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Biomedical subjects

D Ishikawa

Publications and source records attributed to D Ishikawa.

At least 19 recordsLinked to original sources
PubMed2001

EXAFS measurements for liquid Ge-Si alloys.

EXAFS measurements around the Ge-K edge have been carried out for liquid Ge-Si alloys for the first time to investigate the local structure around a Ge atom. To perform the EXAFS measurements for the liquid alloys with high melting temperatures, a new sapphire cell have been developed. The measurements were carried out for the liquid alloys from 10% to 60% of Si and the crystalline ones from 10% to 70% of Si as a reference. EXAFS oscillations, x(k), are observed even at 1480 degrees C for liquid Ge(0.4)Si(0.6). The position of the first peak in the radial distribution function obtained from Fourier transform of x(k) is shifted towards smaller distance for liquid and crystalline alloys with increasing Si concentration. The results of a curve-fit analysis in a harmonic approximation show that Ge-Ge and Ge-Si bonds in the liquid alloys become long with increasing Si concentration while those become slightly short in the crystaline ones.

Journal Article↗
PubMed2000

Suppression of GD1alpha ganglioside-mediated tumor metastasis by liposomalized WHW-peptide.

GD1alpha ganglioside-replica peptides were recently isolated from a phage-displayed random pentadecapeptide library by assaying for inhibition of adhesion of RAW117-H10 lymphosarcoma cells to hepatic sinusoidal microvessel endothelial (HSE) cells. We show here that the Trp-His-Trp (WHW) peptide was identified as a minimal sequence of the GD1alpha-replica peptide WHWRHRIPLQLAAGR. The addition of WHW peptide-attached liposomes displayed efficient inhibition of liver metastasis of RAW117-H10 cells as well as of GD1alpha-mediated adhesion of RAW117-H10 cells to HSE cells in vitro. These results suggest that engineered liposomes for peptide delivery are applicable to treatment for metastasis.

Amino Acid Sequence↗
PubMed1999

Selection of ganglioside GM1-binding peptides by using a phage library.

Ganglioside Gal beta1 --> 3GalNAc beta1 --> 4(NeuAc alpha2 --> 3) Gal beta1 --> 4Glc beta1 -->1'Cer (GM1)-binding peptides were obtained from a phage-displayed pentadecapeptide library by an affinity selection. The selection processes were in situ-monitored by a quartz-crystal microbalance method, on which a ganglioside GM1 monolayer was transferred. After five rounds of biopanning, the DNA sequencing of 18 selected phages showed that only three individual clones were selected. The peptide sequences of the random region were found to be DFRRLPGAFWQLRQP, GWWYKGRARPVSAVA and VWRLLAPPFSNRLLP. Binding constants of these phage clones to the GM1 monolayer were 10(10) M(-1). Three synthetic pentadecapeptides inhibited the binding of cholera toxin B subunit to the GM1 monolayer with an IC50 of 24, 13 and 1.0 microM, respectively. These peptides will be useful for searching functional roles of ganglioside GMI.

Amino Acid Sequence↗
PubMed1999

Epitope mapping of rat neutralizing monoclonal antibody against human immunodeficiency virus type-1 by a phage peptide library: comparison with ELISA using synthetic peptides.

We generated a rat monoclonal antibody (mAb W#10) with the ability to neutralize human immunodeficiency virus type 1IIIB (HIV-1IIIB) infection. The epitope recognized by mAb W#10 was defined as R-I-Q-R-G-P-G by enzyme-linked immunosorbent assay (ELISA) with the use of synthetic peptides. The filamentous phage clones displaying random 15-amino-acid peptides on the amino terminus of the pIII coat protein reacting with mAb W#10 were identified with affinity and immunological selection procedures. Thirteen out of 16 selected phage clones contained the G-X-G-R-X-F sequence in the coat protein region representing significant homology to a part of conserved G-P-G-R-A-F sequence in the V3 loop of various HIV-1 strains. In addition, the phage clones included the G-X-G sequence in the sequence detected by synthetic peptides as the recognition site. The selected phage clones were stained by mAb W#10 specifically and were able to compete with mAb binding to cells expressing viral antigens.

Amino Acid Sequence↗
PubMed1999

[A case of orbital lymphoproliferative lesion diagnosed as malignant lymphoma after recurring 11 years later].

