Sialidase NEU4 is involved in glioblastoma stem cell survival.

The human sialidase, NEU4, has emerged as a possible regulator of neuronal differentiation and its overexpression has been demonstrated to promote the acquisition of a stem cell-like phenotype in neuroblastoma cells. In this paper, we demonstrated that glioblastoma stem cells (GSCs) isolated from glioblastoma multiforme (GBM) cell lines and patients' specimens as neurospheres are specifically marked by the upregulation of NEU4; in contrast, the expression of NEU4 is very low in non-neurosphere-differentiated GBM cells. We showed that NEU4 silencing by miRNA or a chemical inhibitor of its catalytic activity triggered key events in GSCs, including (a) the activation of the glycogen synthase kinase 3ß, with the consequent inhibition of Sonic Hedgehog and Wnt/ß-catenin signalling pathways; (b) the decrease of the stem cell-like gene expression and marker signatures, evidenced by the reduction of NANOG, OCT-4, SOX-2, CD133 expression, ganglioside GD3 synthesis, and an altered protein glycosylation profile; and (c) a significant decrease in GSCs survival. Consistent with this finding, increased NEU4 activity and expression induced in the more differentiated GBM cells by the NEU4 agonist thymoquinone increased the expression of OCT-4 and GLI-1. Thus, NEU4 expression and activity appeared to help to determine the molecular signature of GSCs and to be closely connected with their survival properties. Given the pivotal role played by GSCs in GBM lethality, our results strongly suggest that NEU4 inhibition could significantly improve current therapies against this tumour.

Stem cell mobilization in patients with newly diagnosed multiple myeloma after lenalidomide induction therapy.

Lenalidomide has raised concerns regarding its potential impact on the ability to collect stem cells for autologous stem cell transplantation, especially after prolonged exposure. The use of cyclophosphamide plus granulocyte colony-stimulating factor (G-CSF) to mobilize peripheral blood stem cells may overcome this concern. In newly diagnosed multiple myeloma (MM) patients, we investigated the influence of lenalidomide on stem cell collection. In a prospective study, 346 patients received four cycles of lenalidomide-dexamethasone (Rd). Stem cells were mobilized with cyclophosphamide and G-CSF. Patients failing to collect a minimum of 4 × 10(6) CD34(+)/kg cells received a second mobilization course. After mobilization, a median yield of 8.7 × 10(6) CD34(+)/kg was obtained from patients receiving Rd induction. After first mobilization, inadequate yield was observed in 21% of patients, whereas only 9% of patients failed to collect the target yield after the second mobilization attempt. In conclusion, we confirm that a short induction with lenalidomide allowed sufficient stem cells collection to perform autologous transplantation in 91% of newly diagnosed patients.

Silencing of membrane-associated sialidase Neu3 diminishes apoptosis resistance and triggers megakaryocytic differentiation of chronic myeloid leukemic cells K562 through the increase of ganglioside GM3.

In chronic myeloid leukemia K562 cells, differentiation is also blocked because of low levels of ganglioside GM3, derived by the high expression of sialidase Neu3 active on GM3. In this article, we studied the effects of Neu3 silencing (40-70% and 63-93% decrease in protein content and activity, respectively) in these cells. The effects were as follows: (a) gangliosides GM3, GM1, and sialosylnorhexaosylceramide increased markedly; (b) cell growth and [(3)H]thymidine incorporation diminished relevantly; (c) as mRNA, cyclin D2, and Myc were much less expressed, whereas cyclin D1 was expressed more like its inhibitor p21; (d) as mRNA, pro-apoptotic proteins Bax and Bad increased with concurrent decrease and increase in the anti-apoptotic proteins Bcl-2 and Bcl-XL, respectively; (e) the apoptosis inducers etoposide and staurosporine were active on Neu3 silencing cells but not on mock cells; (f) as mRNA, the megakaryocytic markers CD10, CD44, CD41, and CD61 increased similar to the case of mock cells stimulated with PMA; (g) the signaling cascades mediated by PLC-beta2, PKC, RAF, ERK1/2, RSK90, and JNK were largely activated. The induction of a GM3-rich ganglioside pattern in K562 cells by treatment with brefeldin A elicited a phenotype similar to that of Neu3 silencing cells. In conclusion, upon Neu3 silencing, K562 cells show a decrease in proliferation, propensity to undergo apoptosis, and megakaryocytic differentiation.

