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N Harada

Publications and source records attributed to N Harada.

At least 253 records · Page 14Linked to original sources
PubMed1998

Changes in microtopography across polished resin-dentin interfaces.

PURPOSE: To investigate the resistance of the hybrid layer to polishing and argon-ion etching by measuring the microprofile along the resin-dentin interface. MATERIALS AND METHODS: Mid-coronal human dentin disks were treated with one of three commercially available dentin bonding systems (All-Bond 2, Clearfil Liner Bond II, Scotchbond Multi-Purpose Plus), and bonded together so as to prepare three pairs of specimens. The bonded assemblies were sectioned perpendicular to the adhesive interface and embedded in epoxy resin. The polished interfaces were argon-ion etched through a grid square mesh placed on the interface. A profile of the hybrid layer in the ion-etched and non-etched areas was taken using a surface analyzing scanning electron microscope. RESULTS: The normal mineralized dentin showed no reduction after argon-ion etching. The hybrid layer was reduced more than the adhesive resins during argon-ion etching. Reduction of the hybrid layer by argon-ion etching was greater with phosphoric acid-treated groups than with a self-etching-treated group (P < 0.05). The surface profiles of the non-etched, polished hybrid layer showed a slight concavity, with the deepest point in the middle of the hybrid layer. Argon-ion etching created non-uniform reduction of the hybrid layer, producing the deepest point close to the top of the hybrid layer in all systems.

Analysis of Variance↗
PubMed1997

Adsorption of mutagens to chlorophyllin-chitosan, an insoluble form of chlorophyllin.

Chlorophyllin, a water soluble derivative of chlorophyll is known to suppress the mutagenic and carcinogenic actions of compounds having polycyclic structures, e.g. heterocyclic amines and aflatoxin B1. There is evidence that this suppressing effect arises, at least in part, by a complex formation between the porphyrin-like structure of chlorophyllin and the planar molecular surfaces of these compounds. We report here that chlorophyllin can form an insoluble salt-like material when mixed with chitosan, a polyglucosamine, and that the solid chlorophyllin-chitosan thus prepared can efficiently trap polycyclic mutagenic compounds. The adsorbed polycyclic mutagens were elutable with buffers of acidic pH, but only to small extents. Chlorophyllin-chitosan may be expected to be useful as an intercepting agent against polycyclic mutagens and carcinogens.

Adsorption↗
PubMed1997

The Ras target AF-6 interacts with ZO-1 and serves as a peripheral component of tight junctions in epithelial cells.

The dynamic rearrangement of cell-cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2, and adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell-cell contacts. We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6) as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell-cell contact sites. We investigated roles of Ras and AF-6 in the regulation of cell-cell contacts and found that AF-6 accumulated at the cell-cell contact sites of polarized MDCKII epithelial cells and had a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy revealed a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacted with ZO-1 in vitro. ZO-1 interacted with the Ras-binding domain of AF-6, and this interaction was inhibited by activated Ras. AF-6 accumulated with ZO-1 at the cell-cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells resulted in the perturbation of cell-cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell-cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell-cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras.

Animals↗
PubMed1997

Analysis of the complex formed by Erythrina variegata chymotrypsin inhibitor with chymotrypsin and properties of the peptides prepared from the inhibitor by a limited proteolysis.

