Search PubMed⌕ Search

Biomedical subjects

T Muta

Publications and source records attributed to T Muta.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1997

A peptidyl-prolyl cis/trans-isomerase (cyclophilin G) in regulated secretory granules.

A 27-kDa protein (p27) in horseshoe crab hemocyte that cross-reacts with antiserum against a beta-glucan-sensitive protease zymogen was purified to homogeneity, and its cDNA was cloned. The 1.7-kilobase pair cDNA contains an open reading frame of 660 base pairs, encoding a 23-amino acid signal sequence followed by a mature protein of 197 residues. The sequence of p27 exhibits strong similarity to that of cyclophilin B, a peptidyl-prolyl cis/trans-isomerase. p27 exhibits isomerase activity with a kcat/Km of 0.18 microM-1 s-1 for a peptide substrate; this activity is inhibited by cyclosporin A but is not affected by FK506. Although the p27 precursor possesses an amino-terminal secretory hydrophobic signal sequence, unlike other cyclophilin B molecules, it lacks a conserved carboxyl-terminal endoplasmic reticulum retention signal and it contains a central 8-amino acid insertion. Although p27 is secreted into the culture media of transiently expressed COS cells, it is not detected in horseshoe crab hemolymph plasma but rather is localized to the hemocyte large granules, the regulated secretory granules that are exocytosed upon stimulation. These results indicate that p27 is a new peptidyl-prolyl cis/trans-isomerase in the regulated secretory granules, and is thus designated cyclophilin G. This first report of a cyclophilin homologue in the secretory granule of the horseshoe crab hemocyte suggests that such chaperon-like proteins may constitute a key quality control system for stored proteins in exocytotic granules.

Amino Acid Sequence↗
PubMed1997

p32 protein, a splicing factor 2-associated protein, is localized in mitochondrial matrix and is functionally important in maintaining oxidative phosphorylation.

Human p32, originally cloned as a splicing factor 2-associated protein, has been reported to interact with a variety of molecules including human immunodeficiency virus Tat and complement 1q (C1q). p32 protein is supposed to be in the nucleus and on the plasma membrane for the association with human immunodeficiency virus Tat and C1q, respectively. None of the interactions, however, is proven to have a physiological role. To investigate the physiological function of p32, we determined the intracellular localization of p32. The fractionation of cells, fluorescent immunocytochemistry, and electron microscopic immunostaining show that p32 is exclusively localized in the mitochondrial matrix. We cloned a Saccharomyces cerevisiae homologue of human p32 gene, referred to yeast p30 gene. The yeast p30 protein is also localized in the mitochondrial matrix. The disruption of the p30 gene caused the growth retardation of yeast cells in a glycerol medium but not in a glucose medium, i.e. the impairment of the mitochondrial ATP synthesis. The growth impairment was restored by the introduction of the human p32 cDNA, indicating that p30 is a functional yeast counterpart of human p32. Taken together, both p32 and p30 reside in mitochondrial matrix and play an important role in maintaining mitochondrial oxidative phosphorylation.

Amino Acid Sequence↗
PubMed1997

Xanthogranulomatous pyelonephritis.

A 76-year-old woman presented with spiking fever and right back pain. Ultrasonography (US) revealed that her right kidney was enlarged. Computed tomography (CT) showed the parenchyma was replaced by non-enhancing masses but the overall kidney shape was maintained. These findings were compatible with those of xanthogranulomatous pyelonephritis (XP). The nonenhancing masses on CT coincided with the multiple butter yellow nodules of the resected kidney and microscopically these lesions were proved to be abscesses with xanthoma cells. In this case, the US and CT findings reflected the pathological feature of XP and thus these techniques are thought to be useful for the diagnosis of XP.

Aged↗
PubMed1997

Telomerase activity in pancreatic juice differentiates ductal carcinoma from adenoma and pancreatitis.

Telomerase activity was measured in pancreatic juice obtained by endoscopic retrograde pancreatography from 34 patients (12 with ductal carcinoma, 12 with pancreatic adenoma, and 10 with pancreatitis). The activity in pancreatic juice was expressed as the number of cells of a human pancreatic cancer cell line, MIA PaCa-2, that exhibit an activity equal to that expressed in 1 microg of protein from pancreatic juice. A telomerase ladder was detected in the pancreatic juice obtained from a majority of the patients with ductal adenocarcinoma. The median value of relative telomerase activity in the carcinoma samples was 9.38 (25th percentile, 3.14; 75th percentile, 95.8), a value significantly higher than that derived from patients with either pancreatitis or pancreatic adenoma (P < 0.0001). When a threshold value of relative telomerase activity of 3.00 was used, 75% (9 of 12) of the samples obtained from patients with ductal carcinoma were positive. We conclude that telomerase activity in pancreatic juice differentiates adenocarcinoma from adenoma and pancreatitis and may serve as a useful diagnostic tool.

