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T Ichimura

Publications and source records attributed to T Ichimura.

At least 37 records · Page 2Linked to original sources
PubMed1999

In vitro nuclear assembly with affinity-purified nuclear envelope precursor vesicle fractions, PV1 and PV2.

Nuclear envelope precursor vesicles were affinity purified from a Xenopus egg extract by a chromatin binding method. Vesicles bound to chromatin at 4 degrees C were dissociated with a high salt buffer and further fractionated into nuclear envelope precursor vesicle fractions 1 (PV1) and 2 (PV2) by differential centrifugation. PV1 contained larger vesicles. When chromatin was incubated in a Xenopus egg cytosol fraction supplemented with PV1, vesicles bound to chromatin, fused with each other, formed a bilayered nuclear envelope, and assembled into spherical small nuclei. However, the thus assembled nuclei did not grow to the normal size. Nuclear pore complexes were not found on the thus assembled nuclei. On the other hand, PV2 contained smaller vesicles. PV2 vesicles bound to chromatin, fused little with each other in the Xenopus egg cytosol fraction, and no nuclei were assembled. When PV1 supplemented with PV2 was used for the nuclear assembly reaction, the assembled nuclei grew to the normal size. Nuclear pore complexes existed in the thus assembled nuclear envelopes. These results suggested that 1) two vesicle populations, PV1 and PV2, are necessary for the assembly of normal sized nuclei, 2) PV1 contains a chromatin targeting molecule(s) and membrane fusion machinery, 3) PV2 contains a chromatin targeting molecule(s) and a molecule(s) necessary for nuclear pore complex assembly, and 4) PV1 has the ability to assemble a nuclear membrane, and PV2 is necessary for the assembly of nuclear pore complexes and for nuclei to grow to the normal size. An in vitro nuclear assembly system constituted with affinity-purified vesicle fractions, PV1 and PV2, was established.

Animals↗
PubMed1999

Molecular shape and ATP binding activity of rat p50, a putative mammalian homologue of RuvB DNA helicase.

Based on partial amino acid sequences of p50 purified from a high-salt buffer extract of a rat liver nuclear matrix fraction, p50 cDNA was cloned and sequenced, and its amino acid sequence was predicted. The sequence contained helicase motifs, and showed homology with RuvB DNA helicase of Thermus thermophilus and an open reading frame for an unknown 50.5 k protein of Saccharomyces cerevisiae. p50 was expressed as a GST-fusion protein and antiserum against the protein was generated. p50 was localized to the nuclear matrix by cell fractionation and immunoblotting. p50 bound to ATP-Sepharose beads. Ultracentrifugation and gel filtration analyses showed that p50 in rat liver and Xenopus egg mitotic extracts exists as large complexes corresponding to 697 k and 447 k, respectively. A 50 k protein reactive with p50 antibodies was detected not only in rat liver nuclei, but also in a Xenopus egg cytoplasm fraction and a S. cerevisiae extract. This suggests that this putative DNA helicase is present in a wide variety of species ranging from yeast to mammals.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1999

Molecular cloning of mouse p47, a second group mammalian RuvB DNA helicase-like protein: homology with those from human and Saccharomyces cerevisiae.

A 47k protein (p47) in a high-salt buffer extract of a rat liver nuclear matrix fraction was purified by means of a wheat germ agglutinin affinity column, reversed phase HPLC, and SDS-PAGE, and partial amino acid sequences were analyzed. Based on these sequences, the mouse cDNA of the protein was cloned and sequenced, and its amino acid sequence was deduced. Mouse p47 consists of 463 amino acid residues with a molecular weight of 51,112. The amino acid sequences of human and Saccharomyces cerevisiae p47s were also deduced from the nucleotide sequences of "expressed sequence tag" fragments and genomic DNA, respectively. These sequences contain helicase motifs and show homology to bacterial RuvB DNA helicases acting in homologous recombination. They also show homology with the putative mammalian helicases p50/TIP49 and RUVBL1. Comparison of the amino acid sequences of p47 group proteins and those of p50/TIP49 group proteins revealed the p47 group proteins to comprise a group distinct from the p50/TIP49 proteins. Ultracentrifugation and gel filtration analyses showed that p47 in the rat liver cytosol fraction exists as large complexes of 697k.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1999

Molecular cloning and splicing isoforms of mouse p144, a homologue of CA150.

