Effect of Oil Overlay on Inhibition Potential of Roscovitine in Sheep Cumulus‐Oocyte Complexes

Inhibitors of cyclin‐dependent kinases, as roscovitine, have been used to prevent the spontaneous resumption of meiosis in vitro and to improve the oocyte developmental competence. In this study, the interference of oil overlay on the reversible arrest capacity of roscovitine in sheep oocytes as well as its effects on cumulus expansion was evaluated. For this, cumulus‐oocyte complexes (COCs) were cultured for 20 h in TCM 199 with 10% foetal bovine serum (Control) containing 75 μm roscovitine (Rosco). Subsequently, they were in vitro matured (IVM) for further 18 h in inhibitor‐free medium with LH and FSH. The culture was performed in Petri dishes under mineral oil (+) or in 96 well plates without oil overlay (?) at 38.5°C and 5% CO₂. At 20 and 38 h, the cumulus expansion and nuclear maturation were evaluated under stereomicroscope and by Hoechst 33342 staining, respectively. No group presented cumulus expansion at 20 h. After additional culture with gonadotrophins, a significant rate of COCs from both Control groups (+/?) exhibited total expansion while in both Rosco groups (+/?) the partial expansion prevailed. Among the oocytes treated with roscovitine, 65.2% were kept at GV in the absence of oil overlay while 40.6% of them reached MII under oil cover (p < 0.05). This meiotic arrest was reversible, and proper meiosis progression also occurred in the Control groups (+/?). So, the culture system without oil overlay improved the meiotic inhibition promoted by roscovitine without affecting the cumulus expansion rate or the subsequent meiosis progression.     

TILLING and Ecotilling the Plant Kingdom

The modem crop scientist has a large amount of available nucleotide sequence information to identify genes of potential agronomic importance. Using reverse genetic approaches, specific genes can be disrupted, and hypotheses regarding gene function directly tested in vivo. Although a number of reverse genetic methods have been introduced, many are limited in application because they are organism-specific, expensive, transgenic, or only transiently disrupt gene function. However, traditional mutagenesis using chemical mutagens has been widely used as a forward genetics strategy to create many new crop plant varieties at relatively low cost. Mutagens such as ethyl methanesulphonate （EMS） cause stable point mutations and thus produce an allelic series of truncation and missense changes that can provide a range of phenotypes （Greene et al., 2003）. TILLING （targeting induced local lesions IN genomes） is a high-throughput reverse genetic strategy that combines traditional mutagenesis and SNP discovery methods （Colbert et al., 2001; McCallum et al., 2000）. To identify mutations, target regions of-l.5 kb are amplified with fluorescently labeled gene specific primers. Heteroduplexes are then formed between wild-type and mutant strands, mismatches are cleaved by incubation with a single-strand specific nuclease,     

Mass spectral investigations on trichothecene mycotoxins. II. Detection and quantitation of macrocyclic trichothecenes by gas chromatography/negative ion chemical ionization mass spectrometry

A general, sensitive gas chromatographic/negative ion chemical ionization mass spectrometric (GC/NICIMS) method of analysis was developed for the detection and quantitation of several polar, thermally labile, toxic macrocyclic trichothecenes. The procedure involves the conversion of the molecules to their corresponding alcohols (verrucarols) by alkaline hydrolysis, followed by derivatization of the hydrolysate with heptafluorobutyrylimidazole and analysis by GC/MS technique under negative ion chemical ionization conditions. Nanogram (250 ng) quantities of several macrocyclic trichothecenes with different verrucarol and ester moieties were analyzed successfully with good precision by this procedure. The method was applicable for the accurate determination of at least low ppb levels of these macrocyclic trichothecenes in environmental samples, such as fungal products, fermentation broths, and plant samples. This is the first reported, well developed, sensitive, and applicable method for the detection and quantitation of these compounds in naturally occurring samples.     

Sedimentary aggregates in the Peoria Loess of Nebraska, USA

Loess grain size data used to infer transport direction or wind strength are generally derived from vigorously disaggregated samples. However, these data may not adequately represent the effective particle size distribution during loess transport, if the transported dust contained aggregates of fine-grained material. Thin sections of minimally altered C and BC horizons in the late Pleistocene Peoria Loess of Nebraska, USA, indicate the presence of aggregates with diameters of 30–1000 μm. The larger aggregates (>250 μm) are unlikely to have been transported, and are interpreted as the result of soil faunal activity and other pedogenic processes after deposition. Aggregates smaller than 250 μm could have a similar origin, but laser diffraction particle size analysis suggests that many are sedimentary particles. Comparison of minimally and fully dispersed particle size distributions from each sampling site was used to estimate the modal diameter of aggregates. The aggregate modal diameter becomes finer with decreasing loess thickness, representing increasing distance from the source. A similar trend was observed in the modal diameter of fully dispersed particle size distributions, which represents the mode of sand and silt transported as individual grains. We interpret both trends as the result of sorting during transport, supporting the interpretation that many of the aggregates were transported rather than formed in place. Aggregate content appears to increase with distance from the source, explaining a much more rapid downwind increase in clay content than would be expected if clay were transported as particles smaller than 2 μm diameter. Although the Peoria Loess of Nebraska contains sedimentary aggregates, many of the coarse silt and sand grains in this loess were transported as primary particles, were thoroughly exposed to sunlight and are potentially well suited for luminescence dating.     

Follow-up of mercury levels in fish,human hair and urine in the Madeira and Tapajós basins,Amazon, Brazil

Informal economy of gold mining has contaminated some important river basins in Amazon. Follow-up studies on critical compartments showed some areas with high Hg levels in fish as well as in human hair samples. Average Hg in piscivorous fish in the Madeira river itself was 846 ppb (N=284) with a maximum of 3921 ppb. Mercury in fish from non polluted areas in this basin shows high variability, even for single species. A seasonal variation in Hg content was observed, with higher values at the end of the dry season. In the upper Tapajós basin comparable values were found for fish but with a definite decreasing trend downstream. Average value for piscivorous fish in the whole Tapajós basin is 482 ppb (N=122) with a maximum value of 3770 ppb. Hair Hg was higher in fishing villages in the Tapajós (average: 17 ppm; with N=432 and maximum value of 176 ppm) than in the Madeira (average: 9 ppm; N=169; maximum 71 ppm), and data from some areas of the Tapajós suggest a decrease with time. Mercury was much higher in urine of goldshop workers in Santarém (low Tapajós) than in Alta Floresta (high Tapajbs) and show a decreasing trend in both cases, probably related to the significant decline in gold mining activities during the study period (1986–1994).