Spermine and Spermidine Priming against Botrytis cinerea Modulates ROS Dynamics and Metabolism in Arabidopsis.

Polyamines (PAs) are ubiquitous small aliphatic polycations important for growth, development, and environmental stress responses in plants. Here, we demonstrate that exogenous application of spermine (Spm) and spermidine (Spd) induced cell death at high concentrations, but primed resistance against the necrotrophic fungus Botrytis cinerea in Arabidopsis. At low concentrations, Spm was more effective than Spd. Treatments with higher exogenous Spd and Spm concentrations resulted in a biphasic endogenous PA accumulation. Exogenous Spm induced the accumulation of H2O2 after treatment but also after infection with B. cinerea. Both Spm and Spd induced the activities of catalase, ascorbate peroxidase, and guaiacol peroxidase after treatment but also after infection with B. cinerea. The soluble sugars glucose, fructose, and sucrose accumulated after treatment with high concentrations of PAs, whereas only Spm induced sugar accumulation after infection. Total and active nitrate reductase (NR) activities were inhibited by Spm treatment, whereas Spd inhibited active NR at low concentrations but promoted active NR at high concentrations. Finally, γaminobutyric acid accumulated after treatment and infection in plants treated with high concentrations of Spm. Phenylalanine and asparagine also accumulated after infection in plants treated with a high concentration of Spm. Our data illustrate that Spm and Spd are effective in priming resistance against B. cinerea, opening the door for the development of sustainable alternatives for chemical pesticides.

Immunological evidence for in vivo production of novel advanced glycation end-products from 1,5-anhydro-D-fructose, a glycogen metabolite.

The anhydrofructose pathway is an alternate pathway for glycogen degradation by α-1,4-glucan lyase. The sugar 1,5-anhydro-D-fructose (1,5-AF) acts as the central intermediate of this pathway, but its physiological role of in mammals is unclear. Glycation reactions forming advanced glycation end-products (AGEs) are important in the development of complications of diabetes mellitus. We hypothesized that 1,5-AF may contribute to cellular damage by forming 1,5-AF-derived AGEs (AF-AGEs) with intracellular proteins. To clarify the role of 1,5-AF in protein modification, we created a novel antibody targeting AF-AGEs. Serum albumin modified by AF-AGEs was prepared by incubating rabbit serum albumin (RSA) or bovine serum albumin (BSA) with 1,5-AF. After immunizing rabbits with AF-AGEs-RSA, affinity chromatography of anti-AF-AGE antiserum was performed on a Sepharose 4B column coupled with AF-AGEs-BSA or N-(carboxymethyl)/N-(carboxyethyl)lysine-BSA. A novel immunopurified anti-AF-AGE antibody was obtained and was characterized using a competitive enzyme-linked immunosorbent assay. Then an AF-AGEs assay was established using this immunopurified antibody. This assay was able to detect AF-AGEs in human and animal serum samples. Finally, intracellular accumulation of AF-AGEs was shown to be associated with damage to cultured hepatocytes (HepG2 cells). This is the first report about in vivo detection of AF-AGEs with a novel structural epitope.

A study on hepatopathic, dyslipidemic and immunogenic properties of fructosylated-HSA-AGE and binding of autoantibodies in sera of obese and overweight patients with fructosylated-HSA-AGE.

Over consumption of fructose may lead to obesity and dyslipidemia and cause fructosylation-induced alterations in the structure and function of proteins. The aim of this study was to investigate the role of fructosylated-HSA-AGE in the pathogenesis of fatty liver (NAFLD and NASH) by biochemical, immunological and histological studies. Immunogenicity of fructosylated-HSA-AGE was probed by inducing antibodies in rabbits. Fructosylated-HSA-AGE was found to be highly immunogenic. Furthermore, fructosylated-HSA-AGE caused mild fibrosis with steatosis and portal inflammation of hepatocytes in experimental animals. Liver function test and dyslipidemic parameters in immunized animals were also found to be raised. Ultrasonography, which should form part of the assessment of chronically raised transaminases, shows fatty infiltration. Interestingly, alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, total cholesterol (TC) and triglyceride (TG) profiles confirms USG images of overweight, obese patients. Thus, present study demonstrates that fructosylated-HSA-AGE is hepatotoxic, immunologically active and may cause dyslipidemia.

Fructo-oligosaccharide-Induced Transient Increases in Cecal Immunoglobulin A Concentrations in Rats Are Associated with Mucosal Inflammation in Response to Increased Gut Permeability.

