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Sample records for non-enzymatically glycated peptides

  1. Comprehensive Identification of Glycated Peptides and Their Glycation Motifs in Plasma and Erythrocytes of Control and Diabetic Subjects

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qibin; Monroe, Matthew E.; Schepmoes, Athena A.; Clauss, Therese RW; Gritsenko, Marina A.; Meng, Da; Petyuk, Vladislav A.; Smith, Richard D.; Metz, Thomas O.

    2011-07-01

    Non-enzymatic glycation of proteins is implicated in diabetes mellitus and its related complications. In this report, we extend our previous development and refinement of proteomics-based methods for the analysis of non-enzymatically glycated proteins to comprehensively identify glycated proteins in normal and diabetic human plasma and erythrocytes. Using immunodepletion, enrichment, and fractionation strategies, we identified 7749 unique glycated peptides, corresponding to 3742 unique glycated proteins. Semi-quantitative comparisons revealed a number of proteins with glycation levels significantly increased in diabetes relative to control samples and that erythrocyte proteins are more extensively glycated than plasma proteins. A glycation motif analysis revealed amino acids that are favored more than others in the protein primary structures in the vicinity of the glycation sites in both sample types. The glycated peptides and corresponding proteins reported here provide a foundation for the potential identification of novel markers for diabetes, glycemia, or diabetic complications.

  2. Monitoring the progress of non-enzymatic glycation in vitro

    International Nuclear Information System (INIS)

    Shaw, S.M.; Crabbe, M.J.

    1994-01-01

    The progress of in vitro non-enzymatic glycation of bovine serum albumin was followed by using 14 C-glucose and a nitroblue tetrazolium assay, absorption and fluorescence spectroscopy, SDS gel electrophoresis and protease digestion. The number of adducts detectable using both 14 C-tracers and a fructosamine assay remained low at physiological glucose concentrations, fewer than five adducts being detectable. When glucose concentrations > 1.0 M were used the number of adducts was found to greatly exceed the number of lysyl residues available in BSA, indicative of cross-linking between Maillard products. Incubation of BSA with glucose concentrations of up to 160 mM for one month produced no observable increase in molecular weight by SDS gel electrophoresis, showing that at physiological glucose concentrations, increases in molecular weight were minimal for short incubation periods. Increases in absorption were proportial to both the glucose concentration and the incubation time. Several absorption peaks, at 370, 488 and 554 nm, were consistent in appearance throughout the course of each incubation. Fluorescence spectroscopy of the modified proteins showed a disappearance of the fluorescence associated with peptide bonds and aromatic residues and the appearance of a broad peak at longer wavelengths due to the wide range of absorptive/fluorescent wavelengths of the developing Maillard products. Protease digestion gave similar patterns with non-glycated and glycated protein, suggesting that glycation did not block digestion sites, and that partial digestion did not cause significant further exposure of susceptible sites. Our results show that while glycation ultimately results in protein conformational changes and the formation of large molecular weight species, these occur at a relatively late stage in the maturation of protein Maillard products, after ≥ nine months of incubation with glucose concentration of ≥ 20 mM. Monitoring of AGE maturation in vitro is better

  3. Effects of non-enzymatic glycation in human serum albumin. Spectroscopic analysis

    Science.gov (United States)

    Szkudlarek, A.; Sułkowska, A.; Maciążek-Jurczyk, M.; Chudzik, M.; Równicka-Zubik, J.

    2016-01-01

    Human serum albumin (HSA), transporting protein, is exposed during its life to numerous factors that cause its functions become impaired. One of the basic factors - glycation of HSA - occurs in diabetes and may affect HSA-drug binding. Accumulation of advanced glycation end-products (AGEs) leads to diseases e.g. diabetic and non-diabetic cardiovascular diseases, Alzheimer disease, renal disfunction and in normal aging. The aim of the present work was to estimate how non-enzymatic glycation of human serum albumin altered its tertiary structure using fluorescence technique. We compared glycated human serum albumin by glucose (gHSAGLC) with HSA glycated by fructose (gHSAFRC). We focused on presenting the differences between gHSAFRC and nonglycated (HSA) albumin used acrylamide (Ac), potassium iodide (KI) and 2-(p-toluidino)naphthalene-6-sulfonic acid (TNS). Changes of the microenvironment around the tryptophan residue (Trp-214) of non-glycated and glycated proteins was investigated by the red-edge excitation shift method. Effect of glycation on ligand binding was examined by the binding of phenylbutazone (PHB) and ketoprofen (KP), which a primary high affinity binding site in serum albumin is subdomain IIA and IIIA, respectively. At an excitation and an emission wavelength of λex 335 nm and λem 420 nm, respectively the increase of fluorescence intensity and the blue-shift of maximum fluorescence was observed. It indicates that the glycation products decreases the polarity microenvironment around the fluorophores. Analysis of red-edge excitation shift method showed that the red-shift for gHSAFRC is higher than for HSA. Non-enzymatic glycation also caused, that the Trp residue of gHSAFRC becomes less accessible for the negatively charged quencher (I-), KSV value is smaller for gHSAFRC than for HSA. TNS fluorescent measurement demonstrated the decrease of hydrophobicity in the glycated albumin. KSV constants for gHSA-PHB systems are higher than for the unmodified serum

  4. Destructive effect of non-enzymatic glycation on catalase and remediation via curcumin.

    Science.gov (United States)

    Mofidi Najjar, Fayezeh; Taghavi, Fereshteh; Ghadari, Rahim; Sheibani, Nader; Moosavi-Movahedi, Ali Akbar

    2017-09-15

    Non-enzymatic glycation of proteins is a post-translational modification that is produced by a covalent binding between reducing sugars and amino groups of lysine and arginine residues. In this paper the effect of pathological conditions, derived from hyperglycemia on bovine liver catalase (BLC) as a model protein was considered by measuring enzyme activity, reactive oxygen species (ROS) generation, and changes in catalase conformational properties. We observed that in the presence of glucose, the catalase activity gradually decreased. ROS generation was also involved in the glycation process. Thus, decreased BLC activity was partly considered as a result of ROS generation through glycation. However, in the presence of curcumin the amount of ROS was reduced resulting in increased activity of the glycated catalase. The effect of high glucose level and the potential inhibitory effect of curcumin on aggregation and structural changes of catalase were also investigated. Molecular dynamic simulations also showed that interaction of catalase with curcumin resulted in changes in accessible surface area (ASA) and pKa, two effective parameters of glycation, in potential glycation lysine residues. Thus, the decrease in ASA and increase in pKa of important lysine residues were considered as predominant factors in decreased glycation of BLC by curcumin. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. PSEUDOAFFINITY CHROMATOGRAPHY ENRICHMENT OF GLYCATED PEPTIDES FOR MONITORING ADVANCED GLYCATION END PRODUCTS (AGES IN METABOLIC DISORDERS

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    Rajasekar R. Prasanna

    2016-09-01

    Full Text Available Advanced Glycation End (AGE products are produced due to diabetic progression and they are responsible for many complications in the diabetic disorder. The diabetic progression is measured, particularly following glycated hemoglobin using specific antibodies. However, the most abundant protein in blood, human serum albumin, is also found to be glycated which has a much shorter half life and gives information on short term glycemic control. In addition, glycated albumins are considered as markers of diabetic complications such as nephropathy, peripheral vascular calcification and also in Alzheimer’s disease. The glycation proceeds from the interaction between aldehyde group of sugar and the free amino group of the protein, resulting in the formation of Schiff’s base, which undergoes a series of modifications leading to generation of imidazoyl derivatives of amino acids known as Amadori rearrangement products. The imidazoyl derivatives from arginine and lysine are the most prominent modifications observed in proteins in the presence of reducing sugar and these imidazoyl derivatives have an affinity towards certain transition metal ions. Based on our earlier exhaustive work on trapping the histidine peptides using transition metal ion, Cu(II linked to imino-diacetate complex, we explored Cu(II immobilized metal affinity chromatography (IMAC as a potential tool for specific detection of glycated peptides of human serum albumin. Our results clearly demonstrate that Cu(II IMAC is able to detect glycated peptides very efficiently while the non-glycated forms were not retained on the Cu (II column as confirmed by LC-MS/MS analysis. We further discuss the utility of IMAC technology to enrich the detection of AGE products in plasma. We anticipate that these studies may provide valuable information on understanding disease pathologies and the potential of AGE products as biomarkers of various diseases including neurodegenerative, renal and

  6. Multicenter evaluation of an enzymatic method for glycated albumin.

    Science.gov (United States)

    Paleari, Renata; Bonetti, Graziella; Callà, Cinzia; Carta, Mariarosa; Ceriotti, Ferruccio; Di Gaetano, Nicola; Ferri, Marilisa; Guerra, Elena; Lavalle, Gabriella; Cascio, Claudia Lo; Martino, Francesca Gabriela; Montagnana, Martina; Moretti, Marco; Santini, Gabriele; Scribano, Donata; Testa, Roberto; Vero, Anna; Mosca, Andrea

    2017-06-01

    The use of glycated albumin (GA) has been proposed as an additional glycemic control marker particularly useful in intermediate-term monitoring and in situation when HbA 1c test is not reliable. We have performed the first multicenter evaluation of the analytical performance of the enzymatic method quantILab Glycated Albumin assay implemented on the most widely used clinical chemistry analyzers (i.e. Abbott Architect C8000, Beckman Coulter AU 480 and 680, Roche Cobas C6000, Siemens ADVIA 2400 and 2400 XPT). The repeatability of the GA measurement (expressed as CV, %) implemented in the participating centers ranged between 0.9% and 1.2%. The within-laboratory CVs ranged between 1.2% and 1.6%. A good alignment between laboratories was found, with correlation coefficients from 0.996 to 0.998. Linearity was confirmed in the range from 7.6 to 84.7%. The new enzymatic method for glycated albumin evaluated by our investigation is suitable for clinical use. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Glycated Lysine Residues: A Marker for Non-Enzymatic Protein Glycation in Age-Related Diseases

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    Nadeem A. Ansari

    2011-01-01

    Full Text Available Nonenzymatic glycosylation or glycation of macromolecules, especially proteins leading to their oxidation, play an important role in diseases. Glycation of proteins primarily results in the formation of an early stage and stable Amadori-lysine product which undergo further irreversible chemical reactions to form advanced glycation endproducts (AGEs. This review focuses these products in lysine rich proteins such as collagen and human serum albumin for their role in aging and age-related diseases. Antigenic characteristics of glycated lysine residues in proteins together with the presence of serum autoantibodies to the glycated lysine products and lysine-rich proteins in diabetes and arthritis patients indicates that these modified lysine residues may be a novel biomarker for protein glycation in aging and age-related diseases.

  8. Glycated Lysine Residues: A Marker for Non-Enzymatic Protein Glycation in Age-Related Diseases

    OpenAIRE

    Ansari, Nadeem A.; Moinuddin,; Ali, Rashid

    2011-01-01

    Nonenzymatic glycosylation or glycation of macromolecules, especially proteins leading to their oxidation, play an important role in diseases. Glycation of proteins primarily results in the formation of an early stage and stable Amadori-lysine product which undergo further irreversible chemical reactions to form advanced glycation endproducts (AGEs). This review focuses these products in lysine rich proteins such as collagen and human serum albumin for their role in aging and age-related dise...

  9. Bimolecular interaction of argpyrimidine (a Maillard reaction product) in in vitro non-enzymatic protein glycation model and its potential role as an antiglycating agent.

