Immunological studies in ulcerative colitis: extract, fractionation, and immunological characterization of germ-free rat colon antigen

Intestinal mucins from germ-free rats contained antigens reactive with sera from patients with ulcerative colitis, in addition to human blood group A- and H-like antigens. A crude antigen extract was obtained by phenol-water extraction at 65 °C. Two intestinal glycoproteins were purified from the extract by fractionated ethanol precipitation, ion-exchange chromatography, and gel filtration. The two glycoproteins (2aI and 4aIIb) were homogeneous in regard to electrical charge and molecular size. Both were glycoproteins of the blood group substance type. Component 2aI was very rich in N-acetylgalactosamine and threonine and low in N-acetylglucosamine and sialic acid(s). It had strong blood group A-like activity, weak blood group H activity, and no colon antigen activity as defined by patients' sera. Component 4aIIb was rich in sialic acid(s). About 40% of the sera from patients with ulcerative colitis reacted with this component. No blood group A- or H-like activity could be demonstrated. Colon antigen activity was sensitive to periodate oxidation, but resistant to boiling at neutral pH. It was very sensitive to acid hydrolysis. In fact, colon antigen activity was significantly reduced when subjected to weak acid hydrolysis under conditions which only appeared to release sialic acids.     

A rapid prognostic electrophoretic assay for serum lipid conjugates in carcinoma uterine cervix and breast

The levels of cholesterol and lipid associated sialic acid in sera of patients with carcinoma uterine cervix and breast cancer were determined using agarose electrophoresis and precipitation method. Elevated level of lipid associated sialic acid was found, that was specifically reflected only in LDL and VLDL fractions. In carcinoma uterine cervix patients, cholesterol in low density lipoprotein increased with corresponding decrease in high density lipoprotein and there was no change in total as well as VLDL cholesterol content. But breast cancer patients were found to have higher concentrations of both total and high density lipoprotein cholesterol. A good positive correlation was found between electrophoresis and precipitation methods for the lipoprotein analysis. We recommend electrophoretic method for rapid routine analysis.     

Effect of streptozotocin diabetes on sialic acid content and glycoprotein binding of isolated hepatocytes

The possible correlation between sialic acid content of hepatocyte plasma membranes, and their binding capacity was investigated. For that purpose, [3H] asialotransferrin binding was tested with hepatocytes from both normal and streptozotocin treated rats. The observed decrease in binding capacity was parallel to a decrease in the sialic acid content Insulin therapy restored simultaneously normal blood glucose level, hepatic membrane sialic acid content and binding capacity.     

Sialic acid and carcinoembryonic antigen (CEA) as markers of colon cancer: Preliminary report]

Tumor markers known to date are not sensitive and specific enough to detect malignant tumors. Therefore, attempts to find new markers have led to sialic acid assays in cancer patients. Serum sialic acid, CEA and ESR have been determined in 33 patients with the cancer of the colon. All patients have been divided into four groups, according to TNM cancer staging. Serum sialic acid levels have been increased by 100% of patients in groups I and IV. The most significant correlation was noted between sialic acid levels and ESR. No significant relationship between serum sialic acid and CEA have been noted. No correlation of the colon cancer stage, according to TNM staging, and sialic acid and CEA levels in the peripheral blood has been observed. It seems, however, that serum sialic acid assay may be useful auxiliary technique in the detection and monitoring of patients with colon cancer.     

Autoantibodies to the extracellular matrix microfibrillar protein, fibrillin-1, in patients with scleroderma and other connective tissue diseases.

A duplication in the fibrillin-1 gene has been implicated as the cause of the tight skin 1 (tsk1) phenotype, an animal model of scleroderma or systemic sclerosis (SSc). In addition to the production of abnormal fibrillin-1 protein, the tsk1 mouse also produces autoantibodies to fibrillin-1. Among a population of Choctaw Native Americans with the highest prevalence of SSc yet described, a chromosome 15q haplotype containing the fibrillin-1 gene has been strongly associated with SSc. With a recombinant human fibrillin-1 protein, autoantibodies to fibrillin-1 were detected in the sera of Native American SSc patients that correlated significantly with disease. Abs to fibrillin-1 also were detected in sera from Japanese, Caucasian, and African-American SSc patients. Compared with other ethnic groups, Japanese and Native American SSc patients had significantly higher frequencies of anti-fibrillin-1 Abs. Sera from patients with diffuse SSc, calcinosis, Raynaud's, esophageal dysmotility, sclerodactyly, and telangiectasias syndrome and mixed connective tissue disease also had significantly higher frequencies of anti-fibrillin-1 Abs than sera from controls or patients with other non-SSc connective tissue diseases (lupus, rheumatoid arthritis, and Sj?gren's syndrome). Ab specificity for fibrillin-1 was demonstrated by the lack of binding to a panel of other purified autoantigens. The results presented demonstrate for the first time the presence of high levels of anti-fibrillin-1 Abs in a significant portion of patients with SSc.     

