Conserved roles for Oct4 homologues in maintaining multipotency during early vertebrate development

All vertebrate embryos have multipotent cells until gastrulation but, to date, derivation of embryonic stem (ES) cell lines has been achieved only for mouse and primates. ES cells are derived from mammalian inner cell mass (ICM) tissue that express the Class V POU domain (PouV) protein Oct4. Loss of Oct4 in mice results in a failure to maintain ICM and consequently an inability to derive ES cells. Here, we show that Oct4 homologues also function in early amphibian development where they act as suppressors of commitment during germ layer specification. Antisense morpholino mediated PouV knockdown in Xenopus embryos resulted in severe posterior truncations and anterior neural defects. Gastrulation stage embryos showed reduced expression of genes associated with uncommitted marginal zone cells, while the expression of markers associated with more mature cell states was expanded. Importantly, we have tested PouV proteins from a number of vertebrate species for the ability to substitute Oct4 in mouse ES cells. PouV domain proteins from both Xenopus and axolotl could support murine ES cell self-renewal but the only identified zebrafish protein in this family could not. Moreover, we found that PouV proteins regulated similar genes in ES cells and Xenopus embryos, and that PouV proteins capable of supporting ES cell self-renewal could also rescue the Xenopus PouV knockdown phenotype. We conclude that the unique ability of Oct4 to maintain ES cell pluripotency is derived from an ancestral function of this class of proteins to maintain multipotency.     

Comparison between two classes of selective EP(3) antagonists and their biological activities

Two different series of very potent and selective EP(3) antagonists have been reported: a novel series of ortho-substituted cinnamic acids [Belley, M., Gallant, M., Roy, B., Houde, K., Lachance, N., Labelle, M., Trimble, L., Chauret, N., Li, C., Sawyer, N., Tremblay, N., Lamontagne, S., Carrière, M.-C., Denis, D., Greig, G. M., Slipetz, D., Metters, K. M., Gordon, R., Chan, C. C., Zamboni, R. J. Bioorg. Med. Chem. Lett.2005, 15, 527] and the acylsulfonamides of ortho-(arylmethyl)cinnamates. [(a) Juteau, H., Gareau, Y., Labelle, M., Sturino, C. F., Sawyer, N., Tremblay, N., Lamontagne, S., Carrière, M.-C., Denis, D., Metters, K. M. Bioorg. Med. Chem. 2001, 9, 1977; (b) Juteau, H., Gareau, Y., Labelle, M., Lamontagne, S., Tremblay, N., Carrière, M.-C., Denis, D., Sawyer, N., Metters, K. M. Bioorg. Med. Chem. Lett.2001, 11, 747] The structural differences between the two series, along with their biological activity in vivo, in vitro, and metabolism, are analyzed. Some of those compounds, including hybrids containing the best structural features of both series, possess K(i) as low as 0.6 nM on the EP(3) receptor.     

Identification of an indole series of prostaglandin D2 receptor antagonists

A novel indole series of PGD2 receptor (DP receptor) antagonists is presented. Optimization of this series led to the identification of potent and selective DP receptor antagonists. In particular, antagonists 35 and 36 were identified with Ki values of 2.6 and 1.8 nM, respectively. These two antagonists are also potent in a DP functional assay where they inhibit the PGD2 induced cAMP production in platelet rich plasma with IC50 values of 7.9 and 8.6 nM, respectively. The structure-activity relationships of this indole series of DP receptor antagonists will also be discussed.     

Ligand Reduces Galectin-1 Sensitivity to Oxidative Inactivation by Enhancing Dimer Formation