    Science.gov (United States)

    Bhattacherjee, Abhishek; Dhara, Kaliprasanna; Chakraborti, Abhay Sankar

    2017-09-01

    Non- enzymatic glycation, also known as Maillard reaction, is one of the most important and investigated reactions in biochemistry. Maillard reaction products (MRPs) like protein-derived advanced glycation end products (AGEs) are often referred to cause pathophysiological complications in human systems. On contrary, several MRPs are exogenously used as antioxidant, antimicrobial and flavouring agents. In the preset study, we have shown that argpyrimidine, a well-established AGE, interacts with bovine serum albumin (BSA) and glucose individually in standard BSA-glucose model system and successfully inhibits glycation of the protein. Bimolecular interaction of argpyrimidine with glucose or BSA has been studied independently. Chromatographic purification, different spectroscopic studies and molecular modeling have been used to evaluate the nature and pattern of interactions. Binding of argpyrimidine with BSA prevents incorporation of glucose inside the native protein. Argpyrimidine can also directly entrap glucose. Both these interactions may be associated with the antiglycation potential of argpyrimidine, indicating a beneficial function of an AGE. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Protein Glycation in Diabetes as Determined by Mass Spectrometry

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    Annunziata Lapolla

    2013-01-01

    Full Text Available Diabetes is a common endocrine disorder characterized by hyperglycemia leading to nonenzymatic glycation of proteins, responsible for chronic complications. The development of mass spectrometric techniques able to give highly specific and reliable results in proteome field is of wide interest for physicians, giving them new tools to monitor the disease progression and the possible complications related to diabetes, as well as the effectiveness of therapeutic treatments. This paper reports and discusses some of the data pertaining protein glycation in diabetic subjects obtained by matrix-assisted laser desorption ionization (MALDI mass spectrometry (MS. The preliminary studies carried out by in vitro protein glycation experiments show clear differences in molecular weight of glycated and unglycated proteins. Then, the attention was focused on plasma proteins human serum albumin (HSA and immunoglobulin G (IgG. Enzymatic degradation products of in vitro glycated HSA were studied in order to simulate the in vivo enzymatic digestion of glycated species by the immunological system leading to the highly reactive advanced glycation end-products (AGEs peptides. Further studies led to the evaluation of glycated Apo A-I and glycated haemoglobin levels. A different MALDI approach was employed for the identification of markers of disease in urine samples of healthy, diabetic, nephropathic, and diabetic-nephropathic subjects.

  11. Using an enzymatic galactose assay to detect lactose glycation extents of two proteins caseinate and soybean protein isolate via the Maillard reaction.

    Science.gov (United States)

    Wang, Xiao-Peng; Zhao, Xin-Huai

    2017-06-01

    Glycation of food proteins via the Maillard reaction has been widely studied in the recent years; however, the amount of saccharide connected to proteins is usually not determined. An enzymatic galactose assay was proposed firstly in this study to detect lactose glycation extents of caseinate and soybean protein isolate (SPI) during the Maillard reaction at two temperatures and different times. The separated glycated proteins were hydrolysed to release galactose necessary for the enzymatic assay and glycation calculation. Caseinate and SPI both obtained the highest lactose glycation extents at 100 °C or 121 °C by a reaction time of 180 or 20 min. Short- and long-time reaction resulted in lower glycation extents. During the reaction, three chemical indices (absorbences at 294/490 nm and fluorescence intensities) of reaction mixtures increased continually, but another index reactable NH 2 of glycated proteins showed the opposite trend. In general, changing profiles of the four indices were inconsistent with those profiles of lactose glycation extents of glycated proteins, implying practical limitation of the four indices in studies. This proposed enzymatic assay could directly detect lactose glycation of the two proteins, and thus was more useful than the four chemical indices to monitor glycation of the two proteins. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  12. Application of Ni(II-assisted peptide bond hydrolysis to non-enzymatic affinity tag removal.

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    Edyta Kopera

    Full Text Available In this study, we demonstrate a non-enzymatic method for hydrolytic peptide bond cleavage, applied to the removal of an affinity tag from a recombinant fusion protein, SPI2-SRHWAP-His(6. This method is based on a highly specific Ni(II reaction with (S/TXHZ peptide sequences. It can be applied for the protein attached to an affinity column or to the unbound protein in solution. We studied the effect of pH, temperature and Ni(II concentration on the efficacy of cleavage and developed an analytical protocol, which provides active protein with a 90% yield and ∼100% purity. The method works well in the presence of non-ionic detergents, DTT and GuHCl, therefore providing a viable alternative for currently used techniques.

  13. Plasma disappearance of glycated and non-glycated albumin in type 1 (insulin-dependent) diabetes mellitus

    DEFF Research Database (Denmark)

    Bent-Hansen, L; Feldt-Rasmussen, B; Kverneland, Arne

    1993-01-01

    transport ratio) was determined from the disappearance data. The index was high in control subjects (1.021 +/- 0.0057 (SEM)). This reflects a mean difference between the two escape rates of 2.1% per hour (for comparison the mean of the fractional escape rate of non-glycated albumin of the normal control......The fractional plasma escape rates of glycated and non-glycated albumin have earlier been measured in groups of Type 1 (insulin-dependent) diabetic patients and control subjects. The escape of non-glycated albumin was similar in control subjects and normoalbuminuric patients, but elevated...... in patients with micro or macroalbuminuria. In all groups the escape rate of glycated albumin was lower than that of non-glycated albumin. Glycation increases the anionic charge of albumin. To assay for charge-dependent alterations of transport a selectivity index (non-glycated albumin/glycated albumin...

  14. Evaluation of the site specific protein glycation and antioxidant capacity of rare sugar-protein/peptide conjugates.

    Science.gov (United States)

    Sun, Yuanxia; Hayakawa, Shigeru; Ogawa, Masahiro; Izumori, Ken

    2005-12-28

    Protein-sugar conjugates generated in nonenzymatic glycation of alpha-lactalbumin (LA) with rare sugars [D-allose (All) and D-psicose (Psi)] and alimentary sugars as controls [D-glucose (Glc) and D-fructose (Fru)] were qualitatively determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Mass spectra revealed that the extent of glycation at lysine residues on LA with D-aldose molecules was very much higher than that of glycation with d-ketose molecules. To identify the specific site of glycation, the peptide mapping was established from protease V8 digestion, using a combination of computational cutting of proteins and MALDI-TOF-MS. As compared to peptide mapping, three and seven glycation sites were located in the primary structure of LA-ketose and LA-aldose conjugates, respectively. On the other hand, the antioxidant activities of protein-sugar conjugates and their peptic hydrolysates were investigated by 1,1-diphenyl-2-picrylhydrazyl radical scavenging method. The antioxidant activities of proteins/peptides glycated with rare sugars were significantly higher than those modified with the control sugars. The results indicated that the glycation degree and position were not markedly different between rare sugar and corresponding control sugar, but the antioxidant properties of protein and its hydrolysate were significantly enhanced by modifying with rare sugar.

  15. Vanillin restrains non-enzymatic glycation and aggregation of albumin by chemical chaperone like function.

    Science.gov (United States)

    Awasthi, Saurabh; Saraswathi, N T

    2016-06-01

    Vanillin a major component of vanilla bean extract is commonly used a natural flavoring agent. Glycation is known to induce aggregation and fibrillation of globular proteins such as albumin, hemoglobin. Here we report the inhibitory potential of vanillin toward early and advanced glycation modification and amyloid like aggregation of albumin based on the determination of both early and advanced glycation and conformational changes in albumin using circular dichroism. Inhibition of aggregation and fibrillation of albumin was determined based on amyloid specific dyes i.e., Congo red and Thioflavin T and microscopic imaging. It was evident that vanillin restrains glycation of albumin and exhibits protective effect toward its native conformation. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. A novel monoclonal antibody targeting carboxymethyllysine, an advanced glycation end product in atherosclerosis and pancreatic cancer.

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    Ulrika Wendel

    Full Text Available Advanced glycation end products are formed by non-enzymatic reactions between proteins and carbohydrates, causing irreversible lysine and arginine alterations that severely affect protein structure and function. The resulting modifications induce inflammation by binding to scavenger receptors. An increase in advanced glycation end products is observed in a number of diseases e.g. atherosclerosis and cancer. Since advanced glycation end products also are present in healthy individuals, their detection and quantification are of great importance for usage as potential biomarkers. Current methods for advanced glycation end product detection are though limited and solely measure total glycation. This study describes a new epitope-mapped single chain variable fragment, D1-B2, against carboxymethyllysine, produced from a phage library that was constructed from mouse immunizations. The phage library was selected against advanced glycation end product targets using a phage display platform. Characterization of its binding pattern was performed using large synthetic glycated peptide and protein libraries displayed on microarray slides. D1-B2 showed a preference for an aspartic acid, three positions N-terminally from a carboxymethyllysine residue and also bound to a broad collection of glycated proteins. Positive immunohistochemical staining of mouse atherosclerotic plaques and of a tissue microarray of human pancreatic tumors confirmed the usability of the new scFv for advanced glycation end product detection in tissues. This study demonstrates a promising methodology for high-throughput generation of epitope-mapped monoclonal antibodies against AGE.

  17. Selective detection of carbohydrates and their peptide conjugates by ESI-MS using synthetic quaternary ammonium salt derivatives of phenylboronic acids.

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    Kijewska, Monika; Kuc, Adam; Kluczyk, Alicja; Waliczek, Mateusz; Man-Kupisinska, Aleksandra; Lukasiewicz, Jolanta; Stefanowicz, Piotr; Szewczuk, Zbigniew

    2014-06-01

    We present new tags based on the derivatives of phenylboronic acid and apply them for the selective detection of sugars and peptide-sugar conjugates in mass spectrometry. We investigated the binding of phenylboronic acid and its quaternary ammonium salt (QAS) derivatives to carbohydrates and peptide-derived Amadori products by HR-MS and MS/MS experiments. The formation of complexes between sugar or sugar-peptide conjugates and synthetic tags was confirmed on the basis of the unique isotopic distribution resulting from the presence of boron atom. Moreover, incorporation of a quaternary ammonium salt dramatically improved the efficiency of ionization in mass spectrometry. It was found that the formation of a complex with phenylboronic acid stabilizes the sugar moiety in glycated peptides, resulting in simplification of the fragmentation pattern of peptide-derived Amadori products. The obtained results suggest that derivatization of phenylboronic acid as QAS is a promising method for sensitive ESI-MS detection of carbohydrates and their conjugates formed by non-enzymatic glycation or glycosylation.

  18. Glycation precedes lens crystallin aggregation

    International Nuclear Information System (INIS)

    Swamy, M.S.; Perry, R.E.; Abraham, E.C.

    1987-01-01

    Non-enzymatic glycosylation (glycation) seems to have the potential to alter the structure of crystallins and make them susceptible to thiol oxidation leading to disulfide-linked high molecular weight (HMW) aggregate formation. They used streptozotocin diabetic rats during precataract and cataract stages and long-term cell-free glycation of bovine lens crystallins to study the relationship between glycation and lens crystallin aggregation. HMW aggregates and other protein components of the water-soluble (WS) and urea-soluble (US) fractions were separated by molecular sieve high performance liquid chromatography. Glycation was estimated by both [ 3 H]NaBH 4 reduction and phenylboronate agarose affinity chromatography. Levels of total glycated protein (GP) in the US fractions were about 2-fold higher than in the WS fractions and there was a linear increase in GP in both WS and US fractions. This increase was parallelled by a corresponding increase in HMW aggregates. Total GP extracted by the affinity method from the US fraction showed a predominance of HMW aggregates and vice versa. Cell-free glycation studies with bovine crystallins confirmed the results of the animals studies. Increasing glycation caused a corresponding increase in protein insolubilization and the insoluble fraction thus formed also contained more glycated protein. It appears that lens protein glycation, HMW aggregate formation, and protein insolubilization are interrelated

  19. Effect of Maillard induced glycation on protein hydrolysis by lysine/arginine and non-lysine/arginine specific proteases

    NARCIS (Netherlands)

    Deng, Y.; Wierenga, P.A.; Schols, H.A.; Sforza, S.; Gruppen, H.

