Detection of N-glycolyated gangliosides in non-small-cell lung cancer using GMR8 monoclonal antibody.

Gangliosides are glycosphingolipids found on the cell surface. They act as recognition molecules or signal modulators and regulate cell proliferation and differentiation. N-glycolylneuraminic acid (NeuGc)-containing gangliosides have been detected in some neoplasms in humans, although they are usually absent in normal human tissues. Our aim was to evaluate the presence of NeuGc-containing gangliosides including GM3 (NeuGc) and assess their relationship with the prognosis of non-small-cell lung cancer (NSCLC). NeuGc-containing ganglioside expression in NSCLC tissues was analyzed immunohistochemically using the mouse monoclonal antibody GMR8, which is specific for gangliosides with NeuGc alpha 2,3Gal-terminal structures. On the basis of NeuGc-containing ganglioside expression, we performed survival analysis. We also investigated the differences in the effects of GM3 (N-acetylneuraminic acid [NeuAc]) and GM3 (NeuGc) on inhibition of epidermal growth factor receptor (EGFR) tyrosine kinase in A431 cells. As a result, the presence of NeuGc-containing gangliosides was evident in 86 of 93 (93.5%) NSCLC samples. The NSCLC patients with high NeuGc-containing ganglioside expression had a low overall survival rate and a significantly low progression-free survival rate. In the in vitro study, the inhibitory effect of GM3 on EGFR tyrosine kinase in A431 cells after exposure to GM3 (NeuGc) was lower than that after exposure to GM3 (NeuAc). In conclusion, NeuGc-containing gangliosides including GM3 (NeuGc) are widely expressed in NSCLC, and NeuGc-containing ganglioside expression is associated with patient survival. The difference in the effects of GM3 (NeuGc) and GM3 (NeuAc) on the inhibition of EGFR tyrosine kinase might contribute to improvement in the prognosis of NSCLC patients.

Pulmonary collectins play distinct roles in host defense against Mycobacterium avium.

Pulmonary collectins, surfactant protein A (SP-A) and surfactant protein D (SP-D), play important roles in the innate immunity of the lung. Mycobacterium avium is one of the well-known opportunistic pathogens that can replicate within macrophages. We examined the effects of pulmonary collectins in host defense against M. avium infection achieved via direct interaction between bacteria and collectins. Although both pulmonary collectins bound to M. avium in a Ca(2+)-dependent manner, these collectins revealed distinct ligand-binding specificity and biological activities. SP-A and SP-D bound to a methoxy group containing lipid and lipoarabinomannan, respectively. Binding of SP-D but not SP-A resulted in agglutination of M. avium. A chimeric protein with the carbohydrate recognition domain of SP-D, which chimera revealed a bouquet-like arrangement similar to SP-A, also agglutinated M. avium. The ligand specificity of the carbohydrate recognition domain of SP-D seems to be necessary for agglutination activity. The binding of SP-A strongly inhibited the growth of M. avium in culture media. Although pulmonary collectins did not increase membrane permeability of M. avium, they attenuated the metabolic rate of the bacteria. Observations under a scanning electron microscope revealed that SP-A almost completely covers bacterial surfaces, whereas SP-D binds to certain areas like scattered dots. These observations suggest that a distinct binding pattern of collectins correlates with the difference of their biological activities. Furthermore, the number of bacteria phagocytosed by macrophages was significantly increased in the presence of SP-D. These data indicate that pulmonary collectins play critical roles in host defense against M. avium.

Ex vivo large-scale generation of human red blood cells from cord blood CD34+ cells by co-culturing with macrophages.

We generated red blood cells (RBC) from cord blood (CB) CD34+ cells using a four-phase culture system. We first cultured CB CD34+ cells on telomerase gene-transduced human stromal cells in serum-free medium containing stem cell factor (SCF), Flt-3/Flk-2 ligand, and thrombopoietin to expand CD34+ cells (980-fold) and the total cells (10,400-fold) (first phase). Expanded cells from the first phase were liquid-cultured with SCF, interleukin-3 (IL-3), and erythropoietin (EPO) to expand (113-fold) and differentiate them into erythroblasts (second phase). To obtain macrophages for the next phase, we expanded CD34+ cells from a different donor using the same coculture system. Expanded cells from the first phase were liquid-cultured with granulocyte-macrophage colony stimulating factor, macrophage-colony stimulating factor (M-CSF), IL-3, and SCF to generate monocytes/macrophages (75-fold), which were incubated with type AB serum and M-CSF to fully differentiate them into macrophages. Erythroblasts were then co-cultured with macrophages in the presence of EPO to expand (threefold) and fully differentiate them (61% orthochromatic erythroblasts plus 39% RBC) (third phase). RBC were purified from erythroblasts and debris through a deleukocyting filter to generate 6.0 x 10(12) RBC from 1.0 unit of CB (3.0 transfusable units). Qualitatively, these RBC showed a hemoglobin content, oxygenation of hemoglobin, and in vivo clearance similar to those of adult peripheral RBC. Finally, an almost complete enucleation of orthochromatic erythroblasts (99.4%) was achieved by the cultivation method recently described by Miharada et al. in the absence of macrophages and cytokines (fourth phase). RBC were purified from remnant erythroblasts and debris by passage through a deleukocyting filter to generate 1.76 x 10(13) RBC from 1.0 unit of CB (8.8 transfusable units), the highest yield ever reported. Thus, this method may be useful for generating an alternative RBC supply for transfusions, investigating infectious agents that target erythroid cells, and as a general in vitro hematopoietic model system.

