Biomonitoring of arylamines: haemoglobin adducts of aniline derivatives

Aromatic amines are important intermediates in industrial manufacturing. They are used in a large number of products, such as pesticides, dyes, plastics and pharmaceuticals. The parent arylamines can be metabolically released from these arylamine-based compounds and form DNA and protein adducts after N-oxidation to N-hydroxy arylamines. Aromatic amine derivatives, including the industrial intermediates acetoacetanilide, acetoacet-m-xylidide and N-ethylaniline, were examined for their ability to form Hb adducts in rats as potential biomarkers of exposure. The haemoglobin binding indices (HBI=binding [mmol mol^-1 Hb]/dose [mmol kg^-1 body weight]) of the arylamines were determined 24 h after oral administration to female Wistar rats. The precipitated haemoglobin was dissolved in 0.1 M sodium hydroxide in the presence of internal standards. After hexane extraction the released arylamines were analysed by gas chromatography-mass spectrometry (GC-MS). For aniline released from acetoacetanilide an HBI of 15 and for 2,4-dimethylaniline released from acetoacet-m-xylidide an HBI of 0.129 were determined. The HBIof aniline released from N-ethylaniline was 45.     

Formation of rosettes by nonspecific lymphoid cells which have adhered to antigen-antibody complexes

Rat lymph node-sheep red cell rosettes have been formed through the adherence of nonspecific small lymphocytes and larger cell types to antibody-sheep red cell complexes. Enough antibody to form a significant number of rosettes has been synthesized in cultures, containing draining lymph node cells of immunized rats, within 1 hr of incubation at 37 °C, or during overnight storage at 4 °C. This suggests that conditions which differentiate between specific and nonspecific rosette-forming cells are essential when immunocyto-adherence phenomena are utilized for the study of the specificity of the immune response.     

Use of locally weighted scatterplot smoothing (LOWESS) regression to study selection signatures in Piedmontese and Italian Brown cattle breeds

Selection is the major force affecting local levels of genetic variation in species. The availability of dense marker maps offers new opportunities for a detailed understanding of genetic diversity distribution across the animal genome. Over the last 50 years, cattle breeds have been subjected to intense artificial selection. Consequently, regions controlling traits of economic importance are expected to exhibit selection signatures. The fixation index (F_st) is an estimate of population differentiation, based on genetic polymorphism data, and it is calculated using the relationship between inbreeding and heterozygosity. In the present study, locally weighted scatterplot smoothing (LOWESS) regression and a control chart approach were used to investigate selection signatures in two cattle breeds with different production aptitudes (dairy and beef). F_st was calculated for 42 514 SNP marker loci distributed across the genome in 749 Italian Brown and 364 Piedmontese bulls. The statistical significance of F_st values was assessed using a control chart. The LOWESS technique was efficient in removing noise from the raw data and was able to highlight selection signatures in chromosomes known to harbour genes affecting dairy and beef traits. Examples include the peaks detected for BTA2 in the region where the myostatin gene is located and for BTA6 in the region harbouring the ABCG2 locus. Moreover, several loci not previously reported in cattle studies were detected.     

Vicariance patterns in the Mediterranean Sea: east–west cleavage and low dispersal in the endemic seagrass Posidonia oceanica

Aim The seagrass, Posidonia oceanica is a clonal angiosperm endemic to the Mediterranean Sea. Previous studies have suggested that clonal growth is far greater than sexual recruitment and thus leads to low clonal diversity within meadows. However, recently developed microsatellite markers indicate that there are many different genotypes, and therefore many distinct clones present. The low resolution of markers used in the past limited our ability to estimate clonality and assess the individual level. New high‐resolution dinucleotide microsatellites now allow genetically distinct individuals to be identified, enabling more reliable estimation of population genetic parameters across the Mediterranean Basin. We investigated the biogeography and dispersal of P. oceanica at various spatial scales in order to assess the influence of different evolutionary factors shaping the distribution of genetic diversity in this species. Location The Mediterranean. Methods We used seven hypervariable microsatellite markers, in addition to the five previously existing markers, to describe the spatial distribution of genetic variability in 34 meadows spread throughout the Mediterranean, on the basis of an average of 35.6 (± 6.3) ramets sampled. Results At the scale of the Mediterranean Sea as a whole, a strong east–west cleavage was detected (amova) . These results are in line with those obtained using previous markers. The new results showed the presence of a putative secondary contact zone at the Siculo‐Tunisian Strait, which exhibited high allelic richness and shared alleles absent from the eastern and western basins. F statistics (pairwise θ ranges between 0.09 and 0.71) revealed high genetic structure between meadows, both at a small scale (about 2 to 200 km) and at a medium scale within the eastern and western basins, independent of geographical distance. At the intrameadow scale, significant spatial autocorrelation in six out of 15 locations revealed that dispersal can be restricted to the scale of a few metres. Main conclusions A stochastic pattern of effective migration due to low population size, turnover and seed survival is the most likely explanation for this pattern of highly restricted gene flow, despite the importance of an a priori seed dispersal potential. The east–west cleavage probably represents the outline of vicariance caused by the last Pleistocene ice age and maintained to this day by low gene flow. These results emphasize the diversity of evolutionary processes shaping the genetic structure at different spatial scales.     

Carboxylmethylation of Calmodulin in Cultured Pituitary Cells

We have used fast protein liquid chromatography (FPLC) and reverse-phase HPLC to rapidly resolve carboxylmethylated proteins in cultured pituitary GH3 cells. This procedure preserves labile carboxylmethyl esters, which are lost under the usual procedures employed for protein fractionation. GH3 cells were incubated with [methyl-3H]-methionine in culture and incorporation of label into the soluble fraction, total cell protein, and protein carboxylmethyl esters was determined; protein carboxylmethyl ester formation was shown to be resistant to cycloheximide. Fractionation of protein carboxylmethyl esters from GH3 cells by gel permeation FPLC, anion-exchange FPLC, and reverse-phase HPLC in the presence of calcium and in the presence of EGTA identified two proteins that are major substrates for protein carboxylmethyltransferase and indicated that one of these proteins is calmodulin. Similar results were obtained when a cytosolic fraction from GH3 cells was incubated with S-adenosyl-L-[methyl-3H]methionine. These results indicate that rapid chromatography at low temperature and low pH is useful for the analysis of eucaryotic carboxylmethylated proteins and that contrary to reports obtained in other systems, calmodulin is carboxylmethylated in intact pituitary cells.