Characterization of a novel glycolipid with a difucosylated H-antigen in human blood group O erythrocytes with monoclonal antibody HMMC-1 and its detection in human uterine cervical carcinoma tissues.

Humanized monoclonal antibody HMMC-1 established by immunizing transchromosomal mice with a human uterine endometrial cancer cell line has been found to react with the H-antigen carried on core l O-glycans through cotransfection of glycosyltransferases for O-glycans and inhibition of antibody-binding with synthetic oligosaccharides. However, direct binding analysis of an antibody against glycosphingolipids from human erythrocytes with different ABO blood groups revealed that it was able to bind selectively with polar glycolipids in blood group O, but not blood group A, B and AB erythrocytes. Unexpectedly, typical monofucosylated H-glycolipids, IV2Fucα-nLc4Cer and VI2Fucα-nLc6Cer, which are the precursors for A and B-glycolipids, and were present not only in blood group O, but also A, B and AB-erythrocytes, were not the antigens for the HMMC-1 antibody. The antigen comprised less than 0.001% of the total glycolipids in blood group O-erythrocytes, and was purified by conventional silica gel column chromatography. Structural determination by permethylation, GC-MS, and ESI-TOFMS demonstrated that the structure was a novel glycolipid with a difucosylated H-antigen, Fucα1-2Galß1-4GlcNAcß1-3Gal(2-1αFuc)ß1-4GlcNAcß1-3Galß1-4GlcNAcß1-3Galß1-4Glcß1-1'Cer, VI2,VIII2(Fucα)2-nLc8Cer, whose terminal difucosylated structure was the epitope of the HMMC-1 antibody. The HMMC-1 glycolipid was detected in five out of 29 tissues from patients suffering from uterine cervical carcinomas, irrespective of their ABO-blood groups.

Polyunsaturated fatty acids induce ovarian cancer cell death through ROS-dependent MAP kinase activation.

Free fatty acids not only play a role in cell membrane construction and energy production but also exert diverse cellular effects through receptor and non-receptor mechanisms. Moreover, epidemiological and clinical studies have so far suggested that polyunsaturated fatty acids (PUFAs) could have health benefits and the advantage as therapeutic use in cancer treatment. However, the underlying mechanisms of PUFA-induced cellular effects remained to be cleared. Here, we examined the effects of ω-3 and ω-6 PUFAs on cell death in ovarian cancer cell lines. ω-3 PUFA, docosahexaenoic acid (DHA) and ω-6 PUFA, γ-linolenic acid (γ-LNA) induced cell death in KF28 cells at the levels of physiological concentrations, but not HAC2 cells. Pharmacological and biochemical analyses demonstrated that cell death induced by DHA and γ-LNA was correlated with activation of JNK and p38 MAP kinases, and further an upstream MAP kinase kinase, apoptosis signal-regulating kinase 1, which is stimulated by reactive oxygen species (ROS). Furthermore, an antioxidant vitamin E attenuated PUFA-induced cell death and MAP kinase activation. These findings indicate that PUFA-induced cell death involves ROS-dependent MAP kinase activation and is a cell type-specific action. A further study of the underlying mechanisms for ROS-dependent cell death induced by PUFAs will lead to the discovery of a new target for cancer therapy or diagnosis.

Enhanced fucosylation of GA1 in the digestive tracts of X-ray-irradiated mice.

In mice at 4 days after X-ray-irradiation at 0.5 Gy/min for 16 min, the tissue weights of immune organs, i.e., thymus and spleen, were decreased due to injury to lymphocytes by the X-rays. The resulting immunosuppressive condition allowed the growth of lactobacilli, i.e., L. murinus, which contained LacßTH-DG and possessed the ability to induce transcription of the fucosyltransferase gene for synthesis of FGA1. LacßTH-DG was detected in the jejunal and ileal contents of X-ray-irradiated mice, but not in those of control mice, whereas LacTetH-DG of L. johnsonii was present in the stomach and caecal contents of both mice. The amounts of FGA1 in the duodenal and jejunal tissues of X-ray-irradiated mice increased to 4- and 9-fold of those in controls, respectively. Reflecting the enhanced fucosylation of GA1, the total amounts of FGA1 excreted into the contents of X-ray-irradiated mice were 1.4-times higher than those in controls. Also, when the extent of enhanced fucosylation of GA1 in several regions of the digestive tracts of X-ray-irradiated mice was compared with that in immune deficient nude, scid and pIgR(-/-) mice, the more than 4-fold increases of FGA1 observed in duodenal and jejunal tissues corresponded to those in pIgR(-/-) mice without secretory IgA. Since an increased amount of FGA1 in the small intestine was observed only 4 days after X-ray-irradiation, and diminished synthesis of FGA1 occurred on administration of penicillin and streptomycin, fucosylation of GA1 in the small intestine was revealed to occur quickly in response to a change in the intestinal bacterial population.

