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Biomedical subjects

R Houlgatte

Publications and source records attributed to R Houlgatte.

At least 19 recordsLinked to original sources

Gene expression profiling identifies molecular subgroups among nodal peripheral T-cell lymphomas.

The classification of peripheral T-cell lymphomas (PTCL) is still a matter of debate. To establish a molecular classification of PTCL, we analysed 59 primary nodal T-cell lymphomas using cDNA microarrays, including 56 PTCL and three T-lymphoblastic lymphoma (T-LBL). The expression profiles could discriminate angioimmunoblastic lymphoma, anaplastic large-cell lymphoma and T-LBL. In contrast, cases belonging to the broad category of 'PTCL, unspecified' (PTCL-U) did not share a single molecular profile. Using a multiclass predictor, we could separate PTCL-U into three molecular subgroups called U1, U2 and U3. The U1 gene expression signature included genes known to be associated with poor outcome in other tumors, such as CCND2. The U2 subgroup was associated with overexpression of genes involved in T-cell activation and apoptosis, including NFKB1 and BCL-2. The U3 subgroup was mainly defined by overexpression of genes involved in the IFN/JAK/STAT pathway. It comprised a majority of histiocyte-rich PTCL samples. Gene Ontology annotations revealed different functional profile for each subgroup. These results suggest the existence of distinct subtypes of PTCL-U with specific molecular profiles, and thus provide a basis to improve their classification and to develop new therapeutic targets.

Gene Expression Profiling↗

Feature extraction and signal processing for nylon DNA microarrays.

BACKGROUND: High-density DNA microarrays require automatic feature extraction methodologies and softwares. These can be a potential source of non-reproducibility of gene expression measurements. Variation in feature location or in signal integration methodology may be a significant contribution to the observed variance in gene expression levels. RESULTS: We explore sources of variability in feature extraction from DNA microarrays on Nylon membrane with radioactive detection. We introduce a mathematical model of the signal emission and derive methods for correcting biases such as overshining, saturation or variation in probe amount. We also provide a quality metric which can be used qualitatively to flag weak or untrusted signals or quantitatively to modulate the weight of each experiment or gene in higher level analyses (clustering or discriminant analysis). CONCLUSIONS: Our novel feature extraction methodology, based on a mathematical model of the radioactive emission, reduces variability due to saturation, neighbourhood effects and variable probe amount. Furthermore, we provide a fully automatic feature extraction software, BZScan, which implements the algorithms described in this paper.

Algorithms↗

Gene expression profiling of cancer by use of DNA arrays: how far from the clinic?

DNA arrays allow the simultaneous analysis of expression levels for thousands of genes in normal and pathological tissues and hold great promise in molecular medicine, notably in cancer research. The great biological and clinical diversity present in human tumours is poorly characterised by the current classification systems. DNA arrays can provide a better understanding of oncogenesis, leading to improvements in cancer management. First, the identification of new target genes and pathways will allow the development of specific molecular-based anticancer drugs. Secondly, expression profiles will permit tumour classification in more homogeneous diagnostic and prognostic groups, as well as the identification of new clinically and biologically relevant tumour subclasses. Here, we review the technology and present some cancer studies with promising results. Finally, we discuss some of the issues that must be resolved in the near future, so that DNA arrays can fulfil the aims mentioned above.

Gene Expression Profiling↗

[DNA arrays: technological aspects and applications].

The Human Genome Project has allowed considerable progress in the construction of physical and genetic maps and the identification of genes involved in human sicknesses. The accelerated accumulation of biological information and knowledge is due in large part to the sequencing projects of other organisms, which in fact paved the way for the Human Genome Project. In parallel, recently developed techniques which take advantage of genomic sequences allow large scale molecular analyses resulting in the functional annotation of many of the proteins represented by these genes. This is the goal of functional genomics. These progresses are at the origin of the present revolution in biomedical research. DNA microarrays are playing a dominant role compared to the other developing technologies since they are relatively easy to make and use and are applicable to numerous scientific inquiries. They allow the simultaneous analysis of several thousands of genes in biological samples from sick or healthy tissues, at the genome or transcriptome level. The data obtained is expected to result in major advances in the health sciences. In addition to an improved understanding of the complex molecular interaction networks of healthy cells and tissues, a more precise genetic characterization of the molecular mechanisms involved in pathology should result in the identification of new therapeutic targets and the development of new medicines. The genetic profiles thus obtained should also permit the definition of new pathologic subclasses not recognizable by traditional clinical factors, as well as new markers for susceptibility to certain illnesses, and new prognostic markers or methods of predicting responses to treatment. In this article, we present the different approaches and potential applications of DNA microarray technology, in particular as applied to cancer research.

