Evolution of several cytoskeletal proteins of astrocytes in primary culture: Effect of prenatal alcohol exposure

In the present work we have analyzed, using immunoblotting and immunofluorescence techniques, the evolution of several cytoskeletal proteins during the development of astrocytes in primary culture. The effect of prenatal exposure to alcohol on these proteins was also evaluated. Microtubular protein -tubulin decreased approximately 47% from 4 to 7 days after which its content remained practically constant. Immunofluorescence studies showed also that the content of -tubulin was greater at day 4 of culture. This increase in fluorescence was coincident with the presence of globular particles which were found in interphase astrocytes and stained with both anti - and anti--tubulin. These structures appeared only in proliferating cells. Glial fibrillary acidic protein (GFAP) and vimentin were analyzed as intermediate filament (IF) proteins. GFAP, in cytoskeletal preparations, increased regularly for 14 days followed by a decrease to day 21. In contrast, vimentin showed a progressive increase throughout the entire culture period. Fluorescence studies revealed some differences between the IF distribution patterns of GFAP and vimentin.In astrocytes obtained from rats prenatally exposed to ethanol, decreases in the amounts of all the cytoskeletal proteins studied were found during the entire culture period. In these cells a striking disorganization of cytoskeleton was also observed. The alcohol-induced decrease of GFAP in cultured astrocytes was also found when this protein was studied in preparations from whole brain developed in vivo.Special issue dedicated to Dr. Santiago Grisolia     

Persistence of drug-induced chromosome aberrations in peripheral blood lymphocytes of the rat

We have studied the persistence of pre-clastogenic lesions, detected as induced chromosomal aberrations, in rat peripheral lymphocytes at various time intervals after acute treatment with 3 different antineoplastic drugs: cyclophosphamide (CPA), 5-fluorouracil (5-FU) and adriamycin (AM). Single i.p. doses were administered to groups of rats and heart blood samples from each group were taken after 3, 12, 24 or 48 h or weekly up to 20 weeks later. The cytogenetic analysis was performed on lymphocytes cultured for 33 h after sampling. The results for CPA exposure (10 mg/kg) show that the yield of chromosome aberrations is maximal 3 h after the treatment (20 times the control level). For up to 8 weeks the values remain about 6 times the baseline; afterwards a decrease is observed and the control level is reached after 20 weeks. For 5-FU (50 mg/kg) a remarkable increase (13-fold) in chromosomal damage is observed at the first sampling time. Within 48 h the effect is drastically reduced but persistent (3 times the control level), and the level returns to spontaneous values 1 week later. AM treatment (2 mg/kg) induced an increase of about 8 times the control level at 3 h post exposure. The clastogenic effects remained at a detectable level for 1 week (about 6 times the control level at all sampling times); 2 weeks after the treatment the control level was found. A parallel analysis was performed on bone marrow cells. In this tissue the clastogenic effects of the treatments were maximal, as in lymphocytes, at the first sampling time (20-25 times the control level) and were no longer detectable within 72 h after exposure, irrespective of the administered drug.     

Airborne grass pollen in Granada (Spain)

We have carried out a study on the annual and daily pollen concentrations from Gramineae over four consecutive years in the atmosphere of Granada (Spain). Samples of pollen grains were collected by the volumetric method with the aid of a Burkard sporetrap. Gramineae, according both to their high sensitizing capacity and to data from allergologists, are responsible for many pollinoses diagnosed in this area. In this work, daily pollen levels from April to July are monitored and the variations identified are interpreted in relation to meteorological conditions. Results indicated that the highest airborne concentrations of Gramineae pollen were found in May and June, although the beginning and intensity of pollination have been variable during these 4 years.     

Grain size,sucrose level and starch accumulation in developing rice grain

Five rices (Oryza sativa L.) differing in final grain size were studied at the midmilky stage to determine if any factor could be identified which might limit rate of starch accumulation. Only UDP glucose pyrophosphorylase activity increased with increasing grain size. Detached rice panicles incubated in liquid medium containing 1% sucrose and 0.1% glutamine, in addition to minerals and vitamins, produced grains similar to those on intact plants. Sucrose level (0–1.5%) in the medium determined the extent of dry matter and starch accumulation and influenced physiological development of the ripening grains. Chemical and enzymic composition of the grain were similar to previously reported levels in grains of intact panicles analysed at regular intervals after anthesis. Addition of 3-P glycerate or K⁺ to the medium did not improve dry matter accumulation in the developing grain.     

