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Kumiko Ui-Tei

Publications and source records attributed to Kumiko Ui-Tei.

At least 19 recordsLinked to original sources
PubMed2007

Guidelines for the selection of effective short-interfering RNA sequences for functional genomics.

To avoid long-double-stranded-RNA-dependent interferon response, short-interfering RNAs (siRNAs) are widely used for RNA interference (RNAi) in mammalian cells. siRNA-based RNAi, however, may not be readily available for the large-scale gene silencing essential for systematic functional genomics, because only a limited fraction of siRNAs is capable of inducing effective mammalian RNAi. siRNAs correctly designed for the knockdown of a particular gene may also destroy the functions of unrelated genes. Here, we describe algorithms by which these serious setbacks can be eliminated in mammalian functional genomics using RNAi and a Web-based online software system for computing highly functional siRNA sequences with maximal target-specificity in mammalian RNAi.

Algorithms↗
PubMed2006

siVirus: web-based antiviral siRNA design software for highly divergent viral sequences.

siVirus (http://siVirus.RNAi.jp/) is a web-based online software system that provides efficient short interfering RNA (siRNA) design for antiviral RNA interference (RNAi). siVirus searches for functional, off-target minimized siRNAs targeting highly conserved regions of divergent viral sequences. These siRNAs are expected to resist viral mutational escape, since their highly conserved targets likely contain structurally/functionally constrained elements. siVirus will be a useful tool for designing optimal siRNAs targeting highly divergent pathogens, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus and SARS coronavirus, all of which pose enormous threats to global human health.

Anti-HIV Agents↗
PubMed2006

Molecular cloning and characterization of a novel 3'-phosphoadenosine 5'-phosphosulfate transporter, PAPST2.

Sulfation is an important posttranslational modification associated with a variety of molecules. It requires the involvement of the high energy form of the universal sulfate donor, 3'-phosphoadenosine 5'-phosphosulfate (PAPS). Recently, we identified a PAPS transporter gene in both humans and Drosophila. Although human colonic epithelial tissues express many sulfated glycoconjugates, PAPST1 expression in the colon is trace. In the present study, we identified a novel human PAPS transporter gene that is closely related to human PAPST1. This gene, called PAPST2, is predominantly expressed in human colon tissues. The PAPST2 protein is localized on the Golgi apparatus in a manner similar to the PAPST1 protein. By using yeast expression studies, PAPST2 protein was shown to have PAPS transport activity with an apparent Km value of 2.2 microM, which is comparable with that of PAPST1 (0.8 microM). Overexpression of either the PAPST1 or PAPST2 gene increased PAPS transport activity in human colon cancer HCT116 cells. The RNA interference of the PAPST2 gene in the HCT116 cells significantly reduced the reactivity of G72 antibody directed against the sialyl 6-sulfo N-acetyllactosamine epitope and total sulfate incorporation into cellular proteins. These findings indicate that PAPST2 is a PAPS transporter gene involved in the synthesis of sulfated glycoconjugates in the colon.

Amino Acid Sequence↗
PubMed2006

Essential notes regarding the design of functional siRNAs for efficient mammalian RNAi.

Short interfering RNAs (siRNAs) are widely used to bring about RNA interference (RNAi) in mammalian cells. Numerous siRNAs may be designed for any target gene though most of which would be incapable of efficiently inducing mammalian RNAi. Certain highly functional siRNAs designed for knockout of a particular gene may render unrelated endogenous genes nonfunctional. These major bottlenecks should be properly eliminated when RNAi technologies are employed for any experiment in mammalian functional genomics. This paper thus presents essential notes and findings regarding the proper choice of siRNA-sequence selection algorithms and web-based online software systems.

Journal Article↗
PubMed2005

dsCheck: highly sensitive off-target search software for double-stranded RNA-mediated RNA interference.

Off-target effects are one of the most serious problems in RNA interference (RNAi). Here, we present dsCheck (http://dsCheck.RNAi.jp/), web-based online software for estimating off-target effects caused by the long double-stranded RNA (dsRNA) used in RNAi studies. In the biochemical process of RNAi, the long dsRNA is cleaved by Dicer into short-interfering RNA (siRNA) cocktails. The software simulates this process and investigates individual 19 nt substrings of the long dsRNA. Subsequently, the software promptly enumerates a list of potential off-target gene candidates based on the order of off-target effects using its novel algorithm, which significantly improves both the efficiency and the sensitivity of the homology search. The website not only provides a rigorous off-target search to verify previously designed dsRNA sequences but also presents 'off-target minimized' dsRNA design, which is essential for reliable experiments in RNAi-based functional genomics.

Algorithms↗
PubMed2005

Requirements of genetic interactions between Src42A, armadillo and shotgun, a gene encoding E-cadherin, for normal development in Drosophila.