    2017-01-01

    Enzymatic protein hydrolysis is sensitive to modifications of protein structure, e.g. Maillard reaction. In early stages of the reaction glycation takes place, modifying the protein primary structure. In later stages protein aggregation occurs. The specific effect of glycation on protein

  20. Effect of glycation on α-crystallin structure and chaperone-like function

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    Kumar, P. Anil; Kumar, M. Satish; Reddy, G. Bhanuprakash

    2007-01-01

    The chaperone-like activity of α-crystallin is considered to play an important role in the maintenance of the transparency of the eye lens. However, in the case of aging and in diabetes, the chaperone function of α-crystallin is compromized, resulting in cataract formation. Several post-translational modifications, including non-enzymatic glycation, have been shown to affect the chaperone function of α-crystallin in aging and in diabetes. A variety of agents have been identified as the predominant sources for the formation of AGEs (advanced glycation end-products) in various tissues, including the lens. Nevertheless, glycation of α-crystallin with various sugars has resulted in divergent results. In the present in vitro study, we have investigated the effect of glucose, fructose, G6P (glucose 6-phosphate) and MGO (methylglyoxal), which represent the major classes of glycating agents, on the structure and chaperone function of α-crystallin. Modification of α-crystallin with all four agents resulted in the formation of glycated protein, increased AGE fluorescence, protein cross-linking and HMM (high-molecular-mass) aggregation. Interestingly, these glycation-related profiles were found to vary with different glycating agents. For instance, CML [Nϵ-(carboxymethyl)lysine] was the predominant AGE formed upon glycation of α-crystallin with these agents. Although fructose and MGO caused significant conformational changes, there were no significant structural perturbations with glucose and G6P. With the exception of MGO modification, glycation with other sugars resulted in decreased chaperone activity in aggregation assays. However, modification with all four sugars led to the loss of chaperone activity as assessed using an enzyme inactivation assay. Glycation-induced loss of α-crystallin chaperone activity was associated with decreased hydrophobicity. Furthermore, α-crystallin isolated from glycated TSP (total lens soluble protein) had also increased AGE

  1. Effects of parathyroid hormone on cortical porosity, non-enzymatic glycation and bone tissue mechanics in rats with type 2 diabetes mellitus.

    Science.gov (United States)

    Campbell, G M; Tiwari, S; Hofbauer, C; Picke, A-K; Rauner, M; Huber, G; Peña, J A; Damm, T; Barkmann, R; Morlock, M M; Hofbauer, L C; Glüer, C-C

    2016-01-01

    Type 2 diabetes mellitus increases skeletal fragility; however, the contributing mechanisms and the efficacy of bone-forming agents are unclear. We studied diabetes and parathyroid hormone (PTH) treatment effects on cortical porosity (Ct.Po), non-enzymatic glycation (NEG) and bone mechanics in Zucker diabetic fatty (ZDF) rats. Eleven-week old ZDF diabetic (DB) and non-diabetic (ND) rats were given 75μg/kg PTH (1-84) or vehicle 5days per week over 12weeks. The right femora and L4 vertebrae were excised, micro-CT scanned, and tested in 3-point bending and uniaxial compression, respectively. NEG of the samples was determined using fluorescence. Diabetes increased Ct.Po (vertebra (vert): +40.6%, femur (fem): +15.5% vs. ND group, pbone tissue mechanics where reductions in vertebral maximum strain (-22%) and toughness (-42%) were observed in the DB vs. ND group (pbone mechanics, which were not improved with PTH treatment. PTH therapy alone may worsen diabetic bone mechanics through formation of new bone with high AGEs cross-linking. Optimal treatment regimens must address both improvements of bone mass and glycemic control in order to successfully reduce diabetic bone fragility. This article is part of a Special Issue entitled "Bone and diabetes". Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Advanced glycation end products induce differential structural modifications and fibrillation of albumin

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    Awasthi, Saurabh; Sankaranarayanan, Kamatchi; Saraswathi, N. T.

    2016-06-01

    Glycation induced amyloid fibrillation is fundamental to the development of many neurodegenerative and cardiovascular complications. Excessive non-enzymatic glycation in conditions such as hyperglycaemia results in the increased accumulation of advanced glycation end products (AGEs). AGEs are highly reactive pro-oxidants, which can lead to the activation of inflammatory pathways and development of oxidative stress. Recently, the effect of non-enzymatic glycation on protein structure has been the major research area, but the role of specific AGEs in such structural alteration and induction of fibrillation remains undefined. In this study, we determined the specific AGEs mediated structural modifications in albumin mainly considering carboxymethyllysine (CML), carboxyethyllysine (CEL), and argpyrimidine (Arg-P) which are the major AGEs formed in the body. We studied the secondary structural changes based on circular dichroism (CD) and spectroscopic analysis. The AGEs induced fibrillation was determined by Congo red binding and examination of scanning and transmission electron micrographs. The amyloidogenic regions in the sequence of BSA were determined using FoldAmyloid. It was observed that CEL modification of BSA leads to the development of fibrillar structures, which was evident from both secondary structure changes and TEM analysis.

  3. Posttranslational nitro-glycative modifications of albumin in Alzheimer's disease: implications in cytotoxicity and amyloid-β peptide aggregation.

    Science.gov (United States)

    Ramos-Fernández, Eva; Tajes, Marta; Palomer, Ernest; Ill-Raga, Gerard; Bosch-Morató, Mònica; Guivernau, Biuse; Román-Dégano, Irene; Eraso-Pichot, Abel; Alcolea, Daniel; Fortea, Juan; Nuñez, Laura; Paez, Antonio; Alameda, Francesc; Fernández-Busquets, Xavier; Lleó, Alberto; Elosúa, Roberto; Boada, Mercé; Valverde, Miguel A; Muñoz, Francisco J

    2014-01-01

    Glycation and nitrotyrosination are pathological posttranslational modifications that make proteins prone to losing their physiological properties. Since both modifications are increased in Alzheimer's disease (AD) due to amyloid-β peptide (Aβ) accumulation, we have studied their effect on albumin, the most abundant protein in cerebrospinal fluid and blood. Brain and plasmatic levels of glycated and nitrated albumin were significantly higher in AD patients than in controls. In vitro turbidometry and electron microscopy analyses demonstrated that glycation and nitrotyrosination promote changes in albumin structure and biochemical properties. Glycated albumin was more resistant to proteolysis and less uptake by hepatoma cells occurred. Glycated albumin also reduced the osmolarity expected for a solution containing native albumin. Both glycation and nitrotyrosination turned albumin cytotoxic in a cell type-dependent manner for cerebral and vascular cells. Finally, of particular relevance to AD, these modified albumins were significantly less effective in avoiding Aβ aggregation than native albumin. In summary, nitrotyrosination and especially glycation alter albumin structural and biochemical properties, and these modifications might contribute for the progression of AD.

  4. Inhibition of peptide aggregation by means of enzymatic phosphorylation

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    Kristin Folmert

    2016-11-01

    Full Text Available As is the case in numerous natural processes, enzymatic phosphorylation can be used in the laboratory to influence the conformational populations of proteins. In nature, this information is used for signal transduction or energy transfer, but has also been shown to play an important role in many diseases like tauopathies or diabetes. With the goal of determining the effect of phosphorylation on amyloid fibril formation, we designed a model peptide which combines structural characteristics of α-helical coiled-coils and β-sheets in one sequence. This peptide undergoes a conformational transition from soluble structures into insoluble amyloid fibrils over time and under physiological conditions and contains a recognition motif for PKA (cAMP-dependent protein kinase that enables enzymatic phosphorylation. We have analyzed the pathway of amyloid formation and the influence of enzymatic phosphorylation on the different states along the conformational transition from random-coil to β-sheet-rich oligomers to protofilaments and on to insoluble amyloid fibrils, and we found a remarkable directing effect from β-sheet-rich structures to unfolded structures in the initial growth phase, in which small oligomers and protofilaments prevail if the peptide is phosphorylated.

  5. Enhancing bioactive peptide release and identification using targeted enzymatic hydrolysis of milk proteins.

    Science.gov (United States)

    Nongonierma, Alice B; FitzGerald, Richard J

    2018-06-01

    Milk proteins have been extensively studied for their ability to yield a range of bioactive peptides following enzymatic hydrolysis/digestion. However, many hurdles still exist regarding the widespread utilization of milk protein-derived bioactive peptides as health enhancing agents for humans. These mostly arise from the fact that most milk protein-derived bioactive peptides are not highly potent. In addition, they may be degraded during gastrointestinal digestion and/or have a low intestinal permeability. The targeted release of bioactive peptides during the enzymatic hydrolysis of milk proteins may allow the generation of particularly potent bioactive hydrolysates and peptides. Therefore, the development of milk protein hydrolysates capable of improving human health requires, in the first instance, optimized targeted release of specific bioactive peptides. The targeted hydrolysis of milk proteins has been aided by a range of in silico tools. These include peptide cutters and predictive modeling linking bioactivity to peptide structure [i.e., molecular docking, quantitative structure activity relationship (QSAR)], or hydrolysis parameters [design of experiments (DOE)]. Different targeted enzymatic release strategies employed during the generation of milk protein hydrolysates are reviewed herein and their limitations are outlined. In addition, specific examples are provided to demonstrate how in silico tools may help in the identification and discovery of potent milk protein-derived peptides. It is anticipated that the development of novel strategies employing a range of in silico tools may help in the generation of milk protein hydrolysates containing potent and bioavailable peptides, which in turn may be used to validate their health promoting effects in humans. Graphical abstract The targeted enzymatic hydrolysis of milk proteins may allow the generation of highly potent and bioavailable bioactive peptides.

  6. Enzymatic digestibility of peptides cross-linked by ionizing radiation

    International Nuclear Information System (INIS)

    Dizdaroglu, M.; Gajewski, E.; Simic, M.G.

    1984-01-01

    Digestibility by proteolytic enzymes of peptides cross-linked by ionizing radiation was investigated. Small peptides of alanine and phenylalanine were chosen as model compounds and aminopeptidases and carboxypeptidases were used as proteolytic enzymes. Peptides exposed to γ-radiation in aqueous solution were analysed by high-performance liquid chromatography before and after hydrolysis by aminopeptidase M, leucine aminopeptidase carboxypeptidase A and carboxypeptidase Y. The results obtained clearly demonstrate the different actions of these enzymes on cross-linked aliphatic and aromatic peptides. Peptide bonds of cross-linked dipeptides of alanine were completely resistant to enzymatic hydrolysis whereas the enzymes, except for carboxypeptidase Y, cleaved all peptide bonds of cross-linked peptides of phenylalanine. The actions of the enzymes on these particular compounds are discussed in detail. (author)

  7. Evaluation of Structure, Chaperone-Like Activity and Allergenicity of Reduced Glycated Adduct of Bovine β-casein.

    Science.gov (United States)

    Yousefi, Reza; Ferdowsi, Leila; Tavaf, Zohreh; Sadeghian, Tanaz; Tamaddon, Ali M; Moghtaderi, Mozhgan; Pourpak, Zahra

    2017-01-01

    Milk has a potent reducing environment with an important quantity of sugar levels. In the current study β-casein was glycated in the presence of D-glucose and sodium cyanoborohydride as a reducing agent. Then, the reduced glucitol adduct of β-casein was used for the structural and functional analyses using different spectroscopic techniques. The results of fluorescence and far ultraviolet circular dichroism assessments suggest important structural alteration upon non-enzymatic glycation of β-casein. In addition, the chaperone activity, micellization properties and antioxidant activity of this protein were altered upon glucose modification. Also, as a result of reduced glycation, the allergenicity profile of this protein remained largely unchanged. Additional to its energetic and nutritional values, β-casein has important functional properties. The native structure of this protein is important to perform accurately its biological functions. Non-enzymatic glycation under reducing state was capable to alter both structural and functional aspects of β-casein. Due to effective reducing environment and significant quantity of reducing sugar of human milk, similar structural and functional alterations are most likely to occur upon reducing glycation of β-casein in vivo. Also, these changes might be even intensified during chronic hyperglycemia in diabetic mothers. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  8. Advanced glycation end products in clinical nephrology.

