CENP-A is required for accurate chromosome segregation and sustained kinetochore association of BubR1

CENP-A is an evolutionarily conserved, centromere-specific variant of histone H3 that is thought to play a central role in directing kinetochore assembly and in centromere function. Here, we have analyzed the consequences of disrupting the CENP-A gene in the chicken DT40 cell line. In CENP-A-depleted cells, kinetochore protein assembly is impaired, as indicated by mislocalization of the inner kinetochore proteins CENP-I, CENP-H, and CENP-C as well as the outer components Nuf2/Hec1, Mad2, and CENP-E. However, BubR1 and the inner centromere protein INCENP are efficiently recruited to kinetochores. Following CENP-A depletion, chromosomes are deficient in proper congression on the mitotic spindle and there is a transient delay in prometaphase. CENP-A-depleted cells further proceed through anaphase and cytokinesis with unequal chromosome segregation, suggesting that some kinetochore function remains following substantial depletion of CENP-A. We furthermore demonstrate that CENP-A-depleted cells exhibit a specific defect in maintaining kinetochore localization of the checkpoint protein BubR1 under conditions of checkpoint activation. Our data thus point to a specific role for CENP-A in assembly of kinetochores competent in the maintenance of mitotic checkpoint signaling.     

Brain phenotype of transgenic mice overexpressing cystathionine β-synthase

Background

The cystathionine β-synthase (CBS) gene, located on human chromosome 21q22.3, is a good candidate for playing a role in the Down Syndrome (DS) cognitive profile: it is overexpressed in the brain of individuals with DS, and it encodes a key enzyme of sulfur-containing amino acid (SAA) metabolism, a pathway important for several brain physiological processes.

Methodology/Principal Findings

Here, we have studied the neural consequences of CBS overexpression in a transgenic mouse line (60.4P102D1) expressing the human CBS gene under the control of its endogenous regulatory regions. These mice displayed a ∼2-fold increase in total CBS proteins in different brain areas and a ∼1.3-fold increase in CBS activity in the cerebellum and the hippocampus. No major disturbance of SAA metabolism was observed, and the transgenic mice showed normal behavior in the rotarod and passive avoidance tests. However, we found that hippocampal synaptic plasticity is facilitated in the 60.4P102D1 line.

Conclusion/Significance

We demonstrate that CBS overexpression has functional consequences on hippocampal neuronal networks. These results shed new light on the function of the CBS gene, and raise the interesting possibility that CBS overexpression might have an advantageous effect on some cognitive functions in DS.     

New data on the late Neandertals: Direct dating of the Belgian Spy fossils

In Eurasia, the period between 40,000 and 30,000 BP saw the replacement of Neandertals by anatomically modern humans (AMH) during and after the Middle to Upper Paleolithic transition. The human fossil record for this period is very poorly defined with no overlap between Neandertals and AMH on the basis of direct dates. Four new ¹⁴C dates were obtained on the two adult Neandertals from Spy (Belgium). The results show that Neandertals survived to at least ≈36,000 BP in Belgium and that the Spy fossils may be associated to the Lincombian–Ranisian–Jerzmanowician, a transitional techno‐complex defined in northwest Europe and recognized in the Spy collections. The new data suggest that hypotheses other than Neandertal acculturation by AMH may be considered in this part of Europe. Am J Phys Anthropol, 2009. © 2008 Wiley‐Liss, Inc.     

