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Kota Saito

Publications and source records attributed to Kota Saito.

11 recordsLinked to original sources
PubMed2006

A passive smoking screening program for children.

BACKGROUND: There has been no report to date on mass screening of passive smoking in children using biomarkers. METHOD: To identify children exposed to actual environmental tobacco smoke (ETS), 261 children were divided into the following 3 groups: (A) both parents smoke; (B) one parent smokes; and (C) no parent smokes. Child urinary cotinine measurement and a parent questionnaire were obtained. RESULTS: Urinary cotinine was positive (>10 ng/ml) in 92 (35.2%) of the 261 children. Of the 92 children, 29 were classified into group A, 47 into group B, and 16 into group C. The percentages of children who tested positive for urinary cotinine in groups A, B, and C were 56.9%, 31.1%, and 27.1%, respectively. However, in group B, the percentage of children who tested positive for urinary cotinine was significantly higher if only the mother smoked (47.1%) than if only the father smoked (29.1%) (P<0.05). The mean+SD urinary cotinine level in group A was 12.9+/-6.5 ng/ml, and that in group B was 10.4+/-3.8 ng/ml if the mother smoked and 5.4+/-2.6 ng/ml if the father smoked. CONCLUSIONS: This smoking screening program may be useful in identifying children with actual ETS exposure and motivating their parents to either quit smoking or modify their smoking behavior around children.

Adult↗
PubMed2006

Functional genomics reveals genes involved in protein secretion and Golgi organization.

Yeast genetics and in vitro biochemical analysis have identified numerous genes involved in protein secretion. As compared with yeast, however, the metazoan secretory pathway is more complex and many mechanisms that regulate organization of the Golgi apparatus remain poorly characterized. We performed a genome-wide RNA-mediated interference screen in a Drosophila cell line to identify genes required for constitutive protein secretion. We then classified the genes on the basis of the effect of their depletion on organization of the Golgi membranes. Here we show that depletion of class A genes redistributes Golgi membranes into the endoplasmic reticulum, depletion of class B genes leads to Golgi fragmentation, depletion of class C genes leads to aggregation of Golgi membranes, and depletion of class D genes causes no obvious change. Of the 20 new gene products characterized so far, several localize to the Golgi membranes and the endoplasmic reticulum.

Animals↗
PubMed2005

Purification and analysis of RIN family-novel Rab5 GEFs.

The small GTPase Rab5 plays important roles in membrane budding and trafficking in the early endocytic pathways, and the activation of this GTPase is mediated by several guanine nucleotide exchange factors (GEFs) at each of the transport steps. The RIN family has been identified as GEFs for Rab5 and shown to possess unique biochemical properties. The RIN family preferentially interacts with an activated form of Rab5, although it enhances guanine nucleotide exchange reaction. Moreover, biochemical analysis indicates that the RIN family functions as a tetramer. In this chapter, we describe the isolation of the recombinant RIN family via expression in Spodoptera frugiperda (Sf9) insect cells and in mammalian cells. In addition, functional analysis is also provided to assess the physiological properties of the RIN family.

Animals↗
PubMed2005

Novel role of the small GTPase Rheb: its implication in endocytic pathway independent of the activation of mammalian target of rapamycin.

The Ras-homologous GTPase Rheb that is conserved from yeast to human appears to be involved not only in cell growth but also in nutrient uptake. Recent biochemical analysis revealed that tuberous sclerosis complex (TSC), a GTPase-activating protein (GAP), deactivates Rheb and that phosphatidylinositol 3'-kinase (PI3k)-Akt/PKB kinase pathway activates Rheb through inhibition of the GAP-mediated deactivation. Although mammalian target of rapamycin (mTOR) kinase is implicated in the downstream target of Rheb, the direct effector(s) and exact functions of Rheb have not been fully elucidated. Here we identified that Rheb expression in cultured cells induces the formation of large cytoplasmic vacuoles, which are characterized as late endocytic (late endosome- and lysosome-like) components. The vacuole formation required the GTP form of Rheb, but not the activation of the downstream mTOR kinase. These results suggest that Rheb regulates endocytic trafficking pathway independent of the previously identified mTOR pathway. The physiological roles of the two Rheb-dependent signaling pathways are discussed in terms of nutrient uptake and cell growth or cell cycle progression.

Animals↗
PubMed2004

Genetic dissection of the formation of the forebrain in Medaka, Oryzias latipes.

The forebrain, consisting of the telencephalon and diencephalon, is essential for processing sensory information. To genetically dissect formation of the forebrain in vertebrates, we carried out a systematic screen for mutations affecting morphogenesis of the forebrain in Medaka. Thirty-three mutations defining 25 genes affecting the morphological development of the forebrain were grouped into two classes. Class 1 mutants commonly showing a decrease in forebrain size, were further divided into subclasses 1A to 1D. Class 1A mutation (1 gene) caused an early defect evidenced by the lack of bf1 expression, Class 1B mutations (6 genes) patterning defects revealed by the aberrant expression of regional marker genes, Class 1C mutation (1 gene) a defect in a later stage, and Class 1D (3 genes) a midline defect analogous to the zebrafish one-eyed pinhead mutation. Class 2 mutations caused morphological abnormalities in the forebrain without considerably affecting its size, Class 2A mutations (6 genes) caused abnormalities in the development of the ventricle, Class 2B mutations (2 genes) severely affected the anterior commissure, and Class 2C (6 genes) mutations resulted in a unique forebrain morphology. Many of these mutants showed the compromised sonic hedgehog expression in the zona-limitans-intrathalamica (zli), arguing for the importance of this structure as a secondary signaling center. These mutants should provide important clues to the elucidation of the molecular mechanisms underlying forebrain development, and shed new light on phylogenically conserved and divergent functions in the developmental process.

