Biomedical subjects

Rodney J Ouellette

Publications and source records attributed to Rodney J Ouellette.

9 recordsLinked to original sources

PubMed2006

Data analysis of alternative splicing microarrays.

The importance of alternative splicing in drug and biomarker discovery is best understood through several example genes. For most genes, the identification, detection and particularly quantification of isoforms in different tissues and conditions remain to be carried out. As a result, the focus in drug and biomarker development is increasingly on high-throughput studies of alternative splicing. Initial strategies for the parallel analysis of alternative splicing by microarrays have been recently published. The design specificities and goals of alternative splicing microarrays, in terms of identification and quantification of multiple mRNAs from one gene, are promoting the development of novel methods of analysis.

Algorithms↗

PubMed2006

The 5-HT2A serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin.

Serotonin (5-hydroxytryptamine, 5-HT) has been described as a mitogen in a variety of cell types and carcinomas. It exerts its mitogenic effect by interacting with a wide range of 5-HT receptor types. Certain studies suggest that some selective serotonin re-uptake inhibitors promote breast cancer in animals and humans. This study attempts to clarify the role of serotonin in promoting the growth of neoplastic mammary cells. Expression of the 5-HT(2A) serotoninergic receptor subtype in MCF-7 cells was determined by RT-PCR, Western blotting, and immunofluorescence analysis. The mitogenic effect of 5-HT on MCF-7 cells was determined by means of the MTT proliferation assay. We have demonstrated that the 5-HT(2A) receptor subtype is fully expressed in the MCF-7 human breast cancer cell line, in terms of encoding mRNA and receptor protein. Automated sequencing has confirmed that the 5-HT(2A) receptor present in this cell line is identical to the 5-HT(2A) receptor found in human platelets and in human cerebral cortex. Furthermore, this receptor was found by immunofluorescence to be on the plasma membrane. MTT proliferation assays revealed that 5-HT and DOI, a selective 5-HT(2A) receptor subtype agonist, stimulated MCF-7 cell. These results indicate that 5-HT plays a mitogenic role in neoplastic mammary cells. Our data also indicate that 5-HT exerts this positive growth effect on MCF-7 cells through, in part, the 5-HT(2A) receptor subtype, which is fully expressed in this cell line.

Amphetamines↗

PubMed2006

Optimized hormonal stimulation is critical for production of viable embryos and establishment of subsequent implantation.

PURPOSE: To investigate the effects of follicle number and size at the time of hCG administration, and ovarian stimulation length on the outcome of in vitro fertilization and pregnancy rate. METHODS: During the ovarian stimulation regimen, the follicular number and size were determined by transvaginal ultrasonographic examination. Ovulation was induced as early as three or more follicles were at least 16 mm in their greatest diameter. RESULTS: The fertilization rates were significantly increased with the longer length of stimulation (10-12 days: 75.4% and 13-16 days: 83.2%). However, no significant differences in the chemical pregnancy, clinical pregnancy, and implantation rates were found between 10-12 days (53.7%, 43.9%, and 21.8%) and 13-16 days (50.0%, 43.8%, and 23.4%) of stimulation. There were no significant differences in fertilization and chemical pregnancy rates between two groups with > or = and <18 follicles in the ovaries on the day of hCG (human chorionic gonadotrophin) administration. However, the clinical pregnancy and implantation rates (47.2% and 26.0%) in the group with <18 follicles were significantly higher than those (33.3% and 15.5%) in the group with > or =18 follicles, respectively. CONCLUSIONS: Excessive and rapid ovarian stimulation appears to decrease the survival of embryos at later stages after transfer. The advantage of prolonged stimulation may outweigh the potential adverse effects in some patients.

Chorionic Gonadotropin↗

PubMed2006

Carbohydrate microarrays: survey of fabrication techniques.

Carbohydrate microarrays are being developed in order to decipher the information content of the glycome. This postgenomic activity is necessary because of the complexity of protein biosynthesis and post-translational modifications that cannot currently be detected at the genome level. This review looks, in detail, at the experimental approaches that have been taken in the fabrication and preparation of carbohydrate microarrays, glycan arrays and glyco-chips. Tether structures, glycan solution preparation, detection methods and applications have been gathered together in a tabular format.

Animals↗

PubMed2006

Microarray analysis of alternative splicing.

Alternative splicing, defined as the generation of multiple RNA transcript species from a common mRNA precursor, is one of the mechanisms for the diversification and expansion of cellular proteins from a smaller set of genes. Current estimates indicate that at least 60% of genes in the human genome exhibit alternative splicing. Over the past decade, alternative splicing has increasingly been recognized as a major regulatory process with a critical role in normal development. Furthermore, the importance of alternative splicing in disease development and treatment is starting to be appreciated. Therefore, an increasing number of high-throughput genomics and proteomics studies are being performed in order to delineate (a) the changes in alternative splicing under various conditions; (b) the properties and functions of protein isoforms; and (c) the splicing and alternative splicing regulation process. Strategies for the parallel analysis of alternative splice forms by microarray experiments have been conceived, and examples have been published. In addition to the differences in microarray probe design, the analysis of microarrays with probes for exons, exon/exon junctions as well as specific splice forms is significantly different from the standard experiment. Several methods are being developed in order to address the particular needs of alternative splicing microarrays. Many reviews have already dealt with alternative splicing. However, high-throughput analysis methods that are becoming increasingly popular have not received much attention. Here, we will provide an overview of the tools and analysis methods that were developed specifically for alternative splicing microarrays described in terms of specific experiments.

