Nanoparticle-Aided Detection of Colorectal Cancer-Associated Glycoconjugates of Extracellular Vesicles in Human Serum.

Extracellular vesicles (EVs) are found in all biological fluids, providing potential for the identification of disease biomarkers such as colorectal cancer (CRC). EVs are heavily glycosylated with specific glycoconjugates such as tetraspanins, integrins, and mucins, reflecting the characteristics of the original cell offering valuable targets for detection of CRC. We report here on europium-nanoparticle (EuNP)-based assay to detect and characterize different surface glycoconjugates of EVs without extensive purification steps from five different CRC and the HEK 293 cell lines. The promising EVs candidates from cell culture were clinically evaluated on small panel of serum samples including early-stage (n = 11) and late-stage (n = 11) CRC patients, benign condition (n = 11), and healthy control (n = 10). The majority of CRC cell lines expressed tetraspanin sub-population and glycovariants of integrins and conventional tumor markers. The subpopulation of CD151 having CD63 expression (CD151CD63) was significantly (p = 0.001) elevated in early-stage CRC (8 out of 11) without detecting any benign and late-stage samples, while conventional CEA detected mostly late-stage CRC (p = 0.045) and with only four early-stage cases. The other glycovariant assays such as CEACon-A, CA125WGA, CA 19.9Ma696, and CA 19.9Con-A further provided some complementation to the CD151CD63 assay. These results indicate the potential application of CD151CD63 assay for early detection of CRC patients in human serum.

Glycoconjugates in the detection of alcohol abuse.

Up to 30% of all hospital admissions and health-care costs may be attributable to alcohol abuse. Ethanol, its oxidative metabolites, acetaldehyde and ROS (reactive oxygen species), non-oxidative metabolites of alcohol [e.g. FAEEs (fatty acid ethyl esters)] and the ethanol-water competition mechanism are all involved in the deregulation of glycoconjugate (glycoprotein, glycolipid and proteoglycan) metabolic processes including biosynthesis, modification, transport, secretion, elimination and catabolism. An increasing number of new alcohol biomarkers that are the result of alcohol-induced glycoconjugate metabolic errors have appeared in the literature. Glycoconjugate-related alcohol markers are involved in, or are a product of, altered glycoconjugate metabolism, e.g. CDT (carbohydrate-deficient transferrin), SA (sialic acid), plasma SIJ (SA index of apolipoprotein J), CETP (cholesteryl ester transfer protein), ß-HEX (ß-hexosaminidase), dolichol, EtG (ethyl glucuronide) etc. Laboratory tests based on changes in glycoconjugate metabolism are useful in settings where the co-operativeness of the patient is impaired (e.g. driving while intoxicated) or when a history of alcohol use is not available (e.g. after trauma). In clinical practice, glycoconjugate markers of alcohol use/abuse let us distinguish alcoholic from non-alcoholic tissue damage, having important implications for the treatment and management of diseases.

Eryptosis in hereditary spherocytosis and thalassemia: role of glycoconjugates.

The present work is aimed to study the mechanism of faster erythrocyte clearance in hereditary spherocytosis (HS), a heterogeneous disorders characterized by alterations in the proteins of the red cell membrane skeleton along with different kinds of thalassemia. The maximum exposure of phosphatidylserine (PS) is found in HS compared to those in both α- and ß-thalassemia. Interestingly, in HS more PS exposed cells were found in younger erythrocytes compared to normal and the thalassemics where aged cells showed higher loss of PS asymmetry. Loss of sialic acid and GlcNAc bearing glycoconjugates, presumably the glycophorins, was also found upon aging. The loss of PS asymmetry together with the cell surface glycoproteins mediated by membrane vesiculation, seemed to play key role in early clearance of erythrocytes from circulation following a mechanism similar to HbEß-thalassemia.

FUT3 and FUT2 genotyping and glycoconjugate profile Lewisb as a protective factor to Toxoplasma gondii infection.

