Administration of PLP139-151 primes T cells distinct from those spontaneously responsive in vitro to this antigen

We examined the TCR repertoire used by naive SJL mice in their in vitro spontaneous response to proteolipid protein (PLP) 139-151 by Vbeta-Jbeta spectratyping and compared it to that used after immunization with the peptide. T cells from immunized mice use the public rearrangement Vbeta10-Jbeta1.1, but naive mice do not; in contrast, TCR CDR3-beta rearrangements of Vbeta18-Jbeta1.2 and Vbeta19-Jbeta1.2 consistently are associated with the spontaneous response. T cells involved in spontaneous and induced responses can each recognize PLP(139-151) presented in vivo, but its s.c. administration has different consequences for the two repertoires. Four days after immunization, T cells associated with spontaneous responsiveness appear in the draining lymph nodes but disappear by day 10 and never appear elsewhere. Simultaneously, Vbeta10-Jbeta1.1 T cells are likewise activated in the lymph nodes by day 4 and spread to the spleen by day 10. Eight- to 10-wk-old naive mice use a narrower repertoire of TCRs than do immunized age-matched mice. Induced Vbeta10-Jbeta1.1 T cells home to the CNS during experimental autoimmune encephalomyelitis, whereas we failed to detect Vbeta18-Jbeta1.2 and Vbeta19-Jbeta1.2 TCR rearrangements in the CNS. Thus, we observe that administration of PLP(139-151) primes a T cell repertoire distinct from the one responsible for spontaneous responsiveness. This "immunized" repertoire substitutes for the naive one and becomes dominant at the time of disease onset.     

Suppression of autophagy precipitates neuronal cell death following low doses of methamphetamine

Methamphetamine abuse is toxic to dopaminergic neurons, causing nigrostriatal denervation and striatal dopamine loss. Following methamphetamine exposure, the number of nigral cell bodies is generally preserved, but their cytoplasm features autophagic-like vacuolization and cytoplasmic accumulation of α-synuclein-, ubiquitin- and parkin-positive inclusion-like bodies. Whether autophagy is epiphenomenal or it plays a role in the mechanism of methamphetamine toxicity and, in the latter case, whether its role consists of counteracting or promoting the neurotoxic effect remains obscure. We investigated the signaling pathway and the significance (protective vs . toxic) of autophagy activation and the convergence of the autophagic and the ubiquitin-proteasome pathways at the level of the same intracellular bodies in a simple cell model of methamphetamine toxicity. We show that autophagy is rapidly up-regulated in response to methamphetamine. Confocal fluorescence microscopy and immuno-electron microscopy studies demonstrated the presence of α-synuclein aggregates in autophagy-lysosomal structures in cells exposed to methamphetamine, a condition compatible with cell survival. Inhibition of autophagy either by pharmacologic or genetic manipulation of the class III Phosphatidylinositol-3 kinase-mediated signaling prevented the removal of α-synuclein aggregates and precipitated a bax-mediated mitochondrial apoptosis pathway.     

Activation of human alveolar macrophages via P2 receptors: coupling to intracellular Ca2+ increases and cytokine secretion

Alveolar macrophages play a crucial role in the pathogenesis of inflammatory airway diseases. By the generation and release of different inflammatory mediators they contribute to both recruitment of different leukocytes into the lung and to airway remodeling. A potent stimulus for the release of inflammatory cytokines is ATP, which mediates its cellular effects through the interaction with different membrane receptors, belonging to the P2X and P2Y families. The aim of this study was to characterize the biological properties of purinoceptors in human alveolar macrophages obtained from bronchoalveolar lavages in the context of inflammatory airway diseases. The present study is the first showing that human alveolar macrophages express mRNA for different P2 subtypes, namely P2X(1), P2X(4), P2X(5), P2X(7), P2Y(1), P2Y(2), P2Y(4), P2Y(6), P2Y(11), P2Y(13), and P2Y(14). We also showed that extracellular ATP induced Ca(2+) transients and increased IL-1beta secretion via P2X receptors. Furthermore, extracellular nucleotides inhibited production of IL-12p40 and TNF-alpha, whereas IL-6 secretion was up-regulated. In summary, our data further support the hypothesis that purinoceptors are involved in the pathogenesis of inflammatory lung diseases.     

Lack of genotoxic effects in hematopoietic and gastrointestinal cells of mice receiving chromium(VI) with the drinking water