Adenocarcinoma↗
PubMed1997

Telomerase elevation in pancreatic ductal carcinoma compared to nonmalignant pathological states.

Telomerase activity was measured in surgically resected tissues of 20 human pancreatic ductal carcinomas, 12 adenomas, 5 pancreatitis tissues, 14 normal pancreatic ducts, and 13 normal pancreatic tissues (primarily made up of acinar cells) using a PCR-based telomerase assay. Relative telomerase activity was expressed as the equivalent telomerase intensity of the number of cells of a human pancreatic cancer cell line, MIA PaCa-2, per microgram of protein in the tissue samples. The median value (25th percentile, 75th percentile) of relative telomerase activity in pancreatic carcinomas was 13.2 (3.58, 244), which was significantly higher relative to normal tissues, normal ducts, pancreatitis tissues, and adenomas (P < 0.0001). When the cutoff value of relative telomerase activity was set at 1.00 and 3.00, the positivity rates of telomerase activity in pancreatic ductal carcinomas were 100 and 80%, respectively. Some of the adenoma samples displayed a weak telomerase ladder. However, when semiquantitatively analyzed, the relative telomerase activity of all adenoma tissues was less than 1.00 equivalent cells per microgram protein of the tissues, which was equivalent to the values encountered in normal ducts. Thus, our results indicate that reactivation of telomerase may occur at a late stage of pancreatic ductal carcinogenesis. Therefore, telomerase may be a specific marker for distinguishing pancreatic cancer from pancreatitis and adenomas.

Adenocarcinoma↗
PubMed1996

Crystal structure of a coagulogen, the clotting protein from horseshoe crab: a structural homologue of nerve growth factor.

The clotting cascade system of the horseshoe crab (Limulus) is involved in both haemostasis and host defence. The cascade results in the conversion of coagulogen, a soluble protein, into an insoluble coagulin gel. The clotting enzyme excises the fragment peptide C from coagulogen, giving rise to aggregation of the monomers. The crystal structure of coagulogen reveals an elongated molecule that embraces the helical peptide C fragment. Cleavage and removal of the peptide C would expose an extended hydrophobic cove, which could interact with the hydrophobic edge of a second molecule, leading to a polymeric fibre. The C-terminal half of the coagulogen molecule exhibits a striking topological similarity to the neurotrophin nerve growth factor (NGF), providing the first evidence for a neurotrophin fold in invertebrates. Similarities between coagulogen and Spatzle, the Drosophila ligand of the receptor Toll, suggest that the neurotrophin fold might be considered more ancient and widespread than previously realized.

Amino Acid Sequence↗
PubMed1996

Clotting and immune defense in Limulidae.

The blue blood of the horseshoe crab contains a sophisticated defense system very sensitive to pathogens or foreign materials. The hemocytes circulating in the hemolymph detect trace amounts of LPS molecules on the invading microorganisms and respond quickly to release the granular components into the external milieu. The coagulation system composed of three serine protease zymogens, factor C, factor B, and proclotting enzyme, and a clottable protein, coagulogen, is activated by LPS to form insoluble coagulin gel. The coagulation system also responds to beta-(1,3) glucan through the activation of unique heterodimeric serine protease zymogen, factor G. The pathogens are, thus, engulfed in the gel and subsequently killed by antimicrobial substances with various specificities, which are also released from cells. The horseshoe crab has developed two kinds of serine protease zymogens as biological sensors, factor C and factor G, which are responsive to LPS and beta-(1,3) glucan on the surface of Gram-negative bacteria and fungi, respectively. These are possible invaders for horseshoe crabs and also for most animals including humans. This novel heterodimeric serine protease zymogen, factor G, may open a new way to develop an innovative assay system to quantitate beta-(1,3) glucans. Furthermore, these LPS and beta-(1,3) glucan sensitive factors could be utilized as a unique tool to analyze other biological reactions caused by LPS or the glucan. Although the coagulation reaction in horseshoe crabs is famous, it is not the only defense mechanism of this animal. Many agglutinins are present either in hemolymph plasma or in the cell. The hemolymph plasma also has cytolytic activity against foreign cells. These cellular and humoral defense systems, in concert, defend themselves from invading foreign organisms. Such a sophisticated defense system has allowed the horseshoe crab to survive for more than 200 million years on the earth. Horseshoe crabs are often called ¿living fossils." However, they are not fossils. They are living.

