Synthesis and properties of oligonucleotides containing 5-formyl-2′-deoxycytidine: in vitro DNA polymerase reactions on DNA templates containing 5-formyl-2′-deoxycytidine

Oligodeoxynucleotides (ODNs) containing 5-formyl-2′-deoxycytidine (fC) were synthesized by the phosphoramidite method and subsequent oxidation with sodium periodate. The stabilities of duplexes containing A, G, C or T opposite fC were studied by thermal denaturation. It was found that fC:A, fC:C or fC:T base pairs significantly reduce the thermal stabilities of duplexes. Next, single nucleotide insertion reactions were performed using ODNs containing fC as templates and the Klenow fragment of Escherichia coli DNA polymerase I. It was found that: (i) insertion of dGMP opposite fC appears to be less efficient relative to insertion opposite 5-methyl-2′-deoxycytidine (mC); (ii) dAMP is misincorporated more frequently opposite fC than mC, although the frequency of misincorporation seems to be dependent on the sequence; (iii) TMP is misincorporated more frequently opposite fC than mC. These results suggest that fC may induce the transition mutation C·G→T·A and the transversion mutation C·G→A·T during DNA synthesis.     

Developmental changes in the biochemical and immunological characters of the carbohydrate moiety of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan

Neuroglycan C (NGC), a brain-specific transmembrane proteoglycan, is thought to bear not only chondroitin sulfate but also N- and O-linked oligosaccharides on its core protein. In this study, we isolated and purified NGC from rat brains at various developmental stages by immunoaffinity column chromatography or by immunoprecipitation, and examined the structural characters of its carbohydrate moiety. The chondroitin sulfate disaccharide composition of NGC at postnatal day 10 was significantly different from those of two secreted chondroitin sulfate proteoglycans, neurocan and phosphacan, purified from the brain at the same developmental stage; higher levels of 4-sulfate unit and E unit, a disulfated disaccharide unit, and a lower level of 6-sulfate unit. The levels of both 6-sulfate and E units decreased with a compensatory increase of 4-sulfate unit with postnatal development of the brain. Lectin-blot analysis of the NGC core glycoprotein prepared by chondroitinase digestion confirmed that NGC actually bore both N- and O-linked carbohydrates, and also revealed that lectin-species reactive with NGC did not always recognize other brain-specific proteoglycans, neurocan and phosphacan, and vice versa, even though they were isolated from the brain at the same stage. The reactivity of NGC with lectins and with the HNK-1 antibody markedly changed as the brain matured. These findings indicate that the structure of the carbohydrate moiety of NGC is developmentally regulated, and differs from those of neurocan and phosphacan. The developmentally-regulated structural change of the carbohydrates on NGC may be partly implicated in the modulation of neuronal cell recognition during brain development. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

A Hybrid System Using Both Promoter Activation and Gene Amplification for Establishing Exogenous Protein Hyper-Producing Cell Lines

We previously developed a promoter-activated production (PAP) system using amplified ras oncogene to activate the cytomegalovirus (CMV) promoter controlling the foreign gene in mammalian cells. CHO cells were demonstrated to be suitable for the PAP system. Here, we show that very high-level production of a recombinant protein was achieved when the human CMV promoter was inserted into a glutamine synthetase (GS) minigene expression plasmid, pEE14. A highly productive host CHO cell line, ras clone I containing amplified ras oncogene, was further transfected with the plasmid expressing both hIL-6 gene and GS minigene, and selected with methionine sulphoximine. We were able to establish a hIL-6 hyper-producing cell line, D29, which exhibited a peak productivity rate of approximately 40 μg hIL-6 10^?6 cells day^?1 through a combination of the PAP system and the GS gene amplification system. The cellular productivity of D29 cells was about 13-fold higher than control hIL-6-producing cells derived from CHO cells whose hIL-6 gene was amplified by the GS gene amplification system, and about 5-fold higher than the I13 cells established by the PAP system, which contains amplified ras oncogene and non-amplified hIL-6 gene. When D29 cells were cultured for a month, an accumulation rate of approximately 80 μg hIL-6 ml^?1 per 3 days was achieved on the 9th day. These results indicate that this PAP and GS hybrid system enables the efficient and rapid establishment of recombinant protein hyper-producing cell lines.     

