CD9 is associated with leukemia inhibitory factor-mediated maintenance of embryonic stem cells

Mouse embryonic stem (ES) cells can proliferate indefinitely in an undifferentiated state in the presence of leukemia inhibitory factor (LIF), or differentiate into all three germ layers upon removal of this factor. To determine cellular factors associated with self-renewal of undifferentiated ES cells, we used polymerase chain reaction-assisted cDNA subtraction to screen genes that are expressed in undifferentiated ES cells and down-regulated after incubating these cells in a differentiation medium without LIF for 48 h. The mRNA expression of a tetraspanin transmembrane protein, CD9, was high in undifferentiated ES cells and decreased shortly after cell differentiation. An immunohistochemical analysis confirmed that plasma membrane-associated CD9 was expressed in undifferentiated ES cells but low in the differentiated cells. Addition of LIF to differentiating ES cells reinduced mRNA expression of CD9, and CD9 expression was accompanied with a reappearance of undifferentiated ES cells. Furthermore, activation of STAT3 induced the expression of CD9, indicating the LIF/STAT3 pathway is critical for maintaining CD9 expression. Finally, addition of anti-CD9 antibody blocked ES cell colony formation and reduced cell viability. These results indicate that CD9 may play a role in LIF-mediated maintenance of undifferentiated ES cells.     

Nitrogen transformations in vertical flow systems with and without rice (Oryza sativa) studied with a high-resolution soil–water profiler

Nitrogen transformations were studied in flooded and non-flooded vertical flow columns with and without a rice plant. Influent (average concentration: NH₄⁺-N: 40 mg L^?1; NO₃^?-N: 0.15 mg L^?1; and NO₂^?-N: 4.0 mg L^?1) was supplied at 1.25 cm d^?1 during stage 1 (20 May–5 August) and at 2.50 cm d^?1 at stage 2 (6 August–26 October), which resulted in an average nitrogen loading of 156 g m^?2 during the entire experimental period. Total nitrogen (T-N) removal efficiencies exceeded 90% in vertical flow systems with rice plants. Nitrogen assimilated by the rice plants in the flooded column accounted for 60% of the total input nitrogen, while that in the non-flooded column accounted for 36% of the total input. The remaining nitrogen appeared to be removed through biogeochemical pathways. Although some nitrogen flowed out, most input nitrogen was also removed even in the flooded and non-flooded unplanted columns.A high-resolution vertical distribution investigation showed the changes of nitrogen forms in soil water. In the flooded condition, there were high ammonium and high nitrite concentrations in the upper layers. The concentrations of ammonium and nitrite simultaneously decreased with depth increasing, suggesting that anaerobic ammonia oxidation (anammox) may occur in these anaerobic conditions. In contrast, the distributions of nitrogen in the non-flooded columns with elevated water level suggested that nitrification–denitrification route was the major removal mechanism, whether or not rice plants were present.     

Transforming growth factor-beta1-dependent activation of Smad2/3 and up-regulation of PAI-1 expression is negatively regulated by Src in SKOV-3 human ovarian cancer cells

