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Norie Araki

Publications and source records attributed to Norie Araki.

12 recordsLinked to original sources
PubMed2006

Protein kinase D2 contributes to either IL-2 promoter regulation or induction of cell death upon TCR stimulation depending on its activity in Jurkat cells.

Members of protein kinase D (PKD) family serine/threonine kinases (PKD1, PKD2 and PKD3) are expressed in wide range of cells and regulate various cellular responses including immune responses. We have previously shown that PKD is involved in the signaling pathways of a human CD4(+) T cell clone stimulated with its cognate antigen. Contrary to foregoing publications, PKD1 mRNA was not detected in human T cells, Jurkat cells and mouse thymocytes and splenocytes. Instead, mass-spectrometric and reverse transcription-PCR analyses revealed that PKD2 was predominant in T cells. To investigate the roles of PKD2, wild-type (WT) and constitutively active (CA) PKD2 were expressed in Jurkat cells together with IL-2 promoter-driven reporter gene. Expression of WT-PKD2 enhanced IL-2 promoter activity upon stimulation with anti-CD3 mAb, while expression of CA-PKD2 inhibited IL-2 promoter activity and induced cell death. Although the cell death was suppressed by the treatment with caspase inhibitor, the IL-2 promoter activity was rarely recovered in CA-PKD2-expressing cells upon TCR stimulation. WT-PKD2 localized mainly in the cytoplasm translocated into the nucleus after TCR stimulation, while CA-PKD2 was present in both the cytoplasm and the nuclei before and after stimulation. Proteomic analyses revealed that CA-PKD2 enhanced the amount of phosphorylated SET protein, a histone chaperon that regulates histone acetylation, in Jurkat cells and the recombinant SET protein was phosphorylated by CA-PKD2 in vitro. The data provide a renewing insight into the subset of PKD family kinases expressed in T cells and suggest that PKD2 is involved in IL-2 promoter regulation and cell death depending on its activity upon TCR stimulation.

Animals↗
PubMed2006

AM-3K, an anti-macrophage antibody, recognizes CD163, a molecule associated with an anti-inflammatory macrophage phenotype.

CD163 is a member of the scavenger receptor cysteine-rich superfamily restricted to the monocyte/macrophage lineage and is thought to be a useful marker for anti-inflammatory or alternatively activated macrophages. In this study we used mass spectrometric analysis to determine that the antigen recognized by the antibody AM-3K, which we previously generated as a tissue macrophage-specific monoclonal antibody, was CD163. An anti-inflammatory subtype of macrophages stimulated by dexamethasone or interleukin-10 showed strong reactivity for AM-3K and increased expression of CD163 mRNA. Immunohistochemical staining of routinely processed pathological specimens revealed that AM-3K recognized a specialized subpopulation of macrophages. In granulomatous diseases such as tuberculosis, sarcoidosis, or foreign body reactions, tissue macrophages around granulomas, but not component cells of the granulomas such as epithelioid cells and multinucleated giant cells, showed positive staining for AM-3K. In atherosclerotic lesions, scattered macrophages in diffuse intimal lesions were strongly positive for AM-3K, whereas foamy macrophages in atheromatous plaques demonstrated only weak staining. We therefore suggest that, in routine pathological specimens, AM-3K is a useful marker for anti-inflammatory macrophages because these cells can be distinguished from inflammatory or classically activated macrophages. Because AM-3K cross-reacts with macrophage subpopulations in different animal species including rats, guinea pigs, rabbits, cats, dogs, goats, pigs, bovine species, horses, monkeys, and cetaceans, it will have wide application for detection of CD163 in various animals.

Antigens, CD↗
PubMed2005

The neurofibromatosis type 1 gene product neurofibromin enhances cell motility by regulating actin filament dynamics via the Rho-ROCK-LIMK2-cofilin pathway.

Neurofibromin is a neurofibromatosis type 1 (NF1) tumor suppressor gene product with a domain that acts as a GTPase-activating protein and functions, in part, as a negative regulator of Ras. Loss of neurofibromin expression in NF1 patients is associated with elevated Ras activity and increased cell proliferation, predisposing to a variety of tumors of the peripheral and central nervous systems. We show here, using the small interfering RNA (siRNA) technique, that neurofibromin dynamically regulates actin cytoskeletal reorganization, followed by enhanced cell motility and gross cell aggregation in Matrigel matrix. NF1 siRNA induces characteristic morphological changes, such as excessive actin stress fiber formation, with elevated negative phosphorylation levels of cofilin, which regulates actin cytoskeletal reorganization by depolymerizing and severing actin filaments. We found that the elevated phosphorylation of cofilin in neurofibromin-depleted cells is promoted by activation of a Rho-ROCK-LIMK2 pathway, which requires Ras activation but is not transduced through three major Ras-mediated downstream pathways via Raf, phosphatidylinositol 3-kinase, and RalGEF. In addition, the exogenous expression of the NF1-GTPase-activating protein-related domain suppressed the NF1 siRNA-induced phenotypes. Neurofibromin was demonstrated to play a significant role in the machinery regulating cell proliferation and in actin cytoskeletal reorganization, which affects cell motility and adhesion. These findings may explain, in part, the mechanism of multiple neurofibroma formation in NF1 patients.

