Reduced tubulin tyrosination as an early marker of mercury toxicity in differentiating N2a cells

The aims of this work were to compare the effects of methyl mercury chloride and Thimerosal on neurite/process outgrowth and microtubule proteins in differentiating mouse N2a neuroblastoma and rat C6 glioma cells. Exposure for 4 h to sublethal concentrations of both compounds inhibited neurite outgrowth to a similar extent in both cells lines compared to controls. In the case of N2a cells, this inhibitory effect by both compounds was associated with a fall in the reactivity of western blots of cell extracts with monoclonal antibody T1A2, which recognises C-terminally tyrosinated α-tubulin. By contrast, reactivity with monoclonal antibody B512 (which recognises total α-tubulin) was unaffected at the same time point. These findings suggest that decreased tubulin tyrosination represents a neuron-specific early marker of mercury toxicity associated with impaired neurite outgrowth

The GTPase Rab26 links synaptic vesicles to the autophagy pathway.

Small GTPases of the Rab family not only regulate target recognition in membrane traffic but also control other cellular functions such as cytoskeletal transport and autophagy. Here we show that Rab26 is specifically associated with clusters of synaptic vesicles in neurites. Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles. Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles. Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity. We propose that Rab26 selectively directs synaptic and secretory vesicles into preautophagosomal structures, suggesting the presence of a novel pathway for degradation of synaptic vesicles

Widespread expression of erythropoietin receptor in brain and its induction by injury

Erythropoietin (EPO) exerts potent neuroprotective, neuroregenerative and procognitive functions. However, unequivocal demonstration of erythropoietin receptor (EPOR) expression in brain cells has remained difficult since previously available anti-EPOR antibodies (EPOR-AB) were unspecific. We report here a new, highly specific, polyclonal rabbit EPOR-AB directed against different epitopes in the cytoplasmic tail of human and murine EPOR and its characterization by mass spectrometric analysis of immuno-precipitated endogenous EPOR, Western blotting, immunostaining and flow cytometry. Among others, we applied genetic strategies including overexpression, Lentivirus-mediated conditional knockout of EpoR and tagged proteins, both on cultured cells and tissue sections, as well as intracortical implantation of EPOR-transduced cells to verify specificity. We show examples of EPOR expression in neurons, oligodendroglia, astrocytes and microglia. Employing this new EPOR-AB with double-labeling strategies, we demonstrate membrane expression of EPOR as well as its localization in intracellular compartments such as the Golgi apparatus. Moreover, we show injury-induced expression of EPOR. In mice, a stereotactically applied stab wound to the motor cortex leads to distinct EpoR expression by reactive GFAP-expressing cells in the lesion vicinity. In a patient suffering from epilepsy, neurons and oligodendrocytes of the hippocampus strongly express EPOR. To conclude, this new analytical tool will allow neuroscientists to pinpoint EPOR expression in cells of the nervous system and to better understand its role in healthy conditions, including brain development, as well as under pathological circumstances, such as upregulation upon distress and injury

Cap-Gly Proteins at Microtubule Plus Ends: Is EB1 Detyrosination Involved?

Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with α-tubulin and EB1 through their C-terminal amino acids −EEY. Detyrosination (cleavage of the terminal tyrosine) of α-tubulin by tubulin-carboxypeptidase abolishes CLIP170 binding. Can detyrosination affect EB1 and thus regulate the presence of CLIP170 at microtubule+ends as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue. Over-expressed EB1 was not detyrosinated in cells and chimeric EB1 with the eight C-terminal amino acids of α-tubulin was only barely detyrosinated. Our results indicate that detyrosination regulates CLIPs interaction with α-tubulin, but not with EB1. They highlight the specificity of carboxypeptidase toward tubulin

Tubulin Tyrosination Is Required for the Proper Organization and Pathfinding of the Growth Cone