[Chemical content analysis of specimens of human head temporal portion commercially distributed for otological surgery lessons].

Specimens of the human head temporal portion are provided in the United States and distributed commercially for lessons in otological surgery via the internet. Many otologists have obtained and used these specimens in Japan. According to our chemical content analysis, these specimens were found to contain harmful substances as well as large molecule aldehydes and fatty acid methylesters, which are not or only rarely included in the human cadaveric specimens prepared in Japan. We discuss the suggested treatment of these imported specimens, how trial preparation of specimens are much better than the imported items, and the background of the rapid and wide distribution of the imported items in Japan.

An immunosuppressive effect by synthetic sulfonolipids deduced from sulfonoquinovosyl diacylglycerols of sea urchin.

BACKGROUND: It is important to develop new immunosuppressive agents without clinical drawbacks. In this article, we reveal the possibility of a chemically synthetic sulfonolipid that acts as a novel immunosuppressive drug. METHODS: We evaluated the immunosuppressive effect of 3-O-(6-deoxy-6-sulfono-beta-D-glucopyranosyl)-1,2-di-O-acylglycerol (beta-SQDG) that contains a saturated C18 fatty acid, which is designated as beta-SQDG(18:0) by mixed lymphocyte reaction (MLR) and rat allogeneic skin graft. Then, we investigated the mechanism of immunosuppressive effect of beta-SQDG(18:0). RESULTS: beta-SQDG(18:0) inhibited human MLR in a dose-dependent manner without overt cytotoxic effect and prolonged rat skin allograft rejection in vivo. beta-SQDG(18:0) did not inhibit the direct activation of responder T. This reagent could not affect the expression of either major histocompatibility antigen complex (MHC) class I or class II molecules on the cell surface of the stimulator cells, antigen-presenting cells. In contrast, beta-SQDG(18:0) was demonstrated to inhibit the binding among allogeneic lymphocytes. However, the expression of known cell surface accessory and adhesion molecules, such as CD4, CD28, leukocyte function-associated antigen 1, intercellular adhesion molecule 1, and CTLA-4, was not affected by beta-SQDG(18:0) treatment. CONCLUSIONS: beta-SQDG(18:0) might be a new class of the immunosuppressive reagent, and the inhibition of responder T-lymphocyte activation in MLR by beta-SQDG(18:0) may be responsible for certain three-dimensional structures of this reagent or its quinovose binding to sulfonic acid.

Performance of bipolar forceps during coagulation and its dependence on the tip material: a quantitative experimental assay. Technical note.

The aim of this study was to measure objectively the adherence of burned tissue to bipolar forceps to evaluate the coagulation performance of forceps made of different types of metals. Coagulation performance of bipolar forceps made of gold, titanium, and stainless steel was determined by comparing the amount of protein in the adhered coagulum on the tips. The amount of adhered coagulum was significantly less on the gold-plated bipolar forceps than on those made of the other two materials. The ease with which coagulum could be removed was compared using the cleaning cycle of an ultrasonic rinsing device. This ease of removal was also significant with the gold-plated forceps. Electron microscopy observations of the surface of the forceps tips revealed a significant difference in roughness among these materials, and there were also significant differences in wetting tensions. Measuring adherence based on three different types of roughness and wetting tensions of forceps made from the same metal (titanium) also demonstrated a significant difference in the cleaning cycle. Histological examination of an artery coagulated with the gold-plated bipolar forceps showed that the structure had been completely collapsed without destruction of the layers, whereas arteries coagulated with the other materials revealed severely damaged structures. Adherence to bipolar forceps was dependent on both the material in the tips and the roughness of this material. The gold-plated bipolar forceps demonstrated the best performance.

Phase behavior in multilamellar vesicles of DPPC containing ganglioside GM3 with a C18:1 sphingoid base and a 24:0 acyl chain (GM3(18,24)) observed by X-ray diffraction.