Participation of critical residues from the extreme C-terminal end of the human androgen receptor in the ligand binding function.

A short C-terminal end is present at the end of the human androgen receptor (hAR) similar to that of other steroid receptors. It is located directly after helix 12 of the ligand binding domain and has never been described as being part of the hydrophobic binding pocket. Although some fragmentary data have indicated the involvement of this region in ligand binding, its precise function still remains unclear. To gain deeper insight into the role of the hAR extreme C-terminal end, an extensive mutational analysis was carried out by using site-directed mutagenesis and alanine scanning over the 13-residue C-terminal end region. Both ligand binding and transcriptional activity were tested with each mutant. Our study demonstrates the participation of almost all of the amino acids in this region for the ligand binding function and consequently for the transcriptional activity. A conformational study by limited proteolysis was performed with the mutants that most affected the affinity of the receptor. It was remarkable that the mutants with a low binding affinity adopted an inactive conformation and were either less or not able to undergo a following conformational change to provide the active form of the receptor. Our results demonstrate the importance of hydrophobicity for the function of the C-terminal end with residues located at very precise positions. Especially, both hydrophobicity and aromaticity on position 916 are critical for providing the correct ligand binding conformation of the receptor. Furthermore, this study highlights essential criteria regarding the C-terminal amino acids which could be applied to other steroid receptors.

Competitiveness and conflict behavior in simulation of a social dilemma.

This experiment examined the competitive behavior in a seven-choice Prisoner's Dilemma game of 108 adult students (68 women, 40 men) classified as high, average, or low in competitiveness based on their scores on the Competitiveness Index. Participants were then presented one of three preprogrammed response conditions representing (1) Competitive, (2) De-escalating, or (3) Noncompetitive conflict behavior from a simulated opponent. Participants high in competitiveness engaged in more competitive behavior and reported higher satisfaction with the task than those low in competitiveness. As expected, the Competitive conditions elicited more competitive behavior than Noncompetitive conditions. The results suggest competitive individuals may be particularly susceptible to social cues that trigger competitive behavior.

Cysteines 849 and 942 of human mineralocorticoid receptor are crucial for steroid binding.

To assess the role of each of the four cysteine residues in the hormone binding domain (HBD) of the human mineralocorticoid receptor (hMR), we have separately substituted C808, C849, C910, and C942 into serine. These mutations were created in the G595-K984 hMR receptor fragment which encompasses the DNA binding domain, the hinge region, and the hormone binding domain. Each mutant was further analyzed by steroid binding assays and transactivation assays using wild-type and mutant proteins expressed in vitro in the reticulocyte lysate. While the C910S mutant exhibited similar wild-type G595-K984 receptor binding properties for aldosterone, the C808S mutant affinity was 3.5-fold higher. In contrast, the C849S mutant showed a drastic drop in affinity for aldosterone and the mutant C942S was unable to bind the steroid. Affinities for the antagonist progesterone were also determined. C808S, C849S, and C910S were found to bind progesterone better than aldosterone (about a 2-fold increase in their affinities). Mutant C942S failed to bind any steroid tested (aldosterone, progesterone, cortisol, and the synthetic antagonist RU26752). No change in the specificity toward various agonists and antagonists could be observed with the mutants when compared to the wild-type G595-K984. When transactivation assays were performed, the properties of mutants C808S and C910S were similar to those of the wild-type G595-K984, while mutant C849S showed reduced sensitivity and C942S was devoid of any transcriptional activity. Our data indicate that C849 and C942 are critical for the ligand binding process of hMR. Moreover, C942 might be involved in a direct contact with the 20-keto group of the steroid.

Folding requirements of the ligand-binding domain of the human mineralocorticoid receptor.

