双歧杆菌及中药对小鼠血中抗氧化酶,T3,TSH和吞噬细胞功能的影响

本文用双歧杆菌、中药及复方双歧杆菌制剂饲喂ＢＡＬＢ／ｃ小鼠,发现中药及复方双歧杆菌制剂对老龄鼠血中ＳＯＤ、ＧＳＨ—Ｐｘ活性有明显增高作用,而对ＬＰＯ含量有明显减低作用,双歧杆菌及复方双歧杆菌能增强ＢＡＬＢ／ｃ小鼠内分泌功能及免疫功能,本试验中尚发现双歧杆菌与中药制成的复方双歧杆菌制剂能明显协同促进ＧＳＨ—Ｐｘ及吞噬细胞功能。说明双歧杆菌与一些传统补益类中药具有协同抗衰老作用。     

双歧杆菌对大肠癌细胞ccL187cAMP,cGMP影响的实验研究

双歧杆菌对维持机体正常微生态平衡具有重要作用。本文研究了长双歧杆菌对大肠癌细胞ｃｃＬ１８７内第二信使ｃＡＭＰ、ｃＧＭＰ浓度的影响,对长双歧杆菌的对数期培养物分别进行下列处理：活菌、死菌、代谢产物作用ｃｃＬ１８７细胞２小时后分别收集ｃｃＬ１８７细胞内的ｃＡＭＰ、ｃＧＭＰ浓度。实验表明长双歧杆菌的不同状态及代谢产物对ｃＡＭＰ、ｃＧＭＰ产生显著影响,且存在时间依赖关系,三种处理作用ｃｃＬ１８７细胞内的ｃ     

第二信使系统与消化道平滑肌收缩活动的调节

本主要从三方面介绍了第二信使系统对消化道平滑肌运动的调节作用：（１）ｃＡＭＰ、ｃＧＭＰ作用机制和某些胃肠激素经受体活化后的信息传递机制;（２）ＩＰ３、ＤＡＧ对肌收缩的作用特点和双向调节作用;（３）胞内信使间在调控活动中的相互关系。     

RAPD在双歧杆菌菌种鉴定及分型研究中的应用

采用近年来兴起的一种新的分子生物学分型技术ＲＡＰＤ即ＡＰ－ＰＣＲ对２０株４种不不双歧杆菌进行了基因指纹图谱的构建,其结果不同双歧杆菌种间存在同源性和多态性。本研究结果表明ＲＡＰＤ技术可用于双歧杆菌菌种鉴定及分型。     

RAPD分析快速鉴定双歧杆菌

本文应用ＲＡＰＤ技术,选用１１种引物,以嗜酸乳杆菌为对照,对６种１３珠双歧杆菌菌株基因组ＤＮＡ进行ＰＣＲ扩增,分析其ＤＮＡ指纹图谱,并计算其相似性指数。结果表明,双歧杆菌和非双歧杆菌之间,其相似性指数有显著差异;选择合适的引物进行扩增,双歧杆菌不同种间和同种不同株间可表现不同的ＤＮＡ指纹图谱。本文还对ＲＡＰＤ技术应用于双歧杆菌分类鉴定的可能性进行讨论。     

螺旋藻在体外对双歧杆菌及乳杆菌增殖的促进作用

通过用改良的ＴＰＹ和ＧＡＭ培养基作为螺旋藻的基质,在试管内证实了螺旋藻对双歧杆菌、乳杆菌生长的促进作用。结果显示,钝顶螺旋藻在实验浓度下,对双歧杆菌和乳杆菌的增殖作用与对照组相比有非常显著的差异,这一结果也说明了钝顶螺旋藻在调整肠道菌群,改善肠道内环境方面的作用。     