The stoichiometry of Erythrina variegata chymotrypsin inhibitor (ECI) and chymotrypsin interaction was previously estimated to be 1:2 by a titration of inhibitory activity. In the present study, gel-permeation chromatography and reverse-phase HPLC (RP-HPLC) were employed to analyze the complex formed by the inhibitor and enzyme. The results showed that ECI and chymotrypsin molecules undergo aggregation in the complex-forming buffer simultaneously with a binary complex consisting of one ECI and one chymotrypsin molecules in a soluble form. A mild lysylendopeptidase digestion of ECI produced two peptides in high yield, which were separated by RP-HPLC and characterized in terms of their structures and inhibitory activities. The N-terminal peptide, ECI-(1-107)-peptide, containing the primary reactive site retained a slight inhibitory activity, while the C-terminal peptide, ECI-(108-179)-peptide, exhibited no inhibitory activity. The inhibitory potency of the ECI-(1-107)-peptide was enhanced by the presence of the ECI-(108-179)-peptide in reconstituted mixture. Recovery of the native-like structure of the reconstituted complex was further indicated by fluorescence spectra, which showed strong conformational interaction between the two peptides; their dissociation constant Kd was calculated to be 209 nM. Taken together with the previous result obtained by chymotryptic digestion, it is suggested that the primary binding loop in ECI interacts with chymotrypsin not only by a standard mechanism but also by a non-substrate-like manner. Alternatively, ECI might have an additional binding segment in the N-terminal region which interacts with chymotrypsin by a non-substrate-like manner. Further, it is shown that the C-terminal region may support the native conformation of the binding loop(s) in the N-terminal region as an intramolecular chaperone.

Amino Acid Sequence↗
PubMed1997

Ribosomal protein S7: a new RNA-binding motif with structural similarities to a DNA architectural factor.

BACKGROUND: The ribosome is a ribonucleoprotein complex which performs the crucial function of protein biosynthesis. Its role is to decode mRNAs within the cell and to synthesize the corresponding proteins. Ribosomal protein S7 is located at the head of the small (30S) subunit of the ribosome and faces into the decoding centre. S7 is one of the primary 16S rRNA-binding proteins responsible for initiating the assembly of the head of the 30S subunit. In addition, S7 has been shown to be the major protein component to cross-link with tRNA molecules bound at both the aminoacyl-tRNA (A) and peptidyl-tRNA (P) sites of the ribosome. The ribosomal protein S7 clearly plays an important role in ribosome function. It was hoped that an atomic-resolution structure of this protein would aid our understanding of ribosomal mechanisms. RESULTS: The structure of ribosomal protein S7 from Bacillus stearothermophilus has been solved at 2.5 A resolution using multiwavelength anomalous diffraction and selenomethionyl-substituted proteins. The molecule consists of a helical hydrophobic core domain and a beta-ribbon arm extending from the hydrophobic core. The helical core domain is composed of a pair of entangled helix-turn-helix motifs; the fold of the core is similar to that of a DNA architectural factor. Highly conserved basic and aromatic residues are clustered on one face of the S7 molecule and create a 16S rRNA contact surface. CONCLUSIONS: The molecular structure of S7, together with the results of previous cross-linking experiments, suggest how this ribosomal protein binds to the 3' major domain of 16S rRNA and mediates the folding of 16S rRNA to create the ribosome decoding centre.

Amino Acid Sequence↗
PubMed1997

Human IgGFc binding protein (FcgammaBP) in colonic epithelial cells exhibits mucin-like structure.

Cloning a cDNA for human IgGFc binding protein (FcgammaBP) from human colonic epithelial cells reveals an mRNA and coding region of 17 and 16.2 kilobases, respectively. The predicted amino acid sequence contains 12 occurrences of a 400-amino acid cysteine-rich unit resembling that found in mucin. A motif (CGLCGN) in FcgammaBP is conserved in MUC2 and prepro-von Willebrand factor. The N-terminal 450-amino acid sequences are necessary and sufficient to confer IgG Fc binding activity. FcgammaBP mRNA is expressed only in placenta and colonic epithelial cells. These results suggest that FcgammaBP may play an important role in immune protection and inflammation in the intestines of primates.

Amino Acid Sequence↗
PubMed1997

Platelet endothelial cell adhesion molecule-1 is a major SH-PTP2 binding protein in vascular endothelial cells.

