Identification of calnexin as a binding protein for Amadori-modified glycated albumin.

Albumin modified by Amadori glucose adducts (glycated albumin) selectively binds to glomerular mesangial cells and triggers signal transduction processes that modulate cellular function. To identify glycated albumin binding proteins, we applied membrane extracts prepared from murine mesangial cells to a column of lysine-Sepharose followed by application to an affinity column of fructosyllysine-Sepharose. This procedure yielded an approximately 90 kDa polypeptide that immunoreacted with Amadori-modified but not carbohydrate-free albumin. MALDI mass fingerprinting matched 9 out of 25 peptides with calnexin, and amino acid analysis showed homology with this transmembrane calcium-binding protein of the calreticulin family. These results indicate that one of the mesangial cell receptors for glycated albumin is a calnexin-like protein.

[Evolution of breastfeeding pattern]. / Evolução do padrão de aleitamento materno.

OBJECTIVE: To follow the evolution of the breastfeeding practice among women in a childbirth clinic and evaluate the actions for its promotion. METHODS: Two cohorts of children born in an school hospital of Porto Alegre, Brazil, in different periods of time were compared regarding the prevalence of breastfeeding during the first six months of life, and the percentage of early cessation of breastfeeding. Both were prospective studies, including 202 children in the cohort of 1987, and 187 children in the cohort of 1994. All participants were healthy children, with birth weight of 2500g or more, were being breastfed and their parents were living in the same house. The 1987 study children were followed up through mail, and the 1994 study ones by home visits. RESULTS: Survival analysis showed similar frequencies of breastfeeding in the two cohorts. The prevalence of exclusive breastfeeding, although for a short period in both groups, was higher in the population studied in 1994, especially among children whose mothers were better educated. There was no rate difference of early cessation of breastfeeding between the two cohorts (36% for the 1987 cohort and 39% for the 1994 cohort). CONCLUSIONS: The results revealed a certain degree of apathy in regard of the promotion of breastfeeding during the studied period, giving support to actions to promote breastfeeding, especially among poor families.

Estradiol enhances learning and memory in a spatial memory task and effects levels of monoaminergic neurotransmitters.

The effects of chronic estrogen treatment on radial arm maze performance and on levels of central monoaminergic and amino acid neurotransmitters were examined in ovariectomized (Ovx) rats. In an eight arms baited paradigm, choice accuracy was enhanced following 12 days but not 3 days of treatment. In addition, performance during acquisition of the eight arms baited maze task was better in estrogen-treated Ovx rats than in Ovx rats. Performance of treated rats was also enhanced in win-shift trials conducted 12 days postestrogen treatment. Working, reference, and working-reference memory was examined when four of the eight arms were baited, and only working memory was improved by estrogen and only after long-term treatment. Activity of Ovx rats on an open field, crossings and rearings, was increased at 5 but not at 35 days following estrogen treatment. In medial prefrontal cortex, levels of NE, DA, and 5-HT were decreased but glutamate and GABA levels were not affected following chronic estrogen treatment. Basal forebrain nuclei also showed changes in monoamines following estrogen. Hippocampal subfields showed no effects of estrogen treatment on monoaminergic or amino acid transmitters. Levels of GABA were increased in the vertical diagonal bands following chronic estrogen. Results show that estrogen enhances learning/memory on a task utilizing spatial memory. Effects in Ovx rats appear to require the chronic (several days) presence of estrogen. Changes in activity of both monoaminergic and amino acid transmitters in the frontal cortex and basal forebrain may contribute to enhancing effects of estrogen on learning/memory.

Glycated albumin stimulates fibronectin and collagen IV production by glomerular endothelial cells under normoglycemic conditions.

