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J Beaudoin

Publications and source records attributed to J Beaudoin.

At least 19 recordsLinked to original sources
PubMed2001

A novel copper-regulated promoter system for expression of heterologous proteins in Schizosaccharomyces pombe.

The increasing use of the fission yeast Schizosaccharomyces pombe as a model organism for elucidating the mechanisms of critical biological processes such as cell-cycle control, DNA replication, and stress-mediated signal transduction has fostered the development and utilization of expression systems for gene function analysis. Using the promoter of the ctr4(+) copper transporter gene from S. pombe, we created a series of vectors, named pctr4(+)-X, which regulate the expression of heterologous genes as a function of copper availability. In this system, the addition of copper ions at levels that are non-toxic to yeast cells represses gene expression, while copper deprivation strongly induces gene expression. Conveniently, changes of growth medium or carbon sources are not required to shut down or induce gene expression. The Cu-starvation-mediated inducible expression system is rapid, producing heterologous proteins within 3 h, with sustained expression of proteins that persists for several hours. The pctr4(+)-X expression vectors harbor unique restriction sites constructed in-frame to DNA sequences encoding for epitope tags, which facilitate the detection or purification of the heterologous proteins using commercially available antibodies and affinity columns. Furthermore, the pctr4(+)-X copper-regulatable protein expression vectors have been constructed with three different selectable markers, offering more versatility for studying gene function in fission yeast.

Cation Transport Proteins↗
PubMed2001

The fission yeast copper-sensing transcription factor Cuf1 regulates the copper transporter gene expression through an Ace1/Amt1-like recognition sequence.

Transcriptional regulation of genes encoding critical components of copper transport is essential for copper homeostasis and growth in yeast. Analysis of regulatory regions in the promoter of the ctr4(+) copper transporter gene in fission yeast Schizosaccharomyces pombe reveals the identity of a conserved copper-signaling element (CuSE), which is recognized by the transcription factor Cuf1. We demonstrate that CuSE is necessary for transcriptional activation in response to copper deprivation conditions. Interestingly, the CuSE element bears a strong sequence similarity to the recognition site, denoted MRE (metal regulatory element), which is recognized by a distinct class of copper sensors required for copper detoxification, including Ace1 from Saccharomyces cerevisiae and Amt1 from Candida glabrata. When a consensus MRE from S. cerevisiae is introduced into S. pombe, transcription is induced by copper deprivation in a Cuf1-dependent manner, similar to regulation by Mac1, the nuclear sensor for regulating the expression of genes encoding components involved in copper transport in S. cerevisiae. UV-cross-linking experiments show that the Cuf1 protein directly binds the CuSE. These results demonstrate that the Cuf1 nutritional copper-sensing factor possesses a module that functions similarly to domains found in the Ace1/Amt1 class of metalloregulatory factors, which allows the protein to act through a closely related MRE-like sequence to regulate copper transport gene expression in S. pombe.

Base Sequence↗
PubMed2000

Species-specific regulation of alternative splicing in the C-terminal region of the p53 tumor suppressor gene.

Alternative splicing occurs in the C-terminal region of the p53 tumor suppressor gene between two alternative 3' splice sites in intron 10. This alternative splicing event has been detected in murine cells, but not in rat or human tissues. In this paper, we have characterized the pattern of p53 alternative splicing in cell lines from five different species. Our results confirm that p53 alternative splicing is species-specific, being detected only in cell lines of rodent origin. Using transient transfection assays, we have established that the rat p53 gene undergoes efficient alternative splicing in both mouse and rat cell lines, thus demonstrating that it has all the necessary cis -acting sequences to be alternatively spliced. In contrast, we were unable to detect any usage of the human alternative 3' splice site under the same experimental conditions. Thus, the low levels or absence of alternatively spliced p53 mRNA in rat and human cell lines seems to be the result of different mechanisms. Our results support the hypothesis that there are species-specific mechanisms implicated in the regulation of p53 activity.

Alternative Splicing↗
PubMed1997

Establishment and immunocharacterization of an immortalized pancreatic cell line derived from the H-2Kb-tsA58 transgenic mouse.