The net balance between urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1) has been implicated in tumor cell invasion and metastasis. To elucidate the mechanism of the transforming growth factor-beta1 (TGF-beta1)-dependent up-regulation of PAI-1 expression, we investigated which signaling pathway transduced by TGF-beta1 is responsible for this effect. Here, we show (1) nontoxic concentrations of TGF-beta1 up-regulates uPA expression in HRA and SKOV-3 human ovarian cancer cells, (2) TGF-beta1 activates Smads (phosphorylation of Smad2 and nuclear translocation of Smad3) and subsequently up-regulates PAI-1 expression in HRA cells, whereas TGF-beta1 neither activates Smads nor up-regulates PAI-1 in SKOV-3 cells, (3) pharmacological Src inhibitor PP2 or antisense (AS) c-Src oligodeoxynucleotide (ODN) treatment significantly induces TGF-beta1-dependent activation of Smads, leading to PAI-1 synthesis, compared with controls, in SKOV-3 cells, (4) combination of TGF-beta1 and PP2, which activates PAI-1 expression and reduces uPA expression in SKOV-3, results in decreased invasiveness, (5) pharmacological inhibitors for mitogen-activated protein kinase (MAPK) (PD98059) and phosphoinositide-3-kinase (PI3K) (LY294002 and wortmannin) or AS-PI3K ODN transfection do not affect TGF-beta1-induced Smad signaling and up-regulation of PAI-1 expression in SKOV-3 cells pretreated with PP2, and (6) the induction of PAI-1 protein was partially inhibited by an inhibitor of Sp1-DNA binding, mithramycin, implicating, at least in part, Sp1 in the regulation of this gene by TGF-beta1. In conclusion, TGF-beta1-dependent activation of Smad2/3, leading to PAI-1 synthesis, may be negatively regulated by Src, but not its downstream targets MAPK and PI3K in SKOV-3 cells. These data also reflect the complex biological effect of uPA-PAI-1 system.     

Changes in oligosaccharide expression on plasma membrane of the mouse oocyte during fertilisation and early cleavage

It has been reported that various structural and functional changes occur on the surface of the plasma membrane of the ovum and embryo during fertilisation and cleavage in preparation for implantation. Glycoproteins are thought to be one of the factors in cell attachment. Thus, we investigated the changes in glycoprotein expression on the cell surface membrane of the mouse embryo by using lectins. Among seven types of lectin (ConA, WGA, UEA-I, MPA, LCA, DBA and PNA), the fluorescent intensities of ConA and WGA markedly increased from unfertilised ova to blastocysts. By quantitative analysis using immuno-scanning electron microscopy, the numbers of ConA-gold particles were small until 4-cell cleavage, but increased significantly at the blastocyst stage. In contrast, an increased number of WGA-gold particles was detected even at the 4-cell stage, and this increase continued to the blastocyst stage. From the above observations, we conclude that the numbers of sugar chains bound to both ConA andWGA increases with blastocyst formation and earlier expression is observed with WGA. The present study dearly shows that glycoproteins on the cell membrane surface of the mouse embryo quantitatively increase at the time of implantation, and the possibility has been indicated that glycoproteins are involved in intercellular recognition and adhesion between the embryo and endometrial epithelium.     

Impact of oxidative stress in aged mouse oocytes on calcium oscillations at fertilization

In vivo post-ovulatory aging of oocytes significantly affects the development of oocytes and embryos. Also, oocyte aging alters the regulation of the intracellular calcium concentration, thus affecting Ca(2+) oscillations in fertilized oocytes. Because reactive oxygen species (ROS) are known to significantly perturb Ca(2+) homeostasis mainly through direct effects on the machinery involved in intracellular Ca(2+) storage, we hypothesized that the poor development of aged oocytes that may have been exposed to oxidative stress for a prolonged time might arise from impaired Ca(2+)-oscillation-dependent signaling. The fertilization rates of aged oocytes and of fresh oocytes treated with 100 microM hydrogen peroxide (H(2)O(2)) for 10 min were significantly lower than that of fresh oocytes. Comparing within the fertilized oocytes, blastocyst formation was decreased while embryo fragmentation was increased similarly in the aged and H(2)O(2)-treated fresh oocytes. The frequency of Ca(2+) oscillations was significantly increased whereas the amplitude of individual Ca(2+) transients was lowered in the aged and H(2)O(2)-treated fresh oocytes. The rates of rise and decline in individual Ca(2+) transients were decreased in these oocytes, indicating impaired Ca(2+) handling. When lipid peroxidation was assessed using 4,4-difluoro-5-(4-phenyl-1,3-buttadienyl)-4-bora-3a, 4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY) in unfertilized oocytes placed in a 5% CO(2) in air atmosphere, the green fluorescence (indicating lipid peroxidation) increased faster in the aged oocytes than in the fresh oocytes. Furthermore, the green fluorescence in the aged oocytes was already approximately 20 times higher than that in the fresh oocytes at the beginning of the measurements. These findings support the idea that Ca(2+) oscillations play a key role in the development of fertilized aged oocytes.     