Src42A is one of the two Src homologs in Drosophila. Src42A protein accumulates at sites of cell-cell or cell-matrix adhesion. Anti-Engrailed antibody staining of Src42A protein-null mutant embryos indicated that Src42A is essential for proper cell-cell matching during dorsal closure. Src42A, which is functionally redundant to Src64, was found to interact genetically with shotgun, a gene encoding E-cadherin, and armadillo, a Drosophila beta-catenin. Immunoprecipitation and a pull-down assay indicated that Src42A forms a ternary complex with E-cadherin and Armadillo, and that Src42A binds to Armadillo repeats via a 14 amino acid region, which contains the major autophosphorylation site. The leading edge of Src mutant embryos exhibiting the dorsal open phenotype was frequently kinked and associated with significant reduction in E-cadherin, Armadillo and F-actin accumulation, suggesting that not only Src signaling but also Src-dependent adherens-junction stabilization would appear likely to be essential for normal dorsal closure. Src42A and Src64 were required for Armadillo tyrosine residue phosphorylation but Src activity may not be directly involved in Armadillo tyrosine residue phosphorylation at the adherens junction.

Adherens Junctions↗
PubMed2005

Aph-1 contributes to the stabilization and trafficking of the gamma-secretase complex through mechanisms involving intermolecular and intramolecular interactions.

Gamma-secretase cleaves type I transmembrane proteins, including beta-amyloid precursor protein and Notch, and requires the formation of a protein complex comprised of presenilin, nicastrin, Aph-1, and Pen-2 for its activity. Aph-1 is implicated in the stabilization of this complex, although its precise mechanistic role remains unknown. Substitution of the first glycine within the transmembrane GXXXG motif of Aph-1 causes a loss-of-function phenotype in Caenorhabditis elegans. Here, using an untranslated region-targeted RNA interference/rescue strategy in Drosophila Schneider 2 cells, we show that Aph-1 contributes to the assembly of the gamma-secretase complex by multiple mechanisms involving intermolecular and intramolecular interactions depending on or independent of the conserved glycines. Aph-1 binds to nicastrin forming an early subcomplex independent of the conserved glycines within the endoplasmic reticulum. Certain mutations in the conserved GXXXG motif affect the interaction of the Aph-1.nicastrin subcomplex with presenilin that mediates trafficking of the presenilin.Aph-1.nicastrin tripartite complex to the Golgi. The same mutations decrease the stability of Aph-1 polypeptides themselves, possibly by affecting intramolecular associations through the transmembrane domains. Our data suggest that the proper assembly of the Aph-1.nicastrin subcomplex with presenilin is the prerequisite for the trafficking as well as the enzymatic activity of the gamma-secretase complex and that Aph-1 functions as a stabilizing scaffold in the assembly of this complex.

3' Untranslated Regions↗
PubMed2004

siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference.

siDirect (http://design.RNAi.jp/) is a web-based online software system for computing highly effective small interfering RNA (siRNA) sequences with maximum target-specificity for mammalian RNA interference (RNAi). Highly effective siRNA sequences are selected using novel guidelines that were established through an extensive study of the relationship between siRNA sequences and RNAi activity. Our efficient software avoids off-target gene silencing to enumerate potential cross-hybridization candidates that the widely used BLAST search may overlook. The website accepts an arbitrary sequence as input and quickly returns siRNA candidates, providing a wide scope of applications in mammalian RNAi, including systematic functional genomics and therapeutic gene silencing.

Algorithms↗
PubMed2004

Expression of DDAH1 in chick and rat embryos.

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) is an enzyme that metabolizes methylated arginine to citrulline and methylamine, thus working to produce nitric oxide (NO). We isolated a gene encoding chick DDAH1. In situ hybridization analysis revealed characteristic DDAH1 mRNA expression in the embryonic spinal cord, which was especially strong in the ventral horn and dorsal root ganglion (DRG). DDAH1 was also detected in the brain, kidney, digestive tract, and in other tissues. We examined the expression pattern of DDAH1 in developing rats and compared this with the expression pattern in chicks. The expression pattern in the rats was very similar to that in the chicks, but there were some differences between the chicks and rats in the amount of DDAH1 detected in the heart, liver, lung, and DRG. We also investigated neural nitric oxide synthase (nNOS) mRNA expression patterns in rat embryos. The DDAH1 expression patterns were completely different from nNOS expression patterns. Our study suggests that DDAH1 plays an important role in development.

Aging↗
PubMed2004

Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference.