This study describes the establishment and characterization of an immortalized cell line derived from the pancreas of an adult H-2Kb-tsA58 transgenic mouse. These cells, designated IMPAN for IMmortalized PANcreatic cells, displayed a cobblestone appearance typical of confluent epithelial cells and a distinct polarity in the organization of their cytoplasmic organelles. Immunocytochemical studies revealed that all IMPAN cells stained positively for a wide range of markers characteristic of pancreatic acinar cells, namely the secretory products alpha-amylase, chymotrypsinogen, DNAse, the lectinlike secretory protein PAP (pancreatitis associated protein), and the zymogen granule membrane proteins GP-2 and gp300. They also stained positively for carbonic anhydrase II and cytokeratin 19, two proteins characteristic of pancreatic duct cells, as well as for rab3A, a small GTP-binding protein specifically localized in pancreatic islet cells. No reactivity was ever obtained with insulin antibodies. Taken together, these results show that the IMPAN cells exhibit a phenotype comparable to exocrine pancreatic acinar cells. However the expression of some proteins more specific to duct and islet cells make them similar to in vivo or in vitro growing acinar cells. The cell line should be a valuable model to study the mechanisms of growth, differentiation, and transformation of the exocrine pancreatic acinar cell.

Animals↗
PubMed1996

Cell-specific expression of the ZPK gene in adult mouse tissues.

ZPK is a recently identified human putative protein kinase gene that encodes an unusual serine/threonine kinase containing two potential leucine zipper motifs similar to those found in transcription factors as well as in members of the newly discovered mixed-lineage family of protein kinases. To study the normal biological function of ZPK, we have isolated a mouse ZPK cDNA and examined the pattern of ZPK mRNA expression in adult mouse tissues by Northern blot and in situ hybridization analyses. The predicted open reading frame of this cDNA encodes an 888-amino-acid protein that shares 95% overall identity with its human counterpart. By Northern blot analysis, we detected expression of ZPK mRNA in the brain of adult mice, but not in any other tissue tested. In situ hybridization analysis of mouse brain sections revealed specific association of ZPK mRNA with neuronal cell populations, primarily in the hippocampus, the cerebral cortex, and the Purkinje cell layer of the cerebellum. Interestingly, a remarkable pattern of cell-type-specific expression was also found in the epithelial compartment of various organ systems, including stomach, small intestine, liver, and pancreas, as well as in the seminiferous tubules of mature testes. Taken together, these observations suggest that ZPK could play a role in development, function, and maintenance of a variety of specialized cells.

Amino Acid Sequence↗
PubMed1995

The U1 small nuclear ribonucleoprotein/5' splice site interaction affects U2AF65 binding to the downstream 3' splice site.

In the gene of the neural cell adhesion molecule, the 5' splice site of the alternate exon 18 plays an important role in establishing regulated splicing profiles. To understand how the 5' splice site of exon 18 contributes to splicing regulation, we have investigated the interaction of the U2AF65 splicing factor to pre-mRNAs that contained portions of the constitutive exon 17 or the alternate exon 18 fused to exon 19 and separated by a shortened intron. Despite sharing an identical 3' splice site, only the pre-mRNA that contained a portion of exon 17 and its associated 5' splice site displayed efficient U2AF65 cross-linking. Strikingly, a G-->U mutation at position +6 of the intron, converting the 5' splice site of exon 18 into that of exon 17, stimulated U2AF65 crosslinking. The improved cross-linking efficiency of U2AF65 to a pre-mRNA carrying the 5' splice site of exon 17 required the integrity of the 5' end of U1 but not of U2 small nuclear RNA. Our results indicate that neural cell adhesion molecule 5' splice site sequences influence U2AF65 binding through a U1 small nuclear ribonucleoprotein/U2AF interaction that occurs at the commitment stage of spliceosome assembly, before stable binding of the U2 small nuclear ribonucleoprotein. Thus, the 5' splice sites of exons 17 and 18 differentially affect U2AF65 binding to the 3' splice site of exon 19. Factors that modulate U1 small nuclear ribonucleoprotein binding to these 5' splice sites may play a critical role in regulating exon 18 skipping.

Base Sequence↗
PubMed1989

Modulation of the immunosuppressive activities by blastocoelic fluid during rabbit pregnancy.