Chromium(VI) is genotoxic when tested in vitro or injected parenterally in such a way to escape detoxification mechanisms. However, its genotoxicity and potential carcinogenicity are lost, depending on dose and administration route, due to efficient reduction in body fluids and nontarget cells. Chromium(VI) is a Group 1 IARC carcinogen, but only in the respiratory tract and in well-defined occupational settings that involved heavy exposures. Recently, concern has been expressed that oral chromium(VI) may be a gastric carcinogen. We demonstrated that administration of very high doses of chromium(VI) with the drinking water does not induce any clastogenic effect in hematopoietic cells of adult mice and their fetuses. Thereafter, we investigated whether administration of chromium(VI) with the drinking water may induce local genotoxic effects in the gastrointestinal tract. Sodium dichromate dihydrate was administered to mice for 9 consecutive months, at doses corresponding to 5 and 20 mg chromium(VI)/l, which exceed drinking water standards by 100 and 400 times, respectively. Under these conditions, chromium(VI) failed to enhance the frequency of DNA-protein crosslinks and did not cause oxidative DNA damage, measured in terms of 8-oxo-2'-deoxyguanosine, in the forestomach, glandular stomach and duodenum. When cells from the same organs were isolated and challenged in vitro with chromium(VI), as positive controls, the same genotoxicity biomarkers were evidently affected. Thus, consistently with the knowledge accumulated in 50 years of research on chromium(VI) kinetics and metabolism, oral chromium(VI) appears to be devoid of genotoxicity in the gastrointestinal tract. After 9 months, we did not observe any variation of tumor yield in skin, lung, forestomach, glandular stomach, and duodenum of chromium(VI)-treated mice. These results are discussed in the light of literature data, also including a recent 2-year carcinogenicity study performed by the National Toxicology Program.     

Tuber melanosporum outcrosses: analysis of the genetic diversity within and among its natural populations under this new scenario

Tuber melanosporum is an ectomycorrhizal ascomycete producing edible ascocarps. The prevalent view is that this species strictly selfs, since genetic analyses have never detected heterozygotic profiles in its putatively diploid/dikaryotic gleba. The selfing model has also forged the experimental approaches to assess the population genetic variability. Here, the hypothesis that T. melanosporum outcrosses was tested. To this end, SSR (simple sequence repeats) and ITS (internal transcribed spacer) markers were employed to fingerprint asci and the surrounding gleba within single ascocarps. The distribution of genetic variability was also investigated at different geographical levels using single (SSR and ITS) and multilocus (AFLP, amplified fragment length polymorphism) markers. It is shown that T. melanosporum outcrosses since asci display additional alleles besides those present in the surrounding, uniparental, gleba. Furthermore, SSR and AFLP data reveal a high rate of intrapopulation diversity within samples from the same ground and root apparatus and the highest rate of genetic variability within the southernmost populations of the distributional range. These data call for a profound re-examination of T. melanosporum mating system, life cycle and strategies for managing man-made plantations. They also strongly support the idea that the last glaciation restricted the species distribution to the Italian and Spanish peninsulas.     

Cadmium induces acidosis in maize root cells

* Cadmium (Cd) stress increases cell metabolic demand for sulfur, reducing equivalents, and carbon skeletons, to sustain phytochelatin biosynthesis for Cd detoxification. In this condition the induction of potentially acidifying anaplerotic metabolism in root tissues may be expected. For these reasons the effects of Cd accumulation on anaplerotic metabolism, glycolysis, and cell pH control mechanisms were investigated in maize (Zea mays) roots. * The study compared root apical segments, excised from plants grown for 24 h in a nutrient solution supplemented, or not, with 10 microM CdCl(2), using physiological, biochemical and (31)P-nuclear magnetic resonance (NMR) approaches. * Cadmium exposure resulted in a significant decrease in both cytosolic and vacuolar pH of root cells and in a concomitant increase in the carbon fluxes through anaplerotic metabolism leading to malate biosynthesis, as suggested by changes in dark CO2 fixation, metabolite levels and enzyme activities along glycolysis, and mitochondrial alternative respiration capacity. This scenario was accompanied by a decrease in the net H(+) efflux from the roots, probably related to changes in plasma membrane permeability. * It is concluded that anaplerotic metabolism triggered by Cd detoxification processes might lead to an imbalance in H(+) production and consumption, and then to cell acidosis.     

An 86Y-labeled mirror-image oligonucleotide: influence of Y-DOTA isomers on the biodistribution in rats

A mirror-image oligonucleotide (L-RNA) was radiolabeled with the positron emitting radionuclide (86)Y (t(1/2) = 14.7 h) via the bifunctional chelator approach. DOTA-modification of the L-RNA (sequence: 5'-aminohexyl UGA CUG ACU GAC-3'; MW 3975) was performed using (S)-p-SCN-Bn-DOTA. (86)Y radiolabeling of the DOTA-L-RNA produced more than one species as evidenced by HPLC radiometric detection. For the identification of the (86)Y-labeled L-RNA, the structural analogue nonradioactive precursor [Y((S)-p-NH2-Bn-DOTA)](-) was synthesized. Two coordination isomers were separated via HPLC adopting the square antiprismatic (SAP) and the twisted square antiprismatic (TSAP) geometry, respectively. Their stereochemical configuration in the solution state was assessed by NMR and circular dichroism spectroscopy. Both [Y((S)-p-NH2-Bn-DOTA)](-) isomers were converted into isothiocyanate derivatives [Y((S)-p-SCN-Bn-DOTA)](-) and conjugated to the L-RNA. The identity of the [(86)Y-DOTA]-L-RNA species was finally established by comparison of the radiometric ((86)Y) and UV-visible chromatographic profiles. Biodistribution studies in Wistar rats showed minor changes in the biodistribution profile of the [(86)Y((S)-p-NH2-Bn-DOTA)](-) complex isomers, while no significant differences were observed for the [(86)Y-DOTA]-L-RNA isomers. High renal excretions were found for the [(86)Y((S)-p-NH 2-Bn-DOTA)](-) complex isomers as well as for the L-RNA isomers.