Effective Selection of a Well-Differentiated Type of Human Uterine Endometrial Carcinoma Cells by Transfection of the Sulfotransferase Gene and Possible Association of Sulfoglycolipids With Well-Differentiated Phenotypes.

OBJECTIVES: Sulfatide has been shown to be characteristically increased on the apical surface of the normal endometrium at the secretory phase, and to be related with the formation of the glandular structure and the secretion of mucin from glands for the implantation of a fertilized egg. Additionally, sulfatides are expressed in the well-differentiated type, but not in the poorly differentiated type, of endometrial carcinomas. This suggests that sulfatides are a molecular marker of differentiated phenotypes. To further elucidate the biological significance of sulfoglycolipids, we transfected the sulfotransferase gene into endometrial carcinoma-derived cells without sulfoglycolipids and compared their glycolipid compositions and phenotypes with those of the original cells. MATERIALS AND METHODS: The glycolipid sulfotransferase gene was transfected into endometrial carcinoma-derived SNG-II cells, the resultant transfected cells being found to frequently form a domelike structure, and some of them were selected as SNG-II-GST cells. We compared the glycolipid compositions and phenotypes of SNG-II and SNG-II-GST cells. RESULTS: Although the original SNG-II cells grew in a paving stone pattern, SNG-II-GST cells formed a domelike structure. SNG-II-GST cells exhibited high GST activity and contained sulfoglycolipids, IISO3-LacCer and IISO3-Gg3Cer, which were not found in SNG-II cells. The amounts of sulfoglycolipids in SNG-II-GST cells were 1.5 times higher than those of gangliosides, and the proportions of LacCer and GM3 in SNG-II-GST cells were greatly different from those in SNG-II cells. SNG-II and SNG-II GST cells exhibited poorly differentiated and well-differentiated phenotypes on histochemical examination of cancerous nodules in nude mice. However, by means of an oxygen electrode, SNG-II-GST cells were found to be more resistant to anticancer drugs than SNG-II cells. CONCLUSION: Enhanced expression of sulfoglycolipids in poorly differentiated cells is a feasible means of selecting well-differentiated ones, and sulfoglycolipids are involved in the well-differentiated phenotype like those in the normal endometrium at the secretory phase.

Enhanced expression of unique gangliosides with GM2-determinant in human uterine cervical carcinoma-derived cell lines.

Monoclonal antibody YHD-06 generated by immunization with GM2 reacted with gangliosides with GM2-determinant, i.e., GM2, GalNAc-GM1b and GalNAc-GD1a, among which GalNAc-GD1a was characterized as an antigen of autoimmune peripheral neuropathies including Guillain-Barré syndrome. When glycolipids were examined by TLC-immunostaining with YHD-06 in seven human cervical carcinoma-derived cell lines, GM2 was found in all cell lines, amounting to 15.5 % to 57.5 % of total gangliosides. Whereas GalNAc-GD1a was present in three cell lines, amounting to 5.4-17.5 % of total gangliosides, and GalNAc-GM1b in four cell lines in amounts of less than 2 %. The elevated amounts of gangliosides with GM2 determinant were closely correlated with the relative intensities of gene expression of GalNAc transferase, this being characteristic of cervical carcinoma-derived cells. However, in tissues from patients with several histological types of cervical carcinomas, GM3 was ubiquitously expressed in amounts of more than 66 % of total gangliosides, GM2 was expressed in only five of 15 tissues, and both GalNAc-GM1b and GalNAc-GD1a were not even detected in trace amounts. Since GM1 was detected in all tissues in amounts of less than 0.06 µg/mg dried tissue, all cervical carcinoma tissues were revealed to exhibit GM2 synthesis, indicating that enhanced synthesis of gangliosides with GM2 determinant is a characteristic of cultivated cells in vitro. Similarly, although I(3)SO3-GalCer was not present in the squamous cell carcinoma (SCC) tissues, SCC-derived cells selectively expressed II(3)SO3-LacCer. Since enhanced synthesis of GM2 has been reported in SV-40 virus-transfected fibroblasts, papilloma virus might be involved in the expression of GM2 in cervical carcinoma-derived cells.