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is rapidly tyrosine phosphorylated in mechanically stimulated vascular endothelial cells (ECs). A 65-kDa protein from ECs specifically bound to the c-Src phosphorylated PECAM-1 cytoplasmic domain and was identified as a protein tyrosine phosphatase SH-PTP2 (SHP2, Syp). PECAM-1 was coimmunoprecipitated by anti-SH-PTP2 from EC extracts as a major binding protein, and the level of association increased when PECAM-1 was tyrosine phosphorylated. This association was mediated by SH2 domains of SH-PTP2. A rapid translocation of SH-PTP2 into cell-cell adhesion sites, where PECAM-1 was localized, occurred in mechanically stimulated cells. Our results suggest that PECAM-1 is a component of a mechanosensing machinery acting upstream of SH-PTP2.

Animals↗
PubMed1997

Aromatase in hyperplasia and carcinoma of the human prostate.

The expression and activity of aromatase was evaluated in 19 individuals with benign prostatic hyperplasia (BPH) and 26 prostatic carcinoma (PC) patients to elucidate the possible biological significance of in situ estrogen production in the development of human prostatic disorders. Marked aromatase immunoreactivity was observed in proliferative stromal cells, especially those around hyperplastic glands in 18 (95%) BPH patients and in stromal cells surrounding carcinomatous glands in 18 (69%) PC patient specimens. The percentage of aromatase-positive stromal cells did not differ between BPH and PC. No significant correlation was apparent between the percentage of aromatase-positive cells and either the extent of carcinoma differentiation or surgical stage in the PC patients. Quantitation of aromatase activity by the [3H] water assay yielded values of 27.23 +/- 6.87 and 26.52 +/- 9.12 fmol/hr/mg of protein for BPH (nine patients) and PC (nine patients), respectively. Reverse transcriptase and polymerase chain reaction analysis revealed that the mean aromatase mRNA content was 1.671 +/- 0.82 and 1.11 +/- 0.51 attomole/ng of total RNA (tRNA) for BPH (seven patients) and PC (four patients), respectively. There were no significant differences in aromatase activity or aromatase mRNA concentration between PC and BPH. The alternative use of multiple exons 1 of the aromatase gene was also examined. Predominant aromatase gene transcripts contained exon 1b in three of four of PC specimens and two of three BPH specimens examined, in contrast to the use of exon 1d previously described in normal prostate. Unlike breast and endometrium, therefore, aromatase expression in human prostate was not associated with malignancy. However, overexpression of aromatase, possibly attributable to abnormal gene regulation, may result in estrogen production in situ and play a role in the induction or development of human prostatic disorders.

Aged↗
PubMed1997

Overexpression of cyclin D1 in human pancreatic carcinoma is associated with poor prognosis.

We have investigated the expression of cyclin D1 in adenocarcinoma of the pancreas and the relevance of cyclin D1 expression to clinical outcome. In comparison to normal pancreas, Southern blot analyses revealed amplification of the cyclin D1 coding gene in 25% of the cases, whereas with reverse transcription-PCR, overexpression of mRNA was observed in 82% of the examined tissues. Immunohistochemically, we could demonstrate nuclear overexpression in tumor cells in 68.4%, and this protein accumulation correlated significantly with poor prognosis [median survival, 18.1 versus 10.5 months; P < 0.01 (chi2 test)].

Adult↗
PubMed1997

Functional expression on human trophoblasts of interleukin 4 and interleukin 7 receptor complexes with a common gamma chain.

The gamma chain in the high-affinity receptor complex for interleukin 2(IL-2) is used in receptor complexes for IL-4 and IL-7. We examined expression and function of the gamma chain in trophoblast cells. Flow cytometric studies demonstrated that the human placental cell line and choriocarcinoma cell lines expressed the IL-2R gamma chain. None of these cell lines expressed the IL-2R alpha and beta chains, whereas the alpha chains of IL-4R and IL-7R were present. The gamma chain and IL-4R and IL-7R mRNA were detected in human placental trophoblasts by the in situ hybridization method. The release of human chorionic gonadotropin by trophoblasts was accelerated by IL-4 and IL-7 in a concentration-dependent manner. These findings indicate that IL-4 and IL-7 are involved in trophoblast function.

Antigens, CD↗
PubMed1997

Leukocyte depletion and ONO-5046, a specific inhibitor of granulocyte elastase, prevent a stress-induced decrease in gastric prostaglandin I2 in rats.

