Biomedical subjects

T Haraguchi

Publications and source records attributed to T Haraguchi.

At least 19 recordsLinked to original sources

PubMed2006

SWI/SNF complex is essential for NRSF-mediated suppression of neuronal genes in human nonsmall cell lung carcinoma cell lines.

Mammalian chromatin remodeling factor, SWI/SNF complex contains a single molecule of either Brm or BRG1 as the ATPase catalytic subunit. Here, we show that the SWI/SNF complex forms a larger complex with neuron-restrictive silencer factor (NRSF) and its corepressors, mSin3A and CoREST, in human nonsmall cell lung carcinoma cell lines. We also demonstrate that the strong transcriptional suppression of such neuron-specific genes as synaptophysin and SCG10 by NRSF in these non-neural cells requires the functional SWI/SNF complex; these neuronal genes were elevated in cell lines deficient in both Brm and BRG1, whereas retrovirus vectors expressing siRNAs targeting integral components of SWI/SNF complex (Brm/BRG1 or Ini1) induced expression of these neuronal genes in SWI/SNF-competent cell lines. In cell lines deficient in both Brm and BRG1, exogenous Brm or BRG1 suppressed expression of these neuronal genes in an ATP-dependent manner and induced efficient and specific deacetylation of histone H4 around the NRSF binding site present in the synaptophysin gene by a large complex containing the recruited functional SWI/SNF complex. Patients with Brm/BRG1-deficient lung carcinoma have been reported to carry poor prognosis; derepression of NRSF-regulated genes including these neuron-specific genes could contribute to enhance tumorigenicity and also would provide selective markers for Brm/BRG1-deficient tumors.

Base Sequence↗

PubMed2001

Lead content of brain tissue in diffuse neurofibrillary tangles with calcification (DNTC): the possibility of lead neurotoxicity.

Diffuse neurofibrillary tangles with calcification (DNTC) is a form of presenile dementia, characterized pathologically by fronto-temporal atrophy with neurofibrillary tangles (NFTs), neuropil threads and Fahr-type calcification, in which no senile plaques are observed. As already noted, chronic exposure to lead (Pb) might be one of the etiological factors of Fahr-type calcification. Until now, there have been no reports in which Pb concentration has been quantified in DNTC brains. We examined the concentration of Pb in fresh-frozen brain tissue and in 10% formalin-fixed brain tissue from six cases of DNTC, four cases of Alzheimer's disease, and in nine non-demented elderly controls by flameless atomic absorption spectrometry, and demonstrated a high concentration of Pb in DNTC brains. Although it remains unclear how these findings are related to the formation of NFTs, they suggest that Pb neurotoxicity may be involved in the pathogenesis of DNTC.

Aged↗

PubMed2001

CENP-H, a constitutive centromere component, is required for centromere targeting of CENP-C in vertebrate cells.

CENP-H has recently been discovered as a constitutive component of the centromere that co-localizes with CENP-A and CENP-C throughout the cell cycle. The precise function, however, remains poorly understood. We examined the role of CENP-H in centromere function and assembly by generating a conditional loss-of-function mutant in the chicken DT40 cell line. In the absence of CENP-H, cell cycle arrest at metaphase, consistent with loss of centromere function, was observed. Immunocytochemical analysis of the CENP-H-deficient cells demonstrated that CENP-H is necessary for CENP-C, but not CENP-A, localization to the centromere. These findings indicate that centromere assembly in vertebrate cells proceeds in a hierarchical manner in which localization of the centromere-specific histone CENP-A is an early event that occurs independently of CENP-C and CENP-H.

Amino Acid Sequence↗

PubMed2001

Identification of human endomucin-1 and -2 as membrane-bound O-sialoglycoproteins with anti-adhesive activity.

Using a signal sequence trap method and database search, we identified a series of human cDNAs encoding two structurally related type I membrane proteins of approximately 25 kDa with multiple glycosylation motifs. These genes, termed endomucin-1/-2, are expressed in several human tissues including heart, kidney, and lung. Exogenously expressed human endomucin-1/-2 proteins were modified into 80-120 kDa glycoproteins, which were susceptible to O-sialoglycoprotein endopeptidase digestion. Transient overexpression of endomucin-1/-2 reduced the number of adhesion plaques and reduced cell attachment to the substrate. This phenotype was suppressed by laminin or the protein kinase inhibitor staurosporine. Our findings suggest that human endomucin-1/-2 negatively regulate cell adhesion to the extracellular matrix.

Amino Acid Sequence↗

PubMed2001

Diffuse neurofibrillary tangles with calcification (a form of dementia): X-ray spectrometric evidence of lead accumulation in calcified regions.