Chromosome Mapping↗

[Molecular typing of breast cancer: transcriptomics and DNA microarrays].

Breast cancer is the most frequent and deadly cancer of women. Its great heterogeneity makes prognosis and response to current treatments highly variable and difficult to predict. Mammary oncogenesis remains poorly understood. These issues should benefit from recent development of techniques capable of large-scale molecular analyses. The use of cDNA array techniques allows for the simultaneous analysis of the mRNA expression levels of thousands of genes in mammary tumor cell lines and breast tumors. Expression profiles will help classify tumors and provide new prognostic tools and potential therapeutic targets. They will also boost our knowledge of the molecular events responsible for the development and progression of this cancer.

Breast Neoplasms↗

Gene expression profiling of primary breast carcinomas using arrays of candidate genes.

Breast cancer is characterized by an important histoclinical heterogeneity that currently hampers the selection of the most appropriate treatment for each case. This problem could be solved by the identification of new parameters that better predict the natural history of the disease and its sensitivity to treatment. A large-scale molecular characterization of breast cancer could help in this context. Using cDNA arrays, we studied the quantitative mRNA expression levels of 176 candidate genes in 34 primary breast carcinomas along three directions: comparison of tumor samples, correlations of molecular data with conventional histoclinical prognostic features and gene correlations. The study evidenced extensive heterogeneity of breast tumors at the transcriptional level. A hierarchical clustering algorithm identified two molecularly distinct subgroups of tumors characterized by a different clinical outcome after chemotherapy. This outcome could not have been predicted by the commonly used histoclinical parameters. No correlation was found with the age of patients, tumor size, histological type and grade. However, expression of genes was differential in tumors with lymph node metastasis and according to the estrogen receptor status; ERBB2 expression was strongly correlated with the lymph node status (P < 0.0001) and that of GATA3 with the presence of estrogen receptors (P < 0.001). Thus, our results identified new ways to group tumors according to outcome and new potential targets of carcinogenesis. They show that the systematic use of cDNA array testing holds great promise to improve the classification of breast cancer in terms of prognosis and chemosensitivity and to provide new potential therapeutic targets.

Adult↗

Global analysis of gene expression in cells of the immune system II. Cell-free translation products and high-density filter hybridization data.

We have developed an experimental system for linking information on cell-free transcription and translation products from cDNA clones with data obtained from hybridization signals from complex probes. The work described in this paper consists of two distinct processes, one being the construction of a system of clonal addresses and the other the identification of expressed genes involved in the studied processes. We describe the use of this system to identify genes involved in thymus development. Complex probes from fetal thymuses (GD15, 17 and newborn) of Balb/c mice were used to identify genes, which are up- or downregulated during the process of differentiation. The full set of information is available in the Clone-base of the Basel Institute for Immunology and will be retrievable from the website of the collaborating laboratories.

Animals↗

Definition of the alpha 2 region of HLA-DR molecules involved in CD4 binding.

HLA class II molecules present antigenic peptides to the T cell receptor of CD4+ T lymphocytes and interact with CD4 during the antigen recognition process. A major CD4 binding site encompassing amino acids (aa) 134-148 in the beta 2 domain of HLA-DR has been previously identified and residues located within the alpha 2 subunit of murine MHC class II I-Ad molecules have been shown to contribute to CD4-class II interaction. To characterize the alpha 2 region of HLA-DR molecules involved in the binding of CD4, we have synthesized overlapping linear and cyclic peptides derived from a region encompassing aa 121-143. We demonstrate that two linear peptides (aa 124-138 and 130-143) and a cyclic one (aa 121-138) specifically bind to CD4-sepharose affinity columns. Although cyclic analogues exhibit more ordered populations as detected by circular dichroism measurements, cyclization did not improve the activity of some peptides. Peptide sequence positioning in HLA-DR1 dimer model indicates that alpha 2 residues 124 to 136 form a solvent-exposed loop which faces the beta 2 loop delimited by residues 134-148. These data suggest that one CD4 molecule contacts both alpha 2 and beta 2 loops of the HLA-DR homodimer.

