Dietary advanced glycated end-products and medicines influence the expression of SIRT1 and DDOST in peripheral mononuclear cells from long-term type 1 diabetes patients.

Quantitative polymerase chain reaction was employed to quantify expression of two genes coding for advanced glycation end-product receptors [RAGE ( AGER) and AGER1 ( DDOST)] and of the gene coding the deacetylase SIRT1 ( SIRT1) in peripheral blood mononuclear cells from type 1 diabetes patients without [Group A, n = 35; 28.5 (24-39) years old; median (interquartile interval)] or with at least one microvascular complication [Group B, n = 117; 34.5 (30-42) years old]; 31 healthy controls were also included. In a subgroup of 48 patients, daily advanced glycation end-products intake before blood collection was assessed. Lower expression of DDOST was found in patients than in controls after adjustment for sex, age, use of statins, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers. Higher expressions of AGER, DDOST and SIRT1 were observed in Group A. Stratifying by complications, AGER and DDOST expressions were higher in those without retinopathy and without diabetic kidney disease, respectively, compared to patients with these complications. Patients using statins or angiotensin receptor blockers presented higher expression of DDOST. Expression of SIRT1 was higher in patients consuming ≥12,872 KU daily of advanced glycation end-products. Although AGER, DDOST and SIRT1 are differently expressed in peripheral blood mononuclear cells from type 1 diabetes patients with and without microvascular complications, they are also influenced by dietary advanced glycation end-products and by statins and angiotensin receptor blockers.

Short-term effects of dietary advanced glycation end products in rats.

Dietary advanced glycation end products (AGE) formed during heating of food have gained interest as potential nutritional toxins with adverse effects on inflammation and glucose metabolism. In the present study, we investigated the short-term effects of high and low molecular weight (HMW and LMW) dietary AGE on insulin sensitivity, expression of the receptor for AGE (RAGE), the AGE receptor 1 (AGER1) and TNF-α, F2-isoprostaglandins, body composition and food intake. For 2 weeks, thirty-six Sprague-Dawley rats were fed a diet containing 20% milk powder with different proportions of this being given as heated milk powder (0, 40 or 100%), either native (HMW) or hydrolysed (LMW). Gene expression of RAGE and AGER1 in whole blood increased in the group receiving a high AGE LMW diet, which also had the highest urinary excretion of the AGE, methylglyoxal-derived hydroimidazolone 1 (MG-H1). Urinary excretion of N ε-carboxymethyl-lysine increased with increasing proportion of heat-treated milk powder in the HMW and LMW diets but was unrelated to gene expression. There was no difference in insulin sensitivity, F2-isoprostaglandins, food intake, water intake, body weight or body composition between the groups. In conclusion, RAGE and AGER1 expression can be influenced by a high AGE diet after only 2 weeks in proportion to MG-H1 excretion. No other short-term effects were observed.

[Analysis of antimicrobial resistance of Streptococcus pneumoniae with restriction fragment length polymorphism of pbp2b gene and pulsed-field gel electrophoresis profiles among children].

