Improvement of the antitumor activity of black currant polysaccharide by an enzymatic treatment.

A polysaccharide-rich substance isolated from black currant, named cassis polysaccharide (CAPS), was partially digested with beta-galactosidase from Aspergillus oryzae and its immunostimulatory activity was investigated. The in vitro cytokine-inducing effect of CAPS on RAW264 cells was gradually decreased along with lowering of the average MW of CAPS. In vivo, partially digested CAPS with a mean MW of approximately 20,000 showed the most potent antitumor activity against Ehrlich carcinoma in mice.

Identification and characterization of 125I-insulin-like growth factor-II binding sites on the muscular layer of stem villi vessels of human term placenta.

The primary function of the placenta is to ensure an optimal environment for fetal growth and development. In normal pregnancy, placental vascular tone regulation assures fetus well-being and normal development by maintaining adequate blood flow so as to ensure materno-fetal exchanges. In human placenta, synthesis of insulin-like growth factor (IGF)-II and specific binding sites have been previously characterized in the trophoblast; in contrast, no studies have dealt with this subject in the fetoplacental vascular system, particularly in stem villi vessels. We thus investigated whether membranes of the muscular layer of stem villi vessels contained 125I-IGF-II binding sites. Two complementary approaches were used: 125I-IGF-II binding and affinity cross-linking studies. 125I-IGF-II labeled, in a saturable and noncooperative manner, a single class of high-affinity binding sites characterized by a Kd of 1.24 +/- 0.26 nM (n = 6), a maximum binding capacity (Bmax) of 3.02 +/- 0.45 pmol/mg protein, and a Hill coefficient of 1.00 +/- 0.15. Competitors for 125I-IGF-II binding to membranes were in the order of potency IGF-II > IGF-I. Insulin was not a competitor. Affinity cross-linking of membranes with 125I-IGF-II, followed by SDS-PAGE and autoradiography, revealed two labeled bands: a protein complex of 250 kDa, which corresponds to the type II IGF receptor, and another of 135 kDa, corresponding to the type I IGF receptor. Only IGF-II could displace 125I-IGF-II binding from the major 250-kDa band, while 125I-IGF-II bound to the minor 135-kDa band was displaced by either IGF-I, IGF-II, or insulin. In conclusion, high levels of specific binding sites for 125I-IGF-II are present in the muscular layer of stem villi vessels, which are considered placenta resistance vessels. The involvement of both type I and type II IGF receptors in the growth-promoting action of IGF-II remains to be determined in the fetoplacental vascular system.

Structural definition of a potent macrophage activating factor derived from vitamin D3-binding protein with adjuvant activity for antibody production.

Incubation of human vitamin D3-binding protein (Gc protein), with a mixture of immobilized beta-galactosidase and sialidase, efficiently generated a potent macrophage activating factor, a protein with N-acetylgalactosamine as the remaining sugar. Stepwise incubation of Gc protein with immobilized beta-galactosidase and sialidase, and isolation of the intermediates with immobilized lectins, revealed that either sequence of hydrolysis of Gc glycoprotein by these glycosidases yields the macrophage-activating factor, implying that Gc protein carries a trisaccharide composed of N-acetylgalactosamine and dibranched galactose and sialic acid termini. A 3 hr incubation of mouse peritoneal macrophages with picomolar amounts of the enzymatically generated macrophage-activating factor (GcMAF) resulted in a greatly enhanced phagocytic activity. Administration of a minute amount (10-50 pg/mouse) of GcMAF resulted in a seven- to nine-fold enhanced phagocytic activity of macrophages. Injection of sheep red blood cells (SRBC) along with GcMAF into mice produced a large number of anti-SRBC antibody secreting splenic cells in 2-4 days.

Insulin-like growth factor-I and- II in combination inhibit the release of growth hormone-releasing hormone from the rat hypothalamus in vitro.

This study was designed to investigate the feedback loop between insulin-like growth factor-I (IGF-I) and IGF-II and the hypothalamic hormones growth hormone-releasing hormone (GHRH) and somatostatin (SS) using an in vitro rat hypothalamic model. IGF-I and, to lesser extent, IGF-II, both activate type 1 IGF receptors, while type 2 receptors are activated by IGF-II alone. IGF-I, IGF-II, their various specific analogues (Des[1-3]IGF-I, [Arg54/Arg55]IGF-II and [Leu27]IGF-II), insulin and the type 2 receptor antagonist beta-galactosidase were used on their own or in combination to study their effect on GHRH and SS release. Our results suggest that the simultaneous activation of type 1 and type 2 IGF receptors is needed for the negative feedback effect of IGFs on GHRH release in this in vitro system, in agreement with earlier findings in vivo. Somatostatin was not altered by any combination of peptides.

[Characterization of murine monoclonal antibodies against fetal erythrocytes]. / Caractérisation d'anticorps monoclonaux murins dirigés contre les érythrocytes foetaux.

Balb/c mice were immunized against papain-treated fetal erythrocytes and splenocytes were fused with Sp2/0-Ag-14 myeloma cells. Several hybrids secreting antibodies directed against antigenic determinants predominantly exposed on fetal and cord cells were selected and cloned twice. Antibodies NaM61-1A2 and NaM61-768 (IgM class) were shown to be specific for an endo-beta-galactosidase-sensitive oligosaccharide chain. The antigen, strongly expressed on fetal and cord cells, was identified as the i blood group antigen. The antibodies represent powerful blood group reagents to be use in conventional agglutination techniques as well as in the gel typing system and in indirect flow cytometry. The antibody NaM46-4A8 (IgG class) is specific for an antigenic structure expressed on fetal cells and accessible only after papain, ficin, bromelin and endo-beta galactosidase treatment. The antigen was not identified.

Asymmetric distribution of the phosphatidylinositol-linked phospho-oligosaccharide that mimics insulin action in the plasma membrane.

We have investigated the topography of a glycosyl-phosphatidylinositol implicated in insulin action by a combination of two complementary methods: (a) chemical labelling with a non-permeable (isethionyl acetimidate) and a permeable (ethyl acetimidate) probe; and (b) enzymatic modifications with beta-galactosidase (EC 3.2.1.23) or phosphatidylinositol-specific phospholipase C (EC 3.1.4.3). Using the first approach the majority of the glycosyl-phosphatidylinositol is found in the outer surface of intact hepatocytes, adipocytes, fibroblasts and lymphocytes, but not in erythrocytes which presented only a 20% of the total labelled glycosyl-phosphatidylinositol to the exterior. Upon insulin addition (10 nM), about 60% of the total glycosyl-phosphatidylinositol was hydrolysed in both hepatocytes and adipocytes but not in erythrocytes. In agreement with the extracellular localization in hepatocytes and with the proposed role of this glycolipid in insulin action, treatment of rat hepatocytes with beta-galactosidase from Escherichia coli, an enzyme that hydrolyses the oligosaccharide moiety of the glycosyl-phosphatidylinositol, cleaved 65% of the total glycophospholipid and blocked the effect of insulin (but not of glucagon) on pyruvate kinase (EC 2.7.1.40). Similar treatment with phosphatidylinositol-specific phospholipase C from Bacillus cereus hydrolysed 62% of the total glycosyl-phosphatidylinositol. From the various approaches used it is concluded that the majority of this glycophospholipid is at the outer surface in a variety of insulin-sensitive cells.