    Science.gov (United States)

    Kalousová, M; Zima, T; Tesar, V; Stípek, S; Sulková, S

    2004-01-01

    As a result of oxidative and carbonyl stress, advanced glycation end products (AGEs) are involved in the pathogenesis of severe and frequent diseases and their fatal vascular/cardiovascular complications, i.e. diabetes mellitus and its complications (nephropathy, angiopathy, neuropathy and retinopathy, renal failure and uremic and dialysis-associated complications), atherosclerosis and dialysis-related amyloidosis, neurodegenerative diseases, and rheumatoid arthritis. They are formed via non-enzymatic glycation which is specifically enhanced through the presence of oxidative and carbonyl stress, and their ability to form glycoxidation products in peptide and protein structures finally modulating or inducing biological reactivity. Food can be another source of AGEs; however, high serum AGEs in hemodialysis patients might reflect nutritional status better. Several methods of renal replacement therapy have been studied in connection with the AGE removal, but unfortunately the possibilities are still unsatisfactory even if high flux dialysis, hemofiltration, or hemodiafiltration give better results than conventional low flux dialysis. AGEs are currently being studied in the patients on peritoneal dialysis as their precursors can be formed in the dialysis fluid. AGEs can cause damage to the peritoneum and so a loss of ultrafiltration capacity. Many compounds give promising results in AGE inhibition (inhibition of formation of AGEs, inhibition of their action or degradation of AGEs), are tested for these properties, and eventually undergo clinical studies (e.g. aminoguanidine, OPB-9195, pyridoxamine, antioxidants, N-phenacylthiazolium bromide, antihypertensive drugs, angiotensin-converting enzyme inhibitors and angiotensin II receptor-1 antagonists). Copyright 2004 S. Karger AG, Basel

  9. Advanced glycation end-products (AGES) and heart failure : Pathophysiology and clinical implications

    NARCIS (Netherlands)

    Hartog, Jasper W. L.; Voors, Adriaan A.; Bakker, Stephan J. L.; Smit, Andries J.; van Veldhuisen, Dirk J.

    2007-01-01

    Advanced glycation end-products (AGEs) are molecules formed during a non-enzymatic reaction between proteins and sugar residues, called the Maillard reaction. AGEs accumulate in the human body with age, and accumulation is accelerated in the presence of diabetes mellitus. In patients with diabetes,

  10. THE EFFECTS OF GLYCATION ON THE BINDING OF HUMAN SERUM ALBUMIN TO WARFARIN AND L-TRYPTOPHAN

    OpenAIRE

    Joseph, K.S.; Hage, David S.

    2010-01-01

    Diabetes leads to elevated levels of glucose in blood which, in turn, can lead to the non-enzymatic glycation of serum proteins such as human serum albumin (HSA). It has been suggested that this increase in glycation can alter the ability of HSA to bind to drugs and other small solutes. This study used high-performance affinity chromatography (HPAC) to see if there is any significant change related to glycation in the binding of HSA to warfarin and L-tryptophan, which are often used as probe ...

  11. Transcapillary escape rate and relative metabolic clearance of glycated and non-glycated albumin in type 1 (insulin-dependent) diabetes mellitus

    DEFF Research Database (Denmark)

    Bent-Hansen, L; Feldt-Rasmussen, B; Kverneland, A

    1987-01-01

    The transcapillary escape rate and relative plasma disappearance of glycated and non-glycated albumin were measured in 25 male Type 1 (insulin-dependent) diabetic patients using a double tracer technique. The patients were divided into three groups on the basis of their urinary albumin excretion......: group 1, normal albumin excretion (less than 30 mg/24 h) (n = 8); group 2, microalbuminuria (30-300 mg/24 h) (n = 9); and group 3, clinical nephropathy (greater than 300 mg/24 h) (n = 8). Six male age-matched non-diabetic persons served as control subjects. The transcapillary escape rate of glycated...... significant role in the development of late diabetic microvascular complications....

  12. Glycation alters ligand binding, enzymatic, and pharmacological properties of human albumin.

    Science.gov (United States)

    Baraka-Vidot, Jennifer; Planesse, Cynthia; Meilhac, Olivier; Militello, Valeria; van den Elsen, Jean; Bourdon, Emmanuel; Rondeau, Philippe

    2015-05-19

    Albumin, the major circulating protein in blood plasma, can be subjected to an increased level of glycation in a diabetic context. Albumin exerts crucial pharmacological activities through its drug binding capacity, i.e., ketoprofen, and via its esterase-like activity, allowing the conversion of prodrugs into active drugs. In this study, the impact of the glucose-mediated glycation on the pharmacological and biochemical properties of human albumin was investigated. Aggregation product levels and the redox state were quantified to assess the impact of glycation-mediated changes on the structural properties of albumin. Glucose-mediated changes in ketoprofen binding properties and esterase-like activity were evaluated using fluorescence spectroscopy and p-nitrophenyl acetate hydrolysis assays, respectively. With the exception of oxidative parameters, significant dose-dependent alterations in biochemical and functional properties of in vitro glycated albumin were observed. We also found that the dose-dependent increase in levels of glycation and protein aggregation and average molecular mass changes correlated with a gradual decrease in the affinity of albumin for ketoprofen and its esterase-like property. In parallel, significant alterations in both pharmacological properties were also evidenced in albumin purified from diabetic patients. Partial least-squares regression analyses established a significant correlation between glycation-mediated changes in biochemical and pharmacological properties of albumin, highlighting the important role for glycation in the variability of the drug response in a diabetic situation.

  13. Development of Diagnostic Fragment Ion Library for Glycated Peptides of Human Serum Albumin: Targeted Quantification in Prediabetic, Diabetic, and Microalbuminuria Plasma by Parallel Reaction Monitoring, SWATH, and MSE*

    OpenAIRE

    Korwar, Arvind M.; Vannuruswamy, Garikapati; Jagadeeshaprasad, Mashanipalya G.; Jayaramaiah, Ramesha H.; Bhat, Shweta; Regin, Bhaskaran S.; Ramaswamy, Sureshkumar; Giri, Ashok P.; Mohan, Viswanathan; Balasubramanyam, Muthuswamy; Kulkarni, Mahesh J.

    2015-01-01

    Human serum albumin is one of the most abundant plasma proteins that readily undergoes glycation, thus glycated albumin has been suggested as an additional marker for monitoring glycemic status. Hitherto, only Amadori-modified peptides of albumin were quantified. In this study, we report the construction of fragment ion library for Amadori-modified lysine (AML), N(ε)-(carboxymethyl)lysine (CML)-, and N(ε)-(carboxyethyl)lysine (CEL)-modified peptides of the corresponding synthetically modified...

  14. Betanin reduces the accumulation and cross-links of collagen in high-fructose-fed rat heart through inhibiting non-enzymatic glycation.

    Science.gov (United States)

    Han, Junyan; Tan, Chang; Wang, Yiheng; Yang, Shaobin; Tan, Dehong

    2015-02-05

    We attempted to determine whether betanin (from natural pigments) that has antioxidant properties would be protective against fructose-induced diabetic cardiac fibrosis in Sprague-Dawley rats. Fructose water solution (30%) was accessed freely, and betanin (25 and 100 mg/kg/d) was administered by intra-gastric gavage continuously for 60 d. Rats were sacrificed after overnight fast. The rat blood and left ventricle were collected. In vitro antiglycation assay in bovine serum albumin/fructose system was also performed. In rats treated only with fructose, levels of plasma markers: glucose, insulin, HOMA and glycated hemoglobin rised, left ventricle collagen accumulated and cross-linked, profibrotic factor-transforming growth factor (TGF)-β1 and connective tissue growth factor (CTGF) protein expression increased, and soluble collagen decreased, compared with those in normal rats, showing fructose induces diabetic cardiac fibrosis. Treatment with betanin antagonized the changes of these parameters, demonstrating the antifibrotic role of betanin in the selected diabetic models. In further mechanistic study, betanin decreased protein glycation indicated by the decreased levels of protein glycation reactive intermediate (methylglyoxal), advanced glycation end product (N(ε)-(carboxymethyl) lysine) and receptors for advanced glycation end products (AGEs), antagonized oxidative stress and nuclear factor-κB activation elicited by fructose feeding, suggesting inhibition of glycation, oxidative stress and nuclear factor-κB activation may be involved in the antifibrotic mechanisms. Betanin also showed anitglycative effect in BSA/fructose system, which supported that anitglycation was involved in betanin's protective roles in vivo. Taken together, the potential for using betanin as an auxillary therapy for diabetic cardiomyopathy deserves to be explored further. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  15. Skin beautification with oral non-hydrolized versions of carnosine and carcinine: Effective therapeutic management and cosmetic skincare solutions against oxidative glycation and free-radical production as a causal mechanism of diabetic complications and skin aging.

    Science.gov (United States)

    Babizhayev, Mark A; Deyev, Anatoliy I; Savel'yeva, Ekaterina L; Lankin, Vadim Z; Yegorov, Yegor E

    2012-10-01

    Advanced glycation Maillard reaction end products (AGEs) are causing the complications of diabetes and skin aging, primarily via adventitious and cross-linking of proteins. Long-lived proteins such as structural collagen are particularly implicated as pathogenic targets of AGE processes. The formation of α-dicarbonyl compounds represents an important step for cross-linking proteins in the glycation or Maillard reaction. The purpose of this study was to investigate the contribution of glycation coupled to the glycation free-radical oxidation reactions as markers of protein damage in the aging of skin tissue proteins and diabetes. To elucidate the mechanism for the cross-linking reaction, we studied the reaction between a three-carbon α-dicarbonyl compound, methylglyoxal, and amino acids using EPR spectroscopy, a spectrophotometric kinetic assay of superoxide anion production at the site of glycation and a chemiluminescence technique. The transglycating activity, inhibition of transition metal ions peroxidative catalysts, resistance to hydrolysis of carnosine mimetic peptide-based compounds with carnosinase and the protective effects of carnosine, carcinine and related compounds against the oxidative damage of proteins and lipid membranes were assessed in a number of biochemical and model systems. A 4-month randomized, double-blind, controlled study was undertaken including 42 subjects where the oral supplement of non-hydrolized carnosine (Can-C Plus® formulation) was tested against placebo for 3 months followed by a 1-month supplement-free period for both groups to assess lasting effects. Assessment of the age-related skin parameters and oral treatment efficacy measurements included objective skin surface evaluation with Visioscan® VC 98 and visual assessment of skin appearance parameters. The results together confirm that a direct one-electron transfer between a Schiff base methylglyoxal dialkylimine (or its protonated form) and methylglyoxal is responsible for

  16. Anti-glycated and antiradical activities in vitro of polysaccharides from Ganoderma capense.

    Science.gov (United States)

    Yan, Chunyan; Kong, Fansheng; Zhang, Dezhi; Cui, Jiangxia

    2013-01-01

    Ganoderma capense is a Ganoderma species and is widely used, especially in Asia, as a well-known medicinal mushroom for health-promoting effect and for treatment of chronic diseases, such as diabetes, aging, etc. G. capense is rich of polysaccharide. To isolate the polysaccharides from G. capense and evaluate their anti-glycated and antiradical activities in vitro. The dried powder of submerged fermentation culturing mycelium of G. capense was defatted, extracted with water/alkaline water followed by ethanol precipitation and deproteinated. And four crude polysaccharides, named as GC50, GC70, GC90 and GCB, were obtained. For the first time, the in vitro anti-glycated activities of the four samples were studied by non-enzymatic glycation reaction. Then, the DPPH radical and hydroxyl radical assays were established to estimate the antiradical capacity of the four samples. Meanwhile the contents of polysaccharides were determined by phenol-sulphuric acid colorimetry. Preliminary antiradical in vitro studies indicated that the four crude polysaccharides showed concentration-dependent scavenging abilities on DPPH and hydroxyl radicals. The evaluation of anti-glycation activity suggested that GC70 had good potential for inhibiting the formation of advanced glycation end products. Time- and dose-dependent effects were also observed for all GC70 samples.