UNLABELLED: Streptococcus pneumoniae is a common cause of potentially life-threatening infections such as meningitis, bacteraemia, pneumonia worldwide, for which children of preschool age are at particularly high risk. Since the late 1970s and 1980s, antibiotic resistance among pneumococci has become an emerging problem. Several multidrug-resistant clones have rapidly spread throughout the world. OBJECTIVE: (1) To investigate the prevalence of penicillin and other antibiotics nonsusceptibility among pneumococci. (2) To analyze the correlation of pbp2b amplicon profiles with penicillin resistance. (3) To serotype 31 isolates of penicillin-resistant pneumococci by latex agglutination. (4) To analyze the chromosomal relatedness of serotype 23F and 6 isolates of penicillin-resistant pneumococci by using pulsed-field gel electrophoresis (PFGE) and characterize these isolates in molecular epidemiology. METHODS: (1) Susceptibility was determined by using broth microdilution, E-test, and K-B disk. (2) The correlation of pbp2b amplicon profiles with penicillin resistance was assessed by restriction fragment length polymorphism (RFLP). (3) Serotyping of penicillin-resistant pneumococcal isolates was performed by using latex agglutination. (4) The properties of serotype 23F and 6 isolates of penicillin-resistant pneumococci were assessed by PFGE. RESULTS: S. pneumoniae with increased nonsusceptibility (including intermediate strains and resistant strains) to penicillin G was 9.9% in 1997, 12.6% in 1998, 14.6% in 2000; to cefuroxime 4.2%, 1.5%, 8.2%; to cefotaxime 0.0%, 1.7%, 1.0% respectively. There were no statistically significant differences (P > 0.05). While resistance to erythromycin, trimethoprim-sulfamethoxazole and chloramphenicol increased significantly from 76.8% in 1997 to 87.4% in 2000, from 74.7% to 88.3%, and from 22.6% to 40.8%, respectively (P < 0.05). RFLP analysis of pneumococcal pbp2b-specific amplicons was effective for screening penicillin resistance. Of the 31 strains of penicillin-resistant pneumococci (MICs 0.12 - 2.0 micro g/ml) studied, 6 (19.4%) strains (MICs 0.12 - 0.19 micro g/ml) were serotype 23F and 3 (9.7%) strains (MICs 0.5 - 1.5 micro g/ml) were serotype 6. There were nearly identical susceptibility to antibiotics and identical PFGE patterns in the former, and there were different susceptibility to antibiotics and different PFGE patterns in the latter. Three serotype 6 strains had different susceptibility to antibiotics and different PFGE patterns, which suggested that those strains may be scattered. CONCLUSION: Generally beta-lactams retained their activity against S. pneumoniae in Beijing. Resistance to erythromycin, trimethoprim-sulfamethoxazole, and chloramphenicol increased drastically. RFLP analysis of pneumococcal pbp2b-specific amplicons was effective for screening penicillin resistance. In 6 strains of serotype 23 F there were nearly identical susceptibility to antibiotics and identical PFGE patterns, which suggested the probability that there was a spread of serotype 23F isolates with low-level penicillin resistance in local area.

Aberrant O-linked oligosaccharide biosynthesis in lymphocytes and platelets from patients with the Wiskott-Aldrich syndrome.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immunodeficiency affecting B lymphocytes, T lymphocytes, and platelets. Previous studies on lymphocytes from WAS patients have revealed that leu-kosialin (CD43), a cell-surface glycoprotein bearing approximately 90 O-linked oligosaccharide chains, shows an aberrant electrophoretic mobility. To determine whether this finding reflects a different pattern of O-linked glycosylation in WAS cells, we have compared healthy individuals and WAS patients with respect to glycosyltransferase activities in T lymphocytes, platelets, and Epstein-Barr virus (EBV)-immortalized B cell lines. Stimulation of peripheral T cells from normal individuals in vitro with anti-CD3 antibodies and interleukin-2 was associated with a 3-fold increase in UDP-GlcNAc:Gal beta 3GalNAc-R (GlcNAc to GalNAc) beta 6-N-acetylglucosaminyltransferase (core 2 GlcNAc-T) from 0.8 to 2.2 nmol/mg/h. In contrast, peripheral T lymphocytes from WAS patients showed an inversion of this phenotype with high core 2 GlcNAc-T activity in unstimulated cells (2.3 nmol/mg/h) and a 2-3-fold decrease in activity following stimulation. Core 2 GlcNAc-T activity was also three times higher in platelets from WAS patients than in normal platelets. Glycosyltransferase activities were measured in immortalized B cell lines established from WAS and normal subjects by infection with EBV. Core 2 GlcNAc-T was less than 0.4 nmol/mg/h in WAS EBV-B cell lines compared to 2.4 nmol/mg/h in EBV-B cell lines from healthy individuals, In contrast, CMP-SA:SA alpha 2-3Gal beta 1-3GalNAc-R (where SA represents sialyl (sialic acid to GalNAc) alpha 6-sialyltransferase II activity was 2.0 nmol/mg/h in the WAS EBV-B cell and less than .01 nmol/mg/h in EBV-B cell lines derived from normal subjects. Eleven other glycosyltransferase activities were measured and found to be similar in EBV-B cell lines from WAS and normal individuals. Polylactosamine sequences were much reduced in the O-linked oligosaccharides of CD43 from WAS EBV-B cells consistent with decreased core 2 GlcNAc-T activity and expression of core 1 oligosaccharides in the cells. In conclusion, B cells, T cells, and platelets in WAS patients show abnormal expression of two developmentally regulated glycosyltransferases, consistent with the idea that the WAS immunodeficiency is due to a failure of normal lymphocyte maturation.

