Increased plasma GlyCAM-1, a mouse L-selectin ligand, in response to an inflammatory stimulus.

GlyCAM-1 (glycosylation-dependent cell adhesion molecule-1) is one of the sialomucin-like ligands for L-selectin, which is a member of the selectin family and mediates initial adhesion of leukocytes to specialized high endothelial venules in lymph nodes and venules at sites of inflammation. GlyCAM-1, lacking a transmembrane domain, is supposed to be secreted into the blood. To understand the functional role of secreted GlyCAM-1, we performed sandwich enzyme-linked immunosorbent assay to measure GlyCAM-1 plasma levels after inflammatory stimulus. BALB/c mice were injected with complete Freund's adjuvant (CFA) in the hind footpads; serum levels of GlyCAM-1 and L-selectin bound to GlyCAM-1 and several inflammatory cytokines, including interleukin-6 (IL-6), were measured at various intervals. IL-6 showed a significant increase 3 h after CFA stimulation. GlyCAM-1 was increased at 3 h, reached peak levels at 12 h, and gradually decreased thereafter. Levels of L-selectin bound to the plasma GlyCAM-1 changed over a similar time course, reached peak at 12 h after, and then began to decrease. The binding of L-selectin to plasma GlyCAM-1 was completely eliminated with the presence of ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid, showing the calcium dependency of this binding. These findings show that GlyCAM-1 release is enhanced by inflammatory stimulation and also suggest that released plasma GlyCAM-1 may trap, at least in part, soluble L-selectin shed from stimulated leukocytes to neutralize each other.

Inhibition of Helicobacter pylori sialic acid-specific haemagglutination by human gastrointestinal mucins and milk glycoproteins.

Helicobacter pylori, a human gastric pathogen causing chronic gastritis and duodenal ulcer disease, has been found in large amounts in gastric mucous gel layer. Mucin preparations, separated from human gastric juices and isolated from different colon regions, were examined for their ability to inhibit haemagglutination of H. pylori with the emphasis on evaluating the role of sialic acid-dependent haemagglutinins of the bacteria in colonisation of the stomach. The mucins showed high inhibitory activity for H. pylori, which was significantly decreased after the removal of sialic acids from the mucins. The inhibitory potencies using high molecular mass mucin-like components from bovine milk were comparable with those obtained for gastric mucins, suggesting their possible role in the prevention of H. pylori infection.

Formation of oligosaccharides from lactitol by Aspergillus oryzae beta-D-galactosidase.

Six oligosaccharides were first formed from lactitol by a transgalactosylation reaction catalyzed by Aspergillus oryzae beta-D-galactosidase. From the results of methylation analysis, MS, and 1H- and 13C-NMR studies, it was concluded that these oligosaccharides are O-beta-D-galactopyranosyl-(1----4)-O-beta-D-galactopyranosyl-(1----4)-D- glucitol, O-beta-D-galactopyranosyl-(1----3)-O-beta-D-galactopyranosyl-(1----4)-D- glucitol, O-beta-D-galactopyranosyl-(1----4)-[O-beta-D-galactopyranosyl-(1----6)]- D- glucitol, O-beta-D-galactopyranosyl-(1----6)-O-beta-D-galactopyranosyl-(1---4)- glucitol, O-beta-D-galactopyranosyl-(1----4)-[O-beta-D-galactopyranosyl- (1----5)]-D-glucitol, and O-beta-D-galactopyranosyl-(1----4)-[O-beta-D-galactopyranosyl-(1----1)]- D-glucitol. The last three are newly observed oligosaccharides.

Effects of lactitol-oligosaccharides on calcium and magnesium absorption in rats.

