Masahiko Ikekita

Change in electrophoretic mobility of HL-60RG cells by apoptosis

We measured the electrophoretic mobilities of HL-60RG cells and their apoptotic cells triggered by Actinomycin D as a function of the ionic strength of the suspending medium at pH 7.4. Both types of cells showed negative mobilities. The apoptotic HL-60RG cells exhibited larger mobility values in magnitude than intact HL-60RG cells in the whole range of the electrolyte concentration measured. The obtained data were analyzed via a mobility expression for “soft particles’, that is, colloidal particles with ionpenetrable surface layers. The observed mobility difference between the intact and apoptotic HL-60RG cells was found to be due mainly to the difference in friction exerted by the cell surface layers on the liquid flow around the cells between these two types of cells rather than the difference in charge density in their surface layers. A possible explanation for this mobility change by apoptosis is given.

Observation of tissue prokallikrein activation by some serine proteases, arginine esterases in rat submandibular gland.

Two serine proteases, arginine esterases (esterases I and II) which showed the activity of tissue prokallikrein activation were identified in rat submandibular gland. These enzymes were separated from the homogenate of rat submandibular gland by two successive DEAE-cellulose chromatographies and were further purified and characterized. Esterases I and II were found to be identical with tonin and esterase B-like enzyme, respectively. Both enzymes activated rat urinary prokallikrein at near neutral pH. Esterase B-like enzyme activated rat urinary prokallikrein better than tonin.

Involvement of Galectin-3 with Vascular Cell Adhesion Molecule-1 in Growth Regulation of Mouse BALB/3T3 Cells *□

β-Galactose residues on N-glycans have been implicated to be involved in growth regulation of cells. In the present study we compared the galactosylation of cell surface N-glycans of mouse Balb/3T3 cells between 30 and 100% densities and found the β-1,4-galactosylation of N-glycans increases predominantly in a 100-kDa protein band on lectin blot analysis in combination with digestions by diplococcal β-galactosidase and N-glycanase. When cells at 100% density were treated with jack bean β-galactosidase, the incorporation of 5-bromodeoxyuridine into the cells was stimulated in a dose-dependent manner, suggesting the involvement of the galactose residues in growth regulation of cells. A galactose-binding protein was isolated from the plasma membranes of cells at 100% density by affinity chromatography using an asialo-transferrin-Sepharose column and found to be galectin-3 as revealed by mass spectrometric analysis. The addition of recombinant galectin-3 into cells at 50% density inhibited the incorporation of 5-bromodeoxyuridine in a dose-dependent manner, but the inhibition was prevented with haptenic sugar. An immunocytochemical study showed that galectin-3 is present at the surface of cells at 100% density but not at 30% density where it locates inside the cells. Several glycoproteins bind to a galectin-3-immobilized column, a major of which was identified as vascular cell adhesion molecule (VCAM)-1. Immunocytochemical studies showed that some galectin-3 and VCAM-1 co-localize at the surface of cells at 100% density, indicating that the binding of galectin-3 secreted from cells to VCAM-1 is one of the pathways involved in the growth regulation of Balb/3T3 cells.

Prognostic significance of reduced expression of β‐N‐acetylgalactosaminylated N‐linked oligosaccharides in human breast cancer

Our previous studies showed that expression of the GalNAcβ1→4GlcNAc group on N‐linked oligosaccharides is associated with functional differentiation of the bovine mammary gland. In the present study, the occurrence of the GalNAcβ1→4GlcNAc group was established in human milk proteins and membrane glycoproteins from a human breast cancer cell line, MRK‐nu‐1, by structural analysis of oligosaccharides released by hydrazinolysis. Whether the expression level of the disaccharide group is affected upon malignant transformation was examined in human breast cancer specimens using Wistaria floribunda agglutinin (WFA) which interacts with oligosaccharides with N‐acetylgalactosamine at their termini. Lectin blot analysis of membrane glycoprotein samples from human breast cancer specimens showed that the number of protein bands reacting with WFA, as well as their intensities, are lower in samples from primary carcinoma lesions compared with samples from surrounding normal tissues. No lectin binding was observed when the blots were treated with jack bean β‐N‐acetylhexosaminidase or N‐glycanase, indicating that WFA‐reactive oligosaccharides are N‐linked. A histochemical study of tissue specimens from 92 patients with breast cancer revealed that the reduced WFA staining levels in primary carcinoma lesions correlate with advancing clinical stages and prognostic status (i.e., 58% of patients in a group showing reduced/negative staining died of disease recurrence, whereas more than 90% of those in the positive staining group survived for 5 years after surgery). These results indicate that reduced expression of β‐N‐acetylgalactosaminylated N‐linked oligosaccharides on primary carcinoma lesions predicts a poor prognosis for patients with breast cancer.

