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T Muta

Publications and source records attributed to T Muta.

At least 19 recordsLinked to original sources
PubMed2001

A novel IkappaB protein, IkappaB-zeta, induced by proinflammatory stimuli, negatively regulates nuclear factor-kappaB in the nuclei.

The transcription factor nuclear factor-kappaB (NF-kappaB) plays crucial roles in a wide variety of cellular functions and its activity is strictly regulated by cytosolic inhibitors known as IkappaBs. We here report a new member of the IkappaB protein family, IkappaB-zeta, harboring six ankyrin repeats at its carboxyl terminus. IkappaB-zeta mRNA is strongly induced after stimulation by lipopolysaccharide. The induction of IkappaB-zeta is also observed by stimulation with interleukin-1beta but not by tumor necrosis factor-alpha. In contrast to cytosolic IkappaB-alpha, -beta, and -epsilon, the induced IkappaB-zeta localizes in the nucleus via its amino-terminal region, which shows no homology with other proteins. Transiently expressed IkappaB-zeta inhibits the NF-kappaB activity without affecting the nuclear translocation of NF-kappaB upon stimulation. The expressed IkappaB-zeta preferentially associates with the NF-kappaB subunit p50 rather than p65 and recombinant IkappaB-zeta proteins inhibit the DNA binding of the p65/p50 heterodimer and the p50/p50 homodimer. Thus, IkappaB-zeta negatively regulates NF-kappaB activity in the nucleus, possibly in order to prevent excessive inflammation. Moreover, transfection of IkappaB-zeta renders cells more susceptible to apoptosis induced by tumor necrosis factor-alpha. The proapoptotic activity of IkappaB-zeta further suggests that it might be one of key regulators for inflammation and other biologically relevant processes.

Amino Acid Sequence↗
PubMed2001

Essential roles of CD14 and lipopolysaccharide-binding protein for activation of toll-like receptor (TLR)2 as well as TLR4 Reconstitution of TLR2- and TLR4-activation by distinguishable ligands in LPS preparations.

Although genetic studies have revealed a critical role for the toll-like receptor (TLR) 4 in the biological response to lipopolysaccharide (LPS), the activities of ectopically expressed TLR4 and TLR2 are controversial. We have found that under appropriate transfection conditions, both TLR2 and TLR4 mediate LPS-induced NF-kappaB activation in human embryonic kidney 293 cells. The reconstitution systems we established here allow direct biochemical characterization and comparison of activation of each receptor. TLR4 is approximately 100-fold more sensitive to LPS than TLR2. In contrast to the response to commercial LPS preparations, TLR2 is unresponsive to repurified LPS or synthetic lipid A, indicating the requirement for an additional molecule(s). On the other hand, a lipid A-neutralizing reagent, polymyxin B, blocks the ability of the LPS preparation to stimulate both receptors, suggesting that lipid A is also involved in the activation of TLR2. Mutant TLRs harboring a point mutation in the cytoplasmic domain is inactive in transducing the signal upon stimulation, and act as dominant-negative mutants specifically inhibiting the activation of corresponding type of the receptor but not the other type. Thus, the two receptors are independently activated by distinguishable ligands. Nevertheless, the responses of both TLRs to the LPS preparation are strongly dependent on serum and CD14 and LPS-binding protein are essential for the activation of both of the two receptors. Supporting its functional significance, both receptors are found to associate with CD14.

Acute-Phase Proteins↗
PubMed2001

Activation of retinoic X receptor and peroxisome proliferator-activated receptor-gamma inhibits nitric oxide and tumor necrosis factor-alpha production in rat Kupffer cells.

Activators of peroxisome proliferator-activated receptor gamma (PPAR gamma), which forms a heterodimer with retinoic X receptor (RXR), inhibit the production of certain inflammatory mediators. To clarify the role of the PPAR gamma:RXR signaling pathway in Kupffer cells, we studied the effect of an RXR agonist and PPARgamma agonist on LPS-induced nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production. An RXR-specific agonist, Ro47-5944, and a PPAR gamma-specific agonist, AD4833 (pioglitazone hydrochloride), each inhibited LPS-induced NO and TNF-alpha production. The combined treatment of Ro47-5944 and AD4833 resulted in enhanced inhibition, and suppressed the mRNA levels of NO and TNF-alpha. PPAR gamma:RXR activation did not affect the level of LPS-induced phosphorylation of c-jun N-terminal kinase and p38 mitogen-activated protein kinase. PPAR gamma:RXR activation also did not affect nuclear factor kappa B (NF-kappa B) nuclear translocation nor NF-kappa B and activator protein 1 (AP-1) activation in the electrophoretic mobility-shift assay. Finally, PPAR gamma:RXR activation suppressed the LPS-induced promoter activity of the NF-kappa B-luciferase reporter gene in RAW 264.7 cells. These data imply that PPARgamma:RXR activation suppresses LPS-induced NO and TNF-alpha production in Kupffer cells, and that this inhibition occurred at the transcriptional level. Although no consensus PPAR gamma:RXR-responsive element in the promoter regions of the inducible isoform of nitric oxide synthase (iNOS) and TNF-alpha genes was found, PPAR gamma:RXR may interfere with NF-kappa B and AP-1 transcriptional activity. Our data also suggest a potential therapeutic approach for moderating hepatic injury such as endotoxin shock in which Kupffer cell activation has been implicated.