青春双歧杆菌DM8504菌株对荷瘤小鼠体内NO诱生作用的研究

应用处死的青春双歧杆菌ＤＭ８５０４菌株皮下免疫荷瘤小鼠,按Ｇｒｉｅｓ反应原理测定一氧化氮（ＮＯ）的含量。结果表明,双歧杆菌能提高荷瘤小鼠体内ＮＯ的含量,较对照组显著升高,肿瘤组织的坏死程度在处理组与对照组之间也具有显著性差异。提示：在双歧杆菌抗肿瘤作用中,除ＴＮＦ—α外,ＮＯ也发挥重要作用。     

中药91－4对抗生素相关性腹泻小鼠肠道膜菌群及腔菌群的影响

本实验以肠道菌群检测与电镜观察法,初步研究了中药９１－４对抗生素相关性腹泻（ＡＡＤ）小鼠肠道菌群的影响。结果表明,盐酸林可霉素对小鼠肠道膜菌群、腔菌群干扰显著,乳酸杆菌、双歧杆菌及肠杆菌等比正常对照组明显减少,致小鼠腹泻或肠炎;中药９１－４能够促进肠道乳酸杆菌、双歧杆菌等生长繁殖,对肠道膜菌群、腔菌群均具有调整作用,加速肠粘膜病变的愈合及修复,可有效地控制ＡＡＤ模型小鼠的腹泻症状。     

双歧杆菌合剂促进急性坏死性胰腺炎肠粘膜损伤修复的实验研究

观察双歧杆菌合剂对急性坏死性胰腺炎（ＡＮＰ）犬肠粘膜损伤修复的作用。经主胰管注入牛磺胆酸钠和胰蛋白酶复制犬ＡＮＰ模型,观察ＡＮＰ时及双歧杆菌合剂治疗后肠粘膜组织结构变化,肠组织蛋白、丙二醛（ＭＤＡ）含量及肠通透性改变,检测血中内毒素（ＬＰＳ）水平,脏器细菌培养。结果发现,双歧杆菌合剂治疗后,ＡＮＰ犬肠粘膜损害明显减轻,肠粘膜绒毛宽度、高度和面积显著增加,肠组织蛋白含量增加,肠通透性显著下降,血中ＬＰＳ水平下降１～２倍,脏器细菌易位率减少３７５％。结论：双歧杆菌合剂能显著促进ＡＮＰ时肠粘膜损伤的修复,保护肠屏障功能,减少肠道ＬＰＳ和细菌易位     

环六亚甲基双乙酰胺对胃癌不同细胞周期两条信号系…

环六亚甲基双乙酰胺（ＨＭＢＡ）对ＭＧｃ８０－３不同时相细胞内ｃＡＭＰ－ＰＫＡ与ＤＡＧ－ＰＫＣ两大系统不仅具有正负调控作用,而且其作用具有周期特异性。其中Ｇ１期是最敏感的调控时相,与对照组相比,ｃＡＭＰ水平上升１０２．３％,ＰＫＡ活性升高３４８％,ＤＡＧ含量下降５１．４％,ＰＫＣ活性降低３２．３％;次敏感时相为Ｇ２期;Ｍ期基本没受影响,Ｓ期变化规律不同于其他时相。     

Microbial species involved in production of 1,2-sn-diacylglycerol and effects of phosphatidylcholine on human fecal microbiota

1,2-sn-Diacylglycerols (DAGs) are activators of protein kinase C (PKC), which is involved in the regulation of colonic mucosal proliferation. Extracellular DAG has been shown to stimulate the growth of cancer cell lines in vitro and may therefore play an important role in tumor promotion. DAG has been detected in human fecal extracts and is thought to be of microbial origin. Hitherto, no attempts have been made to identify the predominant fecal bacterial species involved in its production. We therefore used anaerobic batch culture systems to determine whether fecal bacteria could utilize phosphatidylcholine (0.5% [wt/vol]) to produce DAG. Production was found to be dependent upon the presence of the substrate and was enhanced in the presence of high concentrations of deoxycholate (5 and 10 mM) in the growth medium. Moreover, its production increased with the pH, and large inter- and intraindividual variations were observed between cultures seeded with inocula from different individuals. Clostridia and Escherichia coli multiplied in the fermentation systems, indicating their involvement in phosphatidylcholine metabolism. On the other hand, there was a significant decrease in the number of Bifidobacterium spp. in the presence of phosphatidylcholine. Pure-culture experiments showed that 10 of the 12 strains yielding the highest DAG levels (>50 nmol/ml) were isolated from batch culture enrichments run at pH 8.5. We found that the strains capable of producing large amounts of DAG were predominantly Clostridium bifermentans (8 of 12), followed by Escherichia coli (2 of 12). Interestingly, one DAG-producing strain was Bifidobacterium infantis, which is often considered a beneficial gut microorganism. Our results have provided further evidence that fecal bacteria can produce DAG and that specific bacterial groups are involved in this process. Future strategies to reduce DAG formation in the gut should target these species.     

