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International audience; In chronic liver diseases, the accumulation of extracellular matrix leading to fibrosis is caused by myofibroblasts, the origins of which are debatable. We performed a comparative proteomic study to identify markers and gain insight into distinct functions of myofibroblasts derived either from hepatic stellate cells (HSCs) or from portal mesenchymal cells. After isolation from normal liver and culture in similar conditions, myofibroblastic HSCs (MF-HSCs) presented enlarged cytoplasms whereas portal myofibroblasts (PMFs) were more proliferative, and formed more stress fibers. The two cell types were subjected to comparative analyses by 2-D MS/MS. Six proteins were overexpressed in PMFs, with myofibroblast-related typical functions. Among them, cofilin-1 showed the greatest difference in expression and a lower pI than expected. Immunoblot demonstrated higher levels of phosphorylation, a modification of the protein implicated in stress fiber formation. Eleven proteins, mostly involved in stress response, were overexpressed in MF-HSCs. Cytoglobin had the highest level of overexpression, as confirmed by reverse transcription quantitative real-time PCR, immunoblot and immunocytochemical analyses. These results identify cytoglobin as the best marker for distinguishing MF-HSCs from PMFs and suggest different functions for the two cell populations in the liver wound healing response, with a prominent role for PMFs in scar formation.

Background The lancelet amphioxus (Cephalochordata) is a close relative of vertebrates and thus may enhance our understanding of vertebrate gene and genome evolution. In this context, the globins are one of the best studied models for gene family evolution. Previous biochemical studies have demonstrated the presence of an intracellular globin in notochord tissue and myotome of amphioxus, but the corresponding gene has not yet been identified. Genomic resources of Branchiostoma floridae now facilitate the identification, experimental confirmation and molecular evolutionary analysis of its globin gene repertoire. Results We show that B. floridae harbors at least fifteen paralogous globin genes, all of which reveal evidence of gene expression. The protein sequences of twelve globins display the conserved characteristics of a functional globin fold. In phylogenetic analyses, the amphioxus globin BflGb4 forms a common clade with vertebrate neuroglobins, indicating the presence of this nerve globin in cephalochordates. Orthology is corroborated by conserved syntenic linkage of BflGb4 and flanking genes. The kinetics of ligand binding of recombinantly expressed BflGb4 reveals that this globin is hexacoordinated with a high oxygen association rate, thus strongly resembling vertebrate neuroglobin. In addition, possible amphioxus orthologs of the vertebrate globin X lineage and of the myoglobin/cytoglobin/hemoglobin lineage can be identified, including one gene as a candidate for being expressed in notochord tissue. Genomic analyses identify conserved synteny between amphioxus globin-containing regions and the vertebrate β-globin locus, possibly arguing against a late transpositional origin of the β-globin cluster in vertebrates. Some amphioxus globin gene structures exhibit minisatellite-like tandem duplications of intron-exon boundaries ("mirages"), which may serve to explain the creation of novel intron positions within the globin genes. Conclusions The identification of putative orthologs of vertebrate globin variants in the B. floridae genome underlines the importance of cephalochordates for elucidating vertebrate genome evolution. The present study facilitates detailed functional studies of the amphioxus globins in order to trace conserved properties and specific adaptations of respiratory proteins at the base of chordate evolution.

Background The genome of the nematode Caenorhabditis elegans contains more than 30 putative globin genes that all are transcribed. Although their translated amino acid sequences fit the globin fold, a variety of amino-acid substitutions and extensions generate a wide structural diversity among the putative globins. No information is available on the physicochemical properties and the in vivo expression. Results We expressed the globins in a bacterial system, characterized the purified proteins by optical and resonance Raman spectroscopy, measured the kinetics and equilibria of O2 binding and determined the crystal structure of GLB-1* (CysGH2 → Ser mutant). Furthermore, we studied the expression patterns of glb-1 (ZK637.13) and glb-26 (T22C1.2) in the worms using green fluorescent protein technology and measured alterations of their transcript abundances under hypoxic conditions.GLB-1* displays the classical three-over-three α-helical sandwich of vertebrate globins, assembled in a homodimer associated through facing E- and F-helices. Within the heme pocket the dioxygen molecule is stabilized by a hydrogen bonded network including TyrB10 and GlnE7.GLB-1 exhibits high ligand affinity, which is, however, lower than in other globins with the same distal TyrB10-GlnE7 amino-acid pair. In the absence of external ligands, the heme ferrous iron of GLB-26 is strongly hexacoordinated with HisE7, which could explain its extremely low affinity for CO. This globin oxidizes instantly to the ferric form in the presence of oxygen and is therefore incapable of reversible oxygen binding. Conclusion The presented data indicate that GLB-1 and GLB-26 belong to two functionally-different globin classes.

