GM_3在几种哺乳动物肝脏和狗红细胞中的含量及其分离纯化

从正常兔、猪和狗的肝脏及狗红细胞中分离纯化了总神经节苷脂,测定了脂结合唾液酸,进行了高效薄层层析,比较了上述四种组织中GM_3的含量。结果表明狗红细胞中的GM_3的含量较另三种的高,狗肝和兔肝次之,猪肝含量甚微。从狗红细胞中提取和纯化了GM_3,其得量为每毫升压积红细胞351.0μg,纯度为92.2％。     

肝癌中CMP-乙酰神经氨酸:GM_3(α2→8)唾液酸转移酶(GD_3合成酶)的研究及纯化的初步报告

本实验室曾报道在所检测的不同种族来源与不同致癌剂所诱发的肝细胞肝癌中均有神经节苷脂GD_3组份的明显增高,本文就这一现象的机制进行了探讨。实验结果表明在人肝癌手术标本、人肝癌细胞株SMMC,3′-甲基奶油黄(3′Me-DAB)和二乙基亚硝胺(DENA)所诱发的大鼠肝癌以及大鼠肝癌株BERH-2中GD_3合成酶的活性均有不程度的增高,同对GD_3前体的合成酶(GM_3合成酶)的活性也有所增高。这就提示肝癌中GD_3增高的原因之一在于GD_3合成酶的活性增高与前体供应充足的结果。另外,本文还对GD_3合成酶的提纯做了初步尝试。主要采用Tritonx-100抽提和CDP-hydrazide Sepharose 4B亲合层析的方法从二乙基亚硝胺诱发的大鼠肝癌中提纯了GD_3合成酶。提纯倍数为12500倍,产率0.4％。提纯的GD_3合成酶在醋酸纤维膜上经等电聚焦电泳鉴定示单一条带,其pI值为5.25左右。关于糖脂唾液酸转移酶的纯化工作目前还未见报道。     

人正常子宫肌肉的鞘糖脂组成及其在不同生理功能阶段的变化

本文测定了新生儿、生育期、更年期和足月妊娠人子宫肌肉的神经节苷脂(Gg)与中性鞘糖脂(N-GSL)的含量,比较了两种鞘糖脂的HPTLC谱。新生儿期Gg的总含量(以脂结合唾液酸LBSA量表示)最高,每克湿重组织约45.2μg,足月妊娠子宫肌肉中的含量最低,为10.4μg,生育期为32.8μg、更年期为39.5μg。N—GSL的含量却以足月妊娠子宫肌肉中最多,达99.4μg。按HPTLC谱分析子宫肌肉中Gg的主要组分为GD_3和GM_3,在子宫发育成熟与妊娠时,肌肉组织中这两种组分的含量变化明显:生育期样品的GD_3由新生儿的25.4％增加到56.6％(按占LBSA总量的百分比计算),GM_3则由33.2％降至16.9％。此外,GM_1和GD_(1a)也明显减少。N—GSL在生育期CMH、CDH和CTH的含量(按占含糖基量的百分比计算)成倍增加,而含五糖基以上的组分则仅为新生儿子宫的1/5。足月妊娠与新生儿子宫肌肉的两类鞘糖脂的HPTLC谱类似,但前者GT1b占19.4％,明显高于新生儿样品(6.1％)。     

人体子宫内膜中性鞘糖脂和神经节苷脂的含量和组成在月经周期中和妊娠时的变化

从月经周期各期、早妊(6—8周)及足月妊娠子宫内膜中提取、纯化了神经节苷脂(Gls)和中性鞘糖脂(N-GSL),分别测定了其含量。对两类鞘糖脂的组成进行了HPTLC图谱分析。初步观察了不同时期子宫内膜中CMP—NeuAc:LacCer唾液酰转移酶(ST_1)和CMP-NeuAc:CM_3唾液酰转移酶(ST_2)的活性。结果表明:分泌期的GLs总含量低于增生期(P<0.02);分泌期GD_3含量较生长期增多(P<0.01);妊娠后,GM_3含量增加,而GD_3减少;相应地,ST_1活性增高,ST_2活性降低。分泌期CMH含量为增生期的4.7倍。结果提示子宫内膜鞘糖脂含量和组成的变化可能与子宫的功能有关,而且受女性激素水平的影响。     

