Localization of ribosomal genes and nucleolar activity in lymphocytes of swine (Sus scrofa domestica) stimulated by phytohemagglutinins

The Pig chromosomes that contain rDNA sites displayed a polymorphism in the distribution of the genes among the nucleolar organizers located on pairs Nos. 8 and 10. Two, or more often three, active sites were observed in the chromosomes of lymphocytes stimulated with phytohemagglutinin. Only 5% of the metaphases showed a 4th small active site. At the onset of stimulation most cells contained one-two nucleoli; four nucleoli were never observed. After prolonged stimulation, the number of nuclei containing three nucleoli increased. A 4th small nucleolus appeared in a few cells, presumably formed by activation of the smallest rDNA site.     

Morpho-colorimetric characterization by image analysis to identify diaspores of wild plant species

Digital images of ex situ germplasm stored in the Sardinian Germplasm Bank (BG-SAR) were used for the application of image analysis techniques at the Stazione Sperimentale di Granicoltura per la Sicilia. The analysed accessions refer to 148 taxonomic units belonging to 102 genera and 47 families, typical of the Sardinian flora, and of the Mediterranean basin in general.The images of diaspores were acquired by a flatbed scanner and elaborated with a macro specially developed for the morphometric and colorimetric measurements. This method allowed carrying out a database for the characterization of autochthonous germplasm in entry to the bank and the realization of statistic classifiers for the discrimination of genera and species within the following families: Apiaceae, Boraginaceae, Caryophyllaceae, Cistaceae, Fabaceae and Scrophulariaceae. Such classifiers, based on the linear discriminant analysis (LDA) technique and checked by cross-validation, showed a performance included between 74.3% and 96.4%.In addition, for the genus Astragalus, it was possible to elaborate a classifier able to identify very similar taxa of a species complex, obtaining a performance between 83.7% and 100%. Such analysis proved the validity of the methodology also from the taxonomic point of view.Suggestions for subsequent methodological progress, which could offer applications in other research issues, such as ecological analysis, soil seed bank and archaeological botany are proposed.     

JAR human placental choriocarcinoma cells actively synthesize,take up and release polyamines

Uptake of polyamines has been investigated extensively in many cells, but not in placenta, where the polyamine– polyamine oxidase system is supposed to have an immunoregulatory function in pregnancy. Due to the importance of the transfer in this tissue, we have started this study. JAR human placental choriocarcinoma cells in monolayer at confluency were used as a model for measuring the key enzymes of polyamine synthesis and interconversion, rate of uptake and efflux, and the polyamine content. Polyamines were taken up by JAR cells and released by an independent mechanism. Ornithine decarboxylase and spermidine acetyltransferase activities and the rate of transport in and out of the cell were much higher than in other cells, such as L1210 cells. However the systems used for uptake and release appear in many respects to be similar to those observed in L1210 cells, but different from others. The uptake appears to be regulated by an inhibitory protein. Moreover, protein kinase C appears to be involved in the process. The efflux also is regulated as in L1210 cells, through control of H⁺ and Ca²⁺ concentration. In conclusion, this study shows that, in JAR cells, ornithine decarboxylase and spermidine acetyltransferase activities were much higher than in other cells, and so was the rate of transport in and out of the cells. As a result, a much higher polyamine content was observed.     

Selenium and polyamine metabolism: different effect of selenite on liver and bursa of Fabricius ornithine decarboxylase activity

1. When injected i.p., sodium selenite promoted a marked increase of rat liver ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) activities; when administered with the diet for 6 weeks, a less marked increase in liver ODC was observed, whereas SAMDC was not significantly changed. 2. Protein synthesis was involved in the observed modifications. The rate of ODC inactivation was also changed. 3. ODC increase was accompanied by an enhanced putrescine concentration in liver. 4. A marked increase of ODC, accompanied by an enhancement of putrescine, was promoted by selenite (i.p.) also in chicken liver, together with an enhancement of glutathione concentration. Spermidine acetyltransferase (SAT) was also increased. 5. In the bursa of Fabricius, SAT activity was also increased, whereas ODC was decreased. However the expected modifications in polyamine concentration were not observed. 6. Decrease of ODC activity in the bursa was not due to an antizyme. 7. In vitro, selenite concentrations known to inhibit cell proliferation (greater than 1 microgram/ml) inhibited both ODC and SAT activities; at lower concentration, SAT activity was enhanced.     

DNA damage in stomach, kidney, liver and lung of rats treated with atrazine

The genotoxic activity of atrazine, a widely used triazine herbicide, was assayed by the DNA alkaline elution technique in rats given orally a single high dose or repeated daily doses. DNA breaks (and/or alkali-labile lesions) were detected in cell suspensions obtained from stomach, kidney and liver, but not in those from lung.     

