ERRα promotes breast cancer cell dissemination to bone by increasing RANK expression in primary breast tumors.

Bone is the most common metastatic site for breast cancer. Estrogen-related-receptor alpha (ERRα) has been implicated in cancer cell invasiveness. Here, we established that ERRα promotes spontaneous metastatic dissemination of breast cancer cells from primary mammary tumors to the skeleton. We carried out cohort studies, pharmacological inhibition, gain-of-function analyses in vivo and cellular and molecular studies in vitro to identify new biomarkers in breast cancer metastases. Meta-analysis of human primary breast tumors revealed that high ERRα expression levels were associated with bone but not lung metastases. ERRα expression was also detected in circulating tumor cells from metastatic breast cancer patients. ERRα overexpression in murine 4T1 breast cancer cells promoted spontaneous bone micro-metastases formation when tumor cells were inoculated orthotopically, whereas lung metastases occurred irrespective of ERRα expression level. In vivo, Rank was identified as a target for ERRα. That was confirmed in vitro in Rankl stimulated tumor cell invasion, in mTOR/pS6K phosphorylation, by transactivation assay, ChIP and bioinformatics analyses. Moreover, pharmacological inhibition of ERRα reduced primary tumor growth, bone micro-metastases formation and Rank expression in vitro and in vivo. Transcriptomic studies and meta-analysis confirmed a positive association between metastases and ERRα/RANK in breast cancer patients and also revealed a positive correlation between ERRα and BRCA1mut carriers. Taken together, our results reveal a novel ERRα/RANK axis by which ERRα in primary breast cancer promotes early dissemination of cancer cells to bone. These findings suggest that ERRα may be a useful therapeutic target to prevent bone metastases.

Subchondral and trabecular bone metabolism regulation in canine experimental knee osteoarthritis.

OBJECTIVE: To determine trabecular and subchondral bone metabolic changes in experimental canine osteoarthritis (OA). METHODS: OA was induced in 19 dogs by transection of the anterior cruciate ligament (ACL) of the right knee through a stab wound. Dogs were sacrificed at 8 (n=7) and 12 weeks (n=12) after surgery. Non-operated normal dogs (n=6) were used as controls. After sacrifice, samples were obtained from the weight-bearing area of medial tibial plateaus. Explants and cell cultures were prepared from subchondral and trabecular bone. Osteocalcin (Oc), cellular alkaline phosphatase (ALPase), urokinase plasminogen-activator (uPA), prostaglandin E2 (PGE2), metalloproteinase (MMP) and nitric oxide (NO) were measured using standard procedures. RESULTS: ALPase production was significantly increased only at week 12 in subchondral and trabecular bone, while an increase in Oc was noted at week 8. uPA and MMP activity were increased significantly at week 12 in subchondral bone, while PGE2 levels were significantly higher in subchondral and trabecular bone at week 12 compared to normal. A decrease in NO production appeared late at week 12 in trabecular bone, whereas NO levels from subchondral bone were significantly increased compared to normal at week 8. DISCUSSION: Intense bone remodeling takes place in both subchondral and trabecular bone in the knee following ACL transection. This process seems to occur around week 12, although Oc and NO appeared to be involved earlier at 8 weeks. These results suggest that not only subchondral but also trabecular bone metabolism is altered in this OA model.

Identification and differential expression of human collagenase-3 mRNA species derived from internal deletion, alternative splicing, and different polyadenylation and transcription initiation sites.

OBJECTIVE: Collagenase-3 is a metalloprotease that plays a role in tissue remodeling and pathological processes including arthritis. The human gene is transcribed into major (3.0 and 2.5 kb) and minor (2.2/2.0 kb) transcripts, as seen in Northern blot assays. We investigated the possibility that other transcripts, not detectable by Northern blot, were synthesized as either coding or regulatory RNAs that would modulate collagenase-3 expression and function/activity. DESIGN: We screened a cDNA library and total RNA from human chondrocytes by plaque hybridization and RT-PCR, and expressed the transcripts in a cellular environment. The levels of expression of each transcript in normal and osteoarthritic joint cells and cartilage were monitored by RT-PCR. RESULTS: We identified five different collagenase-3 RNA species derived from alternative polyadenylation sites (COL3-APS), internal deletion (COL3-DEL), alternative splicing (COL3-9B/COL3-9B-2), and different transcription initiation site (COL3-ATS and COL3-ATS-INT). Each transcript could be translated in a cellular environment. Interestingly, the proteins translated from the COL3-DEL and COL3-9B-2 transcripts had a modified hemopexin-like domain, suggesting altered collagenolytic activities. The transcript types COL3-APS, COL3-9B-2, and COL3-ATS were up-regulated in the osteoarthritic samples and expressed in the chondrosarcoma cell line SW1353. CONCLUSION: Our data show that the human collagenase-3 gene is subjected to different levels of regulation and constitutes a more complex system than was originally thought.