Structures and phase behavior of multilamellar vesicles of 1,2-dipalmitoyl-L-phosphatidylcholine (DPPC) containing various amount of ganglioside GM3 with a C18:1 sphingoid base and a 24:0 acyl chain (GM3(18,24)) were investigated by small-angle X-ray diffraction. Below 3.5 mol% GM3 content, the phase behavior was similar to that of pure DPPC except for a slight increase of lamellar repeat distance in the L(beta'), the P(beta') and the L(alpha) phases and a decrease of the pretransition temperature. In the range of 4-12 mol% GM3 content, another phase which has larger repeat distances coexisted with the phase observed below 3.5 mol% GM3 content. This has been interpreted that the phase separation into GM3-poor phase (denoted as A-phase) and GM3-rich phase (denoted as B-phase) took place. Above 13 mol% GM3 content, the B-phase became dominant. This phase separation may be related to the formation of GM3-enriched microdomains that had been observed on the cell surfaces which express large amounts of GM3, such as murine B16 melanoma (J. Biol. Chem. 260 (1985) 13328).

Solubilisation and properties of the sialate-4-O-acetyltransferase from guinea pig liver.

The O-acetylation of sialic acids turns out to be one of the most important modifications that influence the diverse biological and pathophysiological properties of glycoconjugates in animals and microorganisms. To understand the functions of this esterification, knowledge of the properties, structures and regulation of expression of the enzymes involved is essential. Attempts to solubilise, purify or clone the gene of one of the sialate-O-acetyltransferases have failed so far. Here we report on the solubilisation of the sialate-4-O-acetyltransferase from guinea pig liver, the first and essential step in the purification and molecular characterisation of this enzyme, by the zwitterionic detergent CHAPS. This enzyme O-acetylates sialic acids at C-4 both free and bound to oligosaccharides, glycoproteins and glycolipids with varying activity, however, gangliosides proved to be the best substrates. Correspondingly, a rapid enzyme test was elaborated using the ganglioside GD3. The soluble O-acetyltransferase maximally operated at 30 degrees C, pH 5.6, and 50-70 mM KCl and K2HPO4 concentrations. The Km values were 3.6 microM for AcCoA and 1.2 microM for GD3. CoA inhibits the enzyme with a Ki value of 14.8 microM. A most important discovery enabling further enzyme purification is its need for an unknown low molecular mass and heat-stable cofactor that can be separated from the crude enzyme preparation by 30 kDa ultrafiltration.

Inhibition of CD62L+ T-cell response in vitro via a novel sulfo-glycolipid, beta-SQAG9 liposome that binds to CD62L molecule on the cell surface.

We previously reported that synthetic sulfo-glycolipid, 3-O-(6-deoxy-6-sulfono-beta-D-glucopyranosyl)-1,2-di-O-acylglycerol (beta-SQDG(18:0)) which was deduced from sulfonoquinovosyl-diacylglycerols of sea urchin possessed immunosuppressive effects, such as human mixed lymphocyte reaction (MLR) and skin allograft in rat, and that these effects were caused by contact inhibition between T-cells and antigen presenting cells (APCs). Here, we investigated the mechanism of these immunosuppressive effects on human MLR by beta-SQAG9 which had been newly synthesized from beta-SQDG(18:0) to improve structural stability in water solution. CD62L+ T-cells in peripheral blood predominantly respond to APCs, and beta-SQAG9 inhibited the response of CD62L+ T-cell subset in human allogeneic MLR. Surprisingly, it was demonstrated that beta-SQAG9 bound to L- and P-selectin (CD62L and P) molecule in vitro. Meanwhile, beta-SQAG9 efficiently formed liposome structure and bound to L-selectin on the cell surface of CD62L+ T-cell subset but might not be incorporated into the cells. Because the immunosuppressive effects of beta-SQAG9 disappeared when beta-SQAG9 liposome was changed to soluble form by detergent, the liposome structure of beta-SQAG9 was presumed to be essential for these effects. These findings suggested beta-SQAG9 to be a novel drug with a unique immunosuppressive action.

Anti-tumor effect of chemically synthesized sulfolipids based on sea urchin's natural sulfonoquinovosylmonoacylglycerols.

We recently reported that 3'-sulfonoquinovosyl-1'-monoacylglycerol (designated A-5) extracted from sea urchin intestine was effective in suppressing the growth of solid tumors. Although the major fatty acid component of A-5 was a saturated C(16) acid, there were five other fatty acids, 14:0, 18:0, 14:1, 16:1, and 18:1, which constitute minor components of A-5. Therefore, it remains unclear as to which of these six fatty acid components of A-5 has the anti-tumor effect. In this study, we synthesized sulfolipids each containing only one of these six fatty acids and tested their cytotoxicity against tumor cells and in vivo anti-tumor effects on nude-mice bearing solid tumors of human lung adenocarcinoma cell line A-549. The IC(50) values of all products against tumor cells were more than 10(-5) M, suggesting weak cytotoxic activity compared with other chemotherapeutic compounds for cancer. On the other hand, in vivo anti-tumor assay showed that sulfoquinovosylmonoacylglycerols (SQMG) composed of 14:1 and 18:1 (designated SQMG(14:1) and SQMG(18:1), respectively) were significantly effective in suppressing the growth of solid tumors. Our data suggested that these two SQMGs had a substantial anti-tumor effect in vivo, and they are of interest as candidate drugs for anti-cancer treatment.