Participation of prostaglandin E receptor EP4 subtype in duodenal bicarbonate secretion in rats

We examined, by using a specific PGE receptor subtype EP4 agonist and antagonist, the involvement of EP4 receptors in duodenal HCO(3)(-) secretion induced by PGE(2) and mucosal acidification in rats. Mucosal acidification was achieved by exposing a duodenal loop to 10 mM HCl for 10 min, and various EP agonists were given intravenously 10 min before the acidification. Secretion of HCO(3)(-) was dose-dependently stimulated by AE1-329 (EP4 agonist), the maximal response being equivalent to that induced by sulprostone (EP1/EP3 agonist) or PGE(2). The stimulatory action of AE1-329 and PGE(2) but not sulprostone was attenuated by AE3-208, a specific EP4 antagonist. This antagonist also significantly mitigated the acid-induced HCO(3)(-) secretion. Coadministration of sulprostone and AE1-329 caused a greater secretory response than either agent alone. IBMX potentiated the stimulatory action of both sulprostone and AE1-329, whereas verapamil mitigated the effect of sulprostone but not AE1-329. Chemical ablation of capsaicin-sensitive afferent neurons did not affect the response to any of the EP agonists used. We conclude that EP4 receptors are involved in the duodenal HCO(3)(-) response induced by PGE(2) or acidification in addition to EP3 receptors. The process by which HCO(3)(-) is secreted through these receptors differs regarding second-messenger coupling. Stimulation through EP4 receptors is mediated by cAMP, whereas that through EP3 receptors is regulated by both cAMP and Ca(2+); yet there is cooperation between the actions mediated by these two receptors. The neuronal reflex pathway is not involved in stimulatory actions of these prostanoids.     

Proteolytic activity of Boophilus microplus Yolk pro-Cathepsin D (BYC) is coincident with cortical acidification during embryogenesis

In a previous report (Parasitology 116 (1998) 525) we isolated and characterized Boophilus Yolk pro-Cathepsin (BYC), an aspartic proteinase precursor from the eggs of the hard tick. The present study was designed to characterize the function of BYC in the consumption of vitellin (VT), the major yolk protein, during embryogenesis. Both purified BYC and total egg homogenate proteolytic activity showed a similar pH dependence profile with an acidic optimum. Purified BYC presented higher activity against VT as a substrate when compared to other proteins. The VT degradation pattern observed in vitro also showed a similar profile to that observed in vivo. Co-localization of BYC and acidic cortical yolk granules was performed by immunocytochemistry and confocal microscopy. Proton-pumping activity of yolk granules in vitro was higher in eggs collected 4 day after oviposition than in newly laid eggs. Taken together, our data suggest that BYC plays a major role in the degradation of VT and that its activity is controlled by acidification of yolk platelets localized at the cortical cytoplasm of the developing Boophilus microplus egg.     

Clearance rates of biotinylated ferritins in canine: A possible physiological role of canine serum ferritin as an iron transporter

To elucidate the physiological role of canine serum ferritin, we measured clearance rates of biotinylated ferritins in beagle. Biotinylated canine tissue ferritins were cleared rapidly from circulation. The clearance time (T_1/2) of liver ferritin (H/L subunit ratio=0.43) was 6.8 to 11.8 min, and that of heart ferritin (H/L=3.69) was 9.3 to 25.0 min. T_1/2 of biotinylated canine liver ferritin was independent of iron content, whereas canine heart apoferritin (T_1/2=31.2 and 32.7 min) was more slowly removed from circulation than the holoferritin. On the other hand, biotinylated recombinant bovine H-chain ferritin homopolymer show a much slower rate of removal (T_1/2=153.8 and 155.0 min) compared with the L-chain ferritin homopolymer (T_1/2=26.4 and 31.3 min). The rapid clearance of canine tissue ferritin suggests that serum ferritin is an iron transporter in canines.     

Origin of higher affinity to RNA of the N-terminal RNA-binding domain than that of the C-terminal one of a mouse neural protein,musashi1, as revealed by comparison of their structures,modes of interaction,surface electrostatic potentials,and backbone dynamics