Diffuse neurofibrillary tangles with calcification (DNTC) is a form of slowly progressive dementia in which no senile plaques are observed. The calcification is one of the most characteristic features of DNTC. We examined the elemental content of certain mineral deposits (lead, magnesium, phosphorus, calcium, iron, copper and zinc) in the calcified and non-calcified regions of eight cases of DNTC, five cases of Alzheimer's disease (AD) and in eight non-demented elderly controls. The study was performed using a combination of scanning electron microscopy and X-ray spectrometry on 10% formalin-fixed brain tissue. A marked abundance of calcium and phosphorus was observed in the calcified regions of DNTC and non-DNTC brains. Although no lead was observed in the non-calcified regions of DNTC and in non-DNTC brains, traces of lead were detected exclusively in the calcified regions of DNTC brains. The implications and possible significance of the lead accumulation in DNTC brains are discussed.

Aged↗

PubMed2001

Familial amyotrophic lateral sclerosis with a novel Leu126Ser mutation in the copper/zinc superoxide dismutase gene showing mild clinical features and lewy body-like hyaline inclusions.

BACKGROUND: Mutations in the SOD1 gene are responsible for approximately 25% of all familial amyotrophic lateral sclerosis (ALS) cases. However, the correlation between the clinical and pathological features and the various SOD1 gene mutations has not been well characterized. OBJECTIVES: To screen the SOD1 gene in search of potential mutations and to obtain clinical and pathological data for 2 Japanese families with ALS. DESIGN: Clinical histories and neurological findings, gross and microscopic pathological features, and DNA analysis of the SOD1 gene. RESULTS: The 2 families with ALS showed a novel missense mutation in the SOD1 gene, which was heterozygous for point mutation TTG to TCG, causing substitution of leucine for serine at codon 126 (Leu126Ser) in exon 5. Clinically, patients showed slower disease progression and lack of upper motor neuron signs. Neuropathologically, the autopsied patient showed the form of familial ALS with posterior column involvement, and the pontocerebellar tract and the dentate nuclei of the cerebellum were also involved. Furthermore, abundant Lewy body-like hyaline inclusions were observed in the affected motor and nonmotor neurons. CONCLUSIONS: Familial ALS with a novel Leu126Ser mutation in the SOD1 gene showed mild clinical features and lack of upper motor neuron signs. We believe that Leu126Ser might be associated with the clinical features and that the mutation site in the SOD1 gene and disease duration might be associated with the formation of Lewy body-like hyaline inclusions.

Adult↗

PubMed2001

Plaque-like structures and arteriosclerotic changes in "diffuse neurofibrillary tangles with calcification" (DNTC).

"Diffuse neurofibrillary tangles with calcification" (DNTC) is a rare form of slowly progressive dementia characterized by temporal or fronto-temporal atrophy with neuronal loss and astrocytosis, neurofibrillary tangles and Fahr-type calcification, but no senile plaques in the cerebral cortex. In patients with DNTC, we detected a novel histopathological abnormality that we termed "plaque-like structures" (PLS). PLS appeared as oval, slightly eosinophilic masses of up to 100 microns in diameter. With methenamine silver stain, the PLS were argyrophilic, and thread-like structures were observed in and around them. Most PLS were observed in deep layers of the cortex and subcortical white matter, and were accompanied by small vessels. They were intimately associated with the small-vessel walls and astrocytes. They were composed of two types of fibers. The first type comprised straight and loosely interwoven fibers about 25-30 nm in diameter, while the other type evoked tangles. These structures have not been found in other neurodegenerative diseases, including Alzheimer's disease. In addition, to evaluate hyaline arteriosclerosis in DNTC, we examined sclerotic changes of the medullary arteries and assessed white matter lesions in affected patients. In three of four patients with DNTC, sclerosis of the medullary arteries was significantly more extensive than in age-matched controls. In all four patients, the severity of white matter lesions was graded as moderate or severe in the temporal lobe and as mild or moderate in the frontal lobe. Arteriosclerotic changes and white matter lesions can occur without hypertension and beta amyloid deposits in DNTC.

Amyloid beta-Peptides↗

PubMed2001

A conserved protein, Nuf2, is implicated in connecting the centromere to the spindle during chromosome segregation: a link between the kinetochore function and the spindle checkpoint.