Glycoprotein glycosyltransferase activities in serum in alcohol-abusing patients and healthy controls.

The activities of glycoprotein sialyl-, galactosyl- and N-acetylglucosaminyl-transferase in serum were examined in 14 alcoholic patients with a median BAC of 65 mmol/l and in 14 healthy age- and sex-matched controls. The median alcohol intake of the patients was 250 g/day for 1-3 weeks. All had elevated levels of carbohydrate-deficient transferrin in serum and normal or near normal liver function tests. The activities of galactosyl- and N-acetylglucosaminyl-transferase at subsaturated substrate concentration were significantly reduced in the patients. Vmax for all three enzymes was 35-55% lower in the patients than in the controls. Ethanol up to 500 mmol/l had no effect on these enzymes in serum. Acetaldehyde up to 200 mumol/l produced a significant dose-dependent inhibition of all three glycosyltransferases ranging from 19 to 44% at 200 mumol/l. Pyridoxal-5'-phosphate had a similar inhibitory effect on all enzyme activities suggesting the presence of lysine in the substrate or acceptor sites, or in the glycoprotein acceptors. The latter finding provides a possible molecular basis for acetaldehyde to modify glycosyl transfer. The results indicate that in man, alcohol abuse is connected with reduced activities of several glycosyltransferases in serum, which may reflect an important mechanism behind the carbohydrate deficiencies in secretory as well as membrane-bound glyconjugates in alcoholic patients.

Purification of H gene-encoded beta-galactoside alpha 1----2 fucosyltransferase from human serum.

The human serum enzyme, beta-galactoside alpha 1----2 fucosyltransferase, presumably blood group H gene-encoded, was purified to homogeneity from serum of AB and mixed secretor phenotype individuals. The purification procedure involved chromatography on phenyl-Sepharose, S-Sepharose, GDP-hexanolamine-Sepharose, and high pressure liquid chromatography gel filtration. The enzyme was purified 10 x 10(6)-fold, with a final specific activity of 23.6 units/mg for the phenyl-beta-O-galactoside acceptor. The apparent Mr of the H gene-encoded beta-galactoside alpha 1----2 fucosyltransferase was determined as 200,000 and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in nonreducing and reducing conditions, respectively. The Mr of native enzyme was found by gel filtration chromatography to be 148,000. The subunit structure as well as the sensitivity of the enzymatic activity to beta-mercaptoethanol suggest that the native enzyme exists in polymeric form of covalently bound subunits. Lectin binding properties of the purified molecule indicate that the enzyme is glycosylated. Another human serum beta-galactoside alpha 1----2 fucosyltransferase, presumably Se gene-encoded, was separated from the H enzyme by adsorption on S-Sepharose cation exchange matrix. A comparison of the kinetic parameters of the initial rate data of both alpha 1----2 fucosyltransferases revealed differences between Km values for various oligosaccharide acceptors. Higher Km values for the phenyl-beta-O-galactoside acceptor and a lower Km for the lacto-N-tetraose-beta-O-PA8 type 1 acceptor for the enzyme that adsorbed to S-Sepharose compared with nonadsorbed enzyme were observed. The two enzymes also were differentiated by binding properties to S-Sepharose and electrophoretic mobilities on native gel electrophoresis. We, therefore, postulate that the enzyme which does not adsorb to S-Sepharose and adsorbed enzyme are structurally different molecules and they represent the H and Se gene-encoded beta-galactoside alpha 1----2 fucosyltransferases, respectively.

An enzyme-linked immunosorbent assay for the measurement of Lewis blood-group alpha-(1----4)-fucosyltransferase activity.

An enzyme-linked immunosorbent assay (ELISA) has been developed for the Lewis blood-group associated alpha-(1----4)-fucosyltransferase activity. Microtiter plates coated with the bovine serum albumin conjugate of a synthetic beta-D-Galp-(1----3)-beta-D-GlcpNAc disaccharide are incubated with a fucosyltransferase preparation in the presence of guanosine 5'-diphosphofucose. The resulting immobilized Lewis-a active trisaccharide beta-D-Galp-(1----3)-[alpha-L-Fucp-(1----4)]-beta-D-GlcpNAc is then detected and quantitated using a monoclonal anti-Lewis-a antibody. Product formation detected in this manner is linear with time, proportional to enzyme activity, and reproducibly quantitated in the 50-400 fmol range.