Metabolic activation of unsaturated derivatives of valproic acid. Identification of novel glutathione adducts formed through coenzyme A-dependent and -independent processes

The ability of 2-n-propyl-4-pentenoic acid (Δ⁴-VPA) and 2-n-propyl-2(E)-pentenoic acid ([E]-Δ²-VPA), two unsaturated metabolites of valproic acid (VPA), to form reactive intermediates, deplete hepatic glutathione (GSH) and cause accumulation of liver triglycerides was investigated in the rat. With the aid of ionspray liquid chromatography-tandem mass spectrometry (LC-MS/MS), three GSH adducts were detected in the bile of Δ⁴-VPA-treated animals and were identified as 4-hydroxy-5-glutathion-S-yl-VPA-γ-lactone, 5-glutathion-S-yl-(E)-Δ³-VPA and 3-oxo-5-glutathion-S-yl-VPA. A fourth conjugate was identified tentatively as 4-glutathion-S-yl-5-hydroxy-VPA. Quantitative analysis of the corresponding N-acetylcysteine (NAC) conjugates in urine indicated that metabolism of Δ⁴-VPA via the GSH-dependent pathways accounted for approximately 20% of an acute dose (100 mg kg⁻¹ i.p.). In contrast, when rats were given an equivalent dose of (E)-Δ²-VPA, only one GSH adduct (5-glutathion-S-yl-(E)-Δ³-VPA) was detected at low concentrations in bile. In vitro experiments with rat liver mitochondria demonstrated that Δ⁴-VPA undergoes coenzyme A- and ATP-dependent metabolic activation in this organelle via the β-oxidation pathway to intermediates which bind covalently to proteins. When liver homogenates and hepatic mitochondria from rats injected with Δ⁴-VPA, (E)-Δ²-VPA or VPA were analyzed for GSH content, it was found that only Δ⁴-VPA depleted GSH pools significantly. Treatment of rats with Δ⁴-VPA and (to a lesser extent) VPA led to an accumulation of liver triglycerides, whereas (E)-Δ²-VPA had no measurable effect. It is concluded that Δ⁴-VPA undergoes metabolic activation by both microsomal cytochrome P-450-dependent and mitochondrial coenzyme A-dependent processes, and that the resulting electrophilic intermediates, which are trapped in part by GSH, may mediate the hepatotoxic effects of this compound. In contrast, (E)-Δ²-VPA is not transformed to any appreciable extent to reactive metabolites, which thus accounts for the apparent lack of hepatotoxicity of this positional isomer in the rat.     

Comparative Analysis of Short- and Long-Term Changes in Gene Expression Caused by Low Water Potential in Potato (Solanum tuberosum) Cell-Suspension Cultures

To dissect the cellular response to water stress and compare changes induced as a generalized response with those involved in tolerance/acclimation mechanisms, we analyzed changes in two-dimensional electrophoretic patterns of in vivo [35S]methionine-labeled polypeptides of cultured potato (Solanum tuberosum) cells after gradual and long exposure to polyethylene glycol (PEG)- mediated low water potential versus those induced in cells abruptly exposed to the same stress intensity. Protein synthesis was not inhibited by gradual stress imposition, and the expression of 17 proteins was induced in adapted cells. Some polypeptides were inducible under mild stress conditions (5% PEG) and accumulated further when cells were exposed to a higher stress intensity (10 and 20% PEG). The synthesis of another set of polypeptides was up-regulated only when more severe water-stress conditions were applied, suggesting that plant cells were able to monitor different levels of stress intensity and modulate gene expression accordingly. In contrast, in potato cells abruptly exposed to 20% PEG, protein synthesis was strongly inhibited. Nevertheless, a large set of polypeptides was identified whose expression was increased. Most of these polypeptides were not induced in adapted cells, but many of them were common to those observed in abscisic acid (ABA)-treated cells. These data, along with the finding that cellular ABA content increased in PEG-shocked cells but not in PEG-adapted cells, suggested that this hormone is mainly involved in the rapid response to stress rather than long-term adaptation. A further group of proteins included those induced after long exposure to both water stress and shock. Western blot analysis revealed that osmotin was one protein belonging to this common group. This class may represent induced proteins that accumulate specifically in response to low water potential and that are putatively involved in the maintenance of cellular homeostasis under prolonged stress.     

A major difference between the divergence patterns within the lines-1 families in mice and voles

L1 retroposons are represented in mice by subfamilies of interspersed sequences of varied abundance. Previous analyses have indicated that subfamilies are generated by duplicative transposition of a small number of members of the L1 family, the progeny of which then become a major component of the murine L1 population, and are not due to any active processes generating homology within preexisting groups of elements in a particular species. In mice, more than a third of the L1 elements belong to a clade that became active approximately 5 Mya and whose elements are > or = 95% identical. We have collected sequence information from 13 L1 elements isolated from two species of voles (Rodentia: Microtinae: Microtus and Arvicola) and have found that divergence within the vole L1 population is quite different from that in mice, in that there is no abundant subfamily of homologous elements. Individual L1 elements from voles are very divergent from one another and belong to a clade that began a period of elevated duplicative transposition approximately 13 Mya. Sequence analyses of portions of these divergent L1 elements (approximately 250 bp each) gave no evidence for concerted evolution having acted on the vole L1 elements since the split of the two vole lineages approximately 3.5 Mya; that is, the observed interspecific divergence (6.7%-24.7%) is not larger than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses showed no clustering into Arvicola and Microtus clades.