Animals↗
PubMed2001

[European-American-type hairy cell leukemia without splenomegaly, treated successfully with deoxycoformycin].

A 55-year-old Japanese man was hospitalized on October 5, 1999, because of high fever. Physical examination revealed neither lymphadenopathy nor hepato-splenomegaly. Laboratory data on admission showed a white blood cell count of 1,580/microliter, a hemoglobin level of 9.1 g/dl, and a platelet count of 113 x 10(3)/microliter. A small percentage of abnormal mononuclear cells were present in the peripheral blood. A bone marrow biopsy specimen demonstrated myelofibrosis and diffuse infiltration of abnormal monoculear cells with a mature B cell phenotype. A bone marrow aspirate showed 29% abnormal mononuclear cells, which had an indented or folded nucleus and reticular nuclear chromatin. Moderate to strong tartarate-resistant acid phosphatase activity was detected in these cells. Although the cytoplasmic projections were poorly preserved in specimens stained with May-Giemsa, fresh preparations showed numerous slender cytoplasmic projections by phase-contrast microscopy. The hairy cells had the phenotype CD5-, CD10-, CD11c+, CD19+, CD20+, CD25+, lambda. The patient was diagnosed as having European-American-type hairy cell leukemia (HCL) without splenomegaly, which is quite rare in Japan. The value of phase-contrast microscopy for recognition of the hairy cells was emphasized. The patient was treated successfully with deoxycoformycin (DCF).

Antibiotics, Antineoplastic↗
PubMed2000

Head-to-tail polymerization of coagulin, a clottable protein of the horseshoe crab.

A clottable protein coagulogen of the horseshoe crab Tachypleus tridentatus is proteolytically converted into an insoluble coagulin gel through non-covalent self-polymerization. Here we identified binding sites for the polymerization. A tryptic fragment, derived from the coagulin polymer chemically cross-linked by a bifunctional cross-linker, was isolated. Amino acid sequence analysis indicated that the fragment consists of two peptides cross-linked between Lys(85) and Lys(156). The two lysine residues are oppositely located at the head and tail regions of the elongated molecule separated by a much greater distance than the length of the cross-linker, which suggests that the cross-linking occurs intermolecularly. Based on the x-ray structural analysis, exposure of a hydrophobic cove on the head in response to the release of peptide C has been postulated (Bergner, A., Oganessyan, V., Muta, T., Iwanaga, S., Typke, D., Huber, R., and Bode, W. (1996) EMBO J. 15, 6789-6797). An octapeptide containing Tyr(136), which occupies the tail end of coagulin, was found to inhibit the polymerization. Replacement of Tyr(136) of the peptide with Ala resulted in loss of the inhibitory activity. These results indicated that the polymerization of coagulin proceeds through the interaction between the newly exposed hydrophobic cove on the head and the wedge-shaped hydrophobic tail.

Alanine↗
PubMed2000

TAK1 mediates an activation signal from toll-like receptor(s) to nuclear factor-kappaB in lipopolysaccharide-stimulated macrophages.

Stimulation of monocytes/macrophages with lipopolysaccharide (LPS) results in activation of nuclear factor-kappaB (NF-kappaB), which plays crucial roles in regulating expression of many genes involved in the subsequent inflammatory responses. Here, we investigated roles of transforming growth factor-beta activated kinase 1 (TGF-TAK1), a mitogen-activated protein kinase kinase kinase (MAPKKK), in the LPS-induced signaling cascade. A kinase-negative mutant of TAK1 inhibited the LPS-induced NF-kappaB activation both in a macrophage-like cell line, RAW 264.7, and in human embryonic kidney 293 cells expressing toll-like receptor 2 or 4. Furthermore, we demonstrated that endogenous TAK1 is phosphorylated upon simulation of RAW 264.7 cells with LPS. These results indicate that TAK1 functions as a critical mediator in the LPS-induced signaling pathway.