Enzymes in cancer.

Serum enzyme measurements are not useful in screening for cancer or in primary diagnosis. Just as is the case for other tumor markers, they have an important role in confirming diagnosis and establishing the stage of disease. They also are useful in predicting prognosis and then in following the course of the disease.

Serum alpha(1----3)-L-fucosyltransferase, carcinoembryonic antigen, and sialyl Lewis X-i antigen levels in lung cancer.

Serum alpha(1----3)-L-fucosyltransferase activity was measured in 58 patients with lung cancer, 27 benign diseases, and in 100 healthy controls. The levels of enzyme activity were significantly higher in the sera of patients with cancer when compared to those in benign diseases and healthy controls. The elevation of the enzyme activity correlated with the clinical stages and to the size of the primary tumors. Follow-up studies with various stages showed that the enzyme activity was useful in tracking the clinical course of disease after surgery. To evaluate the usefulness of this enzyme as a diagnostic marker, carcinoembryonic antigen (CEA) and sialyl Lewis X-i antigen levels were also measured. The results indicate that alpha(1----3)-L-fucosyltransferase could be a more specific tumor marker than such tumor-associated antigens in lung cancer.

Altered expression of leucocyte sialoglycoprotein in Wiskott-Aldrich syndrome is associated with a specific defect in O-glycosylation.

The Wiskott-Aldrich syndrome (WAS) is an X-linked immune deficiency disorder characterized clinically by both lymphocyte and platelet dysfunction. Studies of WAS T lymphocytes have revealed deficient or defective cell surface expression of the highly O-glycosylated leucocyte sialoglycoprotein CD43. To further elucidate the basis for, and functional relevance of, CD43 modifications on WAS lymphocytes, we have studied lymphocytes from two WAS patients with regard to membrane glycoprotein profile and mitogen-induced proliferative responses. CD43 was found to be either absent or altered in size on peripheral blood lymphocytes and lectin-stimulated T cells from both patients. Compared with control cells, the WAS lymphocytes displayed reduced, but measurable proliferative responses to lectins and neuraminidase/galactose oxidase, and virtually no response to periodate, a mitogenic agent which targets sialic acid residues on membrane glycoproteins such as CD43. Analysis of activities of three glycosyltransferases involved in O-glycosylation revealed marked reduction in the level of activity of UDP-N-acetylglucosamine: Gal beta 1-3GalNAc-R beta-1,6-N-acetylglucosamine (beta-1,6-GlcNAc) transferase in one WAS patient and no detectable activity of this enzyme in a second. beta-1,6-GlcNAc transferase activity has recently been shown to increase during T cell activation coincident with changes in the O-linked glycans on CD43. A selective reduction of this glycosyltransferase in WAS lymphocytes suggests that O-linked oligosaccharides may be important to the structure of membrane glycoproteins involved in lymphocyte activation.

Identification of an anti-A and anti-B blood group glycosyltransferase antibody after incompatible bone marrow transplant.

The occurrence of a potent antibody against plasmatic A and B glycosyltransferase activities has been characterized in a patient (blood group A1) transplanted with a bone marrow from a blood group O donor. A and B glycosyltransferases were purified to near homogeneity from plasma of A1 and B blood-group individuals. The half-maximal inhibition of both enzymes was obtained at 1 to 2 micrograms/mL of the post-transplant IgG fraction, prepared by protein A-sepharose chromatography. A and B glycosyltransferases were also recognized by the post-transplant IgG fraction after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by electrophoretic transfer to nitrocellulose membranes.

Human platelets release alpha-6-L-fucosyltransferase upon activation.

Previously we have shown that human platelets release alpha-6-L-fucosyltransferase (EC 2.4.1.68) during coagulation of blood [(1987) Glycoconjugate J. 4, 43-49]. Here we report that agonists which induce platelet aggregation bring about release of the enzyme. In quantitative terms the release of alpha-6-L-fucosyltransferase by washed, aggregated platelets was very similar to that occurring during blood coagulation.

