Minocycline promotes remyelination in aggregating rat brain cell cultures after interferon-γ plus lipopolysaccharide-induced demyelination.

Minocycline has been shown to inhibit microglia reactivity, and to decrease the severity and progression of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. It remained to be examined whether minocycline was also able to promote remyelination. In the present study, myelinating aggregating brain cell cultures were used as a model to study the effects of minocycline on microglial reactivity, demyelination, and remyelination. Cultures were treated simultaneously with two inflammatory agents, interferon-γ (IFN-γ) and lipopolysaccharide (LPS), which caused an inflammatory response accompanied by demyelination. The inflammatory response was characterized by microglial reactivity, upregulation of inflammatory cytokines and iNOS, and increased phophorylation of P38 and P44/42 mitogen activated protein (MAP) kinases. Minocycline inhibited microglial reactivity, and attenuated the increased phophorylation of P38 and P44/42 MAP kinases. Demyelination, determined by a decrease in myelin basic protein (MBP) content and immunoreactivity 48 h after the treatment with the inflammatory agents, was not prevented by minocycline. However, 1 week after demyelination was assessed, the MBP content was restored in presence of minocycline, indicating that remyelination was promoted. Concomitantly, in cultures treated with minocycline, the markers of oligodendrocyte precursors cells (OPCs) and immature oligodendrocytes NG2 and O4, respectively, were decreased compared to cultures treated with the inflammatory agents only. These results suggest that minocycline attenuates microglial reactivity and favors remyelination by enhancing the differentiation of OPCs and immature oligodendrocytes.

Protein kinases expressed in aggregating brain cell cultures during myelination.

Serum-free aggregating rat brain cell cultures provide sufficient cell surface and paracrine interactions between neurons and glial cells for compact myelination. We are interested in the part played in these signalling pathways by protein kinases and have used a PCR cDNA cloning approach to catalogue the protein kinase genes expressed by these cultures. 8 transmembrane protein kinases were identified: IGF1-R, trk B, bFGF-R, c-met, Tyro2, Tyro1, Tyro4 and a novel eck-related gene. The first 4 are receptors for ligands with known trophic functions. Tyro2 is a novel gene related to the EGF-R. The latter 3 belong to the eck gene family of more than 8 highly related putative receptors for, as yet, unknown ligands. 8 cDNAs for intracellular protein kinases were also isolated including 3 novel genes. Ongoing studies are investigating whether these proteins contribute to myelination and/or could be used as therapeutic targets in demyelinating diseases.

Evaluation of the toxicity of different metal compounds in the developing brain using aggregating cell cultures as a model.

To evaluate their toxicity in the developing brain, eight metal compounds, [bismuth sodium tartrate (BiNA-tartrate), CdCl(2), CoCl(2), HgCl(2), dimethyl mercury, NiCl(2), TlCl and triethyltin chloride (TET)] were tested in aggregating cell cultures of foetal rat telencephalon. The compounds were applied to the cultures continuously, either during an early developmental stage (between days 5 and 14) or during and advanced stage of maturation (between days 24 and 34). Changes in the activities of cell type-specific enzymes were used as a criterion for toxicity. A general cytotoxic effect was observed after treatment with either CdCl(2), HgCl(2) or TET at 10(-6)m, and with TlCl at 10(-5)m. Selective effects were found with BiNa-tartrate and dimethylmercury. CoCl(2) did not modify the parameters tested, whereas a stimulant effect was found with NiCl(2). The effects of several compounds were development dependent: HgCl(2), TET and TlCl were more toxic in immature cultures, whereas BiNa-tartrate, dimethylmercury and NiCl(2) were more effective in differentiated cultures.

Neurotoxicity of dibutyltin in aggregating brain cell cultures.

Dibutyltin (DBT) compounds are used primarily as stabilizers for polyvinyl chloride (PVC) plastics. Small quantities can be released from PVC containers into stored liquids. The neurotoxicological potential of DBT was tested in aggregating brain cell cultures after a 10-day treatment with concentrations ranging from 10(-10) to 10(-6)m, either during an early developmental period, or during a phase of advanced maturation. Changes in protein content, DNA labelling and cell type-specific enzyme activities were measured as end points. DBT caused general cytotoxicity at 10(-6)m in both immature and differentiated cultures. At 10(-7)m, it affected the myelin content and the cholinergic neurons in both states of maturation, while GABAergic neurons remained unchanged. Astrocyte and oligodendrocyte markers were diminished at 10(-7)m of DBT exclusively in immature cultures. DBT uptake by undifferentiated and differentiated cells was similar at this concentration. Whereas trimethyltin (TMT) is known to induce gliosis and triethyltin (TET) to cause demyelination and affect GABAergic neurons, DBT appeared to be more toxic than TMT, and to present a distinct toxicological pattern.