  17. Glucagon-like peptide-1 counteracts the detrimental effects of Advanced Glycation End-Products in the pancreatic beta cell line HIT-T 15

    International Nuclear Information System (INIS)

    Puddu, A.; Storace, D.; Durante, A.; Odetti, P.; Viviani, G.L.

    2010-01-01

    Research highlights: → GLP-1 prevents AGEs-induced cell death. → GLP-1 prevents AGEs-induced oxidative stress. → GLP-1 ameliorated AGEs-induced cell dysfunction. → GLP-1 attenuates AGEs-induced RAGE increment. → GLP-1 counteracts AGEs-induced pancreatic cell death and dysfunction. -- Abstract: Advanced Glycation End-Products (AGEs), a group of compounds resulting from the non-enzymatic reaction of reducing sugars with the free amino group of proteins, are implicated in diabetic complications. We previously demonstrated that exposure of the pancreatic islet cell line HIT-T 15 to high concentrations of AGEs significantly decreases cell proliferation and insulin secretion, and affects transcription factors regulating insulin gene transcription. The glucagon-like peptide-1 (GLP-1) is an incretin hormone that increases proinsulin biosynthesis, stimulates insulin secretion, and improves pancreatic beta-cell viability. The aim of this work was to investigate the effects of GLP-1 on the function and viability of HIT-T 15 cells cultured with AGEs. HIT-T 15 cells were cultured for 5 days in presence of AGEs alone, or supplemented with 10 nmol/l GLP-1. Cell viability, insulin secretion, redox balance, and expression of the AGEs receptor (RAGE) were then determined. The results showed that GLP-1 protected beta cell against AGEs-induced cell death preventing both apoptosis and necrosis. Moreover, addition of GLP-1 to the AGEs culture medium restored the redox balance, improved the responsiveness to glucose, and attenuated AGEs-induced RAGE expression. These findings provide evidence that GLP-1 protects beta cells from the dangerous effects of AGEs.

  18. Glycation of extracellular matrix proteins and its role in atherosclerosis 

    Directory of Open Access Journals (Sweden)

    Aleksandra Kuzan

    2012-10-01

    Full Text Available Glycation consists in formation of advanced glycation end-products (AGE during non-enzymatic reaction between reducing sugars and proteins, lipids or nucleic acids. This review is focused mainly on glycation of collagen and its role in acceleration of vascular disease. Collagen is an extracellular matrix protein characterized by unique structure forming fibrils with great anti-tensile and anti-breaking strength. The protein builds the connective tissue and is responsible for biomechanical properties of blood vessels. It is reported that higher content of glycated collagen correlates with lower elasticity and greater toughness of the vessel walls and, as a consequence, a faster rate of atherosclerosis development. Numerous mechanisms connected with AGE formation are involved in atherogenesis, among others: receptor-mediated production of free radicals, triggering an inflammatory process, activation of leukocytes and thrombocytes, facilitation of LDL binding, change in level of growth factors, adhesion molecules, MMP and some other proteins’ expression. The coverages allow the development of therapeutic strategies to prevent or slow down the pathological processes connected with glycation of collagen and other proteins in the artery wall. The main strategies are based on limitation of exogenous AGE, consumption of products which contain rutin, treatment with drugs which inhibit AGE formation, such aspyridoxamine, and chemicals which are able to cleave already formed AGE protein-protein crosslinks, such as ALT-711.

  19. Exploring the antioxidant property of bioflavonoid quercetin in preventing DNA glycation: A calorimetric and spectroscopic study

    International Nuclear Information System (INIS)

    Sengupta, Bidisa; Uematsu, Takashi; Jacobsson, Per; Swenson, Jan

    2006-01-01

    Reducing sugars for example glucose, fructose, etc., and their phosphate derivatives non-enzymatically glycate biological macromolecules (e.g., proteins, DNA and lipids) and is related to the production of free radicals. Here we present a novel study, using differential scanning calorimetry (DSC) along with UV/Vis absorption and photon correlation spectroscopy (PCS), on normal and glycated human placenta DNA and have explored the antioxidant property of the naturally occurring polyhydroxy flavone quercetin (3,3',4',5,7-pentahydroxyflavone) in preventing the glycation. The decrease in the absorption intensity of DNA in presence of sugars clearly indicates the existence of sugar molecules between the two bases of a base pair in the duplex DNA molecule. Variations were perceptible in the PCS relaxation profiles of normal and glycated DNA. The melting temperature of placenta DNA was decreased when glycated suggesting a decrease in the structural stability of the double-stranded glycated DNA. Our DSC and PCS data showed, for the first time, that the dramatic changes in the structural properties of glycated DNA can be prevented to a significant extent by adding quercetin. This study provides valuable insights regarding the structure, function, and dynamics of normal and glycated DNA molecules, underlying the manifestation of free radical mediated diseases, and their prevention using therapeutically active naturally occurring flavonoid quercetin

  20. Anti-glycation and anti-oxidation properties of Capsicum frutescens and Curcuma longa fruits: possible role in prevention of diabetic complication.

    Science.gov (United States)

    Khan, Ibrar; Ahmad, Haroon; Ahmad, Bashir

    2014-09-01

    The accumulation of advanced glycationend products (AGE's) in the body, due to the non-enzymatic glycation of proteins is associated with several pathological conditions like aging and diabetes mellitus. Hence a plant having anti-glycation and anti-oxidation potentials may serve as therapeutic agent for diabetic complications and aging. In this study the anti-glycation and anti-oxidation properties of crude methanolic extracts of fruits of Capsicum frutescens and Curcuma longa were investigated. Among the two C. frutescens had more anti-glycation ability with a minimum inhibitory concentration (MIC50) of 90βg/mLas compared to 324βg/mL MIC50 of C. longa. Curcuma longa had the more anti-oxidation potential i.e. 35.01, 30.83 and 28.08% at 0.5mg, 0.25mg and 0.125mg respectively.

  1. The effect of Cerasus avium stalk extract on albumin glycation reaction

    Directory of Open Access Journals (Sweden)

    Mohadeseh Abdoli

    2014-10-01

    Full Text Available Background: Non-enzymatic glycosylation of proteins is the major cause of diabetic complications. The inhibition of glycation process can reduce complications of diabetes. In the Iranian traditional medicine, the decoction (boiled extraction of Cerasus avium stalk is used as a hypoglycemic agent. The aim of this study was to investigate the in vitro inhibitory effects of decoction and ethanolic and aqueous extracts of Cerasus avium stalk on albumin glycation reaction. Methods: In this experimental study, first, the ethanolic, aqueous and decoction extracts of Cerasus avium stalk were prepared. Then, different concentrations of these extracts were prepared and added to albumin and glucose solutions. Finally, compared to control group that was not treated with any extracts, the albumin glycation rate in the groups treated with various concentrations of extracts was evaluated using TBA (thio-barbituric acid method. Results: The results showed that compared to control group, decoction of Cerasus avium stalk in the concentrations of 20, 10 and 2 mg/dl could reduce albumin glycation to 85.10±1.55, 72.35±1.75 and 51.25±1.22 %, respectively (P>0.001. Moreover, in the concentration of 20 mg/dl, the inhibitory effect of decoction of Cerasus avium stalk on the albumin glycation reaction was higher than those of aqueous (P=0.021 and ethanolic (P=0.009 extracts. Conclusion: The findings showed that the extracs of Cerasus avium stalk, in particular in the decoction form, could significantly reduce the rate of albumin glycation; therefore, it can be used for decreasing diabetes mellitus complications.

  2. Purification and Identification of Antioxidant Peptides from Enzymatic Hydrolysate of Spirulina platensis.

    Science.gov (United States)

    Yu, Jie; Hu, Yuanliang; Xue, Mingxiong; Dun, Yaohao; Li, Shenao; Peng, Nan; Liang, Yunxiang; Zhao, Shumao

    2016-07-28

    The aim of this study was to isolate antioxidant peptides from an enzymatic hydrolysate of Spirulina platensis. A novel antioxidant peptide was obtained by ultrafiltration, gel filtration chromatography, and reverse-phase high-performance liquid chromatography, with the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay used to measure the antioxidant activity, and the sequence was determined to be Pro-Asn-Asn (343.15 Da) by electrospray ionization tandem mass spectrometry. This peptide was synthesized to confirm its antioxidant properties, and it exhibited 81.44 ± 0.43% DPPH scavenging activity at 100 µg/ml, which was similar to that of glutathione (82.63 ± 0.56%). Furthermore, the superoxide anion and hydroxyl free-radical scavenging activities and the SOD activity of the peptide were 47.84 ± 0.49%, 54.01 ± 0.82%, and 12.55 ± 0.75%, respectively, at 10 mg/ml. These results indicate that S. platensis is a good source of antioxidant peptides, and that its hydrolysate may have important applications in the pharmaceutical and food industries.

  3. Effects of insulin therapy on porosity, non-enzymatic glycation and mechanical competence in the bone of rats with type 2 diabetes mellitus.

    Science.gov (United States)

    Campbell, G M; Tiwari, S; Picke, A-K; Hofbauer, C; Rauner, M; Morlock, M M; Hofbauer, L C; Glüer, C-C

    2016-10-01

    Type 2 diabetes mellitus increases skeletal fragility; however, the contributing mechanisms and optimal treatment strategies remain unclear. We studied the effects of diabetes and insulin therapy on non-enzymatic glycation (NEG), cortical porosity (Ct.Po) and biomechanics of the bone tissue in Zucker Diabetic Fatty (ZDF) rats. Eleven-week old ZDF diabetic and non-diabetic rats were given insulin to achieve glycaemic control or vehicle seven days per week over twelve weeks (insulin dose adapted individually 0.5 international units (IU) at week 1 to 13.0IU at week 12). The right femora were excised, micro-CT scanned, and tested in 3-point bending to measure biomechanics. NEG of the midshaft was determined from bulk fluorescence. Diabetes led to increased NEG (+50.1%, p=0.001) and Ct.Po (+22.9%, p=0.004), as well as to reduced mechanical competence (max. stress: -14.2%, p=0.041, toughness: -29.7%, p=0.016) in the bone tissue. NEG and Ct.Po both correlated positively to serum glucose (NEG: R(2)=0.41, p1, Ct.Po: R(2)=0.34, p=0.003) and HbA1c (NEG: R(2)=0.42, p1, Ct.Po: R(2)=0.28, p=0.008) levels, while NEG correlated negatively with bone biomechanics (elastic modulus: R(2)=0.21, p=0.023, yield stress: R(2)=0.17, p=0.047). Twelve weeks of insulin therapy had no significant effect on NEG or Ct.Po, and was unable to improve the mechanical competence of the bone tissue. A reduction of mechanical competence was observed in the bone tissue of the diabetic rats, which was explained in part by increased collagen NEG. Twelve weeks of insulin therapy did not alter NEG, Ct.Po or bone biomechanics. However, significant correlations between NEG and serum glucose and HbA1c were observed, both of which were reduced with insulin therapy. This suggests that a longer duration of insulin therapy may be required to reduce the NEG of the bone collagen and restore the mechanical competence of diabetic bone. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Enzymatic synthesis of tRNA-peptide conjugates and spectroscopic studies of fluorine-modified RNA

    International Nuclear Information System (INIS)

    Graber, D.

