Funerary practices from columnata necropolis. Presentation of the last three burials excavated

We describe in this paper 3 burial practices from the last hunter-gatherers lower Holocene populations of Columnata locality (Tiaret-Algeria). Despite their poor bone condition and some obvious grave disturbances, these individual burials are seen as primary depositions. They were placed in clogged tombs in a dorsal position, with no particular orientation of the body and without any deliberate bone’s modification as noticed with iberomaurusian burials. Bovine horns can be considered as voluntary funerary goods as reported for iberomaurusian or capsian burial unlike the semewhat non-deliberate lithic and faunal remains discovered into the graves. In this respect, grave H1 deposit is a singular case. Another special feature of this site is the stone arrangement in the inner space of H1 and H2. Such an early stonework could be reported according to the graves dates to the 10th and 8th millenia BC. Two of the tombs were built with stones to mark their position which is quite advanced for that time.En este trabajo describimos 3 prácticas de enterramiento de las últimas poblaciones de caza-dores-recolectores y pastores del Holoceno inferior en la localidad de Columnata (Tiaret-Ar-gelia). A pesar de su mal estado de conservación ósea, y algunas obvias perturbaciones graves, estos entierros individuales se consideran como deposiciones primarias. Fueron colocados en tumbas selladas en posición dorsal, sin orientación particular del cuerpo y sin ninguna modificación ósea deliberada como se observa en los entierros iberomaurusienses. Los cuernos de bóvidos pueden considerarse como ajuares funerarios voluntarios como ha sido descrito en enterramientos iberoamericanos y capsienses a diferencia de los restos líticos y faunísticos cuya colocación en la tumba sería no deliberada. Otra característica especial de este sitio es la disposición de las medias en el espacio interior de H1 y H2. este tipo de estructura se ha documentado en tumbas datadas en los milenios 10 y 8. Dos de las tumbas fueron cons-truidas con piedras para marcar su posición, una práctica bastante avanzada para esa época

The Rho-associated protein kinase p160ROCK is required for centrosome positioning

The p160–Rho-associated coiled-coil–containing protein kinase (ROCK) is identified as a new centrosomal component. Using immunofluorescence with a variety of p160ROCK antibodies, immuno EM, and depletion with RNA interference, p160ROCK is principally bound to the mother centriole (MC) and an intercentriolar linker. Inhibition of p160ROCK provoked centrosome splitting in G1 with the MC, which is normally positioned at the cell center and shows little motion during G1, displaying wide excursions around the cell periphery, similar to its migration toward the midbody during cytokinesis. p160ROCK inhibition late after anaphase in mitosis triggered MC migration to the midbody followed by completion of cell division. Thus, p160ROCK is required for centrosome positioning and centrosome-dependent exit from mitosis

Manganese Cytotoxicity Assay on Hippocampal Neuronal Cell Culture

Compared to an in vivo experiment, neuronal cell cultures are immediately accessibleto observation and manipulation. In this protocol, we describe a technique to evaluate thecytotoxicity of a metal, manganese (Mn2+), on hippocampal neuronal cell cultures. Interestingly, this protocol is easily adaptable to any type of primary culture (e.g., cortical neurons) and any type of toxic compound (e.g., chemical product).Fil: Daoust, Alexia. Inserm; Francia. Universite Joseph Fourier; FranciaFil: Saoudi, Yasmina. Inserm; Francia. Universite Joseph Fourier; FranciaFil: Brocard, Jacques. Inserm; Francia. Universite Joseph Fourier; FranciaFil: Collomb, Nora. Inserm; Francia. Universite Joseph Fourier; FranciaFil: Batandier, Cecile. Laboratoire de Bioénergétique Fondamentale et Appliquée; FranciaFil: Bisbal, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra. Universidad Nacional de Córdoba. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Inserm; Francia. Universite Joseph Fourier; FranciaFil: Salome, Murielle. European Synchrotron Radiation Facility; FranciaFil: Andrieux, Annie. Inserm; Francia. Universite Joseph Fourier; FranciaFil: Bohic, Sylvain. Inserm; Francia. Universite Joseph Fourier; Francia. European Synchrotron Radiation Facility; FranciaFil: Barbier, Emmanuel. Inserm; Francia. Universite Joseph Fourier; Franci

BENA435, a new cell-permeant photoactivated green fluorescent DNA probe

N′-(2,8-Dimethoxy-12-methyl-dibenzo [c,h] [1,5] naphthyridin-6-yl)-N,N-dimethyl-propane-1,3-diamine (BENA435) is a new cell-membrane permeant DNA dye with absorption/emission maxima in complex with DNA at 435 and 484 nm. This new reagent is unrelated to known DNA dyes, and shows a distinct preference to bind double-stranded DNA over RNA. Hydrodynamic studies suggest that BENA435 intercalates between the opposite DNA strands. BENA435 fluoresces much stronger when bound to dA/dT rather than dG/dC homopolymers. We evaluated 14 related dibenzonaphthyridine derivatives and found BENA435 to be superior in its in vivo DNA-binding properties. Molecular modelling was used to develop a model of BENA435 intercalation between base pairs of a DNA helix. BENA435 fluorescence in the nuclei of cells increases upon illumination, suggesting photoactivation. BENA435 represents thus the first known cell-permeant photoactivated DNA-binding dye

MAP6-F is a temperature sensor that directly binds to and protects microtubules from cold-induced depolymerization.: Microtubule stabilization by MAP6