Effects of liposomal phophatidylserine on phagocytic uptake of liposomes by macrophage-like HL-60RG cells

Abstract The purpose of this work is to know the effect of surface properties of liposomes on their phagocytic uptake by macrophages. For this, liposomes were prepared by the Bangham technique from the mixture of phosphatidylcholine (PC) and cholesterol (Chol) incorporated either with phosphatidylserine (PS), phosphatidylethanolamine (PE) or phosphatidic acid (PA). The liposomes thus prepared had diameters in the range between 150 and 260 nm. Electric surface properties of the liposomes and the macrophages differentiated from HL-60RG cells were determined by measuring their electrophoretic mobilities. The phagocytic uptake of liposomes with different contents of PS, PE and PA by macrophage-like HL-60RG cells was investigated by measuring oxygen consumption associated with phagocytic uptake. The phagocytic activity was found to be the highest with the PC–Chol liposomes containing 7 mol% PS, but no significant effects were observed with PA- and PE-containing PC–Chol liposomes. As the uptake was independent of the electric surface property of liposomes, PS was concluded to be specifically important for phagocytic activity of macrophages.

Rat Testicular Angiotensin I Converting Enzyme: Purification and Comparison with Rat Pulmonary Enzyme

Enzymological properties of rat testicular angiotensin I converting enzyme (RT-ACE) were compared with those of rat pulmonary angiotensin I converting enzyme (RP-ACE). The molecule of RT-ACE was different from that of RP-ACE with respect to the molecular weight, i.e., the molecular weight of RT-ACE was estimated to be 104 kilo-dalton (kd) and that of RP-ACE (150 kd) on SDS-polyacrylamide gel electrophoresis. On the other hand, the enzymochemical properties of RT-ACE were very similar to those of RP-ACE, with regard to activation by NaCl, optimum pH, Km value for N*-hippuryl-His-Leu-OH hydrolysis and sensitivities to various inhibitors. Therefore, it was speculated that the portions contributing to the appearance of catalytic activity would be similar between RT-ACE and RP-ACE.

Fast Atom Bombardment Mass Spectrometry (FAB-MS): Analysis of Complex Carbohydrate Chains of Tissue Kallikreins

Molecular ions and fragment ions of underivatized and permethylated oligosaccharides derived from human urinary kallikrein and some model glycoproteins gave information about the sugar composition and arrangement of sugars. These ions were conveniently created by FAB-MS in a JEOL JMS-HX110 high field high resolution mass spectrometer. Glycoprotein samples were digested with N-glycanase and the asparagine(Asn)-linked oligosaccharides were separated from polypeptides by Sephadex G-50 chromatography. The mixture of oligosaccharides was converted to the reduced p-aminobenzoic ethyl ester (ABEE) derivative and the components separated by HPLC on an ion-exchange (AX-10) column. Individual components were analyzed by negative FAB-MS using glycerol as a matrix. Permethylated oligosaccharides were also analyzed by positive FAB-MS.

Studies on carbohydrate structure and immunological properties of human urinary kallikreins.

Some studies on carbohydrates structure and immunological properties of human urinary kallikreins purified from the human urine of healthy men (HUK) were investigated. A general technique for fractionating asparagine-linked oligosaccharides of HUK was applied. This involves serial chromatographies on concanavalin A-Sepharose 4B and other lectins bound Agarose. Asparagine-linked main oligosaccharides of both active and inactive types of HUK were mixture of tri- or tetra-antennary complex oligosaccharide having GlcNAC beta 1-4Man beta residue (bisecting GlcNAc) (type I-2), bi-antennary complex oligosaccharide having core Fuc alpha 1-6GlcNAc residue (type III-3), tri- or tetra-antennary complex oligosaccharides having Gal beta 1-4GlcNAc beta 1-4 (Gal beta 1-4GlcNAc beta 1-2)Man alpha-residue (type I-1-A-a) etc. Besides these structural studies, the method to probe the heterogeneous profiles of HUK due to the different structures of carbohydrate chains bound, was devised without the complete purification of urinary kallikrein even with individual subject of not only active but also inactive forms of the kallikrein.

Anti-bradykinin activity found in beet and some of its anti-inflammatory actions

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