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S Hashiguchi

Publications and source records attributed to S Hashiguchi.

At least 19 recordsLinked to original sources
PubMed2004

Molecular characterization of a 10-kDa buckwheat molecule reactive to allergic patients' IgE.

BACKGROUND: Using the sera from buckwheat (BW)-allergic patients, several putative causative molecules were reported. However, few molecules were determined on the molecular structure. We demonstrated in 2000 that the major allergen with 24 kDa (BW24KD) is a legumin-like storage protein. OBJECTIVE: The aim of this study was to isolate and characterize further a major allergen with 10 kDa by molecular cloning. METHODS AND RESULTS: Buckwheat allergens were identified by immunoblotting analysis using sera from 14 allergic and two nonallergic individuals. We identified a protein with 10 kDa (BW10KD) that reacted with immunoglobulin E (IgE) more strongly than with IgG and IgA in 57% of the allergic patients but not with IgE in nonallergic individuals. Analyses were performed by N-terminal amino acid sequencing and molecular cloning. Physiological significance was assessed by an immunoblotting experiment showing that the reactivity of an allergic patient's serum IgE to BW10KD was competitively inhibited by natural BW extracts. CONCLUSION: Molecular cloning experiments indicated that BW10KD as a BW allergen was a member of the 2S-albumin multigene family.

Adolescent↗
PubMed2002

Time-dependent potentiation of the beta-cell is a Ca2+-independent phenomenon.

When isolated rat pancreatic islets are treated with 16.7 mM glucose, a time-dependent potentiation (TDP) of insulin release occurs that can be detected by subsequent treatment with 50 mM KCl. It has been thought that TDP by glucose is a Ca2+-dependent phenomenon and only occurs when exposure to glucose is carried out in the presence of Ca2+. In contrast to this, we now demonstrate TDP under stringent Ca2+-free conditions (Ca2+-free buffer containing 1 mM EGTA). In fact, under these Ca2+-free conditions glucose caused an even stronger TDP than in the presence of Ca2+. TDP induced by glucose in the absence of extracellular Ca2+ was unaffected by inhibitors of protein kinase C (PKC). However, cerulenin or tunicamycin, two inhibitors of protein acylation, eradicated TDP without affecting glucose metabolism. The TDP by glucose was not associated with an increase in the cytosolic free Ca2+ concentration ([Ca2+]i) during subsequent treatment with high K+. Exposure of islets to forskolin under Ca(2+)-free conditions did not cause TDP despite a large increase in the cellular cAMP levels. In conclusion, glucose alone induces TDP under stringent Ca2+-free conditions when [Ca2+]i was significantly lowered. Protein acylation is implicated in the underlying mechanism of TDP.

Acylation↗
PubMed2002

Human single-chain Fv (scFv) antibody specific to human IL-6 with the inhibitory activity on IL-6-signaling.

Human anti-IL-6 antibody may be useful for the immunotherapy of various inflammatory diseases, such as rheumatoid arthritis. Since IL-6 is a growth factor for B cell hybridoma, it is not easy to isolate murine B cell hybridomas producing the anti-IL-6 antibody with the IL-6-signaling inhibitory activity. In this study, the antibody library (Vgamma-Vkappa, Vgamma-Vlambda, Vmu-Vkappa or micro-Vlambda ligated into the pCANTAB 5E phagemid vector) was prepared from peripheral blood mononuclear cells of 20 healthy subjects. The phage display library was panned with an IL-6-coated plastic plate, and the binding specificity was confirmed by ELISA and BIAcore. From the antibody library (Vgamma-Vlambda), five IL-6-specific phage clones were isolated. The effects of the soluble scFvs purified from these phage clones were tested on the growth of the IL-6-dependent human cell line, KT-3. Two of these clones significantly inhibited the growth of KT-3, and three showed no inhibition.

Amino Acid Sequence↗
PubMed2001

Ceramic endoprosthesis for aggressive giant cell tumors near the knee: eight cases followed for more than 10 years.

