Biomedical subjects

Kaoru Saigo

Publications and source records attributed to Kaoru Saigo.

At least 19 recordsLinked to original sources

PubMed2007

Polarized exocytosis and transcytosis of Notch during its apical localization in Drosophila epithelial cells.

Notch (N) and its ligands, Delta (Dl) and Serrate (Ser), are transmembrane proteins that mediate the cell-cell interactions necessary for many cell-fate decisions. In Drosophila, N is predominantly localized to the apical portion of epithelial cells, but the mechanisms and functions of this localization are unknown. Here, we found N, Dl, and Ser were mostly located in the region from the subapical complex (SAC) to the apical portion of the adherens junctions (AJs) in wing disc epithelium. N was delivered to the SAC/AJs in two phases. First, polarized exocytosis specifically delivered nascent N to the apical plasma membrane and AJs in an O-fut1-independent manner. Second, N at the plasma membrane was relocated to the SAC/AJs by Dynamin- and Rab5-dependent transcytosis; this step required the O-fut1 function. Disruption of the apical polarity by Drosophila E-cadherin (DEcad) knock down caused N and Dl localization to the SAC/AJs to fail. N, but not Dl, formed a specific complex with DEcad in vivo. Finally, our results suggest that juxtacrine signaling in epithelia generally depends on the apicobasally polarized structure of epithelial cells.

Adherens Junctions↗

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

Fate determination of Drosophila leg distal regions by trachealess and tango through repression and stimulation, respectively, of Bar homeobox gene expression in the future pretarsus and tarsus.

During tissue patterning, developing fields may be subdivided into several non-overlapping domains by region-specific expression of transcription factors. In Drosophila leg development, the most distal segments, the pretarsus and tarsal segment 5 (ta5), are precisely specified by interactions between tarsus homeobox genes (BarH1 and BarH2) and pretarsus homeobox genes (aristaless, clawless, and Lim1). Here, we demonstrate that trachealess and tango, both encoding bHLH-PAS proteins that are required for the formation of the embryonic tracheal system, are essential for forming two adjacent distal segments of the leg. trachealess is expressed in the pretarsus and ta5, and the concerted action of trachealess and tango seems to modulate the activity of homeobox gene regulatory loops by repressing Bar in the pretarsus and activating Bar in ta5.

Animals↗

PubMed2006

Differential microarray analysis of Drosophila mushroom body transcripts using chemical ablation.

Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the Drosophila brain. As a way to systematically elucidate genes preferentially expressed in MBs, we have analyzed genome-wide alterations in transcript profiles associated with MB ablation by hydroxyurea. We selected 100 genes based on microarray data and examined their expression patterns in the brain by in situ hybridization. Seventy genes were found to be expressed in the posterodorsal cortex, which harbors the MB cell bodies. These genes encode proteins of diverse functions, including transcription, signaling, cell adhesion, channels, and transporters. Moreover, we have examined developmental functions of 40 of the microarray-identified genes by transgenic RNA interference; 8 genes were found to cause mild-to-strong MB defects when suppressed with a MB-Gal4 driver. These results provide important information not only on the repertoire of genes that control MB development but also on the repertoire of neural factors that may have important physiological functions in MB plasticity.

Animals↗

PubMed2006

Fate map of the distal portion of Drosophila proboscis as inferred from the expression and mutations of basic patterning genes.

The late-third-instar labial disc is comprised of two disc-proper cell layers, one representing mainly the ventral half of the anterior compartment (L-layer) and the other, the dorsal half of the anterior compartment and most, if not all, of the posterior compartment (M-layer). In the L-layer, Distal-less represses homothorax whereas no Distal-less-dependent homothorax repression occurs in the M-layer where Distal-less is coexpressed with homothorax. In wild-type labial discs, clawless, one of the two homeobox genes expressed in distal cells receiving maximum (Decapentaplegic+Wingless) signaling activity in leg and antennal discs, is specifically repressed by proboscipedia. A fate map, inferred from data on basic patterning gene expression in larval and pupal stages and mutant phenotypes, indicates the inner surface of the labial palpus, which includes the pseudotracheal region, to be a derivative of the distal portion of the M-layer expressing wingless, patched, Distal-less and homothorax. The outer surface of the labial palpus with more than 30 taste bristles derives from an L-layer area consisting of dorsal portions of the anterior and posterior compartments, each expressing Distal-less. Our analysis also indicates that, in adults and pupae, the anterior-posterior boundary, dividing roughly equally the outer surface of the distiproboscis, runs along the outer circumference of the inner surface of distiproboscis.