Albumin modified by Amadori glucose adducts, formed in increased amounts in diabetes, stimulates the synthesis of matrix by renal glomerular mesangial cells and has been causally linked to the pathogenesis of diabetic nephropathy. However, the effect of glycated albumin on the biology of glomerular endothelial cells, which elaborate a basement membrane that undergoes thickening in diabetes, has not been investigated. We used well-characterized rat glomerular endothelial cells to examine the influence of glycated albumin on the synthesis of extracellular matrix proteins by these cells in culture. Concentrations of glycated albumin that are present in clinical specimens stimulate fibronectin and collagen IV production by glomerular endothelial cells, and this effect is operative under normoglycemic conditions. These results support the hypothesis that increased glycated albumin contributes to glomerular basement membrane thickening in diabetes.

Albumin modified by Amadori glucose adducts activates mesangial cell type IV collagen gene transcription.

The direct relationship between elevated glucose concentrations and accelerated protein glycation has implicated increased glycation as a potential mechanistic link between hyperglycemia and the pathogenesis of diabetic nephropathy. Albumin modified by Amadori glucose adducts has been shown to stimulate collagen secretion by mesangial cells in vitro, and to contribute to the overproduction of glomerular mesangial matrix in vivo. To delineate mechanisms responsible for these effects, we examined the influence of glycated albumin on transcriptional activation of the alpha 1 (IV) collagen gene in renal glomerular mesangial cells. These experiments used a stably transfected reporter mesangial cell line that exhibits responses to media manipulations that are directionally parallel with those of non-transformed mesangial cells, and that expresses luciferase driven by 5'-flanking and first intron regions of the alpha 1 (IV) collagen gene. In these cells, purified glycated albumin stimulated collagen IV gene transcription, whereas glucose-free albumin did not. Further, glycated albumin induced a significant increase in mesangial cell collagen IV mRNA, assessed by Northern blot analysis and quantified by calculation of the ratio of collagen IV mRNA to 18S ribosomal RNA after densitometric scanning. The stimulation of collagen gene transcription and mRNA expression were both prevented by monoclonal antibodies known to specifically recognize Amadori-modified albumin. The findings indicate that glycated albumin promotes mesangial cell transcriptional activation and mRNA expression of the alpha 1 (IV) collagen gene and further implicate increased glycated albumin in diabetes in the pathogenesis of diabetic nephropathy.

Prevention of diabetic nephropathy in db/db mice with glycated albumin antagonists. A novel treatment strategy.

Accelerated protein glycation in diabetes has been mechanistically linked to the pathogenesis of diabetic nephropathy. Because glycated albumin induces abnormalities in cultured mesangial cells that resemble those characterizing the glomerular mesangium in diabetes, and monoclonal antibodies (A717) specific for Amadori-modified glycated albumin prevent these abnormalities, we postulated that in vivo administration of A717 could retard the progression of diabetic nephropathy. To test this hypothesis, diabetic db/db mice and their nondiabetic db/m littermates were treated with eight consecutive weekly injections of 150 micrograms of A717 (Fab fragments) to reduce the elevated plasma glycated albumin concentration, or with irrelevant murine IgG (MIg). Relative to nondiabetics, diabetic mice (MIg treated) manifested proteinuria (3.35 +/- 0.15 vs 0.87 +/- 0.1 mg albumin/mg creatinine), 3.8-fold increase in mesangial matrix fraction, and renal cortical overexpression of mRNAs encoding alpha 1(IV) collagen (2.6-fold increase) and fibronectin (3.8-fold increase). Treatment of db/db mice with A717 significantly reduced the proteinuria (1.52 +/- 0.3 mg/mg creatinine), inhibited mesangial matrix expansion, and attenuated overexpression of matrix mRNAs. The nephropathic protective effects of A717 were independent of any change in blood glucose concentrations. Antibodies unreactive with glycated albumin did not duplicate the beneficial effects of A717. Thus, abrogating the biologic effects of increased glycated albumin with A717 has a salutary influence on the pathogenesis of diabetic nephropathy and has novel therapeutic potential in its management.

