[Non invasive assessment of embryo quality: proteomics, metabolomics and oocyte-cumulus dialogue]. / Approches non invasives de l'embryon : protéomique, métabolomique, dialogue ovocyte-cumulus.

Preimplantation embryo development is one of the key features with implantation itself to achieve a pregnancy. Assisted Reproductive Technologies both in human and animal have improved our knowledge on these events, although it remains elusive to predict embryo potential to give a baby. Among various ways to define embryo viability, noninvasive approaches get a serious advantage linked to the final transfer of the embryo. Techniques devoted to characterize the embryo secretome using proteomic or metabolomic approaches may be non invasive. Based on a direct identification of products of the embryo metabolism or an assessment of profile(s) related with embryo viability, they have greatly improved their sensitivity to allow their use in clinical embryology, once validated. Oocyte-cumulus dialogue, as a key factor for oocyte competence to meiosis and embryo development, was particularly concerned with both genomic and proteomic assessment of cumulus cells. While it is not possible to designate at the time being which among these approaches will be robust and cost-efficient enough to help routinely the clinical embryologist in assisted reproductive techniques (ART), one can predict that our ability to select the "right" embryo will combine morphological criteria already available with validated biomarkers.

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.

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.

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.

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.

Phorbol 12-myristate 13-acetate (PMA) increases the expression of the nerve growth factor (NGF) gene in mouse L-929 fibroblasts.

The rise of the NGF mRNA pool which takes place following exposure of L-929 fibroblasts to serum was prevented in the presence of 5 microM K-252a, a compound which inhibits several species of protein kinase activities. To characterize further this phenomenon, L-929 cells growing in a serum-free medium were exposed to cyclic nucleotide analogs, to a divalent cation ionophore or to the phorbol ester PMA. Only this latter compound induced an enhancement of the NGF mRNA pool, suggesting an involvement of protein kinase C in the upregulation of the NGF transcripts. The effects of PMA or serum also require a synthesis of protein since the level of NGF transcripts remained stable in the presence of cycloheximide.

Abnormal expression of actin in lymphocytes of Alzheimer's disease and Down's syndrome patients.

Alzheimer's disease (AD) is a degenerative disorder of the central nervous system accompanied by several immunological disturbances and a number of common features exist between AD and Down's syndrome (DS). High resolution two-dimensional electrophoresis of lymphocyte proteins demonstrates an actin abnormality in AD and DS: a double actin spot instead of the single spot observed in controls. This dual form was studied by pulse-chase experiments and seems to be related to extracellular factors which influence the post-translational modification of actin. These results agree with the immunological disturbances observed in AD and DS, and with the well established hypothesis that AD is a systemic as well as cerebral disease.

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.

Measurement of nerve growth factor-like immunoreactivity in human brain using an anti-mouse-NGF enzyme immunoassay.

Nerve growth factor (NGF) like immunoreactivity, expressed as mouse 2.5S nerve growth factor equivalents, was evaluated in three structures of the human brain post-mortem using a commercially available enzyme immunoassay. Regional differences in NGF-like immunoreactivity were observed. Highest levels were found in hippocampus (148 pg/g) compared to putamen (76 pg/g) and frontal cortex (34 pg/g). In addition, these results suggest differences in the distribution of brain NGF between human and rodent, where relatively high levels of NGF are found in the cortex.

Activation of nerve growth factor synthesis in primary glial cells by phorbol 12-myristate 13-acetate: role of protein kinase C.

Phorbol 12-myristate 13-acetate (PMA) induces a dramatic production of nerve growth factor (NGF) in primary cultures of newborn mouse astrocytes maintained in a serum-free medium. This stimulation is dose-dependent and a maximal effect on the levels of cell-secreted factor was observed at a concentration of 10 nM. At this concentration, the promoting effect of PMA appears much more important than that elicited by 10% fetal calf serum (FCS) under the same culture conditions. PMA acts primarily on the accumulation of NGF mRNA, which was detected by northern blot analysis after 6 h of treatment. This accumulation may be totally or partially prevented when PMA-treated glial cells are concomitantly exposed to the protein kinase inhibitors H-7, H-9, and to a lesser degree, HA-1004. The known specificity of these inhibitors agrees with the possibility that protein kinase C (PKC), which constitutes so far the sole known target of PMA, represents a key element involved in the stimulation of NGF gene. The role of PKC is further supported by the observation that alpha phorbol didecanoate, which has no activity on PKC, is depleted of effect on the synthesis of NGF. Likewise, 1,2-dioctanoylglycerol (1,2-DOG) has a weak, but significant promoting action on the production of NGF, unlike the 1,3-isomer which is not active on PKC. Finally, a treatment of 15 min with 100 nM PMA is sufficient to stimulate the cells, suggesting that the activation phase of PKC, rather than its down regulation, constitutes an important trigger leading to an increased expression of the NGF gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Levels of nerve growth factor secreted by rat primary fibroblasts and iris transplants are influenced by serum and glucocorticoids.

