[Is there a place for stepped hormone assay in the development of hyperandrogenism?] / Comment j'explore Le dosage hormonal étagé a-t-il une place dans la mise au point des hyperandrogénies ?

Clinical hyperandrogenism is common in women. Nevertheless, it is important to identify the cause. As the hyperandrogenism may be ovarian or adrenal in origin, making the difference requires hormonal testing and ovarian and/or adrenal imaging. We present the case report of a patient explored in our clinic, that illustrates the difficulties to determine the origin of the endocrine disorder. The interest of employing selective ovarian and adrenal venous catheterization to aid in the diagnosis and the localization of the androgen-secreting tumor is discussed.

L'hyperandrogénie clinique est un motif de consultation fréquent. Le diagnostic différentiel permet d'établir l'étiologie parmi les causes ovariennes ou surrénaliennes. Outre le repérage de signes pathognomoniques cliniques, des examens complémentaires biologiques et iconographiques sont nécessaires pour la mise au point. Les difficultés diagnostiques sont illustrées à partir d'un cas clinique traité dans notre institution. L'intérêt du bilan hormonal étagé par cathétérisation des veines ovariennes et surrénaliennes afin de localiser l'origine de la sécrétion hormonale pathologique est discuté.

[Biopsy of suspicious lesions in patients with breast cancer]. / BIOPSIE DES LÉSIONS SUSPECTES CHEZ LES PATIENTS AYANT PRÉSENTÉ UN CANCER DU SEIN.

Discordances between hormone receptors and HER2 status in primary and metastatic breast cancer have been reported by several studies. In this context, systematic biopsies could be clinically relevant in breast cancer to confirm the biological characteristics of a suspicious lesion. In this article, illustrated by 2 case reports and based on a recent review on this topic, we discuss the clinical significance of receptor discordances and possible diagnosis of a secondary primary tumor. The role of these biopsies for the identification of new therapeutic targets is also envisaged as well as underlying mechanisms for receptors' modification like tumoral heterogeneity, clonal selection and technical artifacts.

Abrupt RNA changes precede the first cell division during the differentiation of Trypanosoma brucei bloodstream forms into procyclic forms in vitro.

We have monitored the timing of DNA and RNA synthesis during the synchronous differentiation of Trypanosoma brucei bloodstream forms into procyclic forms in vitro. Both are triggered after a lag period of 4 h and reach a first peak after 9 h. The division of the kinetoplast precedes that of the nucleus by about 4 h. The first cell divisions are observed after 10 h, and the cell number is doubled after 20 h. The total RNA content per cell increases sharply between 4 and 10 h, then progressively decreases as cell division progresses. The increase in RNA content cannot be due solely to accumulation of rRNA since it is also observed for mRNAs such as actin. The VSG mRNA has almost disappeared within 2 h, while the procyclin mRNA accumulates soon after the triggering of differentiation, with a strong peak between 4 and 6 h. At this moment, the amount of procyclin mRNA per cell is at least 20-fold higher than in established procyclic culture forms. The loss of the VSG and the appearance of procyclic-specific proteins essentially occur before the first cell division. These observations contrast with the progressive transition observed when monomorphic slender forms are induced to transform under the same conditions.

Transient adenylate cyclase activation accompanies differentiation of Trypanosoma brucei from bloodstream to procyclic forms.

Pleomorphic bloodstream forms of Trypanosoma brucei differentiate synchronously into procyclic forms when cultivated at 27 degrees C in the presence of citrate/cis-aconitate. The activity of adenylate cyclase was monitored during this process. Two phases of transient stimulation were observed. The first phase occurred 6-10 h after the triggering of differentiation, a period which immediately follows the release of the bulk of the VSG and immediately precedes both the first cell division and the loss of the bloodstream-specific ESAG 4 transmembrane adenylate cyclase. The second phase occurred between 20 and 40 h, when the cells that emerged from the first division began to proliferate. These observations suggest that cAMP may be involved in differentiation/proliferation of the parasite.

Families of adenylate cyclase genes in Trypanosoma brucei.

