Gangliosides with O-Acetylated Sialic Acids in Tumors of Neuroectodermal Origin

Gangliosides, carrying an O-acetylated sialic acid in their carbohydrate moiety, are often found in growing and developing tissues, especially of neuro-ectodermal origin. The most prominent one is 9-O-Ac-GD3, which is considered as an oncofetal marker in animal and human tumors like neuronal tumors, melanoma, basalioma or breast cancer, as well as in psoriatic lesions. Also other gangliosides like GD2 or GT3 were found to be O-acetylated in their terminal sialic acid. In this review we are summarising the occurrence of such gangliosides in normal and transformed tissues and delineate a more general theory that O-acetylated sialic acids in gangliosides are a universal marker for growing cells and tissues.     

Expression of Mouse Sialic Acid on Gangliosides of a Human Glioma Grown as a Xenograft in SCID Mice

Ganglioside sialic acid content was examined in the U87-MG human glioma grown as cultured cells and as a xenograft in severe combined immunodeficiency (SCID) mice. The cultured cells and the xenograft possessed N-glycolylneuraminic acid (NeuGc)-containing gangliosides, despite the inability of human cells to synthesize NeuGc. Human cells express only N-acetylneuraminic acid (NeuAc)-containing gangliosides, whereas mouse cells express both NeuAc- and NeuGc-containing gangliosides. Small amounts of NeuGc ganglioside sialic acid (2-3% of total ganglioside sialic acid) were detected in the cultured cells, whereas large amounts (66% of total ganglioside sialic acid) were detected in the xenograft. The NeuGc in gangliosides of the cultured cells was derived from gangliosides in the fetal bovine serum of the culture medium, whereas that in the U87-MG xenograft was derived from gangliosides of the SCID host. The chromatographic distribution of U87-MG gangliosides differed markedly between the in vitro and in vivo growth environments. The neutral glycosphingolipids in the U87-MG cells consisted largely of glucosylceramide, galactosylceramide, and lactosylceramide, and their distribution also differed in the two growth environments. Asialo-GM1 (Gg4Cer) was not present in the cultured tumor cells but was expressed in the xenograft, suggesting an origin from infiltrating cells (macrophages) from the SCID host. The infiltration of mouse host cells and the expression of mouse sialic acid on human tumor cell glycoconjugates may alter the biochemical and immunogenic properties of xenografts.     

The Cleavage of Nucleic Acids in Reversed Micelles Using Site Specific Endonucleases

Plasmid and λ DNA molecules of between 2.2 and 48.5 kb pairs can be solubilised in n-hexane containing the surfactant sodium dioctyl sulfosuccinate (AOT) and aqueous buffers. Linear λ phage DNA fragments (2.2-23.1 kb pairs) and intact λ bio 1 DNA (48.5 kb pairs) are efficiently cleaved by Bam HI and Em RI in systems containing 100 mM AOT. Under these conditions, λ bio 1 DNA undergoes regioselective restriction by Hind III at only one site but is completely cleaved when the surfactant concentration is lowered to 50 mM. Covalent closed circular plasmid DNA (pUC8, 2.73 kb pairs) is only partially linearised by Eco RI and Bam HI in reversed micelles; Hae II cleavage affords both complete and partial restriction fragments. The results suggest that the tertiary structures adopted by substrate DNA in reversed micelles influence the availability of restriction sites.     

Synthesis and Characteristics of Fluorescent BODIPY-Labeled Gangliosides

A series of new fluorescent-labeled gangliosides bearing the residues of acids labeled by 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene(BODIPY) in the polar or/and apolar moiety were synthesized. These are ganglioside GM1 labeled with the residue of 4,4-difluoro-4-bora-3a,4a-diaza-5,7-dimethyl-s-indacenyl-3-propanoic (BODIPY-FL-propanoic) and -indacenyl-5-pentanoic (BODIPY-FL-pentanoic) acid in the oligosaccharide moiety of the molecule, and ganglioside GD1a labeled with two residues of BODIPY-FL-pentanoic acid in the oligosaccharide moiety and also with the residue of BODIPY-FL-pentanoic acid and the residue of 4,4-difluoro-4-bora-3a,4a-diaza-5-octyl-s-indacenyl-5-pentanoic acid in the ceramide part of the molecule. Some spectral characteristics and the behavior in the model membrane systems of the synthesized probes were studied. In their emission spectra, the BODIPY-labeled gangliosides included into phosphatidylcholine liposomes at high concentrations (>1 mol %) exhibit a long-wavelength maximum (at 630 nm) in addition to the usual maximum (at 510–515 nm).     