Extracellular lipid metabolism influences the survival of ovarian cancer cells.

Lysophosphatidic acid (LPA) is an extracellular lipid mediator consisting of a fatty acid and a phosphate group linked to the glycerol backbone. Here, we show that 1-oleoyl- and 1-palmitoyl-LPA, but not 1-stearoyl- or alkyl-LPA, enhance HNOA ovarian cancer cell survival. Other lysophospholipids with oleic or lauric acid, but not stearic acid, also induce the survival effects. HNOA cells have the lipase activities that cleave LPA to generate fatty acid. Oleic acid stimulates HNOA cell survival via increased glucose utilization. Our findings suggest that extracellular lysolipid metabolism might play an important role in HNOA cell growth.

Changes in bacterial glycolipids as an index of intestinal lactobacilli and epithelial glycolipids in the digestive tracts of mice after administration of penicillin and streptomycin.

The major lipid constituent of symbiotic gram-positive bacteria in animals are phosphatidylglycerol, cardiolipin and dihexaosyl diglycerides (DH-DG), whose hydrophobic structures are characteristic of the environments, and the carbohydrate structures of DH-DGs are bacterial species-characteristic. Immunization of rabbits with intestinal lactobacilli generated antibodies against DH-DGs and their modified structures, among which Galα1-6-substituted DH-DG, i.e., Lactobacillus tetrahexaosyl diglyceride (LacTetH-DG), reacted with antibodies more intensely than DH-DG. Whereas, from the 16S-rRNA sequence, the intestinal lactobacilli in murine digestive tracts were revealed to be L. johnsonii, in which LacTetH-DG is present at the concentration of 2.2 ng per 1 × 10(6) cells. To obtain more accurate estimates of intestinal lactobacilli in several regions of the digestive tract of mice, LacTetH-DG was detected by TLC-immunostaining with anti-Lactobacillus antisera, being found in the stomach, cecum and colon of normal breeding mice, 1.0 × 10(9), 3.5 × 10(9) and 7.4 × 10(9) cells, respectively. Administration of penicillin and streptomycin for 6 days resulted in a reduction in the number of intestinal lactobacilli, the levels being 0 %, 30 % and 4 % of the control ones in the stomach, cecum and colon, respectively, which was associated with the accumulation of the contents in the tracts from the stomach to the cecum and with diarrhea. In addition, a reduced amount of fucosyl GA1 (FGA1) and a compensatory increase in GA1 due to the reduced activity of α1,2-fucosyltransferase in the small intestine and the enhanced discharge of FGA1 into the contents occurred in mice, probably due to the altered population of bacteria caused by administration of penicillin and streptomycin.

Reduced expression of gangliosides with GM2-determinant in cervical carcinoma-derived cells after subcutaneous transplantation into nude mice.

Human cervical carcinoma-derived cell lines have been frequently found to contain gangliosides with GM2-determinant, i.e., GM2, GalNAc-GM1b and GalNAc-GD1a, but GM2 was only detected in 5 of 15 tissues, and GalNAc-GM1b and GalNAc-GD1a were not found in any tissues from patients with several histological types of cervical carcinomas. To further characterize the ganglioside expression in cervical carcinomas, cells were grown by subcutaneous transplantation into nude mice, and gangliosides were quantitated by TLC-immunostaining with the anti-GM2 (YHD-06) antibody and a newly developed anti-GM3 (5H6) antibody, which reacts with GM3 and GM1b, but not with GD1a. Gangliosides with GM2-determinant in cells disappeared in transplanted cells, and the amount of GM3, a precursor for GM2, in transplanted cells was greater than in cultured cells. Also, transplanted cells containing GalNAc-GM1b newly expressed GM1b, suggesting that the activity of GalNAc transferase for synthesis of GalNAc-GM1b is retarded on subcutaneous transplantation. The ganglioside composition, with GM3 as the major one, in the transplanted cells was similar to that in cervical carcinoma tissues, and thus, the expression of gangliosides with GM2-determinant seemed to be accelerated under cell-cultivation conditions.