Lymphopenia in occupational pulmonary silicosis with or without autoimmune disease.

An increased prevalence of autoimmune diseases such as rheumatoid arthritis has been demonstrated in silica-exposed patients. The aim of this study was to determine the peripheral blood lymphocyte phenotype in a population of silicotic workers employed in the slate mines of the district. Silicosis was assessed in 58 patients according to the International Labor Office's criteria. Clinical and biological data including flow cytometric evaluation of the lymphocyte subsets were compared with those from 41 healthy volunteers. The silicotic patients had a higher prevalence of autoimmune diseases (6/58 versus 0/41: P < 0.05) and of elevated antinuclear antibody titres compared to the control group. A very significant decrease of total lymphocyte count (P < 0.001) involving B, T and Natural Killer cells was found in silicotic patients as compared with matched healthy volunteers. A significant increase in the percentage of activated T cells (12.3%) was observed in the silicotic group as compared to 6.5% in the control group (P = 5 x 10(-5)). Our results show that in silicotic patients, the absolute number of circulating lymphocytes is diminished with an increased proportion of activated T cells. Whether these findings could predispose to the development of autoimmune disorders is discussed.

Chondrocyte death in experimental osteoarthritis is mediated by MEK 1/2 and p38 pathways: role of cyclooxygenase-2 and inducible nitric oxide synthase.

OBJECTIVE: To explore the mechanisms responsible for in situ induction of chondrocyte death in experimental dog osteoarthritic (OA) cartilage. The roles of 2 mitogen activated protein kinases (MAPK), MEK 1/2 and p38, nuclear factor-kappaB (NF-kappaB), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and the caspase cascade were investigated. METHODS: OA knee cartilage was obtained from dogs that had received sectioning of the anterior cruciate ligament and were sacrificed 12 weeks after surgery. Cartilage explants were cultured in different inhibitors: Z-DEVD-FMK (caspase 3 inhibitor), Z-LEHD-FMK (caspase 9 inhibitor), PD 98059 (MEK 1/2 inhibitor). SB 202190 (p38 inhibitor), SN-50 (NF-kappaB inhibitor), NS-398 (COX-2 inhibitor), N-iminoethyl-l-lysine (L-NIL) (iNOS inhibitor). Cartilage specimens were stained for TUNEL reaction and immunostained using specific antibodies for caspase 3, COX-2, iNOS, and nitrotyrosine. Morphometric analyses were performed. RESULTS: The significant level of chondrocyte death in OA cartilage was markedly decreased by caspase 3 and caspase 9 inhibitors. The two MAPK inhibitors, but not the NF-kappaB inhibitor, decreased chondrocyte death concomitant with the levels of caspase 3 and iNOS. COX-2 level was reduced by all 3 inhibitors. Specific inhibition of either COX-2 or iNOS reduced the level of chondrocyte death and caspase 3. There was evidence of crosstalk between these 2 latter systems; specific inhibition of COX-2 reduced the iNOS level, and selective inhibition of iNOS reduced COX-2 expression. COX-2 and iNOS seem to function in a positive autoregulatory manner that triggers transcription of their own biosynthetic machinery, since the specific inhibition of each system downregulates its expression. CONCLUSION: This study shows that in the early lesions of experimental OA cartilage in situ, activation of the caspase cascade is responsible for induction of chondrocyte death. Marked inhibition of cell death by caspase inhibitors indicates a significant participation of apoptosis in the phenomenon. This phenomenon is linked to the activation of at least 2 major kinase pathways, MEK 1/2 and p38. These pathways are responsible for upregulating the expression of iNOS and COX-2, each of which seems essential for the induction of apoptosis. Data are provided about possible regulation and interregulation of the COX-2 and iNOS systems by prostaglandin E2 and NO.