Animals↗
PubMed2000

The D-loop structure of human mtDNA is destabilized directly by 1-methyl-4-phenylpyridinium ion (MPP+), a parkinsonism-causing toxin.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine has been reported to cause parkinsonism via its neurotoxic form, 1-methyl-4-phenylpyridinium ion (MPP+), which inhibits complex I of the mitochondrial respiratory chain. Its parkinsonism-causing mechanisms attract a great deal of interest as a model of the disease. Recently, we reported that MPP+ strongly decreases the amount of mtDNA independent of the inhibition of complex I. Maintenance of a proper amount of mtDNA is essential for the normal function of mitochondria as exemplified in many mitochondrial diseases. The most characteristic feature in vertebral mtDNA replication is that H-strand synthesis proceeds displacing the parental H-strand as a long single strand. It forms the D-loop, a triplex replication intermediate composed of the parental L-strand, nascent H-strand and displaced H-strand. Here we show that MPP+ does not inhibit DNA synthesis by DNA polymerase gamma, but rather releases the nascent H-strands from mtDNA both in organello and in vitro. This indicates that MPP+ directly destabilizes the D-loop structure, thereby inhibiting replication. This study raises a new mechanism, i.e. destabilization of replication intermediates, for depletion of mtDNA.

1-Methyl-4-phenylpyridinium↗
PubMed2000

A horseshoe crab receptor structurally related to Drosophila Toll.

Innate immunity against microbial pathogens relies on the pattern recognition of cell wall components on invading microbes. Recent evidence has shown that a mammalian Toll-like receptor (TLR) is activated by bacterial lipopolysaccharides (LPS). The innate immunity in invertebrates is also triggered by LPS, as seen in the hemolymph coagulation in horseshoe crab. We report the cloning of a TLR from the Japanese horseshoe crab Tachypleus tridentatus. A cDNA coding for Tachypleus Toll was isolated from a hemocyte cDNA library and the open reading frame codes for a proprotein including a signal sequence. Like Drosophila Toll, Tachypleus Toll is a type I transmembrane protein with an extracellular domain consisting of two leucine-rich repeats flanked by two cystein-rich clusters and a cytoplasmic domain exhibiting striking similarity with the cytoplasmic domain of interleukin-1 receptor. Tachypleus Toll is most similar to Drosophila Toll in the domain architecture and the overall length.

Animals↗
PubMed1999

Horseshoe crab acetyl group-recognizing lectins involved in innate immunity are structurally related to fibrinogen.

We have characterized and cloned newly isolated lectins from hemolymph plasma of the horseshoe crab Tachypleus tridentatus, which we named tachylectins 5A and 5B (TLs-5). TLs-5 agglutinated all types of human erythrocytes and Gram-positive and Gram-negative bacteria. TLs-5 specifically recognize acetyl group-containing substances including noncarbohydrates; the acetyl group is required and is sufficient for recognition. TLs-5 enhanced the antimicrobial activity of a horseshoe crab-derived big defensin. cDNA sequences of TLs-5 indicated that they consist of a short N-terminal Cys-containing segment and a C-terminal fibrinogen-like domain with the highest sequence identity (51%) to that of mammalian ficolins. TLs-5, however, lack the collagenous domain found in a kind of "bouquet arrangement" of ficolins and collectins. Electron microscopy revealed that TLs-5 form two- to four-bladed propeller structures. The horseshoe crab is equipped with a unique functional homologue of vertebrate fibrinogen, coagulogen, as the target protein of the clotting cascade. Our observations clearly show that the horseshoe crab has fibrinogen-related molecules in hemolymph plasma and that they function as nonself-recognizing lectins. An ancestor of fibrinogen may have functioned as a nonself-recognizing protein.

ABO Blood-Group System↗
PubMed1999

Mitochondrial DNA replication in human T lymphocytes is regulated primarily at the H-strand termination site.