Bacterial species-characteristic profiles of molecular species, and the antigenicity of phospholipids and glycolipids in symbiotic Lactobacillus, Staphylococcus and Streptococcus species.

Human symbiotic bacteria, Lactobacillus reuteri (LR) in the intestines, Staphylococcus epidermidis (SE) in skin and Streptococcus salivalis (SS) in the oral cavity, contain dihexaosyl diglycerides (DH-DG) in concentrations equivalent to those of phosphatidyl glycerol (PG) and cardiolipin (CL), together with mono- to tetrahexaosyl DGs. The molecular species, as the combination of fatty acids in the DG moiety, were revealed to be bacterial species-characteristic, but to be similar between glycolipids and phospholipids in individual bacteria, the major ones being 16:0 and cy19:0 for LR, ai15:0 and ai17:0 for SE, and 16:0 and 18:1 for SS, respectively. The carbohydrate structures of DH-DGs were also bacterial species-characteristic, being Galα1-2Glcα for LR, Glcß1-6Glcß for SE, and Glcα1-2Glcα for SS, respectively. Also, bacterial glycolipids were revealed to provide antigenic determinants characteristic of bacterial species on immunization of rabbits with the respective bacteria. Anti-L. johnsonii antiserum intensely reacted with tri- and tetrahexaosyl DGs, in which Galα was bound to DH-DG through an α1-6 linkage, as well as with DH-DG from LR. Although anti-SE antiserum preferentially reacted with DH-DG from SE, anti-SS antiserum reacted with DH-DG from SS and, to a lesser extent, with DH-DGs from LR and SE. But, both anti-SE and anti-SS antiserum did not react at all with monohexaosyl DG or glycosphingolipids with the same carbohydrates at the nonreducing terminals. In addition, 75 % of human sera, irrespective of the ABO blood group, were found to contain IgM to tri- and tetrahexaosyl DGs from LR, but not to DH-DGs from LR, SE and SS.

Enhanced expression of sulfatide, a sulfated glycolipid, in well-differentiated endometrial adenocarcinoma.

OBJECTIVES: It is well known that a poorly differentiated endometrial adenocarcinoma shows more rapid progression and a worse response to therapy than a well-differentiated endometrial adenocarcinoma. Qualitative and quantitative changes of cell surface glycolipids occur during neoplastic transformation. Sulfatide is one of the sulfated glycolipids in the cell membrane that may have an important role in various functions such as cell adhesion. To examine the molecular background of the morphological and biological features of well-differentiated and poorly differentiated cancer, we measured the levels of lipids, especially glycolipids, in tumor tissues from patients with endometrial carcinoma. MATERIALS AND METHODS: We determined the composition of lipids and glycolipids in tumor tissues, investigated glycosyltransferase messenger RNA expression by the reverse transcription-polymerase chain reaction, and assessed the localization of galactosylceramide sulfotransferase (an enzyme involved in sulfatide biosynthesis) by immunohistochemical staining. RESULTS: No significant differences were observed between well-differentiated and poorly differentiated cancer with respect to the levels of cholesterol ester, cholesterol, phospholipids, cholesterol sulfate, ceramides, neutral glycolipids of the globo series, and GM3 ganglioside. However, the amount of sulfatides in well-differentiated tumors was significantly greater than that in poorly differentiated tumors, which was confirmed by thin-layer chromatography and immunostaining with a monoclonal antisulfatide antibody. Altered expression of sulfatide was found to be secondary to a change of galactosylceramide sulfotransferase messenger RNA expression. Immunohistochemical staining revealed that galactosylceramide sulfotransferase expression was characteristically observed in glandular areas but not in solid areas. CONCLUSION: These findings suggest that sulfatide contributes to the well-differentiated phenotype of endometrial adenocarcinoma and that it is being expressed in normal uterine endometrium at sites of gland formation during the luteal phase, as we have previously reported.

Lewis y regulate cell cycle related factors in ovarian carcinoma cell RMG-I in vitro via ERK and Akt signaling pathways.