急性重复性缺氧小鼠脑神经节苷脂含量的研究

以昆明鼠为实验对象,用CS-9000薄层扫描仪测定急性重复性缺氧小鼠脑组织中神经节苷脂GM1、GD1a、GD1b和GT的各个组分相对百分比及唾液酸的含量.结果发现:与未经缺氧处理的空白对照组(A)比较,缺氧一次的实验对照组(B)及急性重复缺氧4次后饲养2d(天)实验对照组(D)等3组小鼠比急性重复缺氧4次实验组(C)小鼠脑组织中的唾液酸含量显著下降,神经节苷脂GM1与GD1b相对百分比明显降低;神经节苷脂GT相对组分百分比明显上升,神经节苷脂GD1a,还没发现有统计学差异.结果提示脑组织中神经节苷脂可能参与急性重复缺氧小鼠的耐缺氧能力的形成,而且是一个短时间内不能恢复的过程.     

产唾液酸酶微生物的筛选及产酶条件的优化

以无单唾液酸四己糖神经节苷脂（GM1）的神经节苷脂为唯一C源,从土壤中筛选出1株能以多唾液酸神经节苷脂为底物,转化生成GM1的产唾液酸酶微生物,经鉴定为溶黄嘌呤厄菌（Oerskovia xanthineolytica）。利用单因素法和响应面分析法对溶黄嘌呤厄菌产生唾液酸酶的条件进行优化,酶活提高了4.89倍。利用优化后培养条件,以神经节苷脂混合体为底物,经过微生物生物转化后,单唾液酸神经节苷脂GM1的含量从10%提高到83.7%。     

妊娠期家兔子宫内膜的鞘糖脂表达

 妊娠期家兎子宫内膜的神经节苷脂(Gls)的含量明显低于动情期的,而中性鞘糖脂(NGSL)的含量则以妊娠中、晚期的最高,动情期最低。鞘糖脂组成变化最显著的是妊娠早期,由动情期到早孕GM_3从28.0％增加到52.7％,CMH.CDH由未测出分别增加到29.2％和21.9％,而糖链复杂的组分GD_3,GTlb和CPH的百分含量则明显减少,到妊娠中、晚期、短糖链组分逐渐减少,而复杂糖链组分渐增。中期妊娠内膜的(GIs)以GD_3为主要组分,占45％,明显高于其它各期。NGSL在妊娠中、晚期CPH增高达70％,与动情期水平相当。结果提示,妊娠期间子宫内膜的鞘糖脂含量与组成均发生明显变化,这些变化可能与子宫功能密切相关。特别是早孕对的变化,推测与子宫内膜和胚泡的识别,粘连特性的获得有关。     

一些抗癌药物对荷瘤小鼠血清中唾液酸含量的影响

测定荷六种小鼠肿瘤S180肉瘤(实体型和腹水型),腹水肝癌(HepA),艾氏腹水瘤(EC),白血病P388和Lewis肺癌的小鼠腹水和血清中唾液酸含量,结果显示血清中唾液酸含量与肿瘤生长、肿瘤类型有关。腹水中唾液酸含量高,推测肿瘤能比正常组织产生更多唾液酸。对四种腹水肿瘤用阴离子交换树脂层析鉴定,发现HepA腹水中葡萄糖代唾液酸(NcuGc)含量明显低于其它三种腹水瘤。还研究了十几种抗癌药物对荷S180和Lewis肺癌小鼠血清中唾液酸含量的影响。发现吗丙嗪(probimane)和顺铂(DDP)能降低荷瘤小鼠血清中唾液酸含量,提示此二药物在肿瘤治疗中更具选择性。     

自然杀伤细胞和甲状腺细胞膜上的神经节苷脂的研究

本文对NK效应细胞和甲状腺细胞膜上的神经节苷脂进行了研究,采用同位素标记法,免疫组织化学法和ELISA法。结果说明,霍乱肠毒素B亚单位预先处理,人或小鼠的NK效应细胞,就能抑制对相应靶细胞杀伤作用, 随着B亚单位浓度增大,抑制作用增强。霍乱肠毒素能与甲状腺细胞膜结合。神经节苷脂预先与TsAb(LATS-B)结合,就能完全阻断TsAb(LATS-B)与甲状腺细胞膜结合。体外实验(ELISA法)证实TsAb(LATS-B)能与混合的神经节苷脂相结合。但是,CT和TSH仅能部分阻断TsAb与甲状腺细胞膜相结合,说明TsAb(LATS-B)的膜受体是很复杂的,GM_1神经节苷脂不是唯一的成分。     