Four genes for adenylate cyclase have been characterized in Trypanosoma brucei. One of them, esag 4 (for expression site associated gene 4) is present in different VSG (variant surface glycoprotein) gene expression sites and, thus, is only expressed in the bloodstream form of the parasite. The others, termed gresag 4.1, 4.2 and 4.3 (for genes related to esag 4) are expressed in both bloodstream and procyclic forms. In addition, we cloned a esag 4-related gene from T. congolense. Here we characterize the genomic organization of gresag 4.1 and 4.3. While gresag 4.3 is unique, gresag 4.1 exists as a multigenic family of at least nine members located on a 3-Mb chromosome. Six of them are clustered in a region of 300 kb, three copies being tandemly linked. The determination of the nucleotide sequence of a conserved 1.6 kb PstI fragment demonstrated the presence of two separate subgroups in this family. This gene arrangement is present in different isolates of T.b. brucei/rhodesiense/gambiense. Several gresag 4.1 copies are transcribed in both bloodstream and procyclic forms.

Mild acid stress as a differentiation trigger in Trypanosoma brucei.

In vitro differentiation of Trypanosoma brucei from the bloodstream to the procyclic form is efficiently induced by the combination of cold shock from 37 to 27 degrees C and the addition of citrate/cis-aconitate (CCA) to the incubation medium. Here it is reported that exposure of pleomorphic bloodstream trypanosomes to mild acidic conditions (pH 5.5 for 2 h at 37 degrees C) not only accelerated the process of morphological transformation from long slender and intermediate to short stumpy bloodstream forms but also allowed their subsequent differentiation into procyclic forms even in the absence of CCA. This process appeared to involve the glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC), since null GPI-PLC mutants (PLC-) appeared to be largely refractory to acid stress-induced differentiation. However, an effective response was restored upon reintegration of the GPI-PLC gene in the genome (PLC+).

Expression of ras-like proteins in embryonic and adult cells of Xenopus laevis.

A polyclonal antibody raised against v-Ha-ras p21 was purified and its specificity was checked on Ha-ras transformed cell lines. It was used to immunoprecipitate p21 from different Xenopus laevis cell types: brain cells, blood cells, and embryonic material. By one-dimensional Western blot analysis, we show that ras p21 is synthesized very early in oogenesis and accumulates throughout vitellogenesis. The ras p21 content, estimated to be 1.1 ng in the full-grown oocyte, remains constant during oocyte maturation and egg cleavage. Increase in the amount of ras p21 occurs at the beginning of neurulation. Two-dimensional Western blot patterns reveal the presence of multiple molecular forms of p21 in all Xenopus cell types studied. The numerous resolved polypeptides were ascribed to the expression of at least two different ras genes. Furthermore, specific charge modifications of the ras polypeptides are observed in brain, blood, and embryonic cells. During oogenesis and early embryonic development, differences in two-dimensional patterns mainly concern variations in the relative amounts of the different polypeptides. The results are discussed in relation to the well documented synthesis activities of the growing oocyte and of the early developing embryo.

Presence of high molecular weight glycans on the surface of germ and embryonic cells of Xenopus laevis.

Xenopus laevis male germ cells, fertilized eggs and gastrula cells were labelled with 3H labelled sodium borohydride reduction after galactose oxidase treatment. After pronase digestion, the bulk of the label is carried by high molecular weight glycans (greater than or equal to 6,000 D). The high molecular weight of these labelled glycans and their susceptibility to degradation by endo-beta-galactosidase suggest that they may be related to the polylactosaminoglycans.

Injection of an antibody against a p21 c-Ha-ras protein inhibits cleavage in axolotl eggs.

The presence of a ras protein was demonstrated in cleaving axolotl eggs by selective immunoprecipitation with a polyclonal antibody against a peptide encoded by the c-Ha-ras oncogene, cellular homolog of the v-Ha-ras oncogene of Harvey rat sarcoma virus. Injection of this antibody into axolotl oocytes subjected to progesterone treatment does not prevent meiotic maturation. Injection of the same antibody into a blastomere of axolotl eggs at the 2- or 4-cell stage causes cleavage arrest in the descendants of the injected blastomere. Cytological observations of the injected eggs show, in the arrested blastomeres, enlarged nuclei always surrounded by an intact nuclear envelope and containing uncondensed chromatin. The possible role of ras protein in meiosis and mitosis is discussed.

Characterization of yolk DNA from Xenopus laevis oocytes ovulated in vitro.

