Veränderung der Acylneuraminsäuregehalte auf T-Lymphozyten und im Plasma bei Erkrankung an Mamma-Karzinom

Summary Increased sialic acid levels reflecting tumor burden are found on the surface of T-lymphocytes and in the plasma of patients with carcinoma of the mammary gland. The data of the determinations of sialic acid content and distribution on T-cells, using microanalytical methods such as HPLC and a colorimetric test, show that the total sialic acid content is increased by about 60% and that nearly 80–90% of the sialic acids consist of Nacetyl-9-O-acetyl-neuraminic acid, in comparison to the healthy controls (not containing O-acetylated neuraminic acid). Investigations on lymphocytes of malignant melanoma patients show similar changes of sialic acid content and distribution on the cell surface.Increased sialic acid levels are also found in the plasma of patients with cancer but no O-acetylated derivative can be found.Furthermore the examinations show that the separation of the T-lymphocytes from the total lymphocyte fraction is not required. Determination of sialic acids in the total lymphocyte fraction can be a simplification in carrying out further diagnostic investigations.A high level of sialic acids as antirecognition factor seems to be not only a marker of tumor cells but also an attribute of T-lymphocytes, involved in the defence against the malignoma (malignant melanoma, breast cancer).Considering the possible contribution of sialic acid to the immunoregulatory protective mechanism during the first stage of pregnancy, sialic acid content and distribution on T-cells of pregnant women are investigated. Both an increase and a change in the distribution of sialic acids can be excluded.

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Abkürzungen HPLC High performance liquid chromatography - Neu 5 Ac N-Acetyl-neuraminsäure - Neu 5, 9 Ac₂ N-Acetyl-9-O-acetyl-neuraminsäure Diese Arbeit wurde unterstützt von der Deutschen Forschungsgemeinschaft (Fa 45/4).     

Erythrocyte sialic acid content during aging in humans: correlation with markers of oxidative stress

Sialic acids are substituted neuraminic acid derivatives which are typically found at the outermost end of glycan chains on the membrane in all cell types. The role of erythrocyte membrane sialic acids during aging has been established however the relationship between sialic acid and oxidative stress is not fully understood. The present work was undertaken to analyze the relationship between erythrocyte membrane sialic acid with its plasma level, membrane and plasma lipid hydroperoxide levels and plasma total antioxidant capacity. Results show that sialic acid content decreases significantly (P< 0.001) in RBC membrane (r= -0.901) and increases in plasma (r=0.860) as a function of age in humans. Lipid peroxidation measured in the form of hydroperoxides increases significantly (P<0.001) in plasma (r=0.830) and RBC membranes (r=0.875) with age in humans. The Trolox Equivalent Total Antioxidant Capacity (TETAC) of plasma was found to be significantly decreased (P< 0.001, r=-0.844). We observe significant correlations between decrease of erythrocyte membrane sialic acid and plasma lipid hydroperoxide and TETAC. Based on the observed correlations, we hypothesize that increase in oxidative stress during aging may influence the sialic acid decomposition from membrane thereby altering the membrane configuration affecting many enzymatic and transporter activities. Considering the importance of plasma sialic acid as a diagnostic parameter, it is important to establish age-dependent reference.     

Clinical relevance of sialic acids determination in serum and synovial fluid in orthopaedic disorders

Sialic acids, derivatives of neuraminic acid, are present as structural components of mucoprotein mainly in the alpha 1 and alpha 2-globulin regions, and they are known to change in diseases associated with acute inflammation or tissue necrosis. The present study was performed to clarify the significance of measurements of sialic acids in the serum and synovial fluid of patients with diseases in the field of orthopaedic surgery. 1. Serum sialic acids were markedly high in cases of acute pyogenic diseases and showed moderately high values in stage 2 or 3 of rheumatoid arthritis (RA). There was a close correlation with ESR in RA cases and with CRP in cases of acute pyogenic diseases. Differences in the correlation with ESR and CRP were seen in proportion to the severity of the inflammation. 2. Synovial fluid sialic acids were high in cases of RA, and within the normal range in cases of osteoarthritis. The values changed within this range in accordance with treatment. In RA cases, there was a significant correlation with monocytes in the synovial fluid. 3. Serum sialic acids appeared to be sufficiently useful as a parameter of inflammation independent of ESR and CRP, and synovial fluid sialic acids were also considered to be useful for differentiation between RA and OA.     

A histochemical comparison of the O-acylated sialic acids of the epithelial mucins in ulcerative colitis, Crohn''s disease, and normal controls.