International audience; A survey is presented of picosecond kinetics of heme-residue bond formation after photolysis of histidine, methionine, or cysteine, in a broad range of ferrous six-coordinate heme proteins. These include human neuroglobin, a bacterial heme-binding superoxide dismutase (SOD), plant cytochrome b 559, the insect nuclear receptor E75, horse heart cytochrome c and the heme domain of the bacterial sensor protein Dos. We demonstrate that the fastest and dominant phase of binding of amino acid residues to domed heme invariably takes place with a time constant in the narrow range of 5-7 ps. Remarkably, this is also the case in the heme-binding SOD, where the heme is solvent-exposed. We reason that this fast phase corresponds to barrierless formation of the heme-residue bond from a configuration close to the bound state. Only in proteins where functional ligand exchange occurs, additional slower rebinding takes place on the time scale of tens of picoseconds after residue dissociation. We propose that the presence of these slower phases reflects flexibility in the heme environment that allows external ligands (O2, CO, NO, . . .) to functionally replace the internal residue after thermal dissociation of the heme-residue bond.

International audience; In this study, we report on a Cameroonian family from the Ewondo ethnic group, presenting with three oculocutaneous albinism type 2 (OCA2) patients homozygous for the 2.7-kb deletion of the P gene. In one of these patients OCA2 was associated with sickle cell anaemia and in two with the sickle cell trait. We took this opportunity to determine single nucleotide polymorphism (SNP) haplotypes within the P gene in this family in comparison with a group of 53 OCA2 patients homozygous for the same mutation and with a matched unrelated full-coloured control group of 49 subjects, originating from seven different ethnic groups of Southern Cameroon including Ewondo. A combination of five exonic and intronic SNPs in the OCA2 gene was genotyped by sequencing PCR products. We found 3 different haplotypes (TAGCT, TAGTT and TAGCC with frequencies of 0.66, 0.28 and 0.06, respectively) associated with the mutation in the 53 OCA2 patients, while 11 different haplotypes were observed in the control group. These observations suggest that the mutation appeared on the relatively frequent haplotype TAGCT, and that the two other haplotypes are derived from two independent recombination events. These haplotypic data, associated with a value of 1/15,000 for the prevalence of the 2.7-kb mutation, a present effective population size of 10,000,000 for Cameroon and a recombination rate of 0.0031, allowed us to estimate that this mutation originated 4,100-5,645 years ago.

We have examined whether replication of the chicken beta-globin locus changes during differentiation of primary erythroid progenitors into erythrocytes. In undifferentiated progenitors, four principal initiation sites and a replication fork pausing region (RFP) were observed. Forty-eight hours after induction of differentiation, the principal sites were maintained, even in the activated beta(A)-globin gene, some minor sites were enhanced, three new sites appeared and the RFP disappeared. One of the activated origins showed increased histone H3 K9K14 diacetylation, but the others did not. These results demonstrate a broadening of DNA replication origin usage during differentiation of untransformed metazoan cells and indicate that histone H3 diacetylation, other histone modifications so far reported and transcription are not crucial determinants of origin selection in this system.

Sequential, position-selective, Pd-catalyzed cross-coupling reactions of 2,4-dibromo-5-hydroxymethylthiazole provided the scaffold for the synthesis of GW501516, the most potent PPARbeta/delta agonist yet described, and equally selective analogs at the thiazole-C4 position.

The growth factor-independent erythroleukemic cell line ERY-1 was established from the peripheral blood of a 87-year-old woman with chronic myeloid leukemia (CML) in the acute phase. Immunophenotyping showed that fresh leukemic cells were positive for CD13, CD33, CD36 and CD235a (glycophorin A), a phenotype compatible with that of erythroblastic cells. Cytogenetic and fluorescence in situ hybridization (FISH) analysis demonstrated classical t(9;22)(q34;q11) chromosomic translocation associated with a duplication of the BCR-ABL fusion gene. Other cytogenetic abnormalities were detected in all analyzed mitosis, the most frequent being a trisomy of chromosome 8. The established ERY-1 cell line retains these immunophenotypic and cytogenetic features, and light and electron microscopy confirmed the relatively mature erythroblastic phenotype of the cells. In addition, ERY-1 cell line expressed beta-globin mRNA and a non-phosphorylable form of the erythropoietin receptor, even in presence of erythropoietin. Of note, the proliferation of ERY-1 cells was inhibited by TGFbeta1 or STI-571 (Gleevec), without significant induction of further differentiation. In conclusion, ERY-1 is a new growth factor-independent human erythroleukemic cell line with a relatively mature phenotype that may be useful to study the molecular events involved in erythroblastic differentiation.