Inhibition of the UDP-N-Acetylgalactosamine: GM3, N-Acetylgalactosaminyl Transferase by Gangliosides

Gangliosides in the range of 0.1-0.4 mM inhibited the UDP-N-acetylgalactosamine:GM3, N-acetylgalactosaminyl transferase (EC 2.4.1.79) of chicken retina. Other lipids such as phosphatidylethanolamine, sphingomyelin, sulfatides, and phosphatidic acid in concentrations similar to those of gangliosides did not affect the enzyme activity significantly. GM3 has an inhibition capability slightly less than that of gangliosides with two or three sialyl groups in their molecules, while asialo-GM1 is clearly less inhibitory. The inhibitory effect of a constant amount of GT1 ganglioside was higher at low concentrations of membrane preparation, but the inhibition was similar at different concentrations of the substrates GM3 or UDP-N-acetylgalactosamine and at all incubation times studied. The added gangliosides were found attached to the membranes. In this attached state they may act either as substrate or inhibitor. The inhibitory effect of gangliosides was not apparent when a mixture of Triton CF 54-Tween 80 was added to the incubation medium at concentrations greater than 0.33%.     

Effects of Specific Gangliosides on the In Vitro Proliferation of MPTP-Susceptible Cells

Abstract: To determine which portion of a ganglioside molecule might be necessary for the enhancement of recovery from MPTP-induced lesions, the ability of specific gangliosides to stimulate proliferation of MPTP-treated 140–3 cells was investigated. The results indicate that of the gangliosides tested, GM1 was the most effective. Although GD1 a and GT1 b were able to enhance the proliferation of MPTP-treated cells, twice as much GT1b was needed to induce the same effect seen with GM1. In contrast, asialo-GM1, GM2, and GM3 were ineffective at promoting proliferation of MPTP-treated cells. The isolated oligosaccharide of GM1 had little effect. These results indicate that in addition to the sialosyl residue, at least the Gal(β1–3)Gal-NAc portion of the oligosaccharide chain and the ceramide moiety are essential for the induction of proliferation of the MPTP-treated cells. Investigation of the time of addition of GM1 on its ability to counteract the MPTP-induced inhibition of 140–3 cell proliferation indicated that addition of GM1 before or concomitantly with MPTP resulted in a significant reduction in MPTP-induced inhibition of cell proliferation.     

Histochemical Studies on Sialic Acids in the Epididymis During Post-natal Development of the Rat

A variety of sialic acids contained in the rat epididymis during post-natal development were examined by means of lectin and carbohydrate histochemistry. Epididymides from male Sprague– Dawley rats on post-natal days 14, 21, 30, 39, 49, 56 and 70 were fixed in Bouin's fluid and embedded routinely in paraffin wax. Hydrated sections were subjected either to the lectin methods using biotinylated Sambucus sieboldiana lectin or Maackia amurensis lectin or to the selective periodate oxidation– phenylhydrazine–thiocarbohydrazide–silver protein–physical development technique with or without saponification. The results revealed that sialic acids appeared in the epididymal epithelium at day 14, followed by particular distribution patterns corresponding to cell differentiation during days 21–39. High-level O-acetylation of sialic acids was observed in the principal cells of the initial segment and proximal caput after day 39. These results suggest that sialic acids with different linkages and O-acetylation become adult in distribution at the differentiation period under the influence of androgen, before spermatozoa reach the epididymal lumen. Such carbohydrates may be correlated, at least in part, with sperm-binding sialoproteins, which increase dramatically during the window between days 21 and 39.     

