Laboratoire de Biochimie

The genome-wide program of gene expression during the cell division cycle in a human cancer cell line (HeLa) was characterized using cDNA microarrays. Transcripts of >850 genes showed periodic variation during the cell cycle. Hierarchical clustering of the expression patterns revealed coexpressed groups of previously well-characterized genes involved in essential cell cycle processes such as DNA replication, chromosome segregation, and cell adhesion along with genes of uncharacterized function. Most of the genes whose expression had previously been reported to correlate with the proliferative state of tumors were found herein also to be periodically expressed during the HeLa cell cycle. However, some of the genes periodically expressed in the HeLa cell cycle do not have a consistent correlation with tumor proliferation. Cell cycle-regulated transcripts of genes involved in fundamental processes such as DNA replication and chromosome segregation seem to be more highly expressed in proliferative tumors simply because they contain more cycling cells. The data in this report provide a comprehensive catalog of cell cycle regulated genes that can serve as a starting point for functional discovery. The full dataset is available at http://genome-www.stanford.edu/Human-CellCycle/HeLa/.

BACKGROUND: Colon cancer (CC) pathological staging fails to accurately predict recurrence, and to date, no gene expression signature has proven reliable for prognosis stratification in clinical practice, perhaps because CC is a heterogeneous disease. The aim of this study was to establish a comprehensive molecular classification of CC based on mRNA expression profile analyses. METHODS AND FINDINGS: Fresh-frozen primary tumor samples from a large multicenter cohort of 750 patients with stage I to IV CC who underwent surgery between 1987 and 2007 in seven centers were characterized for common DNA alterations, including BRAF, KRAS, and TP53 mutations, CpG island methylator phenotype, mismatch repair status, and chromosomal instability status, and were screened with whole genome and transcriptome arrays. 566 samples fulfilled RNA quality requirements. Unsupervised consensus hierarchical clustering applied to gene expression data from a discovery subset of 443 CC samples identified six molecular subtypes. These subtypes were associated with distinct clinicopathological characteristics, molecular alterations, specific enrichments of supervised gene expression signatures (stem cell phenotype-like, normal-like, serrated CC phenotype-like), and deregulated signaling pathways. Based on their main biological characteristics, we distinguished a deficient mismatch repair subtype, a KRAS mutant subtype, a cancer stem cell subtype, and three chromosomal instability subtypes, including one associated with down-regulated immune pathways, one with up-regulation of the Wnt pathway, and one displaying a normal-like gene expression profile. The classification was validated in the remaining 123 samples plus an independent set of 1,058 CC samples, including eight public datasets. Furthermore, prognosis was analyzed in the subset of stage II-III CC samples. The subtypes C4 and C6, but not the subtypes C1, C2, C3, and C5, were independently associated with shorter relapse-free survival, even after adjusting for age, sex, stage, and the emerging prognostic classifier Oncotype DX Colon Cancer Assay recurrence score (hazard ratio 1.5, 95% CI 1.1-2.1, p = 0.0097). However, a limitation of this study is that information on tumor grade and number of nodes examined was not available. CONCLUSIONS: We describe the first, to our knowledge, robust transcriptome-based classification of CC that improves the current disease stratification based on clinicopathological variables and common DNA markers. The biological relevance of these subtypes is illustrated by significant differences in prognosis. This analysis provides possibilities for improving prognostic models and therapeutic strategies. In conclusion, we report a new classification of CC into six molecular subtypes that arise through distinct biological pathways.

Endocrine disrupting chemicals (EDC) are compounds that alter the normal functioning of the endocrine system of both wildlife and humans. A huge number of chemicals have been identified as endocrine disruptors, among them several pesticides. Pesticides are used to kill unwanted organisms in crops, public areas, homes and gardens, and parasites in medicine. Human are exposed to pesticides due to their occupations or through dietary and environmental exposure (water, soil, air). For several years, there have been enquiries about the impact of environmental factors on the occurrence of human pathologies. This paper reviews the current knowledge of the potential impacts of endocrine disruptor pesticides on human health.