Two histochemical techniques, the PAT/KOH/PAS and the PBT/KOH/PAS, were used to investigate the side chain O-acyl substitution patterns of the sialic acids of the colonic epithelial mucins in cases of ulcerative colitis and Crohn's disease. In both diseases there was, as compared to normal, a reduction in the proportion of sialic acids O-acylated at C7C8, the reduction being greater in ulcerative colitis. Further, there appeared to be an association between the severity of the disease and the reduction in the staining of O-acylated sialic acids. This relationship was more marked in ulcerative colitis. In some cases of both diseases there was evidence for epithelial mucins containing predominantly C7-substituted sialic acids. This study has confirmed our previous conclusion that, in Crohn's disease of the terminal ileum, the disease is associated with an increase in the proportion of sialic acids bearing side chain substituents.     

Structural analogs of sialic acid interfere with the binding of erythrocyte binding antigen-175 to glycophorin A, an interaction crucial for erythrocyte invasion by Plasmodium falciparum

Plasmodium falciparum causes the most virulent form of malaria and remains a major worldwide health problem. The erythrocytic development of P. falciparum relies on parasite invasion of host erythrocytes, a process mediated in part by the interaction of erythrocyte binding antigen 175 (EBA-175) with the erythrocyte receptor glycophorin A (GA). The binding domain of EBA-175 that interacts with glycophorin A is a approximately 330 residues module called F2. Several studies have shown that F2 recognizes both sialic acids and the protein backbone on glycophorin A. Here, we have developed ELISA-based quantitative F2-GA binding assays. We also performed a series of competitive inhibition assays to block the F2-GA interaction using a variety of sialic acid analogs. Our data show that both 2,3-didehydro-2-deoxy-N-acetyl neuraminic acid (DANA) and 3'-N-acetyl neuraminyl-N-acetyl lactosamine are excellent inhibitors of the F2-GA interaction. Moderate levels of inhibition were also observed with monomers or oligomers of N-acetyl neuraminic acid (sialic acid). Furthermore, we show that DANA is able to significantly inhibit the invasion of erythrocytes by P. falciparum. Together, our ELISA-based binding assays and in vitro inhibition of erythrocyte invasion data suggest that small variations in the structures of DANA and related inhibitors can result in even more potent invasion inhibitory activities. Our studies provide a platform for the development of high potency inhibitors of the F2-GA interaction using high throughput drug discovery technologies. Such compounds may form part of inhibitor cocktails, which aim to block invasion of erythrocytes by P. falciparum.     

Attachment of bovine parvovirus to O-linked alpha 2,3 neuraminic acid on glycophorin A

Summary. The bovine parvovirus (BPV) hemagglutinates human erythrocytes by binding to glycophorin A (GPA). The purpose of this study was to determine which carbohydrate on GPA binds BPV. Treatment of GPA with α2,3,-6,-8 neuraminidase eliminated binding of BPV to GPA. Beta-elimination of O-linked sialic acids on GPA eliminated binding, while removal of N-linked carbohydrates using the N-glycosidase PNGase F failed to eliminate binding. Treatment of GPA with a neuraminidase which specifically cleaved α2,3 glycosidic bonds eliminated BPV binding and, following this treatment, virus binding to GPA was restored by reconstitution of α2,3-linked neuraminic acids. These results indicated the O-linked α2,3 neuraminic acids of GPA bind BPV.     

Differences in arachidonic acid metabolism by human myelomonocytic cell lines.

The production of arachidonic acid metabolites by the HL60, ML3, and U937 human phagocyte cell lines was determined after incubation with interferon-gamma (IFN-gamma, 500 U/ml) or vehicle for 4 days. Cells were prelabeled with tritiated arachidonic acid, [3H]AA, for 4 h, and media supernatants were analyzed by high-performance liquid chromatography. None of the cell lines produced [3H]AA metabolites in large amounts during an unstimulated, basal release period (30 or 60 min). In response to 10 microM calcium ionophore A23187 incubation (30 min), undifferentiated and IFN-gamma-differentiated HL60 cells formed both cyclooxygenase products (thromboxane and prostaglandins) and lipoxygenase products (leukotrienes and hydroxyeicosatetraenoic acids). In contrast to the HL60 cells, IFN-gamma-differentiated U937 cells formed primarily cyclooxygenase products and undifferentiated and IFN-gamma-differentiated ML3 cells did not form any [3H]AA metabolites in response to A23187. These results indicate the need to be careful in selecting a cell line for use in a phagocyte assay system when cyclooxygenase and/or lipoxygenase products could influence the assay results.     

