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Biomedical subjects

D Picard

Publications and source records attributed to D Picard.

At least 37 records · Page 2Linked to original sources
PubMed1999

Two compounds commonly used for phospholipase C inhibition activate the nuclear estrogen receptors.

The aminosteroid U73122 is generally used as a specific inhibitor of phosphoinositide specific phospholipase C (PLC) and typically, the structurally related compound U73343 is used as control, since it lacks PLC inhibitory activity. We have found that both compounds possess strong estrogenic activity and that this activity is mediated by the estrogen receptors (ER) alpha and beta. Although no direct evidence for binding of U73122 and U73343 to the ER could be provided, the estrogenic activity of the aminosteroids requires an intact ER hormone binding pocket. Given the chemical structure of the two aminosteroids, they may be converted to an estrogenic derivative by chemical degradation or an enzymatic metabolization reaction. Our data indicate that additional care should be taken in the interpretation of the effects of U73122 in cells expressing ER.

Cell Line↗
PubMed1999

A yeast-based bioassay for the determination of functional and non-functional estrogen receptors.

The response to endocrine therapy of breast cancer is not entirely predictable from hormone receptor status alone since some point mutated or splicing variants of the estrogen receptor (ER) show altered biological activities. In order to characterize the activities of all forms of ER in a heterogeneous breast tumor, a functional assay in Saccharomyces cerevisiae was developed. Total RNA isolated from breast cancer cells and one breast cancer specimen was reverse transcribed and the ER cDNA was amplified by PCR. The products were then cloned into an expression vector by in vivo homologous recombination in yeast. The yeast strain carries a reporter gene ( ADE2 ) coupled to an estrogen response element. Activation of the reporter by ER yielded white colonies whereas lack of ER activity produced red colonies. This permitted the testing for functionality of individual ER molecules and subsequent analysis by rescuing of the ER expression plasmids and complete DNA sequencing. This simple visual test allows discrimination between wild-type ER, constitutively active ER and inactive ER.

Biological Assay↗
PubMed1999

Ligand-independent Activation of Steroid Receptors: New Roles for Old Players.

Steroid receptors are known as ligand-regulated transcription factors, but more and more evidence indicates that several steroid receptors can be activated in the absence of cognate hormone by agents that fall into three separate groups: peptide growth factors, the neurotransmitter dopamine and other agents that activate cAMP-dependent protein kinase A, and the cyclins A and D1.

Journal Article↗
PubMed1999

Alpha-complemented beta-galactosidase. An in vivo model substrate for the molecular chaperone heat-shock protein 90 in yeast.

Intracistronic complementation of N-terminally truncated beta-galactosidase mutants such as M15 by coexpressed alpha-peptide was originally discovered in Escherichia coli and exploited for plasmid cloning as the well-known blue-white screening method. We show here that alpha-complementation also works in the budding yeast Saccharomyces cerevisiae, and that it can be used as a simple nonselective enzymatic marker for a variety of in vivo studies, for example, on the role of molecular chaperones in protein folding and assembly. To be able to induce alpha-complementation post-translationally, we have constructed a hormone-inducible alpha-peptide by fusion of the DNA encoding the alpha-peptide to that of to the hormone binding domain of the estrogen receptor. The accumulation of both subunits, the alpha-peptide and M15, is severely compromised when they are expressed separately, presumably because their hydrophobic surfaces remain exposed. Moreover, alpha-complementation is defective in a strain of S. cerevisiae carrying a point mutant of the molecular chaperone heat-shock protein 90. Heat-shock protein 90, which coprecipitates with M15, might be required in vivo to prevent the degradation of unassembled M15 and to hold it in an interaction-competent conformation.

Blotting, Western↗
PubMed1999

Hsp90 binds and regulates Gcn2, the ligand-inducible kinase of the alpha subunit of eukaryotic translation initiation factor 2 [corrected].

