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Biomedical subjects

C Bolduc

Publications and source records attributed to C Bolduc.

15 recordsLinked to original sources

Reproducibility, bioinformatic analysis and power of the SAGE method to evaluate changes in transcriptome.

The serial analysis of gene expression (SAGE) method is used to study global gene expression in cells or tissues in various experimental conditions. However, its reproducibility has not yet been definitively assessed. In this study, we have evaluated the reproducibility of the SAGE method and identified the factors that affect it. The determination coefficient (R2 ) for the reproducibility of SAGE is 0.96. However, there are some factors that can affect the reproducibility of SAGE, such as the replication of concatemers and ditags, the number of sequenced tags and double PCR amplification of ditags. Thus, corrections for these factors must be made to ensure the reproducibility and accuracy of SAGE results. A bioinformatic analysis of SAGE data is also presented in order to eliminate these artifacts. Finally, the current study shows that increasing the number of sequenced tags improves the power of the method to detect transcripts and their regulation by experimental conditions.

Animals↗

Effects of dihydrotestosterone on adipose tissue measured by serial analysis of gene expression.

Intra-abdominal fat accumulation is related to several diseases, especially diabetes and heart disease. Molecular mechanisms associated with this independent risk factor are not well established. Through the serial analysis of gene expression (SAGE) strategy, we have studied the transcriptomic effects of castration and dihydrotestosterone (DHT) in retroperitoneal adipose tissue of C57BL6 male mice. Approximately 50,000 SAGE tags were isolated in intact and gonadectomized mice, as well as 3 and 24 h after DHT administration. Transcripts involved in energy metabolism, such as glyceraldehyde-3-phosphate dehydrogenase, malic enzyme supernatant, fatty acid synthase, lipoprotein lipase, hormone-sensitive lipase and monoglyceride lipase, were upregulated by DHT. Transcripts involved in adipogenesis, and cell cycle and cell shape organization, such as DDX5, C/EBPalpha, cyclin I, procollagen types I, III, IV, V and VI, SPARC and matrix metalloproteinase 2, were upregulated by DHT. Cell defense, division and signaling, protein expression and many novel transcripts were regulated by castration and DHT. The present results provide global genomic evidence for a stimulation of glycolysis, fatty acids and triacylglycerol production, lipolysis and cell shape reorganization, as well as cell proliferation and differentiation, by DHT. The novel transcripts regulated by DHT may contribute to identify new mechanisms involved in the action of sex hormones and their potential role in obesity.

Adipose Tissue↗

DPCP for the treatment of alopecia areata.

Topical immunotherapy with diphencyprone (DPCP) for the treatment of severe alopecia areata has been used since 1983 and is felt to be the treatment of choice by many dermatologists. Although there have been no major side effects reported since its initial use, there remain some unknowns regarding its safety. Because DPCP has at least a 40% success rate for cosmetically acceptable regrowth in extensive alopecia areata, its availability is an important matter for patients with alopecia areata.

Administration, Topical↗

Calmodulin point mutations affect Drosophila development and behavior.

Calmodulin (CAM) is recognized as a major intermediary in intracellular calcium signaling, but as yet little is known of its role in developmental and behavioral processes. We have generated and studied mutations to the endogenous Cam gene of Drosophila melanogaster that change single amino acids within the protein coding region. One of these mutations produces a striking pupal lethal phenotype involving failure of head eversion. Various mutant combinations produce specific patterns of ectopic wing vein formation or melanotic scabs on the cuticle. Anaphase chromosome bridging is also seen as a maternal effect during the early embryonic nuclear divisions. In addition, specific behavioral defects such as poor climbing and flightlessness are detected among these mutants. Comparisons with other Drosophila mutant phenotypes suggests potential CAM targets that may mediate these developmental and behavioral effects, and analysis of the CAM crystal structure suggests the structural consequences of the individual mutations.

Alleles↗

Spontaneous avoidance behavior in Drosophila null for calmodulin expression.

The regulatory protein calmodulin is a major mediator of calcium-induced changes in cellular activity. To analyze the roles of calmodulin in an intact animal, we have generated a calmodulin null mutation in Drosophila melanogaster. Maternal calmodulin supports calmodulin null individuals throughout embryogenesis, but they die within 2 days of hatching as first instar larvae. We have detected two pronounced behavioral abnormalities specific to the loss of calmodulin in these larvae. Swinging of the head and anterior body, which occurs in the presence of food, is three times more frequent in the null animals. More strikingly, most locomotion in calmodulin null larvae is spontaneous backward movement. This is in marked contrast to the wild-type situation where backward locomotion is seen only as a stimulus-elicited avoidance response. Our finding of spontaneous avoidance behavior has striking similarities to the enhanced avoidance responses produced by some calmodulin mutations in Paramecium. Thus our results suggest evolutionary conservation of a role for calmodulin in membrane excitability and linked behavioral responses.