Musashi1 is an RNA-binding protein abundantly expressed in the developing mouse central nervous system. Its restricted expression in neural precursor cells suggests that it is involved in maintenance of the character of progenitor cells. Musashi1 contains two ribonucleoprotein-type RNA-binding domains (RBDs), RBD1 and RBD2, the affinity to RNA of RBD1 being much higher than that of RBD2. We previously reported the structure and mode of interaction with RNA of RBD2. Here, we have determined the structure and mode of interaction with RNA of RBD1. We have also analyzed the surface electrostatic potential and backbone dynamics of both RBDs. The two RBDs exhibit the same ribo-nucleoprotein-type fold and commonly make contact with RNA on the beta-sheet side. On the other hand, there is a remarkable difference in surface electrostatic potential, the beta-sheet of RBD1 being positively charged, which is favorable for binding negatively charged RNA, but that of RBD2 being almost neutral. There is also a difference in backbone dynamics, the central portion of the beta-sheet of RBD1 being flexible, but that of RBD2 not being flexible. The flexibility of RBD1 may be utilized in the recognition process to facilitate an induced fit. Thus, comparative studies have revealed the origin of the higher affinity of RBD1 than that of RBD2 and indicated that the affinity of an RBD to RNA is not governed by its fold alone but is also determined by its surface electrostatic potential and/or backbone dynamics. The biological role of RBD2 with lower affinity is also discussed.     

Growth and differentiation potential of main- and side-population cells derived from murine skeletal muscle

Skeletal muscle-derived CD34+/45- (Sk-34) cells were identified as a new candidate for stem cells. However, the relationship between Sk-34 cells and side-population (SP) cells is unknown. Here, we demonstrate that Sk-34 cells prepared from murine skeletal muscles consist wholly of main-population (MP) cells. The Sk-34 cells included only a few SP cells (1:1000, SP:MP). Colony-forming units of Sk-34 cells of both SP and MP possessed the same potential to differentiate into adipocytes, endothelial, and myogenic cells and showed the same colony-forming activity (1.6%). In addition, the colony-forming units of the CD34-/45- (double negative: DN) population were found to begin CD34 expression and to possess the potential to differentiate into myogenic and endothelial cells. We also found that expression of CD34 antigen precedes MyoD expression during the myogenic process of DN cells. Furthermore, both Sk-34 and DN cell populations were mostly negative for CD73 (93-95%), whereas the CD45+ cell population was >25% positive for CD73, and this trend was also seen in bone marrow-derived CD45+ cells. These results indicate that the MP cell population is about 99.9% responsible for the reported in vitro myogenic-endothelial responses of skeletal muscle-derived cells.     

Mass isolation of cuticle protein cDNAs from wing discs of Bombyx mori and their characterizations

Multiple cloning of cuticle protein genes was performed by sequencing of cDNAs randomly selected from a cDNA library of wing discs just before pupation, and nine different cuticular protein genes were identified. Thirty-one clones of a cuticle protein gene were identified from the 1050 randomly sequenced clones; about 3% were cuticle protein genes in the W3-stage wing disc cDNA library. The sequence diversity of the deduced amino acid sequences of isolated Bombyx cuticle genes was examined along with the expression profiles. The deduced amino acid sequences of the nine cuticle protein genes contained a putative signal peptide at the N-terminal region and a very conserved hydrophilic region known as the R and R motif. The developmental expression of cuticle genes was classified into two types: pupation (five clones were expressed only around pupation) and pupation and mid-pupal (four clones were expressed around this stage). All the isolated genes were expressed in the head, thoracic, and abdominal regions of the epidermis at different levels around pupation, but no expression was observed in the epidermis at the fourth molting stage.     

Asymmetric catalytic ene-cyclization approach to 2-fluoro-19-nor-1,25-dihydroxyvitamin D(3) A-ring analog with significant transactivation activity

1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)2D3) has been shown to modulate not only proliferation and differentiation, but also apoptosis in malignant cells, indicating that it could be useful for the treatment of cancer and psoriasis. However, little information has been available on the binding conformation of the 1alpha,25(OH)2D3 molecule and its analogs with the vitamin D receptor (VDR). Therefore, we synthesized 2alpha-fluorinated A-ring analogs of 19-nor-1alpha,25(OH)2D3 in order to investigate the VDR-binding conformation of the A-rings on the basis of the (19)F NMR analysis. The 2alpha-fluoro-19-nor-1alpha,25-dihydroxyvitamin D3 A-ring analog thus synthesized via a asymmetric catalytic carbonyl-ene cyclization, shows significant activity in transactivation.     

De novo methylation of MMLV provirus in embryonic stem cells: CpG versus non-CpG methylation

Nuclear factor Y (NF-Y) is a highly conserved trimeric activator that recognizes with high specificity and affinity the widespread CCAAT box promoter element. We previously cloned the genes of 23 NF-Y genes of Arabidopsis thaliana (Gene 264 (2001) 173). Now that the Arabidopsis genome sequencing project is complete, we present the cloning, alignments and expression profiles of the remaining six genes coding for the three NF-Y subunits. Consistent with our previous reports, most of the new members of the three subunits show a unique tissue-specific pattern, while another AtNF-YC9 is rather ubiquitous.