Major gangliosides in normal and pathological human thyroids

The major gangliosides of human thyroids were extracted, purified and then analyzed by gas-liquid chromatography. In normal thyroid, GM3 and GD3 represented about 80% of lipid-bound sialic acid. GM3 contained more than 50% of long-chain fatty acids, whereas GD3 contained mostly short ones. 4D hydroxy sphinganine represented 20% of long-chain base content in both cases. In pathological thyroids (Graves' disease, cancer, toxic adenoma), GM3 represented about 60% of lipid-bound sialic acid; its fatty acid content was mostly short chain fatty acids, as in normal GD3. 4D hydroxy sphinganine proportion was decreased.     

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.     

Gangliosides of hepatoma 27, normal and regenerating rat liver.

The highly malignant rat hepatoma 27 was found to have increased amounts of lipid-bound sialic acid as compared with normal liver whereas in regenerating liver the lipid-bound sialic acid level was reduced. In contrast to the liver the hepatoma contained higher amounts of disialogangliosides and no trisialogangliosides, which are abundant in the liver. The main disialoganglioside of the hepatoma had no analogue among the liver gangliosides and was identified as Gal-GalNAc(AcNeu-AcNeu)-Glc-Cer (GD1b), which in other tissues is known to be a precursor of trisialogangliosides. These findings may be explained by a reduced activity of glycosyltransferases in the hepatoma and apparently do not simply reflect differences in growth rate since the ganglioside pattern of regenerating rat liver was not altered significantly in comparison with the liver. Liver and hepatoma microsomes were found to be enriched in gangliosides as compared with whole cells, liver mitochondria were slightly poorer, while the ganglioside level of hepatoma mitochondria was much higher than that of the hepatoma cells. It thus appears that the existing image of the plasma membranes as the only sites of high ganglioside concentration may not hold true for weakly differentiated hepatomas of high malignancy.     

Isolation and characterization of gangliosides from pig lymphocytes

Two major gangliosides from pig spleen lymphocytes, accounting for 57% of the total lipid-bound sialic acids, were isolated and purified to homogeneity by column chromatography on DEAE-Sephadex and silica gel. They were identified as GM3 (II3Neu5GcLacCer), and GD3 (II3(Neu5Gc)2LacCer), by thin-layer chromatography in comparison with standards and by analysis of the constituent sugars. The major fatty acids of these gangliosides were stearic acid and myristic acid, respectively. In addition to these gangliosides, GD2 and bands comigrating on thin-layer chromatography with authentic GM2, GM1, GD1a and GD1b were found. These compounds also occur in pig peripheral blood lymphocytes, where, however, GD3 represents about 70% of the total lipid-bound sialic acid.     

Sialic acid and neuraminidase activity in the frog oviduct: comparative biochemical investigation in the different tracts during the reproductive cycle

1. A biochemical study was carried out on the protein-bound and lipid-bound sialic acid, and neuraminidase activity in the different tracts of the oviduct of the frog Rana esculenta during the reproductive cycle. 2. Plasma sexual steroids were also investigated by RIA. 3. Fluctuations in neuraminidase activity are related to that of glycoprotein sialic acid and plasma estradiol. Glycolipid sialic acid does not have a close relationship either with neuraminidase or plasma estradiol. 4. Very high plasma concentration of progesterone before ovulation and, on the contrary, its drop after ovulation were observed. 5. The results are discussed and hypotheses advanced to explain fluctuations of the studied parameters during the reproductive cycle.     

The organization of gangliosides and other lipid components in synaptosomal plasma membranes and modifying effects of calcium ion

Synaptosomes were prepared from bovine brain by zonal rotor sucrose density centrifugation. While a major fraction of lipid-bound sialic acid is included uniformly within the synaptosomal distribution profile, the sialoglycoproteins and some gangliosides do not follow this pattern, Exposure to extrasynaptosomal calcium results in alterations in the surface labeling properties of some gangliosides and membrane plasmalogens, suggesting that extrasynaptic Ca²⁺ may influence the conformation of complex lipids in synaptic plasma membranes. The level of intrinsic membrane-associated sialidase activity that liberates sialic acid from these sialoglycoconjugates parallels the synaptosomal buoyant density distribution profile, supporting a view that this enzyme resides in synaptosomal membranes in close association with a sialolipid substrate.     