Galectin-1 (Gal-1) regulates leukocyte turnover by inducing the cell surface exposure of phosphatidylserine (PS), a ligand that targets cells for phagocytic removal, in the absence of apoptosis. Gal-1 monomer-dimer equilibrium appears to modulate Gal-1-induced PS exposure, although the mechanism underlying this regulation remains unclear. Here we show that monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidation. A mutant form of Gal-1, containing C2S and V5D mutations (mGal-1), exhibits impaired dimerization and fails to induce cell surface PS exposure while retaining the ability to recognize carbohydrates and signal Ca²⁺ flux in leukocytes. mGal-1 also displayed enhanced sensitivity to oxidation, whereas ligand, which partially protected Gal-1 from oxidation, enhanced Gal-1 dimerization. Continual incubation of leukocytes with Gal-1 resulted in gradual oxidative inactivation with concomitant loss of cell surface PS, whereas rapid oxidation prevented mGal-1 from inducing PS exposure. Stabilization of Gal-1 or mGal-1 with iodoacetamide fully protected Gal-1 and mGal-1 from oxidation. Alkylation-induced stabilization allowed Gal-1 to signal sustained PS exposure in leukocytes and mGal-1 to signal both Ca²⁺ flux and PS exposure. Taken together, these results demonstrate that monomer-dimer equilibrium regulates Gal-1 sensitivity to oxidative inactivation and provides a mechanism whereby ligand partially protects Gal-1 from oxidation.Immunological homeostasis relies on efficient contraction of activated leukocytes following an inflammatory episode. Several factors, including members of the galectin and tumor necrosis factor families (1, 2), regulate leukocyte turnover by inducing apoptotic cell death. In contrast, several galectin family members, in particular galectin-1 (Gal-1),² uniquely regulate neutrophil turnover by inducing phosphatidylserine (PS) exposure, which normally sensitizes apoptotic cells to phagocytic removal (3, 4), independent of apoptosis, a process recently termed preaparesis (5).Previous studies suggested that dimerization may be required for Gal-1-induced PS exposure, as a mutant form of Gal-1 (mGal-1) containing two point mutations within the dimer interface, C2S and V5D (C2S,V5D), displays impaired Gal-1 dimerization and fails to induce PS exposure (6). However, the manner in which monomer-dimer equilibrium regulates Gal-1 signaling remains unclear. Previous studies suggest that dimerization may be required for efficient cross-linking of functional receptors or the formation of signaling lattices (7–9). Consistent with this, monomeric mutants of several other galectins fail to induce PS exposure or signal leukocytes (4, 8). Gal-1 signaling of PS exposure requires initial signaling events, such as mobilization of intracellular Ca²⁺ followed by sustained receptor engagement (10). Although mGal-1 fails to induce PS exposure (6), whether mGal-1 can induce these initial signaling events remains unknown (10).In addition to directly regulating signaling, monomer-dimer equilibrium may also regulate other aspects of Gal-1 function. Unlike many other proteins involved in the regulation of immunity, Gal-1 displays unique sensitivity to oxidative inactivation (11–15). Although engagement of ligand partially protects Gal-1 from oxidation (15), the impact of Gal-1 oxidation on signaling remains enigmatic. During oxidation, Gal-1 forms three distinct intramolecular disulfide bridges that facilitate profound conformational changes that preclude ligand binding and Gal-1 dimerization (12–14), suggesting that monomerdimer equilibrium may also regulate Gal-1 sensitivity to oxidative inactivation.Previous studies utilized dithiothreitol (DTT) in treatment conditions to protect Gal-1 from oxidative inactivation (16, 17). Indeed, failure to include DTT precluded Gal-1-induced death in T cells (3, 18), suggesting that Gal-1 undergoes rapid oxidation in vivo in the absence of reducing conditions. However, DTT itself can induce apoptosis in leukocytes (19), leaving questions regarding the impact of Gal-1 oxidation on these signaling events. In contrast, recent studies utilizing iodoacetamide-alkylated Gal-1 (iGal-1), previously shown to protect Gal-1 from oxidative inactivation (20–29), demonstrated that DTT actually primes cells to become sensitive to Gal-1-induced apoptosis regardless of Gal-1 sensitivity to oxidation (5).As the engagement of leukocyte ligands requires glycan recognition and oxidation precludes this binding (11, 15), understanding the impact of oxidation on Gal-1 signals will facilitate a greater appreciation of the factors that govern Gal-1 oxidation and therefore function. Our results demonstrate that Gal-1 monomer-dimer equilibrium provides a key regulatory point controlling both Gal-1 sensitivity to oxidation and its ability to signal PS exposure in leukocytes. These results provide novel insights into Gal-1 function and explain at a biochemical level the mechanisms regulating Gal-1 oxidative inactivation and signaling.     

Hypoxia stimulates vesicular ATP release from rat osteoblasts

Many neuronal and non‐neuronal cell types release ATP in a controlled manner. After release, extracellular ATP (or, following hydrolysis, ADP) acts on cells in a paracrine manner via P2 receptors. Extracellular nucleotides are now thought to play an important role in the regulation of bone cell function. ATP (and ADP), acting via the P2Y₁ receptor, stimulate osteoclast formation and activity, whilst P2Y₂ receptor stimulation by ATP (or UTP) inhibits bone mineralization by osteoblasts. We found that rat calvarial osteoblasts released ATP constitutively, in a differentiation‐dependent manner, with mature, bone‐forming osteoblasts releasing up to sevenfold more ATP than undifferentiated, proliferating cells. The inhibitors of vesicular exocytosis, monensin, and N‐ethylmaleimide (1–1,000 µM) inhibited basal ATP release by up to 99%. The presence of granular ATP‐filled vesicles within the osteoblast cytoplasm was demonstrated by quinacrine staining. Exposure to hypoxia (2% O₂) for up to 3 min increased ATP release from osteoblasts up to 2.5‐fold without affecting cell viability. Peak concentrations of ATP released into culture medium were >1 µM, which equates with concentrations known to exert significant effects on osteoblast and osteoclast function. Monensin and N‐ethylmaleimide (100 µM) attenuated the hypoxia‐induced ATP release by up to 80%. Depletion of quinacrine‐stained vesicles was also apparent after hypoxic stimulation, indicating that ATP release had taken place. These data suggest that vesicular exocytosis is a key mediator of ATP release from osteoblasts, in biologically significant amounts. Moreover, increased extracellular ATP levels following acute exposure to low O₂ could influence local purinergic signaling and affect the balance between bone formation and bone resorption. J. Cell. Physiol. 220: 155–162, 2009. © 2009 Wiley‐Liss, Inc.     