The effects of aldosterone are mediated by the mineralocorticoid receptor (MR), a ligand-dependent transcription factor. We investigated the structural determinants for ligand binding to the receptor using a series of human MR (hMR) deletion mutants. These proteins were produced in vitro in rabbit reticulocyte lysate and analyzed for their ability to bind agonists, antagonists, and the heat shock protein hsp90, which is a prerequisite for ligand binding to hMR. Studies on N terminus-truncated hMRs showed that the ligand-binding domain (LBD: amino acids 734-984) has a lower affinity for aldosterone than the entire receptor [dissociation constant (Kd) 2.9 vs. 0.47 nM] and does not interact with hsp90. Addition of the five-amino acid sequence (729-733) upstream from the LBD is necessary for interaction with hsp90, but a larger region is needed for high aldosterone affinity. Deletions at the C-terminal end of the hMR greatly reduced both agonist and antagonist binding: deletion of the last three amino acids reduced the affinity for aldosterone to 1/20 that of the entire protein, and deletion of the last four amino acids completely abolished binding, although the interaction with hsp90 was not affected. These effects can be explained by misfolding of the receptor, since limited proteolysis assays showed that deletions at the C-terminal end of hMR affect the accessibility of the cleavage sites within the DNA-binding domain and the N-terminal part of the hinge region to trypsin. Thus, our results support the idea that a short sequence upstream of the LBD is essential for the interaction of hMR with hsp90 and that the C terminus of hMR and hsp90 are both essential for folding of the receptor in a high-affinity hormone-binding state.

Involvement of the N-terminal region of the human mineralocorticoid receptor hormone-binding domain in agonist and antagonist binding as revealed by a new monoclonal antibody.

To gain a better understanding of the mechanism of binding to the human mineralocorticoid receptor (hMR), we developed a new monoclonal antibody (mAb) raised against the hormone-binding domain (HBD). For this purpose, mice were immunized with a fusion protein including the sequence Thr729-Lys984 of hMR. After ELISA screening, mAb 18C7 was selected for its specificity towards the HBD. This antibody recognized both the denatured and native MR forms, as well as the hetero-oligomeric MR form and the transformed MR state. By using several HBD subfragments, the mAb 18C7 epitope was located in the N-terminal region of the HBD from Thr729 to Leu765. We then studied the effect of the antibody on aldosterone and progesterone binding to the hMR. When 18C7 was incubated with liganded MR, it was able to partly displace (20%) the hormone from its binding site. When 18C7 was incubated with MR before aldosterone or progesterone, the antibody inhibited 75-80% of the binding. The effect of 18C7 on the binding was similar with both hormones. A sucrose gradient analysis indicated the simultaneous presence of two kinds of receptor complexes: the steroid-MR complex and the antibody-MR complex. After its associated proteins, especially the heat-shock protein hsp90, had been cross-linked with the hMR by dimethylpimelimidate, 18C7 was still able to react with the receptor. Our results indicated that the epitope recognized by 18C7 was directly implicated in hormone binding. The lack of steroid binding of HBD mutants with the Thr729-Leu765 sequence deleted [Jalaguier, Mesnier, Léger and Auzou (1996) J. Steroid Biochem. Mol. Biol. 57, 43-50] supports this hypothesis. Because of the similar behaviours of aldosterone and progesterone, we conclude that the N-terminal Thr729-Leu765 region of the HBD is similarly involved in the binding of both hormones.

GEP31, a new gastric epithelial protein, early expressed during ontogenesis.

A set of monoclonal antibodies directed against various gastric markers was produced in order to study the developmental expression of the gastric mucosa. A previously described monoclonal antibody, mab 146.14, labeled the proton pump (H+, K+) ATPase specifically located in gastric parietal cells. Mabs 15.1 and 121.17 revealed the mucus of mucous neck cells and mucous surface cells, respectively. By addition, mab 81.1 was directed against a cell surface membrane protein antigen composed of a major 31 kDa component (called GEP31). Our study mainly focused on the characterization of GEP31. This protein was typically located in the gastrointestinal epithelial tract (stomach, small intestine, colon). Moreover, interesting features were observed during the study of the early ontogenesis of the gastric mucosa. The 31 kDa protein was detected at the onset of gland formation (day 18), and a gradual increase in expression of the protein could be observed between day 18 and day 19. Furthermore, a comparative study of the expression of different terminal differentiation markers of gastric epithelial cells ((H+, K+) ATPase, mucins) during the early period of ontogenesis revealed that GEP31 could be detected well before the appearance of these markers. To our knowledge, GEP31 thus appears as the earliest expressed specific cell surface epithelial gastric antigen described to date. Furthermore, the partial N-terminal amino acid sequence of GEP31 was determined and revealed that it is not a known protein.

Endosulfine, an endogenous peptidic ligand for the sulfonylurea receptor: purification and partial characterization from ovine brain.