A Kunitz-type protease inhibitor,bikunin, inhibits ovarian cancer cell invasion by blocking the calcium-dependent transforming growth factor-beta 1 signaling cascade

Bikunin is a Kunitz-type protease inhibitor, acting at the level of tumor invasion and metastasis. The goal of this study was to investigate the effect of bikunin-dependent signal transduction involved in the expression of a plasminogen activator (PA) system and invasion. We report here the following. 1) The human ovarian cancer cell line HRA produced secreted and cell-associated urokinase-type PA (uPA) and PA inhibitor type 1 (PAI-1). The plasma membrane of the cells showed enzymatically active uPA even in the presence of high level of PAI-1, as measured by zymography, Western blot, chromogenic assay, enzyme-linked immunosorbent assay, and Northern blot. 2) HRA cells leading to invasion are induced through up-regulation of uPA expression. 3) HRA cells specifically released transforming growth factor-beta type 1 (TGF-beta1) participating in an autocrine/paracrine regulation of cell invasion. 4) Elimination of endogenous TGF-beta1 could induce change in uPA/PAI-1 expression, which could in turn modify the invasive behavior of the cells. 5) The constitutive expression of TGF-beta1 as well as up-regulation of the PA system observed in HRA cells was inhibited by preinoculation of the cells with bikunin or calcium channel blocker SK&F 96365 but not with nifedipine or verapamil, with an IC(50) of approximately 100 nm for bikunin or approximately 30 microm for SK&F 96365, respectively, as measured by enzyme-linked immunosorbent assay. Bikunin showed no additive effect on SK&F 96365-mediated suppression of TGF-beta1 expression. 6) The ability of TGF-beta1 to elevate free intracellular Ca(2+), followed by activation of Src and ERK, was reduced by preincubation of the cells with bikunin. In conclusion, bikunin could inhibit the constitutive expression of TGF-beta1 and TGF-beta1-mediated, Src- and ERK-dependent, PA system signaling cascade, at least in part, through inhibition of a non-voltage-sensitive calcium channel.     

Characterization of recombinant skeletal muscle (Ser-2843) and cardiac muscle (Ser-2809) ryanodine receptor phosphorylation mutants

Phosphorylation of the skeletal muscle (RyR1) and cardiac muscle (RyR2) ryanodine receptors has been reported to modulate channel activity. Abnormally high phosphorylation levels (hyperphosphorylation) at Ser-2843 in RyR1 and Ser-2809 in RyR2 and dissociation of FK506-binding proteins from the receptors have been implicated as one of the causes of altered calcium homeostasis observed during human heart failure. Using site-directed mutagenesis, we prepared recombinant RyR1 and RyR2 mutant receptors mimicking constitutively phosphorylated and dephosphorylated channels carrying a Ser/Asp (RyR1-S2843D and RyR2-S2809D) and Ser/Ala (RyR1-S2843A and RyR2-S2809A) substitution, respectively. Following transient expression in human embryonic kidney 293 cells, the effects of Ca2+, Mg2+, and ATP on channel function were determined using single channel and [3H]ryanodine binding measurements. In both assays, neither the skeletal nor cardiac mutants showed significant differences compared with wild type. Similarly essentially identical caffeine responses were observed in Ca2+ imaging measurements. Co-immunoprecipitation and Western blot analysis showed comparable binding of FK506-binding proteins to wild type and mutant receptors. Finally metabolic labeling experiments showed that the cardiac ryanodine receptor was phosphorylated at additional sites. Taken together, the results did not support the view that phosphorylation of a single site (RyR1-Ser-2843 and RyR2-Ser-2809) substantially changes RyR1 and RyR2 channel function.     