Evidence for a ligand receptor system mediating the biologic effects of glycated albumin in glomerular mesangial cells.

Mesangial cells cultured with albumin modified by Amadori glucose adducts exhibit decreased proliferation in association with increased elaboration of Type IV collagen. To test the hypothesis that this modulation of mesangial cell biology is linked to ligand binding, we examined renal glomerular mesangial cells for the expression of receptors that interact with Amadori-modified albumin. Murine mesangial cells bound glycated albumin in a dose-dependent and saturable manner, displaying high and low affinity binding sites. Binding of glycated but not nonglycated albumin was inhibited by monoclonal antibodies that specifically react with albumin containing fructosyllysine groups. LDL containing Amadori glucose adducts did not compete with glycated albumin for binding. These results are consistent with ligand selectively of the binding sites for an Amadori-modified sequence within an albumin domain and suggest that a ligand receptor system mediates the effects of glycated albumin on mesangial cell proliferation and matrix production.

Glycated albumin modified by Amadori adducts modulates aortic endothelial cell biology.

Increased protein glycation has been mechanistically linked to accelerated vascular pathobiology in diabetes. To test the influence of protein modified by Amadori glucose adducts on vascular cell biology, we examined the effect of glycated albumin on replicative capacity and basement membrane collagen production by aortic endothelial cells in culture. Relative to carbohydrate-free albumin, which supported cell proliferation and Type IV collagen synthesis, glycated albumin significantly inhibited 3H-thymidine incorporation and Type IV collagen production. The glycated albumin-induced effects were prevented by monoclonal antibodies (A717) that specifically react with Amadori-modified albumin, but not by IgG that was unreactive with glycated albumin. A717 had no effect on thymidine incorporation or collagen synthesis by cells cultured in the presence of nonglycated albumin. The findings indicate that the interaction of glycated albumin with endothelial cells, which have been shown to display dose-responsive, saturable receptors, limits cell replication and triggers maladaptive biosynthetic programs, which may contribute to degenerative macrovascular disease in diabetes.

Amelioration of diabetic nephropathy by treatment with monoclonal antibodies against glycated albumin.

The pathogenesis of diabetic nephropathy is incompletely understood, but increased nonenzymatic glycation of proteins is considered an important contributory factor. Glycated albumin, which is increased in diabetic sera and is preferentially transported into the renal glomerulus, induces an increase in Type IV collagen production and a decrease in proliferative capacity by mesangial cells in culture. These effects resemble the abnormalities that characterize the glomerular mesangium in diabetes and are prevented by monoclonal antibodies that specifically react with Amadori adducts in glycated albumin. To explore the possibility that the in vitro effects of glycated albumin on mesangial cell biology pertain to the in vivo situation, we examined the effect of treatment with the A717 monoclonal antibodies on glomerular functional and structural changes in a rodent model of genetic diabetes, the db/db mouse. Weekly parenteral antibody administration reduced the elevated albumin excretion and attenuated the mesangial expansion that were observed in the untreated db/db mice that served as controls. Monoclonal antibody treatment also was shown to lower plasma concentrations of glycated albumin in diabetic mice. Thus, reducing glycated albumin concentrations and/or blocking its biologically active epitopes has a salutary influence on the pathogenesis of diabetic nephropathy. The findings indicate that glycated albumin participates in the development of the glomerular lesion in the db/db mouse, and suggest a new approach to the therapy of this complication of diabetes.

Receptors specific for Amadori-modified glycated albumin on murine endothelial cells.

Albumin modified by Amadori glucose adducts has been shown to modulate endothelial and glomerular mesangial cell biology. Recognizing that circulating proteins may trigger cellular events through ligand binding, we examined murine aortic endothelial cells for the expression of a receptor system that recognizes fructosyllysine epitopes in glycated albumin. Endothelial cells contain membrane-associated polypeptides that bind Amadori-modified glycated albumin and exhibit traditional receptor characteristics of dose-responsivity, saturability and ligand specificity. Nonglycated albumin does not compete for binding to the receptor, and the interaction of glycated albumin with its receptor is blocked by a monoclonal antibody that selectively reacts with the Amadori adduct in glycated albumin. The findings suggest that a ligand-receptor system specific to Amadori glucose adducts mediates the biologic effects of glycated albumin.