Amino Acid Sequence↗
PubMed1996

Semi-quantitative analysis of telomerase in pancreatic ductal adenocarcinoma.

Using a polymerase chain reaction-based amplification assay, we measured telomerase activity in surgically resected pancreatic ductal carcinomas (n = 16 cases) and normal ducts (n = 6), comparing findings with the telomerase activity of a human pancreatic cancer cell line, MIA PaCa-2, as a standard, i.e., relative telomerase activity was determined. Telomerase activity was expressed as the equivalent telomerase intensity of the number of cells of MIA PaCa-2 per microgram protein of tissue samples. The median value for telomerase activity in normal pancreatic ducts was 0.13 and the 25th and 75th percentile were 0.01 and 0.76. The median value for telomerase activity in pancreatic ductal adenocarcinoma was 34.7 (25th percentile, 4.98; and 75th percentile, 296), significantly higher than that of normal ducts (P < 0.001). When the cut-off value was set at 1.0 and 3.0, the telomerase positivity rate of pancreatic ductal adenocarcinomas was 100% and 81.3%, respectively. Telomerase may be specific marker for pancreatic ductal carcinomas.

Biomarkers, Tumor↗
PubMed1996

The role of hemolymph coagulation in innate immunity.

Invertebrate animals, which lack adaptive immune systems, have developed defense systems that respond to common antigens on the surface of potential pathogens. Hemolymph coagulation is one such defense system in innate immunity. The discovery of lipopolysaccharide-sensitive and (1-->3)-beta-D-glucan-sensitive serine protease zymogens in horseshoe crab (limulus) hemocytes, both of which trigger the coagulation cascade, has exemplified how the animals detect and respond to foreign materials.

Amino Acid Sequence↗
PubMed1995

Purified horseshoe crab factor G. Reconstitution and characterization of the (1-->3)-beta-D-glucan-sensitive serine protease cascade.

Horseshoe crab hemocyte lysate responds to (1-->3)-beta-D-glucans, initiating an enzymatic cascade, which culuminates in clot formation. We have purified to homogeneity the serine protease zymogen factor G, which is directly activated by (1-->3)-beta-D-glucans and which initiates the hemolymph clotting cascade. Factor G is a heterodimeric protein composed of two noncovalently associated subunits alpha (72 kDa) and beta (37 kDa). In the presence of (1-->3)-beta-D-glucans such as curdlan and paramylon, factor G is autocatalytically activated to an active serine protease named factor G. This activation is accompanied by limited proteolysis of both subunits: the 72-kDa subunit alpha is cleaved to 55-kDa and 17-kDa fragments, and the 37-kDa subunit beta is shortened to 34 kDa. Longer incubations with (1-->3)-beta-D-glucans result in cleavage of the 55-kDa fragment to 46 kDa and the 34-kDa fragment to 32 kDa, with concomitant loss of amidase activity. Reconstitution experiments using purified proteins participating in the hemolymph clotting cascade demonstrate that factor G is capable of activating proclotting enzyme directly, resulting in the conversion of coagulogen to coagulin gel. Thus, purified factor G is shown to be the primary initiator of the (1-->3)-beta-D-glucan-sensitive coagulation pathway in the horseshoe crab hemocyte lysate.

Amino Acid Sequence↗
PubMed1995

cDNA and deduced amino acid sequence of human PK-120, a plasma kallikrein-sensitive glycoprotein.

PK-120 is a substrate for plasma kallikrein (PK), recently purified from human plasma. Here we have established the cDNA sequence for human PK-120 mRNA. The deduced amino sequence of PK-120 revealed that it consists of 902 amino acid residues with a calculated mass of 116,423 Da. The putative cleavage sites by PK have been proposed, suggesting that PK-120 may be a precursor of a bioactive peptide. Most interestingly, PK-120 showed significant sequence identities to heavy chains (HCs) of the inter-alpha-trypsin inhibitor (ITI) superfamily.

Amino Acid Sequence↗