Characterization with monoclonal antibodies of a surface antigen of Plasmodium falciparum merozoites

The merozoite, the extracellular form of the erythrocyte stage of the malarial parasite, invades the erythrocyte and develops intracellularly. Cloned hybridoma cell lines secreting monoclonal antibodies directed against the merozoite surface were selected by indirect immunofluorescent assay by using intact isolated merozoites. Monoclonal antibodies to a 200,000 m.w. merozoite surface antigen were selected and were used to characterize this protein and its role in erythrocyte invasion. Immunoelectron microscopy demonstrated that the antigen was located exclusively on the merozoite surface coat, distributed evenly over the entire surface. The 200,000 m.w. protein incorporated [3H]glucosamine, suggesting that it is a glycoprotein and could be purified to homogeneity by using immuno-affinity chromatography. Freshly isolated, invasive merozoites retained the 200,000 m.w. antigen, but the protein was rapidly cleaved to proteins of 90,000 and 50,000 m.w. when the merozoite was extracellular. The 50,000 m.w. fragment was retained the epitope binding to monoclonal antibody 5B1 and were labeled with [3H]glucosamine. Monoclonal antibodies against the 200,000 m.w. antigen partially inhibited merozoite invasion into erythrocytes.     

Tempting fate: BMP signals for cardiac morphogenesis

Heart muscle cell specification (cardiac myogenesis) and creating the four-chambered heart (cardiac morphogenesis) are subject to regulation, in certain model organisms, by bone morphogenetic proteins and their receptors. Extrapolation to mammals from organisms that develop outside the mother (flies, fish, frogs, and avians) has been confounded by very early lethality-at gastrulation-of many null alleles needed to prove cause-effect relations in this pathway. Here, we describe the use of lineage- or compartment-restricted null alleles as well as hypomorphic alleles, which circumvent these limitations and pinpoint novel essential functions for the bone morphogenetic protein cascade in mammalian cardiac development.     

Cross signaling,cell specificity,and physiology

The literature on intracellular signaltransduction presents a confusing picture: every regulatory factorappears to be regulated by all signal transduction cascades and toregulate all cell processes. This contrasts with the known exquisitespecificity of action of extracellular signals in different cell typesin vivo. The confusion of the in vitro literature is shown to arisefrom several causes: the inevitable artifacts inherent in reductionism,the arguments used to establish causal effect relationships, the use ofless than adequate models (cell lines, transfections, acellular systems, etc.), and the implicit assumption that networks ofregulations are universal whereas they are in fact cell and stagespecific. Cell specificity results from the existence in any cell typeof a unique set of proteins and their isoforms at each level of signal transduction cascades, from the space structure of their components, from their combinatorial logic at each level, from the presence ofmodulators of signal transduction proteins and of modulators ofmodulators, from the time structure of extracellular signals and oftheir transduction, and from quantitative differences of expression ofsimilar sets of factors.

     

Glucocorticoids reduce inflammation in cystic fibrosis bronchial epithelial cells

Reduction of lung inflammation is one of the goals of cystic fibrosis (CF) therapy. Among anti-inflammatory molecules, glucocorticoids (GC) are one of the most prescribed. However, CF patients seem to be resistant to glucocorticoid treatment. Several molecular mechanisms that contribute to decrease anti-inflammatory effects of glucocorticoids have been identified in pulmonary diseases, but the molecular actions of glucocorticoids have never been studied in CF. In the cytoplasm, glucocorticoids bind to glucocorticoid receptor (GR) and then, control NF-κB and MAPK pathways through direct interaction with AP-1 and NF-κB in the nucleus. Conversely, MAPK can regulate glucocorticoid activation by targeting GR phosphorylation. Together these pathways regulate IL-8 release in the lung. Using bronchial epithelial cell lines derived from non CF and CF patients, we analyzed GR-based effects of glucocorticoids on NF-κB and MAPK pathways, after stimulation with TNF-α. We demonstrate that the synthetic glucocorticoid dexamethasone (Dex) significantly decreases IL-8 secretion, AP-1 and NF-κB activity in CF cells in a pro-inflammatory context. Moreover, we show that p38 MAPK controls IL-8 release by determining GR activation through specific phosphorylation on serine 211. Finally, we demonstrate a synergistic effect of dexamethasone treatment and inhibition of p38 MAPK inducing more than 90% inhibition of IL-8 production in CF cells. All together, these results demonstrate the good responsiveness to glucocorticoids of CF bronchial epithelial cells and the reciprocal link between glucocorticoids and p38 MAPK in the control of CF lung inflammation.