Germline mutation in ATR in autosomal- dominant oropharyngeal cancer syndrome

ATR (ataxia telangiectasia and Rad3 related) is an essential regulator of genome integrity. It controls and coordinates DNA-replication origin firing, replication-fork stability, cell-cycle checkpoints, and DNA repair. Previously, autosomal-recessive loss-of-function mutations in ATR have been demonstrated in Seckel syndrome, a developmental disorder. Here, however, we report on a different kind of genetic disorder that is due to functionally compromised ATR activity, which translates into an autosomal-dominant inherited disease. The condition affects 24 individuals in a five-generation pedigree and comprises oropharyngeal cancer, skin telangiectases, and mild developmental anomalies of the hair, teeth, and nails. We mapped the disorder to a ～16.8 cM interval in chromosomal region 3q22-24, and by sequencing candidate genes, we found that ATR contained a heterozygous missense mutation (c.6431A>G [p.Gln2144Arg]) that segregated with the disease. The mutation occurs within the FAT (FRAP, ATM, and TRRAP) domain-which can activate p53-of ATR. The mutation did not lead to a reduction in ATR expression, but cultured fibroblasts showed lower p53 levels after activation of ATR with hydroxyurea than did normal control fibroblasts. Moreover, loss of heterozygosity for the ATR locus was noted in oropharyngeal-tumor tissue. Collectively, the clinicopathological and molecular findings point to a cancer syndrome and provide evidence implicating a germline mutation in ATR and susceptibility to malignancy in humans.     

Macrophages infiltrate the human and rat decidua during term and preterm labor: evidence that decidual inflammation precedes labor

Preterm delivery is the leading cause of perinatal mortality and morbidity. Current tocolytics target myometrial contractions, a late step in the labor cascade. Identifying earlier events in parturition may lead to more effective therapeutic strategies. We hypothesized that inflammatory events in decidua (the maternal-fetal interface), characterized by leucocyte infiltration, are an early event during term and preterm labor (PTL). Leucocyte abundance in decidua of human pregnancies was quantified following term labor and PTL (idiopathic and infection associated), in conjunction with investigation of temporal inflammatory events in rat uterus during the perilabor period and in PTL induced by mifepristone. In human decidua, macrophage numbers were 4-fold higher in term labor (P < 0.01) and 2.5-fold higher in non-infection-associated PTL (P < 0.05) than in term nonlaboring samples. Neutrophil abundance was unchanged with labor but elevated in PTL with infection (5- to 53-fold increase; P < 0.01). T and NK cells were more abundant in idiopathic PTL than TL (P < 0.05). In rat, decidual macrophage infiltration increased 4.5-fold 12 h prior to labor and remained elevated during labor and early postpartum (P < 0.01). Decidual infiltration preceded that of the myometrium and was 4-fold higher (P < 0.01). In rat PTL, decidual macrophage numbers were also elevated (P < 0.01) and exceeded those of the myometrium (P < 0.05). These studies show for the first time that leucocytes infiltrate decidua during labor at term and preterm, supporting a role for leucocyte-derived inflammatory mediators in decidual activation. In the rat, this occurred prior to labor, suggesting it is an early event during parturition and thus a potential target for intervention.     

Failure to Detect the Novel Retrovirus XMRV in Chronic Fatigue Syndrome

Background

In October 2009 it was reported that 68 of 101 patients with chronic fatigue syndrome (CFS) in the US were infected with a novel gamma retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), a virus previously linked to prostate cancer. This finding, if confirmed, would have a profound effect on the understanding and treatment of an incapacitating disease affecting millions worldwide. We have investigated CFS sufferers in the UK to determine if they are carriers of XMRV.

Methodology

Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness and met the CDC criteria for CFS. DNA extracted from blood samples of 186 CFS patients were screened for XMRV provirus and for the closely related murine leukaemia virus by nested PCR using specific oligonucleotide primers. To control for the integrity of the DNA, the cellular beta-globin gene was amplified. Negative controls (water) and a positive control (XMRV infectious molecular clone DNA) were included. While the beta-globin gene was amplified in all 186 samples, neither XMRV nor MLV sequences were detected.

Conclusion

XMRV or MLV sequences were not amplified from DNA originating from CFS patients in the UK. Although we found no evidence that XMRV is associated with CFS in the UK, this may be a result of population differences between North America and Europe regarding the general prevalence of XMRV infection, and might also explain the fact that two US groups found XMRV in prostate cancer tissue, while two European studies did not.