Tumor-related elevation of serum (alpha 1----3)-L-fucosyltransferase activity in gastric cancer.

(alpha 1----3)-L-Fucosyltransferase activity was measured in serum samples from 90 gastric cancer patients, 10 patients with benign diseases and 100 healthy controls. The enzyme activity was significantly elevated in the serum samples of patients with cancer compared to those from patients with benign diseases (P less than 0.01) and healthy controls (P less than 0.001). The elevation of the enzyme activity was found to correlate strongly with the clinical stage of disease. The sensitivity of (alpha 1----3)-L-fucosyltransferase was also demonstrated to be high in comparison with the tumor-associated antigens, such as carcinoembryonic antigen and sialylated Lewis X-i. Follow-up studies of (alpha 1----3)-L-fucosyltransferase in 11 cancer patients with disease at different stages showed that the enzyme activity could be useful for monitoring the post-surgical course of the disease. These results suggest that (alpha 1----3)-L-fucosyltransferase activity has a clinically important potential as a tumor marker in gastric cancer.

Cancer-associated elevation of alpha(1----3)-L-fucosyltransferase activity in human serum.

GDP-fucose:N-acetylglucosaminide alpha(1----3)-L-fucosyltransferase activity was measured in sera of patients with various cancers using a synthetic substrate, N-acetyl-2'-O-methyllactosamine, as an acceptor. One hundred twenty-four of the 169 patients showed significantly high levels of the enzyme activity when compared to healthy controls, irrespective of the location of their tumor. However, enzyme levels were in the normal range in patients with non-neoplastic diseases, such as infectious disease, liver disease, and other inflammatory problems as well as in leukemic patients. The chromatofocusing profile of the enzyme using PBE-94 gel over a pH gradient from pH 6.0 to 4.0 demonstrated that the level of enzyme eluted at pH 5.4 was markedly elevated in the sera of stomach and ovarian cancer patients. A correlation was established between alpha(1----3)-L-fucosyltransferase activity and the presence of malignancy which may be used to evaluate the utility of the enzyme as a tumor marker.

Significance of altered alpha-2-L-fucosyltransferase levels in serum of leukemic patients.

Serum alpha-2-L-fucosyltransferase levels were measured in 20 patients with leukemia. Lower than normal levels were found in patients with acute leukemia and higher than normal activity was demonstrated in patients with chronic granulocytic leukemia. alpha-2-L-Fucosyltransferase activity is expressed in platelets, and in both groups of leukemic patients this enzyme activity in serum was found to correlate directly with the platelet count. Assays on platelets from normal donors and from leukemic patients showed that the changes in level of the enzyme in serum result from changes in platelet numbers and not from alterations in the cellular expression of the transferase in leukemia. alpha-2-L-Fucosyltransferase levels in serum are therefore not per se diagnostic markers of leukemia.

Electrofocusing pattern of fucosyltransferase activity in human leukemic cells.

Fucosyltransferase (FT) activity of normal lymphocytes, normal granulocytes, and various types of human leukemic cells and electrofocusing pattern of FT activity in human leukemic cells and normal lymphocytes were examined using asialofetuin as an acceptor. Levels of FT activity in normal lymphocytes were higher than those of normal granulocytes in which FT activity was almost undetectable. The FT activity was higher in blast cells of acute myeloblastic leukemia and chronic myelogenous leukemia in blast crisis than in blast cells of acute lymphoblastic leukemia and the chronic phase of chronic myelogenous leukemia. The level of FT activity was lower in cells of chronic lymphocytic leukemia than that of normal lymphocytes, but it was higher than that of normal granulocytes. Three main isoelectric forms of FT in leukemic blast cells were identified by isoelectrofocusing, and they each had a characteristic focusing point: around pH 4.5 (peak 1); pH 4.9 (peak 2); and pH 5.2 (peak 3). In blast cells of myeloid leukemia, the activity of peak 3 was markedly higher than those of peaks 1 and 2. In blast cells of lymphoid leukemia, the activity of peak 3 was also the highest, but the activity of peak 2 was higher than that in myeloid blast cells. In normal lymphocytes, the major isoelectric form of FT was focused at around pH 4.9 and peak 3 was undetectable. These results indicated apparent differences not only in FT activity but also in isoelectric forms of FT between myeloid leukemic cells and lymphoid leukemic cells.