Background: The mechanism underlying transient increases in immunoglobulin (Ig) A concentrations in the cecal contents of rats fed fructo-oligosaccharide (FOS) is unclear.Objective: This study was designed to test whether increased IgA concentrations represent one aspect of the inflammatory response to increased permeability induced by FOS in the cecum.Methods: Seven-week-old male Wistar rats were fed a fiber-free semipurified diet (FFP) with or without supplemental FOS (60 g/kg diet) for 9 or 58 d [experiment (expt.) 1], 7 d (expt. 2), or 7 or 56 d (expt. 3). In addition to measuring IgA concentrations in cecal content, we assessed gut permeability, inflammatory responses (expt. 1), the number of IgA plasma cells in the cecal lamina propria, polymeric Ig receptor (pIgR) expression in the cecal mucosa (expt. 2), and the condition of the cecal mucus layer (expt. 3).Results: The cecal IgA concentration in the FOS-fed rats was 15-fold higher than that of the rats fed FFP for 9 d (P < 0.05). Gut permeability estimated by urinary chromium-EDTA excretion, bacterial translocation to mesenteric lymph nodes, myeloperoxidase activity, and expression of inflammatory cytokine genes in the cecal mucosa was greater in the FOS-fed rats than in the rats fed FFP for 9 d. These effects were not observed in the rats fed FOS for 58 d (expt. 1). Accompanying the higher cecal IgA concentration, pIgR protein and the number of IgA plasma cells in the cecal mucosa were higher in the FOS-fed rats than in the rats fed FFP for 7 d (expt. 2). Destruction of the mucus layer on the epithelial surface, as evidenced by Alcian blue staining in the cecal sections, was evident in the rats fed FOS for 7 d, but the mucus layer appeared normal in the rats fed FOS for 56 d (expt. 3).Conclusions: These findings suggest that transient increases in cecal IgA concentrations induced by FOS in rats are associated with mucosal inflammation in response to increased gut permeability; these are presumably evoked by disruption of the cecal mucus barrier. The observed responses could contribute to the maturation of the gut immune system.

Levels of anti-fructose-modified HSA antibodies correlate with disease status in diabetic subjects.

OBJECTIVE: This study aimed to assess the changes induced in HSA upon fructose-modification and to use the modified protein as an antigen for studying the presence of antibodies in diabetic patients. Further, magnitude of oxidative stress was also assessed. METHODS: HSA was modified with fructose, changes induced were studied by DSC measurements and near-UV CD. The binding characteristics of antibodies in the sera of diabetes patients to native and modified-HSA was assessed by ELISA and band shift assay. The oxidative stress in these patients was studied by carbonyl content estimation, FRAP assay and TBARS determination RESULTS: DSC revealed that fructose modified-HSA was more thermostable than its native form. Changes in tertiary structure of fructose-modified HSA were seen in near-UV CD. Patient studies showed that fructose-modified HSA acts as a potent immunogen compared to its native form and the levels of antibodies against fructose-modified HSA served as a parameter for tracking the glycemic control and oxidative stress parameters (carbonyl content, FRAP value and MDA level) in diabetic patients. CONCLUSIONS: Fructose-modification of HSA causes perturbations in its structure and function, thereby, making the protein antigenic besides decreasing its antioxidant capacity. This study suggests that fructose-modified-HSA is an important contributor in diabetic pathophysiology.

Affinity of HIV-1 antibody 2G12 with monosaccharides: a theoretical study based on explicit and implicit water models.

In order to develop potential ligands to HIV-1 antibody 2G12 toward HIV-1 vaccine, binding mechanisms of the antibody 2G12 with the glycan ligand of D-mannose and D-fructose were theoretically examined. D-Fructose, whose molecular structure is slightly different from D-mannose, has experimentally shown to have stronger binding affinity to the antibody than that of D-mannose. To clarify the nature of D-fructose's higher binding affinity over D-mannose, we studied interaction between the monosaccharides and the antibody using ab initio fragment molecular orbital (FMO) method considering solvation effect as implicit model (FMO-PCM) as well as explicit water model. The calculated binding free energies of the glycans were qualitatively well consistent with the experimentally reported order of their affinities with the antibody 2G12. In addition, the FMO-PCM calculation elucidated the advantages of D-fructose over D-mannose in the solvation energy as well as the entropic contribution term obtained by MD simulations. The effects of explicit water molecules observed in the X-ray crystal structure were also scrutinized by means of FMO methods. Significant pair interaction energies among D-fructose, amino acids, and water molecules were uncovered, which indicated contributions from the water molecules to the strong binding ability of D-fructose to the antibody 2G12. These FMO calculation results of explicit water model as well as implicit water model indicated that the strong binding of D-fructose over D-mannose was due to the solvation effects on the D-fructose interaction energy.