Alteration of gangliosides in plasma and red cells of humans bearing melanoma tumors

In melanoma tumor-bearing humans, the levels of lipid-bound sialic acid were significantly elevated in both plasma and erythrocytes. Disialosyllactosylceramide in the plasma and sialosyllactosylceramide in red cells were the gangliosides mainly concerned by the increase, as compared to their concentration in the blood of healthy humans. In surgically treated patients, the plasma gangliosides remained higher than the controls, whereas a downward trend was noticeable in red cells. It is suggested that the occurrence of increased amounts of disialosyllactosylceramide in patients' plasma reflects the previously shown presence of this ganglioside as a major component in the sialic acid-containing glycolipid fraction of malignant melanocytes.     

An improved method for the measurement of total lipid-bound sialic acids after cleavage of α2,8 sialic acid linkage withVibrio cholerae sialidase in the presence of cholic acid,SDS and Ca2+

In the measurement of total lipid-bound sialic acids involving periodic acid oxidation, as in the periodate-resorcinol assay, the inner sialic acids of disialoglycolipids (such as GD₃ and GD₂) are not involved because their 2,8 ketosidic linkages are resistant to periodic acid oxidation, even after acid/enzyme hydrolysis or alkali pretreatment. However, the sialic acids from these glycolipids can be recovered completely after cleavage of 2,8 linkages byV. cholerae sialidase in the presence of cholic acid, sodium dodecyl sulphate and calcium. Interestingly, removal of calcium or detergent(s) or both significantly minimizes the sialidase action on the disialyl residues of these gangliosides. Therefore, we recommend sialidase (Vibrio cholerae) pretreatment of the glycolipids in the presence of cholic acid, SDS and Ca²⁺ for complete recovery of sialic acids from di- and polysialogangliosides and for accurate measurement of total lipid-bound sialic acids by periodate-resorcinol assay.Presented at the Second International Glycobiology Symposium which was held in San Francisco, CA, USA (14 February 1994).     

Antibody-dependent cellular cytotoxicity-inducing antibodies against human immunodeficiency virus. Presence at different clinical stages

The presence of antibodies mediating antibody-dependent cellular cytotoxicity (ADCC) against human immunodeficiency virus (HIV)-infected target cells was investigated with 170 sera from patients with varying severity of HIV infection. Approximately 40% of sera from individuals representing all stages of infection were ADCC-positive when tested against HTLV-IIIB infected 0937 clone 2 target cells. The positive sera had higher HIV antibody titers as measured by enzyme-linked immunosorbent assay compared with ADCC-negative sera. ADCC titers were lower in patients with acquired immune deficiency syndrome than in asymptomatic carriers. This decline in ADCC titer was not correlated with a general decrease of HIV antibodies. No correlation between the CD4:CD8 lymphocyte ratio and ADCC activity was found. The possible beneficial effect of ADCC-inducing antibodies early in infection is discussed in relation to the effect of ADCC-inducing antibodies in other retrovirus systems and to the nature of lentivirus infections.     

D- 二聚体、血流变学指标对结缔组织病患者诊疗价值研究

目的:探讨分析结缔组织病合并多器官损害患者D-二聚体(DD)、血流变学指标(血浆纤维蛋白原/血浆纤维蛋白降解产物FDP、全血黏度、血浆黏度)水平及指标变化对结缔组织病合并多器官损害患者病情活动期的诊疗价值。方法:本研究共纳入120例患者,其中活动组患者60例,病情缓解组患者60例,分别测定二组患者DD、血流变学指标水平,并采用t检验、灵敏度与特异度进行统计学分析与描述。结果:与缓解组比较,活动组DD、纤维蛋白降解产物(FDP)、全血黏度、血浆黏度、γ球蛋白、补体C4、ESR均具有统计学差异(P0.01),补体C3组间差异也具有统计学意义(P0.05)。各指标对于疾病活动性诊断的准确性,其DDFDP补体C3、C4γ-球蛋白血浆黏度全血黏度ESR。结论:结缔组织病合并多器官损害患者,其纤溶水平、微循环状态与疾病活动密切相关。     

Diglyceride kinase from Escherichia coli. Modulation of enzyme activity by glycosphingolipids

Diglyceride kinase was purified from membranes of Escherichia coli K-12 using organic solvents. The enzyme apoprotein depended on lipids, such as cardiolipin (diphosphatidylglycerol), phosphatidylcholine or 1-monooleoylglycerol, for activity with 1,2-dipalmitoylglycerol. Mixed brain cerebrosides and gangliosides as well as defined ganglioside fractions and synthetic lactocerebroside were devoid of lipid cofactor activity. However, all these glycosphingolipids were strong inhibitors of activation by phosphatidylcholine. When cardiolipin was used as lipid activator with the detergent, Triton X-100, as solubilizing agent, the addition of mixed or purified gangliosides first (at about 0.4 mM) resulted in additional activation, but higher ganglioside concentrations were strongly inhibitory. Both effects were absolutely dependent on the presence of lipid-bound sialic acid and were not given by cerebrosides, by free sialic acid or by sialyl-lactose. The stimulating and inhibitory effects of glycosphingolipids could also be demonstrated when 1-monooleoylglycerol was used as substrate, lipid activator and solubilizing agent at the same time. The modulation of kinase activity by glycosphingolipids is discussed at the level of lipid/protein interactions.     