The protein kinase Gcn2 stimulates translation of the yeast transcription factor Gcn4 upon amino acid starvation. Using genetic and biochemical approaches, we show that Gcn2 is regulated by the molecular chaperone Hsp90 in budding yeast Saccharomyces cerevisiae. Specifically, we found that (i) several Hsp90 mutant strains exhibit constitutive expression of a GCN4-lacZ reporter plasmid; (ii) Gcn2 and Hsp90 form a complex in vitro as well as in vivo; (iii) the specific inhibitors of Hsp90, geldanamycin and macbecin I, enhance the association of Gcn2 with Hsp90 and inhibit its kinase activity in vitro; (iv) in vivo, macbecin I strongly reduces the levels of Gcn2; (v) in a strain expressing the temperature-sensitive Hsp90 mutant G170D, both the accumulation and activity of Gcn2 are abolished at the restrictive temperature; and (vi) the Hsp90 cochaperones Cdc37, Sti1, and Sba1 are required for the response to amino acid starvation. Taken together, these data identify Gcn2 as a novel target for Hsp90, which plays a crucial role for the maturation and regulation of Gcn2.

5' Untranslated Regions↗
PubMed1999

A non-radioactive method for inexpensive quantitative RT-PCR.

We present a novel method for quantitative RT-PCR that involves direct incorporation of digoxigenin-11-dUTP (DIG-dUTP) during amplification of cDNAs, separation of RT-PCR products by agarose gel electrophoresis, Southern transfer to a nylon membrane, and chemiluminescent detection with an anti-DIG antibody. The whole procedure can be done in about a day and has the following advantages: It is highly sensitive, specificity is confirmed by monitoring the size of the RT-PCR product, it is non-radioactive, quantitative, and does not require expensive specialized equipment.

Base Sequence↗
PubMed1999

The anti-estrogen hydroxytamoxifen is a potent antagonist in a novel yeast system.

The budding yeast Saccharomyces cerevisiae has been used extensively as a biological 'test tube' to study the regulation of the human estrogen receptor (ER) alpha. However, anti-estrogens, which are of great importance as therapeutic agents and research tools, fail to antagonize the activation by estrogen in yeast. Here, we have surveyed the antagonistic potential of five different anti-estrogens of diverse chemical nature. While they all act as agonists for wild-type ERalpha, we have established a novel yeast assay system for anti-estrogens, in which at least the commonly used anti-estrogen hydroxytamoxifen is a potent antagonist.

Dose-Response Relationship, Drug↗
PubMed1998

Bioactive steroids as contaminants of the common carbon source galactose.

Most inducible expression vectors for the budding yeast Saccharomyces cerevisiae are based on galactose-inducible promoters. Yeast has been increasingly used to study vertebrate steroid receptors because of its powerful genetics. In principle, both regulatory systems are compatible and can be combined in the same strain. However, we found that commercial galactose can be contaminated by bioactive estrogen and progesterone at concentrations that are sufficient to fully activate their cognate receptors. Since steroids can elicit biological responses in pathogenic fungi and possibly other microorganisms, such contaminants in a commonly used fermentable carbon source may need to be screened for.

Drug Contamination↗
PubMed1998

Hsp90 is required for pheromone signaling in yeast.

The heat-shock protein 90 (Hsp90) is a cytosolic molecular chaperone that is highly abundant even at normal temperature. Specific functions for Hsp90 have been proposed based on the characterization of its interactions with certain transcription factors and kinases including Raf in vertebrates and flies. We therefore decided to address the role of Hsp90 for MAP kinase pathways in the budding yeast, an organism amenable to both genetic and biochemical analyses. We found that both basal and induced activities of the pheromone-signaling pathway depend on Hsp90. Signaling is defective in strains expressing low levels or point mutants of yeast Hsp90 (Hsp82), or human Hsp90beta instead of the wild-type protein. Ste11, a yeast equivalent of Raf, forms complexes with wild-type Hsp90 and depends on Hsp90 function for accumulation. For budding yeast, Ste11 represents the first identified endogenous "substrate" of Hsp90. Moreover, Hsp90 functions in steroid receptor and pheromone signaling can be genetically separated as the Hsp82 point mutant T525I and the human Hsp90beta are specifically defective for the former and the latter, respectively. These findings further corroborate the view that molecular chaperones must also be considered as transient or stable components of signal transduction pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Evaluation of gastric emptying function in clinical practice.