The interaction between the ABO, FUT2 and FUT3 genes results in the synthesis of different glycoconjugates profiles expressed in gastrointestinal tract. Moreover, the protozoan Toxoplasma gondii, which causes toxoplasmosis, utilizes this organ as an infection route. We analyzed the frequencies of the different glycoconjugate profiles which were determined by phenotyping ABO and genotyping the status secretor (FUT2; substitution G428A) and Lewis (FUT3; substitution T202C and C314T) histo-blood systems, assessed by PCR-RFLP and PCR-SSP, respectively. A total of 244 pregnant women (G1: Seropositive; G2: Seronegative) for IgG T. gondii antibodies were enrolled. IgG anti-T. gondii antibodies were determined by ELISA. G1 was composed of 158 (64.8%) sample and G2 by 86 (36.2%). The glycoconjugate profile was accessed in 151 seropositive and 85 seronegative samples by the combination of ABO and Lewis phenotyping as well as FUT2 and FUT3 genotyping. In G1, 36 (22.8%) presented the glycoconjugate profile ALeb, 5 (3.3%) A, 13 (8.6) BLeb, 1 (0.6%) B, 41 (27.1%) Leb, 13(8.6%) H, 38(25.2%) Lea and 4 (2.6%) Lec. G2 was composed of 13 (15.3%) of ALeb, 15 (17.6%) BLeb, 1 (1.2%) B, 42 (49,4%) Leb and 14 (16.5) Lea. H and Lec glycoconjugate profiles were not found in G2. The frequencies of the glycoconjugates profiles Leb (p = 0.001) and H (p = 0.005) were significantly different compared between G1 and G2. The glycoconjugate profile H inferred from the ABO phenotyping and FUT3 and FUT2 genotyping is associated with infection by T. gondii in pregnant women and the Leb profile appears to protect the infection by this parasite.

Loss of phospholipid membrane asymmetry and sialylated glycoconjugates from erythrocyte surface in haemoglobin E beta-thalassaemia.

This study aimed to investigate any correlation between the extent of phosphatidylserine (PS) asymmetry and sialylated glycoconjugate levels with the faster clearance of circulating erythrocytes in haemoglobin E (HbE) beta-thalassaemia. Erythrocytes from peripheral blood samples of different HbEbeta-thalassaemia patients showed loss of PS asymmetry measured by annexin V binding using flow cytometry. Maximum PS exposure was found when HbE was 50-60% and HbF was <20% indicating a possible correlation with severity of the disease. Separation of erythrocytes into aged and younger cells showed higher loss of PS asymmetry in the younger erythrocytes of HbEbeta-thalassaemia patients when compared with normal blood, where PS asymmetry was lost only in the older cells. Sialylated glycoconjugate measurement using the lectins wheatgerm agglutinin and pokeweed mitogen showed loss of sialic acid and N-acetyl-D-glucosamine-bearing glycoproteins in the order normal

Abnormalities of erythrocyte glycoconjugates are identical in two families with congenital dyserythropoietic anemia type II with different chromosomal localizations of the disease gene.

We analyzed erythrocyte glycoconjugates in two families with congenital dyserythropoietic anemia type II (CDA-II): family 2 with the typical localization of the disease gene to chromosome 20q11.2 and family 1 in which this localization was excluded. Despite the different genetics, the erythrocyte glycoconjugate abnormalities in the two families were identical suggesting a complex inheritance of CDA-II. We also found that erythrocyte anion exchanger 1 protein is decreased in CDA-II homozygotes and obligate carriers alike.

Serum glycomarkers of endoplasmic reticulum and lysosomal-endosomal system stress in human healthy aging and diseases.

To verify the idea that extracellular free oligosaccharides might be able to reflect the functional status of the endoplasmic reticulum (ER) and lysosomal-endosomal system, HPLC-profiles of serum-derived free oligosaccharides (FOS) in human healthy aging, acute myeloproliferative neoplasms, and cardiovascular pathologies were compared with intracellular glycans. After plasma deproteinization and FOS purification the oligosaccharides were labelled with anthranilic acid, separated into the neutral and charged with QAE Sephadex (Q25-120) chromatography and analysed using high-performance liquid chromatography (HPLC). The charged FOS were digested with a sialidase and compared with free oligosaccharides from transferrin for structural decoding. HPLC-profiles of serum-derived FOS revealed mild delay of the dolichol phosphate cycle in ER, moderate intensification of ER-associated degradation (ERAD) and degradation in endosomal-lysosomal system with aging; an inhibition of the dolichol phosphate cycle, intensification of ERAD and increasing of lysosomal exocytosis in acute myeloproliferative neoplasms; intensification of ERAD and glycocojugate degradation with endosomal-lysosomal system in cardiovascular diseases. As serum free oligosaccharides are able to reflect specifically perturbations in ER and endosomal-lysosomal system under wide range of stressors they can serve as extracellular markers of functionality of these organelles.

Detection of immune-complexed 9-O-acetylated sialoglycoconjugates in the sera of patients with pediatric acute lymphoblastic leukemia.