We previously characterized p144 bearing N-acetylglucosamine residues in a rat liver nuclear matrix fraction. Based on partial amino acid sequences of rat p144, mouse p144 cDNA was cloned and sequenced, and its amino acid sequence was predicted. The sequence revealed that p144 is a rat homologue of CA150, which is a transcription factor involved in Tat-activated human immunodeficiency virus type 1 transcription. The reported human CA150 consists of 1098 amino acids and has a leucine zipper-like motif in its carboxyl-region. However, a clone of mouse p144 cDNA encoded a CA150 consisting of 1,034 amino acids. The mouse CA150 was shorter by 64 amino acids than hitherto known human CA150 and lacked the leucine zipper-like motif. We designated the longer and shorter CA150 species as CA150a and CA150b, respectively. The partial nucleotide sequences of other mouse p144 cDNA clones were examined and it was found that some clones encode CA150a having a leucine zipper-like motif. It was suggested that CA150a and CA150b are splicing isoforms. All rat and mouse tissues examined contained transcripts for both CA150a and CA150b. Both transcripts were detected in human blood and Jurkat cells as well as mouse CD4(+) T-cells, which are the HIV-1-sensitive counterpart in humans.

Amino Acid Sequence↗
PubMed1999

IgG transcytosis is accelerated but its pathway is not disrupted by Brefeldin-A.

To see the action of Brefeldin-A (Bref-A) to transcytosis of the immunoglobulin G (IgG), we have examined the early stage of endocytosis and intracellular transport of IgG and transferrin (Tf) in epithelial cells of the rat yolk sac in culture with and without Bref-A. In the absence of Bref-A, the endocytosed IgG and Tf appeared in large apical endosomes during the first 5-15 min. At 15 min Tf-recycling endosomes and IgG-transferring vesicles also appeared in the apical or basolateral cytoplasm, respectively. At 15 min in the presence of Bref-A, clouds of Tf-recycling endosomes accumulated in the apical cytoplasm, while distinct IgG-transferring vesicles were distributed further to the basolateral cytoplasm. This differential action of Bref-A to IgG-transferring vesicles and Tf-recycling vesicles suggested an unexplored mechanism of sorting and basolateral transport of IgG.

Animals↗
PubMed1999

HIV-1 protease inhibition and anti-HIV effect of natural and synthetic water-soluble lignin-like substances.

Water-soluble lignin extracted from natural sources and dehydrogenated polymers of p-coumaric acid and ferulic acid inhibited HIV-1 protease activity. The dehydrogenated polymers, which are thought to be model compounds for lignin, were synthesized and fractionated into four ranges of molecular mass by ultra-filtration: i.e., over 30 kDa, 30-10 kDa, 10-1 kDa and 1 kDa-500 Da. All of these fractions had HIV-1 protease inhibitory activity. The anti-HIV-1 effect of the smallest mass fractions of the dehydrogenated polymers (1 kDa-500 Da) was also tested, and it was found that these fractions inhibited the replication of HIV-1 in MT-4 cells.

Cell Line↗
PubMed1999

Behavioral factors predicting serum cotinine concentrations of male smokers in a Japanese community.