GM3在几种哺乳动物肝脏和狗红细胞中的含量及其分离纯化

从正常兔、猪和狗的肝脏及狗红细胞中分离纯化了总神经节苷脂,测定了脂结合唾液酸,进行了高效薄层层析,比较了上述四种组织中GM₃的含量。结果表明狗红细胞中的GM₃的含量较另三种的高,狗肝和兔肝次之,猪肝含量甚微。从狗红细胞中提取和纯化了GM₃,其得量为每毫升压积红细胞351.0μg,纯度为92.2％。     

Adaptation of Zajdela ascitic hepatoma cells to monolayer growth: change in the cell ganglioside pattern

The Zajdela hepatoma is a transplantable ascitic tumor of the rat, characterized by a very simple ganglioside pattern, GM3 being the main compound. When these cells are adapted to monolayer culture, they undergo a maturation process and the total cellular ganglioside concentration increases progressively; GM2, GM1 and GD3 amounts rose and GD1a accumulated. These modifications in the ganglioside pattern complexity are not affected by the addition of ascitic fluid to the cultures, nor by growth in serum free, hormone-supplemented medium. They are totally reversible when the cultured hepatoma cells are reinjected into a rat and developed an ascitic tumour. Cell growth control and adhesion processes could be related to the maturation process of these hepatoma cells growing in monolayer, which may constitute a convenient model for further investigations on the regulation of membrane glycolipid composition by the external environment.     

Specific ganglioside changes in extraneural tissues of adult rats with hypothyroidism

Adults rats with hypothyroidism were prepared by administration of 6-propyl-2-thiouracil (PTU) or methimazole, and the tissues were examined for their gangliosides through methods including glycolipid-overlay techniques. Normal thyroid tissue contained GM3, GD3, and GD1a as the major gangliosides, with GM1, GD1b, GT1b, and GQ1b in lesser amounts. The goitrous tissue of PTU-induced hypothyroid rats had higher concentrations of GM1 and GD1a with a concomitant decrease of GM3. The amount of GT3 in thyroid tissue was increased in hypothyroid animals. While normal liver tissue had a complex ganglioside pattern with a- and b-series gangliosides, the PTU-induced hypothyroid tissue showed a simpler ganglioside profile that consisted mainly of a-series gangliosides with almost undetectable amounts of b-series gangliosides. The expression of c-series gangliosides was suppressed in the hypothyroid liver tissue. Heart tissue had higher contents of GM3 and GT3 than control. No apparent change was observed in the compositions of major and c-series gangliosides in other extraneural tissues (i.e., kidney, lung, spleen, thymus, pancreas, testis, skeletal muscle, and eye lenses), and neural tissues (i.e., cerebrum and cerebellum) from PTU-induced hypothyroid rats. The ganglioside changes of thyroid, liver, and heart tissues were reproduced in corresponding tissues of methimazole-induced hypothyroid rats. These results suggest that hypothyroid conditions affect the biosynthesis and expression of gangliosides in specific tissue and cell types.     

Decreased expression of alpha2,8 sialyltransferase and increased expression of beta1,4 N-acetylgalactosaminyltransferase in gastrointestinal cancers

Gangliosides such as GD3, GM2, and GD2 are abundantly expressed on the cell surfaces of various malignant cells, suggesting the potential for anti-ganglioside antibody therapy for tumors. Anti-ganglioside GD2 antibody treatment is currently undergoing clinical trials for melanoma and neuroblastoma. We previously reported high in vivo antitumor effects of anti-GM2 ganglioside antibody against lung cancer. To determine whether anti-GM2 antibody may be clinically indicated for gastrointestinal cancers, we evaluated the mRNA expression of alpha2,8 sialyltransferase, a GD3 synthase, and beta1,4 N-acetylgalactosaminyltransferase (beta1,4 GalNAc-T), a GM2/GD2 synthase, in gastrointestinal cancers. We performed modified semi-quantitative RT-PCR, which reduces complexity incidental to radiolabeling on samples taken from small surgically removed clinical specimens. Stomach (19/22) and colorectal (21/30) cancers showed decreased expression of alpha2,8 sialyltransferase as compared with respective normal tissues (P < 0.05). In contrast, increased expression of beta1,4 GalNAc-T was detected in both types of tumors. Clinicopathological analysis revealed significantly higher expression level of alpha2,8 sialyltransferase in the poorly differentiated than in the well-differentiated stomach cancer group (P < 0.05). Furthermore, the expression level of alpha2,8 sialyltransferase was significantly decreased in male as compared with female colorectal cancer patients (P < 0.05). These results suggest that expression level of GM2 ganglioside is elevated in gastrointestinal cancer, and that anti-GM2 antibody may be applicable to its treatment.     

