PubMed Health⌕ Search

Biomedical subjects

Tsuyoshi Nakagawa

Publications and source records attributed to Tsuyoshi Nakagawa.

17 recordsLinked to original sources
PubMed2006

Aquaporin NIP2;1 is mainly localized to the ER membrane and shows root-specific accumulation in Arabidopsis thaliana.

We investigated a nodulin 26-like protein NIP2;1, which belongs to the third subgroup of Arabidopsis aquaporins. Histochemical analysis of a promoter-beta-glucuronidase fusion revealed the root-specific expression of NIP2;1. The NIP2;1 protein was detected in young roots, but not in leaves, stems, flowers or siliques. The transient expression of NIP2;1 linked with green fluorescent protein in Arabidopsis cultured cells showed its putative endoplasmic reticulum (ER) localization. NIP2;1 expressed in yeast cells had low water channel activity in the membranes. NIP2;1 may function as a water channel and/or ER channel for other small molecules or ions.

Amino Acid Sequence↗
PubMed2006

Cloning and sequencing analysis of alginate lyase genes from the marine bacterium Vibrio sp. O2.

We isolated a new marine bacteria, which displayed alginate-depolymerizing activity in plate assays, from seawater in Mihonoseki Harbor, Japan. Analysis of the 16S ribosomal RNA gene sequence of one of the isolates proved that this alginate-depolymerizing bacterium belonged to the genus Vibrio and it was named Vibrio sp. O2. The alginate lyase genes of Vibrio sp. O2 were cloned and expressed in Escherichia coli. Two alginate lyase-producing clones, pVOA-A4 and pVOA-B5, were obtained. The alginate lyase gene alyVOA from pVOA-A4 was composed of an 858-bp open reading frame (ORF) encoding 285 amino acid residues, while alyVOB from pVOA-B5 was composed of an 828-bp ORF encoding 275 amino acid residues. The degree of identity between the deduced amino acid sequences of AlyVOA or AlyVOB and Photobacterium sp. ATCC43367 alginate poly(ManA)lyase AlxM was 92.3% or 32.6%, respectively. Alginate lyase consensus regions corresponding to the sequences YFKAGXYXQ and RXELR were observed in all three of these sequences. AlyVOA and AlyVOB both degraded polymannuronate in plate assays and were therefore confirmed to be poly(beta-D-mannuronate)lyases.

Amino Acid Sequence↗
PubMed2006

The plastid protein THYLAKOID FORMATION1 and the plasma membrane G-protein GPA1 interact in a novel sugar-signaling mechanism in Arabidopsis.

Mutations in genes encoding components of the heterotrimeric G-protein complex were previously shown to confer altered sensitivity to increased levels of D-glucose. This suggests that G-protein coupling may be a novel sugar-signaling mechanism in Arabidopsis thaliana. THYLAKOID FORMATION1 (THF1) is here demonstrated in vivo as a Galpha interaction partner that functions downstream of the plasma membrane-delimited heterotrimeric G-protein (GPA1) in a D-glucose signaling pathway. THF1 is a plastid protein localized to both the outer plastid membrane and the stroma. Contact between root plastidic THF1 and GPA1 at the plasma membrane occurs at sites where the plastid membrane abuts the plasma membrane, as demonstrated by Förster resonance energy transfer (FRET). A probable role for THF1 in sugar signaling is demonstrated by both biochemical and genetic evidence. Root growth in the thf1-1 null mutant is hypersensitive to exogenous D-glucose, and THF1-overexpressing roots are resistant to inhibition of growth rate by high D-glucose. Additionally, THF1 levels are rapidly degraded by D-glucose but not L-glucose. The interaction between THF1 and GPA1 has been confirmed by in vitro and in vivo coimmunoprecipitation, FRET analysis, and genetic epistasis and provides evidence of a sugar-signaling mechanism between plastids and the plasma membrane.

Amino Acid Sequence↗
PubMed2006

Loss of AtPDR8, a plasma membrane ABC transporter of Arabidopsis thaliana, causes hypersensitive cell death upon pathogen infection.

