Shinsei Gasa

Inhibition of apoptosis by the actin-regulatory protein gelsolin

Gelsolin is an actin‐regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility in vivo through modulation of the actin network. In addition to its actin‐regulatory function, gelsolin has also been proposed to affect cell growth. Our present experiments have tested the possible involvement of gelsolin in the regulation of apoptosis, which is significantly affected by growth. When overexpressed in Jurkat cells, gelsolin strongly inhibited apoptosis induced by anti‐Fas antibody, C2‐ceramide or dexamethasone, without changing the F–actin morphology or the levels of Fas or Bcl‐2 family proteins. Upon the induction of apoptosis, an increase in CPP32(‐like) protease activity was observed in the control vector transfectants, while it was strongly suppressed in the gelsolin transfectants. Pro‐CPP32 protein, an inactive form of CPP32 protease, remained uncleaved by anti‐Fas treatment in the gelsolintransfectants, indicating that gelsolin blocks upstream of this protease. The tetrapeptide inhibitor of CPP32(‐like) proteases strongly inhibited Fas‐mediated apoptosis, but only partially suppressed both C2‐ceramide‐ and dexamethasone‐induced apoptosis. These data suggest that the critical target responsible for the execution of apoptosis may exist upstream of CPP32(‐like) proteases in Jurkat cells and that gelsolin acts on this target to inhibit the apoptotic cell death program.

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 co-culture 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 × 1012 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 × 1013 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.

Anti‐tumor Effect of Chemically Synthesized Sulfolipids Based on Sea Urchin's Natural Sulfonoquinovosylmonoacylglycerols

We recently reported that 3′‐sulfonoquinovosyl‐1′‐monoacylglycerol (designatedA‐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 C16 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 IC50 values of all products against tumor cells were more than 10‐5M, suggesting weak cytotoxic activity compared with other chemotherapeutic compounds for cancer. On the other hand, in vivo anti‐tumor assay showed that sulfoquinovosyl‐monoacylglycerols (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.

Glycolipid composition in bladder tumor: a crucial role of GM3 ganglioside in tumor invasion.

Glycolipids were extracted from primary bladder tumors of 14 patients and 2 normal counterparts. Their expression pattern was assessed by thin‐layer chromatography (TLC). The most remarkable change was massive accumulation of GM3 in superficial bladder tumors compared with invasive tumors. This change was also confirmed by immunohistochemistry using anti‐GM3 monoclonal antibody. The activities of glycosyltransferases responsible for GM3 synthesis (GM3 synthase, Gb3 synthase and GD3 synthase) were consistent with upregulated expression of GM3 in superficial tumors. It was suggested that the marked GM3 accumulation in superficial tumors was caused not only by upregulated GM3 synthase but also by downregulated activities of Gb3 and GD3 synthase. Histopathologic examination revealed an inverse correlation of the amount of GM3 expressed with invasive potential. Exogenously supplemented GM3 suppressed invasion potential in human bladder tumor cell lines (T‐24, KK‐47). These results indicate that the amount of GM3 expressed may serve as an indicator of the invasion potential of bladder tumor. Furthermore, new antiinvasion therapeutics may be possible by administration of GM3.

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. (Cancer Sci 2013; 104: 43–47)

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-&bgr;-D-glucopyranosyl)-1,2-di-O-acylglycerol (&bgr;-SQDG) that contains a saturated C18 fatty acid, which is designated as &bgr;-SQDG(18:0) by mixed lymphocyte reaction (MLR) and rat allogeneic skin graft. Then, we investigated the mechanism of immunosuppressive effect of &bgr;-SQDG(18:0). Results. &bgr;-SQDG(18:0) inhibited human MLR in a dose-dependent manner without overt cytotoxic effect and prolonged rat skin allograft rejection in vivo. &bgr;-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, &bgr;-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 &bgr;-SQDG(18:0) treatment. Conclusions. &bgr;-SQDG(18:0) might be a new class of the immunosuppressive reagent, and the inhibition of responder T-lymphocyte activation in MLR by &bgr;-SQDG(18:0) may be responsible for certain three-dimensional structures of this reagent or its quinovose binding to sulfonic acid.

Differential lipid specificities of the repeated domains of annexin IV.

The roles of the four domains of annexin IV in binding to phospholipids and glycolipids were assessed by analyzing the binding of a group of mutant annexins IV in which one or more of the four domains was inactivated by replacing a critical amino residue(s) (Asp or Glu) with the neutral residue Ala. The data reveal that individual annexin domains may have characteristic affinities for different lipids. In particular, inactivation of the fourth domain inhibits the binding to phosphatidylserine (PS) and phosphatidylinositol (PI) but not to phosphatidylglycerol (PG), suggesting that this domain specifically can accommodate the larger head groups of PS and PI whereas the other three domains may form more restricted binding pockets. In order to block binding to PG, domain 1, or both domains 2 and 3 must be inactivated in addition to domain 4, suggesting that all four domains may be able to accommodate the headgroup of PG to some extent. Binding to acidic glycolipids (sulfatides) was also sensitive to inactivation of domain 4. However, in the case of sulfatides the nature of the binding reaction is fundamentally different compared with the binding to phospholipids since the interaction with sulfatides was highly sensitive to an increase in ionic strength. The binding to sulfatides may depend therefore on charge-charge interactions whereas the binding to phospholipid may involve a more specific interaction between the lipid headgroup and the protein surface, and/or interaction of the protein with the hydrophobic portion of a lipid bilayer.

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 Ca2+-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.

Mucolipidosis III (pseudo-Hurler polydystrophy); clinical studies in aged patients in one family

Three adult patients (38-year-old male, 86-year-old female, and 61-year-old male) in a family with mucolipidosis III (ML-III) were described. They had characteristic features of ML-III and they survived a long time. N-acetylglucosaminyl 1-phosphotransferase activity was low in fibroblasts of a patient, but its residual activity remained at a relatively high level (24.5-35.3% of controls), which may explain the benign clinical course. Odontoid dysplasia and atlanto-axial dislocation was found in one patient, and surgical treatment improved his physical disability. Bilateral carpal tunnel syndrome as well as claw hand deformities were common in all of the patients. The clinical manifestations were important for the diagnosis and the management of the patients.

Characterization of novel mono-O-acetylated GM3s containing 9-O-acetyl sialic acid and 6-O-acetyl galactose in equine erythrocytes

Novel mono-O-acetylated GM3s, one containing 9-O-acetylN-glycolyl neuraminic acid and another containing 6′-O-acetyl galactose, were isolated as a mixture from equine erythrocytes, and the structures were characterized by one- and two-dimensional proton nuclear magnetic resonance (NMR) and fast atom bombardment-mass spectrometry (FAB-MS). The position of theO-acetyl residue was identified by the downfield shift of the methylene protons at C-9 ofN-glycolyl neuraminic acid (9-O-Ac GM3) and C-6 of galactose (6′-O-Ac GM3) in the NMR spectrum, in comparison to the respective non-acetylated counterparts. To confirm the presence of 6′-O-Ac GM3, theO-acetylated GM3 mixture was desialylated withArthrobacter neuraminidase, giving 6-O-acetyl galactosyl glucosylceramide, the structure of which was estimated by NMR and FAB-MS, together with non-acetylated lactosylceramide with a ratio of 1:1.