Effect of harvesting methods, growth conditions and growth phase on diacylglycerol levels in cultured human adherent cells

The cellular mass of sn-1,2-diacylglycerols, which are intracellular second messengers which activate protein kinase C, were quantitatively determined with an enzymatic assay. The method employed to harvest cultured human skin fibroblasts or human epidermal A431 cells prior to extraction of lipid into chloroform/methanol affected diacylglycerol (DAG) levels. Scraping or trypsinization significantly increased DAG levels. A method was devised to allow reliable and reproducible DAG measurements from adherent cells. The addition of methanol prior to scraping was shown to stop cellular metabolism and to permit accurate quantitation. Importantly, this solvent was compatible with cultures grown on plastic. Using this method, growth conditions which could affect DAG levels were investigated. Changes in the osmolality of the culture medium did not affect the DAG levels of A431 cells; exposure of A431 cells to acidic pH or elevated temperature lowered DAG levels. In contrast to fibroblasts, the total DAG levels of A431 cells continued to increase during serum deprivation. The highest DAG levels, normalized to phospholipids, were observed during the exponential growth phase. This ratio dropped when the cultures reached confluency. These experiments also demonstrated that A431 cells possess higher DAG levels than do normal fibroblasts. The function of DAG in cellular regulation is discussed.     

Production of inositol-phospholipid-derived second messengers in a rat natural killer cell line exposed to susceptible tumor targets

It is currently believed that natural killer (NK) cells kill bound target cells by exocytosis of cytotoxic granules via a calcium-dependent process. After confirming that NK-mediated killing was indeed dependent upon extracellular calcium, we investigated the production of inositol-phospholipid-derived second messengers in a rat NK cell line, RNK, upon exposure to susceptible target cells. These messengers, inositol trisphosphate (IP3) and diacylglycerol (DAG), are associated with calcium-dependent secretory processes in a number of cell types. When RNK cells were exposed to susceptible YAC-1 tumor targets significant amounts of both IP3 and DAG were produced. The levels of the membrane phospholipid parent molecules of these second messengers declined in similarly stimulated RNK cells over a comparable time period. Using three different target cell lines, it was found that the levels of DAG that RNK produced in response to the different targets followed the same rank order as their susceptibility to RNK-mediated lysis. These data suggest that IP3 and DAG are produced in NK cells in response to tumor target cells, and these second messengers may have a functional role in NK-mediated killing.     

v-Src increases diacylglycerol levels via a type D phospholipase-mediated hydrolysis of phosphatidylcholine.

Activating the protein-tyrosine kinase of v-Src in BALB/c 3T3 cells results in rapid increases in the intracellular second messenger, diacylglycerol (DAG). v-Src-induced increases in radiolabeled DAG were most readily detected when phospholipids were prelabeled with myristic acid, which is incorporated predominantly into phosphatidylcholine. Consistent with this observation, v-Src increased the level of intracellular choline. No increase in DAG was observed when cells were prelabeled with arachidonic acid, which is incorporated predominantly into phosphatidylinositol. Inhibiting phosphatidic acid (PA) phosphatase, which hydrolyzes PA to DAG, blocked v-Src-induced DAG production and enhanced PA production, implicating a type D phospholipase. Consistent with the involvement of a type D phospholipase, v-Src increased transphosphatidylation activity, which is characteristic of type D phospholipases. Thus, v-Src-induced increases in DAG most likely result from the activation of a type D phospholipase/PA phosphatase-mediated signaling pathway.     