International audienceMicrotubules are dynamic structures that present the peculiar characteristic to be ice-cold labile in vitro. In vivo, microtubules are protected from ice-cold induced depolymerization by the widely expressed MAP6/STOP family of proteins. However, the mechanism by which MAP6 stabilizes microtubules at 4 °C has not been identified. Moreover, the microtubule cold sensitivity and therefore the needs for microtubule stabilization in the wide range of temperatures between 4 and 37 °C are unknown. This is of importance as body temperatures of animals can drop during hibernation or torpor covering a large range of temperatures. Here, we show that in the absence of MAP6, microtubules in cells below 20 °C rapidly depolymerize in a temperature-dependent manner whereas they are stabilized in the presence of MAP6. We further show that in cells, MAP6-F binding to and stabilization of microtubules is temperature- dependent and very dynamic, suggesting a direct effect of the temperature on the formation of microtubule/MAP6 complex. We also demonstrate using purified proteins that MAP6-F binds directly to microtubules through its Mc domain. This binding is temperature-dependent and coincides with progressive conformational changes of the Mc domain as revealed by circular dichroism. Thus, MAP6 might serve as a temperature sensor adapting its conformation according to the temperature to maintain the cellular microtubule network in organisms exposed to temperature decrease

Prion protein expression and functional importance in developmental angiogenesis: role in oxidative stress and copper homeostasis.

International audienceAIM: It has been convincingly shown that oxidative stress and toxicity by deregulated metals, such as copper (Cu), are tightly linked to the development of pre-eclampsia and intrauterine growth retardation (IUGR), the most threatening pathologies of human pregnancy. However, mechanisms implemented to control these effects are far from being understood. Among proteins that bind Cu and insure cellular protection against oxidative stress is the cellular prion protein (PrP(C)), a glycosyl phosphatidyl inositol-anchored glycoprotein, which we reported to be highly expressed in human placenta. Herein, we investigated the pathophysiological role of PrP(C) in Cu and oxidative stress homeostasis in vitro using human placenta and trophoblast cells, and in vivo using three strains of mice (C57Bl6, PrP(C) knockout mice [PrP(-/-)], and PrP(C) overexpressing mice [Tga20]). RESULTS: At the cellular level, PrP(C) protection against oxidative stress was established in multiple angiogenic processes: proliferation, migration, and tube-like organization. For the animal models, lack (PrP(-/-)) or overexpression (Tga20) of PrP(C) in gravid mice caused severe IUGR that was correlated with a decrease in litter size, changes in Cu homeostasis, increase in oxidative stress response, development of hypoxic environment, failure in placental function, and maintenance of growth defects of the offspring even 7.5 months after delivery. INNOVATION: PrP(C) could serve as a marker for the idiopathic IUGR disease. CONCLUSION: These findings demonstrate the stress-protective role of PrP(C) during development, and propose PrP(C) dysregulation as a novel causative element of IUGR

Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends

Tubulin-tyrosine ligase (TTL), the enzyme that catalyzes the addition of a C-terminal tyrosine residue to α-tubulin in the tubulin tyrosination cycle, is involved in tumor progression and has a vital role in neuronal organization. We show that in mammalian fibroblasts, cytoplasmic linker protein (CLIP) 170 and other microtubule plus-end tracking proteins comprising a cytoskeleton-associated protein glycine-rich (CAP-Gly) microtubule binding domain such as CLIP-115 and p150 Glued, localize to the ends of tyrosinated microtubules but not to the ends of detyrosinated microtubules. In vitro, the head domains of CLIP-170 and of p150 Glued bind more efficiently to tyrosinated microtubules than to detyrosinated polymers. In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in both spindle positioning during mitosis and cell morphology during interphase. These results indicate that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus-end tracking proteins and provide explanations for the involvement of TTL in tumor progression and in neuronal organization

Evidence for new C-terminally truncated variants of α- and β-tubulins

New C-terminally truncated α- and β-tubulin variants, both ending with an -EEEG sequence, are identified in vivo: αΔ3-tubulin, which has a specific neuronal distribution pattern (distinct from that of αΔ2-tubulin) and seems to be related to dynamic microtubules, and βΔ4-tubulin, corresponding to β2A/B-tubulin modified by truncation of four C-terminal residues, which is ubiquitously present in cells and tissues. Cellular α-tubulin can bear various carboxy-terminal sequences: full-length tubulin arising from gene neosynthesis is tyrosinated, and two truncated variants, corresponding to detyrosinated and Δ2 α‑tubulin, result from the sequential cleavage of one or two C-terminal residues, respectively. Here, by using a novel antibody named 3EG that is highly specific to the -EEEG C-terminal sequence, we demonstrate the occurrence in neuronal tissues of a new αΔ3‑tubulin variant corresponding to α1A/B‑tubulin deleted of its last three residues (EEY). αΔ3‑tubulin has a specific distribution pattern: its quantity in the brain is similar to that of αΔ2-tubulin around birth but is much lower in adult tissue. This truncated α1A/B-tubulin variant can be generated from αΔ2-tubulin by the deglutamylases CCP1, CCP4, CCP5, and CCP6 but not by CCP2 and CCP3. Moreover, using 3EG antibody, we identify a C‑terminally truncated β-tubulin form with the same -EEEG C-terminal sequence. Using mass spectrometry, we demonstrate that β2A/B-tubulin is modified by truncation of the four C-terminal residues (EDEA). We show that this newly identified βΔ4-tubulin is ubiquitously present in cells and tissues and that its level is constant throughout the cell cycle. These new C-terminally truncated α- and β-tubulin variants, both ending with -EEEG sequence, are expected to regulate microtubule physiology. Of interest, the αΔ3-tubulin seems to be related to dynamic microtubules, resembling tyrosinated-tubulin rather than the other truncated variants, and may have critical function(s) in neuronal development