Eight patients with locally aggressive giant cell tumor were treated with marginal resection and implantation of a ceramic endoprosthesis with a non-hinged knee joint. The patients have been followed-up for between 10 to 16 years. Newly formed bone appeared at the junction between implant and cortex and in 2 cases subsidence of the implant was seen. All the patients had good function of the knee and no revision was needed.

Adult↗
PubMed2001

Acridine orange induces binucleation in chondrocytes.

OBJECTIVE: Although it is well known that binuclear cells commonly appear among the chondrocytes of normal cartilages as well as among neoplastic chondrocytes of chondrosarcomas, the mechanism of binucleation is still unclear. Therefore, this study was undertaken to clarify the mechanism of binucleation in chondrocytes, using primary culture cells of growth plate cartilage. DESIGN: These chondrocytes were exposed to acridine orange (AO) which is a fluorescent dye for differentiating certain DNAs and RNAs in nuclei and cytoplasm, and which inhibits mitosis. After exposure to 0.5 microg/ml AO, for 0, 6, 24, 48, and 96 h, the following parameters were investigated: (1) cell growth rate (GR); (2) frequency of hyperdiploid cells (%HDC) by DNA cytofluorometry; (3) mitotic index (MI); (4) BrdU labeling index (LI); (5) frequency of binuclear cells (%BNC). RESULTS: Compared with the control cells, which were cultured in AO-free medium, the GR was remarkably inhibited at 24 h. MI was also decreased from 6 to 24 h, and LI decreased at 48 h. However, these parameters were recovered at 96 h. The %HDC was increased from 6 to 96 h, and the %BNC was also increased to a maximum of six times that of the control cells at 96 h. DISCUSSION: These results suggested that the binuclear cells observed among the cultured chondrocytes may be formed from G2 arrested cells by amitotic nuclear division, but not by mitosis without cytoplasmic division or cell fusion.

Acridine Orange↗
PubMed2001

DNA cytofluorometric analysis of chondrocytes in human articular cartilages under normal aging or arthritic conditions.

OBJECTIVE: Since most chondrocytes in articular cartilage are in the resting phase (G0) of the cell cycle, it has been difficult to investigate their cell kinetics using 3H-thymidine autoradiography, or immunohistochemistry. In the present study, DNA cytofluorometry, which is useful to analyse the cell kinetics even for such inactive cell populations as in the G0 phase, was applied to human chondrocytes of the articular cartilages under normal aging and pathologic conditions such as osteoarthritis (OA), rheumatoid arthritis (RA), and aseptic necrosis (AN). DESIGN: The human articular cartilages for the study were obtained from autopsy and surgical materials. Fifty joints were used for the study of aging, 54 for the study of OA, 20 for studying RA, and 10 for AN study. The isolated chondrocytes were quickly prepared from fresh articular cartilages, using a combination method of enzymatic digestion with papain and collagenase, followed by mechanical cell separation by churning and homogenization. RESULTS: The DNA histograms obtained by cytofluorometry with propidium-iodide staining showed that most chondrocytes had diploid DNA content (2c) in all cartilages studied, suggesting that they were in the G0 phase. However, there were a few chondrocytes having tetraploid DNA content (4c) in the normally aged articular cartilages, and there were some cells having DNA content between 2c and 4c in the diseased cartilages. The former cells were considered to be G0-phase cells of the 4c chondrocytes, while the latter cells were considered to be in the DNA synthetic (S) phase or G2-phase of the 2c chondrocytes. The frequency of 4c chondrocytes in aged cartilage was significantly increased, compared to that in the young cartilage. In contrast to the normal cartilage, the frequency of S- and G2-phase cells, which was expressed as the S- G2 index, in diseased cartilages (OA, RA and AN) was significantly high (P< 0.0001). In OA cartilage, the S-G2 index was much higher in the severe or moderate stage than in the mild stage, suggesting that the chondrocytes in clusters may actively proliferate. CONCLUSION: These results showed that in normal articular cartilages most chondrocytes are in the G0 phase, while some became 4c polyploid cells, and that these G0-phase chondrocytes had a potential to proliferate under diseased conditions.