Isolation and Characterization of Neisseria meningitidis Groups A, C, X, and Y Polysaccharide Antigens

To prepare Neisseria meningitidis groups A, C, X, and Y polysaccharide antigens, culture supernatant fluids were subjected to serial processes of salt precipitation, alkaline hydrolysis, ethyl alcohol precipitation, and Sephadex G-200 chromatography. This method resulted in the isolation of large quantities of group antigens. All are acidic polysaccharides, the group C antigen being a polymer of n-acetyl neuraminic acid. Thiobarbituric acid assay failed to reveal sialic acids in the other group antigens. Protein was undetectable by absorption at 280 nm or by Folin analysis. These antigens are of similar molecular size, the majority of which are excluded by Sephadex G-200. They migrate in the upper one-third of sucrose density gradients and are retained by 5% acrylamide gel. All are highly group-specific and react only with homologous hyperimmune antisera in hemagglutination, complement fixation, and immunodiffusion systems. As little as 0.03 μmoles of n-acetyl neuraminic acid in group C antigen inhibits the hemagglutination of group C-sensitized red cells. All antigens are immunogenic in rabbits. These techniques afford a simplified method for the production of relatively large yields of highly specific group antigens which participate in multiple immunologic systems.     

雷公藤多甙诱导人前骨髓白血病细胞的凋亡

探讨雷公藤多甙诱导人前骨髓白血病细胞凋亡的作用。方法将ＨＬ－６０与雷公藤多甙、泼尼松共同培养,观察细胞的细胞凋亡形态学变化,如细胞膜泡样变、细胞浆及细胞核内染色质固缩、凋亡小体出现以及ＤＮＡ规律性断裂。（２）细胞周期分析提示作用首先影响增殖期细胞,并且呈剂量、时间依赖性。（３）雷公藤多甙诱导ＨＬ－６０细胞凋亡的作用较泼尼松强;两种药物间无协同作用。结论体外实验发现雷公藤多甙具有诱导细胞凋亡的作用,     

Antigenic studies on an enzymatically sialylated carbohydrate: NeuAc(alpha 2-3)Gal(beta 1-3)GalNAc

Sialic acid residues are often the end moiety of the carbohydrate chain of biologically important glycoconjugates. It is difficult to study sialylated glycoconjugates because the purification of these compounds is often laborious yielding only very small amounts of oligosaccharides for study. Chemical synthesis of sialylated compounds is complicated by the labile nature of the sialic acid bond. In both of these cases the sialylated compounds would need to be conjugated to a polypeptide to be an effective immunogen, and again, such conjugation is fraught with problems due to the instability of the sialic acid linkage. We have developed a combined enzymatic and synthetic route for obtaining quantities of sialylated carbohydrates conjugated to a protein carrier in amounts sufficient for antigenic studies. The notable novelty of this protocol is the addition of sialic acid after the carbohydrate-protein conjugation step. Antiserum to the compounds was developed and after absorption, antibodies that demonstrate a requirement for sialic acid for their binding were produced and studied. CA 125 has been shown to be a prognostically significant marker for ovarian adenocarcinoma. The nature of the epitope involved has been analyzed with conflicting results. To attempt to resolve this conflict, we initiated studies on sialylated antigens with NeuAc alpha 2-3Gal beta 1-3GalNAc. This trisaccharide occupies the terminal region in a series of complex carbohydrates which have been suggested to be involved as the epitope. Hanisch et al. reported that the neuraminic acid was important for the reaction.     

Influenza virus hemagglutinins differentiate between receptor determinants bearing N-acetyl-, N-glycollyl-, and N,O-diacetylneuraminic acids

This report examines the ability of three sialic acids (SA), N-acetylneuraminic acid (NeuAc), N-glycollylneuraminic acid (NeuGc), and 9-O-acetyl-N-acetylneuraminic acid (9-O-Ac-NeuAc), to serve as receptor determinants for 18 human and animal influenza type A viruses. Viruses were compared by agglutination of receptor-modified erythrocytes containing either the Sa alpha 2,6Gal or the SA alpha 2,3Gal linkages with each of the three sialic acids. Individual isolates differed markedly in their ability to agglutinate cells containing NeuAc, NeuGc, and 9-O-Ac-NeuAc. The results suggest that recognition of the various sialic acids is an important factor in analysis of the receptor specificity of influenza virus hemagglutinins.     

