Antagonistic effect of ganglioside GM1 and GM3 on the activity and conformation of sarcoplasmic reticulum Ca(2+)-ATPase.

It was found that rabbit skeletal muscle sarcoplasmic reticulum (SR) contained two main gangliosides: NeuNAc alpha 2-->3 Gal beta 1-->4 Glc beta 1-->1'ceramide (GM3) and Gal beta 1-->3 GalNAc beta 1-->4(NeuNAc alpha 2-->3) Gal beta 1-->4 Glc beta 1-->1'ceramide (GM1), and that the most abundant ganglioside GM3 could positively modulate the SR Ca(2+)-ATPase activity. In this paper, the effect of GM1 on Ca(2+)-ATPase was further investigated and compared with that of GM3. The study demonstrates that GM1 has an opposite effect with respect to GM3 on the activity of SR Ca(2+)-ATPase. Using assays, including intrinsic and time-resolved fluorescence and fluorescence quenching, the conformational changes of SR Ca(2+)-ATPase induced by GM1 and GM3 were compared. Obtained results indicate that GM1 could make the Ca(2+)-ATPase molecules less compact in the hydrophilic domain but more compact in the hydrophobic domain, while GM3 makes the enzyme more compact in both the hydrophilic and hydrophobic domain. Homogeneous GM1 and GM3 with the same ceramide moiety had similar effects on SR Ca(2+)-ATPase activities compared to their natural counterparts, suggesting that the carbohydrate chain may be the key moiety of the ganglioside molecule to be responsible for the difference of the effect on enzyme activity.

Mechanistic study of modulation of SR Ca2+-ATPpase activity by gangliosides GM1 and GM3 through some biophysical measurements.

On the basis of confirming the antagonistic effects of GM1 and GM3 on the activity of Ca2+-ATPase, we further demonstrated that some of the components of these two gangliosides, including sialic acid (NeuNAc), asialo-GM1, asialo-GM3 and ceramide, failed to show any effects on the activity of Ca2+-ATPase. Thus it is apparent that the intact molecules of these two gangliosides with their specific conformations were needed to perform their effects on Ca2+-ATPase. From the fluorescence resonance energy transfer measurements, the energy transfer between Cys 670/674 and Lys 515 was decreased by GM1 and increased by GM3, indicating GM1 induced the conformation of the hydrophilic region of Ca2+-ATPase to be less compact, while GM3 induced it to be more compact. From the CD spectra measurements, GM1 and GM3 both reduced the content of alpha-helical structures of Ca2+-ATPase, but GM1 caused a stronger decrease than that of GM3. Using DPH as the probe, we found that the membrane lipid fluidity of the proteoliposomes containing Ca2+-ATPase was decreased by GM1 and tend to increase by GM3.

Induction of nitric oxide production by ganglioside GM3 in murine peritoneal macrophages activated for tumor cytotoxicity.

BACKGROUND: Recently, it was found that ganglioside GM3, known as a potent monocytic cell differentiation inducer, mimicked bacterial lipopolysaccharide (LPS) in the activation of macrophage-mediated tumor cytotoxicity (MTC). Since GM3 and LPS share partial structural and functional similarity, we postulate that the GM3-induced MTC was due to the induction of nitric oxide(NO) production in murine peritoneal macrophages (M phi s). MATERIALS RESULTS, CONCLUSIONS: Ganglioside GM3 was highly purified from canine erythrocytes by the authors. Murine peritoneal M phi s were used as the effector cells, and mouse ascites hepatoma cell line HCa-F25/16A3-F as the target cells. We found that: GM3 induced NO production by macrophages in a time- and dose-dependent manner; GM3 and IFN-gamma synergistically increased NO production; GM3 markedly enhanced MTC; both NO production and MTC were significantly inhibited by aminoguanidine. These results demonstrated for the first time that ganglioside GM3 in vitro induces the production of nitric oxide by macrophages, and the GM3-induced MTC is NO-dependent.

Cholecystokinin antisense RNA increases the analgesic effect induced by electroacupuncture or low dose morphine: conversion of low responder rats into high responders.

The analgesic effects of the rat in response to electroacupuncture (EA) or low-dose morphine (3 mg/kg) show marked individual variations. In the midbrain periaqueductal gray (PAG) of the rat, the content of the neuropeptide cholecystokinin octapeptide (CCK-8) was found to be significantly higher in the low responder (LR) rats as compared to that in the high responders (HR). Since PAG has been shown to be a strategic site for CCK-8 to exert an anti-opioid action, a high CCK content in PAG may account for the low analgesic responsiveness to EA and morphine. In order to block the expression of the gene encoding preproCCK in the brain, antisense CCK expression vector pSV2-CCKAS was microinjected into the lateral cerebral ventricle of the rat, leading to a decrease of the CCK-mRNA as well as the CCK-8 content in rat brain. This effect started 4 days after the intracerebroventricular (i.c.v.) injection of the antisense expression vector, and lasted no more than 1 week. This procedure was shown to be very effective in converting LR rats into HR for EA analgesia and morphine analgesia, and also delayed the development of tolerance elicited by prolonged EA stimulation or repeated morphine administration. The time course of the augmentation of opioid analgesia (4-6 days after the i.c.v. injection of the expression vector) paralleled the decrease of the brain CCK-8 content. The results argue that blocking the CCK gene expression in the brain may tilt the balance between opioid and anti-opioid peptides in favor of the former, thus strengthening the EA analgesia and morphine analgesia, and delaying the development of opioid tolerance.

The role of ganglioside GM3 in the modulation of conformation and activity of sarcoplasmic reticulum Ca(2+)-ATPase.