Changes in ganglioside contents, plasma sialic acid and cAMP levels in experimental hepatoma in mice

The present study was designed to assess whether changes in glycolipids and cyclic AMP contents might serve as markers for the diagnosis of malignancy in the liver. The experimental model was a transplantable murine hepatoma. Experimental mice were divided into three groups: (1) a therapeutic group, which had been transplanted with hepatoma and treated with the antimetabolism drug 5-flurouracil (0.2 mg/day i.p.), (2) a control group, which had been transplanted with hepatoma and treated with 0.2 ml 0.9% NaCl/day and (3) a normal group of mice. The ganglioside and cAMP contents in the hepatoma tissue, plasma cAMP, total- and lipid-bound sialic acid levels and red blood cell membrane sialic acid levels were determined. Results showed that the ganglioside content, total and lipid-bound sialic acid levels in the control group were significantly higher than those in the livers of normal mice (p < 0.01) while these respective values in the therapeutic group were significantly lower than those in the control group (p < 0.01). The cAMP levels of tumor tissues and plasma in the control group were lower than those in normal mice. No significant difference in red blood cell membrane sialic acid content was observed between the therapeutic and control groups though levels for both were higher than those in normal mice. These results indicate that ganglioside content and sialic acid levels in hepatoma tissues were significantly elevated, and cAMP levels in hepatoma tissues were significantly decreased during proliferation and abnormal differentiation.     

Biologically active sphingolipids in transplantable tumors of different histogenesis

The composition of biologically active sphingolipids in hepatoma 22, breast adenocarcinoma, Lewis lung carcinoma, large intestine adenocarcinoma, cervical carcinoma, and melanomas M3 and B16 was compared to elucidate the similarity and differences in sphingolipids of subcutaneously transplantable murine tumors. The sphingolipid composition of the tumors was found to widely vary. The sphingomyelin, ceramide, glucosyl- and lactosylceramide, and ganglioside GD3 contents in hepatoma 22 are higher than those in normal tissue. No common regularities for tumors of different origin were observed in the ratios of bioeffectors inhibiting proliferation and stimulating apoptosis and bioeffectors stimulating cell growth and survival. However, the Cer/(GlcCer + LacCer) ratios were very low and practically equal in two melanoma strains, which probably indicates the degree of tumor malignancy. The results suggest that the content and composition of sphingolipids in tumors depend on their histogenesis.     

GANGLIOSIDE1−COMPOSITION OF BRAIN IN TAY-SACHS DISEASE: INCREASED AMOUNTS OF GD2 AND N-ACETYL-β-D-GALACTOSAMINYL GD1a GANGLIOSIDE

Abstract— The ganglioside composition of the brain of a patient with Tay-Sachs disease (TS-brain) was determined by a newly developed ganglioside-mapping procedure and compared with that of an age-matched control brain. GM2 ganglioside was the predominant component in TS-brain and the following gangliosides were also found, GM1, GD1a, GD1b and GT1 (major gangliosides in normal brain), and GM3, GD3, GD2 and GD1a-GAN (minor or undetectable components of normal brain). Individual gangliosides were isolated by column chromatography using a combination of DEAE-Sepharose, Iatrobeads and Silica Gel 60 and their structures were confirmed by comparing them with authentic standards using TLC, analysing their carbohydrate compositions by gas-liquid chromatography and cleaving them sequentially with glycosidases. The amounts of individual components were measured by quantitative densitometric scanning of the thin-layer plates. As a reflection of myelin breakdown, no sialosylgalactosyl ceramide was detectable in TS-brain. Although the total amounts of all gangliosides except GM2 in TS-brain were low, there were normal molar ratios of the main gangliosides in normal brain, that is, GM1, GD1a, GD1b and GT1. In comparison with the amount of GDla ganglioside, the amounts of GM2, GD2 and GD1a-GAN, which contain N-acetylgalactosamine as a terminal carbohydrate residue, were all elevated in TS-brain. The long chain bases of individual gangliosides contained both C-18 and C-20 sphingosine in different ratios and the ratio of C-20 to C-18 increased in the gangliosides in the order: GM2 < GM1 < GD1a < GD1a-GAN < GD1b < GT1 in both normal brain and TS-brain. In contrast, GD2 and GD3 gangliosides consisted mainly of C-18 sphingosine. The C-20 to C-18 ratios of individual gangliosides in the TS-brain were lower than those of age-matched control brain. Hexosaminidase from Turbo cornutus showed the same specific activity and K_m value in catalysing the cleavage of terminal N-acetylgalactosaminyl residues from GM2, GD2 and GD1a-GAN, suggesting that the brain gangliosides that increase in Tay-Sachs disease may be cleaved by the same enzyme.     