BACKGROUND: Most primary lymphoproliferative lesions in the ocular adnexa, including the eyelid, conjunctiva, and orbit, are diagnosed as low-grade malignant lymphomas. Recurrence and dissemination of these tumors are rare in Japan. The long-term prognosis for this disorder still remains to be clarified. CASE AND METHOD: A 53-year-old woman was first referred to us for right orbital tumor in 1986. After subtotal resection of the tumor, the patient received no additional treatment. She visited us in 1997 with the complaint of bilateral orbital tumor. Biopsied specimens were examined histologically using hematoxylin-eosine and immunohistological staining. Southern blot hybridization was used to detect immunoglobulin gene rearrangement. The paraffin-embedded specimen obtained in 1986 was also examined for immunoglobulin gene rearrangement using nested polymerase chain reaction technique. FINDINGS: The specimens from 1997 and 1986 were both diagnosed as lymphoid type of inflammatory pseudotumor, based on polyclonal B cell immunohistological staining. Immunoglobulin gene rearrangement was present in both specimens. CONCLUSION: The orbital tumor resected in 1986 was a low-grade malignant lymphoma which disseminated systemically 11 years later. This case shows a long-term course of orbital lymphoproliferative lesion with positive immunoglobulin gene rearrangement. It also shows the importance of follow-up over 10 years in the case of low-grade malignant lymphoma of the ocular adnexa.

Diagnosis, Differential↗
PubMed1998

GD1alpha-replica peptides functionally mimic GD1alpha, an adhesion molecule of metastatic tumor cells, and suppress the tumor metastasis.

A novel peptide technology to produce mimicking peptides of carbohydrate moiety (which we propose to name glyco-replica peptides) is a useful tool to elucidate the functions of glycoconjugate. Carbohydrate moiety of ganglioside GD1alpha functions as a molecule involved in the adhesion between murine highly metastatic lymphoma RAW117-H10 cells and hepatic sinusoidal endothelial (HSE) cells. To prepare peptides which mimic the carbohydrate structure of GD1alpha, phage clones expressing peptides which bound to a monoclonal antibody against GD1alpha (KA17) were isolated from a phage-displayed random peptide library. Four phage clones having affinity to the monoclonal antibody KA17 were isolated, and these clones showed inhibitory effect on the binding of KA17 to GD1alpha. The amino acid sequences of the displayed pentadecamers were determined, and one of the phages displaying sequence WHWRHRIPLQLAAGR bound to HSE cells directly and showed the highest inhibitory effect on the adhesion between RAW117-H10 cells and HSE cells. The synthesized peptides having the same sequences to the displayed 15mers in the four isolated phage clones also showed the inhibitory effect on the adhesion of RAW117-H10 cells to HSE cells, and, again, the WHWRHRIPLQLAAGR peptide showed the highest inhibitory effect. Furthermore, intravenous injection of the peptide brought almost complete inhibition of the metastasis of RAW117-H10 cells to lung and spleen, and about 50% inhibition of the liver metastasis. These results indicate that GD1alpha plays an important role for metastasis of RAW117-H10 cells, and the peptides obtained by the present procedure are able to mimic the functional role of the glycoconjugate.

Amino Acid Sequence↗
PubMed1998

Assembly of flammutoxin, a cytolytic protein from the edible mushroom Flammulina velutipes, into a pore-forming ring-shaped oligomer on the target cell.

Flammutoxin has been previously isolated as a cardiotoxic and cytolytic polypeptide of 22 or 32 kDa from the fruiting bodies of the edible mushroom Flammulina velutipes. In the present study, we purified flammutoxin as a single haemolytic protein of 31 kDa and studied the mode of its cytolytic action. (1) Flammutoxin caused efflux of potassium ions from human erythrocytes and swelling of the cells before haemolysis. (2) Flammutoxin did not lyse human erythrocytes in the presence of non-electrolytes with hydrodynamic diameters of >5.0 nm, although it caused leakage of potassium ions and swelling of the cells under the same conditions. (3) Experiments including solubilization of cell-bound toxin with 2% (w/v) SDS at 20 degrees C and subsequent Western immunoblots showed that flammutoxin formed a band corresponding to 180 kDa under the conditions where it lysed erythrocytes. (4) Electron microscopy of flammutoxin-treated human erythrocytes revealed the presence of a ring-shaped structure with outer and inner diameters of 10 and 5 nm, respectively, on the cells. (5) A ring-shaped toxin oligomer of the same dimensions was solubilized from the toxin-treated human erythrocytes with 2% (w/v) SDS at 20 degrees C and isolated by a sucrose-gradient ultracentrifugation. These data indicated that flammutoxin assembles into a ring-shaped oligomer possessing a hydrophilic pore of 4-5 nm on target cells.

Basidiomycota↗
PubMed1998

[A case of syringomatous carcinoma derived from the lid].