Plants contain a large number of ATP-binding cassette (ABC) transporters belonging to different subclasses. AtPDR8 is the only member of the pleiotropic drug resistance (PDR) ABC transporter subclass in Arabidopsis that is constitutively highly expressed. In transgenic Arabidopsis plants harboring the AtPDR8 promoter fused to beta-glucuronidase (GUS), reporter expression was shown to be strong in the stomata and hydathode. In the stomata, transcripts of AtPDR8 were particularly frequent in the cells surrounding air spaces. Subcellular fractionation and immunochemical analysis showed that AtPDR8 was localized in the plasma membrane. When a knockout mutant of AtPDR8 (atpdr8) was infected with bacterial and oomycete pathogens, the plants exhibited chlorotic lesions and a hypersensitive response (HR)-like cell death. Cell death was detected in the atpdr8 mutants within 10 h of infection with the virulent bacterial pathogen, Pseudomonas syringae. As a result, the growth of P. syringae in the leaves of the atpdr8 mutant was reduced to 1% of that in the wild type. The defense response genes, PR-1, PR-2, PR-5, VPEgamma, AtrbohD and AtrbohF were highly expressed when the mutant plants were grown under non-sterile conditions. The expression of the AtPDR8 gene was enhanced by infection of virulent and avirulent bacterial pathogens. Our results indicate that AtPDR8 is a key factor controlling the extent of cell death in the defense response and suggest that AtPDR8 transports some substance(s) which is closely related to the response of plants to pathogens.

ATP-Binding Cassette Transporters↗
PubMed2005

zds1, a novel gene encoding an ortholog of Zds1 and Zds2, controls sexual differentiation, cell wall integrity and cell morphology in fission yeast.

While screening for genes that reverse the sporulation-deficient phenotype of the ras1delta diploid Schizosaccharomyces pombe strain, we identified zds1. This gene shares sequence homology with the ZDS1 and ZDS2 genes from Saccharomyces cerevisiae, which appear to be involved in multiple cellular events. Expression of Zds1 in ras1delta diploid cells elevated their sporulation rate from 0.3 to 11.2%. Expression of the Zds1 C-terminal region increased the sporulation rate further (to 21.9%) while introduction of the Zds1 N-terminal region had no effect. zds1 expression did not induce sporulation in strains with mutations in genes participating in the downstream MAP kinase cascade. The zds1-disrupted strain is sensitive to CaCl2, and this effect is suppressed by the C-terminal region of Zds1. The growth of the zds1delta strain is markedly inhibited by cold temperatures, while its viability decreased in the stationary phase. Moreover, the zds1delta strain is round in shape and very sensitive to zymolyase, and its cell wall becomes thicker than that of wild type. Thus, zds1 must be required to maintain cell wall integrity. The Zds1-GFP fusion protein localized to the cytosol, the septum, and the cell cortex. Its localization in the septum was dependent on its C-terminal region. Overexpression of the C-terminal region of Zds1 induced multi-septa and abnormal zygotes. We propose that the C-terminal region is the functional domain of Zds1 while the N-terminal region is a negative regulatory region. Thus, Zds1 is involved in multiple cellular events in fission yeast, including sexual differentiation, Ca2+ tolerance, cell wall integrity, viability in the stationary phase, and cell morphology.

Adaptor Proteins, Signal Transducing↗
PubMed2005

The Arabidopsis R2R3 MYB proteins FOUR LIPS and MYB88 restrict divisions late in the stomatal cell lineage.

The two guard cells of a stoma are produced by a single symmetric division just before terminal differentiation. Recessive mutations in the FOUR LIPS (FLP) gene abnormally induce at least four guard cells in contact with one another. These pattern defects result from a persistence of precursor cell identity that leads to extra symmetric divisions at the end of the cell lineage. FLP is likely to be required for the correct timing of the transition from cell cycling to terminal differentiation. FLP encodes a two-repeat (R2R3) MYB protein whose expression accumulates just before the symmetric division. A paralogous gene, MYB88, overlaps with FLP function in generating normal stomatal patterning. Plants homozygous for mutations in both genes exhibit more severe defects than flp alone, and transformation of flp plants with a genomic MYB88 construct restores a wild-type phenotype. Both genes compose a distinct and relatively basal clade of atypical R2R3 MYB proteins that possess an unusual pattern of amino acid substitutions in their putative DNA binding domains. Our results suggest that two related transcription factors jointly restrict divisions late in the Arabidopsis thaliana stomatal cell lineage.

Amino Acid Substitution↗
PubMed2005

Characterization and dynamic analysis of Arabidopsis condensin subunits, AtCAP-H and AtCAP-H2.

