Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; Cell Line, Tumor ; Doxorubicin ; pharmacology ; Gangliosides ; Humans ; NF-kappa B ; metabolism

Gangliosides have been suggested to play important roles in various functions such as adhesion, cell differentiation, growth control, and signaling. Mouse follicular development, ovulation, and luteinization during the estrous cycle are regulated by several hormones and cell-cell interactions. In addition, spermatogenesis in seminiferous tubules of adult testes is also regulated by several hormones, including follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and cell-cell interactions. The regulation of these processes by hormones and cell-cell interactions provides evidence for the importance of surface membrane components, including gangliosides. During preimplantation embryo development, a mammalian embryo undergoes a series of cleavage divisions whereby a zygote is converted into a blastocyst that is sufficiently competent to be implanted in the maternal uterus and continue its development. Mouse embryonic stem (mES) cells are pluripotent cells derived from mouse embryo, specifically, from the inner cell mass of blastocysts. Differentiated neuronal cells are derived from mES cells through the formation of embryonic bodies (EBs). EBs recapitulate many aspects of lineage-specific differentiation and temporal and spatial gene expression patterns during early embryogenesis. Previous studies on ganglioside expression during mouse embryonic development (including during in vitro fertilization, ovulation, spermatogenesis, and embryogenesis) reported that gangliosides were expressed in both undifferentiated and differentiated (or differentiating) mES cells. In this review, we summarize some of the advances in our understanding of the functional roles of gangliosides during the stages of mouse embryonic development, including ovulation, spermatogenesis, and embryogenesis, focusing on undifferentiated and differentiated mES cells (neuronal cells).
Animals ; *Cell Differentiation ; *Embryonic Development ; Embryonic Stem Cells/*cytology/metabolism ; Gametogenesis ; Gangliosides/*metabolism ; Mice ; Urogenital System/cytology/embryology/metabolism

OBJECTIVETo compare the substrate specificity of three murine GDP fucose: beta-galactoside alpha1,2-fucosyltransferases (alpha1,2-FT).

METHODSThree members of MFUT- I, -II and -III, coding for a alpha1,2-FT, a GDP-fucose, were cloned from a cDNA of murine small intestine by reverse transcription-polymerase chain reaction. The coding regions were ligated into mammalian expression vector pcDNA 3.1 (pcDNA3.1-MFUT-I, pcDNA3.1-MFUT- II , and pcDNA3.1-MFUT- III) and were transiently transfected into COS-7 cells using a cellphect transfection kit. Then the cells were analyzed for expression and function of alpha1,2-FT and the substrate specificity of three alpha1,2-FT was compared.

RESULTSMFUT- I, -II, and -III exhibited sequence homology with human H (77%), Se (79%), and Sec1 (75%) genes, respectively. COS-7 cells transfected with pcDNA3.1-MFUT- I and pcDNA3.1-MFUT- II showed alpha1,2-FT activity, but no activity was detected in COS-7 cells transfected with pcDNA3.1- MFUT-III. MFUT- II showed alpha1,2-FT activity with both asialo-monosialoteterahexosyl ganglioside (GA1) and monosialoteterahexosyl ganglioside (GM1) as substrates to produce fucosyl GA1(FGA1) and fucosyl GM1(FGM1), respectively, but MFUT- I only showed alpha1,2-FT activity with GA1. The relative activity of MFUT- II with GA1 was 80-90-folds higher compared with MFUT- I, and the relative activity of MFUT- II with GA1 was 10-20-folds higher than that of GM1. The fucosyltransferase encoded by the MFUT- II gene showed the enzyme activity not only responsible for the synthesis of type 4-H antigens FGA1 and FGM1, but also responsible for the synthesis of type 1-H and 2-H antigens with lactotetraosylceramide and neolactotetraosylceramide as substrates.

CONCLUSIONMFUT- II is the main alpha1,2-FT in mouse and MFUT- II can product type 4-H antigen FGA1 and FGM1, but MFUT- I only synthesizes FGA1. MFUT-III has no alpha1,2-FT activity.

Animals ; Antigens, Bacterial ; biosynthesis ; COS Cells ; Cercopithecus aethiops ; Cloning, Molecular ; Fucosyltransferases ; chemistry ; genetics ; metabolism ; Gangliosides ; metabolism ; Mice ; Substrate Specificity ; Transfection