In vivo dual inhibition of cyclooxygenase and lipoxygenase by ML-3000 reduces the progression of experimental osteoarthritis: suppression of collagenase 1 and interleukin-1beta synthesis.

OBJECTIVE: To study the therapeutic effectiveness of ML-3000, a new antiinflammatory drug that has balanced dual inhibitory activity against 5-lipoxygenase and cyclooxygenase, on the development of lesions in the experimental osteoarthritis (OA) dog model, and to determine the action of ML-3000 on the synthesis of collagenase 1 in cartilage and interleukin-1beta (IL-1beta) in synovial membrane. METHODS: The anterior cruciate ligament of the right stifle joint of 21 mongrel dogs was sectioned with a stab wound. Dogs were divided into 3 groups: group 1 (n = 7) received placebo; groups 2 (n = 7) and 3 (n = 7) were treated with therapeutic dosages of oral ML-3000 at 2.5 mg/kg/day and 5 mg/kg/day, respectively. The dogs began receiving medication the day after surgery and were killed 8 weeks later. The size and grade of cartilage erosions on both the condyles and plateaus were evaluated, and the severity of the cartilage lesions and synovial inflammation was examined histologically. Levels of collagenase 1 in cartilage and IL-1beta in the synovial membrane were measured by immunohistochemistry. In addition, levels of prostaglandin E2 (PGE2) in the synovial fluid and leukotriene B4 (LTB4) in cultured synovial membrane explants were determined using specific enzyme immunoassays. RESULTS: Serum levels of ML-3000 in treated dogs were within the therapeutic range. ML-3000 significantly decreased the size and grade of the cartilage lesions in tibials and plateaus, compared with placebo. At the histologic level, the severity of cartilage lesions was also decreased in the ML-3000-treated dogs versus the placebo-treated dogs in both the condyles and the plateaus. All 3 OA groups exhibited a notable and similar level of synovial inflammation. ML-3000 significantly decreased the level of PGE2 in synovial fluid and LTB4 production by synovium. It also markedly reduced the levels of collagenase 1 in cartilage and IL-1beta in synovial membrane. CONCLUSION: ML-3000 significantly reduced the development of lesions in experimental dog OA. The drug acts by reducing the synthesis of the inflammation mediators PGE2 and LTB4 and catabolic factors such as collagenase 1 and IL-1beta, which are known to play an important role in the pathophysiology of OA lesions. The effect of the drug on catabolic factors could possibly be related to its inhibitory action on LTB4 synthesis.

Transforming growth factor-beta induced collagenase-3 production in human osteoarthritic chondrocytes is triggered by Smad proteins: cooperation between activator protein-1 and PEA-3 binding sites.

OBJECTIVE: To examine the signaling pathways leading to transforming growth factor-beta (TGF-beta) induced collagenase-3 production in human osteoarthritic (OA) chondrocytes, as well as the transcription factors and their binding sites involved in the transcriptional control of collagenase-3 gene. METHODS: Identification of the TGF-beta signaling pathway was by Western immunoblotting using specific antibodies for the phosphorylated forms of p44/42 and p38 MAPK, SAPK/JNK, and the Smad2 protein. Electromobility shift assays (EMSA) were carried out for activator protein- (AP-1), polyomavirus enhancer A (PEA-3), activin-response-element-like, Smad-binding-element-like, and TGF-beta inhibitory element oligonucleotides. Supershift assays using antibodies to the Jun, Fos, and Smad families of proteins were used for identification of transcription factors. Chondrocyte transfections were also performed using the -133CAT collagenase-3 promoter plasmid (containing PEA-3, AP-1, and TATA sites) and mutated AP-1 and PEA-3 sites. RESULTS: The primary target of TGF-beta induced collagenase-3 in OA chondrocytes was the Smad2 protein, with significant phosphorylation within 5 min. Contrasting with the Smad2, the untreated OA chondrocytes already had detectable levels of the phosphorylated forms of p38 and p44/42 MAPK. Of the oligonucleotides tested, EMSA revealed that TGF-beta treated OA chondrocyte proteins bound only to the AP-1 and PEA-3. Supershifts with the AP-1 oligonucleotide showed the presence of the Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) proteins in the untreated and TGF-beta treated OA chondrocytes, whereas only Smad proteins (Smad2, 3, 4) were present in the AP-1 binding proteins from the TGF-beta treated chondrocytes. The AP-1 mutation decreased both basal (95%) and TGF-beta induced (99%) collagenase-3 production, whereas the PEA-3 mutation decreased the basal (15%) but more significantly (50%) the TGF-beta induced transcription. CONCLUSION: Smad proteins are the main cytoplasmic signaling pathways in TGF-beta stimulated collagenase-3 in OA chondrocytes. The AP-1 site appears critical for upregulation of collagenase-3 production, but TGF-beta stimulation requires both AP-1 and PEA-3 sites for optimal response.