Novel structural and functional mode of a knot essential for RNA binding activity of the Esa1 presumed chromodomain

Chromodomains are methylated histone binding modules that have been widely studied. Interestingly, some chromodomains are reported to bind to RNA and/or DNA, although the molecular basis of their RNA/DNA interactions has not been solved. Here we propose a novel binding mode for chromodomain-RNA interactions. Essential Sas-related acetyltransferase 1 (Esa1) contains a presumed chromodomain in addition to a histone acetyltransferase domain. We initially determined the solution structure of the Esa1 presumed chromodomain and showed it to consist of a well-folded structure containing a five-stranded β-barrel similar to the tudor domain rather than the canonical chromodomain. Furthermore, the domain showed no RNA/DNA binding ability. Because the N-terminus of the protein forms a helical turn, we prepared an N-terminally extended construct, which we surprisingly found to bind to poly(U) and to be critical for in vivo function. This extended protein contains an additional β-sheet that acts as a knot for the tudor domain and binds to oligo(U) and oligo(C) with greater affinity compared with other oligo-RNAs and DNAs examined thus far. The knot does not cause a global change in the core structure but induces a well-defined loop in the tudor domain itself, which is responsible for RNA binding. We made 47 point mutants in an esa1 mutant gene in yeast in which amino acids of the Esa1 knotted tudor domain were substituted to alanine residues and their functional abilities were examined. Interestingly, the knotted tudor domain mutations that were lethal to the yeast lost poly(U) binding ability. Amino acids that are related to RNA interaction sites, as revealed by both NMR and affinity binding experiments, are found to be important in vivo. These findings are the first demonstration of how the novel structure of the knotted tudor domain impacts on RNA binding and how this influences in vivo function.     

Synthesis of nuclease-resistant siRNAs possessing benzene-phosphate backbones in their 3'-overhang regions

We describe the synthesis and silencing activities of siRNA possessing N(1)-[3,5-bis(hydroxymethyl)phenyl]thymine (b(t)) in their 3'-overhang regions. We found that an siRNA possessing b(t) in the 3'-overhang region was more effective than an siRNA with natural nucleosides and the siRNA possessing 1,3-bis(hydroxymethyl)benzene (b) without a nucleobase at the 3'-overhang region in in vitro experiment using HeLa cells system. Furthermore, the RNA possessing b(t) at its 3'-end was more resistant to nucleolytic hydrolysis by snake venom phosphodiesterase (a 3'-exonuclease) than the RNA possessing the natural nucleoside 2'-deoxythymidine at the 3'-end. Thus, the compound b(t) will be a novel 3'-overhang moiety that can enhance the silencing activity and nuclease-resistant property of siRNAs.     

Apoptosis of odontoclasts under physiological root resorption of human deciduous teeth

This study was designed to establish the apoptosis of odontoclasts during physiological root resorption of human deciduous teeth. Deciduous teeth were fixed, decalcified, and embedded in paraffin for immunohistochemical (IHC) observations and in Epon for transmission electron microscopy (TEM). Apoptotic cells were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick-end labeling (TUNEL), and then tartrate-resistant acid phosphatase (TRAP) activity was determined on the same sections. Epon-embedded specimens were sectioned serially into 0.5-μm semithin sections; some of these sections were re-embedded in Epon, sectioned into 0.1-μm ultrathin sections, and observed by TEM. IHC revealed that the nuclei of TRAP-positive odontoclasts on the dentine were generally TUNEL-negative. Around these odontoclasts, a few TRAP-positive structures were present together with TUNEL-positive structures, e.g., a TRAP-positive structure with one TUNEL-positive nucleus, a TRAP-positive structure with one TUNEL-positive nucleus plus one or two TUNEL-negative nuclei, or a TRAP-positive structure with no nucleus. By TEM, some odontoclasts showed nuclear fragments including compacted chromatin. The results suggest that, during apoptosis, odontoclasts fragment into variously sized cellular parts including three or fewer nuclei. This study was supported by a grant from the Japanese Ministry of Education, Science, Sports, Culture, and Technology (grant no. 16591819) and by a grant from the Ministry of Education, Science, Sports, Culture, and Technology to promote multidisciplinary research projects (2003).     