Condensin complexes are thought to play essential roles in mitotic chromosome assembly and segregation in eukaryotes. To date, two condensin complexes (condensin I and II) have been identified. Both complexes contain two structural maintenance of chromosome (SMC) subunits and three non-SMC subunits. In plants, little is known about the localization and function of all the condensin subunits. Here, we report the analyses on the localization of a non-SMC subunit of Arabidopsis condensin I and II, AtCAP-H, and AtCAP-H2, respectively. Our study indicated that localization of AtCAP-H and AtCAP-H2 is dynamically changed through the mitotic cell cycle using GFP-tagged AtCAP-H and AtCAP-H2 in tobacco cultured cells. They are localized at mitotic chromosomes from prometaphase to telophase. However, their localization in interphase is quite different. AtCAP-H was mainly found in the cytoplasm whereas AtCAP-H2 was mainly found in a nucleolus. It is revealed using GFP-tagged deletion mutant s of AtCAP-H that the kleisin-gamma middle domain (GM domain) is a unique domain only in AtCAP-H, responsible for chromosomal localization. We propose that the GM domain of CAP-H is essential for its chromosomal localization at mitosis and thus proper function of CAP-H. Differences in localization of AtCAP-H and AtCAP-H2 at interphase also suggest their functional differentiation.

Adenosine Triphosphatases↗
PubMed2005

Inverse correlation between the expression of O6-methylguanine-DNA methyl transferase (MGMT) and p53 in breast cancer.

BACKGROUND: Expression of O(6)-methylguanine-DNA methyl transferase (MGMT), a DNA repair protein, has been associated with tumor resistance to alkylating agents such as cyclophosphamide. Promoter hypermethylation of MGMT is a strong predictor of survival, and expression of mutant p53 protein may be associated with downregulated MGMT expression in brain tumors. In order to clarify further the correlation between MGMT and p53 expression, we investigated the expression levels of both MGMT and p53 in breast cancer. METHODS: MGMT and p53 expression was examined in tissues from 48 consecutive cases of primary breast cancer patients using immunohistochemical staining with antibodies specific for MGMT and p53. The prognosis for survival was analyzed based on MGMT and p53 immunoreactivity and clinicopathological characteristics. RESULTS: Expression of either MGMT or p53 was classified as negative or positive based on immunohistochemical staining. The expression of MGMT showed a significant inverse correlation with three clinicopathological characteristics, the status of the estrogen receptor, local recurrence and distant metastasis. There was a significant correlation between the expression level of p53 and the lymphatic involvement, estrogen receptor status and distant metastasis. MGMT and p53 were correlated with distant metastasis and local recurrence, but Kaplan-Meier curves did not show a significant difference. MGMT immuno-negative specimens showed a significantly higher expression of p53 (P = 0.026, chi(2) test). CONCLUSION: p53 may be associated with the regulation of MGMT expression in breast cancers.

Adult↗
PubMed2004

Identification and subcellular localization of two solanesyl diphosphate synthases from Arabidopsis thaliana.

Two solanesyl diphosphate synthases, designated SPS1 and SPS2, which are responsible for the synthesis of the isoprenoid side chain of either plastoquinone or ubiquinone in Arabidopsis thaliana, were identified. Heterologous expression of either SPS1 or SPS2 allowed the generation of UQ-9 in a decaprenyl diphosphate synthase-defective strain of fission yeast and also in wild-type Escherichia coli. SPS1-GFP was found to localize in the ER while SPS2-GFP localized in the plastid of tobacco BY-2 cells. These two different subcellular localizations are thought to be the reflection of their roles in solanesyl diphosphate synthesis in two different parts: presumably SPS1 and SPS2 for the side chains of ubiquinone and plastoquinone, respectively.

Alkyl and Aryl Transferases↗
PubMed2004

Zinc transporter of Arabidopsis thaliana AtMTP1 is localized to vacuolar membranes and implicated in zinc homeostasis.

Cation diffusion facilitator (CDF) proteins belong to a family of heavy metal efflux transporters that might play an essential role in homeostasis and tolerance to metal ions. We investigated the subcellular localization of Arabidopsis thaliana AtMTP1, a member of the CDF family, and its physiological role in the tolerance to Zn using MTP1-deficient mutant plants. AtMTP1 was immunochemically detected as a 43 kDa protein in the vacuolar membrane fractioned by sucrose density gradient centrifugation. The expression level of AtMTP1 in suspension-cultured cells was not affected by the Zn concentration in the medium. When AtMTP1 fused with green fluorescent protein was transiently expressed in protoplasts prepared from Arabidopsis suspension-cultured cells, green fluorescence was clearly observed in the vacuolar membrane. A T-DNA insertion mutant line for AtMTP1 displays enhanced sensitivity to high Zn concentrations ranging from 200 to 500 microM, but not to Zn-deficient conditions. Mesophyll cells of the mtp1-1 mutant plants grown in the presence of 500 microM Zn were degraded, suggesting that Zn at high concentrations causes serious damage to leaves and that AtMTP1 plays a crucial role in preventing this damage in plants. Thus we propose that AtMTP1 is localized in the vacuolar membrane and is involved in sequestration of excess Zn in the cytoplasm into vacuoles to maintain Zn homeostasis.