Modulation of TIMP-1 synthesis by antiinflammatory cytokines and prostaglandin E2 in interleukin 17 stimulated human monocytes/macrophages.

OBJECTIVE: To examine the regulation of tissue inhibitor of metalloproteinase 1 (TIMP-1) synthesis by interleukin 17 (IL-17) stimulated human monocytes/macrophages in primary culture in the presence of prostaglandin E2 (PGE2) and antiinflammatory cytokines, and to compare this with the regulation of matrix metalloproteinase (MMP-9) production. METHODS: IL-17 stimulated human monocytes isolated from the peripheral blood of healthy donors were cultured in the presence of PGE2, cyclic adenosine monophosphate (cAMP) mimetics (IBMX, cAMP, forskolin, cholera toxin), or antiinflammatory cytokines (IL-4, IL-10, IL-13), or with protein kinase inhibitors of diverse specificity. MMP-9 and TIMP-1 were measured using specific ELISA, while expression of specific messenger RNA was determined by Northern blotting. RESULTS: IL-17 stimulated an increased level of MMP-9 production relative to TIMP-1 production in monocytes/macrophages. Stimulation was accompanied by upregulation of specific MMP-9 mRNA expression relative to TIMP-1 mRNA. Exogenous PGE2, cAMP, and cAMP-mimetics completely inhibited both basal and IL-17 induced MMP-9 synthesis, while only IL-17 induced TIMP-1 synthesis was abrogated. The same effect was found for the antiinflammatory cytokines. Both basal and IL-17 induced production of TIMP-1 involved p42/44 and p38 kinases and nuclear factor kappaB signaling pathways. CONCLUSION: The excess of MMP-9 over TIMP-1 production, and decreased inhibition of MMP-9 activity in chronic rheumatoid diseases, may result in cartilage degradation and joint destruction.

The induction of cell death in human osteoarthritis chondrocytes by nitric oxide is related to the production of prostaglandin E2 via the induction of cyclooxygenase-2.

There is increasing evidence suggesting that chondrocyte death may contribute to the progression of osteoarthritis (OA). This study focused on the characterization of signaling cascade during NO-induced cell death in human OA chondrocytes. The NO generator, sodium nitroprusside (SNP), promoted chondrocyte death in association with DNA fragmentation, caspase-3 activation, and down-regulation of Bcl-2. Both caspase-3 inhibitor Z-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-CH2F and caspase-9 inhibitor Z-Leu-Glu(OCH3)-His-Asp(OCH3)-CH2F prevented the chondrocyte death. Blocking the mitogen-activated protein kinase pathway by the mitogen-activated protein kinase kinase 1/2 inhibitor PD98059 or p38 kinase inhibitor SB202190 also inhibited the SNP-mediated cell death, suggesting possible requirements of both extracellular signal-related protein kinase 1/2 and p38 kinase for the NO-induced cell death. Furthermore, the selective inhibition of cyclooxygenase (COX)-2 by NS-398 or the inhibition of COX-1/COX-2 by indomethacin blocked the SNP-induced cell death. The chondrocyte death induced by SNP was associated with an overexpression of COX-2 protein (as determined by Western blotting) and an increase in PGE2 release. PD98059 and SB202190, but neither Z-DEVD FMK nor Z-LEHD FMK completely inhibited the SNP-mediated PGE2 production. Analysis of interactions between PGE2 and the cell death showed that PGE2 enhanced the SNP-mediated cell death, whereas PGE2 alone did not induce the chondrocyte death. These data indicate that NO-induced chondrocyte death signaling includes PGE2 production via COX-2 induction and suggest that both extracellular signal-related protein kinase 1/2 and p38 kinase pathways are upstream signaling of the PGE2 production. The results also demonstrate that exogenous PGE2 may sensitize human OA chondrocytes to the cell death induced by NO.