Dual Catalytic Activity of Hydroxycinnamoyl-Coenzyme A Quinate Transferase from Tomato Allows It to Moonlight in the Synthesis of Both Mono- and Dicaffeoylquinic Acids

Tomato (Solanum lycopersicum), like other Solanaceous species, accumulates high levels of antioxidant caffeoylquinic acids, which are strong bioactive molecules and protect plants against biotic and abiotic stresses. Among these compounds, the monocaffeoylquinic acids (e.g. chlorogenic acid [CGA]) and the dicaffeoylquinic acids (diCQAs) have been found to possess marked antioxidative properties. Thus, they are of therapeutic interest both as phytonutrients in foods and as pharmaceuticals. Strategies to increase diCQA content in plants have been hampered by the modest understanding of their biosynthesis and whether the same pathway exists in different plant species. Incubation of CGA with crude extracts of tomato fruits led to the formation of two new products, which were identified by liquid chromatography-mass spectrometry as diCQAs. This chlorogenate:chlorogenate transferase activity was partially purified from ripe fruit. The final protein fraction resulted in 388-fold enrichment of activity and was subjected to trypsin digestion and mass spectrometric sequencing: a hydroxycinnamoyl-Coenzyme A:quinate hydroxycinnamoyl transferase (HQT) was selected as a candidate protein. Assay of recombinant HQT protein expressed in Escherichia coli confirmed its ability to synthesize diCQAs in vitro. This second activity (chlorogenate:chlorogenate transferase) of HQT had a low pH optimum and a high K_m for its substrate, CGA. High concentrations of CGA and relatively low pH occur in the vacuoles of plant cells. Transient assays demonstrated that tomato HQT localizes to the vacuole as well as to the cytoplasm of plant cells, supporting the idea that in this species, the enzyme catalyzes different reactions in two subcellular compartments.The importance of plant-based foods in preventing or reducing the risk of chronic disease has been widely demonstrated (Martin et al., 2011, 2013). In addition to vitamins, a large number of other nutrients in plant-based foods promote health and reduce the risk of chronic diseases; these are often referred to as phytonutrients. The presence of phytonutrients in fruit and vegetables is of significant nutritional and therapeutic importance, as many have been found to possess strong antioxidant activity (Rice-Evans et al., 1997). Phenolics are the most widespread dietary antioxidants and caffeoylquinic acids, such as chlorogenic acid (CGA), dicaffeoylquinic acids (diCQAs), and tricaffeoylquinic acids (triCQAs), play important roles in promoting health (Clifford, 1999; Niggeweg et al., 2004). CGA limits low density lipid oxidation (Meyer et al., 1998), diCQAs possess antihepatotoxic activity (Choi et al., 2005), and triCQAs reduce the blood Glc levels of diabetic rats (Islam, 2006). diCQA derivatives have been shown to protect humans from various kinds of diseases; diCQAs suppress melanogenesis effectively (Kaul and Khanduja, 1998), show anti-inflammatory activity in vitro (Peluso et al., 1995), and exhibit a selective inhibition of HIV replication (McDougall et al., 1998). The physiological effects of caffeoylquinic acid derivatives with multiple caffeoyl groups are generally greater than those of monocaffeoylquinic acids, perhaps because the antioxidant activity is largely determined by the number of hydroxyl groups present on the aromatic rings (Wang et al., 2003; Islam, 2006). Furthermore, both diCQAs and triCQAs may function as inhibitors of the activity of HIV integrase, which catalyzes the insertion of viral DNA into the genome of host cells (McDougall et al., 1998; Slanina et al., 2001; Gu et al., 2007).CGA is the major soluble phenolic in Solanaceous crops (Clifford, 1999) and the major antioxidant in the average U.S. diet (Luo et al., 2008), while different isomers of diCQAs have been identified in many crops such as coffee (Coffea canephora), globe artichoke (Cynara cardunculus), tomato (Solanum lycopersicum), lettuce (Lactuca sativa), and sweet potato (Ipomoea batatas; Clifford, 1999; Islam, 2006; Moco et al., 2006, 2007; Moglia et al., 2008). In tomato, CGA accounts for 75% and 35% of the total phenolics in mature green and ripe fruit, respectively, amounting to 2 to 40 mg 100 g^–1 dry weight (DW), although levels decline after ripening and during postharvest storage (Slimestad and Verheul, 2009). diCQAs and triCQAs also accumulate in tomato fruit (diCQAs, approximately 2 mg 100 g^–1 DW; and triCQAs, 1–2 mg 100 g^–1 DW; Chanforan et al., 2012).Three pathways (Villegas and Kojima, 1986; Hoffmann et al., 2003; Niggeweg et al., 2004) have been proposed for the synthesis of CGA: (1) the direct pathway involving caffeoyl-CoA transesterification with quinic acid by hydroxycinnamoyl-Coenzyme A:quinate hydroxycinnamoyl transferase (HQT; Niggeweg et al., 2004; Comino et al., 2009; Menin et al., 2010; Sonnante et al., 2010); (2) the route by which p-coumaroyl-CoA is first transesterified with quinic acid via hydroxycinnamoyl-Coenzyme A transferase (HCT) acyltransferase (Hoffmann et al., 2003; Comino et al., 2007), followed by the hydroxylation of p-coumaroyl quinate to 5-caffeoylquinic acid, catalyzed by C3′H (p-coumaroyl-3-hydroxylase; Schoch et al., 2001; Mahesh et al., 2007; Moglia et al., 2009); and (3) the use of caffeoyl-glucoside as the acyl-donor (Villegas and Kojima, 1986). In tomato, the synthesis of CGA involves transesterification of caffeoyl-CoA with quinic acid by HQT (Niggeweg et al., 2004).To date, it is not clear whether diCQAs are derived directly from the monocaffeoylquinic acids (such as CGA) through a second acyltransferase reaction involving an acyl-CoA or not, although their structural similarity provides good a priori evidence supporting this hypothesis. Recently the in vitro synthesis of 3,5-diCQA from CGA and CoA by HCT from coffee has been reported (Lallemand et al., 2012). By contrast, in sweet potato, an enzyme that catalyzes the transfer of the caffeoyl moiety of CGA to another molecule of CGA, leading to the synthesis of isochlorogenate (3,5-di-O-caffeoylquinate), has been described, but the corresponding gene has not been identified (Villegas and Kojima, 1986).We report a chlorogenate:chlorogenate transferase (CCT) activity leading to the synthesis of diCQAs in tomato fruits and describe how alternative catalysis, by a single enzyme, leads to the production of both CGA and diCQA in different cellular compartments.     