Rabbit sarcoplasmic reticulum does contain trace amounts of gangliosides, and the main species is GM3. Incorporation of GM3 into the SR vesicles or addition of it to the soybean phospholipid used for reconstitution of proteoliposomes obviously increased ATP hydrolysis, as well as, Ca2+ uptake activity of sarcoplasmic reticulum Ca(2+)-ATPase. Conformation changes of Ca(2+)-ATPase induced by GM3 were also observed by circular dichroism, intrinsic fluorescence and fluorescence quenching measurements.

Effect of ganglioside GM3 on the activity and conformation of reconstituted Ca2+-ATPase.

Trace amounts of gangliosides were found in rabbit skeletal muscle sarcoplasmic reticulum and their main part was shown, by high performance thin layer chromatography. to be GM3. Addition of GM3 to the soybean phospholipids used for reconstitution of proteoliposomes markedly increased ATP hydrolysis as well as Ca2+ uptake activity of sarcoplasmic reticulum Ca2+-ATPase incorporated into the proteoliposomes. Conformation changes of Ca2+-ATPase induced by GM3 were also observed by intrinsic fluorescence and circular dichroism measurements.

Isolation and ex vivo expansion of CD34+ cells from cord blood using dextran sedimentation and avidin column selection.

Human umbilical cord bloods were fractionated by unit gravity sedimentation in 1% (v/v) dextran, followed by immunoaffinity selection for CD34+ stem and progenitor cells. Dextran sedimentation alone enabled recovery of more than 80% of the nucleated cells present and 90% of the CD34+ cells, as determined by flow cytometry. The addition of an immunoaffinity selection step for CD34+ cells resulted in a 134-fold enrichment for CD34+ cells, with a mean yield of 64 +/- 15%. The resultant CD34+ population contained almost half the CFU-GM activity initially present in the cord bloods and could be expanded ex vivo in liquid culture.

Glycosphingolipids of rabbit endometrium and their changes during pregnancy.

The glycolipids of nonpregnant and pregnant rabbit endometrium were characterized using a combination of biochemical and immunochemical techniques. Quantitative analyses indicated a 70% decline in acidic glycolipid (ganglioside) content during early pregnancy (day 6), and a 2.5-fold increase in neutral glycolipid content during later pregnancy (day 26). The major gangliosides of rabbit endometrium were identified by thin-layer chromatography as GM3 and GD3, with minor amounts of GM1, GD1a and GT1b. The major neutral glycolipids were identified similarly as globo-series structures Gb3 and Gb4. Monoclonal antibodies (mAbs) directed to glycolipid antigens permitted the detection of additional glycolipid species, including sialylated, sulfated and fucosylated lacto-series structures. Difucosyl Ley structure (defined by mAb AH-6) and sulfated-galactosyl structure (defined by mAb VESP 6.2) were identified by indirect immunofluorescence along the luminal surface of the endometrium during the implantation period. Rapid changes in the glycolipid composition of endometrial cells during early pregnancy may facilitate embryo adhesion and trophectoderm outgrowth during implantation.

Effect of a cell differentiation inducer, ganglioside GM3, on the neutral glycosphingolipid composition and cell membrane fluidity of a human promyelocytic leukemia cell line HL-60.

In this paper the authors confirmed the finding reported by Nojiri et al that exogenous ganglioside GM3 could induce HL-60 cells to differentiate along the monocytic-macrophage route with significant inhibition of proliferation. It was found that after differentiation the neutral GSL content of CDH and CMH increased, while that of CXH, a polyhexosyl ceramide with a TLC mobility slower than CQH, decreased significantly. The membrane fluidity of HL-60 cells decreased after GM3 treatment.

Glycosphingolipids of human myometrium and endometrium and their changes during the menstrual cycle, pregnancy and ageing.

The glycolipid composition of human myometrium and endometrium was examined at various stages of maturation and reproduction. The major neutral glycolipids of both myometrium and endometrium were identified by high-performance thin-layer chromatography as globo-series glycolipids, Gb3 and Gb4. The major acidic glycolipids (gangliosides) were identified similarly as GM3 and GD3, with lesser amounts of GM1, GD1a, and GT1b. During pregnancy, GD3 expression declined in both myometrium and endometrium, whereas GM3 expression increased. Reciprocal changes in GM3/GD3 expression were mirrored by appropriate changes in the glycosyltransferases required for their synthesis; alpha 2----3sialyltransferase activity increased approximately 3-fold during pregnancy, while alpha 2----8sialyltransferase activity declined to about 20%. The results focus attention on the glycolipids of uterine tissues, their regulation, and their possible role in reproduction and fertility.

The relationship between nutritional antioxidants and serum lipid peroxides in cancer patients.

A new parameter, the ratio of lipid peroxide and vitamins E and C [LPO/(VE + VC)], has been proposed and used to reflect the balance between lipid peroxidation and antioxidation capability of cancer patients and of healthy human controls. The effects of vitamins E, C, and selenium on the serum LPO level in mice bearing Ascites Hepatomas (H22) have also been examined. The results showed that the average of LPO/(VE + VC) ratios in cancer patients (135 cases) was significantly higher than that of the normal controls (222 cases). The authors suggest that this ratio might be used as one of the parameters for early diagnosis and prognosis of diseases (including cancers) caused by free radicals and lipid peroxides. The results also showed that antioxidants - Se(Na2SeO3, 1mg/kg) or vitamin E (5mg/kg) could markedly decrease the level of serum LPO in the tumor-bearing animals. A smaller dose of VE (1mg/kg) and doses of Vc up to 300mg/kg showed no effect on the serum LPO levels when given separately. However, synergistic effects were observed when any 2 out of 3 or three nutrients were given together. Those with three nutrients significantly lowered the serum LPO level. These antioxidants also inhibited the proliferation of tumour cells.