Although childhood acute lymphoblastic leukemia (ALL) is highly responsive to chemotherapy, reliable techniques are needed to determine treatment outcome. Over expression of 9-O-acetylated sialoglycoconjugates (9-OAcSGs) on lymphoblasts and concomitant anti-9-OAcSGs was found to have a diagnostic and prognostic potential. However, the presence of circulatory immune-complexed antigens remains unknown. The present study was aimed to evaluate whether immune-complexed 9-OAcSGs can be harnessed for better disease management. Immune-complexed antigens were evaluated in ALL sera (n=262) by a Dot-blot using a 9-OAcSAalpha2-6GalNAc-specific lectin, Achatinin-H. Using three serum samples, the inter- and intra-assay imprecision was evaluated as 11-13% and 7-11%, respectively. The recovery of spiked 9-OAcSGs was 84.2-95.4%. The central 95% reference interval for immune-complexed 9-OAcSGs in normal human sera (NHS, n=144) was 2.9-3.4 mug/ml irrespective of sex and age. At disease presentation, the immune-complexed 9-OAcSGs were fivefold higher than NHS, decreased with remission induction and importantly, reappeared with clinical relapse. Sera from patients with other hematological disorders (n=86) showed negligible levels. The Dot-blot demonstrated the potential application of immune-complexed antigen as a disease-specific marker and its efficacy as a sensitive and specific method that could serve as an economical yet effective index for monitoring disease status.

Validation of a commercial serodiagnostic kit for diagnosing pulmonary Mycobacterium avium complex disease.

SETTING: A commercial serodiagnostic kit for diagnosing pulmonary disease due to Mycobacterium avium complex (MAC-PD) was developed and launched in Japan in 2011. OBJECTIVE: To evaluate the performance of this kit in routine clinical settings. METHODS: In this retrospective single-centre study, data on serum levels of anti-glycopeptidolipid (GPL) core IgA antibody (U/ml) measured using the kit were analysed in patients diagnosed with MAC-PD according to American Thoracic Society criteria, in those with pulmonary tuberculosis (PTB) or pulmonary M. kansasii disease and in healthy volunteers. RESULTS: The anti-GPL-core IgA antibody levels of serum were significantly higher (P < 0.0001) in patients with MAC-PD (n = 485) than in those with PTB (n = 133) or pulmonary M. kansasii disease (n = 23) or in healthy subjects (n = 265). When the cut-off level was set at 0.7 U/ml, the sensitivity and specificity were respectively 78.6% and 96.9%. Higher antibody levels were observed in patients with greater extent of disease on chest computed tomography (P < 0.0001). CONCLUSIONS: The serodiagnostic kit revealed good sensitivity and specificity. The antibody levels may reflect disease activity. Additional work is needed to determine whether the diagnostic assay could be used in conjunction with current diagnostic criteria to improve the diagnosis of MAC-PD.

Structural Changes of Erythrocyte Surface Glycoconjugates after Treatment with Medicinal Mushrooms.

Under conditions of chronic hyperglycemia there is dysregulation of ion homeostasis, violation of redox metabolism and functioning of membrane enzymes, as well as changes in the structural and functional states of erythrocyte membranes. As a result, the aggregation ability of erythrocytes increased and their deformability decreased. These changes lead to complications to microcirculation blood flow and provoke the development of vascular complications caused by diabetes mellitus. This study investigated the effect of the medicinal mushrooms Agaricus brasiliensis and Ganoderma lucidum on the structure of carbohydrate determinants of surface membrane glycoconjugates of rat peripheral blood erythrocytes under both normal conditions and streptozotocin-induced diabetes mellitus. The research was carried out using Wistar outbred white rats. Diabetes was induced by streptozotocin intraperitoneally injected once at a dose of 50 mg/kg body weight. The mushroom preparations were orally administered at a dose of 1 g/kg for 14 days. The treatment of diabetic rats by submerged culture mycelium powder restored the physiological balance between sialylation and desialylation processes, renewed the membrane surface charge of red blood cells, normalized aggregation properties, and caused the structural recovery of oligosaccharide chains of erythrocyte membrane surface glycoconjugates. The discovered changes show an improvement in the erythrocyte functional state and rejuvenation of their population caused by biologically active compounds of the studied medicinal mushrooms.

Analysis of glycoconjugates in patients with oral squamous cell carcinoma.

BACKGROUND: Our aim was to examine the levels of glycoconjugates in plasma, erythrocyte membranes and buccal mucosa of healthy subjects and oral cancer patients. SUBJECTS AND METHODS: This study was conducted on 48 adult male oral cancer patients with various clinical stages (stage II to stage IV; 16 of each) and 16 disease-free healthy subjects who underwent surgical removal of impacted teeth or vestibuloplasty without inflammation. RESULTS: The plasma and tumor tissues glycoconjugates levels were significantly increased, whereas the erythrocyte membranes glycoconjugates were significantly decreased in oral cancer patients as compared to healthy subjects. The levels of glycoconjugates were gradually increased from stage II to stage IV in plasma and tumor tissues and decreased in erythrocyte membranes from stage II to stage IV of oral cancer patients. CONCLUSION: The increased plasma glycoconjugates can be due to the expense of erythrocyte membrane glycoconjugates or tumor tissue itself.