OBJECTIVE: To investigate the effect of Lewis y overexpression on the expression of proliferation-related factors in ovarian cancer cells. METHODS: mRNA levels of cyclins, CDKs, and CKIs were measured in cells before and after transfection with the α1,2-fucosyltransferase gene by real-time PCR, and protein levels of cyclins, CDKs and CKIs were determined in cells before and after gene transfection by Western blot. RESULTS: Lewis y overexpression led to an increase in both mRNA and protein expression levels of cyclin A, cyclin D1 and cyclin E in ovarian cancer cells, decrease in both mRNA and protein expression levels of p16 and p21, and decrease of p27 at only the protein expression level without change in its mRNA level. There were no differences in proteins and the mRNA levels of CDK2, CDK4 and CDK6 before and after gene transfection. Anti-Lewis y antibody, ERK and PI3K pathway inhibitors PD98059 and LY294002 reduced the difference in cyclin and CKI expression caused by Lewis y overexpression. CONCLUSION: Lewis y regulates the expression of cell cycle-related factors through ERK/MAPK and PI3K/Akt signaling pathways to promote cell proliferation.

Excretion into feces of asialo GM1 in the murine digestive tract and Lactobacillus johnsonii exhibiting binding ability toward asialo GM1. A possible role of epithelial glycolipids in the discharge of intestinal bacteria.

In the digestive tract of mice (HR-1, 5 months old, ♀), asialo GM1 (GA1) exhibiting receptor activity toward several intestinal bacteria was preferentially expressed in the small intestine. Also, less than 10% of GA1 in the small intestine was converted into fucosylated and sulfated derivatives, but it was completely converted to fucosyl GA1 (FGA1) in the stomach, cecum and colon. Among the lipid components in these tissues, glycolipids other than Forssman antigen and cholesterol sulfate (CS) were present in the digestive tract contents. However, sulfated GA1, sulfatide and fucosyl GM1 in the gastro-intestinal contents were not present in the cecal and colonic contents, in which the major glycolipids were ceramide monohexoside (CMH), GA1 and FGA1. The total amount of GA1 in the whole contents was 20% of that in the tissues. Thus, glycolipids were stable during the process of digestion, and excreted from the body together with cholesterol and CS. On the other hand, Lactobacillus johnsonii (LJ), whose receptor is GA1, was detected in the cecal and colonic contents on sequential analysis of 16S-ribosomal RNA and TLC-immunostaining of antigenic glycolipids with anti-LJ antiserum. LJ was found to comprise 20% of the total bacteria cultured in the lactobacillus medium under aerobic conditions, and to be present in the cecal and colonic contents, 9.8 × 10(7) cells versus 37 µg GA1 and 1.4 × 10(8) cells versus 49 µg GA1, respectively. Thus, GA1 in the contents might facilitate the discharge of intestinal bacteria by becoming attached them to prevent their irregular diffusion.

Study on the expression and clinical significances of lewis y antigen and integrin αv, ß3 in epithelial ovarian tumors.

OBJECTIVE: To detect the expression and clinical significances of Lewis y antigen and integrin αv, ß3 in epithelial ovarian tumors, and to explore the expression correlation between Lewis y antigen and integrin αv, ß3. METHODS: Immunohistochemical staining was performed in 95 cases of epithelial ovarian cancer, 37 cases of borderline tumors, 20 cases of benign tumors, and 20 cases of normal ovarian tissue, for the detection of Lewis y antigen and integrin αv, ß3 expressions, and to analyze the relationship between Lewis y antigen and integrin, and the relationship between clinical and pathological parameters of ovarian cancer. In addition, immunofluorescence double labeling was utilized to detect the expression correlation between Lewis y antigen and integrin αv, ß3 in ovarian cancer. RESULTS: In epithelial ovarian tumors, the expression rate of Lewis y antigen was 81.05%, significantly higher than that of borderline (51.53%) (P < 0.05) and benign (25%) (P < 0.01) tumors, and normal ovarian tissues (0) (P < 0.01). The expression rate of integrin αv, ß3 in malignant epithelial ovarian tumors was 78.95% and 82.11%, respectively, significantly higher than that of the borderline (45.94%, 40.54%) (both P < 0.05), benign group (10.00%, 15.00%) (both P < 0.01) and normal ovary group (5%, 15%) (both P < 0.01). CONCLUSIONS: Lewis y and integrins αv, ß3 are relevant to pelvic and abdominal diffusion and metastasis of ovarian cancer cells, suggesting that these two molecules mediate a boosting function for tumor metastasis.

The stimulation of IGF-1R expression by Lewis(y) antigen provides a powerful development mechanism of epithelial ovarian carcinoma.

