Hétérogénéité structurale du récepteur d'oestrogènes dans le cancer mammaire humain

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Importance of A/B and C domains of the estrogen receptor for its adsorption to hydroxylapatite

Regulatory properties of estrogen receptor (ER) result from the existence of functional domains within its primary structure. Thus, A/B and C domains which are rich in tyrosyl residues control gene expression while the E domain confers estrogen binding capacity. Hydroxylapatite (HAP) is known to adsorb ER. Scatchard plot analysis of [3H]estradiol binding patterns of HAP batches to which cytosolic ER had been adsorbed revealed that AB and/or C domains are mainly responsible for this property. Thus, treatment of these batches with the tyrosine reagent tetranitromethane (TNM) led to a dramatic release of adsorbed receptors. This did not occur with ER preparations devoid of exposed ABC domains obtained by selective immunoextraction with H-226 anti-ER monoclonal antibody prior to HAP assay. KCl treatment (500 mM) of HAP batches also led to a release of bound receptors especially those devoid of exposed ABC domains. Such binding characteristics were also found with full length and truncated ERs produced in yeast: the full length receptor strongly interacted with HAP while the truncated receptor devoid of AB and C domains displayed only a weak adsorption. Additional investigation revealed that estradiol binding to cytosolic ER does not modify its reactivity towards TNM.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Major molecular weight heterogeneity of estrogen receptor from breast cancer is not related to neoplasia

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Inhibition of estradiol binding to its receptor by the cupric ion

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Decrease of hormone binding capacity of estrogen receptor by calcium

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Comparison of Different Methods of Isolation of DNA of Commonly Encountered Candida Species and Its Quantitation by Using a Real-Time PCR-Based Assay

Molecular diagnosis based on genomic amplification methods such as real-time PCR assay has been reported as an alternative to conventional culture for early detection of invasive candidiasis. However, a major limitation of the molecular method is the difficulty associated with breaking fungal cell walls since the DNA extraction step still requires more than half of a working day. It has been suggested that PCR detection of free template DNA in serum is preferred over the use of whole blood for the diagnosis of systemic candidiasis. In this study, two conventional procedures (the first [the HLGT method] consists of boiling sera in an alkaline guanidine-phenol-Tris reagent, and the second [the PKPC method] uses proteinase K digestion, followed by organic extraction) and three commercially available kits for DNA isolation were evaluated for sensitivity, purity, cost, and use of template for most clinically important Candida species in a TaqMan-based PCR assay. To optimize these procedures, we evaluated the effect of adding 0.5% bovine serum albumin to DNA extracts and found that it decreased the effects of inhibitors. The QIAamp DNA blood kit did significantly shorten the duration of the DNA isolation but was among the most expensive procedures. Furthermore, the QIAamp DNA blood kit proved to be as sensitive as the HLGT DNA isolation method for PCR amplification from 52 serum samples from hematology or oncology patients with clinically proven or suspected systemic Candida infections. All PCR-positive samples showed approximately the same Candida species load by both procedures (100% correspondence), whereas one discordant result was obtained between PCR and blood culture

Real-time PCR for determining capsular serotypes of Haemophilus influenzae.

A two-step real-time PCR assay targeting all six capsulation loci of Haemophilus influenzae (i.e. serotypes a to f) was developed and compared with a previously published qualitative PCR assay by using 131 H. influenzae clinical isolates. There was a 98.5% concordance between the two tests. The sensitivity of detection of capsular type-specific reference strains of H. influenzae a to c (10(1) CFU/PCR) was higher than that for type e (10(3) CFU/PCR) and types d and f (10(4) CFU/PCR), and a broader dynamic range was obtained (5 to 8 log(10) units). No cross-reaction was observed with bacteria commonly isolated from the respiratory tract. We showed that both PCR assays are more reliable than slide agglutination serotyping. The real-time PCR-based assay seems to be an alternative of choice for the epidemiological follow-up of H. influenzae invasive infections.Journal Articleinfo:eu-repo/semantics/publishe

Evolution towards hormone independence of the MXT mouse mammary tumor is associated with a gradual change in its estrogen receptor molecular polymorphism.

Using a method based on [3H]tamoxifenaziridine ([3H]TAZ) labeling, sequential immunoadsorption with anti-ER monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and fluorography, we observed a striking change inthe estrogen receptor (ER) electrophoresis pattern of the transplantable MXT mouse mammary tumor. Early, ER "rich" tumors (approximately 100 fmol/mg prot) displayed classical cytosolic 67 and 50 KDa bands. These bands disappeared in favor of a "cytosolic" 35 KDa band during progression towards undifferentiated ER "poor" tumors (approximately 25 fmol/mg prot). Although we can not rule out that this 35 KDa peptide results from in vivo ER proteolysis, it seems unique in view of the following: 1. It is immunoadsorbed not only by an anti-ER monoclonal antibody (H-222) directed to the hormone-binding domain, but also by an anti-ER monoclonal antibody (H-226) which interacts with an epitope in the A/B region close to the DNA-binding domain and is mainly exposed under activation conditions. 2. It does not bind [3H]estradiol([3H]E2) and a tentative to restore its [3H]E2 binding capacity with calmodulin and ATP was unsuccessful. The observation of similar approximately 35 KDa ERs in the nuclear fraction of early tumor transplants and in control uterus suggests that this peptide is already in an activated form. Structural alterations of ER and/or associated "anchorage" nuclear proteins may beat the origin of its cytosolic localization. Moreover, the fact that the addition of calmodulin and ATP to late MXT transplants cytosols fails to increase their [3H]E2 binding capacity indicates that the low ER content of these tumors does not result from a deficiency in the phosphorylation status of the receptor.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Practical use of GenoType® HelicoDR, a molecular test for Helicobacter pylori detection and susceptibility testing.

Compared to culture-based method, the sensitivity, specificity, positive, and negative predictive values of the GenoType(®) HelicoDR for detecting Helicobacter pylori resistance were, respectively, 100, 86.2, 89.7%, and 100% to clarithromycin as well as 82.6, 95.1, 90.5%, and 90.7% to fluoroquinolones. This molecular assay detected a mixture of genotypes and could successfully analyze biopsies without transport/storage limitations.Comparative StudyEvaluation StudiesJournal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe