Biomedical subjects

K Miyake

Publications and source records attributed to K Miyake.

At least 73 records · Page 4Linked to original sources

PubMed2000

Regulatory roles for CD14 and phosphatidylinositol in the signaling via toll-like receptor 4-MD-2.

The complex consisting of Toll-like receptor 4 (TLR4) and associated MD-2 signals the presence of lipopolysaccharide (LPS) when it is expressed in cell lines. We here show that normal human mononuclear cells express TLR4 and signal LPS via TLR4. CD14 is a molecule that binds to LPS and facilitates its signaling. Little is known, however, about the relationship of CD14 with TLR4-MD-2. We show that CD14 helps TLR4-MD-2 to sense and signal the presence of LPS. CD14 has also been implicated in recognition of apoptotic cells, which leads to phagocytosis without activation. Membrane phospholipids such as phosphatidylserine (PS) or phosphatidylinositol (PtdIns) are thought to serve as the ligands for CD14 in apoptotic cells. We find that PtdIns acts as an LPS antagonist in the signaling via TLR4-MD-2. TLR4-MD-2 seems to discriminate LPS from phospholipids. The signaling via TLR4-MD-2 is thus regulated by CD14 and phospholipid such as PtdIns.

Animals↗

PubMed2000

Non-CD28 costimulatory molecules present in T cell rafts induce T cell costimulation by enhancing the association of TCR with rafts.

While CD28 functions as the major T cell costimulatory receptor, a number of other T cell molecules have also been described to induce T cell costimulation. Here, we investigated the mechanisms by which costimulatory molecules other than CD28 contribute to T cell activation. Non-CD28 costimulatory molecules such as CD5, CD9, CD2, and CD44 were present in the detergent-insoluble glycolipid-enriched (DIG) fraction/raft of the T cell surface, which is rich in TCR signaling molecules and generates a TCR signal upon recruitment of the TCR complex. Compared with CD3 ligation, coligation of CD3 and CD5 as an example of DIG-resident costimulatory molecules led to an enhanced association of CD3 and DIG. Such a DIG redistribution markedly up-regulated TCR signaling as observed by ZAP-70/LAT activation and Ca2+ influx. Disruption of DIG structure using an agent capable of altering cholesterol organization potently diminished Ca2+ mobilization induced by the coligation of CD3 and CD5. This was associated with the inhibition of the redistribution of DIG although the association of CD3 and CD5 was not affected. Thus, the DIG-resident costimulatory molecules exert their costimulatory effects by contributing to an enhanced association of TCR/CD3 and DIG.

Adjuvants, Immunologic↗

PubMed2000

Mouse toll-like receptor 4.MD-2 complex mediates lipopolysaccharide-mimetic signal transduction by Taxol.

Taxol, an antitumor agent derived from a plant, mimics the action of lipopolysaccharide (LPS) in mice but not in humans. Although Taxol is structurally unrelated to LPS, Taxol and LPS are presumed to share a receptor or signaling molecule. The LPS-mimetic activity of Taxol is not observed in LPS-hyporesponsive C3H/HeJ mice, which possess a point mutation in Toll-like receptor 4 (TLR4); therefore, TLR4 appears to be involved in both Taxol and LPS signaling. In addition, TLR4 was recently shown to physically associate with MD-2, a molecule that confers LPS responsiveness on TLR4. To determine whether TLR4.MD-2 complex mediates a Taxol-induced signal, we constructed transformants of the mouse pro-B cell line, Ba/F3, expressing mouse TLR4 alone, both mouse TLR4 and mouse MD-2, and both mouse MD-2 and mouse TLR4 lacking the cytoplasmic portion, and then examined whether Taxol induced NFkappaB activation in these transfectants. Noticeable NFkappaB activation by Taxol was detected in Ba/F3 expressing mouse TLR4 and mouse MD-2 but not in the other transfectants. Coexpression of human TLR4 and human MD-2 did not confer Taxol responsiveness on Ba/F3 cells, suggesting that the TLR4. MD-2 complex is responsible for the species specificity with respect to Taxol responsiveness. Furthermore, Taxol-induced NFkappaB activation via TLR4.MD-2 was blocked by an LPS antagonist that blocks LPS-induced NFkappaB activation via TLR4.MD-2. These results demonstrated that coexpression of mouse TLR4 and mouse MD-2 is required for Taxol responsiveness and that the TLR4.MD-2 complex is the shared molecule in Taxol and LPS signal transduction in mice.

Animals↗

PubMed2000

Structure and function of a novel coliphage-associated sialidase.

A coliphage named 63D, isolated previously, associated sialidase as a component of phage particles. In order to localize the enzyme in phage particles, phages were partially destroyed by sonication, and the disrupted particles were size fractionated using a sucrose density gradient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme assay and electron micrography of the fractions revealed the enzyme to be composed of four identical subunits with a molecular mass of 90 kDa, and the subunits were cross-linked by disulfide bonds. Electron micrographic observation indicated that six enzyme molecules were localized in a phage tail plate as a hexagonal array.

Cell Fractionation↗

PubMed2000

Molecular cloning and characterization of a novel bacteriophage-associated sialidase.

Bacteriophage 63D, previously isolated from sewage, is associated with alpha-2,8-linked polysialic acid degrading activity. We cloned a DNA fragment containing the sialidase gene from a 63D phage genomic library and the enzyme was functionally expressed in Escherichia coli. Determination of the nucleotide sequence of the fragment revealed that it contained one open reading frame (ORF) coding for a 108-kDa polypeptide consisting of 984 amino acid residues. The fragment had promoter sequences similar to the E. coli consensus promoters for sigma70. The deduced amino acid sequence of the central region of the ORF showed homology to those of phages K1F (51.6% identity) and PK1E (51.7% identity) endosialidases. Two Asp-box motifs that are widely found in sialidases were conserved. Purification of the soluble enzyme from lysed culture broth of infected E. coli yielded a 90-kDa protein upon SDS polyacrylamide gel electrophoresis, suggesting that the primary translational product is processed to the mature 90-kDa protein. The molecular mass of the enzyme was determined as 360 kDa by gel filtration, indicating that the native enzyme was probably a tetramer of identical 90-kDa subunits.

Journal Article↗

PubMed2000

Transcriptional regulation of nir and nor operons of Paracoccus denitrificans.

Transcripts of nitrite reductase (nir) and nitric oxide reductase (nor) operons of Paracoccus denitrificans were expressed only under anaerobic conditions in the presence of potassium nitrite. The nir and nor operons produced at least two transcription products. Large transcripts seemed to contain whole operons and small ones seemed to contain the genes encoding denitrifying enzymes. All transcription start sites were detected 41.5 or 42.5 bp downstream from the center of FNR boxes.

Journal Article↗

PubMed2000

Attachment and culture. Security in the United States and Japan.

Attachment theorists maintain that cultural differences are relatively minor, and they focus on universals. Here the authors highlight evidence of cultural variations and note ways in which attachment theory is laden with Western values and meaning. Comparisons of the United States and Japan highlight the cultural relativity of 3 core hypotheses of attachment theory: that caregiver sensitivity leads to secure attachment, that secure attachment leads to later social competence, and that children who are securely attached use the primary caregiver as a secure base for exploring the external world. Attachment theorists use measures of sensitivity, competence, and secure base that are biased toward Western ways of thinking: The measures emphasize the child's autonomy, individuation, and exploration. In Japan, sensitivity, competence, and secure base are viewed very differently, calling into question the universality of fundamental tenets of attachment theory. The authors call for an indigenous approach to the psychology of attachment.

Adult↗

PubMed2000

Induction of cyclooxygenase-2 in mesothelial cells in peritonitis caused by perforated ulcers--an immunohistochemical study in humans.

BACKGROUND: Increasing evidence suggests that mesothelial cells contribute to the control of inflammation in the peritoneal cavity by secreting prostaglandins. A study has shown that cyclooxygenase (COX)-2 knockout mice die partly as a result of peritonitis. AIM: To investigate the expression and location of COX in peritonitis associated with peptic ulcer perforation. METHODS: Gastric and duodenal tissues were collected intraoperatively from nine and four patients, respectively, and immunohistochemical staining for COX-1 and COX-2 was performed. RESULTS: Histologically, all patients had severe peritonitis around the perforation sites, into which many inflammatory cells and fibroblasts had infiltrated, and reactive mesothelial cells exhibited hyperplastic change. The COX-1 protein was not detected, whereas COX-2 was abundant in reactive mesothelial cells near the perforation site and disappeared away from the site. Macrophages and fibroblasts around the perforation site also revealed immunostaining for COX-2. CONCLUSIONS: Our results showed that COX-2 protein is induced in mesothelial cells, as well as in macrophages and fibroblasts, in inflamed peritoneal tissues associated with peptic ulcer perforation, suggesting involvement of COX-2 in tissue repair.

Adult↗

PubMed2000

A short-term eradication therapy for Helicobacter pylori acute gastritis.

BACKGROUND AND AIMS: Acute gastritis, caused by an initial infection of Helicobacter pylori (H. pylori), may resolve spontaneously, but the infection sometimes becomes chronic. We examined the efficacy of a short-term H. pylori eradication therapy on acute gastritis. METHODS: Among the 15 patients with hemorrhagic acute gastritis who were randomly allocated to group A (eradication therapy) or group B (Lansoprazole, LPZ), 10 of them started to receive treatment within 1 day after the disease onset. The other five patients began the eradication therapy 4-6 days after disease onset (group C). Eradication therapy consisted of a daily oral administration of each of 30 mg lansoprazole (LPZ), once a day; 400 mg clarithromycin, twice a day; 1000 mg amoxicillin, twice a day; and 300 mg rebamipide, three times a day, for one week. If the endoscopy was normal, medication was stopped for the following 4 weeks before gastric endoscopy was performed again in order to assess H. pylori eradication. RESULTS: All group A patients were cured after the 1-week treatment and therefore, they became H. pylori negative. Group B and C patients had erosions or ulcers after the 1-week treatment and so received an additional 3-week administration of LPZ. Four weeks later, their gastritis was cured and except for one group B patient, they became H. pylori-negative. CONCLUSION: In patients with acute gastritis, caused by an initial H. pylori infection, eradication therapy was efficacious in achieving early healing. This therapy should be started as soon as possible after disease onset.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed2000

Improvement of solubility and oral bioavailability of rutin by complexation with 2-hydroxypropyl-beta-cyclodextrin.

The object of this study was to enhance the solubility, dissolution rate, and oral bioavailability of rutin by complexation with 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CyD). The interaction of rutin with cyclodextrins (CyDs) was evaluated by the solubility, and ultraviolet (UV) and circular dichroism (CD) spectrophotometries. The chemical and enzymatic stability of rutin was examined in an alkaline buffer solution and in rat small intestinal homogenates, respectively. Dissolution rates of rutin and its CyD complexes were measured by the dispersed amount method. In vivo absorption studies of rutin after oral administration via conventional tablet containing rutin alone or its beta-CyD complexes was performed on beagle dogs. The stability constants calculated from the phase solubility method increased in the order of HP-gamma-CyD < G2-beta-CyD < beta-CyD < HP-beta-CyD. Spectroscopic studies also revealed that HP-beta-CyD and beta-CyD formed a relatively more stable inclusion complex with rutin. The dissolution rates of rutin increased by the complexation with CyDs in the order of rutin alone < HP-beta-CyD < or = beta-CyD. HP-beta-CyD inhibited the hydrolysis of rutin in the alkaline buffer solution and the small intestinal homogenates of rats, suggesting that HP-beta-CyD may stabilize rutin in a gastrointestinal tract after oral administration. When the tablet containing rutin or its beta-CyD complexes was administered to beagle dogs, the plasma levels of homovanillic acid (HVA) (a major stable metabolite of rutin) after oral administration of HP-beta-CyD complex were much higher than in either that of rutin alone or in its beta-CyD complex. The in vivo absorption study suggests that HP-beta-CyD increased the oral bioavailability of rutin from the gastrointestinal tracts of beagle dogs because of the increase in solubility, faster dissolution rate, and gastrointestinal stability. HP-beta-CyD has a significant advantage with respect to providing high aqueous solubility while maintaining a lack of toxicity in oral pharmaceutical preparations of rutin.

2-Hydroxypropyl-beta-cyclodextrin↗

PubMed2000

Transforming growth factor-beta1 stimulates contraction of human glioblastoma cell-mediated collagen lattice through enhanced alpha2 integrin expression.

Rapid invasiveness is a feature of the highly malignant glioblastoma tumor and is closely related to patient prognosis. The interaction between extracellular matrix (ECM) and cell surface receptors such as integrin heterodimers play a key role in the process of tumor invasion. We investigated the effects of transforming growth factor-beta1 (TGF-beta1), which is a mitogenic factor for glial cells, on integrin expression in T98G human glioblastoma cells using an in vitro model 3-dimensional collagen lattice. Exogenously applied TGF-beta1 dose-dependently enhanced collagen lattice contraction. Among the inhibitory antibodies tested against alpha integrin subunits, the anti-alpha2 antibody, P1-E6, alone prevented the enhanced contractile response by TGF-beta1, whereas any alpha integrin antibody (including P1-E6) had little effect on lattice contraction when cultured without TGF-beta1. RT-PCR analysis revealed that TGF-beta1 strongly increased alpha2 integrin transcript level. Furthermore, pretreatment with antisense phosphorothioate oligodeoxynucleotides against human alpha2 integrin using hemagglutinating virus of Japan (HVJ) liposome-mediated transfer prevented the effects of TGF-beta1 and also reduced the lattice contraction even in the absence of TGF-beta1. This data indicates that increased expression of alpha2 integrin is responsive to enhanced collagen lattice contraction by TGF-beta1. We suggest that TGF-beta1 exerts its effects on the invasive property of glioblastoma cells via upregulation of the alpha2 integrin subunit expression.

Antigens, CD↗

PubMed2000

Increased incidence of follicular lymphoma in the duodenum.

The incidence of indolent lymphomas in the lymph nodes and extranodal regions is quite different. Follicular lymphoma (FL) is most common in the nodes, and it seems to be least common in the gastrointestinal (GI) tract, where mucosa-associated lymphoid tissue lymphoma arises most frequently. The authors report that the incidence of FL is unexpectedly high in the duodenum compared with other portions of the GI tract. FL was detected in only eight of 222 cases of GI lymphoma (3.6%). However, five cases of FL arose in the duodenum, which accounted for 38.5% of 13 duodenal lymphomas. Only in two patients did FL arise in either the stomach or the colorectum, and in the remaining patients FL was widespread with lymphomatous polyposis. Duodenal FL was composed of neoplastic follicles with small cleaved cells in dominance, and the immunophenotype of the lymphoma cells was CD10+, BCL-2+, CD20+, CD75+, CD79+, CD3-, CD5-, cyclin D1-, CD23-, and CD45RO-. All the patients were women age 37 to 66 years (average age, 52.4 yrs). In all patients the lymphoma was present around the ampulla of Vater, and four of five patients showed multiple small-size polyps. Although lymphoma cell infiltration was confined to the submucosa in the four patients examined, the regional lymph nodes were involved partially in two patients without distant metastasis. All patients are alive at 2 to 50 months of follow up (average, 27 mos), which is comparable with the prognosis for indolent nodal lymphomas. These results suggest that the duodenum has a distinct background of histogenesis of the lymphomas and that biopsy specimens from the duodenum with multiple polyps should be examined carefully.

Adult↗

PubMed2000

Mechanism of the incidental production of a melanin-like pigment during 6-demethylchlortetracycline production in Streptomyces aureofaciens.

The secondary metabolite 6-demethylchlortetracycline (6-DCT), which is produced by Streptomyces aureofaciens, is used as a precursor of semisynthetic tetracyclines. Strains that produce 6-DCT also produce a melanin-like pigment (MP). The correlation between MP production and 6-DCT production was investigated by using S. aureofaciens NRRL 3203. Production of both MP and 6-DCT was repressed by phosphate or ammonium ions, suggesting that syntheses of these compounds are controlled by the same regulators. Ten chlortetracycline-producing recombinants were derived from 6-DCT-producing mutant NRRL 3203 by gene replacement. All of the recombinants produced chlortetracycline but not MP, indicating that MP production is the results of a defect in the 6-methylation step and suggesting that the polyketide nonaketideamide is a common intermediate leading to MP as well as 6-DCT. To further examine the possibility that MP might be synthesized via the 6-DCT-biosynthetic pathway, mutants defective in 6-DCT biosynthesis were derived from a 6-DCT producer. Some of these mutants were able to produce MP, while others, including mutants with mutations in the gene encoding anhydrotetracycline oxygenase, an enzyme catalyzing the penultimate step in the pathway, produced neither 6-DCT nor MP. Production of 6-DCT and production of MP were restored simultaneously by integrative transformation with the corresponding 6-DCT-biosynthetic genes, indicating that some of 6-DCT-biosynthetic enzymes are indispensable for MP production. These findings suggest that a defect in the 6-methylation step results in redirection of carbon flux from a certain intermediate in the 6-DCT-biosynthetic pathway to a shunt pathway and results in MP production.

Anti-Bacterial Agents↗

PubMed2000

Toll-like receptors confer responsiveness to lipopolysaccharide from Porphyromonas gingivalis in human gingival fibroblasts.

Gingival fibroblasts produce proinflammatory cytokines in response to lipopolysaccharide (LPS) from periodontopathic bacteria. Recently it has become evident that the human homologue of Drosophila Toll can transduce intracellular signaling by LPS stimulation. Toll-like receptors (TLRs) have been identified in myeloid cells; however, their role in nonmyeloid cells such as gingival fibroblasts has not been fully elucidated. Here, we report that human gingival fibroblasts constitutively express TLR2 and TLR4 and that their levels of expression are increased by stimulation with LPS from Porphyromonas gingivalis. Upregulated expression of interleukin-6 gene and protein in fibroblasts stimulated with LPS is inhibited by anti-TLR4 antibody. These findings suggest that TLRs may confer responsiveness to LPS in gingival fibroblasts.

Cells, Cultured↗

PubMed2000

Localisation of cyclooxygenase 1 and cyclooxygenase 2 in Helicobacter pylori related gastritis and gastric ulcer tissues in humans.

BACKGROUND: Prostaglandin endoperoxide synthase/cyclooxygenase (COX) is the key enzyme in gastric mucosal protection and repair but its cellular localisation in the human stomach is still unclear. AIMS: To investigate immunohistochemically the cellular distribution of COX-1 and COX-2 proteins in the human stomach with or without gastritis or ulceration. PATIENTS AND METHODS: Tissues were obtained by surgical resection of gastric ulcers associated with perforation (n = 9) or by biopsy from Helicobacter pylori positive patients with gastric ulcers (n = 45) and H pylori negative healthy subjects (n = 15). COX expression was detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and light and electron microscopic immunohistochemistry. RESULTS: COX-2 mRNA and protein were detected in gastric ulcer tissues but not in intact gastric mucosa. COX-1 mRNA and protein were detected in the intact mucosa. COX-2 immunostaining was exclusively localised in macrophages and fibroblasts between necrotic and granulation tissues of the ulcer bed. The percentage of COX-2 expressing cells was significantly higher in open than in closed ulcers, and in gastritis than in gastric mucosa without H pylori infection. COX-1 immunoreactivity localised in lamina propria mesenchymal cells was similar in various stages of ulcer disease and in intact gastric mucosa. Electron microscopic immunohistochemistry revealed both COX-1 and COX-2 on the luminal surfaces of the endoplasmic reticulum and nuclear envelope of macrophages and fibroblasts. CONCLUSIONS: Our results showed that COX-2 protein was induced in macrophages and fibroblasts in gastric ulcers and H pylori related gastritis, suggesting its involvement in the tissue repair process.