Els joves que viuen entre cultures: un nou repte educatiu

Aquest article és una aproximació a l'estudi de la mal anomenada segona generació d'immigrants per a poder comprendre com viuen aquests joves entre cultures. ¿Quins sentiments i conductes genera aquesta situació? ¿Amb qui i quan se veu en la necessitat d'identificar-se amb un o altre grup? ¿Com es situa amb respecte a la cultura majoritària i al grup familiar d'origen? ¿Quins són els seus grups de participació i pertenença? ¿Quines respostes reben del seu entorn? ¿Aquesta situació és vivida com un avantatge o com un desavantatge?. En definitiva, es tracta d'abordar i analitzar comprensivament les avantatges i les dificultats d'aquesta generació que viu "entre cultures" i s'ofereix un model explicatiu denominat "Model de pertenença i competències culturals". També s'exposen algunes recomanacions educatives amb l'objectiu d'optimitzar els processos educatius i que les avantatges de les seves competències pluriculturals puguin ser aplocables a tot l'alumnat e incorporat al currículum del futur.

Carbonic anhydrase activity in primary sensory neurons. II. Influence of environmental factors on the phenotypic expression of the enzyme in dissociated cultures of chicken dorsal root ganglion cells.

Neuronal subpopulations of dorsal root ganglion (DRG) cells in the chicken exhibit carbonic anhydrase (CA) activity. To determine whether CA activity is expressed by DRG cells maintained in in vitro cultures, dissociated DRG cells from 10-day-old chick embryos were cultured on a collagen substrate. The influence exerted by environmental factors on the enzyme expression was tested under various conditions of culture. Neuron-enriched cell cultures and mixed DRG-cell cultures (including numerous non-neuronal cells) were performed either in a defined medium or in a horse serum-supplemented medium. In all the tested conditions, subpopulations of cultured sensory neurons expressed CA activity in their cell bodies, while their neurites were rarely stained; in each case, the percentage of CA-positive neurons declined with the age of the cultures. The number and the persistence of neurons possessing CA activity as well as the intensity of the reaction were enhanced by addition of horse serum. In contrast, the expression of the neuronal CA activity was not affected by the presence of non-neuronal cells or by the rise of CO2 concentration. Thus, the appearance and disappearance of neuronal subpopulations expressing CA activity may be decisively influenced by factors contained in the horse serum. The loss of CA-positive neurons with time could result from a cell selection or from genetic repression. Analysis of the time curves does not support a preferential cell death of CA-positive neurons but suggests that the eventual conversion of CA-positive neurons into CA-negative neurons results from a loss of the enzyme activity. These results indicate that the phenotypic expression of cultured sensory neurons is dependent on defined environmental factors.

Activity-dependent phosphorylation of SNAP-25 in hippocampal organotypic cultures.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is thought to play a key role in vesicle exocytosis and in the control of transmitter release. However, the precise mechanisms of action as well as the regulation of SNAP-25 remain unclear. Here we show by immunoprecipitation that activation of protein kinase C (PKC) by phorbol esters results in an increase in SNAP-25 phosphorylation. In addition, immunochemical analysis of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels shows that SNAP-25 focuses as three or four distinct spots in the expected range of molecular weight and isoelectric point. Changing the phosphorylation level of the protein by incubating the slices in the presence of either a PKC agonist (phorbol 12,13-dibutyrate) or antagonist (chelerythrine) modified the distribution of SNAP-25 among these spots. Phorbol 12,13-dibutyrate increased the intensity of the spots with higher molecular weight and lower isoelectric point, whereas chelerythrine produced the opposite effect. This effect was specific for regulators of PKC, as agonists of other kinases did not produce similar changes. Induction of long-term potentiation, a property involved in learning mechanisms, and production of seizures with a GABA(A) receptor antagonist also increased the intensity of the spots with higher molecular weight and lower isoelectric point. This effect was prevented by the PKC inhibitor chelerythrine. We conclude that SNAP-25 can be phosphorylated in situ by PKC in an activity-dependent manner.