OBJECTIVE: This study aimed to measure and correlate the expression of insulin-like growth factor receptor-1 (IGF-1R) and the Lewis(y) antigen in ovarian cancer cell lines and tissue samples. METHODS: Reverse transcriptase PCR (RT-PCR), Western blotting, immunoprecipitation, immunohistochemistry, and immunofluorescence double-labeling techniques were applied to detect and measure the expression of Lewis(y) and IGF-1R. RESULTS: In α1,2-fucosyltransferase (α1,2-FT)-transfected cells, IGF-1R expression was significantly upregulated compared with cells that do not overexpress α1,2-FT (P < 0.05). The amount of Lewis(y) expressed on IGF-1R increased 1.81-fold in α1,2-FT-overexpressing cells (P < 0.05), but the ratio of Lewis(y) expressed on IGF-1R to total IGF-1R was unaltered between two cells (P > 0.05). In malignant epithelial ovarian tumors, the positivity rates of Lewis(y) and IGF-1R detection were 88.3% and 93.33%, respectively, which is higher than the positivity rates in marginal (60.00% and 63.33%, all P < 0.05), benign (33.00% and 53.33%, all P < 0.01), and normal (0% and 40%, all P < 0.01) ovarian samples. No correlations were detected in positivity rates of Lewis(y) or IGF-1R expression with respect to clinicopathological parameters in ovarian cancers (all P > 0.05). Both IGF-1R and Lewis(y) were highly expressed in ovarian cancer tissues, and their expression levels were positively correlated (P < 0.05). CONCLUSION: Overexpression of Lewis(y) results in overexpression of IGF-1R. Both IGF-1R and Lewis(y) are associated with the occurrence and development of ovarian cancers.

Increase in docetaxel-resistance of ovarian carcinoma-derived RMG-1 cells with enhanced expression of Lewis Y antigen.

Epithelial carcinomas of the ovary exhibit the highest mortality rate among gynecologic malignancies. Studies found that the metabolism of glycolipids or carbohydrates is associated with acquirement of anticancer drug-resistance by cancer cells. This study was to characterize possible involvement of Lewis Y (Le(Y)) antigen in the drug-resistance of cancer cells. We transfected the α1,2-fucosyltransferase gene into human ovarian carcinoma-derived RMG-1 cells and established RMG-1-hFUT cells with enhanced expression of Le(Y). We determined the effects of docetaxel on the survival of cells by MTT assaying and observed the apoptosis of cells in the presence of docetaxel by flow cytometric analysis and by transmission electron microscopy. Plasma membranes and intracellular granules in RMG-1-hFUT cells were stained with anti-Le(Y) antibody, the intensity of the staining was higher than that in control cells. The RMG-1-hFUT cells exhibited higher resistance to docetaxel than the control cells with regard to the docetaxel concentration and time course. After treatment with 10 µg/mL docetaxel for 72 h, the control cells, but not RMG-1-hFUT, contained abundant positively stained cell debris due to disintegration of the cytoskeleton. On transmission electron microscopy, although the control cells treated with docetaxel as above showed the following morphology, i.e., absence of villi, cells shrunken in size, pyknosis, agglutinated chromatin and cell buds containing nuclei in the process of apoptosis, the RMG-1-hFUT cells showed only agglutinated chromatin and vacuoles in the cytoplasm. In summary, cells with enhanced expression of Le(Y) were shown to acquire docetaxel-resistance, indicating the possible involvement of glycoconjugates in the drug-resistance.

Lewis (y) antigen overexpression increases the expression of MMP-2 and MMP-9 and invasion of human ovarian cancer cells.

Lewis (y) antigen is a difucosylated oligosaccharide present on the plasma membrane, and its overexpression is frequently found in human cancers and has been shown to be associated with poor prognosis. Our previous studies have shown that Lewis (y) antigen plays a positive role in the process of invasion and metastasis of ovarian cancer cells. However, the mechanisms by which Lewis (y) antigen enhances the invasion and tumor metastasis are still unknown. In this study, we established a stable cell line constitutively expressing Lewis (y) antigen (RMG-1-hFUT) by transfecting the cDNA encoding part of the human α1,2-fucosyltransferase (α1,2-FUT) gene into the ovarian cancer cell line RMG-1, and investigated whether Lewis (y) antigen regulates the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9, and tissue inhibitors of metalloproteinases (TIMP-1) and TIMP-2. We found that RMG-1-hFUT cells exhibited higher invasive capacities than their control cells. In addition, expression of TIMP-1 and TIMP-2 was down-regulated and expression of MMP-2 and MMP-9 was up-regulated. Anti-Lewis (y) antigen antibody treatment significantly reversed the expression of TIMP-1, TIMP-2, MMP-2 and MMP-9. Taken together, we provide the first evidence that down-regulation of TIMP-1 and TIMP-2 and up-regulation of MMP-2 and MMP-9 represents one of the mechanisms by which Lewis (y) antigen promotes cell invasion.