Immunomodulation of gibberellin biosynthesis using an anti-precursor gibberellin antibody confers gibberellin-deficient phenotypes

Immunomodulation is a means to modulate an organism's function by antibody production to capture either endogenous or exogenous antigens. We have recently succeeded in obtaining gibberellin (GA)-deficient phenotypes in Arabidopsis thaliana by using anti-bioactive GA antibodies. In this study, a single-chain antibody (scFv) against GA(24), a precursor GA, was utilized to repress the biosynthesis of bioactive gibberellins. Stable accumulation of the scFv in endoplasmic reticulum (ER) was achieved by being produced as a fusion with GFP as well as KDEL ER-retention signal. The transgenic plants showed GFP fluorescence in the reticulate cortical ER network in epidermal cells. The GFP-scFv fusion produced in plants maintained its binding activity. The transgenic plants showed GA-deficient phenotypes, including reduced rosette leaf development, delayed flower induction and reduced stem elongation of the main culm, especially in the early stage of inflorescence growth. Contrarily, stem elongation of the main culm at a later stage, or that of lateral shoots was much less affected by scFv production. These phenotypes were different from anti-bioactive GA scFv-producing lines, whose stem elongation was continuously repressed throughout the inflorescence development. The GA-deficient phenotypes were recovered by treatment with GA(24) and bioactive GA(4), the latter being more effective. The transgenic lines contained conspicuously higher endogenous GA(24) and clearly less GA(4) than wild-type plants. The expression of GA 20-oxidase and GA 3-oxidase genes, which are feedback-regulated by GA signaling, were up-regulated in those plants. These results demonstrate that the scFv trapped GA(24) in ER and inhibited metabolism of GA(24) to bioactive GA(4).     

Copper(II)-cis,cis-1,3,5-triaminocyclohexane complex-promoted hydrolysis of dipeptides: kinetic, speciation and structural studies

The hydrolysis of glycylglycine (GylGly), glycyl-L-leucine (GlyLeu), L-leucylglycine (LeuGly) and glycyl-DL-serine (GlySer) promoted by a copper(II)- cis, cis-1,3,5-triaminocyclohexane complex [Cu(II)TACH] was investigated at 70 degrees C and pH 7-10, using HPLC. The observed pseudo-first-order rate constants (k(obs)) and rate enhancing factors (REF) were as follows: 4.1x10(-3 )h(-1)(REF=23) for GylGly, 1.6x10(-3 )h(-1)(REF=21) for GlyLeu, 5.1x10(-3 )h(-1)(REF=64) for LeuGly and 9.2x10(-2 )h(-1)(REF=47) for GlySer [pH 8.1, dipeptide 2 mM, copper(II) 2 mM and TACH 2 mM]. Based on the pH dependence and dipeptide concentration dependence of the initial rates and speciation of the Cu(II)-TACH-dipeptide system at 25 degrees C and I=0.1, the reactions proceed via the formation of a ternary complex [Cu(TACH)(dipeptide)](+) as an intermediate followed by OH(-)-dependent and OH(-)-independent paths to give amino acid(s). GylGly, GlyLeu and LeuGly preferred the OH(-)-dependent path, while GlySer preferred the OH(-)-independent path. The latter can be explained by the intramolecular attack of the amide carbonyl group coordinated with its oxygen atom by the OH group in the serine residue. The X-ray crystal structure of [Cu(TACH)(GlyGly)]BPh(4).MeOH confirmed that GlyGly coordinates to copper(II) ion with its terminal amino N and amide O atoms. The crystal structures of [Cu(TACH)(Gly)]BPh(4) and [Cu(2)(TACH)(2)(OH)(2)](ClO(4))(2).NaClO(4).H(2)O are also reported.