Identification of aortic endothelial cell binding proteins for Amadori adducts in glycated albumin.

The ability of glycated serum proteins, which exist in vivo predominantly as Amadori adducts, to influence cell biology suggests the existence of cell binding proteins that recognize glucose adducts in nonenzymatically glycated proteins. To explore this possibility, we applied detergent extracts of aortic endothelial cells to HPLC and to an affinity column of glycated human albumin immobilized onto Sepharose, which was eluted with alkaline pH. HPLC fractionation revealed a peak reactive in ELISA with glycated but not nonglycated albumin. Two polypeptides of approximately 110 kDa and 205 kDa were identified when the affinity bound proteins were electrophoresed, transferred to immunoblotting membranes, and reacted with an enzyme conjugate of glycated albumin containing Amadori adducts. These polypeptides did not react with enzyme conjugate of carbohydrate-free albumin, confirming their binding specificity for glucose modified albumin and absence of co-identity with previously described albumin-binding proteins. The molecular weights of the glycated albumin binding polypeptides are distinct from those described for bovine albumin modified by advanced glycation end products or by formaldehyde, indicating that Amadori adducts in glycated albumin are recognized by unique endothelial cell binding sites.

Purified haemoglobin preparations in the evaluation of HbA1c determination by ion exchange chromatography.

The use of ion exchange resins for the estimation of HbA1c in clinical samples rests on the assumption that HbA1c is effectively and efficiently separated from other N-terminally modified haemoglobins and from HbA0. To test this assumption, we applied highly purified preparations of HbA(1a+1b, HbA1c and HbA0 to ion exchange minicolumns, using conditions of application simulating actual blood samples and the first and second elution buffers provided by the manufacturer. The authenticity and purity of the applied haemoglobin preparations were documented by high performance liquid chromatography, gel electrophoresis and carbohydrate content. About 40% of the applied HbA(1a+1b) eluted in the first fraction; 45% eluted in the second fraction, and 10% to 15% required 1 mol/L NaCl to elute from the column. Of the applied HbA1c, 65-80% eluted where expected in the second fraction, about 20% required 1 mol/L NaCl to elute from the column, and the remainder eluted with HbA(1a+1b). Some 3-6% of pure HbA0 applied to minicolumns emerged in the second fraction, with the remainder eluting as expected after making the buffer 1 mol/L in NaCl. The results indicate that the fraction eluting from ion exchange minicolumn chromatography that is designated 'HbA1c' contains HbA(1a+1b), and that a substantial portion of the HbA1c in an applied sample does not elute in this fraction.

Production and characterization of monoclonal antibodies against non-A1c glycated hemoglobin.

Hybridomas secreting monoclonal antibodies specific for hemoglobin nonenzymatically glycated in the non-A1c position were produced by fusion of SP 2/0 myeloma cells with spleen cells from BALB/c mice immunized with nonenzymatically glycated hemoglobin prepared from human erythrocytes. Wells containing hydridomas secreting antibodies against glycohemoglobin were identified by binding, in an enzyme-linked immunosorbent assay, to purified glycated hemoglobin. The colony designated E85, which secreted antibodies discriminating between glycated versus unglycated hemoglobin, was cloned at least four times by limiting dilution and used for further study, performed with purified monoclonal antibody. Specificity of E85 was demonstrated by immunoblotting and by ELISA, wherein the monoclonal antibody reacted with glycated hemoglobin but not with hemoglobin A1c or with unglycated hemoglobin. Immunoblotting of human plasma with E85 on nitrocellulose yielded no reactive proteins, indicating site specificity for glycated epitopes residing in hemoglobin but not in other nonenzymatically glycated proteins present in plasma. E85 differs from other antibodies raised against glycated hemoglobin and other glycated proteins, which recognize hemoglobin glycated at the N terminal valine of the beta chain (HbA1c) or which recognize glycated residues only after reductive conversion to glucitollysine and which do not discriminate between different glycated proteins. Thus, this report describes the establishment of the first hybridoma secreting monoclonal antibody raised against a physiologic (unreduced) form of non-A1c glycohemoglobin, and for the glycated epitope when it resides in glycohemoglobin but not in other proteins or in hemoglobin A1c.