Spermatogenesis-associated changes of fucosylated glycolipids in murine testis.

By targeted deletion of either the FUT1- or FUT2-gene for α1,2-fucosyltransferase, expression of FGM1 and FGA1, in murine testis was revealed to be sustained through unique interchangeability of the genes, indicating their significant roles for spermatogenesis. Accordingly, we examined the amounts of FGM1 and FGA1 in the testes of mice at 1-42 days after birth in comparison to those of several glycolipids including seminolipid. Although Forssman antigen and GM1 were present in relatively constant amounts during the period examined, GM3, which was the major one at 1 day, quickly decreased during development and had completely disappeared at 4 weeks. The following glycolipids were expressed in stage-specific manners, FGM1 for primary spermatocytes at 1 week, a seminolipid for secondary spermatocytes at 2 weeks, and GM3 lactone and FGA1 for spermatids and spermatozoa at 3 weeks. In fact, immunohistochemical staining with anti-FGM1 and anti-FGA1 antibodies demonstrated that FGM1 and FGA1 were distributed in the spermatocytes, and the spermatids and spermatozoa, respectively, and FGA1, together with seminolipid, were the immunogenic markers of spermatozoa. Thus, the fucosylation of glycolipids is a spermatogenesis-associated event, which should occur even with use of either the FUT1- or FUT2-gene.

Contribution of glycolipids to species-specific antigens on erythrocytes of several animal species as to recognition of antigens with rabbit anti-glycolipids and anti-erythrocyte antisera.

Because anti-glycolipid antibodies are involved in the onset of several neurological diseases, the reactivity of glycolipids on erythrocytes and the probability of generating the antibodies were determined to clarify the contribution of glycolipids as antigens. Anti-erythrocyte antisera reacted with the following glycolipids in a species-specific manner, i.e. blood group A-active glycolipid for man, Forssman glycolipid for sheep, Gg(3)Cer for guinea pig, and Gg(4)Cer and fucosyl GM1 for rat, and the hemolytic activities of the anti-erythrocyte antisera were attenuated by absorption of the antisera with liposomes prepared from the lipids of erythrocytes to the following levels, 94.5% for man, 24.5% for sheep, 17.5% for guinea pig, and 54.5% for rat. These species-specific glycolipids on erythrocytes reacted well with the respective anti-glycolipid antisera, but Gb(4)Cer in man and GM1 in rat were shown to be cryptic on immunization with erythrocytes, indicating that the contribution of glycolipids as erythrocyte antigens differs among animal species.

Phosphatidyl ethanolamine with increased polyunsaturated fatty acids in compensation for plasmalogen defect in the Zellweger syndrome brain.

To elucidate the neuropathological mechanism of Zellweger syndrome (ZS), we studied changes in the molecular species of glycerophospholipids in the cerebral tissue by thin-layer chromatography (TLC) and fast atom bombardment mass spectrometry (FABMS). First, we estimated the amount of plasmalogens by TLC. Plasmalogen-type phosphatidyl ethanolamine (PE) accounted for 30% of the total PE in the control brain, but was absent in the ZS brain. Plasmalogen-type phosphatidyl choline (PC) was undetectable in both control and ZS brains. Next, we analyzed plasmalogen-type PE by FABMS. Oleic (18:1), arachidonic (20:4) and docosapentanoic (22:5) acids were present in the control gray matter, but not in the ZS gray matter. In compensation for the defect of plasmalogen, the level of diacyl PE with polyunsaturated fatty acids, 20:4, 22:4, 22:5 and 22:6, was higher in the ZS brain than that in the control brain. These results indicate an alteration in the molecular species of PE, which may cause abnormal neural membrane fluidity and excessive vulnerability to oxygen stress.