    2010-01-01

    The research presented in this thesis concerns the enzymatic synthesis of artificially modified tRNA, in particular the preparation of non-hydrolysable tRNA-peptide conjugates. Another focus is on NMR-spectroscopic investigations of fluorine-modified RNA. In both projects, chemical methods were developed to address specific RNA-biological research questions. In the first part of this thesis the preparation of tRNA-peptide conjugates with a non-hydrolysable 3'-amide linkage is presented. These molecules are of high relevance for the characterization of ribosomal processes that occur in the peptidyl transferase center (such as peptide bond formation, peptide release, or translocation) using X-ray crystallography and biochemical methods. First, a novel concept to prepare chemically modified ('labeled') tRNA was elaborated based on the combination of solid-phase synthesis and enzymatic ligation. Thereby, a variety of differently labeled tRNAs was achieved. Moreover, the most successful high-yield ligation sites were identified to be situated within the TΨ C-loop. Optimization of the synthesis and the corresponding HPLC-purification of the conjugates were initially conducted with puromycin derivatized tRNA. In the course of this project, also two tRNAs with a ribose 3'-amino group at the terminal adenosine A76 were synthesized. For that purpose a protection group pattern had to be developed to obtain a functionalized solid-support bound to 3'-amino-3'-deoxyadenosine which was appropriate for RNA solid-phase synthesis. The successful preparation of tRNA-peptide conjugates was accomplished in cooperation with Holger Moroder and Jessica Steger (Micura group) who contributed short synthetic RNA-peptide conjugates. These fragments represented the tRNA 3'-termini that were required for exploring the new ligation strategies for non-hydrolisable tRNA - a main aim of this thesis. If the 5'-fragments are synthesized by solid-phase synthesis or in vitro transcription they do not

  5. In situ characterization of advanced glycation end products (AGEs) in collagen and model extracellular matrix by solid state NMR.

    Science.gov (United States)

    Li, R; Rajan, R; Wong, W C V; Reid, D G; Duer, M J; Somovilla, V J; Martinez-Saez, N; Bernardes, G J L; Hayward, R; Shanahan, C M

    2017-12-14

    Non-enzymatic glycation of extracellular matrix with (U- 13 C 5 )-d-ribose-5-phosphate (R5P), enables in situ 2D ssNMR identification of many deleterious protein modifications and crosslinks, including previously unreported oxalamido and hemiaminal (CH 3 -CH(OH)NHR) substructures. Changes in charged residue proportions and distribution may be as important as crosslinking in provoking and understanding harmful tissue changes.

  6. Comparison of non-volatile umami components in chicken soup and chicken enzymatic hydrolysate.

    Science.gov (United States)

    Kong, Yan; Yang, Xiao; Ding, Qi; Zhang, Yu-Yu; Sun, Bao-Guo; Chen, Hai-Tao; Sun, Ying

    2017-12-01

    Umami taste is an important part to the taste of chicken. To isolate and identify non-volatile umami compounds, fractions from chicken soup and hydrolysate were prepared and analyzed. Amino acids were analyzed by amino acid analyzer. Organic acids and nucleotides were determined by ultra-performance liquid chromatography. Separation procedures utilizing ultrafiltration, Sephadex G-15 and reversed-phase high-performance liquid chromatography were used to isolate umami taste peptides. Combined with sensory evaluation and LC-Q-TOF-MS, the amino acid sequences of 12 oligopeptides were determined. The amount of taste compounds was higher in chicken enzymatic hydrolysate than that of chicken soup. Eight oligopeptides from chicken enzymatic hydrolysate were identified, including Ala-Asp, Ala-Met, His-Ser, Val-Glu, Ala-Glu, Asp-Ala-Gly, Glu-Asp and Ala-Glu-Ala. Four oligopeptides from chicken soup were identified, including Val-Thr, Ala-His, Ala-Phe and Thr-Glu. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. DAF in diabetic patients is subject to glycation/inactivation at its active site residues.

    Science.gov (United States)

    Flückiger, Rudolf; Cocuzzi, Enzo; Nagaraj, Ram H; Shoham, Menachem; Kern, Timothy S; Medof, M Edward

    2018-01-01

    Decay accelerating factor (DAF or CD55) is a cell associated C3 and C5 convertase regulator originally described in terms of protection of self-cells from systemic complement but now known to modulate adaptive T cell responses. It is expressed on all cell types. We investigated whether nonenzymatic glycation could impair its function and potentially be relevant to complications of diabetes mellitus and other conditions that result in nonenzymatic glycation including cancer, Alzheimer's disease, and aging. Immunoblots of affinity-purified DAF from erythrocytes of patients with diabetes showed pentosidine, glyoxal-AGEs, carboxymethyllysine, and argpyrimidine. HPLC/MS analyses of glucose modified DAF localized the sites of AGE modifications to K 125 adjacent to K 126 , K 127 at the junction of CCPs2-3 and spatially near R 96 , and R 100 , all identified as being critical for DAF's function. Functional analyses of glucose or ribose treated DAF protein showed profound loss of its regulatory activity. The data argue that de-regulated activation of systemic complement and de-regulated activation of T cells and leukocytes could result from non-enzymatic glycation of DAF. Copyright © 2017. Published by Elsevier Ltd.

  8. Metal nanostructures for non-enzymatic glucose sensing

    International Nuclear Information System (INIS)

    Tee, Si Yin; Teng, Choon Peng; Ye, Enyi

    2017-01-01

    This review covers the recent development of metal nanostructures in electrochemical non-enzymatic glucose sensing. It highlights a variety of nanostructured materials including noble metals, other transition metals, bimetallic systems, and their hybrid with carbon-based nanomaterials. Particularly, attention is devoted to numerous approaches that have been implemented for improving the sensors performance by tailoring size, shape, composition, effective surface area, adsorption capability and electron-transfer properties. The correlation of the metal nanostructures to the glucose sensing performance is addressed with respect to the linear concentration range, sensitivity and detection limit. In overall, this review provides important clues from the recent scientific achievements of glucose sensor nanomaterials which will be essentially useful in designing better and more effective electrocatalysts for future electrochemical sensing industry. - Highlights: • Overview of recent development of metal nanostructures in electrochemical non-enzymatic glucose sensing. • Special attention is focussed on noble metals, other transition metals, bimetallic systems, and their hybrid with carbon-based nanomaterials. • Merits and limitations of various metal nanostructures in electrochemical non-enzymatic glucose sensing. • Strategies to improve the glucose sensing performance of metal nanostructures as electrocatalysts.

  9. Anti-Inflammatory and Antioxidant Properties of Peptides Released from β-Lactoglobulin by High Hydrostatic Pressure-Assisted Enzymatic Hydrolysis.

    Science.gov (United States)

    Bamdad, Fatemeh; Bark, Seonghee; Kwon, Chul Hee; Suh, Joo-Won; Sunwoo, Hoon

    2017-06-07

    β-lactoglobulin hydrolysates (BLGH) have shown antioxidant, antihypertensive, antimicrobial, and opioid activity. In the current study, an innovative combination of high hydrostatic pressure and enzymatic hydrolysis (HHP-EH) was used to increase the yield of short bioactive peptides, and evaluate the anti-inflammatory and antioxidant properties of the BLGH produced by the HHP-EH process. BLG was enzymatically hydrolyzed by different proteases at an enzyme-to-substrate ratio of 1:100 under HHP (100 MPa) and compared with hydrolysates obtained under atmospheric pressure (AP-EH at 0.1 MPa). The degree of hydrolysis (DH), molecular weight distribution, and the antioxidant and anti-inflammatory properties of hydrolysates in chemical and cellular models were evaluated. BLGH obtained under HHP-EH showed higher DH than the hydrolysates obtained under AP-EH. Free radical scavenging and the reducing capacity were also significantly stronger in HHP-BLGH compared to AP-BLGH. The BLGH produced by alcalase (Alc) (BLG-Alc) showed significantly higher antioxidant properties among the six enzymes examined in this study. The anti-inflammatory properties of BLG-HHP-Alc were observed in lipopolysaccharide-stimulated macrophage cells by a lower level of nitric oxide production and the suppression of the synthesis of pro-inflammatory cytokines. Peptide sequencing revealed that 38% of the amino acids in BLG-HHP-Alc are hydrophobic and aromatic residues, which contribute to its anti-inflammatory properties. Enzymatic hydrolysis of BLG under HHP produces a higher yield of short bioactive peptides with potential antioxidant and anti-inflammatory effects.

  10. Fucoidan as an inhibitor of thermally induced collagen glycation examined by acetate electrophoresis.

    Science.gov (United States)

    Pielesz, Anna; Paluch, Jadwiga

    2014-08-01

    Non-enzymatic glycation (Maillard reaction) in vitro could be a simple method to obtain glycoconjugates for studying their biological properties. Hence, fucoidan was retained by acetate electrophoresis indicating a strong interaction with the protein. A loss of colour in fucoidan bands was found for samples incubated with collagen as compared with samples of free fucoidan. Also under in vitro conditions at 100°C - simulating a sudden burn incident - fucoidan binds with collagen as a result of the Maillard reaction. In contrast, the colour of the fucoidan bands intensified for samples incubated with collagen, with the addition of glucose. Electrophoretic analyses were carried out after heating the samples to a temperature simulating a burn incident. The bands were found to intensify for samples incubated with collagen during a 30-day-long incubation. Thus, spontaneous in vitro glycation - i.e. without the addition of glucose - was confirmed. This process is highly intensified both by the temperature and time of incubation. For a sample incubated in vitro in a fucoidan solution containing glucose, glycation was confirmed in a preliminary FTIR and acetate electrophoresis examinations, occurring in collagen obtained from chicken skins. In particular, a new band emerging around 1746 cm(-1) was observed for above samples, as was its increasing intensity, as compared with samples without the addition of glucose. In the collagen glycation assay, while glucose reacts with collagen and forms cross-linked aggregates, fucoidan decreases the process of aggregation and recovery of native collagen. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Implication of advanced glycation end products (Ages) and their receptor (Rage) on myocardial contractile and mitochondrial functions.

    Science.gov (United States)

    Neviere, Remi; Yu, Yichi; Wang, Lei; Tessier, Frederic; Boulanger, Eric

    2016-08-01

    Advanced glycation end products (AGEs) play an important role for the development and/or progression of cardiovascular diseases, mainly through induction of oxidative stress and inflammation. AGEs are a heterogeneous group of molecules formed by non-enzymatic reaction of reducing sugars with amino acids of proteins, lipids and nucleic acids. AGEs are mainly formed endogenously, while recent studies suggest that diet constitutes an important exogenous source of AGEs. The presence and accumulation of AGEs in various cardiac cell types affect extracellular and intracellular structure and function. AGEs contribute to a variety of microvascular and macrovascular complications through the formation of cross-links between molecules in the basement membrane of the extracellular matrix and by engaging the receptor for advanced glycation end products (RAGE). Activation of RAGE by AGEs causes up regulation of the transcription factor nuclear factor-κB and its target genes. of the RAGE engagement stimulates oxidative stress, evokes inflammatory and fibrotic reactions, which all contribute to the development and progression of devastating cardiovascular disorders. This review discusses potential targets of glycation in cardiac cells, and underlying mechanisms that lead to heart failure with special interest on AGE-induced mitochondrial dysfunction in the myocardium.

  12. Glycation inhibitors extend yeast chronological lifespan by reducing advanced glycation end products and by back regulation of proteins involved in mitochondrial respiration.