Animals↗

A second maternally expressed Drosophila gene encodes a putative RNA helicase of the "DEAD box" family.

Recently, a family of proteins containing the conserved motif Asp-Glu-Ala-Asp, the "DEAD box" proteins, has been identified. This family is typified by the eukaryotic translation initiation factor eIF4A, and its members are believed to share the functional property of ATP-dependent RNA unwinding. One of the previously identified members of this family (vasa) is the product of a maternally expressed gene from Drosophila melanogaster that is known to play a role in the formation of the embryonic body plan. We report here the isolation of a Drosophila gene that has an mRNA expression pattern somewhat similar to that of vasa and also encodes a DEAD box protein. We have termed this gene ME31B to reflect its maternal (ovarian germ-line) expression and its location within the 31B chromosome region. Comparisons with the other members of this family reveal that although ME31B is most like the protein Tif1/Tif2, which probably represents the Saccharomyces cerevisiae version of eIF4A, it is unlikely that ME31B represents the Drosophila eIF4A protein per se. A search for mutations in the ME31B gene has established that the P element which causes the female-sterile mutation flipper lies in the 3' flank of the ME31B gene.

Amino Acid Sequence↗

Beta-tubulin mutants of the unicellular green alga Chlamydomonas reinhardtii.

Two beta-tubulin mutants of the unicellular green alga Chlamydomonas reinhardtii have been isolated on the basis of altered sensitivity to the growth-inhibitory effect of colchicine. The two mutations of colR4 and colR15 have been found to be tightly linked, mapping to a previously unmarked site in linkage group XII. The drug-resistance phenotypes of both mutations segregated in genetic crosses with the presence of distinct, acidic variant beta-tubulin isoforms found assembled into the microtubules of the flagella. Analysis of the in vitro translation products of total poly(A)+ RNA from the mutants provided evidence that the variant proteins are altered primary beta-tubulin gene products. Compared to wild type, strains carrying the mutations expressed an increased resistance to the inhibitory effects of colchicine in clonal growth, flagellar assembly, and germination of meiotic products, suggesting that the beta-tubulin altered in the mutants participates in multiple microtubule functions.

Chlamydomonas↗

Biosynthesis of kitasamycin (leucomycin) by leucine analog-resistant mutants of Streptomyces kitasatoensis.

The biosynthesis of kitasamycin in Streptomyces kitasatoensis B-896 was profoundly influenced by the addition of precursors to complex and defined media: l-valine and l-leucine directed biosynthesis towards the pairs A(4)/A(5) (R(2) = butyryl) and A(1)/A(3) (R(2) = isovaleryl), respectively, and total kitasamycin titers were doubled and quadrupled, respectively. S. kitasatoensis B-896 was very resistant (>20 mg/ml) to alpha-aminobutyric acid, an analog of l-valine, but very susceptible to l-leucine analogs 5', 5', 5'-trifluoroleucine and 4-azaleucine (5 to 10 mug/ml). The inhibition by 4-azaleucine could be reversed by l-leucine, but by none of the other amino acids of the pyruvate family or the amino acids of the aspartate pathway. 4-Azaleucine-resistant mutants were isolated which in the absence of any precursors overproduced l-leucine and a kitasamycin complex mainly consisting of the pair A(1)/A(3). These 4-azaleucine-resistant mutants are presumed to be regulatory mutants in which alpha-isopropylmalate synthase, the first enzyme of the l-leucine pathway, has become either derepressed or desensitized to leucine feedback inhibition. l-Leucine-regulatory mutants have economic value: in the absence of expensive precursors, they produce a kitasamycin complex in which the most potent pair A(1)/A(3) is dominant and the least active components are absent.

Anti-Bacterial Agents↗

Antimycin A fermentation. I. Production and selection of strains.