Adolescent↗
PubMed2001

Peptide mimics of monocyte chemoattractant protein-1 (MCP-1) with an antagonistic activity.

In this study, we attempted to analyze the peptide motifs recognized by 24822.111 and F9, monoclonal antibodies (mAbs) that inhibit the chemotactic activity of monocyte chemoattractant protein-1 (MCP-1), a member of the CC subfamily of chemokines. We isolated phage clones from a phage display library and identified six peptide motifs. One of these clones, C27, was strongly and specifically recognized by 24822.111 mAb, while another, G25, was similarly recognized by F9 mAb. Both the C27 motif and the G25 motif contain two cysteines in their sequences and have little homology to the primary amino acid sequence of MCP-1. These clones, however, bound to THP-1 cells, and the binding was competitively inhibited by MCP-1. The clones strongly inhibited the MCP-1-induced chemotaxis of human monocytes. The synthetic and intramolecularly disulfide-linked peptides of C27 and G25 (sC27 and sG25) also inhibited the chemotaxis induced by MCP-1, while their derivatives with serine in place of cysteine did not, suggesting the importance of the loop structure for the inhibition. These results suggest that sC27 and sG25 may mimic the MCP-1-binding domain to the MCP-1 receptor.

Amino Acid Sequence↗
PubMed2001

Multiple sclerosis with caudate lesions on MRI.

A 31-year-old woman displayed sleepiness and impairment of recent memory. T2-weighted MRI revealed high signal intensity lesions in the bilateral basal ganglia, thalamus, and brainstem. Although remission was achieved with corticosteroid therapy, she again displayed memory dysfunction and emotional disturbance one year later, at which time MRI disclosed new lesions in the right caudate nucleus and left frontal white matter. Corticosteroid therapy lead to improvement, and she suffered no recurrence on maintenance steroid therapy. These findings suggest that caudate lesions do occur in multiple sclerosis, the manifestations of which can be abulia and memory dysfunction, as in the present case.

Adrenal Cortex Hormones↗
PubMed2001

Studies on anti-MRSA parenteral cephalosporins. IV. A novel water-soluble N-phosphono type prodrug for parental administration.

A systematic approach for improving the water-solubility of anti-MRSA (methicillin-resistant Staphylococcus aureus) cephalosporin derivatives is described. We first tried to improve the water-solubility of 7beta-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2(Z)-fluoromethoxyiminoacetamido]-3-[(E)-2-(1-methylimidazo[1,2-b]pyridazinium-6-yl)thiovinyl]-3-cephem-4-carboxylate (1a) by substitution of the C-3' pharmacophore. Replacement of the C-3' pharmacophore with a 1-methyl-4-pyridinio group improved the water-solubility without decreasing the anti-MRSA activity. Furthermore, we applied the N-modified prodrug strategy to the C-7 acyl group in order to enhance the water-solubility drastically. Among the compounds prepared, the N-phosphono type prodrugs 2a(1-methylimidazo[1,2-b]pyridazinium derivative) and 2b (1-methyl-4-pyridinio derivative) showed water-solubility appropriate for a product intended for intravenous injection and in vivo anti-MRSA activity comparable to that of vancomycin.

Animals↗
PubMed2001

A new hypoglycemic agent, JTT-608, evokes protein kinase A-mediated Ca(2+) signaling in rat islet beta-cells: strict regulation by glucose, link to insulin release, and cooperation with glucagon-like peptide-1(7-36)amide and pituitary adenylate cyclase-activating polypeptide.