The detection of double-stranded (ds) DNA by SYBR Green I (SG) is important in many molecular biology methods including gel electrophoresis, dsDNA quantification in solution and real-time PCR. Biophysical studies at defined dye/base pair ratios (dbprs) were used to determine the structure-property relationships that affect methods applying SG. These studies revealed the occurrence of intercalation, followed by surface binding at dbprs above approximately 0.15. Only the latter led to a significant increase in fluorescence. Studies with poly(dA)* poly(dT) and poly(dG)* poly(dC) homopolymers showed sequence-specific binding of SG. Also, salts had a marked impact on SG fluorescence. We also noted binding of SG to single-stranded (ss) DNA, although SG/ssDNA fluorescence was at least approximately 11-fold lower than with dsDNA. To perform these studies, we determined the structure of SG by mass spectrometry and NMR analysis to be [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]. For comparison, the structure of PicoGreen (PG) was also determined and is [2-[N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]+. These structure-property relationships help in the design of methods that use SG, in particular dsDNA quantification in solution and real-time PCR.

BACKGROUND AND METHODS: The implications of the detection of residual disease after treatment of acute lymphoblastic leukemia (ALL) are unclear. We conducted a prospective study at 11 centers to determine the predictive value of the presence or absence of detectable residual disease at several points in time during the first six months after complete remission of childhood ALL had been induced. Junctional sequences of T-cell-receptor or immunoglobulin gene rearrangements were used as clonal markers of leukemic cells. Residual disease was quantitated with a competitive polymerase-chain-reaction (PCR) assay. Of 246 patients enrolled at diagnosis and treated with a uniform chemotherapy protocol, 178 were monitored for residual disease with one clone-specific probe (in 74 percent) or more than one probe (in 26 percent). The median follow-up period was 38 months. RESULTS: The presence or absence and level of residual leukemia were significantly correlated with the risk of early relapse at each of the times studied (P<0.001). PCR measurements identified patients at high risk for relapse after the completion of induction therapy (those with > or =10(-2) residual blasts) or at later time points (those with > or =10(-3) residual blasts). Multivariate analysis showed that as compared with immunophenotype, age, risk group (standard or very high risk), and white-cell count at diagnosis, the presence or absence and level of residual disease were the most powerful independent prognostic factors. CONCLUSIONS: Residual leukemia after induction of a remission is a powerful prognostic factor in childhood ALL. Detection of residual disease by PCR should be used to identify patients at risk for relapse and should be taken into account in considering alternative treatment.

Progressive loss of T cell functionality is a hallmark of chronic infection with human immunodeficiency virus 1 (HIV-1). We have identified a novel population of dysfunctional T cells marked by surface expression of the glycoprotein Tim-3. The frequency of this population was increased in HIV-1-infected individuals to a mean of 49.4 +/- SD 12.9% of CD8(+) T cells expressing Tim-3 in HIV-1-infected chronic progressors versus 28.5 +/- 6.8% in HIV-1-uninfected individuals. Levels of Tim-3 expression on T cells from HIV-1-infected inviduals correlated positively with HIV-1 viral load and CD38 expression and inversely with CD4(+) T cell count. In progressive HIV-1 infection, Tim-3 expression was up-regulated on HIV-1-specific CD8(+) T cells. Tim-3-expressing T cells failed to produce cytokine or proliferate in response to antigen and exhibited impaired Stat5, Erk1/2, and p38 signaling. Blocking the Tim-3 signaling pathway restored proliferation and enhanced cytokine production in HIV-1-specific T cells. Thus, Tim-3 represents a novel target for the therapeutic reversal of HIV-1-associated T cell dysfunction.