Glucosamine sulfate modulates dysregulated activities of human osteoarthritic chondrocytes in vitro.

OBJECTIVE: The efficacy of glucosamine sulfate (GS) in the symptomatic treatment of patients with osteoarthritis (OA) is suggested to be mediated by still unknown effects on the altered OA cartilage. DESIGN: Using human OA chondrocytes in culture, the effects of GS on protein synthesis, caseinase, collagenase, phospholipase A2 (PLA2) and protein kinase C (PKC) activities as well as production of nitric oxide and cyclic AMP were studied in both cells and culture medium. RESULTS: GS significantly reduced PLA2 activity, and more modestly collagenase activity, in the OA chondrocytes in a dose-dependent manner. By contrast, PLA2 and collagenase activity of the culture medium was not modified. No effects on caseinase activity was seen. GS significantly and dose-dependently increased protein synthesis. GS did not modify nitric oxide and cAMP production but significantly increased PKC production. CONCLUSION: GS modified cultured OA chondrocyte metabolism by acting on PKC, cellular PLA2, protein synthesis and possibly collagenase activation. Extrapolation of the effect to the in-vivo situation remains hypothetical but they might represent some possible mechanisms of action of the drug in human.

The presence of cytoplasmic antineutrophil cytoplasmic antibodies (C-ANCA) in the course of subacute bacterial endocarditis with glomerular involvement, coincidence or association?

Antineutrophil cytoplasmic antibodies positivity with cytoplasmic pattern (C-ANCA) and proteinase-3 (PR-3) specificity was found in two patients with both subacute bacterial endocarditis (SBE) and glomerular involvement. Renal biopsy showed membranoproliferative glomerulonephritis in one case and focal segmental glomerulonephritis in the second case. Immunofluorescence study showed granular immune deposits in both cases evocating immune complex glomerulonephritis. Renal and biological manifestations disappeared with clinical improvement secondary to antibiotherapy. Physicians have to consider the possible occurrence of such C-PR-3 ANCA, claimed to be specific markers for Wegener's granulomatosis, in infectious diseases such as SBE. Hence we focus on the necessity of performing a renal biopsy with light microscopy and immunofluorescence studies in all patients with ANCA associated glomerular disease.

Phospholipase A2 activity in herniated lumbar discs. Clinical correlations and inhibition by piroxicam.

STUDY DESIGN: Prospective study of phospholipase A2 activity in the serum and intervertebral discs of patients undergoing surgery for sciatica due to disc herniation. OBJECTIVES: To determine correlations between herniated disc phospholipase A2 and clinical, radiographic, and anatomic signs of common sciatica; to evaluate serum phospholipase A2 activity as a marker of disc phospholipase A2; and to investigate the in vivo effect of piroxicam on disc phospholipase A2. SUMMARY OF BACKGROUND DATA: Several studies suggest disc inflammation as a mechanism of sciatica due to disc herniation, and phospholipase A2 emerges as a key enzyme of cartilage and disc tissues. METHODS: Phospholipase A2 activity was determined, using the degradation of a specific substrate, in the serum and discs of 31 patients (14 treated with acetaminophen and 17 treated with piroxicam) undergoing surgery for sciatica due to lumbar disc herniation. Visual analog scale for pain, Dallas Pain Questionnaire, Lasègue's sign, radiographic stage of degeneration of the herniated disc, volume of disc herniation shown by computed tomography, and surgical findings were recorded. RESULTS: Disc phospholipase A2 activity was independent of the patient's age or sex, the radiologic stage of disc degeneration, and the volume of the herniation, and showed no significant correlation with Lasègue's sign or pain measured on a visual analog scale. The correlation between disc phospholipase A2 and the Dallas category of items measuring the impact of pain on daily activities approached the level of significance (P = 0.07). Disc phospholipase A2 activity was significantly higher in cases of sequestrated discs than in other herniations. Disc phospholipase A2 was significantly correlated with serum phospholipase A2, and was significantly lower in patients treated with piroxicam than in those treated with acetaminophen. CONCLUSIONS: Disc phospholipase A2 is thought to participate in the physiopathology of sciatica and to bemodulated by nonsteroidal anti-inflammatory drug therapy. Serum phospholipase A2 is suggested as a biologic marker of disc inflammation in patients with sciatica.