Amino Acid Sequence↗

The Genexpress IMAGE knowledge base of the human brain transcriptome: a prototype integrated resource for functional and computational genomics.

Expression profiles of 5058 human gene transcripts represented by an array of 7451 clones from the first IMAGE Consortium cDNA library from infant brain have been collected by semiquantitative hybridization of the array with complex probes derived by reverse transcription of mRNA from brain and five other human tissues. Twenty-one percent of the clones corresponded to transcripts that could be classified in general categories of low, moderate, or high abundance. These expression profiles were integrated with cDNA clone and sequence clustering and gene mapping information from an upgraded version of the Genexpress Index. For seven gene transcripts found to be transcribed preferentially or specifically in brain, the expression profiles were confirmed by Northern blot analyses of mRNA from eight adult and four fetal tissues, and 15 distinct regions of brain. In four instances, further documentation of the sites of expression was obtained by in situ hybridization of rat-brain tissue sections. A systematic effort was undertaken to further integrate available cytogenetic, genetic, physical, and genic map informations through radiation-hybrid mapping to provide a unique validated map location for each of these genes in relation to the disease map. The resulting Genexpress IMAGE Knowledge Base is illustrated by five examples presented in the printed article with additional data available on a dedicated Web site at the address http://idefix.upr420.vjf.cnrs.fr/EXPR++ +/ welcome.html.

Brain Chemistry↗

Mapping of 59 EST gene markers in 31 intervals spanning the human X chromosome.

The positioning of Expressed Sequence Tags (ESTs) constitutes an important step towards a functional map of the human genome, including candidate genes for human genetic disorders that have been localized by linkage analysis. We localized 59 ESTs on the human X chromosome, including 44 derived from infant brain and 15 from adult muscle cDNA libraries. Localizations by a somatic cell hybrid panel were refined for five cDNAs by mapping them in yeast artificial chromosome (YAC) contigs.

Animals↗

Regional assignment of 64 human gene transcripts on chromosome 8.

A panel of somatic cell hybrids specific for human chromosome 8 was used to localize 64 expressed sequence-tagged site (ESTS) markers to six individual regions by PCR amplification. Nine ESTS correspond to 8 known human genes and 6 others show similarities with vertebrate genes, whereas the remaining 49 ESTS markers correspond to novel genes with no database similarities. These gene transcript markers will contribute to the developing physical and expression maps of chromosome 8, and to the search for candidate genes for various human diseases.

Animals↗

A gene map of the human genome.

The human genome is thought to harbor 50,000 to 100,000 genes, of which about half have been sampled to date in the form of expressed sequence tags. An international consortium was organized to develop and map gene-based sequence tagged site markers on a set of two radiation hybrid panels and a yeast artificial chromosome library. More than 16,000 human genes have been mapped relative to a framework map that contains about 1000 polymorphic genetic markers. The gene map unifies the existing genetic and physical maps with the nucleotide and protein sequence databases in a fashion that should speed the discovery of genes underlying inherited human disease. The integrated resource is available through a site on the World Wide Web at http://www.ncbi.nlm.nih.gov/SCIENCE96/.

Amino Acid Sequence↗

Novel gene transcripts preferentially expressed in human muscles revealed by quantitative hybridization of a high density cDNA array.