J. Chemistry of rock-forming minerals K. Volcanic emanations, by

OBJECTIVE: Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor expressed in tissues with high oxidative activity that plays a central role in metabolism. In this work, we investigated the effect of hepatocyte PPARα on non-alcoholic fatty liver disease (NAFLD). DESIGN: We constructed a novel hepatocyte-specific PPARα knockout (Pparα(hep-/-)) mouse model. Using this novel model, we performed transcriptomic analysis following fenofibrate treatment. Next, we investigated which physiological challenges impact on PPARα. Moreover, we measured the contribution of hepatocytic PPARα activity to whole-body metabolism and fibroblast growth factor 21 production during fasting. Finally, we determined the influence of hepatocyte-specific PPARα deficiency in different models of steatosis and during ageing. RESULTS: Hepatocyte PPARα deletion impaired fatty acid catabolism, resulting in hepatic lipid accumulation during fasting and in two preclinical models of steatosis. Fasting mice showed acute PPARα-dependent hepatocyte activity during early night, with correspondingly increased circulating free fatty acids, which could be further stimulated by adipocyte lipolysis. Fasting led to mild hypoglycaemia and hypothermia in Pparα(hep-/-) mice when compared with Pparα(-/-) mice implying a role of PPARα activity in non-hepatic tissues. In agreement with this observation, Pparα(-/-) mice became overweight during ageing while Pparα(hep-/-) remained lean. However, like Pparα(-/-) mice, Pparα(hep-/-) fed a standard diet developed hepatic steatosis in ageing. CONCLUSIONS: Altogether, these findings underscore the potential of hepatocyte PPARα as a drug target for NAFLD.

Research Article| May 01 1954 Chromatographic determination of cystine as cysteic acid E. Schram; E. Schram 1Laboratory of Biochemistry, Faculty of Medicine, University of Brussels Search for other works by this author on: This Site PubMed Google Scholar S. Moore; S. Moore * 1Laboratory of Biochemistry, Faculty of Medicine, University of Brussels Search for other works by this author on: This Site PubMed Google Scholar E. J. Bigwood E. J. Bigwood 1Laboratory of Biochemistry, Faculty of Medicine, University of Brussels Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1954) 57 (1): 33–37. https://doi.org/10.1042/bj0570033 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation E. Schram, S. Moore, E. J. Bigwood; Chromatographic determination of cystine as cysteic acid. Biochem J 1 May 1954; 57 (1): 33–37. doi: https://doi.org/10.1042/bj0570033 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search © 1954 CAMBRIDGE UNIVERSITY PRESS1954 Article PDF first page preview Close Modal You do not currently have access to this content.

The adipose differentiation-related protein (ADRP) was first characterized as a mRNA induced early during adipocyte differentiation (Jiang, H. P., and G. Serrero. 1992. Proc. Natl. Acad. Sci. USA. 89:7856-7860). The present study demonstrates that ADRP mRNA is expressed in a variety of tissues and cultured cell lines. Immunocytochemical examination revealed that ADRP localizes to neutral lipid storage droplets in cultured murine 3T3-L1 adipocytes, murine MA-10 Leydig cells, Chinese hamster ovary (CHO) fibroblasts, and human HepG2 hepatoma cells; the association of ADRP with lipid droplets was confirmed by subcellular fractionation of MA-10 Leydig cells. In addition to ADRP, steroidogenic cells and adipocytes express the perilipins, a family of lipid droplet-associated proteins that share a highly related sequence domain with ADRP. ADRP and perilipins co-localize on lipid droplets in MA-10 Leydig cells. While ADRP was found on small lipid droplets in 3T3-L1 preadipocytes and early differentiated adipocytes, it was absent in maturing adipocytes. In contrast, perilipins were absent early during differentiation, but were found on small and large lipid droplets at later stages. The transition in surface protein composition of adipocyte lipid droplets from ADRP to perilipins occurred 3 days after the initiation of differentiation when cells displayed co-localizatioin of both proteins on the same lipid droplets. The specific localization of adipose differentiation-related protein to lipid droplets in a wide variety of cells suggests that ADRP plays a role in management of neutral lipid stores.