Atherogenesis. An epidemiological model based on the presence of unnatural trans and cis isomers of unsaturated fatty acids in the maternal diet and in mothers milk

The hypothesis that the presence of unnatural trans and cis isomers of unsaturated fatty acids in the maternal diet and in human mothers milk could be responsible for initiating atherosclerosis in utero or in infants is proposed. It is suggested that the key etiological factor involved in the formation of atherosclerotic plaques could be uncontrolled division of smooth muscle cells of the intima resulting from the intracellular excess of linoleic acid and deficiency of its metabolites gamma-linolenic acid and dihomogamma-linolenic acid. This imbalance is brought about by competitive inhibition of the enzyme delta-6-desaturase by unnatural trans and cis unsaturated fatty acids. Delta-6-desaturase is the enzyme responsible for converting linoleic acid to dihomogamma-linolenic acid. The cellular presence of unnatural trans and cis isomers of unsaturated fatty acids would therefore enhance increased levels of linoleic acid and deficiency of its metabolites gamma-linolenic acid and dihomogamma-linolenic acid. It is proposed that prophylaxis against the effects of delta-6-desaturase inhibition could be achieved by the adoption of an Eskimo-like diet containing the essential fatty acid metabolites gamma-linolenic acid and/or dihomogamma-linolenic acid and eicosapentaenoic acid per se in high concentrations.     

Effect of Neuraminidase on Lymphoid Cells

After neuraminidase treatment the electrophoretic mobility of mouse lymphocytes (B and T cells) and thymocytes was reduced to a similar value, .a diminution of about 40%, 60%, and 40%, respectively. Thus B and T cells and thymocytes may have a common surface charge after removal of sialic acid. The percentage of cells binding anti-thymocyte serum was not changed; thus sialic acid does not seem to be an important part of the specific antigen of lymphoid cells. Similar amounts of sialic acid were released from B and T Ceils, whereas less than half of this was released from the thymocytes. Thus, whereas thymocytes are characterized by their low electrophoretic mobility and low sialic acid release and T cells by high electrophoretic mobility and high sialic acid release, B-cells in contrast show a low electrophoretic mobility and high sialic acid release.     

Genetically encoding unnatural amino acids for cellular and neuronal studies

Proteins participate in various biological processes and can be harnessed to probe and control biological events selectively and reproducibly, but the genetic code limits the building block to 20 common amino acids for protein manipulation in living cells. The genetic encoding of unnatural amino acids will remove this restriction and enable new chemical and physical properties to be precisely introduced into proteins. Here we present new strategies for generating orthogonal tRNA-synthetase pairs, which made possible the genetic encoding of diverse unnatural amino acids in different mammalian cells and primary neurons. Using this new methodology, we incorporated unnatural amino acids with extended side chains into the K+ channel Kv1.4, and found that the bulkiness of residues in the inactivation peptide is essential for fast channel inactivation, a finding that had not been possible using conventional mutagenesis. This technique will stimulate and facilitate new molecular studies using tailored unnatural amino acids for cell biology and neurobiology.     

Chimpanzee models for human disease and immunobiology

Summary: Chimpanzees have greater than 98% genomic sequence homology with humans but have significantly more favorable reponses to human imunodeficiency virus (HIV)-1 and hepatitis B virus (HBV) and an apparently low incidence of epithelial malignancy. Although there are few shared major histocompatibility complex (MHC) alleles between human and chimp, there is considerable overlap in binding repertoires for epitopes of HIV-1 and HBV. This indicates that differences in viral handling may be due to involvement of cells other than T lymphocytes. Similar mechanisms may be involved in host response to dysplastic or malignant cells. In seeking to understand these differences, most attention has been focused on comparing and contrasting well-characterized steps in immune response. As an additional possibility, alterations in cell–cell interactions dependent upon sialic acid binding proteins known to be involved in immune responses should also be considered. The lack of a particular sialic acid structure (N-glycolyl neuraminic acid, or Neu5Gc) in humans, due to a gene mutation in an essential synthetic enzyme, has potentially altered the kinetics of cellular responses dependent upon these lectins. The absence of Neu6Gc represents the only known major biochemical difference between humans and chimpanzees.     

Surface carbohydrate composition of murine thymocytes and of lymph node cells from normal and athymic (nude) mice.

Surface glycoproteins (papain digests) have been isolated from lymph node cells of normal mice which contain mainly T cells, and from lymph node cells of nude (athymic) mice, which essentially represent B cells. Gaschromatographic analysis revealed that the glycoproteins from the lymph node cells of the euthymic mice contain less galactose than the glycoproteins from lymph node cells of the athymic mice, but contain still more galactose than glycoproteins from thymocytes. Lymph node cells from both sources contain about equal amounts of neuraminic acid, while thymocytes contain slightly less sialic acid. The observed differences provide a molecular explanation for the different reactivity of murine B cells and T cells towards soybean agglutinine and other galactose-binding plant lectins.     