    Science.gov (United States)

    Kazi, Rubina S; Banarjee, Reema M; Deshmukh, Arati B; Patil, Gouri V; Jagadeeshaprasad, Mashanipalya G; Kulkarni, Mahesh J

    2017-03-06

    Advanced Glycation End products (AGEs) are implicated in aging process. Thus, reducing AGEs by using glycation inhibitors may help in attenuating the aging process. In this study using Saccharomyces cerevisiae yeast system, we show that Aminoguanidine (AMG), a well-known glycation inhibitor, decreases the AGE modification of proteins in non-calorie restriction (NR) (2% glucose) and extends chronological lifespan (CLS) similar to that of calorie restriction (CR) condition (0.5% glucose). Proteomic analysis revealed that AMG back regulates the expression of differentially expressed proteins especially those involved in mitochondrial respiration in NR condition, suggesting that it switches metabolism from fermentation to respiration, mimicking CR. AMG induced back regulation of differentially expressed proteins could be possibly due to its chemical effect or indirectly by glycation inhibition. To delineate this, Metformin (MET), a structural analog of AMG and a mild glycation inhibitor and Hydralazine (HYD), another potent glycation inhibitor but not structural analog of AMG were used. HYD was more effective than MET in mimicking AMG suggesting that glycation inhibition was responsible for restoration of differentially expressed proteins. Thus glycation inhibitors particularly AMG, HYD and MET extend yeast CLS by reducing AGEs, modulating the expression of proteins involved in mitochondrial respiration and possibly by scavenging glucose. This study reports the role of glycation in aging process. In the non-caloric restriction condition, carbohydrates such as glucose promote protein glycation and reduce CLS. While, the inhibitors of glycation such as AMG, HYD, MET mimic the caloric restriction condition by back regulating deregulated proteins involved in mitochondrial respiration which could facilitate shift of metabolism from fermentation to respiration and extend yeast CLS. These findings suggest that glycation inhibitors can be potential molecules that can be used

  13. Consequential secondary structure alterations and aggregation during prolonged casein glycation.

    Science.gov (United States)

    Jindal, Supriya; Naeem, Aabgeena

    2013-05-01

    Non-enzymatic glycosylation (glycation) of casein is a process used not just to ameliorate the quality of dairy products but also to increase the shelf life of canned foods, including baby milk supplements. Incubation of κ-casein with reducing sugars for 15 days at physiological temperature showed the formation of a molten globule state at day 9 and 12 during fructation and glucation respectively. This state exhibits substantial secondary structure and maximum ANS binding. Later on, glycation resulted in the formation of aggregates at day 12 in presence of fructose and day 15 in presence of glucose. Aggregates possess extensive β-sheet structure as revealed by far-UV CD and FTIR. These aggregates showed altered tryptophan environment, decrease ANS binding relative to molten globule state and increase in Thioflavin T fluorescence. Aggregates were also accompanied by the accumulation of AGEs, indicative of structural damage to the protein and formation of potentially harmful species at the physiological level. Fructose was more reactive than glucose and thus caused early and significant changes in the protein. From our studies, we conclude that controlling the extent of the Maillard reaction in the food industry is essential to counter its negative effects and expand its safety spectrum.

  14. Comparison of the role that entropy has played in processes of non-enzymatic and enzymatic catalysis

    International Nuclear Information System (INIS)

    Dixon Pineda, Manuel Tomas

    2012-01-01

    The function that entropy has played is compared in processes of non-enzymatic and enzymatic catalysis. The processes followed are showed: the kinetics of the acid hydrolysis of 3-pentyl acetate and cyclopentyl acetate catalyzed by hydrochloric acid and enzymatic hydrolysis of ethyl acetate and γ-butyrolactone catalyzed by pig liver esterase. The activation parameters of Eyring were determined for each process and interpreted the contribution of the entropy of activation for catalysis in this type of model reactions. (author) [es

  15. A Perspective on Reagent Diversity and Non-covalent Binding of Reactive Carbonyl Species (RCS and Effector Reagents in Non-enzymatic Glycation (NEG: Mechanistic Considerations and Implications for Future Research

    Directory of Open Access Journals (Sweden)

    Kenneth J. Rodnick

    2017-06-01

    Full Text Available This perspective focuses on illustrating the underappreciated connections between reactive carbonyl species (RCS, initial binding in the nonenzymatic glycation (NEG process, and nonenzymatic covalent protein modification (here termed NECPM. While glucose is the central species involved in NEG, recent studies indicate that the initially-bound glucose species in the NEG of human hemoglobin (HbA and human serum albumin (HSA are non-RCS ring-closed isomers. The ring-opened glucose, an RCS structure that reacts in the NEG process, is most likely generated from previously-bound ring-closed isomers undergoing concerted acid/base reactions while bound to protein. The generation of the glucose RCS can involve concomitantly-bound physiological species (e.g., inorganic phosphate, water, etc.; here termed effector reagents. Extant NEG schemes do not account for these recent findings. In addition, effector reagent reactions with glucose in the serum and erythrocyte cytosol can generate RCS (e.g., glyoxal, glyceraldehyde, etc.. Recent research has shown that these RCS covalently modify proteins in vivo via NECPM mechanisms. A general scheme that reflects both the reagent and mechanistic diversity that can lead to NEG and NECPM is presented here. A perspective that accounts for the relationships between RCS, NEG, and NECPM can facilitate the understanding of site selectivity, may help explain overall glycation rates, and may have implications for the clinical assessment/control of diabetes mellitus. In view of this perspective, concentrations of ribose, fructose, Pi, bicarbonate, counter ions, and the resulting RCS generated within intracellular and extracellular compartments may be of importance and of clinical relevance. Future research is also proposed.

  16. Glycation in human fingernail clippings using ATR-FTIR spectrometry, a new marker for the diagnosis and monitoring of diabetes mellitus.

    Science.gov (United States)

    Coopman, Renaat; Van de Vyver, Thijs; Kishabongo, Antoine Sadiki; Katchunga, Philippe; Van Aken, Elisabeth H; Cikomola, Justin; Monteyne, Tinne; Speeckaert, Marijn M; Delanghe, Joris R

    2017-01-01

    Although HbA1c is a good diagnostic tool for diabetes, the precarity of the health system and the costs limit the use of this biomarker in developing countries. Fingernail clippings contain ±85% of keratins, which are prone to glycation. Nail keratin glycation may reflect the average glycemia over the last months. We explored if attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) can be used as a non-invasive tool for assessing glycation in diabetes. Using ATR-FTIR spectroscopy, glycation and deglycation experiments with fructosamine 3-kinase allowed to identify the spectrum that corresponds with keratin glycation in fingernail clippings. Clippings of 105 healthy subjects and 127 diabetics were subjected to the standardized ATR-FTIR spectroscopy method. In vitro glycation resulted in an increased absorption at 1047cm -1 . Following enzymatic deglycation, this peak diminished significantly, proving that the AUC between 970 and 1140cm -1 corresponded with glycated proteins. Within-run CV of the assay was 3%. Storage of nail clippings at 37°C for 2weeks did not significantly change results. In diabetics, glycated nail protein concentrations (median: 1.51μmol/g protein, IQR: 1.37-1.85μmol/g protein) were significantly higher than in the controls (median: 1.19μmol/g protein, IQR: 1.09-1.26μmol/g protein) (p<0.0001). ROC analysis yielded an AUC of 0.92 at a cut-off point of 1.28μmol/g nail (specificity: 82%; sensitivity: 90%). No correlation was observed between the glycated nail protein concentrations and HbA1c. Protein glycation analysis in fingernails with ATR-FTIR spectroscopy could be an alternative affordable technique for diagnosing and monitoring diabetes. As the test does not consume reagents, and the preanalytical phase is extremely robust, the test could be particularly useful in developing countries. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  17. High volumetric power density, non-enzymatic, glucose fuel cells.

    Science.gov (United States)

    Oncescu, Vlad; Erickson, David

    2013-01-01

    The development of new implantable medical devices has been limited in the past by slow advances in lithium battery technology. Non-enzymatic glucose fuel cells are promising replacement candidates for lithium batteries because of good long-term stability and adequate power density. The devices developed to date however use an "oxygen depletion design" whereby the electrodes are stacked on top of each other leading to low volumetric power density and complicated fabrication protocols. Here we have developed a novel single-layer fuel cell with good performance (2 μW cm⁻²) and stability that can be integrated directly as a coating layer on large implantable devices, or stacked to obtain a high volumetric power density (over 16 μW cm⁻³). This represents the first demonstration of a low volume non-enzymatic fuel cell stack with high power density, greatly increasing the range of applications for non-enzymatic glucose fuel cells.

  18. Effect of methylglyoxal on the physico-chemical and biological properties of low-density lipoprotein

    NARCIS (Netherlands)

    Schalkwijk, C.G.; Vermeer, M.A.; Stehouwer, C.D.A.; Koppele, J. te; Princen, H.M.G.; Hinsbergh, V.W.M. van

    1998-01-01

    In patients with diabetes, non-enzymatic glycation of low-density lipoprotein (LDL) has been suggested to be involved in the development of atherosclerosis. α-Dicarbonyl compounds were identified as intermediates in the non-enzymatic glycation and increased levels were reported in patients with

  19. Enzymatic characterization of peptidic materials isolated from aqueous solutions of ammonium cyanide (pH 9) and hydrocyanic acid (pH 6) exposed to ionizing radiation.

    Science.gov (United States)

    Niketic, V; Draganić, Z; Nesković, S; Draganić, I

    1982-01-01

    The enzymatic digestion of some radiolytically produced peptidic materials was examined. The substrates were compounds isolated from 0.1 molar solutions of NH4CN (pH 9) and HCN (pH 6), after their exposure to gamma rays from a 60Co source (15-20 Mrad doses). Commercial proteolytic enzymes pronase and aminopeptidase M were used. The examined materials were of composite nature and proteolytic action was systematically observed after their subsequent purification. In some fractions the effect was found to be positive with up to 30% of peptide bonds cleaved with respect to the amino acid content. These findings support our previous conclusions on the free radical induced formation of peptidic backbones without the intervention of amino acids. Some side effects were also noted which might be of interest in observations on enzymatic cleavage of other composite peptidic materials of abiotic origin.

  20. Peptide synthesis by enzymatic catalysis: new application to the total radiosynthesis of the tritiated leucine-enkephalin hormone, using Y carboxypeptidase

    International Nuclear Information System (INIS)

    Hellio, F.

    1986-01-01

    A new method of enzymatic labelling of peptide hormones is described. The enzyme used, a protease, Y carboxypeptidase is able, in some conditions, to catalyze the formation of peptide bounds. This property has been used for the synthesis of a pentapeptide, the tritiated leucine-enkephalin, with the incorporation of every radioactive amino acid. The specific radioactivity of the labelled molecule is 139 Ci/mmole and its biological properties (receptor binding and immunoreactivity) are identical with native leucine-enkephalin properties [fr

  1. Glycation and transglutaminase mediated glycosylation of fish gelatin peptides with glucosamine enhance bioactivity.

    Science.gov (United States)

    Hong, Pui Khoon; Gottardi, Davide; Ndagijimana, Maurice; Betti, Mirko

    2014-01-01

    A mixture of novel glycopeptides from glycosylation between cold water fish skin gelatin hydrolysates and glucosamine (GlcN) via transglutaminase (TGase), as well as glycation between fish gelatin hydrolysate and GlcN were identified by their pattern of molecular distribution using MALDI-TOF-MS. Glycated/glycosylated hydrolysates showed superior bioactivity to their original hydrolysates. Alcalase-derived fish skin gelatin hydrolysate glycosylated with GlcN in the presence of TGase at 25°C (FAT25) possessed antioxidant activity when tested in a linoleic acid oxidation system, when measured according to its 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity and when tested at the cellular level with human hepatocarcinoma (HepG2) cells as target cells. In addition, Alcalase-derived glycosylated hydrolysates showed specificity toward the inhibition of Escherichia coli (E. coli). The Flavourzyme-derived glycopeptides prepared at 37°C (FFC37 and FFT37) showed better DPPH scavenging activity than their native hydrolysates. The glycated Flavourzyme-derived hydrolysates were found to act as potential antimicrobial agents when incubated with E. coli and Bacillus subtilis. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. Advanced glycation endproducts form during ovalbumin digestion in the presence of fructose: Inhibition by chlorogenic acid.

    Science.gov (United States)

    Bains, Yasmin; Gugliucci, Alejandro; Caccavello, Russell

    2017-07-01

    One mechanism by which fructose could exert deleterious effects is through intestinal formation and absorption of pro-inflammatory advanced glycation endproducts via the Maillard reaction. We employed simulated stomach and duodenum digestion of ovalbumin (OVA) to test the hypothesis that advanced glycation endproducts (AGEs) are formed by fructose during simulated digestion of a ubiquitous food protein under model physiological conditions. OVA was subjected to simulated gastric and intestinal digestion using standard models, in presence of fructose or glucose (0-100mM). Peptide fractions were analyzed by fluorescence spectroscopy and intensity at Excitation: λ370nm, Emission: λ 440nm. AGE adducts formed between fructose and OVA, evidenced by the peptide fractions (fructose-AGE formation on a ubiquitous dietary protein under model physiological conditions. Our study also suggests ways to decrease the damage: enteral fructose-AGE formation may be partially inhibited by co-intake of beverages, fruits and vegetables with concentrations of phenolics high enough to serve as anti-glycation agents. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. PSA-alpha-2-macroglobulin complex is enzymatically active in the serum of patients with advanced prostate cancer and can degrade circulating peptide hormones.