In the present study, the relationship between short interfering RNA (siRNA) sequence and RNA interference (RNAi) effect was extensively analyzed using 62 targets of four exogenous and two endogenous genes and three mammalian and Drosophila cells. We present the rules that may govern siRNA sequence preference and in accordance with which highly effective siRNAs essential for systematic mammalian functional genomics can be readily designed. These rules indicate that siRNAs which simultaneously satisfy all four of the following sequence conditions are capable of inducing highly effective gene silencing in mammalian cells: (i) A/U at the 5' end of the antisense strand; (ii) G/C at the 5' end of the sense strand; (iii) at least five A/U residues in the 5' terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nt in length. siRNAs opposite in features with respect to the first three conditions give rise to little or no gene silencing in mammalian cells. Essentially the same rules for siRNA sequence preference were found applicable to DNA-based RNAi in mammalian cells and in ovo RNAi using chick embryos. In contrast to mammalian and chick cells, little siRNA sequence preference could be detected in Drosophila in vivo RNAi.

Animals↗
PubMed2004

Active sites in the carboxyl-terminal region of the laminin alpha chain in Drosophila neuronal cell spreading.

An established Drosophila neuronal cell line (BG2-c6) proved to be useful to analyze laminin-mediated cell spreading and signal transduction [Takagi et al. (2000) Biochem Biophys Res Commun 270:482-487]. Here, we report, in addition to the whole molecule, the truncated alpha chain of Drosophila laminin (containing the entire carboxyl-terminal globular domain) and two dodecapeptides corresponding to the cell-binding sites identified in the alpha1 chain of mouse laminin were also active to stimulate BG2-c6 cell spreading. Our previous study [Takagi et al. (1996) J Biol Chem 271:18074-18081] revealed that these recombinant protein and synthetic peptides promoted neurite outgrowth in the primary cell culture system prepared from Drosophila embryo. Therefore, the similar effects by these proteins and peptides suggest the presence of a common mechanism of laminin and neuronal cell interaction working in both primary and established cells. One of the two active peptides contains the sequence SIKVGV. Its murine counterpart carries the sequence SIKVAV by which the interaction of laminin and cells is mediated. Furthermore, laminin-dependent BG2-c6 cell spreading was inhibited by heparin. This observation suggests that cell surface glycoproteins participate in the interaction of laminin and BG2-c6 cells.

Amino Acid Sequence↗
PubMed2003

Characterization of voltage-gated calcium currents in gonadotropin-releasing hormone neurons tagged with green fluorescent protein in rats.

Functional analysis of GnRH neurons is limited, although these neurons play an important role in neuroendocrine regulation. Therefore, we decided to conduct cell physiological analysis of GnRH neurons. To identify GnRH neurons, we tagged the neurons with green fluorescence protein by a transgenic technique. A dispersed culture of GnRH neurons was prepared from the transgenic rats. After overnight culture, a perforated patch clamp was applied to the identified GnRH neurons to analyze the Ca2+ currents. In neonatal GnRH neurons, high voltage-activated Ca2+ currents were clearly observed, but low voltage-activated Ca2+ current was negligible. Nimodipine (L-type channel blocker) and omega-conotoxin GVIA (N-type channel blocker) each attenuated the current by approximately 20%. The R-type channel blocker SNX-482 attenuated the current by approximately 55%. Inhibition by the P/Q-type channel blocker omega-agatoxin IVA was small. In GnRH neurons around puberty, however, both high and low voltage-activated Ca2+ currents were observed. Inhibitions by nifedipine, omega-conotoxin GVIA, and SNX-482 were similar to those in the neonatal neurons, whereas the inhibition by omega-agatoxin IVA was clearly seen in 40-61% of the GnRH neurons examined. These results indicate that GnRH neurons functionally express L-, N-, P/Q-, R-, and T-type channels. Expressions of P/Q- and T-type channels are developmentally regulated.

Animals↗
PubMed2003

Short-interfering-RNA-mediated gene silencing in mammalian cells requires Dicer and eIF2C translation initiation factors.

RNA interference (RNAi) is the process of long, double-stranded (ds), RNA-dependent posttranscriptional gene silencing (PTGS). In lower eukaryotes, dsRNA introduced into the cytoplasm is cleaved by the RNaseIII-like enzyme, Dicer, to 21-23 nt RNA (short interfering [si] RNA), which may serve as guide for target mRNA degradation. In mammals, long-dsRNA-dependent PTGS is applicable only to a limited number of cell types, whereas siRNA synthesized in vitro is capable of effectively inducing gene silencing in a wide variety of cells. Although biochemical and genetic analyses in lower eukaryotes showed that Dicer and some PIWI family member proteins are essential for long-dsRNA-dependent PTGS, little is known about the molecular mechanisms underlying siRNA-based PTGS. Here, we show that Dicer and eIF2C translation initiation factors belonging to the PIWI family (eIF2C1-4) play an essential role in mammalian siRNA-mediated PTGS, most probably through synergistic interactions. Immunoprecipitation experiments suggest that, in human and mouse cells, complex formation occurs between Dicer and eIF2C1 or 2 and that the PIWI domain of eIF2C is essential for the formation of this complex.