Antigenicity and functional properties of ß-lactoglobulin conjugated with fructo-oligosaccharides in relation to conformational changes.

Bovine ß-lactoglobulin (ß-LG) was conjugated with fructo-oligosaccharides (FOS) by Maillard reaction to investigate the relationship among antigenicity, functional properties, and conformational changes of ß-LG. When comparing the antigenicity of ß-LG conjugated with FOS at different ratios, the lowest antigenicity of ß-LG was observed at a ratio of 1:4, which was about 7 times lower than that of the control ß-LG. Thus, the ratio of 1:4 was chosen to conjugate ß-LG with FOS, and the functional properties and conformational changes of ß-LG-FOS conjugates were investigated. The functional properties (solubility, emulsifying ability, and emulsion stability) of ß-LG were enhanced after conjugation with FOS. Furthermore, the molecular weight of ß-LG increased from 18.4 to 19.9 kDa after conjugation with FOS, as evaluated by sodium dodecyl sulfate-PAGE and mass spectrometry. Partial unfolding of ß-LG occurred after conjugation with FOS, as reflected by the quenching of fluorescence, the red-shift of fluorescence spectra, and the increase of ß-strands, which may contribute to the decrease in antigenicity and the improvement of functional properties.

Effects of short-chain galacto- and long-chain fructo-oligosaccharides on systemic and local immune status during pregnancy.

Nondigestible oligosaccharides can positively influence health via various mechanisms. During pregnancy, supplementation of nondigestible oligosaccharides has positive effects on hypertension and metabolism and may be used to ameliorate pregnancy-related metabolic disturbances. In the nonpregnant state, nondigestible oligosaccharides have been shown to induce a tolerogenic immune response mediated by T-regulatory cells. Since relatively little is known about the effects of nondigestible oligosaccharides on the immune system during pregnancy, pregnant mice were supplemented with a specific mixture of short-chain galacto- and long-chain fructo-oligosaccharides (scGOS/lcFOS; ratio 9:1). Systemic and local immune parameters were analyzed on day 18 of pregnancy. This study shows that, compared with virgin mice, scGOS/lcFOS supplementation appears to elicit a more tolerogenic immune reaction in pregnant mice and supplementation does not increase the Th1-dependent delayed type hypersensitivity response in pregnant mice as it does in virgin mice.

Immunological and physicochemical properties of cervical ovulatory mucus.

Selected biochemical and immunological parameters of cervical ovulatory mucus collected at ovulation have been determined. These include the levels of glucose, fructose, immunoglobulins, C3, hormones, prostaglandin E2, Th1 and Th2 cytokines and antibodies to sperm (and the local effect of hydrocortisone treatment on their levels), zona pellucida, phospolipids and laminin-1. The results of these studies are relevant to the problems associated with fertility disorders and repeated loss of pregnancy.

A nonself sugar mimic of the HIV glycan shield shows enhanced antigenicity.

Antibody 2G12 uniquely neutralizes a broad range of HIV-1 isolates by binding the high-mannose glycans on the HIV-1 surface glycoprotein, gp120. Antigens that resemble these natural epitopes of 2G12 would be highly desirable components for an HIV-1 vaccine. However, host-produced (self)-carbohydrate motifs have been unsuccessful so far at eliciting 2G12-like antibodies that cross-react with gp120. Based on the surprising observation that 2G12 binds nonproteinaceous monosaccharide D-fructose with higher affinity than D-mannose, we show here that a designed set of nonself, synthetic monosaccharides are potent antigens. When introduced to the terminus of the D1 arm of protein glycans recognized by 2G12, their antigenicity is significantly enhanced. Logical variation of these unnatural sugars pinpointed key modifications, and the molecular basis of this increased antigenicity was elucidated using high-resolution crystallographic analyses. Virus-like particle protein conjugates containing such nonself glycans are bound more tightly by 2G12. As immunogens they elicit higher titers of antibodies than those immunogenic conjugates containing the self D1 glycan motif. These antibodies generated from nonself immunogens also cross-react with this self motif, which is found in the glycan shield, when it is presented in a range of different conjugates and glycans. However, these antibodies did not bind this glycan motif when present on gp120.

Colocalization of polyol-metabolizing enzymes and immunological detection of fructated proteins in the female reproductive system of the rat.