毛细管电泳飞摩尔水平分析糖蛋白中唾液酸

利用毛细管电泳分析唾液酸2-氨基吖啶酮(AMAC)衍生物方法, 可在飞摩尔水平分析糖蛋白中唾液酸. 重组人促红细胞生成素(rhu-EPO)、人尿胰蛋白酶抑制剂(hu-UTI)中唾液酸分析结果与文献值符合较好; 而牛α₁-酸性糖蛋白(α₁-AGP)分析结果与早期文献值相比, 存在一定差异, 并发现该糖蛋白中除含有5-N-乙酰氨基唾液酸(Neu5Ac)外, 还含数量与Neu5Ac相当的5-N-乙醇酰氨基唾液酸(Neu5Gc).     

Synthesis and Evaluation of Protein Conjugates of GM3 Derivatives Carrying Modified Sialic Acids as Highly Immunogenic Cancer Vaccine Candidates

GM3, a sialylated trisaccharide antigen expressed by a number of tumors, is an attractive target in the design of therapeutic cancer vaccines. However, a serious problem associated with GM3 is that it is poorly immunogenic. To overcome this problem for the development of GM3-based cancer vaccines, four GM3 derivatives, including 5'-N-p-methylphenylacetyl, 5'-N-p-methoxyphenylacetyl, 5'-N-p-acetophenylacetyl and 5'-N-p-chlorophenylacetyl GM3, were synthesized and then coupled to a carrier protein, keyhole limpet haemocyanin (KLH). The resultant glycoconjugates were evaluated as vaccines in mouse and compared to the KLH conjugate of 5'-N-phenylacetyl GM3 (GM3NPhAc), a highly immunogenic GM3 derivative that was previously investigated as a vaccine candidate. All of the four new GM3 derivatives were proved to be more immunogenic than GM3NPhAc and elicit very strong T cell-dependent immune responses desirable for cancer immunotherapy. It was concluded that the new GM3 derivatives can form promising vaccine candidates that may be used to combine with cell glycoengineering for cancer immunotherapy.     