Animals↗
PubMed2004

Mutations affecting liver development and function in Medaka, Oryzias latipes, screened by multiple criteria.

We report here mutations affecting various aspects of liver development and function identified by multiple assays in a systematic mutagenesis screen in Medaka. The 22 identified recessive mutations assigned to 19 complementation groups fell into five phenotypic groups. Group 1, showing defective liver morphogenesis, comprises mutations in four genes, which may be involved in the regulation of growth or patterning of the gut endoderm. Group 2 comprises mutations in three genes that affect the laterality of the liver; in kendama mutants of this group, the laterality of the heart and liver is uncoupled and randomized. Group 3 includes mutations in three genes altering bile color, indicative of defects in hemoglobin-bilirubin metabolism and globin synthesis. Group 4 consists of mutations in three genes, characterized by a decrease in the accumulation of fluorescent metabolite of a phospholipase A(2) substrate, PED6, in the gall bladder. Lipid metabolism or the transport of lipid metabolites may be affected by these mutations. Mutations in Groups 3 and 4 may provide animal models for relevant human diseases. Group 5 mutations in six genes affect the formation of endoderm, endodermal rods and hepatic bud from which the liver develops. These Medaka mutations, identified by morphological and metabolite marker screens, should provide clues to understanding molecular mechanisms underlying formation of a functional liver.

Animals↗
PubMed2004

A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes.

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

Animals↗
PubMed2003

RIN3: a novel Rab5 GEF interacting with amphiphysin II involved in the early endocytic pathway.

The small GTPase Rab5, which cycles between active (GTP-bound) and inactive (GDP-bound) states, plays essential roles in membrane budding and trafficking in the early endocytic pathway. However, the molecular mechanisms underlying the Rab5-regulated processes are not fully understood other than the targeting event to early endosomes. Here, we report a novel Rab5-binding protein, RIN3, that contains many functional domains shared with other RIN members and additional Pro-rich domains. RIN3 displays the same biochemical properties as RIN2, the stimulator and stabilizer of GTP-Rab5. In addition, RIN3 exhibits its unique intracellular localization. RIN3 expressed in HeLa cells localized to cytoplasmic vesicles and the RIN3-positive vesicles contained Rab5 but not the early endosomal marker EEA1. Transferrin appeared to be transported partly through the RIN3-positive vesicles to early endosomes. RIN3 was also capable of interacting via its Pro-rich domain with amphiphysin II, which contains SH3 domain and participates in receptor-mediated endocytosis. Interestingly, cytoplasmic amphiphysin II was translocated into the RIN3- and Rab5-positive vesicles when co-expressed with RIN3. These results indicate that RIN3 biochemically characterized as the stimulator and stabilizer for GTP-Rab5 plays an important role in the transport pathway from plasma membrane to early endosomes.

Animals↗
PubMed2002

Di-Ras, a distinct subgroup of ras family GTPases with unique biochemical properties.

The small GTPase Ras family regulates a variety of cell functions including proliferation and differentiation. Here we have identified novel Ras members, human Di-Ras1 and Di-Ras2, belonging to a distinct branch of the GTPase family. Di-Ras1 and Di-Ras2 specifically expressed in heart and brain share 30-40% overall identity with other members of Ras family, however, they have the following characteristic substitutions at highly conserved regions among the Ras family. 1) Thr-63 and Ser-65 in Di-Ras are substituted for Ala-59 and Gln-61 positions in Ha-Ras, respectively, that are known to be critical for GTP hydrolysis. 2) Within the effector domains, Di-Ras has Ile at a position corresponding to Asp-33 in Ha-Ras, which is important for its interaction with the downstream effector Raf. As predicted by these substitutions, Di-Ras has only a quite low level of GTPase activity and exists predominantly as a GTP-bound form upon its expression in living cells. Moreover, Di-Ras fails to interact with the Ras-binding domain of Raf, resulting in no stimulation of mitogen-activated protein kinase. Interestingly, introduction of Di-Ras into HEK293T cells induces large cellular vacuolation. These findings raise the possibility that Di-Ras might regulate cell morphogenesis in a manner distinct from other members of Ras family.

Amino Acid Sequence↗
PubMed2001

A novel binding protein composed of homophilic tetramer exhibits unique properties for the small GTPase Rab5.

The small GTPase Rab family, which cycles between GTP-bound active and GDP-bound inactive states, plays an important role in membrane trafficking. Among them, Rab5 is involved in early endocytic pathway, and several Rab5-binding proteins have been identified as regulators or effectors to coordinate the docking and fusion processes of endocytic vesicles. We describe a novel binding protein exhibiting unique biochemical properties for Rab5. The Rab5-binding protein enhances GDP-GTP exchange reaction on Rab5 but preferentially interacts with its GTP-bound form. Gel filtration and immunoprecipitation analyses indicate that the Rab5-binding protein functions as a tetramer composed of anti-parallel linkage of two parallel dimers. These results suggest that the newly identified protein may function as an upstream activator and/or downstream effector for Rab5 in endocytic pathway. Possible roles of the quaternary structure have been discussed in terms of the Rab5-mediated signaling.

Amino Acid Sequence↗