3T3 Cells↗
PubMed2003

Molecular cloning and characterization of avian bombesin-like peptide receptors: new tools for investigating molecular basis for ligand selectivity.

(1) Bombesin (BN), originally isolated from amphibians, is structurally related to a family of BN-like peptides found in mammals, which include gastrin-releasing peptide (GRP) and neuromedin B (NMB). These peptides have important effects on secretion, smooth muscle contraction, metabolism and behavior. Here we report cloning and characterization of two subtypes of BN-like peptide receptors in Aves. (2) The amino-acid sequence of chick GRP-R (chGRP-R) is highly identical with mammalian and amphibian GRP-R, and this receptor showed high affinity for GRP, BN and synthetic bombesin agonist, [D-Phe(6), beta-Ala(11), Phe(13), Nle(14)]bombesin(6-14) ([FAFNl]BN(6-14)). The chGRP-R gene was localized to chicken chromosome 1q23distal-q24proximal, where chick homologs of other human X-linked genes have also been mapped. (3) ChBRS-3.5, having sequence similarities to both mammalian bombesin-like peptide receptor subtype-3 and amphibian bombesin-like peptide receptor subtype-4, showed high affinity for [FAFNl]BN(6-14), moderate affinity for BN, but low affinity for both GRP and NMB. (4) Expression of both receptors was detected in brain, but only chGRP-R was expressed in gastrointestinal (GI) tissues. (5) When expressed in Chinese hamster ovary K1 cells, these receptors mediate intracellular calcium mobilization upon agonist stimulation. These results suggest that a novel BN peptide may occur in Aves as an endogenous ligand for chBRS-3.5. (6) The receptor sequences responsible for ligand selectivities were discussed and this knowledge about avian BN-like peptide receptors will help us to understand the molecular basis for agonist sensitivities of BN-like peptide receptors.

Amino Acid Sequence↗
PubMed2002

Replacement of midgut epithelium in the greater wax moth, Galleria mellonela, during larval-pupal moult.

The epithelium of larval midgut of the greater wax moth, Galleria mellonela, was replaced during the larval-pupal moult. The development of this moth was tentatively divided into 11 stages, from the full-grown larva of last instar to the 4-day-old pupa. The midgut at each stage was observed for (1) overall structure, (2) the position of goblet cells, and (3) the appearance of the yellow body. Light microscopy revealed that cell death in the midgut began in a cocoon-spinning larva (stage II), when pigments in the stemmata started to migrate. Before drastic remodeling started to occur, cytoplasmic projections in the goblet cavities were transformed. The larval midgut changed markedly at stage III, when the pigments left the stemmata. The epithelium of the larval midgut dropped as a whole into the lumen, transforming into the yellow body. Simultaneously, a pupal midgut epithelium developed. Electron microscopy of the columnar cells of a stage III larva showed that microvilli and mitochondria looked normal even though the nucleus with condensed heterochromatin resembled an apoptotic nucleus of vertebrate and higher plant cells. Caspase-3-like protease activity was restricted to the larval midgut and increased in parallel with the formation of the yellow body. The results indicate that the replacement of the larval midgut is facilitated by a typical apoptotic process.

Animals↗
PubMed2002

The promoter region of the human BUBR1 gene and its expression analysis in lung cancer.

Mitotic checkpoint impairment is present in human lung cancers with chromosomal instability (CIN). Spindle-checkpoint genes have been reported to be mutated in several human cancers, but these mutations are infrequent. Recent reports suggest that the hBUBR1 gene may play an important role in mitotic checkpoint control and in mitotic checkpoint impairment in human cancers. We analyzed the expression of hBUBR1 in lung cancer cell lines using real time quantitative RT-PCR. The expression of BUBR1 was found to be up-regulated in all of these cell lines. In addition, we cloned and characterized the promotor region of hBUBR1 and determined its genomic structure, which includes 23 exons. The open reading frame (ORF) of the hBUBR1 gene comprises exons 1 through 23. There are GC-rich regions located at the flanking region and about 150 bp upstream from exon 1. The promoter region (424 bp upstream from exon 1) showed promoter activity and includes multiple transcription factor consensus binding motifs, including those for Sp1, Nkx-2, CdxA, SRY, MyoD, Ik-2, HNF-3b, Staf, Oct-1, Nkx-2, v-Myb, and AML 1a. Multiple pathways leading to activation of those binding factors may contribute to hBUBR1 gene transcription. Knowledge of the genomic structure and the promoter region of the hBUBR1 gene will facilitate investigation of its role in mitotic checkpoint control and tumor progression in human cancers.

Cell Cycle Proteins↗