Quinolinic acid is an "excitotoxic" metabolite and an agonist of N-methyl-D-aspartate receptors. Of patients infected with human immunodeficiency virus type 1 (HIV-1) who were neurologically normal or exhibited only equivocal and subclinical signs of the acquired immunodeficiency syndrome (AIDS) dementia complex, concentrations of quinolinic acid in cerebrospinal fluid (CSF) were increased twofold in patients in the early stages of disease (Walter Reed stages 1 and 2) and averaged 3.8 times above normal in later-stage patients (Walter Reed stages 4 through 6). However, in patients with either clinically overt AIDS dementia complex, aseptic meningitis, opportunistic infections, or neoplasms, CSF levels were elevated over 20-fold and generally paralleled the severity of cognitive and motor dysfunction. CSF concentrations of quinolinic acid were significantly correlated to the severity of the neuropsychological deficits. After treatment of AIDS dementia complex with zidovudine and treatment of the opportunistic infections with specific antimicrobial therapies, CSF levels of quinolinic acid decreased in parallel with clinical neurological improvement. By analysis of the relationship between levels of quinolinic acid in the CSF and serum and integrity of the blood-brain barrier, as measured by the CSF:serum albumin ratio, it appears that CSF levels of quinolinic acid may be derived predominantly from intracerebral sources and perhaps from the serum. While quinolinic acid may be another "marker" of host- and virus-mediated events in the brain, the established excitotoxic effects of quinolinic acid and the magnitude of the increases in CSF levels of the acid raise the possibility that quinolinic acid plays a direct role in the pathogenesis of brain dysfunction associated with HIV-1 infection.

The high degree of tubulin heterogeneity in neurons is controlled mainly at the posttranslational level. Several variants of alpha-tubulin can be posttranslationally labeled after incubation of cells with [3H]acetate or [3H]glutamate. Peptides carrying the radioactive moiety were purified by high-performance liquid chromatography. Amino acid analysis, Edman degradation sequencing, and mass spectrometric analysis of these peptides led to the characterization of a posttranslational modification consisting of the successive addition of glutamyl units on the gamma-carboxyl group of a glutamate residue (Glu445). This modification, localized within a region of alpha-tubulin that is important in the interactions of tubulin with microtubule-associated proteins and calcium, could play a role in regulating microtubule dynamics.

Fragile X syndrome, a leading cause of inherited mental retardation, is attributable to the unstable expansion of a CGG-repeat within the FMR1 gene that results in the absence of the encoded protein. The fragile X mental retardation protein (FMRP) is a ribosome-associated RNA-binding protein of uncertain function that contains nuclear localization and export signals. We show here detailed cellular localization studies using both biochemical and immunocytochemical approaches. FMRP was highly expressed in neurons but not glia throughout the rat brain, as detected by light microscopy. Although certain structures, such as hippocampus, revealed a strong signal, the regional variation in staining intensity appeared to be related to neuron size and density. In human cell lines and mouse brain, FMRP co-fractionated primarily with polysomes and rough endoplasmic reticulum. Ultrastructural studies in rat brain revealed high levels of FMRP immunoreactivity in neuronal perikarya, where it is concentrated in regions rich in ribosomes, particularly near or between rough endoplasmic reticulum cisternae. Immunogold studies also provided evidence of nucleocytoplasmic shuttling of FMRP, which was localized in neuronal nucleoplasm and within nuclear pores. Moreover, labeling was observed in large- and small-caliber dendrites, in dendritic branch points, at the origins of spine necks, and in spine heads, all known locations of neuronal polysomes. Dendritic localization, which was confirmed by co-fractionation of FMRP with synaptosomal ribosomes, suggests a possible role of FMRP in the translation of proteins involved in dendritic structure or function and relevant for the mental retardation occurring in fragile X syndrome.