Increase in antimycin A production was achieved through a parallel strain and medium improvement program: a 125-fold augmentation (75 to 9,500 mug/ml) was obtained. The selective system included antimycin A productivity, conidiation, sensitivity to ultraviolet radiation, growth rate and yield, and absence of pigment and actinomycin D production. Among the original strains tested one natural isolate possessed high productivity and several of the above characteristics, and was selected for mutagenesis. Spontaneous and induced variability was then exploited in isolating high-producing strains. The first mutagen used was ultraviolet radiation; it was replaced by ethylenimine when it became no longer efficient in increasing variability. As new, high producers were isolated, the medium was modified to best suit their requirements for still higher productivity. The critical environmental factors were absence of phosphate and organic salts, concentration of the nitrogen source and ratio organic/inorganic nitrogen, ratio ammonium sulfate/calcium carbonate, and addition of slowly utilizable carbon sources, such as lactose and oil; optimum temperature and initial pH were 25 degrees C and 7.0. Aeration/agitation requirements of improved strains were high. Fermentation was characterized by abrupt pH changes which impaired rapid accumulation of the antibiotic. Antimycin A was produced during both the trophophase and idiophase.

Antimycin A↗

Tissue distribution and cytofluorometric analysis of oral mucosal T cells in the BALB/c mouse.

The incidence and distribution of Thy-1.2+, Lyt-2.2+ and L3T4+ cells in the murine oral mucosa were investigated using qualitative and quantitative approaches. From immunostaining of frozen tissue sections, it appeared that the majority of oral T cells are located either in the epithelium or within the minor salivary gland network. The occurrence of Thy-1.2+, L3T4+ and Lyt-2.2+ cells at these sites points to two strategic lines of defence in the event of mucosal infections or aggression. A quantitative analysis of oral T-cell subsets was made possible by optimizing an enzymatic digestion procedure which preserves all three T-cell surface markers. Flow cytometric analysis of oral mucosal cells demonstrated that the helper phenotype is about twice as numerous as the cytotoxic/suppressor phenotype in the mucosa. Furthermore, in single cell suspensions, virtually all Thy-1+ cells were either L3T4+ or Lyt-2.2+ in the mucosa and in the spleen. From this frequency analysis and our previous studies, we conclude that T cells are a major component of the oral immune system, being 2-3 times as numerous as B cells or macrophages. Present data on the spatial distribution and characteristic ratio of T-cell subsets assess the basal activity of the local T-cell populations in healthy animals and lay the basis for comparative studies of both qualitative and quantitative variations occurring during mucosal infections or autoimmune reactions.

Animals↗

Management of androgenetic alopecia.

Androgenetic alopecia is by far the most common cause of hair loss. It affects approximately 50% of men by the age of 50 and 20 to 53% of women by the age 50. Although it is a medically benign condition, it is a significant psychosocial issue for many patients. Various different treatment options are now available for androgenetic alopecia. The best treatment option for women with androgenetic alopecia Ludwig stage I and II is minoxidil 5% solution. If it is not effective after 1 year, antiandrogens can be tried, but there are no large studies showing their efficacy and they have considerable adverse effects. Also, for patients with alopecia that is unresponsive to treatment or with Ludwig stage III, hair transplantation can be offered if the occipital donor area is sufficient. For men, we always offer minoxidil or finasteride therapy and leave the choice of therapy to the patient. Some patients may prefer a systemic agent, whereas others may favor a topical agent. If the condition is not stabilized after 1 year or if the patient wants greater hair density, hair transplantation can be discussed. There have been tremendous advances in the treatment of hair loss in recent years and the future is very encouraging. As our knowledge of androgenetic alopecia pathophysiology increases, novel targeted treatments will potentially be developed.

Alopecia↗

Receptors for the Fc portion of immunoglobulins (FcR) on murine oral mucosal T cells.

The incidence and distribution of immunoglobulin receptors on oral mucosal T cells were investigated. Enrichment of either Thy-1.2, L3T4 or Lyt-2.2-positive cells was achieved by the use of a panning procedure following cell extraction by an optimized digestion method that preserved all three T-cell markers. The percentage of cells possessing Fc receptors was determined by using immunoglobulin-coated (IgM, IgG or IgA) fluorescent microspheres in a multipoint rosetting assay. We report that approximately 60% of Thy-1.2, L3T4 and Lyt-2.2 positive cells bear Fc gamma R whereas less than 5% of T cells of any subset bear Fc alpha R or Fc mu R. In frozen tissue sections, Fc gamma R+ cells were mainly scattered throughout the basal and suprabasal epithelium and were observed at a lower frequency in the minor salivary gland network. From their distribution, it is anticipated that Fc gamma R+ cells may be involved in the surveillance of the epithelium while the minor Fc alpha R+ L3T4+ T lymphocyte population may promote the expression of sIgA by resident sIgM-bearing B cells, and their differentiation into IgA plasma cells.

Animals↗