International audienceBACKGROUND: During development, neuronal growth cones integrate diffusible and contact guidance cues that are conveyed to both actin and microtubule (MT) cytoskeletons and ensure axon outgrowth and pathfinding. Although several post-translational modifications of tubulin have been identified and despite their strong conservation among species, their physiological roles during development, especially in the nervous sytem, are still poorly understood. METHODOLOGY/FINDINGS: Here, we have dissected the role of a post-translational modification of the last amino acid of the alpha-tubulin on axonal growth by analyzing the phenotype of precerebellar neurons in Tubulin tyrosin ligase knock-out mice (TTL(-/-)) through in vivo, ex vivo and in vitro analyses. TTL(-/-) neurons are devoid of tyrosinated tubulin. Their pathway shows defects in vivo, ex vivo, in hindbrains open-book preparations or in vitro, in a collagen matrix. Their axons still orient toward tropic cues, but they emit supernumerary branches and their growth cones are enlarged and exhibit an emission of mis-oriented filopodia. Further analysis of the TTL(-/-) growth cone intracellular organization also reveals that the respective localization of actin and MT filaments is disturbed, with a decrease in the distal accumulation of Myosin IIB, as well as a concomitant Rac1 over-activation in the hindbrain. Pharmacological inhibition of Rac1 over-activation in TTL(-/-) neurons can rescue Myosin IIB localization. CONCLUSIONS/SIGNIFICANCE: In the growth cone, we propose that tubulin tyrosination takes part in the relative arrangement of actin and MT cytoskeletons, in the regulation of small GTPases activity, and consequently, in the proper morphogenesis, organization and pathfinding of the growth cone during development

SUSTAINABILITY COST ACCOUNTING - PART 2: A CASE STUDY IN THE SOUTH AFRICAN PROCESS INDUSTRY

<p>ENGLISH ABSTRACT: A Sustainability Cost Accounting (SCA) procedure has been introduced that expresses the impacts on sustainable development associated with a developed technology, by means of a common financial denominator. This paper uses a case study to demonstrate and assess the SCA procedure, which considers the construction and operation of a hypothetical Gas-to-Liquid (GTL) fuelmanufacturing facility at a specific location in South Africa. The SCA indicators show that the negative environmental impacts associated with the GTL technology outweigh the internal economic benefits for the company. However, a net positive social benefit is associated with the technology, which decision-makers should consider with respect of the overall sustainability of the technology. Certain limitations of the SCA procedure are highlighted, and recommendations are made to develop such a methodology further.</p><p>AFRIKAANSE OPSOMMING: ’n Volhoubaarheid Koste-Rekeningkunde prosedure (VKR) word voorgestel waarvolgens die impakte van ’n ontwikkelde tegnologie op volhoubaarheid in ’n gemeenskaplike finansiële waarde aangegee kan word. ’n Gevallestudie word hier gebruik om die VKR-prosedure te demonstreer. Die gevallestudie beskryf die konstruksie en bedryf van ’n hipotetiese Gas-tot-Vloeistof brandstofvervaardigingsfasiliteit (GTV) in ‘n spesifieke area van Suid-Afrika. Die VKR-indikators dui aan dat die negatiewe omgewingsimpakte van die GTV tegnologie tot ‘n geringe mate groter is as die interne ekonomiese voordele vir die maatskappy. Die tegnologie het wel oorwegende positiewe sosiale voordele wat besluitnemers in ag moet neem wanneer die globale volhoubaarheid van die tegnologie ge-assesseer word. Sekere beperkinge van die VKR-prosedure word uitgelig en voorstelle word gemaak om dié tipe metodologie verder te ontwikkel.</p&gt

Radiation-induced segregation and precipitation in molybdenum-rhenium alloys

Specimens of Mo-7 at. % Re and Mo-30 at. % Re were irradiated with 1.8 MeV /sup 4/He/sup +/ ions at elevated temperatures. Radiation-induced segregation of Re was measured during irradiation by in situ Rutherford backscattering spectrometry. Segregation of the undersized Re atoms in the same direction as the defect fluxes, i.e., toward the external surface, was observed. The amount of Re enrichment in the near-surface region was measured as a function of temperature and of dose at a calculated near-surface displacement rate near 1 x 10/sup -4/dpa/s. Segregation was observed at temperatures from 800 to 1500/sup 0/C in Mo-7Re, and from 850 to 1225/sup 0/C in Mo-30Re. Irradiated disks were examined by transmission electron microscopy. Precipitates of Chi phase were observed on grain boundaries, or in a thin layer at the irradiated surface in Mo-30Re after irradiation at temperatures from 750 to 1075/sup 0/C. Frequently, Chi precipitates formed with a crystallographic twin orientation with respect to the host matrix. No voids were observed for doses up to 1.6 dpa