A set of 1091 human skeletal muscle cDNA clone inserts representing more than 800 human gene transcripts were spotted as PCR products at high density on nylon membranes. Replicas of the filters were hybridized in stringent conditions with 33P-radiolabeled cDNA probes transcribed from skeletal muscle poly(A)+ RNA. Hybridization signals were collected on phosphor screens and processed using a software specifically adapted for this application to identify and quantitate each spot. Parameters likely to influence the hybridization signal intensity were assessed to eliminate artifacts. Each clone was assigned to one of four intensity classes reflecting the steady-state level of transcription of the corresponding gene in skeletal muscle. Differential expression of specific gene transcripts was detected using complex cDNA probes derived from nine different tissues, allowing assessment of their tissue specificity. This made it possible to identify 48 novel gene transcripts (including 7 homologous or related to known sequences) with a muscle-restricted pattern of expression. These results were validated through the analysis of known muscle-specific transcripts and by Northern analysis of a subset of the novel gene transcripts. All these genes have been registered in the Genexpress Index, such that sequence, map, and expression data can be used to decipher their role in the physiology and pathology of human muscles.

Blotting, Northern↗

Molecular basis of T lymphocyte CD4 antigen functions.

The T cell surface antigen CD4 plays a pivotal role in the MHC class II-restricted response of specific T lymphocytes and serves as the major receptor of human immunodeficiency viruses (HIV). Recent studies have shown the high complexity of CD4 functions in physiological and pathological conditions. We report here a short review of recent developments in the field and discuss the structural features which regulate the functions mediated by the CD4 coreceptor in mature T lymphocytes.

Binding Sites↗

Characterization of the Pasteurella multocida skp and firA genes.

A 2.9-kb fragment of the Pasteurella multocida (Pm) genome encoding proteins p25 (25 kDa) and p28 (28 kDa) has previously been cloned and expressed in Escherichia coli (Ec). In the present paper, the nucleotide (nt) sequence of a 1.8-kb subfragment encoding the two proteins is described. The cloned fragment contains three open reading frames (ORFs). ORF1 is incomplete. ORF2 is homologous to the skp gene of Ec. ORF3 overlaps ORF2 and is highly homologous to the firA gene of Ec. The skp and firA genes are part of an operon governing the first steps of lipid A synthesis. Comparing the nt sequence with the N-terminal sequences of p25 and p28 revealed that the two proteins are encoded by ORF2 (skp). The preprotein p28 is converted into p25 by cleavage of a 23-amino-acid leader peptide. Though it serologically cross-reacts with porin H of Pm, p25 is not related to known bacterial porins.

Acyltransferases↗

Competition of HLA-DR and a beta 2 domain peptide for HIV envelope glycoprotein gp120 binding to CD4.

HLA class II molecules and the HIV envelope glycoprotein gp120 are ligands of CD4. Reciprocal interaction sites have been well characterized for gp120 and CD4, but require further definition for HLA class II and CD4. A major CD4 binding site encompassing amino acids 134-148 in the beta 2 domain of HLA-DR has been previously identified. Recently, we have shown, by extensive characterization of mutated CD4 molecules expressed in COS cells, that HLA class II antigens interact mainly through the HIV gp120 binding site and possibly through a second minor interaction site mapping on the same face of the molecule. Based on the direct binding in vitro of iodinated affinity-purified HLA-DR1 molecules to polystyrene immobilized human IgG3-CD4, we now report on reciprocal binding inhibition of gp120, HLA-DR1 and the DR beta 2 synthetic peptide to CD4. The results strongly suggest that gp120 and the beta 2 region (amino acids 134-148) of HLA-DR1 bind mainly to the same part of CD4 domain 1 and that the CD4 binding site of HLA-DR requires the existence of a class II homodimer. In that case, alpha 2 chain residues might interact with CD4 residues different from those involved in the binding of gp120 but located close to them in the first domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

The Genexpress Index: a resource for gene discovery and the genic map of the human genome.

Detailed analysis of a set of 18,698 sequences derived from both ends of 10,979 human skeletal muscle and brain cDNA clones defined 6676 functional families, characterized by their sequence signatures over 5750 distinct human gene transcripts. About half of these genes have been assigned to specific chromosomes utilizing 2733 eSTS markers, the polymerase chain reaction, and DNA from human-rodent somatic cell hybrids. Sequence and clone clustering and a functional classification together with comprehensive data base searches and annotations made it possible to develop extensive sequence and map cross-indexes, define electronic expression profiles, identify a new set of overlapping genes, and provide numerous new candidate genes for human pathologies.

Amino Acid Sequence↗