Amino Acid Sequence↗
PubMed2004

Double-counting of intracardiac electrogram during biatrial pacing.

BACKGROUND: Biatrial pacing has a significant problem with memory function that misinterprets normal sinus rhythm as atrial tachyarrhythmias and in addition estimation of the atrial pacing thresholds (biatrial and uniatrial pacing thresholds) is sometimes difficult because of small P waves. METHODS AND RESULTS: The intracardiac electrograms recorded by a pacemaker in 10 patients (age, 66.7+/-10.7 (SD) years) with implanted biatrial pacemakers were analyzed. Atrial sensing within the atrial refractory period after atrial pacing was counted in 6 of the 10 patients (timing of the double counting was 143+/-64 ms) when pacing failed in the left or right atrium. Atrial sensing within the atrial refractory period after atrial pacing disappeared when biatrial pacing was successfully performed. Atrial double-counts depend on interatrial conduction delay. The memory function of implanted pacemaker devices misinterpreted normal sinus rhythm as atrial tachyarrhythmias because of atrial double-counts. On the other hand, the biatrial pacing threshold was easily recognized using this phenomenon. CONCLUSIONS: The memory function of pacemaker devices is unreliable because of atrial double-counting during sinus rhythm in patients with biatrial pacing. However, the biatrial pacing threshold is easily checked using this phenomenon.

Aged↗
PubMed2004

Genetic analysis of chs1+ and chs2+ encoding chitin synthases from Schizosaccharomyces pombe.

To explore the function of chitin in Schizosaccharomyces pombe, we have cloned chs1+ and chs2+, encoding putative chitin synthases, based on sequences in the Sanger Centre database. The synthetic lethal phenotype of the S. cerevisiae chs1 chs2 chs3 mutant was complemented by expression of S. pombe chs1+ or chs1+, indicating that both chs1+ and chs2+ in fact encode chitin synthase. The homothallic Deltachs1 strain formed abnormal asci that contained 1, 2, or 3 spores, while the Deltachs2 strain had no noticeable phenotype. The chs1 chs2 double disruptant looked similar phenotypically to the Deltachs1 strain. The Chs2-GFP fusion protein predominantly localized at the septum after the septum was formed during vegetative growth. The level of chs2+ mRNA increased just before the septum was formed. Levels of Chs2-13Myc synthesis also changed during the cell cycle. Thus, chs1+ is required for proper spore formation, and chs2+ is perhaps involved in septum formation.

Benzenesulfonates↗
PubMed2004

A novel gene, msa1, inhibits sexual differentiation in Schizosaccharomyces pombe.

Sexual differentiation in the fission yeast Schizosaccharomyces pombe is triggered by nutrient starvation or by the presence of mating pheromones. We identified a novel gene, msa1, which encodes a 533-aa putative RNA-binding protein that inhibits sexual differentiation. Disruption of the msa1 gene caused cells to hypersporulate. Intracellular levels of msa1 RNA and Msa1 protein diminished after several hours of nitrogen starvation. Genetic analysis suggested that the function of msa1 is independent of the cAMP pathway and stress-responsive pathway. Deletion of the ras1 gene in diploid cells inhibited sporulation and in haploid cells decreased expression of mating-pheromone-induced genes such as mei2, mam2, ste11, and rep1; simultaneous deletion of msa1 reversed both phenotypes. Overexpression of msa1 decreased activated Ras1(Val17)-induced expression of mam2. Phenotypic hypersporulation was similar between cells with deletion of only rad24 and both msa1 and rad24, but simultaneous deletion of msa1 and msa2/nrd1 additively increased hypersporulation. Therefore, we suggest that the primary function of Msa1 is to negatively regulate sexual differentiation by controlling the expression of Ste11-regulated genes, possibly through the pheromone-signaling pathway.

Amino Acid Sequence↗
PubMed2002

Two glutamic acids in chitosanase A from Matsuebacter chitosanotabidus 3001 are the catalytically important residues.