The centromere is crucial for the proper segregation of chromosomes in all eukaryotic cells. We identified a centromeric protein, Nuf2, which is conserved in fission yeast, human, nematode, and budding yeast. Gene disruption of nuf2+ in the fission yeast Schizosaccharomyces pombe caused defects in chromosome segregation and the spindle checkpoint: the mitotic spindle elongated without segregating the chromosomes, indicating that spindle function was compromised, but that this abnormality did not result in metaphase arrest. Certain nuf2 temperature-sensitive mutations, however, caused metaphase arrest with condensed chromosomes and a short spindle, indicating that, while these mutations caused abnormalities in spindle function, the spindle checkpoint pathway remained intact. Metaphase arrest in these cells was dependent on the spindle checkpoint component Mad2. Interestingly, Nuf2 disappeared from the centromere during meiotic prophase when centromeres lose their connection to the spindle pole body. We propose that Nuf2 acts at the centromere to establish a connection with the spindle for proper chromosome segregation, and that Nuf2 function is also required for the spindle checkpoint.

Amino Acid Sequence↗

PubMed2001

Distinct functional domains in emerin bind lamin A and DNA-bridging protein BAF.

Loss of emerin, a lamin-binding nuclear membrane protein, causes Emery-Dreifuss muscular dystrophy. We analyzed 13 site-directed mutations, and four disease-causing mutations that do not disrupt emerin stability or localization. We show that emerin binds directly to barrier-to-autointegration factor (BAF), a DNA-bridging protein, and that this binding to BAF requires conserved residues in the LEM-motif of emerin. Emerin has two distinct functional domains: the LEM-domain at the N-terminus, which mediates binding to BAF, and a second functional domain in the central region, which mediates binding to lamin A. Disease mutation Delta95-99 mapped to the lamin-binding domain and disrupted lamin A binding in vitro. Two other disease-linked residues, Ser54 and Pro183, mapped outside the BAF and lamin-binding domains, suggesting that emerin may have additional functional domains relevant to disease. The disease-linked emerin proteins all remained active for binding to BAF, both in vitro and in vivo, suggesting that disease can result from the loss of specific molecular interactions between emerin and either lamin A or putative novel partner(s). The demonstration that emerin binds directly to BAF, coupled to similar results for LAP2, provides proof in principle that all LEM-domain nuclear proteins can interact with BAF, with interesting implications for chromatin attachment to the nuclear envelope.

Amino Acid Sequence↗

PubMed2001

BAF is required for emerin assembly into the reforming nuclear envelope.

Mutations in emerin cause the X-linked recessive form of Emery-Dreifuss muscular dystrophy (EDMD). Emerin localizes at the inner membrane of the nuclear envelope (NE) during interphase, and diffuses into the ER when the NE disassembles during mitosis. We analyzed the recruitment of wildtype and mutant GFP-tagged emerin proteins during nuclear envelope assembly in living HeLa cells. During telophase, emerin accumulates briefly at the 'core' region of telophase chromosomes, and later distributes over the entire nuclear rim. Barrier-to-autointegration factor (BAF), a protein that binds nonspecifically to double-stranded DNA in vitro, co-localized with emerin at the 'core' region of chromosomes during telophase. An emerin mutant defective for binding to BAF in vitro failed to localize at the 'core' in vivo, and subsequently failed to localize at the reformed NE. In HeLa cells that expressed BAF mutant G25E, which did not show 'core' localization, the endogenous emerin proteins failed to localize at the 'core' region during telophase, and did not assemble into the NE during the subsequent interphase. BAF mutant G25E also dominantly dislocalized LAP2beta and lamin A from the NE, but had no effect on the localization of lamin B. We conclude that BAF is required for the assembly of emerin and A-type lamins at the reforming NE during telophase, and may mediate their stability in the subsequent interphase.

Amino Acid Sequence↗

PubMed2001

Mitotic specific phosphorylation of serine-1212 in human DNA topoisomerase IIalpha.

It is known that topoisomerase IIalpha is phosphorylated by several kinases. To elucidate the role of phosphorylation of topoisomerase IIalpha in the cell cycle, we have examined the cell cycle behavior of phosphorylated topoisomerase IIalpha in HeLa cells using antibodies against several phospho-oligopeptides of this enzyme. Here we demonstrate that serine1212 in topoisomerase IIalpha is phosphorylated only in the mitotic phase. Using an antibody against an oligopeptide containing phosphoserine-1212 in topoisomerase IIalpha (PS1212), subcellular localization of topoisomerase IIalpha phosphorylated at serine1212 was examined by indirect immunofluorescence staining, and compared with that of overall topoisomerase IIalpha. Serine1212-phosphorylated topoisomerase IIalpha was localized specifically on mitotic chromosomes, but not on interphase chromosomes; this result contrasts with overall topoisomerase IIalpha which was observed on chomosomes in both interphase and mitosis. Serine1212-phosphorylated topoisomerase lIalpha first appeared on chromosome arms in prophase, became concentrated on the centromeres in metaphase, and disappeared in early telophase. In addition, ICRF-193, a catalytic inhibitor of topoisomerase II, prevented accumulation of serine1212-phosphorylated topoisomerase IIalpha at the centromeres. These results indicate that serine1212 of topoisomerase IIalpha is phosphorylated specifically during mitosis, and suggest that the serine1212-phosphorylated topoisomerase IIalpha acts on resolving topological constraint progressively from the chromosome arm to the centromere during metaphase chromosome condensation.