A 73-year-old male presented with a slowly growing tumor in the right lower eyelid of one year's duration. The condition had been diagnosed elsewhere as poorly differentiated squamous cell carcinoma by biopsy. The residual tumor progressed rapidly and metastatized to the ipsilateral preauricular lymph nodes. We performed orbital evisceration and radical neck resection. Microscopically, the tumor showed small epithelial nests, cords, and ductal structure buried in abundant collagenous stroma. Besides focal squamous differentiation with keratinization in the surface area, the tumor showed, as cardinal features, ductal differentiation possessing comma-like extensions identical to syringoma. Cellular atypia, invasive growth pattern, and remarkable perineural invasion were suggestive of malignancy. These features led to the diagnosis of syringomatous carcinoma. Immunohistologically, the tumor showed positive staining for cytokeratine and epithelial membrane antigen (EMA). It was mostly negative for S-100 and carcinoembryonic antigen (CEA).

Aged↗
PubMed1997

Preparation of peptides which mimic glycosphingolipids by using phage peptide library and their modulation on beta-galactosidase activity.

We describe the use of a phage-displayed random pentadecamer peptide library for searching glycosphingolipid mimicking peptides. Two phage clones (AD-1 and AD-2) were selected by biopanning using monoclonal antibody AD117m, directed to lactotetraosylceramide (Lc4Cer). The amino acid sequences of the selected clones showed high homology (VPPXFXXXY) in 9-mer. Three phage clones were selected by using monoclonal antibody H11, directed to neolactotetraosylceramide (nLc4Cer), the linkage isomer of Lc4Cer, and the displayed amino acid sequences were compared. One of these peptides showed the same amino acid sequence as that of AD-2 except for one amino acid substitution. Pentadecamer, 9-mer and point mutated 9-mer peptides were synthesized on the basis of the displayed amino acid sequences. Binding activity of the peptides to the monoclonal antibodies or Ricinus communis lectin showed that 9-mer peptides are enough to mimic the epitope carbohydrate structure. Furthermore, six of the synthesized peptides inhibited Jack bean beta-galactosidase activity towards nLc4Cer at a high concentration of the enzyme, whereas at lower enzyme concentrations some peptides showed potent activation of the enzyme activity. This is the first report of carbohydrate mimicking peptides which modulate glycosidase activity.

Amino Acid Sequence↗
PubMed1997

TLC blotting: application to microscale analysis of lipids and as a new approach to lipid-protein interaction.

A simple method for the transfer of phospholipids and glycosphingolipids from a high-performance thin-layer chromatography (HPTLC) plate to a polyvinylidene difluoride (PVDF) membrane, called thin-layer chromatography (TLC) blotting, and its application in lipid research are described. Most of the lipids developed on the HPTLC plate are blotted quantitatively. Detection of the lipids on the membrane is done by chemical and immunological staining. This method makes it possible to purify individual lipid components separated on an HPTLC plate and to determine the structures of lipids in limited biological samples when combined with direct mass spectrometric analysis (TLC blotting/MS). A binding assay and the identification of ligands for microorganisms can be made using TLC blotting/MS. In addition, simple methods for the detection of enzymes on a substrate-immobilized PVDF membrane are described.

Animals↗
PubMed1997

Ganglioside GD1alpha functions in the adhesion of metastatic tumor cells to endothelial cells of the target tissue.

We studied the role of glycosphingolipids expressed on the cell surfaces of a metastatic tumor cell line. Glycosphingolipid compositions of the low-metastatic murine lymphosarcoma cell line RAW117-P and its sub-line, RAW117-H10, which shows higher metastatic potential for the liver than P cells, were compared. Both types of cells had LacCer, Gg3Cer, and Gg4Cer as the major neutral glycosphingolipids and GM1b and GD1alpha as the gangliosides. There are differences in glycosphingolipid contents, the neutral glycosphingolipid contents in the parental cells being 1.5-fold higher than that in the variant ones. In contrast, the level of GD1alpha in H10 cells was twice as much as that in the P cells; however, the expression of other gangliosides was down-regulated. On the basis of the results of glycosphingolipid analysis, we investigated the functional role of GD1alpha in H10 cells in the adhesion of the tumor cells to the target tissue by using hepatic sinusoidal endothelial (HSE) cells. GD1alpha and GM1b inhibited the adhesion when HSE cells were incubated prior to coculture with the tumor cells. This inhibitory effect by GD1alpha and GM1b was observed within 30 min after addition of H10 cells to HSE cells and was dose dependent. GD1alpha showed a higher inhibitory effect on the adhesion than GM1b, whereas other glycosphingolipids showed no inhibitory effect. Anti-GD1alpha monoclonal antibody also inhibited the adhesion between the H10 and HSE cells. When cultured without fetal bovine serum for 30 min in a various glycosphingolipids-coated dish for bacterial culture, HSE cells adhered to the area coated with GD1alpha but not to areas coated with other glycosphingolipids. HSE cell adhesion depended on the amount of GD1alpha coated on the plate. These data indicate that GD1alpha functions as an adhesion molecule in the process of metastasis of H10 cells.

Animals↗
PubMed1996

Tyrosine phosphorylation of protein kinase C delta-isoform and its association with membrane.

