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.

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.

[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.

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.

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.

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.

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.

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.

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.

A simple and quantitative purification of glycosphingolipids and phospholipids by thin-layer chromatography blotting.

A new and simple method for purifying glycosphingolipids and phospholipids by using "TLC blotting" was established. Glycosphingolipids separated by two-dimensional thin-layer chromatography (TLC) were made visible with primuline reagent, and then bands were marked with a drawing colored pencil. The glycosphingolipids that separated on the HPTLC plate were transferred by TLC blotting to a polyvinylidene difluoride membrane together with the color marks.

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.