GM_3、GD_3作用下SMMC－7721肝癌细胞内磷脂酰肌醇（1，4，5）三

外源性ＧＭ３（１０ｎｍｏｌ／ｍＬ）、ＧＤ３（１ｎｍｏｌ／ｍＬ）可使ＳＭＭＣ－７７２１人肝癌培养细胞内钙浓度呈快速的短暂升高,其到达峰值时间为４５秒,一次作用后,内钙水平于２－３ｍｉｎ内恢复至对照水平。在一定时间间隔中连续几次加入ＧＭ３或ＧＤ３后内钙水平的变化表明,ＧＭ３所引起的［Ｃａ２＋］ｉ的增加依赖于内质网钙贮的释放和细胞外钙的流入;而ＧＤ３增加［Ｃａ２＋］ｉ与此二系统无关。进一步研究表明,在细胞内钙达峰值时,１０ｎｍｏｌ／ｍＬＧＭ３可使ＩＰ３（１,４,５）浓度增加９．３倍,ｃＡＭＰ浓度增加８２％;１ｎｍｏｌ／ｍＬＧＤ３反使Ｉｐ３浓度增加１．２倍,提示ＧＭ３、ＧＤ３升高内钙的不同机制。     

Enhancement of metastatic potential of mouse B16-melanoma cells to lung after treatment with gangliosides of B-16-melanoma cells of higher metastatic potential to lung

Mouse B16LuF1 melanoma cells of lower metastatic potential to lung were treated in vitro with same concentration (50 microM) of gangliosides isolated from B16LuF5, B16LuF9 or B16LuF10 cells with higher metastatic potential to lung (LuF1< LuF5< LuF9< LuF10) and injected to groups of normal mice through tail vein. The number of metastatic tumor nodules formed in lung increased in mice receiving B16LuF5, B16LuF9 and B16LuF10-ganglioside-treated B16LuF1 cells compared to mice receiving B16LuF1 cells without any ganglioside treatment. Metastatic potential of B16LuF1 cells gradually increased after treatment with gangliosides of B 16-melanoma cells of increasing metastatic potential to lung. The six major gangliosides isolated from B16LuF10 cells corresponded with standard gangliosides GT1b, GD1b, GD1a, GM1, GM2 and GM3 respectively on TLC-analysis. When B16LuF1 cells were treated in vitro with each of these six individual gangliosides and injected to groups of normal mice through tail vein the number of tumor nodules formed in lung varied. The four groups of mice receiving B16LuF1 cells treated with each of four gangliosides corresponding to GT1b, GD1b, GD1a or GM1 produced lung metastasis comparable to that of untreated control group. Only remaining two gangliosides which corresponded with standard gangliosides GM2 and GM3 increased metastatic potential of B16LuF1 cells. Thus, these results indicated that gangliosides GM2 and GM3 of B16-melanoma cells are definitely associated with metastatic potential of these tumor cells.     

Growth- and development-dependent expression of gangliosides in rat hepatocytes and liver tissues.

Expression of gangliosides in the liver was examined in primary cultures of hepatocytes from adult rats and liver tissues from rats of different ages. Hepatocytes were isolated from 7-week-old rat liver and cultured in L-15 medium containing insulin, dexamethasone and 10% fetal bovine serum. Hepatocytes proliferated only on the first day, and then ceased proliferation. The content of GD3 and GD1a increased during the period of active proliferation and reached a nearly constant level, whereas GM1, GD1b, GT1b, and GQ1b gradually increased throughout culture. Addition of EGF to the culture medium caused significant increases in the content of GD3, and to a lesser degree of GM3, but exhibited little effect on the expression of other ganglioside species. The specific induction of GD3 and GM3 expression by EGF was reproduced under serum-free conditions, despite the lack of hepatocyte proliferation. Expression of gangliosides in cultured hepatocytes was also modulated by cell density; higher cell density brought about increased content of GM1, GD1a, GD1b, GT1b, and GQ1b with concomitant reduction of GM3 in cells. The composition of gangliosides in liver tissues demonstrated a unique developmental pattern. GD3 and GD1a were strongly expressed in E-16 embryonic tissue and rapidly decreased with increasing age. GD1b, GT1b, and GQ1b were found only in postnatal liver tissues. These findings suggest that the expression of gangliosides in rat hepatocytes and liver tissues are regulated by growth- and development-dependent factors.