Adult↗

PubMed2000

Patching plasma membrane disruptions with cytoplasmic membrane.

Vesicle-vesicle fusion initiated in cell cytoplasm by high Ca(2+) can rapidly erect large membrane boundaries. These might be used as a 'patch' for resealing plasma membrane disruptions. Three central predictions of this 'patch' hypothesis are here established in sea urchin eggs. First, we show that surface markers for plasma membrane protein and lipid are initially absent over disruption sites after resealing is complete. Second, we demonstrate that resealing capacity is strongly dependent upon local availability of fusion competent cytoplasmic organelles, specifically the reserve or yolk granule. Lastly, we demonstrate that the reserve granule is capable of rapid (t(1/2) <1 second), Ca(2+)-regulated (high threshold) fusion capable of erecting large (>1000 microm(2)), continuous membrane boundaries. Production of patch vesicles for resealing may proceed by an 'emergency' fusion mechanism distinct from that utilized for the much slower, highly regulated, cytosol-requiring organelle-organelle fusion events typical of constitutive membrane trafficking pathways.

Animals↗

PubMed2000

The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2).

Mechanisms of drug resistance other than P-glycoprotein are of increasing interest as the list of newly identified members of the ABC transport family has grown. We sought to characterize the phenotype of the newly discovered ABC transporter encoded by the mitoxantrone resistance gene, MXR, also known as ABCP1 or BCRP. The pharmacodynamics of mitoxantrone and 12 other fluorescent drugs were evaluated by confocal microscopy in four multidrug-resistant human colon (S1) and breast (MCF-7) cancer cell lines. We utilized two sublines, MCF-7 AdVp3000 and S1-M1-80, and detected overexpression of MXR by PCR, immunoblot assay and immunohistochemistry. These MXR overexpressing sublines were compared to cell lines with P-glycoprotein- and MRP-mediated resistance. High levels of cross-resistance were observed for mitoxantrone, the anthracyclines, bisantrene and topotecan. Reduced levels of mitoxantrone, daunorubicin, bisantrene, topotecan, rhodamine 123 and prazosin were observed in the two sublines with high MXR expression. Neither the P-glycoprotein substrates vinblastine, paclitaxel, verapamil and calcein-AM, nor the MRP substrate calcein, were extruded from MCF-7 AdVp3000 and S1-M1-80 cells. Thus, the multidrug-resistant phenotype due to MXR expression is overlapping with, but distinct from, that due to P-glycoprotein. Further, cells that overexpress the MXR protein seem to be more resistant to mitoxantrone and topotecan than cells with P-glycoprotein-mediated multidrug resistance. Our studies suggest that the ABC half-transporter, MXR, is a potent, new mechanism for conferring multiple drug resistance. Definition of its mechanism of transport and its role in clinical oncology is required.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

The effects of a novel cyclohexane dicarboximide derivative, ST-6, on hypoxia/reoxygenation injury in perfused rat heart.

The present study was undertaken to test if some cyclohexane dicarboximide derivatives may have a cardioprotective effect against hypoxia/reoxygenation injury. Isolated rat hearts were subjected to 20-min of hypoxia followed by 45-min reoxygenation, and their recovery of post-hypoxic cardiac contractile function was examined. Treatment with agents was carried out from 3 min after the onset of hypoxia to the end of hypoxia (17 min during hypoxia). Among the 17 compounds, 2-[4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidinyl]butyl]hexahydro-1H-i soindol-1,3(2H)-dione (ST-6) showed a significant enhancement of post-hypoxic contractile force. This was associated with attenuation of the releases of creatine kinase and purine nucleosides and bases from the perfused heart. Hypoxia-induced increase in myocardial sodium and decrease in potassium ion content was suppressed by ST-6 treatment. The results suggest that ST-6 is capable of protecting the heart against hypoxia/reoxygenation injury possibly through a mechanism by which sodium overload during hypoxia is suppressed.

Animals↗