Antibodies, Monoclonal↗

PubMed2000

Tau pathology in diffuse neurofibrillary tangles with calcification (DNTC): biochemical and immunohistochemical investigation.

Diffuse neurofibrillary tangles with calcification (DNTC) represents a primary and sporadic presenile dementia that is characterized by temporal or fronto-temporal atrophy with diffuse neurofibrillary tangles (NFTs), neuropil threads and Fahr-type calcification without senile plaques. We examined the tau pathology in five autopsy cases of DNTC by immunoblotting and immunohistochemistry using phosphorylation-dependent and -independent anti-tau antibodies. The pattern of staining for different epitopes of beta-amyloid (A beta) was also investigated. NFTs were immunopositive with all the anti-tau antibodies used in this study. On the immunoblots, sarkosyl-insoluble tau appeared as three major bands of 60, 64 and 68 kDa, and as a minor band at 72 kDa. The majority of extracellular NFTs were weakly immunopositive only with the antibody recognizing the 40 carboxyl-terminal of A beta in DNTC. These results suggest that Alzheimer's disease-like tau pathology could exist independently of A beta deposits in DNTC.

Aged↗

PubMed2000

Tau-negative astrocytic star-like inclusions and coiled bodies in dementia with Lewy bodies.

To evaluate glial lesions in cases of dementia with Lewy bodies (DLB), we studied the brains of four patients with DLB. Astrocytic star-like inclusions, which resembled tufted astrocytic fibrillary tangles in shape, were found in the cortex of two of these cases. In addition, coiled bodies were found in the white matter of the cerebrum in two cases. The astrocytic star-like inclusions were immunohistochemically negative for tau protein, ubiquitin and alpha-synuclein. The coiled bodies were immunohistochemically negative for tau protein but immunopositive for ubiquitin and alpha-synuclein. These results suggest that in DLB a primary degenerative process takes place in both glial cells and neurons.

Aged↗

PubMed2000

Large-scale screening of intracellular protein localization in living fission yeast cells by the use of a GFP-fusion genomic DNA library.

BACKGROUND: Intracellular localization is an important part of the characterization of a gene product. In an attempt to search for genes based on the intracellular localization of their products, we constructed a green fluorescent protein (GFP)-fusion genomic DNA library of S. pombe. RESULTS: We constructed the S. pombe GFP-fusion genomic DNA library by fusing, in all three reading frames, random fragments of genomic DNA to the 5' end of the GFP gene in such a way that expression of potential GFP-fusion proteins would be under the control of the own promoters contained in the genomic DNA fragments. Fission yeast cells were transformed with this plasmid library, and microscopic screening of 49 845 transformants yielded 6954 transformants which exhibited GFP fluorescence, of which 728 transformants showed fluorescence localized to distinct intracellular structures such as the nucleus, the nuclear membrane, and cytoskeletal structures. Plasmids were isolated from 516 of these transformants, and a determination of their DNA sequences identified 250 independent genes. The intracellular localizations of the 250 GFP-fusion constructs was categorized as an image database; using this database, DNA sequences can be searched for based on the localizations of their products. CONCLUSIONS: A number of new intracellular structural components were found in this library. The library of GFP-fusion constructs also provides useful fluorescent markers for various intracellular structures and cellular activities, which can be readily used for microscopic observation in living cells.

Amino Acid Sequence↗

PubMed2000

An autopsy case of postencephalitic parkinsonism of von Economo type: some new observations concerning neurofibrillary tangles and astrocytic tangles.