The conversion of group B red blood cells into group O by an alpha-D-galactosidase from taro (Colocasia esculenta).

An alpha-D-galactosidase (EC 3.2.1.21), capable of converting group B into group O red cells, was isolated from the stem portion of taro. It was purified about 3000 fold by gel filtration and ion-exchange chromatography. The blood group-converting activity was demonstrated by hemolysis and hemagglutination studies. This activity is comparable to that of alpha-D-galactosidase isolated from coffee beans. Taro alpha-D-galactosidase also hydrolyzes (1----4)- and (1----6)-linked alpha-D-galactopyranosyl groups from D-galactose-containing glycoconjugates. Taro alpha-D-galactosidase has a low Km value (0.28mM), a low molecular weight (40,000), and a neutral optimal pH (6.0). At a final enzyme concentration of 30 units/mL in the incubation mixture, the conversion of group B into group O activity was completed within two hours, without apparent changes in the shape of the red cells.

Effect of fluororotic acid on the sialylation of the chicken liver, hepatoma MC-29 and serum glycoconjugates.

Fluororotic acid, an orotic acid analogue which interferes with the nucleotide and RNA biosynthesis, was tested to determine its effect on: a) 14C-glucosamine and 14C-N-acetylmannosamine incorporation into acid-soluble nucleotide-sugars and into b) glycoconjugates from chicken liver and hepatoma induced by the avian leukosis strain Mc-29. In vivo metabolism studies indicated that this agent alters the nucleotide biosynthesis in both tissues investigated and causes a decrease in the sialylation rate of liver, hepatoma and serum glycolipids and glycoproteins.

Electrospray ionization-tandem mass spectrometry for the analysis of tryptophan derivatives in food.

Our knowledge about bioactive tryptophan derivatives in the diet is rather limited. Consequently, our attention was focused on the efficient profiling, i.e. the structure specific detection, of novel tetrahydro-beta-carbolines and tryptophan glycoconjugates in food samples. Applying HPLC-MS/MS for screening and structural characterization, numerous products derived from the reaction of tryptophan with alpha-oxo acids and carbohydrates could be identified by means of neutral loss scanning. Subsequently, product ion experiments followed by the synthesis of the respective reference compounds accomplished structure elucidation of tryptophan derivatives.

Serum glycoconjugates in patients with anemia and myeloid leukemia.

Because of carbohydrate alterations in malignant cells, serum glycoproteins have drawn considerable attention. In the current investigation we determined total sialic acid (TSA), lipid bound sialic acid (LSA), protein bound hexoses (galactose + mannose), fucose, hexosamines (galactosamine + glucosamine) and mucoid protein concentrations in the serum of patients with anemia and myeloid leukemia. The results were compared with those obtained in healthy individuals. In the leukemia patients we observed significant increases in glycoconjugates compared with the controls (P less than 0.001), and in TSA and fucose levels compared with the anemia patients (P less than 0.001). LSA and hexosamine levels were significantly lower in anemia patients with respect to the leukemia patients (P less than 0.01 and P less than 0.05 respectively), whereas levels of mucoid proteins and hexoses did not show significant differences. Except for hexosamines, all the markers tested were significantly elevated in the anemia patients compared with the controls. The present study suggests that the glycoconjugates investigated might be useful biochemical markers for differentiating anemic from leukemic conditions.

[Conjugates of amino acids with glucosamine isolated from the plasma of rats with Guerin carcinoma]. / Issledovanie kon''iugata aminokislot s gliukozaminom, vydelennogo iz plazmy krovi krys s kartsinomoi Gerena.

The conjugate of amino acids and glucosamine (glycamine) was purified to homogeneity from blood plasma of rats with Guerin carcinoma. The NH2-groups of amino acids in the glycamine are free. Amino acids are linked to glucosamine via carboxyl groups. 8 amino acids residues were identified in the glycamine molecule (Glu-2, Ser-2, Gly-3, Lys-1). Spatial atomic-molecular model of the glycamine molecule was developed. Two glucosamine molecules are linked to diaminosaccharide via (1-4)-bond, 8 amino acid molecules are linked to diaminosaccharide via 6 ester and 2 pseudopeptide bonds.

Serum and tissue glycoconjugates, digoxin and magnesium levels in chronic calcific pancreatitis.