Fibronectin gene expression in proliferating, quiescent, and SV40-infected mouse kidney cells

To study alterations in cellular gene expression in mouse kidney cell cultures infected with simian virus 40 (SV40) or polyomavirus, we performed a differential screening of a mouse kidney cDNA library with probes prepared from mRNAs of virus-infected and mock-infected cells. We isolated and characterized cDNA recombinant pKT13 which detected increased mRNA levels in infected cells. Sequence analysis of pKT13 revealed close to 100% homology with the 3'-end of mouse fibronectin (FN) mRNA. Since primary cultures of baby mouse kidney cells have been extensively characterized in our laboratories, we studied FN gene expression at different stages of uninfected and virus-infected cultures. High levels of FN and of its mRNA were found in the kidneys of suckling mice, while in primary cultures of proliferating epithelial kidney cells the expression of FN was very low until the cultures became confluent. Thereafter FN increased and reached high levels in cells which were irreversibly arrested in phase Go and which had apparently exhausted their finite division potential. Infection of confluent cultures with polyomavirus or SV40 resulted in a further stimulation of FN gene expression. However, during abortive infection with SV40, FN mRNA and FN levels decreased with emergence of transformed cells and were low in an established SV40-transformed mouse kidney cell line. These changes in FN gene expression suggest that high levels of FN might be indicative in vivo for terminal differentiation and in vitro for cellular senescence.     

Vanadate,alcohol or both increase passive membrane permeability of neuro-2a cells; Lesser sensitivity of Hep-2 cells

Neuro-2a cells incubated for 1 hour with 0.1 mM vanadate showed an increase in cell membrane permeability. This effect is dose dependent, e.g. with 0.01 mM, 0.1 mM and 1 mM vanadate, there was {20, 30 and 40% increase. In contrast, no alteration in permeability was observed in HEp-2 cells under the same conditions.Ethanol (3%, 1 h incubation) also enhanced membrane permeability. The increase was also greater with Neuro-2a cells ({80%) than with HEp-2 cells (～30%). When the cells were incubated with ethanol plus vanadate (0.1 mM), there was a marked potentiation ({200%) in cell membrane permeability in Neuro-2a cells, and again a lesser increase in permeability ({50%) with HEp-2 cells.These results seem to be due to a preferential effect of vanadate on passive permeability of Neuro-2a cells because parallel measurements demonstrate equal inhibition of (Na⁺K) ATPase with both Neuro-2a and HEp-2 cells.     

Reduction and cyclization in biotransformation of carbonyl compounds by cultured cells of Taxus species

Cultured plant cells from Taxus brevifolia Nutt and Taxus globosa Schltdl were investigated as biocatalysts using exogenous substrates. Production of highly specific metabolites by these species prompted us to analyse their synthetic potential. Whole cells suspensions have the capacity to chemoselectively reduce ethyl acetoacetate to ethyl 3-hydroxybutyrate chemo- and stereoselectively reduce rac-2-benzoylcyclohexanone to (1R, 2S)- and (1S, 2S)-2-hydroxycyclohexylphenylmethanones, and to cyclize N-phthaloyl-L-glutamine to thalidomide.     

Application of CO2 carbon stable isotope analysis to ant trophic ecology

Stable isotope analysis of animal tissues is commonly used to infer diet and trophic position. However, it requires destructive sampling. The analysis of carbon isotopes from exhaled CO₂ is non-invasive and can provide useful ecological information because isotopic CO₂ signatures can reflect the diet and metabolism of an animal. However, this methodology has rarely been used on invertebrates and never on social insects. Here, we first tested whether this method reflects differences in δ¹³C-CO₂ between workers of the Mediterranean ant Crematogaster scutellaris (Olivier) (Hymenoptera: Formicidae, Crematogastrini) fed with sugar from beet (C3; Beta vulgaris L., Amaranthaceae) or cane (C4; Saccharum officinarum L., Poaceae). We found that a significant difference can be obtained after 24 h. Consequently, we used this technique on wild co-occurring ant species with different feeding preferences to assess their reliance on C3 or C4 sources. For this purpose, we sampled workers of C. scutellaris, the invasive garden ant Lasius neglectus (van Loon et al.) (Lasiini), and the harvester ant Messor capitatus (Latreille) (Stenammini). No significant differences in their carbon isotopic signatures were recorded, suggesting that in our study site no niche partitioning occurs based on the carbon pathway, with all species sharing similar resources. However, further analysis revealed that M. capitatus, a seed-eating ant, can be regarded as a C3 specialist, whereas L. neglectus and C. scutellaris are generalists that rely on both C3 and C4 pathways, though with a preference for the former. Our results show that this methodology can be applied even to small animals such as ants and can provide useful information on the diets of generalist omnivores.