Lactitol-oligosaccharide (LO) was prepared from lactitol by transglycosylation reaction with Aspergillus oryzae beta-galactosidase. LO is resistant to metabolism in the small intestine but not in the large intestine. The effects of LO, lactose (Lac), lactitol (Lacol) and galactooligosaccharide (GL) on calcium and magnesium absorption were determined by feeding 8-week-old Sprague-Dawley male rats diets containing 5% of the above carbohydrates for two weeks. The results obtained were as follows. 1) A significant increase of calcium absorption was observed in the LO diet. 2) A significant increase of magnesium absorption was observed in the LO, GL and Lacol diets. 3) The concentration of total volatile fatty acids (VFA) in the cecal contents increased significantly in the LO, GL and Lacol diets. The main constituent of VFA in the cecal contents was found to be acetic acid. 4) The correlations between calcium and magnesium absorption ratios and total VFA concentration in the cecum were found to be significantly related. These findings suggest that LO is metabolized to VFA, among which acetic acid concentration seems to have the most stimulatory effect on the absorption of calcium.

Effects of lactitol-oligosaccharides on the intestinal microflora in rats.

Lactitol-oligosaccharide (LO) was prepared from lactitol by a transgalactosylation reaction catalyzed by Aspergillus oryzae beta-galactosidase. The utilization of LO by human intestinal bacteria, the digestion of LO by rat jejunum mucosal homogenates and the effects of LO on the intestinal microflora in rats were compared with those of lactitol. 1) LO was utilized in vitro by Bifidobacterium, but lactitol was not utilized. 2) Neither LO nor lactitol were digested by rat jejunum mucosal homogenates. 3) A significant increase in the fecal counts of Bifidobacterium was observed in the LO diets. 4) The concentration of organic acids in feces and cecal contents significantly increased in the LO diets. 5) The concentration of fecal putrefactive products significantly decreased in the LO and lactitol diets. These findings suggest that LO is effective for improving intestinal conditions.

[A case of primary anterior mediastinal embryonal carcinoma: report of a case].

A 15-year-old man was admitted to our hospital because of chest pain. The chest x-ray film and CT scan revealed a large anterior superior mediastinal mass. The serum alpha-fetoprotein (AFP) value was raised. Percutaneous biopsy of the tumor suggested embryonal carcinoma. The tumor was totally removed. Postoperatively combination chemotherapy including CDDP was performed. However the patient died of tumor recurrence 8 months after operation. AFP is very useful for its diagnosis and the follow-up of the clinical course.

[The extracellular pH and membrane fluidity on intracellular retention of adriamycin].

Using flow cytometry, we evaluated the effect of extracellular pH on intracellular retention of adriamycin (ADR) in K562 cells. The intracellular retention of ADR at pH 7.60 increased markedly compared to the level at pH 7.30. The cell membrane fluidity was measured by spin labeled electron spin resonance techniques. The cell membrane fluidity at pH 7.60 decreased significantly in comparison with those at pH 7.45 and 7.30. The antiproliferative effect of ADR at pH 7.60 was significantly augmented compared to those at pH 7.45 and 7.30. These results suggested that the augmentation of ADR-induced antiproliferative effect by extracellular alkalic shift was caused by membrane rigidity of lipid bilayer, resulting in the decrease of ADR efflux due to less function of P-glycoprotein.

Cell membrane fluidity in K562 cells and its relation to receptor expression.

Cell membrane fluidity (CMF) and transferrin receptor (Tf-R) expression were investigated in K562 cells, a human chronic myelocytic leukemia cell line, treated by gamma-interferon (IFN-gamma). CMF was increased using spin-labeled electron spin resonance techniques, and Tf-R expression was measured by flow cytometric analysis with an EPICS-750 flow cytometer/cell sorter. Treatment of K562 cells in suspension culture with IFN-gamma for as long a time as 6 hr caused an increase in CMF, and then returned to the level of control cells at 12 hr. Conversely, by 24 hr after the beginning of treatment, the rigidity of CMF was increased. Thus, the changes of IFN-gamma-induced CMF was biphasic. While the early change of CMF is related to signal generation and transmission, the later change may reflect changes in lipid compositions and/or cytoskeletal complexes of the plasma cell membrane. A significant increase of Tf-R after 6 hr and 24 hr in number was obtained by treatment of K562 cells with IFN-gamma, but at 12 hr the number of Tf-R did not differ from the control. These results suggested that the early phase of upregulation of Tf-R induced by IFN-gamma was caused by increased CMF, and the late phase of upregulation of Tf-R was due to increased rigidity of CMF. In conclusion, the state of CMF associated with a certain receptor expression in cells is not rigid and can be modulated to some extent by exogenous influences. This may open possibilities of some adjuvant therapeutic measures in malignant diseases by increasing the antigenicity of tumor cells.