To construct an East Asia mitochondrial DNA (mtDNA) phylogeny, we sequenced the complete mitochondrial genomes of 672 Japanese individuals (http://www.giib.or.jp/mtsnp/index_e.html). This allowed us to perform a phylogenetic analysis with a pool of 942 Asiatic sequences. New clades and subclades emerged from the Japanese data. On the basis of this unequivocal phylogeny, we classified 4713 Asian partial mitochondrial sequences, with <10% ambiguity. Applying population and phylogeographic methods, we used these sequences to shed light on the controversial issue of the peopling of Japan. Population-based comparisons confirmed that present-day Japanese have their closest genetic affinity to northern Asian populations, especially to Koreans, which finding is congruent with the proposed Continental gene flow to Japan after the Yayoi period. This phylogeographic approach unraveled a high degree of differentiation in Paleolithic Japanese. Ancient southern and northern migrations were detected based on the existence of basic M and N lineages in Ryukyuans and Ainu. Direct connections with Tibet, parallel to those found for the Y-chromosome, were also apparent. Furthermore, the highest diversity found in Japan for some derived clades suggests that Japan could be included in an area of migratory expansion to Continental Asia. All the theories that have been proposed up to now to explain the peopling of Japan seem insufficient to accommodate fully this complex picture.

The vagus nerve (VN) is a link between the brain and the gut. The VN is a mixed nerve with anti-inflammatory properties through the activation of the hypothalamic-pituitary-adrenal axis by its afferents and by activating the cholinergic anti-inflammatory pathway through its efferents. We have previously shown that VN stimulation (VNS) improves colitis in rats and that the vagal tone is blunted in Crohn's disease (CD) patients. We thus performed a pilot study of chronic VNS in patients with active CD. Seven patients under VNS were followed up for 6 months with a primary endpoint to induce clinical remission and a secondary endpoint to induce biological (CRP and/or fecal calprotectin) and endoscopic remission and to restore vagal tone (heart rate variability). Vagus nerve stimulation was feasible and well-tolerated in all patients. Among the seven patients, two were removed from the study at 3 months for clinical worsening and five evolved toward clinical, biological, and endoscopic remission with a restored vagal tone. These results provide the first evidence that VNS is feasible and appears as an effective tool in the treatment of active CD.

BACKGROUND: Polycystic ovarian morphology (PCOM) at ultrasound is currently used in the diagnosis of polycystic ovary syndrome (PCOS). We hypothesized that the previously proposed threshold value of 12 as an excessive number of follicles per ovary (FN) is no longer appropriate because of current technological developments. In this study, we have revisited the thresholds for FN and for the serum Anti-Müllerian hormone (AMH) level (a possible surrogate for FN) for the definition of PCOM. METHODS: Clinical, hormonal and ultrasound data were consecutively recorded in 240 patients referred to our department between 2008 and 2010 for exploration of hyperandrogenism (HA), menstrual disorders and/or infertility. RESULTS: According to only their symptoms, patients were grouped as: non-PCOS without HA and with ovulatory cycles (group 1, n = 105), presumption of PCOS with only HA or only oligo-anovulation (group 2, n = 73) and PCOS with HA and oligo-anovulation (group 3, n = 62). By cluster analysis using androgens, LH, FSH, AMH, FN and ovarian volume, group 1 appeared to be constituted of two homogeneous clusters, most likely a non-PCOM non-PCOS subgroup (n = 66) and a PCOM, non-PCOS (i.e. asymptomatic) subgroup (n = 39). Receiver operating characteristic curve analysis was applied to distinguish the non-PCOM non-PCO members of group 1 and to group 3. For FN and serum AMH respectively, the areas under the curve were 0.949 and 0.973 and the best compromise between sensitivity (81 and 92%) and specificity (92 and 97%) was obtained with a threshold values of 19 follicles and 35 pmol/l (5 ng/ml). CONCLUSIONS: For the definition of PCOM, the former threshold of >12 for FN is no longer valid. A serum AMH >35 pmol/l (or >5 ng/ml) appears to be more sensitive and specific than a FN >19 and should be therefore included in the current diagnostic classifications for PCOS.