Neurostatin: cellular sources and cellular targets of the inhibitor

Neurostatin, a modified ganglioside in mammalian brain, is highly immunogenic. Fusion of spleen cells of Balb/C mice immunized with bovine neurostatin with SP(2)O myeloma cells, led to hybridoma producers of anti-neurostatin antibodies. Two,monoclonal, IgG secreters were selected that recognized the inhibitor in blots and tissue of cow, rat and pig. The lack of species specificity indicated that the molecular structure of the inhibitory epitope is similar in these mammals. That epitope was also present in other gangliosides of the b series, defining a new regulatory system of glial-cell proliferation and apoptosis. The antibodies further revealed the neuronal origin of neurostatin.     

Alterations of Endogenous Cytokinins in Transgenic Plants Using a Chimeric Isopentenyl Transferase Gene

Cytokinins, a class of phytohormones, appear to play an important role in the processes of plant development. We genetically engineered the Agrobacterium tumefaciens isopentenyl transferase gene, placing it under control of a heat-inducible promoter (maize hsp70). The chimeric hsp70 isopentenyl transferase gene was transferred to tobacco and Arabidopsis plants. Heat induction of transgenic plants caused the isopentenyl transferase mRNA to accumulate and increased the level of zeatin 52-fold, zeatin riboside 23-fold, and zeatin riboside 5[prime]-monophosphate twofold. At the control temperature zeatin riboside and zeatin riboside 5[prime]-monophosphate in transgenic plants accumulated to levels 3 and 7 times, respectively, over levels in wild-type plants. This uninduced cytokinin increase affected various aspects of development. In tobacco, these effects included release of axillary buds, reduced stem and leaf area, and an underdeveloped root system. In Arabidopsis, reduction of root growth was also found. However, neither tobacco nor Arabidopsis transgenic plants showed any differences relative to wild-type plants in time of flowering. Unexpectedly, heat induction of cytokinins in transgenic plants produced no changes beyond those seen in the uninduced state. The lack of effect from heat-induced increases could be a result of the transient increases in cytokinin levels, direct or indirect induction of negating factor(s), or lack of a corresponding level of competent cellular factors. Overall, the effects of the increased levels of endogenous cytokinins in non-heat-shocked transgenic plants seemed to be confined to aspects of growth rather than differentiation. Since no alterations in the programmed differentiation pattern were found with increased cytokinin levels, this process may be controlled by components other than absolute cytokinin levels.     

Specific Prenylation of Tomato Rab Proteins by Geranylgeranyl Type-II Transferase Requires a Conserved Cysteine-Cysteine Motif

Posttranslational isoprenylation of some small GTP-binding proteins is required for their biological activity. Rab geranylgeranyl transferase (Rab GGTase) uses geranylgeranyl pyrophosphate to modify Rab proteins, its only known substrates. Geranylgeranylation of Rabs is believed to promote their association with target membranes and interaction with other proteins. Plants, like other eukaryotes, contain Rab-like proteins that are associated with intracellular membranes. However, to our knowledge, the geranylgeranylation of Rab proteins has not yet been characterized from any plant source. This report presents an activity assay that allows the characterization of prenylation of Rab-like proteins in vitro, by protein extracts prepared from plants. Tomato Rab1 proteins and mammalian Rab1a were modified by geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate. This modification required a conserved cysteine-cysteine motif. A mutant form lacking the cysteine-cysteine motif could not be modified, but inhibited the geranylgeranylation of its wild-type homolog. The tomato Rab proteins were modified in vitro by protein extract prepared from yeast, but failed to become modified when the protein extract was prepared from a yeast strain containing a mutant allele for the [alpha] subunit of yeast Rab GGTase (bet4 ts). These results demonstrate that plant cells, like other eukaryotes, contain Rab GGTase-like activity.     