Antidiabetic sulfonylureas act through receptors coupled to ATP-dependent potassium channels. Using the binding of [3H]glibenclamide, a highly potent sulfonylurea, to rat brain membranes to follow the purification procedure, we extracted from ovine brain, purified, and partially characterized two peptides that are endogenous ligands for the central nervous system sulfonylurea receptors. These peptides, referred to as alpha and beta endosulfine, differ by their isoelectric points, the beta form being more basic. Each form of endosulfine is recognized equally by the sulfonylurea receptors from the central nervous system and from insulin-secreting beta cells. In the same concentration range that is active on the receptors, beta endosulfine releases insulin from a beta-cell line. Endosulfine is a good candidate for being implicated in the physiology of beta cells and their disorders (e.g., type II diabetes) and in certain pathologies related to modifications of ion fluxes.

An endogenous ligand for the central sulfonylurea receptor.

An endogenous ligand for the rat central sulfonylurea receptor has been evidenced in the rat central nervous system. The characteristics of this ligand (extractibility, non-dialysability, chromatographic behaviour on different media, sensitivity to proteases) indicate that it is a neutral to slightly basic peptide.

A binding site for [3H]glipizide in the rat cerebral cortex.

[3H]glipizide, a 2nd generation hypoglycemic sulfonylurea, binds specifically to rat cerebral cortex membranes in a time- and temperature-dependent way. The binding is saturable and reversible. The maximal binding capacity is 110 fmol/mg protein and the dissociation constant 1.5 nM. The binding site was destroyed by proteolytic and lipolytic enzymes suggesting a lipoprotein nature. Active analogs of sulfonylureas are characterized by IC50 values in the cerebral cortex which parallel their insulinotropic activity. In the cerebral cortex, adenylate cyclase was not stimulated by glipizide but sulfonylureas could inhibit, at high doses, the cAMP-dependent phosphodiesterase. This central binding site for glipizide displays the characteristics of the recognition moiety of a biological receptor.

[Demonstration of a specific receptor site for glucagon-37 (oxyntomodulin/bioactive enteroglucagon) in rat oxyntic glands]. / Mise en évidence d'un site récepteur spécifique du glucagon-37 (oxyntomoduline/enteroglucagon bioactif) dans les glandes oxyntiques de rat.

In this work, we show directly the existence of specific binding sites for Glucagon-37 (G-37) in isolated oxyntic glands from rat fundic mucosa. The binding of 125I-Glucagon-37 was inhibited by G-37 with a dissociation constant KD = 2.6 X 10(-9) M (high affinity binding capacity = 85 fmol/mg of protein) and by pancreatic Glucagon (G-29) with a KD = 2.2 X 10(-8) M. These results confirm the tissue specificity of G-37, evidenced in vitro on the cyclic AMP production by the same glands and in vivo on the inhibition of gastric acid secretion. They suggest that these activities are related to the G-37-binding on this novel receptor site for Glucagon-related peptides, which is distinct from receptors for the main stimulants of acid secretion, such as Gastrin, Histamine or cholinergic agents.

[Orthotopic liver graft with aortic clamping. Experimental study in dogs (author's transl)]. / La greffe orthotopique du foie sous clampage aortique. Etude expérimentale chez le chien.

Orthotopic liver transplants were conducted in 15 dogs, with aortic clamping during the anhepatic time of the operation and without venous shunting. Aortic clamping lasted for between 30 and 42 minutes. Immunosuppressant treatment was not given. Eight dogs died within 2 hours after the operation, 5 from haemorrhage and 3 accidentally. Seven animals survived for between 6 hours and 11 days: 3 died within 24 hours from haemorrhage, 2 from hepatic failure between the 2nd and 3rd day. One animal died on the 7th day from an acute intestinal invagination, the dog surviving for the longest period eventually dying after rejection of the transplant. These results demonstrate, as in other reported series, that the most frequent cause of death is the haemorrhagic diathesis, probably as a result of poor graft conservation. Dogs tolerate the supracoeliac aorta clamp both from the renal and intestinal points of view ; spinal cord tolerance to the ischaemia is less evident as paraplegia of the hindquarters was noted in one animal in the group. Aortic clamping considerably reduces operative time, as it avoids the need to construct a mesentericocaval anastomosis and a femorojugular shunt. It also avoids splanchnic blood sequestration and the risk of reducing cardia filling during clamping of the inferior vena cava.