Locus control regions (LCR) were first defined by their theoretical ability to enhance the expression of linked genes in a tissue-specific, position-independent and copy-number dependent manner. In fact, few of the so-called LCR identified completely fulfil this definition. For example, the regulatory elements located in 5' (Emu) and 3' (HS3a; HS1,2; HS3b; HS4) of the IgH locus display some properties of a LCR but lack a copy-number dependence and sometimes display position effects in transgenes. In order to study whether addition of insulators would allow to overcome such problems in transgenes, we studied constructs harboring a V(H) promoter-green fluorescent protein reporter gene linked to the 3' and/or 5' IgH elements, surrounded or not with the chicken beta-globin 5'HS4 insulator. When flanked with insulators it appeared that either 3' IgH and 5' IgH regulatory elements now behave as true LCR elements and noticeably display copy-number dependence in transfected pre-B or B cell lines.

A locus control region (LCR) containing four transcriptional enhancers lies downstream of the IgH chain locus. We studied transgenes carrying a 3(')IgH LCR-driven GFP reporter gene for expression and B cell differentiation stage specificity. We also compared transgenes that were or were not flanked by two copies of the beta-globin HS4 insulator, an element defined by its ability to protect transgenes from the influences of surrounding genes at the insertion site. Results indicate that insulators are instrumental in sustaining GFP expression in GFP-3(')LCR transgenic mice when they were included. Flow cytometry experiments reported a strictly B cell specific GFP expression from pre-B cells in bone marrow to mature B cells in spleen. Despite addition of 5(')HS4 insulators to the GFP-3(')LCR construct, complete transgene silencing occurred in some transgenic lines and was systematically observed in ageing animals from all lines.

Summary The hexamethylene bisacetamide(HMBA) induced differentiation of murine erythroleukemia cells is inhibited by dexamethasone (DEX). We show here that addition of hemin (100 pM) to cells cultured with HMBA (4mM) and DEX (100 μM) leads to an appearance of hemoglobin containing cells. However these cells cultured with HMBA, DEX, and hemin are not reprogrammed to limited growth. DEX does not inhibit the synthesis of the nuclear protein IP25. Hence commitment to terminal differentiation is controlled by at least one heme-independant event (other than the induction of IP25) which is inhibited by DEX.

International audience; Normal c-myc RNAs are very unstable with a half-life of less than 30 min whereas those rearranged in 5', as found in Burkitt's lymphomas and mouse plasmacytomas, are significantly more stable. To learn about the sequence determinants controlling their turnover, we have studied naturally occurring and artificially constructed c-myc RNAs rearranged in 5' or 3'. The first conclusion is that sequences necessary for rapid c-myc RNAs turnover are localized in their 3' untranslated region. The second conclusion is that stabilization of truncated c-myc RNAs in tumors does not result from deletion of the non-coding first exon but rather from its replacement by intronic and/or exogenous sequences. This latter conclusion rests on two lines of evidence: (i) deleting the 5' rearranged sequences from the relatively stable MOPC 315 RNA restores its complete instability (pSV c-myc 1); (ii) reciprocally, appending intron 1 sequences 5' to otherwise unstable germline c-myc exons 2 and 3 have a dramatic stabilizing effect (pIM 0).

International audience; The fos proto-oncogene is rapidly and transiently expressed in resting cells exposed to growth stimulation. This gene is down-regulated at least at two levels: transcriptional repression and mRNA degradation. To determine the sequences and the structures involved in mRNA instability, we analyzed in mouse Ltk- cells various fos/beta-globin constructs for their transcriptional activity and the half-lives of the corresponding RNAs. In these cells, rabbit beta-globin genes under the control of a 500 bp fos SRE (serum responsive element)/promoter region are transiently transcribed within 30 min after stimulation. Analysis of the decay kinetics of RNA originating from these constructs led to the following conclusions with respect to the nature of c-fos destabilizer elements: (i) 100 bases from c-fos 3' untranslated region are able to confer instability when inserted into a normally stable beta-globin RNA; (ii) however, the degradation is more rapid when the complete untranslated region is inserted; (iii) rapid mRNA breakdown requires more determinants than two AUUUA motives and is associated with a reduction in size, presumably due to a poly(A) shortening; (iv) remarkably, c-fos destabilizing sequences remain active even when part of the coding sequence.