Study of O-sialylation of glycoproteins in C6 glioma cells treated with retinoic acid.

When treated with retinoic acid in vivo, C6 glioma cells show an enhancement of CMP-Neu5Ac:Gal beta 1-3 GalNAc-R alpha-2,3 sialyltransferase activity. A 300 kDa glycoprotein was detected by lectin affinoblotting in retinoic acid-treated C6 cells which stained weakly or not at all in control cells. Comparative studies with different lectins demonstrated that this glycoprotein contains alpha 2,3 Neu5Ac Gal-GalNAc O-glycan moieties. Cultures in the presence of an inhibitor of O-glycan synthesis (N-acetylgalactosaminide alpha-O-benzyl) demonstrated that enhancement of staining of the 300 kDa glycoprotein was not due to the increase of the alpha 2,3 sialytransferase but to the de novo synthesis of the polypeptide chain of this glycoprotein.

[Critical study of the characterization of hyperandrogenism in a group of obese women]. / Etude critique de la caractérisation de l'hyperandrogénie dans un groupe de femmes obèses.

The authors report the hormonologic characteristics of 20 obese and hirsute women meeting the criteria for adrenaltype hyperandrogenism, suppressible by dexamethasone, without hyperprolactinemia and without any late developing partial enzyme block appearance. The laboratory profile of these women differed from that of a group of women with type 1 polycystic ovaries syndrome. In this same group obese women in whom LH/FSH ratio was below 1, there was evidence under baseline conditions of a moderate increase in testosterone and delta 4-androstenedione in relation to increased plasma levels of DHA and SDHA, plasma delta 4 and delta 5-androgen levels falling precipitalely during the dexamethasone suppression test. The ACTH stimulation test revealed greater reactivity for 17 hydroxy-pregnenolone (p < 0.001) and less for 21-deoxycortisol than in the control group of normal women (p < 0.01). The essentially adrenal origin of plasma hyperandrogenism in certain cases of obesity is discussed. Insulin could increase adrenal sensitivity to ACTH and its possible action in vivo on the activity of adrenal enzymes requires clarification. The accumulation of certain androgens in the adrenal cortex could also be responsible for dysregulation of 3 beta ol-dehydrogenase and 11-hydroxylase.

Study of retinoic acid effect upon retinoic acid receptors beta (RAR-beta) in C6 cultured glioma cells.

Using monoclonal antibodies against the RAR-alpha and RAR-beta retinoic receptors, we demonstrated that these receptors were present together in C6 glioma cells as two isoforms of 50 and 55 kDa. For RAR-beta, the 50 kDa isoform predominated (60 to 80% of the total of the two isoforms). After a treatment for 48 h with retinoic acid 10 microM, the 55 kDa form was enhanced, while no effect was observed either on RAR-alpha isoforms from C6 cells and on both RAR-alpha and RAR-beta forms from neuroblastoma SKN SH SY5Y used as a control. Using purified neuronal and glial rat brain nuclei, we showed that the 55 kDa isoform from RAR-beta predominated in glial cells. These results suggest that retinoic acid treatment of C6 cells led to a partial differentiation, the enhancement of the heavy form of RAR-beta being a marker of this phenomenon.

[Diagnostic strategy in infertility due to hyperandrogenism. Development of a decision tree]. / Stratégie diagnostique au cours de l'infertilité avec hyperandrogénie. Elaboration d'un arbre décisionnel.

The detection of clinical hyperandrogenism in women presenting with infertility requires detailed hormonal investigations using the decisional plan suggested here. Initial studies including measurement of plasma androgen, gonadotrophic hormones and prolactin levels, may be sufficient to reveal an adrenal origin or pure ovarian origin. Non-tumor androgenic hypercorticism is seen classically in late-presenting enzyme deficits, but also in other situations: excessive adrenarche, hyperprolactinemia, obesity, chronic stress. The immediate Synacthene test can then eliminate diagnostic uncertainties if it leads to the discovery of appearances of 21- or 11-hydroxylase or 3 beta-ol dehydrogenase blocks. Intense virilisation in a woman with a testosterone level above 2 ng/ml (7 nM/l) should lead to suspicion of an androgen-secreting tumor of the ovary or adrenal. CT scan of the abdomen and true pelvis is essential here since it may reveal the presence of an adrenal or ovarian mass. If no morphological abnormality is shown by this investigation, an endocrine lesion of a small ovary should be strongly suspected, the demonstration of which requires isotope techniques and/or catheterisation of the ovarian veins. Two situations also exist which are responsible for severe hyperandrogenism but less alarming in terms of their course and significance: certain homozygous forms of 21-hydroxylase deficit diagnosed late and ovarian hyperthecosis. It may happen that these hormonal investigations do not suffice alone to determine the precise origin of hyperandrogenism and its cause. The dexamethasone adrenal suppression test is useful in the diagnosis of type II micropolycystic dystrophy, in order to define the essentially ovarian, adrenal or mixed origin of hyperandrogenism.