Temporal variation in immunosuppressive activity was determined in biological samples such as embryo-foetal fluids (blastocoelic- or amino-allantoic fluid) and blood collected from pregnant and pseudopregnant rabbits. Each of the fluids to be analyzed was pre-incubated with mitogen stimulated human lymphocytes for 48 h and then inhibition of [3H]thymidine incorporation or IL-2 receptor expression was estimated. Both means of assessing immunosuppression indicated variations in the suppressive activity throughout pregnancy. This was observed in embryo-foetal fluids but not in autologous peripheral blood nor in homologous pseudopregnant blood. At days 9-13 of pregnancy, the immunosuppressive effects of blastocoelic fluids were higher than that of the autologous sera, reached a peak at days 12 and 13 and declined thereafter, to reach the lowest levels. In order to further characterize the biological activity of day-12 blastocoelic fluid and autologous serum, they were submitted to ultracentrifugation. No suppressive activity could be demonstrated in the lipoprotein fractions. But all the activity was found in the protein fraction. Precipitation with cold ethanol confirmed that the biologically active compound was a protein. Furthermore, results obtained after ultrafiltration suggest biologically active compounds of high mol. wt (greater than 300 kDa). From the above findings, we can suggest that in the rabbit, there is no pregnancy specific systemic immunosuppression. We can also infer that (1) the immuno-tolerance of the mother towards the embryo is more due to a localized effect; (2) this effect decreases with the progression of gestation and (3) a high mol. wt factor is responsible for the immunosuppression.

Allantois↗
PubMed1987

Phosphorylation of purified insulin receptor by cAMP kinase.

Highly purified insulin receptor was shown to be a substrate for cAMP kinase. Approximately 1 phosphate was incorporated per molecule of receptor, and the cAMP kinase's affinity for the receptor was at least as high as its affinity for histone. The sites phosphorylated by cAMP kinase seemed distinct from those phosphorylated by the protein kinase C. Phosphorylation by cAMP kinase had no effect on the ability of several monoclonal antibodies to recognize the receptor or on the insulin-binding activity of the receptor. However, cAMP phosphorylation partially inhibited the tyrosine kinase activity of the receptor (approximately 25%). These results suggest that catecholamine-induced resistance to insulin may be partly due to a direct phosphorylation of the receptor by cAMP kinase and a subsequent inhibition of the ability of the receptor kinase to be activated by insulin.

Humans↗
PubMed1986

A solid-phase competitive radioimmunoassay for the insulin receptor.

A radioimmunoassay for the insulin receptor has been developed. In this assay, unlabeled receptor competes with 125I-labeled receptor for binding to monoclonal anti-receptor antibodies immobilized on microtiter wells coated with affinity-purified anti-mouse immunoglobulin G. This assay was highly reproducible and could detect 7 ng (14 fmol) of insulin receptor. By utilizing monoclonal antibodies to various antigenic regions of the receptor, different parts of the receptor molecule could be examined. By utilizing antibodies to the cytoplasmic domain of the receptor, an assay was developed which was not influenced by the presence of insulin and could equally detect the insulin receptor from different species (rat and human) and different tissues (placenta and brain). By utilizing antibodies to an autophosphorylation site of the receptor, the assay was shown capable of detecting the extent of phosphorylation of the receptor. Finally, this assay was utilized to monitor the decrease in insulin receptors in lysates of insulin-treated human lymphocytes. This radioimmunoassay should be useful for monitoring both the number and status of the insulin receptor under a variety of physiological conditions.

Animals↗
PubMed1986

Purification and characterization of the human brain insulin receptor.

The insulin receptor from human brain cortex was purified by a combination monoclonal antibody affinity column and a wheat germ agglutinin column. This purified receptor preparation exhibited major protein bands of apparent Mr = 135,000 and 95,000, molecular weights comparable to those for the alpha and beta subunits of the purified human placental and rat liver receptors. A minor protein band of apparent Mr = 120,000 was also observed in the brain receptor preparation. Crosslinking of 125I-insulin to all three receptor preparations was found to preferentially label a protein of apparent Mr = 135,000. In contrast, cross-linking of 125I-labeled insulin-like growth factor I to the brain preparation preferentially labeled the protein of apparent Mr = 120,000. The purified brain insulin receptor was found to be identical with the placental insulin receptor in the amount of neuraminidase-sensitive sialic acid and reaction with three monoclonal antibodies to the beta subunit of the placental receptor. In contrast, a monoclonal antibody to the insulin binding site recognized the placental receptor approximately 300 times better than the brain receptor. These results indicate that the brain insulin receptor differs from the receptor in other tissues and suggests that this difference is not simply due to the amount of sialic acid on the receptor.