昆明山海棠对中国仓鼠V79细胞二酰基甘油含量的影响

本研究以昆明山海棠根部水抽提物(Tripterygium Hypoglaucum(Level)Hutch,THH)处理中国仓鼠V79细胞,通过检测V79细胞C-M细胞频率以及二酰基甘油(1,2-diacylgcerol,DAG)的含量测定,分析了THH诱发非整倍体与细胞醇磷酯信号通路的关系.结果指出THH能在1mg/ml、2mg/ml两个剂量上使V79细胞的DAG含量显著升高(P＜0.001),并明显的提高C-M细胞频率(P＜0.05),提示肌醇酯信号通路是介导THH诱发非整倍体的途径之一.     

Diacylglycerol formation from phosphatidylcholine in angiotensin II-stimulated vascular smooth muscle cells.

In cultured vascular smooth muscle cells (VSMC), angiotensin II (Ang II) induces a biphasic diacylglycerol (DAG) formation peaking at 15 sec and 5 min. Although it has been well established that the first peak is produced by the hydrolysis of inositol 4,5-bisphosphate (PIP2), the origin of the second DAG peak has never been examined in detail. In the present paper, we provide evidence that the second peak of DAG formation in Ang II-stimulated VSMC originates mainly from PC.     

Receptor-linked early events induced by vasoactive intestinal contractor (VIC) on neuroblastoma and vascular smooth-muscle cells.

Vasoactive intestinal contractor (VIC) caused a series of biochemical events, including the temporal biphasic accumulation of 1,2-diacylglycerol (DAG), transient formation of Ins(1,4,5)P3, and increase in intracellular free Ca2+ [( Ca2+]i) in neuroblastoma NG108-15 cells. In these cellular responses, VIC was found to be much more potent in NG108-15 cells than in cultured rat vascular smooth-muscle cells. The single cell [Ca2+]i assay revealed that in the presence of nifedipine (1 microM) or EGTA (1 mM), the peak [Ca2+]i declined more rapidly to the resting level in VIC-stimulated NG108-15 cells, indicating that the receptor-mediated intracellular Ca2+ mobilization is followed by Ca2+ influx through the nifedipine-sensitive Ca2+ channel. Pretreatment with pertussis toxin only partially decreased Ins(1,4,5)P3 generation as well as the [Ca2+]i transient induced by VIC, whereas these events induced by endothelin-1 were not affected by the toxin, suggesting involvement of distinct GTP-binding proteins. The VIC-induced transient Ins(1,4,5)P3 formation coincident with the first early peak of DAG formation suggested that PtdIns(4,5)P2 is a principal source of the first DAG increase. Labelling studies with [3H]myristate, [14C]palmitate and [3H]choline indicated that in neuroblastoma cells phosphatidylcholine (PtdCho) was hydrolysed by a phospholipase C to cause the second sustained DAG increase. Down-regulation of protein kinase C (PKC) by prolonged pretreatment with phorbol ester markedly prevented the VIC-induced delayed DAG accumulation. Furthermore, chelation of intracellular CA2+ completely abolished the second sustained phase of DAG production. These findings suggest that PtdCho hydrolysis is responsible for the sustained production of DAG and is dependent on both Ca2+ and PKC.     