Actin Depolymerizing Factors↗
PubMed2005

Observation of the intracellular behavior of recombinant Yersinia pseudotuberculosis invasin protein.

In this study, we observed the intracellular behavior of recombinant invasin, a 103-kDa outer membrane protein of Yersinia pseudotuberculosis. To mimic the in vivo behavior of bacterial invasin, a polyvalent form of invasin was generated by incubation of biotinylated GST-fused invasin C-terminal portion protein (GST-INVS) with avidin. Several experiments confirmed that the recombinant invasin could consistently reproduce the invasin-mediated entry to mammalian epithelial cells. We analyzed the molecular kinetics of polyvalent INVS by western blotting, (125) I-uptake, and immunofluorescent microscopy. The internalized polyvalent INVS was rapidly translocated to the RIPA-insoluble (polymerized-actin enriched) fraction and formed cytoplasmic vesicles, while monovalent invasin did not show such kinetics. From these observations, we concluded that our bacterial-free system is able to analyze the action of invasin for Yersinia pseudotuberculosis entry.

Adhesins, Bacterial↗
PubMed2004

Role of TAFII-17, a VDR binding protein, in the increased osteoclast formation in Paget's Disease.

UNLABELLED: In contrast to normal OCL precursors, pagetic OCL precursors express MVNP and form OCL at physiologic concentrations of 1,25(OH)2D3, as do normal OCL precursors transfected with the MVNP gene. Using a GST-VDR chimeric protein, we identified TAFII-17 as VDR binding protein expressed by pagetic OCL precursors and MVNP transduced normal OCL precursors. TAF(II)-17 was in part responsible for the increased 1,25(OH)2D3 responsivity of pagetic OCL precursors. INTRODUCTION: Pagetic osteoclasts (OCLs) and their precursors express measles virus nucleocapsid protein (MVNP) and form large numbers of OCLs at low concentrations of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Similarly, normal OCL precursors transfected with MVNP also form OCLs at low concentrations of 1,25(OH)2D3. These results suggest that expression of MVNP in OCL precursors enhances vitamin D receptor (VDR)-mediated gene transcription. MATERIALS AND METHODS: To determine the mechanism for the increased OCL formation capacity of pagetic OCL precursors in response to 1,25(OH)2D3, lysates from pagetic and MVNP-transduced normal OCL precursors were incubated with a GST-VDR chimeric protein. RESULTS: A 17-kDa peptide that bound VDR was detected in MVNP-transduced cells and pagetic OCL precursors treated with 1,25(OH)2D3. This peptide was identified as TAFII-17, a component of the TFIID transcription complex. Expression of increased levels of TAFII-17 in cells allowed TAFII-17 to bind to VDR at low concentrations of 1,25(OH)2D3. An antisense oligonucelotide (AS-ODN) to TAFII-17 significantly decreased OCL formation in response to 1,25(OH)2D3 in pagetic but not normal marrow cultures by approximately 40%. Transfection of TAFII-17 or MVNP into NIH3T3 cells increased VDR transcriptional activity as measured by DR-3 reporter assays. CONCLUSION: These data show that expression of the MVNP gene in OCL precursors results in increased levels of TAFII-17. TAFII-17 can bind VDR at low concentrations of 1,25(OH)2D3. These results suggest that MVNP expression in Paget's OCL precursors increases expression of a component(s) of the VDR transcription complex that can increase OCL formation.

Bone Marrow Cells↗
PubMed2004

PKA phosphorylation and 14-3-3 interaction regulate the function of neurofibromatosis type I tumor suppressor, neurofibromin.

Neurofibromin, a neurofibromatosis type I (NF1) tumor suppressor gene product, has a domain acting as a GTPase activating protein and functions in part as a negative regulator of Ras. Loss of neurofibromin expression in NF1 patients is associated with elevated Ras activity and increased cell proliferation. Therefore, regulation of the function of neurofibromin is heavily involved in cell growth and differentiation. In the present study, we identified a novel cellular neurofibromin-associating protein, 14-3-3, which belongs to a highly conserved family of proteins that regulate intracellular signal transduction events in all eukaryotic cells. The interaction of 14-3-3 is mainly directed to the C-terminal domain (CTD) of neurofibromin, and the cAMP-dependent protein kinase (PKA)-dependent phosphorylation clustered on CTD-Ser (2576, 2578, 2580, 2813) and Thr (2556) is required for the interaction. Interestingly, the increased phosphorylation and association of 14-3-3 negatively regulate the function of neurofibromin. These findings indicate that PKA phosphorylation followed by 14-3-3 protein interaction may modulate the biochemical and biological functions of neurofibromin.

