Identification and targeted disruption of the mouse gene encoding ESG1 (PH34/ECAT2/DPPA5)

Background  

Embryonic stem cell-specific gene (ESG) 1, which encodes a KH-domain containing protein, is specifically expressed in early embryos, germ cells, and embryonic stem (ES) cells. Previous studies identified genomic clones containing the mouse ESG1 gene and five pseudogenes. However, their chromosomal localizations or physiological functions have not been determined.     

Inhibitory effect of ribbon-type NF-kappaB decoy oligodeoxynucleotides on osteoclast induction and activity in vitro and in vivo

In this study we examined the effect of ribbon-type (circular-type) NF-kappaB decoy oligodeoxynucleotides (RNODN) on osteoclast induction and activity. We extracted bone marrow cells from the femurs of rats and incubated non-adherent cells with receptor activator of nuclear factor kappaB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). First, transfer efficiency into osteoclasts and their precursors, resistance to exonuclease, and binding activity of decoy to NF-kappaB were examined. Next, to examine the effect of RNODN on osteoclast induction and activity, osteoclast differentiation and pit formation assays were performed. RNODN were injected into the ankle joints of rats with collagen-induced arthritis. Joint destruction and osteoclast activity were examined by histological study. The resistance of RNODN to exonuclease and their binding activity on NF-kappaB were both greater than those of phosphorothionated NF-kappaB decoy oligodeoxynucleotides. The absolute number of multinucleate cells scoring positive for tartrate-resistant acid phosphatase was significantly decreased in the RNODN-treated group. The average calcified matrix resorbed area was significantly decreased in the RNODN-treated group. Histological study showed marked suppression of joint destruction and osteoclast activity by intra-articular injection of RNODN. These results suggest the inhibitory effect of RNODN on the induction and activity of osteoclasts. Direct intra-articular injection of RNODN into the joints may be an effective strategy for the treatment of arthritis.     

Distribution of retinylester-storing stellate cells in the arrowtooth halibut, Atheresthes evermanni

Hepatic stellate cells play a major role in retinylester storage in mammals, but the retinoid-storing state in nonmammalian vertebrates remains to be elucidated. In this study, we examined retinoids and retinoid-storing cells in the arrowtooth halibut, Atheresthes evermanni. High-performance liquid chromatography analyses revealed the highest concentrations of stored retinoids (retinol and retinylester, 6199 nmol/g) in the pyloric cecum, a teleost-specific organ protruding from the intestine adjacent to the pylorus. Considerable amounts of retinoids were also stored in the intestine (3355 nmol/g) and liver (1891 nmol/g), and small amounts in the kidney (102 nmol/g). Very small amounts or no retinoids were detected in the heart, gill, skeletal muscle, and gonads (less than 2 nmol/g). Use of gold chloride staining and fluorescence microscopy to detect retinoid autofluorescence showed that, in the pyloric cecum and intestine, retinoid-storing cells were localized in the lamina propria mucosae. Under electron microscopy, cells containing well-developed lipid droplets, which are common morphological characteristics of the hepatic stellate cells of mammals, were observed in the lamina propria mucosae of the pyloric cecum. Thus, the distribution of stellate cells with retinoid-storing capacity differs between this halibut and mammals, suggesting that the retinoid-storing site has shifted during vertebrate evolution.     

Development of a new genotoxicity test system with Salmonella typhimurium OY1001/1A2 expressing human CYP1A2 and NADPH-P450 reductase.

In order to develop a new tester strain detecting environmental promutagens and procarcinogens, we introduced two plasmids into Salmonella typhimurium TA1535; one contains the cDNAs of human cytochrome P450 (P450 or CYP) 1A2 and NADPH-P450 reductase and the other (pOA101) a umuC"lacZ fusion gene. The newly developed tester strain, S. typhimurium OY1001/1A2, was found to express P450 at a level of 0.15 nmol/ml in whole cell culture. Membrane fractions, when isolated from this tester strain, contained 0.04 P450 nmol/mg protein and a reductase activity of 170 nmol cytochrome c reduced/min/mg protein and were active in catalyzing CYP1A2-dependent 7-ethoxyresorufin O-deethylation and metabolic activation of heterocyclic aromatic amines to DNA-damaging products in a conventional tester S. typhimurium NM2009 strain, only when NADPH was added as a reducing equivalent. In the OA1002/1A2 strain, heterocyclic aromatic amines (e.g., IQ, MeIQ, and MeIQx) were found to be activated to reactive metabolites that cause induction of umuC gene expression in a dose-dependent manner, without addition of external NADPH. These results indicate that the newly established strain can be of use to detect mutagenic and carcinogenic potencies of environmental chemicals without addition of metabolic activation system.     