Stem cells are used for the investigation of developmental processes at both cellular and organism levels and offer tremendous potentials for clinical applications as an unlimited source for transplantation. Gangliosides, sialic acid-conjugated glycosphingolipids, play important regulatory roles in cell proliferation and differentiation. However, their expression patterns in stem cells and during neuronal differentiation are not known. Here, we investigated expression of gangliosides during the growth of mouse embryonic stem cells (mESCs), mesenchymal stem cells (MSCs) and differentiated neuronal cells by using high-performance thin-layer chromatography (HPTLC). Monosialoganglioside 1 (GM1) was expressed in mESCs and MSCs, while GM3 and GD3 were expressed in embryonic bodies. In the 9-day old differentiated neuronal cells from mESCs cells and MSCs, GM1 and GT1b were expressed. Results from immunostaining were consistent with those observed by HPTLC assay. These suggest that gangliosides are specifically expressed according to differentiation of mESCs and MSCs into neuronal cells and expressional difference of gangliosides may be a useful marker to identify differentiation of mESCs and MSCs into neuronal cells.
Neurons/*cytology/*metabolism ; Mice ; Mesenchymal Stem Cells/*cytology/*metabolism ; Gangliosides/*metabolism ; Embryonic Stem Cells/*cytology/*metabolism ; Cells, Cultured ; Cell Differentiation ; Animals

Neuronal apoptosis induced by amyloid beta-peptide (A beta) plays an important role in the pathophysiology of Alzheimer's disease (AD). However, the molecular mechanism underlying A beta-induced apoptosis remains undetermined. The disialoganglioside GD3 involves ceramide-, Fas- and TNF-alpha-mediated apoptosis in lymphoid cells and hepatocytes. Although the implication of GD3 has been suggested, the precise role of GD3 in A beta-induced apoptosis is still unclear. Here, we investsigated the changes of GD3 metabolism and characterized the distribution and trafficking of GD3 during A beta-induced apoptosis using human brain-derived TE671 cells. Extracellular A beta induced apoptosis in a mitochondrial-dependent manner. GD3 level was negligible in the basal condition. However, in response to extracellular A beta, both the expression of GD3 synthase mRNA and the intracellular GD3 level were dramatically increased. Neosynthesized GD3 rapidly accumulated in cell surface lipid microdomains, and was then translocated to mitochondria to execute the apoptosis. Disruption of membrane lipid microdomains with methyl-beta-cyclodextrin significantly prevented both GD3 accumulation in cell surface and A beta-induced apoptosis. Our data suggest that rapidly accumulated GD3 in plasma membrane lipid microdomains prior to mitochondrial translocation is one of the key events in A beta-induced apoptosis.
Amyloid beta-Peptides/*pharmacology ; *Apoptosis ; Cell Line ; Gangliosides/*metabolism/physiology ; Humans ; Membrane Microdomains/*metabolism ; Mitochondria/*metabolism ; Sialyltransferases/genetics/metabolism ; beta-Cyclodextrins/pharmacology

AC133 Antigen ; Animals ; Antigens, CD ; metabolism ; Biomarkers, Tumor ; metabolism ; Breast Neoplasms ; metabolism ; pathology ; CD24 Antigen ; metabolism ; CD55 Antigens ; metabolism ; Female ; Gangliosides ; metabolism ; Glycoproteins ; metabolism ; Hedgehog Proteins ; metabolism ; Humans ; Hyaluronan Receptors ; metabolism ; Isoenzymes ; metabolism ; Neoplastic Stem Cells ; metabolism ; Octamer Transcription Factor-3 ; metabolism ; Peptides ; metabolism ; Receptors, Notch ; metabolism ; Retinal Dehydrogenase ; metabolism ; Signal Transduction ; Wnt Signaling Pathway

By combining interleukin2 (IL-2) with a tumor specific antibody, immunotherapy of tumors may become more effective in the future. Anti-GD2 single chain antibody directed to the extracellular domain of GD2 disialoganglioside can result in an antitumor response in some pateins with tumors expressing GD2. In this study, the fusion protein consisting of GD2 single chain antibody (ScFv) and IL-2(Ala125) was constructed. Anti-GD2 ScFv and IL-2 genes were obtained by PCR, then the ScFv-IL-2 gene was constructed by over lap PCR. The gene was inserted into the pMD18-T easy vector. Genes from pMD18-T -vector were inserted into expression vector pSE380. Recombinant expression vector was identified by restriction enzyme-cutting and then was transformed into BL21. SDS-PAGE and Western blot analysis confirmed that the transformed E. Coli BL21 could express ScFv-IL-2 fusion-proteins and the molecular weight is 43 kDa. The fusion protein was purified by affinity chromatograph and Sephacryl S-200HR then was identified through ELISA. The results show that the fusion protein retains the activities of both antigen binding and IL-2.
Antibodies ; genetics ; metabolism ; Antibodies, Monoclonal ; biosynthesis ; genetics ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gangliosides ; immunology ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Interleukin-2 ; biosynthesis ; genetics ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics

This study sought to construct a recombinant vector that expresses anti-GD2/anti-CD16 bispecific single-chain antibody(sc-BsAb), and to assess its biological activities. The anti-GD2 gene and the anti-CD16 gene (NM3E2) were obtained using PCR amplification technique, and then the fusion gene was constructed by overlapping PCR. The sc-BsAb gene was subcloned into the pET-22b(+) plasmid from the pMD18-T easy vector by digestion with NcoI, Hind III restriction endonucleases, whose sites exist in both the vectors. Then the combinant plasmids were transferred into E. coli BL21 (DE3). The expression product in the periplasmic was analyzed by both SDS-PAGE and Western blot technique, then was purified with Ni2+ -NTA superflow affinity chromatography. It was demonstrated that the linker in the sc-BsAb fusion protein is SerGly4Ser. and the molecular is 53 KD.
Antibodies, Bispecific ; biosynthesis ; genetics ; Antibodies, Neoplasm ; biosynthesis ; genetics ; Base Sequence ; Cell Line, Tumor ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gangliosides ; immunology ; HeLa Cells ; Humans ; Melanoma ; pathology ; Molecular Sequence Data ; Receptors, IgG ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; genetics

In brain tissue, astrocytes play defensive roles in central nervous system integrity by mediating immune responses against pathological conditions. Type I phosphatidylinositol 4-phosphate 5-kinase alpha (PIP5Kalpha) that is responsible for production of phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) regulates many important cell functions at the cell surface. Here, we have examined whether PIP5Kalpha is associated with astrocyte inflammatory responses. Gangliosides are releasable from damaged cell membranes of neurons and capable of inducing inflammatory responses. We found that treatment of primary cultured astrocytes with gangliosides significantly enhanced PIP5Kalpha mRNA and protein expression levels. PI(4,5)P2 imaging using a fluorescent tubby (R332H) expression as a PI(4,5)P2-specific probe showed that ganglioside treatment increased PI(4,5)P2 level. Interestingly, microRNA-based PIP5Kalpha knockdown strongly reduced ganglioside-induced transcription of proinflammatory cytokines IL-1beta and TNFalpha. PIP5Kalpha knockdown also suppressed ganglioside-induced phosphorylation and nuclear translocation of NF-kappaB and the degradation of IkappaB-alpha, indicating that PIP5Kalpha knockdown interfered with the ganglioside-activated NF-kappaB signaling. Together, these results suggest that PIP5Kalpha is a novel inflammatory mediator that undergoes upregulation and contributes to immune responses by facilitating NF-kappaB activation in ganglioside-stimulated astrocytes.
Animals ; Astrocytes/*metabolism ; Cells, Cultured ; Gangliosides/*metabolism ; Gene Knockdown Techniques ; Inflammation/*metabolism ; Interleukin-1/metabolism ; NF-kappa B/metabolism ; Phosphotransferases (Alcohol Group Acceptor)/*metabolism ; RNA, Messenger/*genetics/metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Tumor Necrosis Factor-alpha/metabolism ; Up-Regulation

The human colorectal carcinoma-associated GA733 antigen epithelial cell adhesion molecule (EpCAM) was initially described as a cell surface protein selectively expressed in some myeloid cancers. Gangliosides are sialic acid-containing glycosphingolipids involved in inflammation and oncogenesis. We have demonstrated that treatment with anti-EpCAM mAb and RAW264.7 cells significant inhibited the cell growth in SW620 cancer cells, but neither anti-EpCAM mAb nor RAW264.7 cells alone induced cytotoxicity. The relationship between ganglioside expression and the anti-cancer effects of anti-EpCAM mAb and RAW264.7 was investigated by high-performance thin-layer chromatography. The results demonstrated that expression of GM1 and GD1a significantly increased in the ability of anti-EpCAM to inhibit cell growth in SW620 cells. Anti-EpCAM mAb treatment increased the expression of anti-apoptotic proteins such as Bcl-2, but the expression of pro-apoptotic proteins Bax, TNF-alpha, caspase-3, cleaved caspase-3, and cleaved caspase-8 were unaltered. We observed that anti-EpCAM mAb significantly inhibited the growth of colon tumors, as determined by a decrease in tumor volume and weight. The expression of anti-apoptotic protein was inhibited by treatment with anti-EpCAM mAb, whereas the expression of pro-apoptotic proteins was increased. These results suggest that GD1a and GM1 were closely related to anticancer effects of anti-EpCAM mAb. In light of these results, further clinical investigation should be conducted on anti-EpCAM mAb to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer.
Animals ; Antibodies, Monoclonal/*immunology/*therapeutic use ; Antigens, Neoplasm/*immunology ; Apoptosis/drug effects ; Cell Adhesion Molecules/*immunology ; Cell Line ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Colon/drug effects/immunology/metabolism/pathology ; Colonic Neoplasms/*drug therapy/genetics/*immunology/pathology ; Gangliosides/genetics/*immunology ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; Male ; Mice ; Mice, Inbred BALB C