The most unique feature in the replication of mitochondrial DNA (mtDNA) is that most of the newly synthesized heavy strands (H-strands) terminate prematurely, resulting in the formation of displacement loop (D-loop) strands. Only the H-strand which proceeds past the termination site is a true nascent H-strand leading to the overall replication on a circular mtDNA molecule. The physiological significance of the D-loop formation has long been unclear. To examine the role of premature termination in mtDNA replication, we therefore developed a method for selectively measuring both the total amount of nascent H-strands and the amount of true nascent H-strands using ligation-mediated polymerase chain reaction, which, for the first time, enabled us to estimate the frequency of premature termination. The stimulation of cell proliferation with interleukin 2 and phytohemagglutinin in human peripheral T lymphocytes caused an increase in the net replication rate of mtDNA. In stimulated cells, in comparison to resting ones, the amount of true nascent H-strands increased approx. 2.6-fold while the total amount of nascent H-strands remained unchanged, indicating that premature termination decreased while the initiation of replication remained the same. Our findings thus demonstrate the first clear example that premature termination plays a primary role in the up-regulation of the net rate of mtDNA replication in human cells.

Aphidicolin↗
PubMed1999

Pulmonary surfactant protein A modulates the cellular response to smooth and rough lipopolysaccharides by interaction with CD14.

Pulmonary surfactant protein A (SP-A) plays an important part in Ab-independent host defense mechanisms of the lung. In this study we investigated how SP-A interacts with distinct serotypes of bacterial LPS and modulates LPS-elicited cellular responses. SP-A bound to rough forms but not to smooth forms of LPS. In the macrophage-like cell line U937, SP-A inhibited mRNA expression and secretion of TNF-alpha induced by smooth LPS, but rough LPS-induced TNF-alpha expression was unaffected by SP-A. When U937 cells and rat alveolar macrophages were preincubated with SP-A, smooth LPS failed to induce TNF-alpha secretion, whereas rough LPS-induced TNF-alpha secretion was modestly increased. To clarify the mechanism by which SP-A modulates LPS-elicited cellular responses, we further examined the interaction of SP-A with CD14, which is known as a major LPS receptor. Western blot analysis revealed that CD14 was one of the SP-A binding proteins isolated from solubilized U937 cells. In addition, SP-A directly bound to recombinant soluble CD14 (rsCD14). When rsCD14 was preincubated with SP-A, the binding of rsCD14 to smooth LPS was significantly reduced but the association of rsCD14 with rough LPS was augmented. These results demonstrate the different actions of SP-A upon distinct serotypes of LPS and indicate that the direct interaction of SP-A with CD14 constitutes a likely mechanism by which SP-A modulates LPS-elicited cellular responses.

Animals↗
PubMed1999

R-Loop in the replication origin of human mitochondrial DNA is resolved by RecG, a Holliday junction-specific helicase.

Stable RNA-DNA hybrids (R-loops) prime the initiation of replication in Escherichia coli cells. The R-loops are resolved by Escherichia coli RecG protein, a Holliday junction specific helicase. A stable RNA-DNA hybrid formation in the mitochondrial D-loop region is also implicated in priming the replication of mitochondrial DNA. Consistent with this hypothesis, the 3' ends of the mitochondrial R-loop formed by in vitro transcription are located close to the initiation sites of the mitochondrial DNA replication. This mitochondrial R-loop is resolved by RecG in a dose-dependent manner. Since the resolution by RecG requires ATP, the resolution is dependent on the helicase activity of RecG. A linear RNA-DNA heteroduplex is not resolved by RecG, suggesting that RecG specifically recognizes the higher structure of the mitochondrial R-loop. This is the first example that R-loops of an eukaryotic origin is sensitive to a junction-specific helicase. The resolution of the mitochondrial R-loop by RecG suggests that the replication-priming R-loops have a common structural feature recognized by RecG.

Bacterial Proteins↗
PubMed1999

Pulmonary thromboembolism after spinal instrumentation surgery.

A 57-year-old woman was hospitalized because of gait disturbance and dysuria. Close examination revealed a cauda equina tumor at the level of L2 and L3. Tumor resection was performed, with posterolateral fusion and spinal instrumentation. On the eleventh day after the surgery, she experienced dyspnea and chest pain during standing and walking exercise. Pulmonary thromboembolism was diagnosed, based on: (1) blood gas analysis findings of hypoxemia and (2) defective images in both of the upper lobes on urgent pulmonary blood flow scintigram. Her clinical status improved with urgent thrombolytic therapy (with tisokinase and urokinase) and anticoagulation therapy (with heparin and warfarin), and her life was saved. When pulmonary thromboembolism occurs, early diagnosis by pulmonary blood flow scintigram and early thrombolytic and anticoagulative therapies are necessary. Special attention should be paid to symptoms of pulmonary thromboembolism in patients after spinal surgery.