To examine whether activated leukocytes may impair the endothelial production of prostaglandin (PG) I2, an important cytoprotective agent in gastric mucosa, we investigated the effects of leukocyte depletion and ONO-5046, a specific inhibitor of granulocyte elastase, on the gastric level of this prostaglandin and gastric mucosal injury in rats subjected to water-immersion restraint stress (WIR). Gastric 6-keto-PGF1 alpha was increased after 30 min of WIR, followed by a decrease to below baseline after 6 h of stress. Gastric levels of 6-keto-PGF1 alpha in leukopenic animals or animals pretreated with ONO-5046 after 1 h of stress were significantly higher than those of controls, levels after 6 h of stress were not lower than those preceding stress. Leukocytopenia or ONO-5046 significantly inhibited WIR-induced gastric mucosa lesion formation. Iloprost, a stable derivative of PGI2, prevented stress-induced lesions. These results suggest that activated leukocytes may play an important role in stress-induced gastric mucosal lesion formation by inhibiting production of PGI2.

6-Ketoprostaglandin F1 alpha↗
PubMed1997

Crystallization and preliminary X-ray crystallographic study of the ribosomal protein S7 from Bacillus stearothermophilus.

Overproduction and crystallization of Bacillus stearothermophilus ribosomal protein S7 (BstS7), a primary 16S rRNA binding protein and also a translational repressor protein, have been performed to analyze its three-dimensional structure by X-ray crystallography. Ribosomal protein BstS7 was expressed in the cytoplasmic fraction of the E. coli cells and purified to homogeneity. This recombinant BstS7 was used to produce crystals with P2(1) symmetry that diffracted to 2.5 A resolution which are suitable for high-resolution X-ray crystallographic analysis.

Crystallization↗
PubMed1997

Oxidized lipoproteins found in patients with NIDDM stimulate radical-induced monocyte chemoattractant protein-1 mRNA expression in cultured human endothelial cells.

Although oxidized low density lipoprotein (LDL) exists in plasma from diabetic patients, there are few studies on its biological activity. Thus, we investigated the biological potency of LDL plus intermediate density lipoprotein fraction isolated from 12 non-diabetic and 24 non-insulin-dependent diabetic subjects of similar age and body mass index, in order to induce monocyte chemoattractant protein-1 (MCP-1) mRNA expression in cultured human endothelial cells. MCP-1 mRNA content in the cells exposed to the lipoproteins isolated from the diabetic patients was significantly higher than that from the control subjects (p < 0.001). The increment of MCP-1 mRNA content was positively correlated with not only HbA1c (r = 0.58, p < 0.0001) but also lysophosphatidylcholine (LPC) content in the lipoprotein (r = 0.46, p < 0.005) and was negatively correlated with diene formation lag time as a marker of oxidizability of the lipoprotein (r = -0.33, p < 0.05). Treatments of the cells with either 50 mumol/l probucol, 50 mumol/l alpha-tocopherol, or 0.1 mmol/l deferoxamine suppressed the increase in MCP-1 mRNA content induced by diabetic lipoproteins, respectively. Furthermore, the diabetic lipoproteins activated nuclear transcription factor NF-kappa B in the cells, which was inhibited by pre-treatment of cells with 50 mumol/l probucol. These data indicate that oxidatively modified lipoproteins found in diabetic plasma stimulate MCP-1 gene expression in endothelial cells. The LPC content which reflects oxidative modification of lipoprotein is at least a possible marker of biological activity to increase an atherogenic cytokine in endothelial cells.

Antioxidants↗
PubMed1997

Role of granulocyte elastase in indomethacin-induced gastric mucosal lesion formation in rats.

