90%, while reducing water consumption by as much as 86%. SRI yields were correlated with available soil K and the difference between SRI and the conventional system yields was positively correlated with extractable Ca, Mg and Mn. The results of this study indicate that SRI is a promising rice production system for smallholder farmers in rural Panama farming under Panamanian soil conditions.     

Performance and selectivity of floral attractant-baited traps targeted for cetoniin scarabs (Coleoptera: Scarabaeidae) in Central and Southern Europe

Trapping tests were performed concurrently in several countries of Central and Southern Europe throughout the spring and summer of 2008, to study the selectivity and performance of floral attractant-baited traps developed for catching Epicometis (Tropinota) hirta Poda (CA-baited traps), Cetonia a. aurata L./Potosia (Protaetia) cuprea Fabr. (ME-baited traps) or Oxythyrea funesta Poda (PH-baited traps) (Coleoptera: Scarabaeidae, Cetoniinae). Amongst the species caught, E. hirta showed strongest attraction to the CA-baited and ME-baited traps. O. funesta was mostly caught by PH-baited traps. In capturing C. a. aurata and P. cuprea, the ME-baited trap appeared to be the most efficient. At two sites in Italy, a related scarab, Tropinota squalida Scop. (Coleoptera: Scarabaeidae, Cetoniinae), was attracted in similar numbers to both CA-baited and ME-baited traps. For the scarab Valgus hemipterus L. (Coleoptera: Scarabaeidae, Valginae), ME-baited traps proved to be the most attractive.     

Identification of four novel members of Kunitz-like α-amylase inhibitors family from Delonix regia with activity toward Coleopteran insects

Crop improvement generally focuses on yield, seed quality and nutritional characteristics, rather than resistance to biotic and abiotic stresses. A clear consequence of this approach is the absence of natural anti-feedant toxins in some improved seed materials, allowing predation of commercial crops by insect herbivores. Cowpea (Vigna unguiculata), commonly cultivated by small farmers, is particularly affected by insect-pests that reproduce and develop inside stored seeds. One alternative to conventional pesticides for pest control is the use of biotechnological tools, such as the digestive enzyme inhibitors, that could be introduced in transgenic crops to enhance resistance. In this study, it was verified that the in vivo bioassays using artificial seeds containing 0.5%, 1.0% and 1.5% (w/w) of Delonix regia rich fraction, containing α-amylase inhibitors with effectiveness toward insect α-amylases and other sources, caused remarkable reduction in development and increased mortality of Callosobruchus maculatus cowpea weevil and to cotton boll weevil Anthonomus grandis. Therefore, attempts were made to isolated those inhibitors by SP-Sepharose ion exchange chromatography followed by high performance liquid chromatography on a Vydac C18-TP analytical column. Four inhibitor peaks were obtained with molecular masses of 6.0, 20 and 24 kDa. Their N-termini showed high sequence similarities with Kunitz-like inhibitor family members. These results provide evidence that D. regia synthesizes a multiple family of Kunitz-like α-amylase inhibitors, with different molecular masses and a wide biotechnological potential to control insect-pests.     

Label-Free Darkfield-Based Technique to Assist in the Detection of <Emphasis Type="Italic">Giardia</Emphasis> Cysts

Giardia is a pathogenic protozoan that has caused giardiasis outbreaks worldwide, and this water-borne disease may occur due to faults in water supply and treatment systems. To support surveillance and control programs, the monitoring of this parasite is essential in water samples. Although Giardia cyst detection is usually based on standard light microscopy, the resulting low contrasted cysts together with a wide array of particles of comparable size to the target protozoan demands a high level of observer skill and experience, as well as a long time to process the individual samples. The visualization of this protozoan can be significantly improved by using staining procedures, e.g., Lugol’s iodine in brightfield or fluorescence-based methods such as fluorescence-isothiocyanate (FITC) and 4′,6-diamidino-2-phenylindole (DAPI). However, the significant costs make fluorescence microscopy difficult to be performed in less-developed countries. Accordingly, the present study evaluated the effectiveness of inexpensive darkfield (DF) setups to acquire higher contrasted images of unstained cyst samples as a direct and rapid method for monitoring protozoa. Two low-cost custom-made DF illumination modes, transmitted and reflected, were evaluated on a standard light microscope. Examination of purified Giardia cyst suspensions with both DF setups revealed a direct correlation between morphological appearance and uptake of DAPI. Images captured under transmitted illumination showed higher contrast and sharpness when compared to the reflected images. DF microscopy might provide a simple, direct, and inexpensive method for observing Giardia cysts, which shows basic aspects of their intracellular structure, although the applicability of the method to raw water concentrates remains to be demonstrated.