14-3-3 Proteins↗
PubMed2003

Neurofibromatosis type I tumor suppressor neurofibromin regulates neuronal differentiation via its GTPase-activating protein function toward Ras.

Neurofibromin, the neurofibromatosis type 1 (NF1) gene product, contains a central domain homologous to a family of proteins known as Ras-GTPase-activating proteins (Ras-GAPs), which function as negative regulators of Ras. The loss of neurofibromin function has been thought to be implicated in the abnormal regulation of Ras in NF1-related pathogenesis. In this study, we found a novel role of neurofibromin in neuronal differentiation in conjunction with the regulation of Ras activity via its GAP-related domain (GRD) in neuronal cells. In PC12 cells, time-dependent increases in the GAP activity of cellular neurofibromin (NF1-GAP) were detected after NGF stimulation, which were correlated with the down-regulation of Ras activity during neurite elongation. Interestingly, the NF1-GAP increase was due to the induction of alternative splicing of NF1-GRD type I triggered by the NGF-induced Ras activation. Dominant-negative (DN) forms of NF1-GRD type I significantly inhibited the neurite extension of PC12 cells via regulation of the Ras state. NF1-GRD-DN also reduced axonal and dendritic branching/extension of rat embryonic hippocampal neurons. These results demonstrate that the mutual regulation of Ras and NF1-GAP is essential for normal neuronal differentiation and that abnormal regulation in neuronal cells may be implicated in NF1-related learning and memory disturbance.

Alternative Splicing↗
PubMed2003

Tissue culture methods to study neurological disorders: establishment of immortalized Schwann cells from murine disease models.

Previously, the authors have established spontaneously immortalized cell lines from long-term cultures of normal adult mouse Schwann cells. Establishment of such Schwann cell lines derived from murine disease models may greatly facilitate studies of the cellular mechanisms of their peripheral nervous system lesions in the relevant diseases. Recently, the authors have established immortalized Schwann cell lines derived from Niemann-Pick disease type C mice (NPC; spm/spm) and globoid cell leukodystrophy mice (twitcher). In the present study, long-term cultures were maintained of Schwann cells derived from dorsal root ganglia and consecutive peripheral nerves of another NPC mouse (npc(nih)/npc(nih), npc(nih)/+), myelin P0 protein-deficient mice (P0-/-, P0+/-) with their wild-type littermates (P0+/+), and neurofibromatosis type 1 gene (NF1)-deficient mice (Nf1(FCr)/+) for 8-10 months, and immortalized cell lines from all these animals established spontaneously. These cell lines had spindle-shaped Schwann cell morphology and distinct Schwann cell phenotypes and retained genomic and biochemical abnormalities, sufficiently representing the in vivo pathological features of the mutant mice. These immortalized Schwann cell lines can be useful in studies of nervous system lesions in these mutant mice and relevant human disorders.

Animals↗
PubMed2002

WARTS tumor suppressor is phosphorylated by Cdc2/cyclin B at spindle poles during mitosis.

Identification of physiological substrates for Cdc2/cyclin B is crucial for understanding the functional link between mitotic events and Cdc2/cyclin B activation. A human homologue of the Drosophila warts tumor suppressor, termed WARTS, is a serine/threonine kinase and a dynamic component of the mitotic apparatus. We have found that Cdc2/cyclin B forms a complex with a fraction of WARTS in the centrosome and phosphorylates the Ser613 site of WARTS during mitosis. Immunocytochemical analysis has shown that the S613-phosphorylated WARTS appears in the spindle poles at prometaphase and disappears at telophase. Our findings suggest that Cdc/cyclin B regulates functions of WARTS on the mitotic apparatus.

Amino Acid Sequence↗
PubMed2002

Proteomic analysis of human brain identifies alpha-enolase as a novel autoantigen in Hashimoto's encephalopathy.

Hashimoto's encephalopathy (HE) is a rare autoimmune disease associated with Hashimoto's thyroiditis (HT). To identify the HE-related autoantigens, we developed a human brain proteome map using two-dimensional electrophoresis and applied it to the immuno-screening of brain proteins that react with autoantibodies in HE patients. After sequential MALDI-TOF-MASS analysis, immuno-positive spots of 48 kDa (pI 7.3-7.8) detected from HE patient sera were identified as a novel autoimmuno-antigen, alpha-enolase, harboring several modifications. Specific high reactivities against human alpha-enolase were significant in HE patients with excellent corticosteroid sensitivity, whereas the patients with fair or poor sensitivity to the corticosteroid treatment showed less reactivities than cut-off level. Although a few HT patients showed faint reactions to alpha-enolase, 95% of HT patients, patients with other neurological disorders, and healthy subjects tested were all negative. These results suggest that the detection of anti-alpha-enolase antibody is useful for defining HE-related pathology, and this proteomic strategy is a powerful method for identifying autoantigens of various central nervous system diseases with unknown autoimmune etiologies.

Amino Acid Sequence↗