Anticoagulants↗
PubMed1999

1-Methyl-4-phenylpyridinium ion (MPP+) selectively inhibits the replication of mitochondrial DNA.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine is known to cause Parkinsonism in its neurotoxic form, 1-methyl-4-phenylpyridinium ion (MPP+). We have previously reported that MPP+ decreases the content of mitochondrial DNA (mtDNA) independently of the inhibition of complex I in human cells [Miyako, K., Kai, Y., Irie, T., Takeshige, K., and Kang, D. (1997) J. Biol. Chem. 272, 9605-9608]. Here we study the mechanism causing the decrease in mtDNA. MPP+ inhibits the incorporation of 5-bromo-2'-deoxyuridine into mtDNA but not into nuclear DNA, indicating that MPP+ inhibits the replication of mtDNA but not that of the nuclear genome. The replication of mtDNA is initiated by the synthesis of the heavy strand switched from the transcription of the light strand. MPP+ decreases the nascent heavy strands per mtDNA and increases the transcript of the ND6 gene, encoded on light strand, per mtDNA. The amount of mitochondrial transcription factor A is not decreased. These data suggest that the transcription is not inhibited and therefore the transition from transcription to replication of mtDNA is lowered in the MPP+-treated cells. Electron microscopy shows that the number of mitochondria is not decreased in the MPP+-treated cells, suggesting that MPP+ does not affect the overall biogenesis of mitochondria. Thus, MPP+ selectively inhibits the replication of mtDNA.

1-Methyl-4-phenylpyridinium↗
PubMed1999

Lipopolysaccharide stimulates HepG2 human hepatoma cells in the presence of lipopolysaccharide-binding protein via CD14.

Lipopolysaccharide (LPS)-binding protein (LBP), an opsonin for activation of macrophages by bacterial LPS, is synthesized in hepatocytes and is known to be an acute phase protein. Recently, cytokine-induced production of LBP was reported to increase 10-fold in hepatocytes isolated from LPS-treated rats, compared with those from normal rats. However, the mechanism by which the LPS treatment enhances the effect of cytokines remains to be clarified. In the present study, we examined whether LPS alone or an LPS/LBP complex directly stimulates the hepatocytes, leading to acceleration of the cytokine-induced LBP production. HepG2 cells (a human hepatoma cell line) were shown to express CD14, a glycosylphosphatidylinositol-anchored LPS receptor, by both RT/PCR and flow cytometric analyses. An LPS/LBP complex was an effective stimulator for LBP and CD14 production in HepG2 cells, but stimulation of the cells with either LPS or LBP alone did not significantly accelerate the production of these proteins. The findings were confirmed by semiquantitative RT/PCR analysis of mRNA levels of LBP and CD14 in HepG2 cells after stimulation with LPS alone and an LPS/LBP complex. In addition, two monoclonal antibodies (mAbs) to CD14 (3C10 and MEM-18) inhibited LPS/LBP-induced cellular responses of HepG2 cells. Furthermore, prestimulation of HepG2 cells with LPS/LBP augmented cytokine-induced production and gene expression of LBP and CD14. All these findings suggest that an LPS/LBP complex, but not free LPS, stimulates HepG2 cells via CD14 leading to increased basal and cytokine-induced LBP and CD14 production.

Acute-Phase Proteins↗
PubMed1998

New types of clotting factors and defense molecules found in horseshoe crab hemolymph: their structures and functions.

Invertebrate animals, which lack adaptive immune systems, have developed defense systems, so-called innate immunity, that respond to common antigens on the surface of potential pathogens. One such defense system is involved in the cellular responses of horseshoe crab hemocytes to invaders. Hemocytes contain two types, large (L) and small (S), of secretory granules, and the contents of these granules are released in response to invading microbes via exocytosis. Recent biochemical and immunological studies on the granular components of L- and S-granules demonstrated that the two types of granules selectively store granule-specific proteins participating in the host defense systems. L-Granules contain all the clotting factors essential for hemolymph coagulation, protease inhibitors including serpins and cystatin, and anti-lipopolysaccharide (LPS) factor and several tachylectins with LPS binding and bacterial agglutinating activities. On the other hand, S-granules contain various new cysteine-rich basic proteins with antimicrobial or bacterial agglutinating activities, such as tachyplesins, big defensin, tachycitin, and tachystatins. The co-localization of these proteins in the granules and their release into the hemolymph suggest that they serve synergistically to construct an effective host defense system against invaders. Here, the structures and functions of these new types of defense molecules found in the Japanese horseshoe crab (Tachypleus tridentatus) are reviewed.

Animals↗