High molecular weight DNA was isolated from the yolk platelets of Xenopus laevis oocytes ovulated in vitro. Yolk DNA has the same buoyant density in CsCl gradients as mitochondrial and nuclear DNAs, and, like them, it is double-stranded. Yolk DNA behaves like nuclear DNA, and not like mitochondrial DNA, upon renaturation after denaturation. The molecules are always linear. Cytochemical and biochemical controls preclude the possibility that the yolk DNA might be contaminated by nuclear or mitochondrial DNA. We conclude that the yolk DNA is an endogenous component of the yolk platelets.

A novel heterodimeric transferrin receptor encoded by a pair of VSG expression site-associated genes in T. brucei.

In T. brucei, a transferrin-binding protein has been found to share sequence homology with pESAG-7 and -6, the products of two related genes present in the VSG gene polycistronic transcription unit. When expressed in Xenopus oocytes, they appear as N-glycosylated proteins secreted in the medium (pESAG-7) and GPI anchored to the membrane (pESAG-6). These proteins are able to homo- or heterodimerize, probably through association in the same orientation. Only heterodimers can bind Tf, possibly two molecules per dimer. A comparison of Tf binding to pESAG-7/6-expressing oocytes and trypanosomes suggests that pESAG-7/6 is the Tf receptor of the parasite. In trypanosomes, the majority of pESAG-7/6 is released from the membrane and associates, together with Tf, with a glycosylated matrix present in the lumen of the flagellar pocket. Both pESAG-7/6 and Tf are internalized via coated pits and vesicles. These observations suggest a novel mode of Tf binding and uptake in trypanosomes.

Expression of a variant surface glycoprotein of Trypanosoma gambiense in procyclic forms of Trypanosoma brucei shows that the cell type dictates the nature of the glycosylphosphatidylinositol membrane anchor attached to the glycoprotein.

Procyclic forms of Trypanosoma brucei have been genetically modified to express the major metacyclic variant surface glycoprotein (VSG variant AnTat 11.17) of Trypanosoma gambiense. The VSG is expressed in an intact membrane-bound form that can be detected over the entire plasma membrane, together with procyclin, and as a series of lower-molecular-mass fragments that are mostly soluble degradation products. The presence of degraded VSG in the cells and the culture medium suggests that VSG is not efficiently processed and/or efficiently folded when expressed in procyclic cells. The level of procyclin expressed on the surface of these cells is slightly reduced, although there is no difference in procyclin mRNA levels. The intact membrane-bound form of the VSG is N-glycosylated with oligomannose structures and contains a glycosylphosphatidylinositol (GPI) membrane anchor that can be biosynthetically labelled with [3H]ethanolamine. The anchor is sensitive to mammalian GPI-specific phospholipase D but, like the anchor of procyclin, it is resistant to the action of bacterial phosphatidylinositol-specific phospholipase C. This pattern of phospholipase sensitivity suggests that the GPI anchor acquired by VSG when expressed in procyclics is acylated on the inositol ring and therefore resembles a procyclic procyclin-type anchor rather than a trypomastigote VSG-type anchor with respect to the lipid structure. The VSG expressed in procyclics was sensitive to the action of a mixture of sialidase, beta-galactosidase and beta-hexosaminidase, suggesting that the VSG GPI anchor also contains a sialylated polylactosamine side-chain modification similar to that described for procyclin. These results indicate that the nature of the protein expressed has little influence on the post-translational modifications performed in the secretory pathway of procyclic trypanosomes.

Characterization of the ligand-binding site of the transferrin receptor in Trypanosoma brucei demonstrates a structural relationship with the N-terminal domain of the variant surface glycoprotein.

The Trypanosoma brucei transferrin (Tf) receptor is a heterodimer encoded by ESAG7 and ESAG6, two genes contained in the different polycistronic transcription units of the variant surface glycoprotein (VSG) gene. The sequence of ESAG7/6 differs slightly between different units, so that receptors with different affinities for Tf are expressed alternatively following transcriptional switching of VSG expression sites during antigenic variation of the parasite. Based on the sequence homology between pESAG7/6 and the N-terminal domain of VSGs, it can be predicted that the four blocks containing the major sequence differences between pESAG7 and pESAG6 form surface-exposed loops and generate the ligand-binding site. The exchange of a few amino acids in this region between pESAG6s encoded by different VSG units greatly increased the affinity for bovine Tf. Similar changes in other regions were ineffective, while mutations predicted to alter the VSG-like structure abolished the binding. Chimeric proteins containing the N-terminal dimerization domain of VSG and the C-terminal half of either pESAG7 or pESAG6, which contains the ligand-binding domain, can form heterodimers that bind Tf. Taken together, these data provided evidence that the T.brucei Tf receptor is structurally related to the N-terminal domain of the VSG and that the ligand-binding site corresponds to the exposed surface loops of the protein.