Animals↗

PubMed2006

Control of axonal sprouting and dendrite branching by the Nrg-Ank complex at the neuron-glia interface.

Neurons are highly polarized cells with distinct subcellular compartments, including dendritic arbors and an axon. The proper function of the nervous system relies not only on correct targeting of axons, but also on development of neuronal-class-specific geometry of dendritic arbors [1-4]. To study the intercellular control of the shaping of dendritic trees in vivo, we searched for cell-surface proteins expressed by Drosophila dendritic arborization (da) neurons [5-7]. One of them was Neuroglian (Nrg), a member of the Ig superfamily ; Nrg and vertebrate L1-family molecules have been implicated in various aspects of neuronal wiring, such as axon guidance, axonal myelination, and synapse formation [9-12]. A subset of the da neurons in nrg mutant embryos exhibited deformed dendritic arbors and abnormal axonal sprouting. Our functional analysis in a cell-type-selective manner strongly suggested that those da neurons employed Nrg to interact with the peripheral glia for suppressing axonal sprouting and for forming second-order dendritic branches. At least for the former role, Nrg functioned in concert with the intracellular adaptor protein Ankyrin (Ank) [13]. Thus, the neuron-glia interaction that is mediated by Nrg, together with Ank under some situations, contributes to axonal and dendritic morphogenesis.

Animals↗

PubMed2006

GATAe-dependent and -independent expressions of genes in the differentiated endodermal midgut of Drosophila.

Two sequentially-expressed GATA factor genes, serpent (srp) and GATAe, are essential for development of the Drosophila endoderm. The earliest endodermal GATA gene, srp, has been thought to specify the endodermal fate, activating the second GATA gene GATAe, and the latter continues to be expressed in the endodermal midgut throughout life. Previously, we proposed that GATAe establishes and maintains the state of terminal differentiation of the midgut, since some functional genes in the midgut require GATAe activity for their expression. To obtain further evidence of the role of GATAe, we searched for additional genes that are expressed specifically in the midgut in late stages, and examined responses of a total of selected 15 genes to the depletion and overexpression of GATAe. Ten of the 15 genes failed to be expressed in the embryo deficient for GATAe activity, but, the other five genes did not require GATAe. Instead, srp is required for activating the five genes. These observations indicate that GATAe activates a major subset of genes in the midgut, and some other pathway(s) downstream of srp activates other genes.

Animals↗

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

Crystallization and preliminary X-ray analysis of the PIN domain of human EST1A.

Human EST1A (ever shorter telomeres 1A) is associated with most or all active telomerase in cell extracts and is involved either directly or indirectly in telomere elongation and telomere capping. The C-terminal region of EST1A contains the PIN (PilT N-terminus) domain, a putative nuclease domain. The PIN domain of human EST1A was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group C2, with unit-cell parameters a = 107.3, b = 51.6, c = 100.5 angstroms, beta = 119.3 degrees, and diffracted X-rays to 1.8 angstroms resolution. The asymmetric unit contained two molecules of the PIN domain and the solvent content was 57%.

Crystallization↗

PubMed2006

Three-dimensional structure of AzoR from Escherichia coli. An oxidereductase conserved in microorganisms.

The crystal structure of AzoR (azoreductase) has been determined in complex with FMN for two different crystal forms at 1.8 and 2.2 A resolution. AzoR is an oxidoreductase isolated from Escherichia coli as a protein responsible for the degradation of azo compounds. This enzyme is an FMN-dependent NADH-azoreductase and catalyzes the reductive cleavage of azo groups by a ping-pong mechanism. The structure suggests that AzoR acts in a homodimeric state forming the two identical catalytic sites to which both monomers contribute. The structure revealed that each monomer of AzoR has a flavodoxin-like structure, without the explicit overall amino acid sequence homology. Superposition of the structures from the two different crystal forms revealed the conformational change and suggested a mechanism for accommodating substrates of different size. Furthermore, comparison of the active site structure with that of NQO1 complexed with substrates provides clues to the possible substrate-binding mechanism of AzoR.