Synthesis and Translocation of Gangliosides and Glycoproteins During Urethane Anesthesia

In this work, we have studied (a) the contents of gangliosides, glycoproteins, and phospholipids of the vesicle and plasma membrane fractions from brains of anesthetized and control rats and chickens and (b) the labeling of gangliosides and glycoproteins in the retina ganglion cell layer and optic tectum of urethane-anesthetized and control chickens after intraocular injection of a labeled N-acetylneuraminic acid precursor and the distribution of the label after subcellular fractionation. We found an increase in the content of gangliosides relative to protein in the vesicle fraction of both anesthetized rats and chickens relative to their controls. Other values were not affected by anesthesia. These results do not reflect a faster synthesis of gangliosides stimulated by urethane, because their rate of labeling was diminished in anesthetized animals. During the 4-h period after the animals were injected intraocularly with the radioactive precursor, the highest values of ganglioside-specific radioactivity were found in the vesicle fraction of control and anesthetized animals; at longer intervals, the specific radioactivity of the vesicle and plasma membrane fractions became rather similar. These data are in accordance with previous studies from this laboratory suggesting that the synthesis of the carbohydrate chain of gangliosides is regulated by the physiological demands made by the neurotransmitting system.     

The Fluidity of the Bacterial Outer Membrane Is Species Specific

The outer membrane (OM) is an essential barrier that guards Gram-negative bacteria from diverse environmental insults. Besides functioning as a chemical gatekeeper, the OM also contributes towards the strength and stiffness of cells and allows them to sustain mechanical stress. Largely influenced by studies of Escherichia coli, the OM is viewed as a rigid barrier where OM proteins and lipopolysaccharides display restricted mobility. Here the discussion is extended to other bacterial species, with a focus on Myxococcus xanthus. In contrast to the rigid OM paradigm, myxobacteria possess a relatively fluid OM. It is concluded that the fluidity of the OM varies across environmental species, which is likely linked to their evolution and adaptation to specific ecological niches. Importantly, a fluid OM can endow bacteria with distinct functions for cell-cell and cell-environment interactions.     

末端脱氧核苷酸转移酶在生物传感及核酸合成领域的应用*

末端脱氧核苷酸转移酶(terminal deoxynucleotidyl transferase, TdT)是聚合酶X家族中的一员,与典型的DNA聚合酶不同,TdT以恒温的无模板依赖的方式催化脱氧核糖核苷三磷酸(dNTP)聚合到寡核苷酸的3'羟基端来合成DNA。并且TdT对底物的耐受性高具有聚合修饰型dNTP的能力,如荧光修饰的dNTP、生物素修饰的dNTP,甚至人工碱基均可作为其良好底物。TdT的这些生化特性使其被广泛的应用在生物传感和核酸合成领域中,促进了许多基于核酸的工具和方法的发展,并为酶促从头合成DNA技术的发展奠定基础。介绍了TdT的性质,重点总结了它在其介导的生物检测技术、核酸的修饰技术以及酶促合成DNA技术三个方面的核心作用、目前面临的挑战以及未来研究的方向,以期促进TdT在生物传感器和核酸合成中的进一步应用。     

用混合氨基酸接肽的方法合成OX型肽亚库

肽库合成是药物研究组合化学策略的重要技术之一。建立合成OX型肽亚库的固相合成方法 ,接肽反应采用等摩尔的Fmoc氨基酸混合物和DIC HOBt缩合方法 ,以高浓度的缩合剂和不断缩减溶剂的策略促进偶联反应进行完全。产物的氨基酸组成分析结果显示 ,所使用的常见氨基酸都能以相近的摩尔比例接肽至X位置。推测经多次接肽后最终形成的肽亚库中 ,含低活力氨基酸较多的肽其浓度虽然会较低一些 ,但影响不会太大 ,且本合成方法成本相对较低 ,故可为抗原表位分析、多肽药物筛选及构效关系分析提供一种有用的工具。