Stimulation of CHO cells stably overexpressing the delta-isoform of protein kinase C (delta PKC) by phorbol ester resulted in the tyrosine phosphorylation and association of the enzyme with particulate fraction. This tyrosine phosphorylation of delta PKC occurred preferentially in the enzyme prephosphorylated at serine/threonine residue(s). The enzymatic activity of tyrosine-phosphorylated delta PKC was dependent on both phospholipid and diacylglycerol which is the same as the non-tyrosine-phosphorylated form, and no significant difference was observed between the two forms for their kinetic properties and specific activities. When delta PKC was phosphorylated at tyrosine in vitro, phosphatidylserine and either diacylglycerol or phorbol ester were needed for the maximal rate of the reaction, suggesting that the tyrosine phosphorylation is a consequence of, but not a prerequisite for the enzyme activation. The tyrosine-phosphorylated delta PKC was associated with the particulate fraction, and presumably exists as a large complex in the membrane of the stimulated cells. It may be possible that the tyrosine phosphorylation is related to sustained activation and/or targeting of the delta PKC isoform.

Animals↗
PubMed1995

New methods using polyvinylidene difluoride membranes to detect enzymes involved in glycosphingolipid metabolism.

Two new methods are described using polyvinylidene difluoride (PVDF) membranes to detect enzymes involved in glycosphingolipid metabolism. One is the detection of enzymes on a PVDF membrane to which glycosphingolipids have been transferred from an HPTLC-plate by TLC blotting. The glycosphingolipids on the membrane were incubated with an enzyme preparation, and the resulting product was detected by immunostaining with a monoclonal antibody directed to the product. IV(3)NeuAc(alpha)Lc(4)Cer that had been transferred to a PVDF membrane was incubated with Clostridium perfringens sialidase. Lc(3)Cer then was transferred to the membrane, and the whole incubated with bovine milk beta1-4 galactosyltransferase, after which the product, nLc(4)Cer, was detected by immunostaining with the monoclonal antibody H11 that recognizes the GAl(beta)1-4GlcNAc(beta)1-3Gal structure of neolactoseries glycosphingolipids. This method detects glycosphingolipid-metabolizing enzymes that produce the same epitope. The second method is the detection of enzymes located on a polyacrylamide gel. A sialidase preparation from C. perfringens was subjected to native polyacrylamide electrophoresis then transferred to a PVDF membrane impregnated with IV(3)NeuAc(alpha)nLc(4)Cer as the enzyme substrate. The membrane then was incubated, and the resulting product, nLc(4)Cer, detected by immunostaining with the monoclonal antibody H11. The area stained shows the location of the sialidase on the polyacrylamide gel. The method is effective for determining the apparent molecular weight(s) of the enzyme(s) in the crude enzyme preparation. This was demonstrated by using crude sialidase preparation from C. perfringens.

Chromatography, Thin Layer↗
PubMed1995

Direct mass spectrometric analysis of glycosphingolipid transferred to a polyvinylidene difluoride membrane by thin-layer chromatography blotting.

A simple, rapid method for the analysis of glycosphingolipid that combines "thin-layer chromatography (TLC) blotting" and mass spectrometry is reported. Glycosphingolipids developed by TLC were transferred to a polyvinylidene difluoride membrane by TLC blotting, after which the glycosphingolipid band on the membrane was excised and placed on a mass spectrometer probe tip, and a few microliters of triethanolamine was added as the matrix. The sample was analyzed by secondary ion mass spectrometry. About 1 microgram of glycosphingolipid subjected to TLC can be analyzed. The major advantage of this method is that glycosphingolipid can be analyzed structurally without purification by repeated column chromatography.

Animals↗
PubMed1994

Blotting of glycolipids and phospholipids from a high-performance thin-layer chromatogram to a polyvinylidene difluoride membrane.

A simple method of blotting glycosphingolipids from a high-performance thin-layer chromatography (HPTLC) plate to a polyvinylidene difluoride (PVDF) membrane is described. The developed HPTLC plate is dipped in a solvent mixture (isopropanol/0.2% CaCl2/methanol, 40/20/7 by volume) for blotting, after which first a PVDF membrane and then a glass microfiber filter is placed on the plate. The assemblage then is pressed for 30 s with heating. Most of the glycosphingolipids that are separated on the HPTLC plate can be blotted quantitatively and detected with the reagents used on the plate. Detection of the glycosphingolipids on the membrane was confirmed to be more sensitive than that on the HPTLC plate by both chemical visualization and immunological staining. The glycosphingolipids blotted on the membrane could be reextracted. Blotting of phospholipids can be done by the same method. The results suggest that this method can be used in the purification and characterization of glycosphingolipids and in the detection of proteins which recognize glycosphingolipids and phospholipids.

Animals↗