In vitro effects of phosphatidylcholine and transgalactooligosaccharides on the production of 1,2-sn-diacylglycerol by Bifidobacterium longum biovar infantis

Aims: To investigate the production of the tumour promoter 1,2-sn-diacylglycerol (DAG) by a human gut isolate of Bifidobacterium longum biovar infantis. Methods and Results: Bifidobacterium longum biovar infantis was grown in vitro using anaerobic static batch cultures in the presence of phosphatidylcholine (PC) and trans-galactooligosaccharides (TOS). Production of DAG was found to be dependent upon the presence of PC, while TOS had a reducing effect. Considerable differences in morphology, growth and metabolic end products from the micro-organism were observed under the different culture conditions. Conclusions: Our results have provided evidence that B. longum biovar infantis can produce DAG in vitro and that a prebiotic exerted a reducing effect upon this production. Significance and Impact of the Study: The results presented in this study demonstrate an ability of ostensibly beneficial member of the colonic environment to produce unwanted compounds under certain conditions. Therefore, it may be important that a combination of substrates and other factors are assessed when studying the behaviour of any bacterial group or species, especially when designing the dietary interventions.     

Activation of latent EBV via anti-IgG-triggered, second messenger pathways in the Burkitt's lymphoma cell line Akata

Anti-IgG treatment activated latent EBV genomes in 50 to 70% of the cells of the Burkitt's lymphoma cell line Akata. The EBV-activating role of intracellular Ca2+, as potentiated by diacylglycerol (DAG) and suppressed by cAMP, was analyzed in the cells through effects of agonists and antagonists of these second messenger pathways. Early Ag (EA) was induced in 10% of cells with the calcium ionophore A23187 (A23187). EA induction with anti-IgG or A23187 was blocked by a calmodulin antagonist, trifluoperazine. The DAG pathway had a potentiating but not direct effect on EBV activation because: 1) the DAG analog, dioctanoylglycerol (diC8), an agonist for protein kinase C, alone induced only 2% EA-positive cells, 2) diC8 synergized with A23187 for EA induction, and 3) the protein kinase C antagonist, staurosporine, almost completely inhibited EA induction by anti-IgG. When cells were reincubated in medium with fresh diC8 and A23187 at 3, 6, 9, and 12 h, EA induction at 24 h reached the levels seen with anti-IgG stimulation. A cAMP-mediated pathway suppressed EBV activation because dibutyryl cAMP or 8-bromo-cAMP, plus blockage of phosphodiesterase by theophylline, or use of forskolin, inhibited EA induction with anti-IgG. Although the principal stimulatory role in EBV activation of a Ca2(+)-mediated, second messenger pathway, as synergized by DAG and inhibited by cAMP, was established, we did not explain the significant lag in EA induction by A23187 and diC8 as compared with anti-IgG induction of EA. We conclude that EBV genome activation with anti-IgG is mediated by Ca2+/calmodulin and DAG pathways in Akata cells, that the cAMP pathway suppresses EA induction by anti-IgG, and that a mechanism regulating the speed of EA induction remains unexplained.     

Diacylglycerol kinase epsilon, but not zeta, selectively removes polyunsaturated diacylglycerol, inducing altered protein kinase C distribution in vivo

Porcine aortic endothelial cells have previously been shown to contain particularly high basal levels of polyunsaturated diacylglycerol (DAG) together with a very high degree of membrane-associated protein kinase C (PKC), which is largely insensitive to further activation (Pettitt, T. R., Martin, A., Horton, T., Liossis, C., Lord, J. M., and Wakelam, M. J. O. (1997) J. Biol. Chem. 272, 17354-17359). To investigate the possibility that the high polyunsaturated DAG levels were constitutively activating PKC, we transfected porcine aortic endothelial cells with two different forms of human diacylglycerol kinase, epsilon and zeta. In vitro, the former is specific for polyunsaturated structures, whereas the latter shows no apparent selectivity. Overexpression of DAGKepsilon specifically reduced the level of polyunsaturated DAG in the transfected cells while having little effect on the more saturated structures. It also caused the redistribution of PKCalpha and epsilon from the membrane to the cytosol. Overexpression of DAGKzeta caused a general reduction in DAG levels but had little effect on PKC distribution. These results for the first time show that DAGKepsilon specifically phosphorylates polyunsaturated DAG in vivo and that in so doing it regulates PKC localization and activity. This provides support for the proposal that it is the polyunsaturated DAGs that function as messengers and convincing evidence for DAGKepsilon being a physiological terminator of DAG second messenger signaling.