Amino Acid Sequence↗
PubMed1986

Development of a reverse enzymoallergosorbent test (REAST) to detect timothy-specific IgE antibodies. Comparison with RAST.

Radioallergosorbent test (RAST) for the measurement of IgE antibodies has been introduced more than 15 years ago and a number of technical modifications have since improved its sensitivity and reproducibility. The test has been applied to the diagnosis of allergy and to determine changes in the levels of IgE antibodies following immunotherapy. However, specific IgG antibodies are raised during such a therapy and can interfere with the RAST. We have developed a reverse enzymoallergosorbent test (REAST) where microtiter plates are first coated with a purified polyclonal anti-IgE antibody, then with the serum to test and finally with peroxidase-labeled antigen. This assay is antigen specific as shown by the significant inhibition of binding of the labeled antigen in presence of unlabeled specific antigen (greater than 95%) and the absence of inhibition in presence of irrelevant antigens. The values found in atopic patients (85 subjects) were significantly higher than in the non-atopic donors (35 subjects) (1.14 U +/- 1.20 vs. 0.01 U +/- 0.02, P less than 0.0005) and there was a good correlation with the Pharmacia RAST (P less than 0.0005). The levels of specific IgE by both REAST and RAST correlated well with the clinical symptomatology.

Allergens↗
PubMed1986

Protein kinase C directly phosphorylates the insulin receptor in vitro and reduces its protein-tyrosine kinase activity.

The beta subunit of purified insulin receptor is phosphorylated on a serine residue by purified preparations of protein kinase C (ATP: protein phosphotransferase, EC 2.7.1.37). This phosphorylation is inhibited by antibodies to protein kinase C and stimulated by phospholipids, diacylglycerol, and Ca2+. The phosphorylation of the receptor by protein kinase C does not affect its insulin-binding activity but does inhibit by 65% the receptor's intrinsic tyrosine-specific protein kinase activity (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112). These results indicate that activators of protein kinase C, such as phorbol esters, desensitize cells to insulin by direct protein kinase C action on the insulin receptor.

Animals↗
PubMed1986

Effects of H1 and H2 receptor agonists on nonspecific proliferative response of human peripheral blood lymphocytes.

Histamine is known to modulate immune responses through the induction of suppressor cell subsets. The inhibition studies with antagonists suggest that the H2 agonist accounts for most of the suppression. This work studies the effects of various concentrations of 3-methyl histamine (as negative control), histamine, and pure H1 (2-methyl histamine, 2-pyridyl ethylamine) and H2 (4-methyl histamine, dimaprit) receptor agonists on the mitogenic (phytohemagglutinin A) proliferative response of normal human lymphocytes. At high concentrations of agonists (10(-3), 10(-4) M) the suppression induced by the two types of agonists is comparable to that of histamine. At lower concentrations (10(-6) M) the suppression is seen only in the presence of the H2 agonist. The suppression induced by the two agonists is generally reversed in the presence of an H2 receptor antagonist. The H1 receptor antagonist did not abolish and even increased the suppression induced by histamine and the two agonists.

Cimetidine↗
PubMed1985

A rubella outbreak among dental students: description of the outbreak and analysis of control measures.

From February to April 1982, rubella was diagnosed in 17 Washington University dental students. The affected students represented 4.8% of all dental students and 21% of susceptible students. Because a high likelihood of rubella transmission was perceived, three adjacent university hospitals undertook an emergency program to ensure immunity to rubella in all personnel. The program reached 84.6% of all target personnel, but only 36% of physicians (p less than 0.001). We estimated that the program increased the level of rubella immunity from 92.2% to 98.1%, at a total cost of $140,274 of which $29,990 was in excess of ordinary expenditures. Our experience indicates that schools training health professionals face the possibility of rubella outbreaks unless they ensure rubella immunity in all students. The experience also supports the value of ongoing programs to ensure rubella immunity in hospital personnel. Without such programs, hospitals may be forced to undertake emergency programs that are likely to be disruptive and expensive and may have relatively little immediate measurable impact.