An autopsied case of postencephalitic parkinsonism of von Economo type with a 71-year duration is reported. Several cases of postencephalitic parkinsonism of von Economo type have been reported in Japan but this is the first reported case from western Japan. The patient was a Japanese man who was 74 years of age at the time of death. He developed encephalitis of unknown etiology at the age of 3 years. The first symptom was antisocial behavior, which developed at 30 years of age. At the age of 40 years, the patient showed progressive parkinsonism. Neuropathological findings disclosed marked neuronal loss with gliosis in the substantia nigra, locus ceruleus, and raphe nuclei, as well as the appearance of neurofibrillary tangles in the aforementioned areas. There were also widespread tuft-shaped astrocytes (Tu-SA) in the central nervous system, including the thalamus. Tuft-shaped astrocytes are considered to represent non-reactive astrocytes because the distributions of neurofibrillary tangles (NFT) and Tu-SA are clearly different. Therefore, the primary astrocytic lesions in postencephalitic parkinsonism of von Economo type may be more widespread. Ultrastructurally, the Tu-SA consisted of straight filaments, 15 nm in width, which formed tight bundles. Ultrastructurally, NFF in this case revealed paired helical filaments but straight filaments, 15 nm in width, which were also found in the neurons of the substantia nigra.

Aged↗

PubMed2000

Live fluorescence imaging reveals early recruitment of emerin, LBR, RanBP2, and Nup153 to reforming functional nuclear envelopes.

We determined the times when the nuclear membrane, nuclear pore complex (NPC) components, and nuclear import function were recovered during telophase in living HeLa cells. Simultaneous observation of fluorescently-labeled NLS-bearing proteins, lamin B receptor (LBR)-GFP, and Hoechst33342-stained chromosomes revealed that nuclear membranes reassembled around chromosomes by 5 minutes after the onset of anaphase (early telophase) whereas nuclear import function was recovered later, at 8 minutes. GFP-tagged emerin also accumulated on chromosomes 5 minutes after the onset of anaphase. Interestingly, emerin and LBR initially accumulated at distinct, separate locations, but then became uniform 8 minutes after the onset of anaphase, concurrent with the recovery of nuclear import function. We further determined the timing of NPC assembly by immunofluorescence staining of cells fixed at precise times after the onset of anaphase. Taken together, these results showed that emerin, LBR, and several NPC components (RanBP2, Nup153, p62), but not Tpr, reconstitute around chromosomes very early in telophase prior to the recovery of nuclear import activity.

Biological Transport↗

PubMed1999

Mre11 is essential for the maintenance of chromosomal DNA in vertebrate cells.

Yeast Mre11 functions with Rad50 and Xrs2 in a complex that has pivotal roles in homologous recombination (HR) and non-homologous end-joining (NHEJ) DNA double-strand break (DSB) repair pathways. Vertebrate Mre11 is essential. Conditionally, MRE11 null chicken DT40 cells accumulate chromosome breaks and die upon Mre11 repression, showing frequent centrosome amplification. Mre11 deficiency also causes increased radiosensitivity and strongly reduced targeted integration frequencies. Mre11 is, therefore, crucial for HR and essential in mitosis through its role in chromosome maintenance by recombinational repair. Surprisingly perhaps, given the role of Mre11 in yeast NHEJ, disruption of NHEJ by deletion of KU70 greatly exacerbates the effects of MRE11 deficiency, revealing a significant Mre11-independent component of metazoan NHEJ.

Animals↗

PubMed1999

Function of a minus-end-directed kinesin-like motor protein in mammalian cells.

CHO2 is a mammalian minus-end-directed kinesin-like motor protein present in interphase centrosomes/nuclei and mitotic spindle fibers/poles. Expression of HA- or GFP-tagged subfragments in transfected CHO cells revealed the presence of the nuclear localization site at the N-terminal tail. This domain becomes associated with spindle fibers during mitosis, indicating that the tail is capable of interaction with microtubules in vivo. While the central stalk diffusely distributes in the entire cytoplasm of cells, the motor domain co-localizes with microtubules throughout the cell cycle, which is eliminated by mutation of the ATP-binding consensus motif from GKT to AAA. Overexpression of the full-length CHO2 causes mitotic arrest and spindle abnormality. The effect of protein expression was first seen around the polar region where microtubule tended to be bundled together. A higher level of protein expression induces more elongated spindles which eventually become disorganized by loosing the structural integrity between microtubule bundles. Live cell observation demonstrated that GFP-labeled microtubule bundles underwent continuous changes in their relative position to one another through repeated attachment and detachment at one end; this results in the formation of irregular number of microtubule focal points in mitotic arrested cells. Thus the primary action of CHO2 appears to cross-link microtubules and move toward the minus-end direction to maintain association of the microtubule end at the pole. In contrast to the full-length of CHO2, overexpression of neither truncated nor mutant polypeptides resulted in significant effects on mitosis and mitotic spindles, suggesting that the function of CHO2 in mammalian cells may be redundant with other motor molecules during cell division.

Animals↗