Cigarette-derived toxic substances are inhaled along with the nicotine that is absorbed to satisfy the smoker's physical demand. Therefore, serum cotinine, a metabolite of nicotine, may be considered to be an indirect marker of absorption of the other toxic substances from smoking. However, few studies have examined factors related to serum cotinine concentrations in natural settings. The authors, therefore, have studied relations among patterns of smoking behavior and serum cotinine concentrations of community residents. Subjects were 60 smoking men living in the town of Yamasaki, Hyogo Prefecture, Japan. Number of daily cigarettes, depth of inhalation, hours from the last cigarette smoked and the total nicotine tolerance score were significantly, while neither nicotine-yields nor butt length of a discarded cigarette was correlated with serum cotinine concentrations. Multiple regression analysis confirmed that depth of inhalation and hours from the last cigarette smoked were independently significant. This finding suggests that attention only to self-reported daily number of cigarettes smoked may not be sufficient to detect smokers who are actually at high risk. Healthcare workers should also pay attention to smokers' patterns of smoking, particularly depth of inhalation.

Adult↗
PubMed1998

Kidney injury molecule-1 (KIM-1), a putative epithelial cell adhesion molecule containing a novel immunoglobulin domain, is up-regulated in renal cells after injury.

We report the identification of rat and human cDNAs for a type 1 membrane protein that contains a novel six-cysteine immunoglobulin-like domain and a mucin domain; it is named kidney injury molecule-1 (KIM-1). Structurally, KIM-1 is a member of the immunoglobulin gene superfamily most reminiscent of mucosal addressin cell adhesion molecule 1 (MAdCAM-1). Human KIM-1 exhibits homology to a monkey gene, hepatitis A virus cell receptor 1 (HAVcr-1), which was identified recently as a receptor for the hepatitis A virus. KIM-1 mRNA and protein are expressed at a low level in normal kidney but are increased dramatically in postischemic kidney. In situ hybridization and immunohistochemistry revealed that KIM-1 is expressed in proliferating bromodeoxyuridine-positive and dedifferentiated vimentin-positive epithelial cells in regenerating proximal tubules. Structure and expression data suggest that KIM-1 is an epithelial cell adhesion molecule up-regulated in the cells, which are dedifferentiated and undergoing replication. KIM-1 may play an important role in the restoration of the morphological integrity and function to postischemic kidney.

Amino Acid Sequence↗
PubMed1998

Correlation between the growth of uterine leiomyomata and estrogen and progesterone receptor content in needle biopsy specimens.

OBJECTIVE: To examine the relationship between estrogen receptor (ER) and progesterone receptor (PR) content in needle biopsy specimens and the growth of uterine leiomyomata after biopsy. DESIGN: Prospective clinical study. SETTING: University teaching hospital. PATIENT(S): Thirty-one women with uterine leiomyomata and a normal menstrual cycle. INTERVENTION(S): Transcervical needle biopsy of uterine leiomyomata. MAIN OUTCOME MEASURE(S): The relationships between histologic features (smooth muscle content, immunohistochemical expression of ER and PR) and the percent increase over a 12-month observation period in the volume of the largest myoma nodule measured by magnetic resonance imaging were analyzed. RESULT(S): Both the density and intensity of immunohistochemical staining of PRs in uterine leiomyoma tissue showed significant positive correlation with leiomyoma growth. CONCLUSION(S): The growth of uterine leiomyomata can be determined by histologic and immunohistochemical analysis of needle biopsy specimens from uterine leiomyomata.

Adult↗
PubMed1998

Mucin-producing pancreatic tumors: comparison of MR cholangiopancreatography with endoscopic retrograde cholangiopancreatography.

PURPOSE: To compare magnetic resonance cholangiopancreatography (MRCP) with endoscopic retrograde cholangiopancreatography (ERCP) in the detection of mucin-producing pancreatic tumors. MATERIALS AND METHODS: The authors retrospectively reviewed MRCP and ERCP images obtained in 28 patients with mucin-producing pancreatic tumors. Detectability of the pancreatic duct and its branches, intraductal cystic lesions, and intracystic nodules or septa was assessed. RESULTS: MRCP depicted cystic dilated ductal branches significantly better than did ERCP (P < .001). The difference in the number of nodules or septa detected with MRCP compared with ERCP was not significant. MRCP, however, simultaneously showed not only the main pancreatic duct but also the cystic lesions; this was not always possible with ERCP. CONCLUSION: MRCP appears to be more effective and less invasive than ERCP to evaluate changes in the size and extent of tumors and to determine if new lesions appear, as well as to follow up mucin-producing pancreatic tumors.