Amino Acid Sequence↗

PubMed2006

Temporal regulation of late expression of Bar homeobox genes during Drosophila leg development by Spineless, a homolog of the mammalian dioxin receptor.

The spatial and temporal regulation of genes encoding transcription factors is essential for the proper development of multicellular organisms. In Drosophila leg development, the distal-most tarsus (ta5) is specified by the strong expression of a pair of Bar homeobox genes in late third instar. This expression is regulated under the control of the ta5 enhancer activated by Bar. No activation of the ta5 enhancer, however, occurs in early third instar when considerable Bar is produced. The ta5 enhancer was comprised of a basal enhancer required for driving Bar expression and a negative regulatory motif serving as a binding site for the heterodimer of Spineless and Tango, homologs of the mammalian dioxin receptor and aryl hydrocarbon nuclear translocator, respectively. The spineless and tango were essential for suppressing the basal enhancer activation in early third instar. The spineless was transiently expressed in early third instar in the Bar expression domain. ta5 Bar expression may thus be temporally regulated through transient inhibition of premature activation of the basal enhancer via specific binding of the Spineless/Tango heterodimer to the negative regulatory motif in early third instar and subsequent release from the inhibition due to the disappearance of spineless expression at later stages.

Animals↗

PubMed2006

Crystallization and preliminary X-ray analysis of the C-terminal RNase III domain of human Dicer.

Human Dicer protein contains two RNase III domains (RNase IIIa and RNase IIIb) which are involved in the production of short interfering RNAs (siRNAs). The C-terminal RNase III domain (RNase IIIb) of human Dicer was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group C222(1), with unit-cell parameters a = 88.6, b = 199.7, c = 119.6 angstroms, and diffracted X-rays to 2.0 angstroms resolution. The asymmetric unit contained three molecules of the RNase IIIb and the solvent content was 67%.

Cloning, Molecular↗

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

Crystallization and preliminary X-ray analysis of AzoR (azoreductase) from Escherichia coli.

AzoR (azoreductase), an FMN-dependent NADH-azo compound oxidoreductase from Escherichia coli, has been crystallized in the presence of FMN by the sitting-drop vapour-diffusion method using 2-propanol as a precipitant. AzoR catalyzes the reductive cleavage of azo groups. The crystals were found to diffract X-rays to beyond 1.8 A resolution using a synchrotron-radiation source. The crystals belonged to the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 92.2, c = 51.9 A. The crystals are expected to contain one subunit of the homodimer in the asymmetric unit (VM = 2.6 A3 Da(-1)) and to have a solvent content of 51.6%. Data sets were also collected from heavy-atom derivatives for use in phasing. As a result, crystals soaked in a solution containing K2PtCl4 for 23 d were found to be reasonably isomorphous to the native crystals and the presence of Pt atoms could be confirmed. The data sets from the native crystals and the K2PtCl4-derivatized crystals are being evaluated for use in structure determination by single isomorphous replacement with anomalous scattering.

2-Propanol↗

PubMed2005

A concerted action of a paired-type homeobox gene, aristaless, and a homolog of Hox11/tlx homeobox gene, clawless, is essential for the distal tip development of the Drosophila leg.

The subdivision of the developing field by region-specific expression of genes encoding transcription factors is an essential step during appendage development in arthropod and vertebrates. In Drosophila leg development, the distal-most region (pretarsus) is specified by the expression of homeobox genes, aristaless and Lim1, and its immediate neighbor (distal tarsus) is specified by the expression of a pair of Bar homeobox genes. Here, we show that one additional gene, clawless, which is a homolog of vertebrate Hox11/tlx homeobox gene family and formerly known as C15, is specifically expressed in the pretarsus and cooperatively acts with aristaless to repress Bar and possibly to activate Lim1. Similar to aristaless, the maximal expression of clawless requires Lim1 and its co-factor, Chip. Bar attenuates aristaless and clawless expression through Lim1 repression. Aristaless and Clawless proteins form a complex capable of binding to specific DNA targets, which cannot be well recognized solely by Aristaless or Clawless.

Animals↗