Epimerization of moxalactam by albumin and simulation of in vivo epimerization by a physiologically based pharmacokinetic model

We investigated the mechanism of epimerization (R to S or S to R) of moxalactam in serum of rats, dogs, and humans. The epimerization of moxalactam occurred in the serum of these animals, but not in the serum filtrate. The albumin fraction of human serum purified by gel filtration catalysed the epimerization of moxalactam at an identical rate to serum, but other fractions (i.e., lipoproteins and globulins) showed slower epimerization. alpha 1-acid glycoprotein, which was eluted in the same fraction with albumin by G-200 gel filtration, did not epimerize moxalactam. The presence of 2 mM warfarin decreased the binding of R- and S-moxalactam and decreased the epimerization of moxalactam in human serum. These results demonstrate moxalactam was epimerized on the warfarin binding site on albumin in serum. Additionally, a physiologically based pharmacokinetic model shows that the epimerization of moxalactam after administration in dogs is simulated by the epimerization in serum.     

Characteristics of the production of active oxygen species from adherent and non-adherent neutrophils

Our objective was to evaluate the characteristics of the production of AOS from the neutrophils that had adhered to the endothelial cells, fibronectin or polystyrene, using the method of electron paramagnetic resonance (EPR) spin trapping. Neutrophils and endothelial cells were isolated from human venous blood and umbilical veins, respectively. AOS production from neutrophils was not elicited only by adhesion. The stimulation of adherent neutrophils with phorbol myristate acetate (PMA) induced the production of AOS. The production of AOS from adherent neutrophils to endothelial cells, but not to fibronectin or polystyrene, decreased with the interval time between the adhesion and the stimulation by PMA. The amount of AOS produced by the neutrophils adherent to fibronectin or polystyrene was maintained for one hour after stimulation with PMA, whereas that by suspended neutrophils gradually decreased with the time after stimulation. Results indicate that adherent and non-adherent neutrophils exhibit differing time course of AOS production.     

Identification of BAIAP2 (BAI-associated protein 2), a novel human homologue of hamster IRSp53, whose SH3 domain interacts with the cytoplasmic domain of BAI1.

BAI1 (brain-specific angiogenesis inhibitor 1) was originally isolated as a p53-target gene specifically expressed in brain. To clarify its function, we have been searching for cellular proteins that associate with the cytoplasmic domain of BAI1. Using its intracellular carboxyl terminus as "bait" in a yeast two-hybrid system, we isolated a cDNA clone named BAIAP2 whose nucleotide sequence would encode a 521-amino acid protein showing significant homology to a 58/53-kDa substrate of insulin-receptor kinase in the hamster. As the expression profile of BAIAP2 examined by Northern blot analysis was almost identical to that of BAI1, BAIAP2 appears to be active mainly in neurons. In vitro binding assays confirmed that a proline-rich cytoplasmic fragment of BAI1 interacted with the Src homology 3 (SH3) domain of BAIAP2. Double-color immunofluorescent analysis revealed that BAIAP2 was localized at the cytoplasmic membrane when it was coexpressed with BAI1 in COS-7 cells; BAIAP2 not associated with BAI1 was diffused in the cytoplasm. Predominant localization of BAI1 protein in a sub-cellular fraction enriched in growth cones indicated a possible role of BAI1 as a cell adhesion molecule inducing growth cone guidance. As a protein partner of BAI1, BAIAP2 may represent an important link between membrane and cytoskeleton in the process of neuronal growth.