Adenocarcinoma, Papillary↗
PubMed1998

An optimal cutoff point of expired-air carbon monoxide levels for detecting current smoking: in the case of a Japanese male population whose smoking prevalence was sixty percent.

An optimal cutoff point of expired-air carbon monoxide (Ex-CO) for detecting smokers should be determined in terms of its sensitivity and specificity and the prevalence of smoking in the target population. The purpose of this study is to determine the optimal cutoff point of Ex-CO for detecting smoking males in a Japanese community whose smoking prevalence was over 50%. Among free-living residents in a rural population, "true smokers" determined by presence of cotinine in serum were 61% (n = 94). When Ex-CO at 7 ppm or over differentiated "smokers" from "non-smokers", sensitivity and specificity for detecting smokers was 0.93 and 0.95, respectively, which comprised the best Youden's index. This setting also produced the minimum percentage of misclassified cases. In conclusion, 7 ppm of Ex-CO, which is exceptionally low value relative to the western standard, appears to be the most optimal cutoff point for a survey in a population with such high smoking prevalence.

Breath Tests↗
PubMed1998

[Transcytosis of immunoglobulin IgG].

Recent studies on cellular mechanism of transmission of maternal IgG are briefly reviewed. The mechanism has been studied in rodent intestine and yolk sac, where the role of neonate Fc receptors (FcRn) in the transcellular transport of IgG is now well understood. FcRn binds IgG on the cell surface (intestine) or in endosomes (yolk sac) at around pH 6.0. The IgG-FcRn complex is sorted into vesicles from the apical endosome, and transported basolaterally to release IgG extracellularly. In the human placenta, Fc gamma RIII and MHCI-related human Fc receptors (hFcRn) are expressed in the syncytiotrophoblast and are involved in the specific binding and transcytosis of IgG. Small GTP-binding proteins rab4 and/or rab5A are immunohistochemically localized on recycling and/or sorting endosomes, suggesting their involvement in forming vesicles transporting the IgG-FcR complex. Several other GTP-binding proteins and SNAREs are assumed to regulate this process, but remain to be identified. The cellular mechanism of IgG transcytosis, including endocytosis, sorting, fusion and exocytosis may be elucidated in terms of regulator proteins in near future.

Animals↗
PubMed1997

Methylmercury alters the tyrosination status of tubulin in the brains of acutely intoxicated rats.

Tyrosination/detyrosination, a post-translational modification at the carboxyl terminus of alpha-tubulin, was investigated in the brain cytosol fraction of rats treated with methylmercury (MeHg) chloride (10 mg/kg per day, for 7 days). The amount of detyrosinated tubulin species, determined as the incorporation of 14C-tyrosine at the carboxyl-terminal end of alpha-tubulin, was significantly decreased throughout the experimental period of MeHg intoxication. Furthermore, the activity of tubulin-tyrosine ligase, as well as the amounts of tyrosinatable tubulin determined and calculated by a method involving pancreatic carboxypeptidase A, also decreased in the latent and symptomatic periods. Tubulin-tyrosine carboxypeptidase activity did not change during the MeHg intoxication. The total amounts of alpha- and beta-tubulins, as determined by densitometry and immunoblotting, did not show significant changes during the intoxication. These results suggest that MeHg treatment may produce perturbation of cellular activities associated with the tubulin/microtubule system by altering the tyrosination status of tubulin in the rat brain.

Animals↗
PubMed1997

14-3-3 protein binds to insulin receptor substrate-1, one of the binding sites of which is in the phosphotyrosine binding domain.