Epstein-Barr virus (EBV) is a herpesvirus associated with nasopharyngeal carcinoma (NPC), gastric carcinoma (GC), and other malignancies. EBV is the first human virus found to express microRNAs (miRNAs), the functions of which remain largely unknown. We report on the regulation of a cellular protein named p53 up-regulated modulator of apoptosis (PUMA) by an EBV miRNA known as miR-BART5, which is abundantly expressed in NPC and EBV-GC cells. Modulation of PUMA expression by miR-BART5 and anti-miR-BART5 oligonucleotide was demonstrated in EBV-positive cells. In addition, PUMA was found to be significantly underexpressed in approximately 60% of human NPC tissues. Although expression of miR-BART5 rendered NPC and EBV-GC cells less sensitive to proapoptotic agents, apoptosis can be triggered by depleting miR-BART5 or inducing the expression of PUMA. Collectively, our findings suggest that EBV encodes an miRNA to facilitate the establishment of latent infection by promoting host cell survival.

t(8;21) is one of the most frequent translocations associated with acute myeloid leukemia. It produces a chimeric protein, acute myeloid leukemia-1 (AML-1)-eight-twenty-one (ETO), that contains the amino-terminal DNA binding domain of the AML-1 transcriptional regulator fused to nearly all of ETO. Here we demonstrate that ETO interacts with the nuclear receptor corepressor N-CoR, the mSin3 corepressors, and histone deacetylases. Endogenous ETO also cosediments on sucrose gradients with mSin3A, N-CoR, and histone deacetylases, suggesting that it is a component of one or more corepressor complexes. Deletion mutagenesis indicates that ETO interacts with mSin3A independently of its association with N-CoR. Single amino acid mutations that impair the ability of ETO to interact with the central portion of N-CoR affect the ability of the t(8;21) fusion protein to repress transcription. Finally, AML-1/ETO associates with histone deacetylase activity and a histone deacetylase inhibitor impairs the ability of the fusion protein to repress transcription. Thus, t(8;21) fuses a component of a corepressor complex to AML-1 to repress transcription.

Recent developments in next-generation sequencing have enabled whole-genome profiling of nucleosome organizations. Although several algorithms for inferring nucleosome position from a single experimental condition have been available, it remains a challenge to accurately define dynamic nucleosomes associated with environmental changes. Here, we report a comprehensive bioinformatics pipeline, DANPOS, explicitly designed for dynamic nucleosome analysis at single-nucleotide resolution. Using both simulated and real nucleosome data, we demonstrated that bias correction in preliminary data processing and optimal statistical testing significantly enhances the functional interpretation of dynamic nucleosomes. The single-nucleotide resolution analysis of DANPOS allows us to detect all three categories of nucleosome dynamics, such as position shift, fuzziness change, and occupancy change, using a uniform statistical framework. Pathway analysis indicates that each category is involved in distinct biological functions. We also analyzed the influence of sequencing depth and suggest that even 200-fold coverage is probably not enough to identify all the dynamic nucleosomes. Finally, based on nucleosome data from the human hematopoietic stem cells (HSCs) and mouse embryonic stem cells (ESCs), we demonstrated that DANPOS is also robust in defining functional dynamic nucleosomes, not only in promoters, but also in distal regulatory regions in the mammalian genome.

Fabry disease (FD) is an X-linked genetic disorder caused by the deficient activity of lysosomal α-galactosidase (α-Gal). While males are usually severely affected, clinical presentation in female patients may be more variable ranging from asymptomatic to, occasionally, as severely affected as male patients. The aim of this study was to evaluate the existence of skewed X-chromosome inactivation (XCI) in females with FD, its concordance between tissues, and its contribution to the phenotype. Fifty-six females with FD were enrolled. Clinical and biological work-up included two global scores [Mainz Severity Score Index (MSSI) and DS3], cardiac magnetic resonance imaging, measured glomerular filtration rate, and measurement of α-Gal activity. XCI was analyzed in four tissues using DNA methylation studies. Skewed XCI was found in 29% of the study population. A correlation was found in XCI patterns between blood and the other analyzed tissues although some punctual variability was detected. Significant differences in residual α-Gal levels, severity scores, progression of cardiomyopathy and deterioration of kidney function, depending on the direction and degree of skewing of XCI were evidenced. XCI significantly impacts the phenotype and natural history of FD in females.