The expression of aldose reductase (AR) and sorbitol dehydrogenase (SDH), which, in concert, catalyze the conversion of glucose to fructose via sorbitol, in the rat ovary, oviduct, and uterus, was investigated by immunohistochemical and biochemical analyses. The activities and protein levels of AR and SDH were higher in the ovary than in the oviduct and uterus. A strong immunoreactivity to the anti-AR antibody was observed in granulosa cells and epithelia of the oviduct, endometrium, and endometrial glands, and virtually the same tissues were strongly stained with the anti-SDH antibody. The application of an anti-fructated lysine antibody, which detects an adduct of fructose with the epsilon-amino group of lysine in proteins, in this study detected marked staining mainly in the egg and luminal surface of the oviductal epithelia. Collectively, these data indicate that fructose is produced by coordinately expressed AR and SDH in the egg and epithelia of the oviduct and suggest that the resulting sorbitol and fructose can be used as energy sources for spermatozoa motility during the fertilization process. The abundance of AR compared with SDH suggests that it also plays an additional role in the reproductive system, which might include a source of reducing power and protection against toxic carbonyl compounds.

Immunological detection of fructated proteins in vitro and in vivo.

An antibody has been raised against fructated lysine in proteins by immunizing fructated lysine-conjugated ovalbumin in rabbits. The affinity-purified antibody specifically recognized proteins incubated with fructose but not with other reducing sugars such as glucose, galactose or ribose, as judged by immunoblotting and ELISA techniques. Competitive binding to this antibody was observed specifically by fructated lysine but not by glucated lysine, glucose, fructose or lysine. The antibody binds specifically to fructated lysine residues in the protein but not to borohydride-reduced material or advanced glycation end products, indicating that the antibody recognizes only the reducing, carbonyl-containing forms produced in the early stage of the fructation reaction. When BSA was incubated with various concentrations of fructose, the reactivity of the antibody increased in a dose- and time-dependent manner. When soluble proteins prepared from either normal or streptozotocin-induced diabetic rat eyes were analysed by ELISA with this antibody, an increase in the reactive components was observed as a function of aging as well as under diabetic conditions. Western blotting analysis showed that lens crystallin reacted highly with this antibody. Because fructose is biosynthesized largely through the polyol pathway, which is enhanced under diabetic conditions, and lens is known to have a high activity of enzymes in this pathway, this antibody is capable of recognizing fructated proteins in vivo. Thus it is a potentially useful tool for investigating two major issues that seem to be involved in diabetic complications, namely the glycation reaction and the polyol pathway.

Human serum antibodies to melibiose and other carbohydrates.

Human sera were screened by passive haemagglutination for antibodies to various sugars. A high incidence of antibodies to melibiose was observed. There were also a few antibodies to other sugars and to dextran.

Binding studies on anti-fructofuranan mouse myeloma immunoglobulins A47N, A4, U61, and E109.

Four murine myeloma immunoglobulins, A4, A47N, U61, and E109, have been studied for their binding affinities with inulin and a series of oligosaccharides derived from inulin. The results indicate that the combining site of these immunoglobulins shows highest complementarity for a trifructofuranosyl sequence (A4 and A47N) and a tetrafructofuranosyl sequence (U61 and E109). The size of the combining area of the immunoglobulin E109 derived from the antigenic determinant (approximately 15 X 14 X 10 A) agrees well with the size observed on a hypothetical space model of the Fv portion of E109 (Potter, M., Rudikoff, S., Padlan, E. A., and Vrana, M. (1976), Antibodies in Human Diagnosis and Therapy, Haber, E., and Krause, R.M., Ed., New York, N.Y., Raven Press).

Mechanism of the anti-tumour effect of glucans and fructosans: a comparison with C. parvum.

The anti-tumour activity induced by glucans (lentinan, yeast cell walls, pseudonigeran, dextran, DEAE-dextran and dextran sulphate) and fructosans (levan and carboxymethyl-levan) was compared with the activity of C. parvum. The following effects on tumour systems in CBA mice were assayed: (a) adjuvant activity on the immune response against tumour-specific transplantation antigens (TSTA) with a methylcholanthrene-induced fibrosarcoma; (b) cytostatic activity of peritoneal macrophages against radiation-induced leukaemia cells; and (c) inhibition of tumour nodule formation in the lungs following i.v. injection of fibrosarcoma cells. All the polysaccharides induced cytostatic macrophages, but the dextrans and levans did so only after i.p. and not i.v. injection. Only lentinan, yeast cell walls and pseudonigeran were active in the lung-nodule inhibition test; and only lentinan and dextran sulphate showed slight adjuvant activity for TSTA. It is concluded that the anti-tumour activity induced by these polysaccharides is predominantly non-specific macrophage-mediated and much weaker than that found with C. parvum.