Simultaneous but independent activation of adenylate cyclase and glycosylphosphatidylinositol-phospholipase C under stress conditions in Trypanosoma brucei.

Previous observations suggested a concomitant relationship between the release of the variant surface glycoprotein (VSG) and the activation of adenylate cyclase in the bloodstream form of the parasitic protozoan Trypanosoma brucei. In order to evaluate this hypothesis, adenylate cyclase activity was measured in live trypanosomes subjected to different treatments known to induce the shedding of the VSG coat, namely low pH and trypsin digestion. In both cases adenylate cyclase activation occurred in parallel with the release of the VSG. The latter was found to be mediated by the glycosylphosphatidylinositol-specific phospholipase C that cleaves the glycosylphosphatidylinositol anchor of the protein (VSG lipase). Furthermore, both adenylate cyclase and VSG release were activated by the incubation of trypanosomes with specific inhibitors of protein kinase C, suggesting a repressive role for protein kinase C on both VSG lipase and adenylate cyclase activities. Significantly, in mutant trypanosomes lacking VSG lipase, adenylate cyclase was activated under conditions where VSG release did not occur. Moreover,VSG release was also found to occur in the absence of activation of the cyclase, as observed in the presence of low concentration of the thiol modifying reagent p-chloromercuriphenylsulfonic acid. These observations provide the first demonstration that release of the VSG in response to cellular stress is mediated by the VSG lipase and that while both release of the VSG and activation of adenylate cyclase occur in response to the same stimuli they are not obligatorily coupled.

A role for the dynamic acylation of a cluster of cysteine residues in regulating the activity of the glycosylphosphatidylinositol-specific phospholipase C of Trypanosoma brucei.

The glycosylphosphatidylinositol-specific phospholipase C or VSG lipase is the enzyme responsible for the cleavage of the glycosylphosphatidylinositol anchor of the variant surface glycoprotein (VSG) and concomitant release of the surface coat in Trypanosoma brucei during osmotic shock or extracellular acidic stress. In Xenopus laevis oocytes the VSG lipase was expressed as a nonacylated and a thioacylated form. This thioacylation occurred within a cluster of three cysteine residues but was not essential for catalytic activity per se. These two forms were also detected in trypanosomes and appeared to be present at roughly equivalent amounts. A reversible shift to the acylated form occurred when cells were triggered to release the VSG by either nonlytic acid stress or osmotic lysis. A wild type VSG lipase or a gene mutated in the three codons for the acylated cysteines were reinserted in the genome of a trypanosome null mutant for this gene. A comparative analysis of these revertant trypanosomes indicated that thioacylation might be involved in regulating enzyme access to the VSG substrate.

Characterization of a novel alanine-rich protein located in surface microdomains in Trypanosoma brucei.

Heterologous expression in COS cells followed by orientation-specific polymerase chain reaction to select and amplify cDNAs encoding surface proteins in Trypanosoma brucei resulted in the isolation of a cDNA ( approximately 1.4 kilobase) which encodes an acidic, alanine-rich polypeptide that is expressed only in bloodstream forms of the parasite and has been termed bloodstream stage alanine-rich protein (BARP). Analysis of the amino acid sequence predicted the presence of a typical NH(2)-terminal leader sequence as well as a COOH-terminal hydrophobic extension with the potential to be replaced by a glycosylphosphatidylinositol anchor. A search of existing protein sequences revealed partial homology between BARP and the major surface antigen of procyclic forms of Trypanosoma congolense. BARP migrated as a complex, heterogeneous series of bands on Western blots with an apparent molecular mass ( approximately 50-70 kDa) significantly higher than predicted from the amino acid sequence ( approximately 26 kDa). Confocal microscopy demonstrated that BARP was present in small discrete spots that were distributed over the entire cellular surface. Detergent extraction experiments revealed that BARP was recovered in the detergent-insoluble, glycolipid-enriched fraction. These data suggested that BARP may be sequestered in lipid rafts.