A new nonsulfonylurea oral hypoglycemic agent, JTT-608, has been reported to stimulate insulin release at elevated, but not low, glucose concentrations and consequently not to induce hypoglycemia in rats. Accordingly, this drug is potentially a safer antidiabetic agent than sulfonylureas. To explore the mechanisms underlying this glucose-dependent insulinotropism, the present study investigated the effects of JTT-608 on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and protein kinase A (PKA) activity in rat islet beta-cells by microfluorometry using, respectively, fura-2 and a fluorescence PKA substrate, DR II. In the presence of glucose at normal and elevated concentrations (5.0-16.7 mM) JTT-608 (30-1000 microM) concentration dependently increased [Ca(2+)](i) in up to 88% of single beta-cells, whereas at lower glucose concentrations (2.8 and 4.2 mM) it had little effect. The [Ca(2+)](i) responses were inhibited under Ca(2+)-free conditions and by nitrendipine, an L-type Ca(2+) channel blocker. JTT-608 rapidly activated PKA and a PKA inhibitor, H89, inhibited [Ca(2+)](i) responses to JTT-608. JTT-608 also stimulated insulin release from rat islets in a glucose- and Ca(2+)-dependent manner. The glucose-unresponsive beta-cells, which failed to respond to 8.3 mM glucose with increases in [Ca(2+)](i), were frequently recruited to [Ca(2+)](i) increases by JTT-608. JTT-608 also induced oscillations of [Ca(2+)](i). Glucagon-like peptide-1(7-36)amide (GLP-1), pituitary adenylate cyclase-activating polypeptide (PACAP), and acetylcholine (ACh) enhanced the action of JTT-608 on [Ca(2+)](i). In conclusion, JTT-608 evokes PKA-mediated Ca(2+) influx and Ca(2+) signaling in rat islet beta-cells in a glucose-regulated manner, which may account for its glucose-dependent insulinotropism. JTT-608 and neurohormones may cooperatively activate islet beta-cells under physiological conditions.

Acetylcholine↗
PubMed2000

Bilateral metachronous periosteal tibial amyloid tumors.

Localized primary periosteal amyloid tumors are extremely rare. A case of bilateral tibial amyloid tumor is presented. A 62-year-old woman initially presented with a painful mass in the anterior aspect of the right leg. There was no evidence of underlying systemic disease, including chronic infection or malignancy. Based on the results of resistance with Congo red staining to treatment with potassium permanganate and positivity for kappa light chain, we classified this particular case as AL-type amyloidosis. The patient noticed a swelling in the opposite leg 2 years later. The second tumor was also an AL-type amyloidoma. Amyloid tumors are generally solitary. This is the first case of bilateral periosteal amyloid tumors of the AL-type occurring in the tibiae.

Amyloidosis↗
PubMed2000

Development of bone canaliculi during bone repair.

We recently found that silver impregnation staining with protargol (silver protein), that is, a modified Bodian method, is useful for histologically identifying the details of bone canaliculi structure, using thin sections of decalcified bone tissues. With this staining method, we conducted the present study to assess the development of bone canaliculi during the process of intramembranous ossification using a fracture-like stimulation model of the rat femur. After making a drill-hole in the cortex of the rat femur, decalcified thin sections were obtained after 3, 5, 7, and 14 days by the standard paraffin-embedding procedure. Silver staining for bone canaliculi was performed using our previously reported technique. The results showed that woven bone covered the fracture surface of the cortex after 5 days, then immature lamellar bone attached to the woven bone after 7 days, and finally the lamellar bone matured and became thick with appositional growth after 14 days. The osteocytes in the woven bone appeared at an early stage of bone repair and developed a few canaliculi that were short and irregularly distributed in the osteoid matrix, while the osteocytes in the lamellar bone at a late stage formed many bone canaliculi that were long and regularly distributed in mature bone matrix. Therefore, we concluded that woven bone osteocytes may be necessary for induction of the lamellar bone osteocytes followed by active appositional growth of the lamellar bone at the early stage of bone repair, and also that both bone tissues could be clearly distinguished from one another based on the pattern of development of bone canaliculi by the osteocytes, as seen with the use of our sensitive staining method.