Alternative Splicing↗

PubMed2005

Determination of tumour marker genes from gene expression data.

Cancer classification has traditionally been based on the morphological study of tumours. However, tumours with similar histological appearances can exhibit different responses to therapy, indicating differences in tumour characteristics on the molecular level. Thus, development of a novel, reliable and precise method for classification of tumours is essential for more successful diagnosis and treatment. The high-throughput gene expression data obtained using microarray technology are currently being investigated for diagnostic applications. However, these large datasets introduce a range of challenges, making data analysis a major part of every experiment for any application, including cancer classification and diagnosis. One of the major concerns in the application of microarrays to tumour diagnostics is the fact that the expression levels of many genes are not measurably affected by carcinogenic changes in the cells. Thus, a crucial step in the application of microarrays to cancer diagnostics is the selection of diagnostic marker genes from the gene expression profiles. These molecular markers give valuable additional information for tumour diagnosis, prognosis and therapy development.

Biomarkers, Tumor↗

PubMed2004

Human Pax-5 C-terminal isoforms possess distinct transactivation properties and are differentially modulated in normal and malignant B cells.

The transcription factor Pax-5 occupies a central role in B cell differentiation and has been implicated in the development of B cell lymphoma. The transcriptional activation function of Pax-5 requires an intact N-terminal DNA-binding domain and is strongly influenced by the C-terminal transactivation domain. We report the identification and characterization of five human Pax-5 isoforms, which occur through the alternative splicing of exons that encode for the C-terminal transactivation domain. These isoforms arise from the inclusion or exclusion of exon 7, exon 8, and/or exon 9. Three of the Pax-5 isoforms generate novel protein sequences rich in proline, serine, and threonine amino acids that are the hallmarks of transactivation domains. The Pax-5 isoforms are expressed in peripheral blood mononuclear cells, cancerous and non-cancerous B cell lines, as well as in primary B cell lymphoma tissue. Electrophoretic mobility shift assays demonstrate that the isoforms possess specific DNA binding activity and recognize the PAX-5 consensus binding sites. In reporter assays using the CD19 promoter, the transactivation properties of the various isoforms were significantly influenced by the changes in the C-terminal protein sequence. Finally, we demonstrate, for the first time, that human Pax-5 isoform expression is modulated by specific signaling pathways in B lymphocytes.

Alternative Splicing↗

PubMed2004

RT-PCR for mammaglobin genes, MGB1 and MGB2, identifies breast cancer micrometastases in sentinel lymph nodes.

In the present study, we examined the expression of the mammaglobin genes, MGB1 and MGB2, in the sentinel lymph nodes (SLNs) of patients with breast cancer and compared our results with the histologic status of the same SLNs. Compared with immunohistochemical staining for cytokeratin 8, which detected metastases in 17 of 42 patients, reverse transcription-polymerase chain reaction (RT-PCR) for MGB1 or MGB2 genes was positive in 22 patients. The concordance between the expression of any mammaglobin and histologic status was 79% (33/42), with a sensitivity of 88% and specificity of 72%. The detection of patients with metastases was more sensitive when testing for both MGB1 and MGB2 (P < .0001) rather than MGB2 (P < .0005) or MGB1 (P < .05) alone. The increased detection rate relative to histologic examination suggests that using RT-PCR for the mammaglobin genes might identify patients at higher risk compared with patients with negative RT-PCR results.

Adult↗

PubMed2003

Investigation of interaction between Pax-5 isoforms and thioredoxin using de novo modelling methods.

Pax-5 transcription factor plays a crucial role in B-cell development, activation and differentiation. In murine B-cells four different isoforms of Pax-5 have been identified, and their role in the regulation of the activity of the wild-type protein was revealed although still not fully understood. Using theoretical methods, we investigated the properties of one region of the Pax-5e and Pax-5d isoforms (named UDE domain) and we present a possible theoretical model for the interaction of this domain with thioredoxin that have been previously postulated based on the experimental results. Domain UDE (MW 4.8 kDa) is characterised by an extremely high ratio of positively charged residues (8) in comparisons to negatively charged amino acids (3), as well as unusually large concentrations of prolines (11.6%) and cysteines (4.7%). This is indicative of its role in protein-protein interaction. The experimental 3D structure for either UDE domain or for any analogous sequence is not yet available, and therefore we resorted to various bioinformatics methods in order to predict the secondary and 3D structure from the primary sequence of UDE. Physicochemical properties of the predicted UDE structure gave more indication about possibilities for UDE-thioredoxin binding. In addition, UDE domain was shown to have both sequence and structure analogous to a segment of NAD-reducing hydrogenase HOXS a subunit which is believed to interact with thioredoxin. These studies showed that the UDE domain in Pax-5d and Pax-5e represents an ideal binding site for thioredoxin and we developed a model of UDE-TRX complex with two disulphide bridges. The active site of thioredoxin remained exposed after binding to UDE in this model and therefore binding of thioredoxin to Pax-5d could explain the unexpectedly high resistance of this isoform to oxidation. The complex between thioredoxin and Pax-5e can be a method for transportation of thioredoxin into the nucleus and also into the the vicinity of Pax-5a, explaining the observed activator role of Pax-5e.

Amino Acid Sequence↗