KulTIC 08. Paradoxes del contacte entre cultures: l'ús pedagògic de la webquest

A Catalunya, la presència de persones immigrades d'arribada recent ha significat un canvi substancial en les percepcions individuals i les representacions socials sobre la diversitat cultural. Aquest canvi ha resultat més destacat entre la població jove, en fase de construcció de la seva identitat, que ha hagut d'abordar les paradoxes del contacte entre cultures a partir d'estratègies de relació poc estructurades i planificades amb aquesta finalitat. La recerca KulTic¿08 ha explorat l'impacte de determinades paradoxes que es produeixen en el contacte amb altres realitats diferents a la pròpia i ha aplicat experimentalment un material en webquest, amb un enfocament socioafectiu i cognitiu i seguint els plantejaments pedagògics de l'aprenentatge cooperatiu.

KulTIC 08. Paradoxes del contacte entre cultures: l'ús pedagògic de la webquest

A Catalunya, la presència de persones immigrades d'arribada recent ha significat un canvi substancial en les percepcions individuals i les representacions socials sobre la diversitat cultural. Aquest canvi ha resultat més destacat entre la població jove, en fase de construcció de la seva identitat, que ha hagut d'abordar les paradoxes del contacte entre cultures a partir d'estratègies de relació poc estructurades i planificades amb aquesta finalitat. La recerca KulTic¿08 ha explorat l'impacte de determinades paradoxes que es produeixen en el contacte amb altres realitats diferents a la pròpia i ha aplicat experimentalment un material en webquest, amb un enfocament socioafectiu i cognitiu i seguint els plantejaments pedagògics de l'aprenentatge cooperatiu.

Sucrose metabolizing enzymes in cell suspension cultures of Bauhinia forficata, Curcuma zedoaria and Phaseolus vulgaris

The objective of this work was to study the activity of sucrose metabolizing enzymes in extracts of cell suspension cultures of Bauhinia forficata Link, Curcuma zedoaria Roscoe and Phaseolus vulgaris L. Invertase pathway was identified in the three studied species. Sucrose synthase pathway was also responsible for sucrose metabolism in Curcuma zedoaria and Phaseolus vulgaris cells. Activity values higher than 300 nmol min-1 mg-1 of protein were found for acid and neutral invertases, UDPglucose pyrophosphorylase and phosphoglucomutase in the cell extract of the three plant species. Sucrose synthase showed low activity in Bauhinia forficata cells. As sucrose concentration in the culture medium decreased, sucrose synthase activity increased in C. zedoaria and P. vulgaris cells. The glycolytic enzymes activity gradually reduced at the end of the culture period, when carbohydrate was limited.

Comparison of the developmental effects of two mercury compounds on glial cells and neurons in aggregate cultures of rat telencephalon.

A three-dimensional cell culture system was used as a model to study the influence of low levels of mercury in the developing brain. Aggregating cell cultures of fetal rat telencephalon were treated for 10 days either during an early developmental period (i.e., between days 5 and 15 in vitro) or during a phase of advanced maturation (i.e., between days 25 and 35) with mercury. An inorganic (HgCl2) and an organic mercury compound (monomethylmercury chloride, MeHgCl) were examined. By monitoring changes in cell type-specific enzymes activities, the concentration-dependent toxicity of the compounds was determined. In immature cultures, a general cytotoxicity was observed at 10(-6) M for both mercury compounds. In these cultures, HgCl2 appeared somewhat more toxic than MeHgCl. However, no appreciable demethylation of MeHgCl could be detected, indicating similar toxic potencies for both mercury compounds. In highly differentiated cultures, by contrast, MeHgCl exhibited a higher toxic potency than HgCl2. In addition, at 10(-6) M, MeHgCl showed pronounced neuron-specific toxicity. Below the cytotoxic concentrations, distinct glia-specific reactions could be observed with both mercury compounds. An increase in the immunoreactivity for glial fibrillary acidic protein, typical for gliosis, could be observed at concentrations between 10(-9) M and 10(-7) M in immature cultures, and between 10(-8) M and 3 x 10(-5) M in highly differentiated cultures. A conspicuous increase in the number and clustering of GSI-B4 lectin-binding cells, indicating a microglial response, was found at concentrations between 10(-10) M and 10(-7) M. These development-dependent and cell type-specific effects may reflect the pathogenic potential of long-term exposure to subclinical doses of mercury.