ASC is an activating adaptor for NF-kappa B and caspase-8-dependent apoptosis

ASC is a pro-apoptotic protein containing a pyrin domain (PD) and a caspase-recruitment domain (CARD). A previous study suggests that ASC interacts with Ipaf, a member of the Apaf-1/Nod1 protein family. However, the functional relevance of the interaction has not been determined. Here, we report that co-expression of ASC with Ipaf or oligomerization of ASC induces both apoptosis and NF-kappa B activation. Apoptosis induced through ASC was inhibited by a mutant form of Caspase-8 but not by that of Caspase-1. The PD of ASC physically interacted with Caspase-8 as well as with pyrin, the familial Mediterranean fever gene product. Caspase-8 deficiency rescued mouse fibroblasts from apoptosis induced by ASC oligomerization. Pyrin disrupted the interaction between ASC and Caspase-8, and inhibited both apoptosis and NF-kappa B activation induced by ASC. These findings suggest that ASC is a mediator of NF-kappa B activation and Caspase-8-dependent apoptosis in an Ipaf signaling pathway.     

Novel binding sites of 15-deoxy-Delta12,14-prostaglandin J2 in plasma membranes from primary rat cortical neurons

15-Deoxy-Delta12,14-prostaglandin J2 (15d-Delta12,14-PGJ2) is an endogenous ligand for a nuclear peroxysome proliferator activated receptor-gamma (PPAR). We found novel binding sites of 15d-Delta12,14-PGJ2 in the neuronal plasma membranes of the cerebral cortex. The binding sites of [3H]15d-Delta12,14-PGJ2 were displaced by 15d-Delta12,14-PGJ2 with a half-maximal concentration of 1.6 microM. PGD2 and its metabolites also inhibited the binding of [3H]15d-Delta12,14-PGJ2. Affinities for the novel binding sites were 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. Other eicosanoids and PPAR agonists did not alter the binding of [3H]15d-Delta12,14-PGJ2. In primary cultures of rat cortical neurons, we examined the pathophysiologic roles of the novel binding sites. 15d-Delta12,14-PGJ2 triggered neuronal cell death in a concentration-dependent manner, with a half-maximal concentration of 1.1 microM. The neurotoxic potency of PGD2 and its metabolites was also 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. The morphologic and ultrastructural characteristics of 15d-Delta12,14-PGJ2-induced neuronal cell death were apoptotic, as evidenced by condensed chromatin and fragmented DNA. On the other hand, we detected little neurotoxicity of other eicosanoids and PPAR agonists. In conclusion, we demonstrated that novel binding sites of 15d-Delta12,14-PGJ2 exist in the plasma membrane. The present study suggests that the novel binding sites might be involved in 15d-Delta12,14-PGJ2-induced neuronal apoptosis.     

Purification and characterization of formate oxidase from a formaldehyde-resistant fungus

A formate oxidase activity was found in the crude extract of a formaldehyde-resistant fungus isolated from soil. The fungus was classified and designated as Aspergillus nomius IRI013, which could grow on a medium containing up to 0.45% formaldehyde and consumed formaldehyde completely. The specific activity of formate oxidase in the extract of the fungus grown on formaldehyde was found to be considerably higher than that in the extracts of the fungus grown on formate and methanol. Formate oxidase from the fungus grown on formaldehyde was purified to homogeneity. The enzyme had a relative molecular mass of 100000 and was composed of two apparently identical subunits that had a relative molecular mass of 59000. The enzyme showed the highest activity using formate as substrate. Hydrogen peroxide was formed during the oxidation of formate. The Michaelis constant for formate was 15.9 mM; highest enzyme activity was found at pH 4.5-5.0. The enzyme activity was strongly inhibited by NaN(3), p-chloromercuribenzoate and HgCl(2).