Immune response in mice and lack of response in guinea-pigs and in rabbits to DNP on polysaccharide.

The immunogenicity of three DNP-LE conjugates possessing different numbers of haptenic determinants per molecule of carrier (DNP 3-LE, DNP6-LE, DNP 13-LE) has been tested in mice. All conjugates induce only 19S anti-DNP antibodies, but the lowest conjugate is more immunogenic. Lack of response to DNP-LE is observed in guinea-pigs and in rabbits, animals in which native LE is not immunogenic. The immune response is not affected in mice tolerant to LE, when immunization is performed intravenously. These results are discussed with a view to determine the role played by a T-independent carrier in the antibody synthesis against a hapten.

Binding properties of immunoglobulin combining sites specific for terminal or nonterminal antigenic determinants in dextran.

Binding constants of the dextran-reactive BALB/c mouse IgA myeloma proteins W3129 and QUPC 52 have been determined for each member of the isomaltose series of oligosaccharides and for methyl alphaDglucoside. Protein W3129 has maximum complementarity for isomaltopentaose (IM5) deltaf degrees = 7,180 cal/mol) with 55-60% of the total binding energy directed against methylalphaDglucoside. Protein QUPC 52 gives maximum binding with isomaltohexaose (IM6) (deltaF degrees = -5,340 cal/mol) and has about 70% of its total binding energy for isomaltotriose (IM3), but at most only 5% for isomaltose (IM2) or methyl alphaDglucoside. Protein W3129 precipitates with branched dextrans high in alpha (1 yields 6) linkages and reacts with but does not precipitate a synthetic alpha (1 yields 6)-linked linear dextran. Protein QUPC 52 precipitates both branched and linear dextrans. Thus, the immunodominant group for protein W3129 is mimicked by methyl alphaDglucoside and this protein reacts exclusively at the terminal nonreducing ends of alpha (1 yields 6)-linked dextran chains. Protein QUPC 52 has an immunodominant group which is expressed by IM3 but not smaller oligosaccharides and this protein can react at nonterminal locations along alpha (1 yields 6)-linked dextran chains. Precipitation of linear dextran seems to be a valid although not quantitative assay for antidextrans with nonterminal specificity. Quantitative precipitin reactions with branched and linear dextrans suggest that alpha (1 yields 6)-specific human antidextrans are mixtures of molecules having terminal and nonterminal specificities and that the fraction of each type can vary among individuals. Rabbit antisera against IM3 or IM6 coupled to bovine serum albumin also appear to contain antibodies with nonterminal specificity for dextran chains although a large fraction has terminal specificity. Low molecular weight clinical dextran N-150N (congruent to 60,000) reacted more like linear dextran than like its parent native-branched dextran B512. This is thought to result from an abundance of nonterminal determinants in clinical dextran N-150N but a very small number of functional terminal determinants per molecule. An appreciation of terminal and nonterminal specificities and of the different immunodominant structures in isomaltosyl chains has proven to be of a great value in understanding the immunochemical reactions of dextrans. Moreover, certain previous findings with fructosan-reactive mouse myeloma proteins and human antilevans (55, 84) also suggest terminal and nonterminal specificities for levan chains.

Multiple individual and cross-specific indiotypes on 13 levan-binding myeloma proteins of BALB/c mice.

13 leven-binding myeloma proteins (LBMP) of BALB/c origin were classified into two groups with different binding specificities; one group of 11 proteins bound beta2 leads to 1 fructosans, a second group of two proteins bound fructosans probably of beta2 leads to 6 linkage. Anti-idiotypic sera prepared to 10 of the proteins in the appropriate strains of mice identified numerous idiotypic determinants. Each protein used for immunization had its own unique individual idiotypic specificities (IdI) and in addition most of the proteins carried two-nine cross-specific or shared idiotypes (IdX) that were found only among LBMP, and not found in 106 non-LBMP. Most of the IdX determinants and only four of the IdI determinants of the beta2 leads to 1 fructosan binding group were located in the antigen-binding site. The multiplicity of antigenic differences in this functionally related group of immunoglobulins reveals an unexpected degree of heterogeneity in V-regions that appears to be unrelated to binding.