Antibodies, Viral↗
PubMed1985

Expression of a functional human insulin receptor from a cloned cDNA in Chinese hamster ovary cells.

We have placed human insulin receptor cDNA into a vector under the control of the simian virus 40 (SV40) early promoter and tested its function by transient expression in microinjected Xenopus oocytes and by expression in stably transformed CHO cells. The precursor and the alpha and beta subunits of the receptor were detected by immunoprecipitation from extracts of these cells. The human insulin receptor expressed in CHO cells specifically binds 125I-labeled insulin but not insulin-like growth factor I, displays insulin-stimulated autophosphorylation of the beta subunit, and mediates insulin-stimulated 2-deoxyglucose uptake. We conclude that the human insulin receptor is synthesized, processed normally, and functional in this heterologous cell system.

Animals↗
PubMed1985

Absence of correlation between the degree of alveolitis and antibody levels to Micropolysporum faeni.

Specific IgG antibody (Ab) levels to Micropolysporum faeni (MF) antigen were measured in three study groups: 29 farmers of whom one had acute, two chronic and 26 with a past history of Farmer's lung disease (FLD), 91 clinically asymptomatic but exposed farmers and 23 unexposed donors. This study confirms the greater sensitivity of enzyme linked immunosorbent assay (ELISA) over double immunodiffusion (DID): 34 (37%) sera negative by DID were positive by ELISA and only two (4%) were positive by DID and negative by ELISA. Antibody levels to MF were higher in the serum of farmers whether symptomatic (113.2 +/- 196.1 u) or not (25.2 +/- 64.7 u) than in that of unexposed donors (1.05 +/- 1.46 u), (P less than 0.001). In a subgroup of farmers, antibody levels were compared in the serum and bronchoalveolar lavage (BAL) fluid; the levels were higher in the serum than in BAL fluids in all farmers except the only patient with acute FLD. A significant correlation was seen between Ab levels of the serum and BAL fluids. Finally, no correlation was seen between the serum antibody levels and the total number of cells or the percentage of lymphocytes found in BAL fluids, either in the subjects with a history of FLD or the asymptomatic group.

Antibodies, Bacterial↗
PubMed1984

Abnormal bronchoalveolar lavage in asymptomatic dairy farmers. Study of lymphocytes.

Bronchoalveolar lavage (BAL) was performed on 24 asymptomatic dairy farmers. Thirteen had serum precipitins to Micropolyspora faeni (MF) antigens (Group 1), and 11 were seronegative control subjects (Group 2). All were nonsmokers and had no history of previous lung disease. Thirteen of 24 subjects (9 in Group 1 and 4 in Group 2) had a high percentage of lymphocytes (greater than or equal to 20%) in their BAL. The T-lymphocyte subpopulations as estimated by OKT3, OKT4, and OKT8 monoclonal antibody reactivity were measured in peripheral blood lymphocytes; OKT3 = 58.5 +/- 15.6% for Group 1, and 58.5 +/- 8.7% for Group 2; OKT4 = 40.6 +/- 10.7% and 39.9 +/- 10.0%; OKT8 = 21.5 +/- 10.6% and 22.4 +/- 8.0%, respectively (p = NS). These lymphocyte characteristics were also similar when subjects with a high percentage of lymphocytes in BAL were compared to those with a normal percentage. Specific (MF-coated) chicken erythrocyte lymphocytotoxicity (Group 1, 45.2 +/- 29.5%, Group 2, 49.2 +/- 23.4%), and nonspecific lymphocytotoxicity (Group 1, 43.9 +/- 28.6%, Group 2, 37.9 +/- 18.0%) were also similar. We conclude that a large number of asymptomatic dairy farmers have an increased percentage of lymphocytes in their BAL ("alveolitis") and that peripheral blood lymphocytes in these subjects have normal subpopulations, as assessed by monoclonal antibodies, and normal lymphocytotoxicity.

Adult↗