Alterations of membrane fluidity in K562 cells exposed to the anticancer drug adriamycin.

The incubation of K562 cells with adriamycin resulted in a decrease in cell membrane fluidity as measured by electron spin resonance using the paramagnetic probe 5-doxylstearic acid. Coincidently, the antiproliferative effect of adriamycin was progressively inhibited as the concentration of adriamycin was increased. The results indicate that adriamycin induces changes in the plasma membrane of K562 cells after exposure to a low level of this agent.

Formation of oligosaccharides from lactose by Bacillus circulans beta-galactosidase.

Eleven oligosaccharides formed by a transglycosylation reaction during lactose hydrolysis with Bacillus circulans beta-galactosidase were purified by gel permeation chromatography, charcoal chromatography, and HPLC. From the results of methylation analysis, and MS and NMR studies, it was concluded that these oligosaccharides were beta-D-Galp-(1-->3)-D-Glc, beta-D-Galp-(1-->6)-D-Glc, beta-D-Galp-(1-->2)-D-Glc, beta-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-D-Glc, beta-D-Galp-(1-->6)-[beta-D-Galp-(1-->2)]-D-Glc, beta-D-Galp-(1-->6)-[beta-D-Galp-(1-->4)]-D-Glc, beta-D-Galp-(1-->4)-beta-D-Galp-(1-->3)-D-Glc, beta-D-Galp-(1-->4)-beta-D-Galp-(1-->2)-D-Glc, beta-D-Galp-(1-->4)-[beta-D-Galp-(1-->2)]-D-Glc, beta-D-Galp-(1-->4)-beta-D-Galp-(1-->6)-D-Glc, beta-D-Galp-(1-->6)[beta-D-Galp-(1-->3)]-D-Glc. The last five are newly observed oligosaccharides. The results of a use test (in vitro) by human intestinal bacteria showed that the oligosaccharides containing lactose units were predominantly used by human intestinal bifidobacteria.

Modification of sialic acids by 9-O-acetylation is detected in human leucocytes using the lectin property of influenza C virus.

Influenza C virus spike glycoprotein HEF specifically recognizes glycoconjugates containing 9-O-acetyl-N-acetylneuraminic acid. The same protein also contains an esterase activity. Taking advantage of these two properties, influenza C virus was used as a very sensitive probe for the detection of traces of 9-O-acetyl-N-acetylneuraminic acid in human leucocytes. The binding of influenza C virus to leucocyte glycoproteins and gangliosides separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and thin-layer chromatography, respectively, was assayed using a chromogenic esterase substrate. In this way, glycoproteins of B-lymphocytes and T-lymphocytes were found to contain 9-O-acetylated sialic acids. Of the various 9-O-acetylated gangliosides detected, one had the characteristics of 9-O-acetylated GD3. The identification of 9-O-acetylated sialic acids on distinct glycoproteins and glycolipids should be helpful in assigning a physiological role to this sugar.

Milk basic protein promotes bone formation and suppresses bone resorption in healthy adult men.

Milk contains several components effective for bone health. In the previous in vitro and in vivo studies, we have shown that milk whey protein, especially its basic protein fraction (milk basic protein [MBP]), promoted bone formation and suppressed bone resorption. This present study examines the effect of MBP on the biochemical markers of bone metabolism in healthy adult men. Experimental beverages containing MBP (300 mg of MBP a day) were given to 30 normal healthy adult men for 16 days. The serum osteocalcin concentration had increased significantly after 16 days of ingesting the experimental beverage containing MBP. Urinary cross-linked N-teleopeptides of type-I collagen (NTx) excretion had decreased significantly after 16 days of ingesting MBP. The urinary NTx excretion was related to the serum osteocalcin concentration after 16 days of ingestion. These results suggest that MBP promoted bone formation and suppressed bone resorption, while maintaining the balance of bone remodeling.