Previous work performed with mouse fibroblast-like L cells has shown that the level of expression of NGF gene is modulated in these transformed cells by the composition of the growth medium. Glucocorticoids were found to exert a down-regulation on NGF production, while serum stimulated the synthesis of the factor. The contrasting effects of serum and dexamethasone were further investigated in cultures of primary rat fibroblasts or in iris transplants. ELISA assays of NGF released by fibroblasts or by transplanted irides showed that both experimental systems responded to dexamethasone by a 4-5-fold decrease of the amounts of secreted factor. Half-maximal effect took place at a concentration of 3-5 X 10(-9) M, a value close to the dissociation constant of the glucocorticoid receptor in fibroblasts. The glucocorticoid did not influence the secretion of macromolecules. Assays of NGF mRNA performed at a concentration of 10(-7) M dexamethasone indicated that the steroid decreased the pool of NGF transcripts in either experimental systems. In contrast to dexamethasone, serum induced a 4-fold enhancement of the amounts of factor secreted by fibroblasts. This effect was reproduced with serum that was previously heat-treated at mild acidic pH, or with a macromolecular fraction of this heat-treated serum which contains an effector promoting NGF synthesis in L cells. The fact that promotion of NGF synthesis takes place in primary cells raises the possibility that this process may also occur in vivo, for instance following disruption of vasculature, as a part of a wound mechanism. Data collected with iris transplants provide some support to this interpretation.(ABSTRACT TRUNCATED AT 400 WORDS)

[DNA arrays: technological aspects and applications]. / Puces à ADN: technologie et 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.

[Molecular typing of breast cancer: transcriptomics and DNA microarrays]. / Typage moléculaire du cancer du sein: transcriptome et puces á ADN.

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.

Inhibition of GST-pi nuclear transfer increases mantle cell lymphoma sensitivity to cisplatin, cytarabine, gemcitabine, bortezomib and doxorubicin.

PURPOSE: Mantle cell lymphoma (MCL) is a chemoresistant lymphoma overexpressing the class pi glutathione-S-transferase (GST-pi). The nuclear localisation of GST-pi is induced by chemotherapy and is correlated to cell resistance. In this study, the effect of the Agaricus bisporus lectin (ABL), a GST-pi nuclear transfer inhibitor, on the chemosensitivity of MCL cells was investigated. METHODS: The proliferation of three MCL cell lines was evaluated in the presence of doxorubicin (DOX), cisplatin (CDDP), cytarabine (Ara-C), gemcitabine (GEM) or bortezomib with or without ABL pre-treatment. RESULTS: The cytotoxic activities of CDDP, Ara-C, GEM and bortezomib were increased in all cell lines. The DOX cytotoxic activity was enhanced in two of three cell lines. The inhibition of GST-pi nuclear transfer led to the potentialisation of all drug combinations. CONCLUSION: The inhibition of the nuclear transfer of GST-pi increases the MCL sensitivity to DOX, CDDP, Ara-C, GEM and bortezomib, alone or in combination.

Isolation and analysis of differentially expressed genes in Penicillium glabrum subjected to thermal stress.

Penicillium glabrum is a filamentous fungus frequently involved in food contamination. Numerous environmental factors (temperature, humidity, atmospheric composition, etc.) or food characteristics (water activity, pH, preservatives, etc.) could represent potential sources of stress for micro-organisms. These factors can directly affect the physiology of these spoilage micro-organisms: growth, conidiation, synthesis of secondary metabolites, etc. This study investigated the transcriptional response to temperature in P. glabrum, since this factor is one of the most important for fungal growth. Gene expression was first analysed by using suppression subtractive hybridization to generate two libraries containing 445 different up- and downregulated expressed sequence tags (ESTs). Expression of these ESTs was then assessed for different thermal stress conditions, with cDNA microarrays, resulting in the identification of 35 and 49 significantly up- and downregulated ESTs, respectively. These ESTs encode heat-shock proteins, ribosomal proteins, superoxide dismutase, trehalose-6-phosphate synthase and a large variety of identified or unknown proteins. Some of these may be molecular markers for thermal stress response in P. glabrum. To our knowledge, this work represents the first study of the transcriptional response of a food spoilage filamentous fungus under thermal stress conditions.

Microarray analysis refines classification of non-medullary thyroid tumours of uncertain malignancy.

Conventional histology failed to classify part of non-medullary thyroid lesions as either benign or malignant. The group of tumours of uncertain malignancy (T-UM) concerns either atypical follicular adenomas or the recently called 'tumours of uncertain malignant potential'. To refine this classification we analysed microarray data from 93 follicular thyroid tumours: 10 T-UM, 3 follicular carcinomas, 13 papillary thyroid carcinomas and 67 follicular adenomas, compared to 73 control thyroid tissue samples. The diagnosis potential of 16 selected genes was validated by real-time quantitative RT-PCR on 6 additional T-UM. The gene expression profiles in several groups were examined with reference to the mutational status of the RET/PTC, BRAF and RAS genes. A pathological score (histological and immunohistochemical) was estimate for each of the T-UM involved in the study. The correlation between the T-UM gene profiles and the pathological score allowed a separation of the samples in two groups of benign or malignant tumours. Our analysis confirms the heterogeneity of T-UM and highlighted the molecular similarities between some cases and true carcinomas. We demonstrated the ability of few marker genes to serve as diagnosis tools and the need of a T-UM pathological scoring.