二十碳五烯酸联合视黄酸对HL-60细胞增殖与分化功能的影响

目的研究二十碳五烯酸 ( eicosapentaenoic acid,EPA)及其联合视黄酸 ( retinoic acid,RA)对白血病细胞增殖与分化功能的影响。方法采用 MTT法测定细胞增殖功能 ,NBT还原实验鉴定细胞分化 ,高效液相色谱法分析细胞内 RA代谢。结果与对照组相比 ,EPA组细胞增殖抑制率为 2 4 .3 8% ,RA组为 3 5 .74 % ,EPA+ RA组为 4 2 .75 % ;NBT还原实验 ,EPA+ RA组 OD值约为对照组的 5 .9倍 ,而 RA组为 2 .6倍 ,EPA组为 1.3倍 ;分析 HL- 60细胞及其培养介质中 RA含量 ,发现 EPA+ RA组高于 RA组。结论 EPA可抑制 HL- 60细胞增殖和诱导其分化 ,与 RA联合应用能明显增强 HL- 60细胞的增殖抑制及诱导分化效应 ,这种联合效应可能与 EPA减缓 HL - 60细胞内 RA的代谢相关。     

Sialic acid recognition is a key determinant of influenza A virus tropism in murine trachea epithelial cell cultures

Influenza A virus interacts with specific types of sialic acid during attachment and entry into susceptible cells. The precise amino acids in the hemagglutinin protein that control sialic acid binding specificity and affinity vary among antigenic subtypes. For H3 subtypes, amino acids 226 and 228 are critical for differentiating between α2,3- and α2,6-linked forms of sialic acid (SA). We demonstrate that position 190 of the HA from A/Udorn/307/72 (H3N2) plays an important role in the recognition of α2,3-SA, as changing the residue from a glutamic acid to an aspartic acid led to alteration of red blood cell hemagglutination and a complete loss of replication in differentiated, murine trachea epithelial cell cultures which express only α2,3-SA. This amino acid change had a minimal effect on virus replication in MDCK cells, suggesting subtle changes in receptor recognition by the H3 hemagglutinin can lead to significant alterations in cell and species tropism.     

二十碳五烯酸联合视黄酸对HL-60细胞凋亡相关基因表达的影响

目的 研究二十碳五烯酸(EPA)是否协同视黄酸(RA)影响HL-60细胞的凋亡及相应分子机制。方法 流式细胞仪(FCM)检测细胞周期相分布以测定细胞增殖和凋亡功能；Western blot法分析bcl-2和caspase—3表达。结果 与单独应用相比，EPA和RA联合应用可增强HL-60细胞的调亡效应，以及下调节bcl—2和增强caspase-3基因表达。结论 EPA和RA联合应用对HL-60细胞凋亡的增强效应，可能与它们增强caspase-3和降低bcl—2基因表达相关。     

抑制mel18表达增强桂皮醛诱导的HL60细胞分化

目的:研究下调mel18基因表达对桂皮醛(CA)诱导HL60细胞分化的影响。方法:用低浓度CA和反式维甲酸(ATRA)处理HL60细胞。用shRNAmel18质粒及对照质粒shRNALuc包装慢病毒,用病毒感染HL60细胞。低浓度CA和ATRA作用于病毒感染的HL60细胞,流式细胞术检测细胞周期和细胞表面分化抗原表达,Western blot法检测MEL18蛋白、cyclin D1、p27的表达和Akt的磷酸化水平。结果:低浓度CA和ATRA增加HL60细胞的粒细胞分化抗原CD11b表达,MEL18蛋白表达下降。shmel18病毒感染的HL60细胞(shmel18-HL60细胞)MEL18蛋白表达下降,而对照病毒感染的HL60细胞(sh Luc-HL60细胞)MEL18蛋白表达无明显变化。shmel18-HL60细胞的CD11b表达率明显增高,细胞阻滞于G1期;经低浓度CA处理后,shmel18-HL60细胞的CD11b表达率进一步增高。shmel18-HL60细胞Akt的磷酸化水平及cyclin D1表达明显下降,而p27表达则显著升高。结论:抑制mel18基因表达导致HL60细胞向成熟粒细胞方向分化,mel18基因可能通过PI3K/Akt信号途径调控cyclin D1和p27的表达,影响HL60细胞分化。而CA可能通过抑制mel18基因表达从而通过PI3K/Akt途径促进HL60细胞分化。