To investigate whether granulocyte elastase may be involved in indomethacin-induced gastric mucosal injury, we examined the effects of the granulocyte elastase inhibitors ONO-5046 and L-658,758 on gastric mucosal lesion formation in rats given indomethacin. Both gastric mucosal lesion formation and gastric mucosal vascular damage were markedly attenuated in animals with leukocytopenia and in those given granulocyte elastase inhibitors. The administration of indomethacin significantly increased gastric myeloperoxidase activity, a measure of leukocyte accumulation, 3 hours after the administration of indomethacin compared with activity in animals receiving saline solution. The administration of ONO-5046 or L-658,758 significantly prevented this increase. Histologic examinations revealed submucosal edema and marked infiltration by leukocytes, as well as widespread necrosis with loss of surface epithelium. ONO-5046 and L-658,758 markedly prevented these histologic changes. Although cimetidine significantly prevented mucosal lesion formation, it did not inhibit either granulocyte elastase release from activated neutrophils in vitro or gastric accumulation of leukocytes in vivo. These results suggest that granulocyte elastase as well as gastric acid may play an important role in the pathologic process by which indomethacin induces gastric mucosal lesions.

Animals↗
PubMed1997

Novel role of prostacyclin in stress-induced gastric mucosal lesion formation in rats.

We investigated the novel role of prostacyclin (PGI2) in gastric mucosal lesion formation induced by stress in rats. Gastric 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha) levels were significantly increased 30 minutes after water-immersion restraint stress (WIR). Subcutaneous indomethacin (IM) (5 mg/kg) inhibited this increase but significantly exacerbated gastric mucosal lesion formation in rats subjected to WIR. Although gastric myeloperoxidase (MPO) activity was not increased by WIR, it significantly increased with time after WIR in animals pretreated with IM. NS-398, a selective inhibitor of cyclooxygenase-2, did not inhibit the WIR-induced increase in gastric 6-keto-PGF1alpha. Neither the gastric lesion index nor gastric MPO activity were affected in animals pretreated with NS-398 and subjected to WIR. WIR-induced mucosal lesion formation was significantly inhibited in animals given iloprost, a stable analog of PGI2, and in those with nitrogen mustard-induced leukocytopenia. Iloprost prevented the gastric leukocyte accumulation and exacerbation of gastric mucosal lesions induced by IM in animals subjected to WIR. These IM-induced events also were prevented in animals subjected to WIR with nitrogen mustard-induced leukocytopenia. These observations implicate leukocytes in the process leading to gastric mucosal lesions induced by WIR. The increase in WIR-induced gastric PGI2 synthesis, mainly mediated by cyclooxygenase-1, appears important in preventing lesion formation, not only by maintaining gastric mucosal blood flow but also by inhibiting leukocyte activation.

6-Ketoprostaglandin F1 alpha↗
PubMed1997

Hyperdiploid myeloma cell as an indicator of poor prognosis and drug refractoriness.

Although almost 40% of patients with multiple myeloma respond to initial chemotherapy, myeloma with no response to initial chemotherapy remains a serious problem. To understand the characteristics of drug-refractoriness of myeloma, fresh tumor cells from 13 untreated myeloma patients were fixed and stained with anti-human immunoglobulins and propidium iodide for subsequent flow cytometric analysis of DNA content. More than 10% of myeloma cells were hyperdiploid in eight cases (hyperdiploid + cases) while less than 10% of myeloma cells were hyperdiploid in five cases (hyperdiploid - cases). The proportion of hyperdiploid cells among all myeloma cells was highly correlated with incidence of myeloma cells with morphologically abnormal nuclei such as those with multiple-nuclei or convoluted nuclei (P = 0.001). Among the eight hyperdiploid + cases, two (2/8) showed good response to subsequent chemotherapy while four of five hyperdiploid - cases (4/5) responded well. Cases with poor response had more hyperdiploid myeloma cells (average 25.7% of all myeloma cells) than sensitive cases (average 6.8%), suggesting a contribution of hyperdiploid myeloma cells to primary drug resistance (P = 0.065). The 3 year survival rate of hyperdiploid+cases was 0% while that of the control group was 41.9%. These results suggest that myeloma cells with abnormal nuclear morphology may show hyperdiploidy and poor response to chemotherapy.

Adult↗