In this retrospective analysis, we compared different methods to evaluate gastric emptying function, aiming to improve the sensitivity and the clinical availability of our diagnostic testing. In the first study, we compared, in 72 patients clinically suspected of gastroparesis, the emptying of a meal containing two solid nutrients with different disintegration rates: 111In-labeled scrambled eggs and 99Tc-labeled liver cubes. Gastric emptying of 111In-labeled egg was delayed in 12 of our patients and the evacuation of the 99Tc-labeled liver was prolonged in 19 patients. The choice of the nutrient was not important for the identification of diabetic gastroparesis (43% vs 57%; NS), but it was determinant in the case of patients suspected of idiopathic gastroparesis (12% were positive with the egg and 25% with the liver; P < 0.05). In the second study, we compared two different diagnostic methods in 46 patients: a simple radiological detection of the gastric emptying of radiopaque pellets, and the scintigraphic emptying of a solid meal containing 99Tc-labeled liver cubes. Both tests correlated perfectly in 78% of our patients. In 15% of the population (six of these seven patients were diabetics suspected of gastroparesis) the scintigraphic method was normal, while the evacuation of radiopaque pellets was delayed. For clinical purposes, we therefore propose: (1) the scintigraphic method should use liver rather than egg as a radiolabeled tracer in order to improve the sensitivity of the test for detection of gastroparesis; and (2) the radiological detection of radiopaque markers is a reliable and convenient method for the detection of gastroparesis in clinical practice. It is possibly more sensitive than scintigraphy.

Adult↗
PubMed1997

Restorative treatments received by children covered by a universal, publicly financed, dental insurance plan.

OBJECTIVES: This study sought to identify risk markers associated with the provision of new restorations in children and to investigate whether the carious status of a tooth surface is associated with the restorative decisions of dentists. METHODS: A total of 911 schoolchildren in grades one, two, and three were randomly selected from the island of Montreal, Quebec, Canada. Dental examinations were carried out in 1990, 1991, and 1992. Tooth surfaces of first permanent molars were classified as sound, noncavitated, and cavitated. The carious status of a tooth was matched with restorative decisions reported to the insurance board. RESULTS: The presence of a carious cavity was a strong risk marker for placement of new restorations (odds rations > or = 4.11). After one year, less than 2 percent of sound tooth surfaces of first permanent molars were restored and about 21 percent of noncavitated tooth surfaces were restored. When new class I restorations placed in maxillary first permanent molars within 3-6 months after the baseline examination were evaluated, we found that between 73 percent and 86 percent of these new restorations were placed in sound or noncavitated tooth surfaces. A similar trend also was observed in mandibular first permanent molars. Poor agreement between epidemiologic diagnosis and restorative decisions was found. The restorative profile of dentists was a significant risk marker for placement of new restorations. CONCLUSION: The majority of new restorations in first permanent molars were placed in sound and noncavitated tooth surfaces because of the ubiquitous prevalence of these tooth surfaces and the validity problems of current caries diagnosis methods.

Child↗
PubMed1997

Functional analysis of the yeast 40 kDa cyclophilin Cyp40 and its role for viability and steroid receptor regulation.

We have identified and characterized a homolog of the 40 kDa cyclophilins in the budding yeast Saccharomyces cerevisiae. At the amino acid level, this novel yeast cyclophilin, termed Cyp40, is 47% identical to human cyclophilin-40. Recombinant Cyp40 produced in bacteria has a peptidyl-prolyl cis-trans isomerase activity with a catalytic efficiency (k[cat]/K[m]) of 0.5 x 10(6)M(-1)s(-1), which can be inhibited by cyclosporin A with an IC50 value of 60nM. Using a polyclonal antibody against Cyp40 we have found that Cyp40 is predominantly cytoplasmic, and that its expression is induced 3-4-fold by heat shock. Moreover, Cyp40 can be coprecipitated from yeast extracts with the cytosolic molecular chaperone Hsp90. Surprisingly, a Cyp40-deficient yeast strain is fully viable at normal and elevated temperatures. Cyp40 is also dispensable for normal regulation of vertebrate steroid receptors in yeast. While other immunophilins could conceivably compensate a Cyp40 defect, our results are compatible with the notion that immunophilins may be fortuitous partners in the biochemically established steroid receptor-Hsp90 complex.