BACKGROUND: Endogenous or exogenous digoxin can lead to membrane Na+,K+-ATPase inhibition and hypomagnesemia. Low magnesium levels can lead to increased glycosaminoglycans (GAG) concentration in many organs. AIM: To measure the serum levels of pancreatic GAG and glycoproteins, two major components of the extracellular matrix, in patients with chronic calcific pancreatitis (CCP). Serum levels of magnesium and digoxin were also assessed. METHODS: Patients with CCP and age- and sex-matched healthy control subjects (15 each) were studied. Serum GAG, Mg and digoxin levels were measured. RBC membrane Na+,K+-ATPase activity was also assessed. Pancreatic tissue obtained at autopsy from seven patients with CCP and sex- and age-matched healthy subjects who had died in accidents were also tested for GAG and glycoproteins. RESULTS: Total GAG levels were significantly increased in the serum and pancreas of patients with CCP. This was associated with lower serum Mg levels, increased serum digoxin levels and decreased RBC membrane Na+,K+-ATPase activity. CONCLUSION: Exogenous or endogenous digoxin-induced hypomagnesemia and the consequent altered glycoconjugate metabolism may be important in the pathogenesis of CCP.

[ELISA of IgM antibodies to Thomsen-Friedenreich (TF) hapten in cancer diagnostics: comparison of data obtained with four TF-glycoconjugates]. / Immunofermentnyi analiz IgM antitel k gaptenu Tomsena-Fridenreikha (TF) v onkodiagnostike. Sravnenie dannykh, poluchennykh s pomoshch'iu chetyrekh TF-glikokon"iugatov.

The level of IgM antibodies to the Thomsen-Friedenreich hapten (TF) relative to the total IgM level in the blood sera of gastric and breast carcinoma patients and healthy persons was determined using enzyme-linked immunosorbent assay. The following TF glycoconjugates were tested: TF-polyacrylamide (PAA)(with 10 mol.% of TF hapten Gal beta 1-3GalNAc alpha 1-O(CH2)3NH per number of monomeric units in the polyacrylamide), TF-human serum albumin (HSA)(Gal beta 1-3GalNAc alpha 1-O-p-C6H4-HSA containing approximately 15 carbohydrate residues per HSA molecule), asialo-kappa-caseinoglycopeptide, and asialoglycophorin. The total IgM level was determined using antibodies to the mu-chain of human IgM. The statistically significant difference between cancer patients and healthy donors was revealed with two conjugates: TF-PAA and TF-HSA. In the case of TF-PPA, the sensitivity of the assay was 75-83%, and the specificity was 77%. Thus, TF-PAA is the most suitable conjugate for measuring the level of serum anti-TF-IgM antibodies.

Glycoconjugate profile in plasma and erythrocytes of gastric cancer patients.

The present study has examined the glycoconjugate profile in plasma and erythrocyte membranes of 24 adult male gastric cancer patients and an equal number of age and sex-matched controls. Protein-bound hexose, hexosamine and sialic acid were significantly increased in plasma and erythrocytes of gastric cancer patients compared to controls. Elevation of glycoconjugates in circulation is suggested to be a result of increased shedding by the tumor cells or increased synthesis by liver, due to acute phase response.

Effect of luteolin on the levels of glycoproteins during azoxymethane-induced colon carcinogenesis in mice.

Luteolin (LUT), a bioflavonoid has been used as a chemopreventive agent world-wide against chemically induced cancer. Hence we designed an experiment to assess chemopreventive action of LUT on lipid peroxidation (LPO) and glycoconjugates in azoxymethane (AOM)-induced colon carcinogenesis. Colon cancer was induced by 15 mg/body kg. body weight of AOM and administration of LUT (at the dose of 1.2 mg/kg. body weight) was till end of the study. Analysis of lipid peroxidative end products such as protein carbonyl (PC), malonadehyde (MDA) and conjucated dienes (CD) demonstrated significant increase in in AOM-induced animals with reduction by LUT (p<0.05) . Increased levels of glycoconjugates such as hexose, hexosamine, sialic acid, fucose and mucoprotein were analyzed in serum and colon tissues examined histopathologically by periodic acid Schiff's (PAS) staining were also reversed by LUT l(p<0.05) . The secondary marker of colon cancer mucin depleted foci (MDF) was assessed in control and experimental group of animals. A characteristic increase of MDF was observed in AOM- induced colon cancer animals. Treatment with LUT decreased the incidence of MDF. These results suggest that LUT alters the expression of glycoconjugates and suppress colon cancer. Hence, we speculate that LUT can be used as a chemopreventive agent to treat colon cancer.