Serum contains a macromolecular effector promoting the synthesis of nerve growth factor (NGF) in L cells.

Addition of serum to the culture medium of murine L cells increased both the cellular level of NGF mRNA and the secretion of mature factor. Stimulation of NGF production by the serum was dose-dependent and appeared mediated by some specific factor(s). After gel filtration chromatography of serum, most of the biological activity formed a major peak with an apparent MW of about 160 kDa. This promoting factor was sensitive to heat at neutral pH, but resisted after heating at pH4. An activity inducing NGF synthesis, and displaying a comparable thermal sensitivity was also detected in Cohn fraction IV of human or bovine plasma.

Dexamethasone rapidly reduces the expression of the beta-NGF gene in mouse L-929 cells.

Mouse L-929 cells were treated with dexamethasone, and the cellular levels of beta-NGF mRNA were estimated by hybridization of the RNAs with a beta-NGF cDNA probe. The results revealed that the glucocorticoid decreased specifically, in a dose-dependent manner, the pool of beta-NGF transcripts. After 4 h, L-929 cells cultured with 10(-7) M dexamethasone contained one-fifth as much beta-NGF mRNA as untreated control cells, and as little as one-tenth as much when the glucocorticoid concentration was 10(-6) M. The effect of the hormone became maximal after 8 h of treatment. Amounts of beta-NGF secreted by the cells during 24 h were measured with a two-site enzyme immunoassay. They also appeared reduced in cultures exposed to the glucocorticoid. These data indicate that dexamethasone controls negatively the expression of the beta-NGF gene in L-929 cells at some pre-translational level.

Synthesis and secretion of beta-nerve growth factor by mouse teratocarcinoma cell lines.

Using a cDNA probe and a two-site enzyme immunoassay, beta-nerve growth factor (beta NGF) synthesis was monitored in several mouse teratocarcinoma cell lines. Trace amounts of NGF mRNA were detected in the embryonal carcinoma (EC) PCC4, F9 and 1003 clones, whereas the myocardial (PCD1), myogenic (1168) and adipogenic (1246) clones contained significantly higher levels of NGF mRNA and secreted mature beta NGF peptide in the culture medium. The 1003, 1168 and 1246 strains were derived from the same teratocarcinoma cell line and their ability or inability to synthesize the neurotrophic factor may reflect a developmental decision for divergent differentiation programs. Induction of NGF mRNA and protein synthesis was observed in a differentiated derivative of an SV40-transformed F9 clone which expresses the viral T antigen. Southern blot analysis of the genomic DNAs revealed no structural alterations of the NGF locus between teratocarcinoma cells that express the NGF gene and those that do not. Similar analysis of the DNA methylation pattern in C-C-G-G sequences using the Hpa II and Msp I isoschizomers indicated no methylation changes of the NGF gene in the teratocarcinoma DNAs. At least two, and probably all four, of the already mapped Msp I sites within the NGF gene are methylated in all teratocarcinoma DNAs examined, as well as in the male mouse submaxillary gland DNA, the organ richest in this factor.

Secretion of nerve growth factor in cultures of glial cells and neurons derived from different regions of the mouse brain.

The regional ability of central neurons and glial cells to produce nerve growth factor (NGF) was studied in vitro. NGF secretion was compared in cultures of perinatal astrocytes or embryonic neurons that were derived from various mouse brain structures. No regional differences were detected among cultures of post-natal day 2 glial cells of hippocampal, cortical, striatal, or mesencephalic origin. In all cases, levels of NGF released by the cells were very similar. They were closely correlated to the growth rate as shown by the fact that exponentially growing cells produced relatively more factor than did confluent cells, a finding in agreement with previous observations. Unlike growth-phase cells, primary astrocytes immediately plated at high cell density did not secrete any assayable factor before the 7th day of culture. Levels of NGF found during the following days remained low. In contrast, striking differences were observed among cultures of embryonic neurons. NGF was found in relatively large amounts in cultures of embryonic day 17 or 19 striatal neurons, whereas media conditioned by neurons from the mesencephalon, cortex, or septum contained much less factor. Amounts of NGF assayed in cultures of hippocampal neurons varied with the time of sampling of this brain structure. Levels of factor were significantly higher in media conditioned by embryonic day 19 neurons than in media of embryonic day 17 neurons. However, amounts of NGF found in supernatants of hippocampal neurons remained smaller than those present in cultures of striatal nerve cells. Altogether, the results suggest that, in addition to astrocytes, central neurons may also synthesize and secrete NGF in vitro and that this phenomenum is dependent on both the origin and the developmental stage of the neuronal population.