Animals↗
PubMed2000

Watershed infarction associated with dementia and cerebral atrophy.

A 63-year-old man was admitted with progressive left hemiparesis and left homonymous hemianopsia of 1 month's duration. During the 2 months before admission, he had suffered from slowly progressive dementia. The diagnosis of right-sided watershed (WS) infarction was made. He exhibited slow progression of dementia and cerebral atrophy during the period of observation after discharge. There was a positive relationship between cerebral atrophy and the degree of dementia. In the present case, WS infarction caused by right internal carotid artery occlusion might be related to dementia and cerebral atrophy.

Atrophy↗
PubMed2000

Osteoblastic differentiation and P-glycoprotein multidrug resistance in a murine osteosarcoma model.

A recent study of multidrug resistance (MDR) 1 gene transfected osteosarcoma cells found a cause-effect relationship between increased expression of P-glycoprotein (P-gp) and a low aggressive phenotype. However, several experimental and clinical studies have observed contradictory findings in that P-gp expression has been associated with tumour progression. In the present study, we characterized P-gp-positive and P-gp-negative single-cell clones of a murine osteosarcoma, to further investigate the relationship between P-gp expression and changes in cell phenotype. Although these clones were all selected by doxorubicin (DOX) exposure, they were heterogeneous with respect to MDR1 gene expression. The P-gp-positive clones revealed MDR phenotype, whereas the P-gp-negative clones showed no resistance to drugs. Morphological and functional analysis showed that both the P-gp-positive and P-gp-negative clones were more differentiated than the parent cells in terms of enhanced activity of cellular alkaline phosphatase, an increase in well-organized actin stress fibres and enhanced osteogenic activity. Moreover, these subclones all displayed a decrease in malignant potential such as oncogenic activity, tumour growth rate and metastatic ability, regardless of their P-gp status. These results indicate that the observed osteoblastic differentiation and less aggressive phenotype in DOX-selected osteosarcoma cells may not only be explained by the direct effect of P-gp, and accordingly, consideration of the effect of DOX, as well as P-gp, appears to be important.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Intranasal sensitization of Japanese cedar pollen by the co-administration of low doses of cholera toxin but not its recombinant B subunit to mice.

We evaluated the effects of cholera toxin (CT) and the B subunit of cholera toxin (CTB) on the intranasal sensitization of Japanese cedar pollen (JCP) in mice. JCP suspended in phosphate-buffered saline was administered into the nostrils of mice in combination with varying doses of CT or recombinant CTB(r-CTB) once a week for 5 weeks. Antibody responses specific to sugi basic protein (SBP) were monitored by ELISA for seven weeks. The sensitization of JCP alone did not induce IgG1, IgG2b, IgG2a, IgE or IgA. In contrast, sensitization of JCP in combination with CT (JCP/CT) elicited the prominent production of SBP-specific IgG1 and low levels of IgG2b and IgG2a on Day 49. IgE production was detected only in the serum of mice which were treated with JCP/CT, and not under any other protocol. Using spleen cells from these mice, cytokine production was examined by ELISA in culture supernatants after they had been stimulated in vitro with major cedar pollen allergens, Cry j 1, Cry j 2 or SBP. Notable responses were an increase of IFN-gamma as well as IL-4 in JCP/CT-sensitized cells stimulated with Cry j 2, but not in those stimulated with Cry j 1. No significant differences were detected in IL-5 production among the experimental groups. Histopathological examination, however, showed that eosinophil infiltration was evident in the nasal mucosa of the JCP/CT-sensitized mice following challenge with JCP/CT, but weak with BSA/CT or CT alone. Thus, the immunological and histological analyses indicated that the co-administration of a low dose of CT in combination with JCP allows the induction of pollen-allergic states in mice.

Adjuvants, Immunologic↗