Chitosanase is the glycolytic enzyme that hydrolyzes the glucosamine GlcN-GlcN bonds of chitosan. To determine the catalytically important residues of chitosanase A (ChoA) from Matsuebacter chitosanotabidus 3001, we performed both site-directed and random mutagenesis of choA, obtaining 31 mutants. These mutations indicated that Glu-121 and Glu-141 were catalytically important residues, as mutation at these sites to Ala or Asp drastically decreased the enzymatic activity to 0.1-0.3% of that of the wild type enzyme. Glu-141 mutations remarkably decreased kinetic constant k(cat) for hydrolysis of chitosan, meanwhile Glu-121 mutations decreased the activities to undeterminable levels, precluding parameter analysis. No hydrolysis of (GlcN)(6) was observed with the purified Glu-121 mutant and extremely slow hydrolysis with the Glu-141 mutant. We also found that Asp-139, Asp-148, Arg-150, Gly-151, Asp-164, and Gly-280 were important residues for enzymatic activities, although they are not directly involved in catalysis. In addition, mutation of any of the six cysteine residues of ChoA abrogated the enzymatic activity, and Cys-136 and Cys-231 were found to form a disulfide bond. In support of the significance of the disulfide bond of ChoA, chitosanase activity was impaired on incubation with a reducing agent. Thus, ChoA from M. chitosanotabidus 3001 uses two glutamic acid residues as putative catalytic residues and has at least one disulfide bond.

Bacterial Proteins↗
PubMed2002

The 14-3-3 proteins Rad24 and Rad25 negatively regulate Byr2 by affecting its localization in Schizosaccharomyces pombe.

In Schizosaccharomyces pombe, rad24 and rad25 have been identified to be homologous to mammalian 14-3-3 genes and found to be involved in many cellular events, including checkpoint and meiosis. In the present study, we present evidences that Rad24 and Rad25 act as negative regulators of Byr2 (mitogen-activated protein kinase [MAPK] kinase kinase). Overexpression of rad24 or rad25 reduced mating and sporulation in homothallic wild-type cells. In contrast, the mating and sporulation efficiency of rad24- or rad25-null cells was higher than that of wild-type cells. Deletion of rad24 or rad25 increased sporulation efficiency in ras1-null diploid cells but not in byr2-, ste4-, byr1-, and spk1-null cells. Rad24 and Rad25 had no effect on the activity of constitutively active Byr1(S214DT218D). Rad24 and Rad25 bound to both the N-terminal and the C-terminal domains of Byr2 when these bacterially expressed proteins were examined. The formation of complexes in vivo between Byr2 and either Rad24 or Rad25 was also confirmed by immunocoprecipitation. Furthermore, we showed negative regulation of Byr2 by Rad25, by monitoring the mRNA level of mam2, which is regulated by both the Ras1/MAPK pathway and ste11, in various combinations of mutants. In addition, the cellular localization of Byr2 in living cells was observed by using fusion to green fluorescent protein. Byr2 was mainly localized in the cytoplasm during vegetative growth and then concentrated at the plasma membrane in response to nitrogen starvation. Deletion of rad24 or rad25 fastened the timing of Byr2 translocation. Our results are consistent with the hypothesis that one of the roles of 14-3-3 is to keep Byr2 in the cytoplasm and to affect the timing of Byr2 translocation in response to sexual developmental signal.

14-3-3 Proteins↗
PubMed1998

Identification of a CAP (adenylyl-cyclase-associated protein) homologous gene in Lentinus edodes and its functional complementation of yeast CAP mutants.

The adenylyl-cyclase-associated protein, CAP, was originally identified in yeasts as a protein that functions in both signal transduction and cytoskeletal organization. This paper reports the identification of a cDNA and genomic DNA that encodes a CAP homologue from the mushroom Lentinus edodes. The L. edodes cap gene contains eight introns and an ORF encoding a 518 amino acid protein. The L. edodes CAP is 35.5% and 40.9% identical at the amino acid level with Saccharomyces cerevisiae CAP and Schizosaccharomyces pombe CAP, respectively. The C-terminal domain shows greater homology (39-46% identity) with yeast CAPs than does the N-terminal domain (27-35% identity). Southern blotting and Northern blotting results suggest that L. edodes cap is a single-copy gene and uniformly expressed. Expression of the L. edodes CAP in both Schiz. pombe and Sacch. cerevisiae complemented defects associated with the loss of the C-terminal domain function of the endogenous CAP. By using a yeast two-hybrid assay, an interaction was demonstrated between the L. edodes CAP and Schiz. pombe actin. This result and the functional complementation test indicate that CAP from L. edodes has a conserved C-terminal domain function.

Adaptor Proteins, Signal Transducing↗