Insulin binding to its receptor induces the phosphorylation of cytosolic substrates, insulin receptor substrate (IRS)-1 and IRS-2, which associate with several Src homology-2 domain-containing proteins. To identify unique IRS-1-binding proteins, we screened a human heart cDNA library with 32P-labeled recombinant IRS-1 and obtained two isoforms (epsilon and zeta) of the 14-3-3 protein family. 14-3-3 protein has been shown to associate with IRS-1 in L6 myotubes, HepG2 hepatoma cells, Chinese hamster ovary cells, and bovine brain tissue. IRS-2, a protein structurally similar to IRS-1, was also shown to form a complex with 14-3-3 protein using a baculovirus expression system. The amount of 14-3-3 protein associated with IRS-1 was not affected by insulin stimulation but was increased significantly by treatment with okadaic acid, a potent serine/threonine phosphatase inhibitor. Peptide inhibition experiments using phosphoserine-containing peptides of IRS-1 revealed that IRS-1 contains three putative binding sites for 14-3-3 protein (Ser-270, Ser-374, and Ser-641). Among these three, the motif around Ser-270 is located in the phosphotyrosine binding domain of IRS-1, which is responsible for the interaction with the insulin receptor. Indeed, a truncated mutant of IRS-1 consisting of only the phosphotyrosine binding domain retained the capacity to bind to 14-3-3 protein in vivo. Finally, the effect of 14-3-3 protein binding on the insulin-induced phosphorylation of IRS-1 was investigated. Phosphoamino acid analysis revealed that IRS-1 coimmunoprecipitated with anti-14-3-3 antibody to be weakly phosphorylated after insulin stimulation, on tyrosine as well as serine residues, compared with IRS-1 immunoprecipitated with anti-IRS-1 antibody. Thus, the association with 14-3-3 protein may play a role in the regulation of insulin sensitivity by interrupting the association between the insulin receptor and IRS-1.

14-3-3 Proteins↗
PubMed1997

The 14-3-3 protein binds its target proteins with a common site located towards the C-terminus.

The 14-3-3 protein family binds a variety of proteins in cell-signaling pathways, but the structural elements necessary for the ligand binding are poorly understood. Here we demonstrate that the 'box-1' region, which spans residues 171-213 in the eta-isoform and was previously identified as the binding site of 14-3-3 to the phosphorylated tryptophan hydroxylase, plays a critical role in the interaction with many target proteins. Using a series of truncated 14-3-3 mutants, we show that the mutant 167-213 carrying box-1 binds bacurovirus-expressed Raf-1 and Bcr protein kinases to the similar extent as the full-length 14-3-3 in a phosphorylation-dependent manner, while the mutants lacking this region abolish the binding activity. Furthermore, the box-1 region also appears essential for binding of 14-3-3 to more than 40 phosphoproteins found in the brainstem extract. These results suggest that the box-1 region, consisting of helices 7 and 8 in the tertiary structure, is a common structural element whereby the 14-3-3 protein binds many, if not all, target proteins.

14-3-3 Proteins↗
PubMed1997

Apical tubular network in the rat kidney proximal tubule cells studied by thick-section and scanning electron microscopy.

The apical cytoplasm of several absorbing epithelia contains well-developed apical tubules (AT) which contribute to membrane recycling from endocytic vacuoles to the apical cell membrane. In this study, we examined three-dimensional structures of the AT in rat kidney proximal tubule cells by transmission and scanning electron microscopy. In thin sections, the AT appeared as straight tubules with a rather constant diameter (70-90 nm), but others were curved and, occasionally, branching. No AT were labeled with the marker for the external cell surface (ruthenium red) or exhibited histochemical enzyme activity for lysosomal hydrolase (acid phosphatase). After intravenous injection of horseradish peroxidase, it was absorbed in the kidney proximal tubule cells and the AT were labeled with HRP reaction products. Stereo-viewing of the labeled AT in thick sections revealed that they formed an interconnected tubular network. Scanning electron microscopy allowed a three-dimensional view of the AT, in which a network of branching and anastomosing tubules was revealed. These observations indicate that the AT are intracellular endosomal compartments which form an extensive tubular network in the apical cytoplasm. The possibility that this apical tubular network serves as a large membrane store for membrane recycling is discussed.

Acid Phosphatase↗