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.     

Mechanistic study of the oxidation of caffeic acid by digital simulation of cyclic voltammograms

The oxidation mechanism of caffeic acid (CAF) has been studied by means of cyclic voltammetry with the plastic formed carbon or glassy carbon electrode. CAF gives a well-developed two-electron reversible wave in acidic media, whereas it shows an irreversible behavior, i.e., a decrease of the rereduction peak, in less acidic media, suggesting that the oxidation of CAF follows an irreversible chemical reaction(s). Digital simulation analyses based on different oxidation mechanisms have been performed for the voltammograms obtained with the GC electrode in 1:1 (v/v) water:ethanol solutions. The results clearly show that the seeming two-electron oxidation of CAF occurs stepwise via one-electron processes, each of which follows an irreversible chemical reaction. It has also been suggested that the semiquinone radical as an intermediate of the one-electron oxidation should play an important role in the oxidation reaction. Evaluations of the rate constants for the chemical reactions have further suggested that the chemical reactions are dimerization reactions.     

Polymorphisms of the TNF gene and the TNF receptor superfamily member 1B gene are associated with susceptibility to ulcerative colitis and Crohn's disease, respectively

The importance of tumor necrosis factor (TNF)-alpha and the TNF receptor gene polymorphisms in the etipathogenesis of inflammatory bowel disease (IBD) has not been elucidated. DNA from peripheral blood samples was obtained from 124 patients with Crohn's disease (CD), 106 patients with ulcerative colitis (UC), and 111 unrelated healthy controls. We examined two single nucleotide polymorphisms (SNPs) of the TNF-alpha gene, TNF (-308 G/A and -238 G/A), an SNP of the TNF receptor superfamily member 1A gene, TNFRSF1A(also known as TNFR1), at codon 12 in exon 1 (CCA/CCG), and two SNPs of the 1B gene, TNFRSF1B (also known as TNFR2), (1466 A/G and 1493 C/T). There was a difference in the carrier frequency for haplotype AG (-308 A, -238 G) between UC patients and the controls (OR=4.76, 95% CI=1.53-14.74, P<0.01). We found a significant difference in carrier frequency for haplotype AT (1466 A, 1493 T) of the TNFRSF1B gene between CD patients and the controls (OR=2.13, 95% CI=1.08-4.21, P<0.05). The significance proved to be greater in CD patients with both internal and external fistula (OR=4.8, 95% CI=1.73-13.33, P<0.01), and in those who were poor responders ( n=22) to our treatments, which consisted of nutritional therapy, medical therapy and surgical therapy (OR=9.24, 95% CI=3.37-25.36, P<0.001). This study suggests that one of the genes responsible for UC may be the TNF gene, or an adjacent gene, and that TNFRSF1B gene polymorphisms contribute greatly to the increased onset risk of CD and to the disease behavior.     

rugose (rg), a Drosophila A kinase anchor protein,is required for retinal pattern formation and interacts genetically with multiple signaling pathways

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the EGFR- and Notch-mediated signaling pathways. Our results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the EGFR and Notch signaling pathways.