Purification of glycated hemoglobin free of hemoglobin A1c and its use to produce monoclonal antibodies specific for deoxyfructosyllysine [correction of deoxyfructosyllsine] residues in glycohemoglobin.

Hemoglobin nonenzymatically glycated at E-amino groups of lysine residues was purified from human erythrocyte lysates and used for immunization of BALB/c mice. Hybridomas secreting monoclonal antibodies for glycated hemoglobin were produced by fusion of mouse spleen cells with SP 2/0 myeloma cells. Immunoblotting with purified monoclonal antibody demonstrated specificity for glycated hemoglobin, with no reaction with HbAO. Glycated hemoglobin was effectively separated from other hemoglobins upon application of erythrocyte lysates to an affinity column of monoclonal antibody immobilized onto Sepharose 4B. A small fraction of purified HbA1c adsorbed to the monoclonal antibody affinity column, indicating that glycation can occur at both E-amino lysine and N-terminal valine positions in the same molecule. HbA1c did not react with the antibody after removal by immunoadsorption of molecules containing glycated lysine, confirming specificity of the antibody for deoxyfructosyl-lysine residues. The findings indicate that these monoclonal antibodies are site specific for glycated lysine amino groups in hemoglobin, and can provide rapid and efficient separation and identification of glycated hemoglobin in human erythrocyte lysates.

Evaluation of the effect of aldose reductase inhibition on increased basement membrane collagen fluorescence in diabetic rats.

1. It has been proposed that increased fructose contributes to the formation of fluorescent pigments in diabetic tissues. 2. Since the aldose reductase inhibitor sorbinil lowers glomerular fructose concentrations, we examined the effect of sorbinil on the formation of advanced glycation end products in glomerular basement membrane of streptozotocin diabetic rats. 3. Treatment with sorbinil for 30 days after induction of diabetes did not influence the increased fluorescence observed in collagen from glomerular basement membrane of untreated diabetic rats. 4. The results suggest that nonenzymatic glycation by fructose is not a major contributor to the formation of fluorescent advanced glycation end products in basement membrane in experimental diabetes.

Effect of alpha-glucosidase inhibition on the nonenzymatic glycation of glomerular basement membrane.

1. The effect of the alpha-glucosidase inhibitor Acarbose on integrated glycemic control and on nonenzymatic glycation of glomerular basement membrane was examined in streptozotocin diabetic rats. 2. Treatment with Acarbose for 8 weeks after induction of diabetes significantly reduced the level of HbA1c and of glomerular basement membrane glycation. 3. Acarbose exerts a significant antihyperglycemic effect and has a salutary influence on the nephropathic process in experimental diabetes.

Undersulfation of glomerular basement membrane heparan sulfate in experimental diabetes and lack of correction with aldose reductase inhibition.