Preferential reactivity of autoantibodies in murine lupus NZB mice to neuraminidase-treated monosialogangliosides on B cells of mouse spleen

When analyzed by flow cytometry, reactivity of IgM autoantibodies in sera from NZB mice to spleen B cells, but not to T cells, from BALB/c mice was remarkably increased after treatment of the cells with Vibrio cholerae neuraminidase. By TLC immunostaining with the antibodies, neither neutral nor acidic glycosphingolipids from both BALB/c and NZB mouse spleens were found to be reactive, but after neuraminidase treatment of the TLC plate, prior to the immunostaining, three components became reactive. All of the reactive glycosphingolipids were found to carry a single sialic acid residue and were at a concentration less than 1.3% of the total lipid-bound sialic acids. Their mobilities on TLC plate were close to those of IV3 NeuAcnLc4Cer, IV3 NeuAcII3 NeuAcGg4Cer, and IV3 NeuAcII3 NeuAc2Gg4Cer. In addition, the monosialogangliosides, which became reactive with the autoantibodies after neuraminidase treatment, were found to be predominantly distributed on B cells from BALB/c mice spleen, but not on T cells by TLC immunostaining. These studies demonstrate that the majority of IgM autoantibodies to spleen lymphocytes in sera from NZB mice might react preferentially to terminal sugar residues of three new glycosphingolipids masked by a single sialic acid on B cells, but not on T cells.     

Lipid composition of bloodstream forms of Trypanosoma brucei brucei

The qualitative and quantitative lipid composition of bloodstream forms of Trypanosoma brucei brucei S42 was compared with that of rat plasma and erythrocytes. The concentrations of lipid-bound phosphate and lipid-bound sialic acid in the trypanosomes were markedly higher than those of the rodent tissues. There was no trace in the trypanosomes of dolichol or dolichol phosphate. Trypanosomes contained two unidentified lipids: an acidic phospholipid and a highly polar glycolipid.     

Diglyceride kinase from Escherichia coli: Modulation of enzyme activity by glycosphingolipids

Diglyceride kinase was purified from membranes of Escherichia coli K-12 using organic solvents. The enzyme apoprotein depended on lipids, such as cardiolipin (diphosphatidylglycerol), phosphatidylcholine or 1-monooleoylglycerol, for activity with 1,2-dipalmitoylglycerol. Mixed brain cerebrosides and gangliosides as well as defined ganglioside fractions and synthetic lactocerebroside were devoid of lipid cofactor activity. However, all these glycosphingolipids were strong inhibitors of activation by phosphatidylcholine. When cardiolipin was used as lipid activator with the detergent, Triton X-100, as solubilizing agent, the addition of mixed or purified gangliosides first (at about 0.4 mM) resulted in additional activation, but higher ganglioside concentrations were strongly inhibitory. Both effects were absolutely dependent on the presence of lipid-bound sialic acid and were not given by cerebrosides, by free sialic acid or by sialyl-lactose. The stimulating and inhibitory effects of glycosphingolipids could also be demonstrated when 1-monooleoylglycerol was used as substrate, lipid activator and solubilizing agent at the same time. The modulation of kinase activity by glycosphingolipids is discussed at the level of lipid/protein interactions.     

The importance of sialic acid,pH and ion concentration on the interaction of uromodulin and complement factor H

Uromodulin (UMOD) can bind complement factor H (cFH) and inhibit the activation of complement alternative pathway (AP) by enhancing the cofactor activity of cFH on degeneration of C3b. UMOD, an N-glycans-rich glycoprotein, is expressed in thick ascending limb of Henle's loop where the epithelia need to adapt to gradient change of pH and ion concentration. ELISA-based cofactor activity of cFH and erythrocytes haemolytic assay was used to measure the impact of native and de-glycosylated UMOD on the functions of cFH. The binding assay was performed under different pH and ion concentrations, using ELISA. The levels of sialic acid on UMOD, from healthy controls and patients with chronic kidney disease (CKD), were also detected by lectin-ELISA. It was shown that removal of glycans decreased the binding between UMOD and cFH and abolished the ability of enhancing C3b degradation. In acidic condition, the binding became stronger, but it reduced as sodium concentration increased. A significant decrease of α-2,3 sialic acids on UMOD was observed in CKD patients compared with that of healthy individuals. The sialic acids on UMOD, local pH and sodium concentration could impact the binding capacity between UMOD and cFH and thus regulate the activation of complement AP.