Amino Acid Isomerases↗
PubMed1997

School-related issues among HIV-infected children.

OBJECTIVE: Many children with human immunodeficiency virus (HIV) infection are surviving long enough to reach school age. This study describes issues related to school attendance and disclosure of HIV infection in a population of HIV-infected children. METHODS: A statewide pediatric HIV surveillance system was used to collect data on school-age (>/=5 years old) HIV-infected children. In addition, HIV clinic nurses familiar with the child's history participated in a cross-sectional survey that collected information on school-related issues during the 1993-1994 school year. RESULTS: Of the 92 school-age children, only 3 were too ill to attend school. Another 5 children were home-schooled. Of the 84 who attended school outside the home, 25% had severe symptoms of HIV infection (Centers for Disease Control and Prevention [CDC] clinical category C). Absence from school ranged from less than 2 weeks during the year for half of the children (51%) to more than 8 weeks for 9 children (12%). Twenty-nine percent of the children received medication in school, usually administered by the school nurse. Over two thirds of the 50 children ages 5 to 10 years had not been told that they had HIV infection. Only 1 of the 20 children more than 10 years of age was not aware of her HIV infection. For 53% of the children attending school, no school personnel had been informed of the child's HIV infection. Administration of HIV medications at school, age of child, and treatment at one particular HIV clinic were associated with the parents' decision to inform school personnel. In the 47% of cases where the school had been informed, school nurses were most frequently notified, followed by principals and teachers. CONCLUSION: Only 3% of school-age children were too ill to attend school, and almost all were enrolled in public schools. The number of HIV-infected children reaching school age will continue to grow, and public schools will bear the responsibility for educating these children. Health care providers will increasingly be called upon for guidance by both educators and families to assure that HIV-infected children receive the best education possible.

Absenteeism↗
PubMed1997

Methodological validation and clinical usefulness of carbon-14-urea breath test for documentation of presence and eradication of Helicobacter pylori infection.

UNLABELLED: A simple [14C]urea breath test (C-14-UBT) was validated with aims of determining accuracy in documenting both the presence and proof of eradication of Helicobacter pylori infection. METHODS: Fifty-six dyspeptic patients had endoscopy with biopsies and C-14-UBT. Eleven biopsy-proven H. pylori-negative patients allowed C-14-UBT normal value determination. Forty-three patients with recurrent peptic ulcer disease and biopsy-proven H. pylori infection were included in an antimicrobial eradication protocol. Endoscopy with biopsies and C-14-UBT were done again 8 wk after initiation of treatment in 35 patients. For C-14-UBT, 185 kBq (5 microCi) of [14C]urea was swallowed. Breath samples obtained up to 20 min were counted to calculate AS20, [(% 14CO2 dose excreted/mmol of CO2) x kg] at 20 min. Combined histologic and microbiologic analyses of antral biopsies were used as a gold standard. RESULTS: The positivity value was set as AS20 > 0.33% (mean + 3 s.d. of AS20 in H. pylori-negative patients). Diagnosis of H. pylori infection was correct with C-14-UBT in 55/56 patients (44 true-positive, 11 true-negative and 1 false-negative; sensitivity = 98%; specificity = 100%). As a proof of eradication, C-14-UBT correctly classified 33/35 patients (5 true-positive, 28 true-negative and 2 false-positive; sensitivity = 100%; specificity = 93%). The C-14-UBT global performance yielded sensitivity, specificity and accuracy of 98%, 95% and 97%, respectively. A significant correlation (r = 0.84) was found between AS20 and the number of H. pylori colonies on culture. CONCLUSION: This C-14-UBT is highly accurate both for diagnosis and proof of eradication of H. pylori infection and reflects the antral bacterial load. It is simple, fast and inexpensive, and it is therefore suitable for clinical practice.

Breath Tests↗