In this study we examined the effect of experimental diabetes and of treatment with an aldose reductase inhibitor on the level of sulfation of glomerular basement membrane (GBM) heparan sulfate, the principal glycosaminoglycan in this extracellular matrix. Glycosaminoglycans were isolated from GBM purified from control, streptozocin-induced diabetic, and sorbinil-treated diabetic rats and analyzed for sulfate and uronate content. Glomerular yields from diabetic kidneys were greater than those from control animals, but the amount of sulfate per glomerulus in diabetic samples, both untreated and sorbinil treated, did not differ significantly from that in control samples. However, the sulfate-to-uronate ratio in heparan sulfate isolated from diabetic GBM (0.34 +/- 0.08) was significantly less than in control samples (0.69 +/- 0.11), and treatment of diabetic rats with an aldose reductase inhibitor did not correct this reduced ratio (0.36 +/- 0.06). The results indicate that there is an undersulfation of heparan sulfate of GBM in experimental diabetes, an abnormality that may contribute to loss of anionic sites and decreased charge selectivity of the glomerular filtration barrier. The findings further suggest that this abnormality results from disturbances in glycosaminoglycan synthesis and/or metabolism in diabetes that are independent of polyol-pathway activation in the renal glomerulus.

Analysis of glycosaminoglycans in bovine retinal microvessel basement membrane.

Glycosaminoglycans (GAG) were isolated from bovine retinal microvessel basement membrane (RMV-BM) and quantitatively analyzed using a recently described competitive binding assay that is specific for and sensitive to nanogram amounts of heparan and chondroitin sulfates. Treatment of osmotically lysed retinal microvessels with the ionic detergent deoxycholate (DOC), required for liberation of the extracellular matrix for plasma membrane lipoproteins and purification of the insoluble matrix, solubilized less than 5% of the GAG in the water-insoluble material. Total GAG content in the DOC-insoluble basement membranes was approx. 0.52 micrograms/mg dry weight; about 70% of the measurable GAG was resistant to both chondroitinase ABC and chondroitinase AC digestion and was sensitive to nitrous acid treatment, indicating its heparan sulfate nature. Cellulose acetate electrophoresis revealed two bands, one of which had an electrophoretic mobility similar to heparan sulfate standard and was sensitive to nitrous acid; the other migrated in the same position as chondroitin sulfate standard and was sensitive to chondroitinase ABC and chondroitinase AC digestion. These results provide evidence that RMV-BM contains chondroitin sulfate(s) as well as heparan sulfate, and offer the first quantitative analysis of GAG in this extracellular matrix.

Disturbances in glomerular basement membrane glycosaminoglycans in experimental diabetes.

Glycosaminoglycans (GAGs) were purified from basement membranes isolated from glomeruli of control and streptozocin-induced diabetic rats and were quantitatively analyzed with a recently described competitive binding assay that is specific for and sensitive to microgram amounts of chondroitin and heparan sulfate. Total GAG content in glomeruli from diabetic rats and in the basement membranes prepared from these samples (17.22 +/- 1.45 and 6.56 +/- 0.49 micrograms/10(5) glomeruli, respectively was significantly less than that found in comparable control preparations (43.71 +/- 3.35 and 16.05 +/- 1.41 micrograms/10(5) glomeruli, respectively). The portion of total GAG in the water-soluble fraction recovered after osmotic lysis of isolated glomeruli was also markedly decreased in diabetic samples (26.11 +/- 4.55 vs. 3.30 +/- 0.32 micrograms/10(5) glomeruli, control vs. diabetic). Treatment of lysed glomeruli with the ionic detergent deoxycholate, required for liberation of the extracellular matrix from plasma membrane lipoproteins and purification of the insoluble glomerular basement membrane (GBM), resulted in solubilization of approximately 10% of the water-insoluble GAG in control samples but greater than 50% in diabetic membranes. Heparan sulfate comprised greater than 90% of the GAGs in both control and diabetic GBM, defined as the water- and detergent-insoluble matrix. The findings clearly demonstrate that the GAG content of GBM is diminished in experimental diabetes and provide evidence that the reduction in GBM anionic sites associated with diabetes derives from a decrease in the constituent GAGs of this extracellular matrix. The results further suggest that the interaction between GBM and populations of GAG associated with the surface of plasma membranes of adjacent cells is disturbed in diabetes.