[Hormonal profile of idiopathic gynecomastia in young adults. Apropos of 488 cases]. / Profil hormonal des gynécomasties idiopathiques de l'adulte jeune. A propos de 488 cas.

Gynecomastia, a very frequent disorder, is present in almost 40% of young men. In this population the investigations often fail to find any aetiology, therefore defining idiopathic gynecomastia. The aim of this work is to compare clinical and hormonal characteristics of 488 subjects with gynecomastia to 41 healthy controls. Their are many explanations for the occurrence of idiopathic gynecomastia, including modification of hormonal balance, change of aromatase activity, or a receptor anomaly. Our works demonstrate a significant decrease in mean testosteronemia, linked to a high prevalence of incipient hypogonadism in the studied population, especially in patients with an history of testicular disease.

Study of O-glycan sialylation in C6 cultured glioma cells: regulation of a beta-galactoside alpha 2,3 sialyltransferase activity by Ca2+/calmodulin antagonists and phosphatase inhibitors.

We have demonstrated that the alpha 2,3 sialyltransferase (alpha 2,3 ST) from C6 cultured glioma cells was inhibited in vivo by W-7 and related Ca2+/Calmodulin (Ca/CaM) antagonists while protein kinase C effectors had no effect. Dephosphorylation of alpha 2,3 ST by the wide specificity alkaline phosphatase led to inactivation indicating that the enzyme is phosphorylated. The serine/threonine protein phosphatase inhibitors okadaic acid and Calyculin A led also to an inhibition of alpha 2,3 ST activity. In addition, Ca/CaM antagonists and phosphatase inhibitors led both to an inhibition of a alpha 2,3 sialoglycoprotein from C6 glioma cells as demonstrated with lectin affinity blotting. A concerted regulatory mechanism with phosphorylation/dephosphorylation of alpha 2,3 ST is then postulated.

Study of O-glycan sialylation in C6 cultured glioma cells: evidence for post-translational regulation of a beta-galactoside alpha 2,3 sialyltransferase activity by N-glycosylation.

We have studied the Gal beta 1-3GalNAc-R alpha 2,3 sialyltransferase from C6 glioma cells transferring Neu5Ac from CMP-Neu5Ac onto O-glycans of glycoproteins. Using synchronized C6 glioma cells, we showed that the alpha 2,3 sialyltransferase activity was inhibited by tunicamycin to a greater extend than DNA and protein biosynthesis suggesting inhibition of N-glycosylation of this enzyme. Additional demonstration of N-glycosylation of the alpha 2,3 sialytransferase was provided through ConA-Sepharose binding. Treatment of partially purified alpha 2,3 sialytransferase by peptide-N-glycosidase F showed a significative inhibition demonstrating that N-glycan moiety is required for complete activity of the C6 glioma cell alpha 2,3 sialyltransferase.

Effect of retinoic acid on two glycosyltransferase activities in C6 cultured glioma cells.

1. Activity of two glycosyltransferases was studied in retinoic acid-treated C6 cultured glioma cells. 2. The beta-galactoside alpha 2,3-sialyltransferase transferring N-acetylneuramin onto the O-glycans residues of glycoproteins was activated up to twice after chronic treatment (from 24 to 96 hr) with all-trans retinoic acid. 3. No effect was observed for shorter treatments. 4. On the opposite, the N-glycan galactosyltransferase activity remained unchanged whatever the length of retinoic acid treatment was. 5. The activatory effect was not dependent on isomery, as all-trans and 13-cis retinoic acid isomers were both activators of the C6 glioma cell sialyltransferase. 6. Measurement of adhesion of retinoic acid-treated cells using labelled plasma membranes showed an enhancement of adhesion in correlation with enhancement of sialyltransferase activity.