    Science.gov (United States)

    Kostova, Maya B; Brennen, William Nathaniel; Lopez, David; Anthony, Lizamma; Wang, Hao; Platz, Elizabeth; Denmeade, Samuel R

    2018-08-01

    Prostate cancer cells produce high levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the tumor microenvironment but is presumed to be enzymatically inactive in the blood due to complex formation with serum protease inhibitors α-1-antichymotrypsin and α-2-macroglobulin (A2M). PSA-A2M complexes cannot be measured by standard ELISA assays and are also rapidly cleared from the circulation. Thus the exact magnitude of PSA production by prostate cancer cells is not easily measured. The PSA complexed to A2M is unable to cleave proteins but maintains the ability to cleave small peptide substrates. Thus, in advanced prostate cancer, sufficient PSA-A2M may be in circulation to effect total A2M levels, levels of cytokines bound to A2M and hydrolyze small circulating peptide hormones. Total A2M levels in men with advanced prostate cancer and PSA levels above 1000 ng/mL were measured by ELISA and compared to controls. Additional ELISA assays were used to measure levels of IL-6 and TGF-beta which can bind to A2M. The ability of PSA-A2M complexes to hydrolyze protein and peptide substrates was analyzed ± PSA inhibitor. Enzymatic activity of PSA-A2M in serum of men with high PSA levels was also assayed. Serum A2M levels are inversely correlated with PSA levels in men with advanced prostate cancer. Il-6 Levels are significantly elevated in men with PSA >1000 ng/mL compared to controls with PSA PSA-A2M complex in serum of men with PSA levels >1000 ng/mL can hydrolyze small fluorescently labeled peptide substrates but not large proteins that are PSA substrates. PSA can hydrolyze small peptide hormones like PTHrP and osteocalcin. PSA complexed to A2M retains the ability to degrade PTHrP. In advanced prostate cancer with PSA levels >1000 ng/mL, sufficient PSA-A2M is present in circulation to produce enzymatic activity against circulating small peptide hormones. Sufficient PSA is produced in advanced prostate cancer to alter

  4. Alzheimer's disease against peptides products of enzymatic cleavage APP protein: Biological, pathobiological and physico-chemical properties of fibrillating peptides.

    Science.gov (United States)

    Marszałek, Małgorzata

    2017-05-17

    Various peptides products of enzymatic cleavage of key for Alzheimer's disease Amyloid Precursor Protein (APP) are well known, but still are matter of scientific debate. The Aβ type products are especially challenging for experimental and medical research. This paper outlines several, still poorly known, biological and medical processes such as peptides biology, i.e., formation, biodistribution, translocation, transport and finally removal from brain compartments and body fluids like Intracellular Fluid (ICF), Cerebrospinal Fluid (CSF), Interstitial Fluid (ISF), blood serum or urine. In addition, the following studies concerning AD patients might prove challenging and simultaneously promising: peptides translocation through Blood-Brain - Barrier (BBB) and Blood-Cerebrospinal Fluid Barrier (BCSFB) and their removal from the brain according to a new concept of glymphatic system; - diagnostic difficulties that stem from physico-chemical properties and the nature of proteins or fibrillating peptides itself like low concentration, short half-live and from experimental-technical problems as well like high adsorption or low solubility of Aβ, tau or amylin. The study of diagnostic parameters is very important, as it may better reflect early changes before the disease develops; one such parameter is the Aβ42/Aβ40 ratio, or the ratio with the total tau concentration combination and other new biomarkers like Aβ1-38; other factors include oxidative stress and inflammation process proteins, complement factor H, alpha-2-macroglobulin, or clusterin. The study of various forms of pathological amyloid deposits that emerge in different but specific brain regions AD patients seems to be crucial as well. The composition of the first initial pathological, pre-fibrillating monomers of fibrillating peptides and their role in AD development and disease progression have been described as well. They are even more challenging for science and simultaneously might be more promising in

  5. Advanced glycation end products impair glucose-induced insulin secretion from rat pancreatic β-cells.

    Science.gov (United States)

    Hachiya, Hiroyuki; Miura, Yoshikazu; Inoue, Ken-Ichi; Park, Kyung Hwa; Takeuchi, Masayoshi; Kubota, Keiichi

    2014-02-01

    Advanced glycation end products (AGEs) are derivative compounds generated from non-enzymatic glycosylation and oxidation. In comparison with glucose-derived AGEs (Glu-AGEs), glyceraldehyde-derived AGEs (Glycer-AGEs) have stronger toxicity to living systems. In this study, we compared the effects of Glu-AGE and Glycer-AGE on insulin secretion. Rat pancreatic islets were isolated by collagenase digestion and primary-cultured in the presence of 0.1 mg/ml bovine serum albumin (BSA) or 0.1 mg/ml Glu-AGE or Glycer-AGE-albumin. After 48 h of culture, we performed an insulin secretion test and identified the defects by a battery of rescue experiments [corrected]. Also, mRNA expression of genes associated with insulin secretion was measured. Insulin secretion induced by a high glucose concentration was 164.1 ± 6.0, 124.4 ± 4.4 (P < 0.05) and 119.8 ± 7.1 (P < 0.05) μU/3 islets/h in the presence of BSA, Glu-AGE, and Glycer-AGE, respectively. Inhibition of insulin secretion by Glu-AGE or Glycer-AGE was rescued by a high extracellular potassium concentration, tolbutamide and α-ketoisocaproic acid, but not by glyceraldehyde, dihydroxacetone, methylpyruvate, glucagon-like peptide-1 and acetylcholine. Glu-AGE or Glycer-AGE reduced the expression of the malate dehydrogenase (Mdh1/2) gene, which plays a critical role in the nicotinamide adenine dinucleotide (NADH) shuttle. Despite its reported cytotoxicity, the effects of Glycer-AGE on insulin secretion are similar to those of Glu-AGE. © 2013 Japanese Society of Hepato-Biliary-Pancreatic Surgery.

  6. Recent advances in α-synuclein functions, advanced glycation, and toxicity: implications for Parkinson's disease.

    Science.gov (United States)

    Guerrero, Erika; Vasudevaraju, P; Hegde, Muralidhar L; Britton, G B; Rao, K S

    2013-04-01

    The toxicity of α-synuclein in the neuropathology of Parkinson's disease which includes its hallmark aggregation has been studied scrupulously in the last decade. Although little is known regarding the normal functions of α-synuclein, its association with membrane phospholipids suggests its potential role in signaling pathways. Following extensive evidences for its nuclear localization, we and others recently demonstrated DNA binding activity of α-synuclein that modulates its conformation as well as aggregation properties. Furthermore, we also underscored the similarities among various amyloidogenic proteins involved in neurodegenerative diseases including amyloid beta peptides and tau. Our more recent studies show that α-synuclein is glycated and glycosylated both in vitro and in neurons, significantly affecting its folding, oligomeric, and DNA binding properties. Glycated α-synuclein causes increased genome damage both via its direct interaction with DNA and by increased generation of reactive oxygen species as glycation byproduct. In this review, we discuss the mechanisms of glycation and other posttranslational modifications of α-synuclein, including phosphorylation and nitration, and their role in neuronal death in Parkinson's disease.

  7. A high throughput screening assay for identifying glycation inhibitors on MALDI-TOF target.

    Science.gov (United States)

    Zhang, Qiuting; Tu, Zongcai; Wang, Hui; Fan, Liangliang; Huang, Xiaoqin; Xiao, Hui

    2015-03-01

    The Maillard reaction plays an important role in the food industry, however, the deleterious effects generated by the advanced glycation end-products (AGEs) have been well recognized. Many efforts have been made to seek new AGE inhibitors, in particular those natural ones without adverse effect. We have developed a rapid, mass spectrometry based, on-plate screening assay for novel AGE inhibitors. The glycation reaction, inhibition feedback as well as the subsequent MALDI mass spectrometric analysis occurred on one single MALDI plate. At 1:10 M ratio of peptide to sugar, as little as 4h incubation time allowed the screening test to be ready for analysis. DSP, inhibition and IC50 were calculated to evaluate selected inhibitors and resulting inhibition efficiencies were consistent with available references. We demonstrated that this method provide a potential high throughput screening assay to analyze and identify the anti-glycation agents. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. Production of antihypertensive peptides by enzymatic zein hydrolysate from maize-zea mays ssp. mexicana introgression line

    International Nuclear Information System (INIS)

    Wang, L.; Zhang, X.; Qiao, Y.; Qu, M.

    2014-01-01

    Teosintes are essential gene reservoir for maize breeding improvement, among which Zea mays ssp. mexicana has many valuable traits deserved to be transferred into maize genetic background. In this study, one maize-teosinte introgression line SD00100 was selected from the population of Zea mays ssp. mexicana as wild parent. This introgression line manifested the outstanding agricultural traits similar to maize parent Ye 515 and alien genetic material was identified by genomic in situ hybridization (GISH). To produce bioactive peptides with potent angiotensin converting enzyme (ACE) inhibitory activity, zein extracted from endosperm meal was then undergone enzymatic hydrolysis with thermolysin and the hydrolysate was then filtered through a 3 kDa cut-off membrane. ACE inhibitory activity of permeate from Ye 515 and SD00100 was evaluated by RP-HPLC. The IC50 values of the peptides obtained from maize parent and the introgression line were 96.9 micro g/ml and 22.9 micro g/ml, respectively, with significant difference between them. Our results showed that an outstanding inbred maize line was obtained for production of antihypertensive peptides as well as for further development of functional food. (author)

  9. Design of non-aggregating variants of Aβ peptide

    Energy Technology Data Exchange (ETDEWEB)

    Caine, Joanne M., E-mail: jo.caine@csiro.au [CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, Victoria 3052 (Australia); Preventative Health Flagship, 343 Royal Parade, Parkville, Victoria 3052 (Australia); CRC for Mental Health, Level 2, 161 Barry Street, Carlton South, Victoria 3053 (Australia); Churches, Quentin; Waddington, Lynne [CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, Victoria 3052 (Australia); Preventative Health Flagship, 343 Royal Parade, Parkville, Victoria 3052 (Australia); Nigro, Julie; Breheney, Kerry [CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, Victoria 3052 (Australia); Preventative Health Flagship, 343 Royal Parade, Parkville, Victoria 3052 (Australia); CRC for Mental Health, Level 2, 161 Barry Street, Carlton South, Victoria 3053 (Australia); Masters, Colin L. [CRC for Mental Health, Level 2, 161 Barry Street, Carlton South, Victoria 3053 (Australia); Florey Institute for Neuroscience and Mental Health, 30 Royal Parade, Parkville, Victoria 3052 (Australia); Nuttall, Stewart D. [CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, Victoria 3052 (Australia); Preventative Health Flagship, 343 Royal Parade, Parkville, Victoria 3052 (Australia); CRC for Mental Health, Level 2, 161 Barry Street, Carlton South, Victoria 3053 (Australia); Streltsov, Victor A., E-mail: victor.streltsov@csiro.au [CSIRO Materials Science and Engineering, 343 Royal Parade, Parkville, Victoria 3052 (Australia); Preventative Health Flagship, 343 Royal Parade, Parkville, Victoria 3052 (Australia); CRC for Mental Health, Level 2, 161 Barry Street, Carlton South, Victoria 3053 (Australia)

    2014-10-24

    Highlights: • Non-aggregating, non-toxic variants of Aβ peptide were designed using Aβ structure. • Mutations reduce aggregation by stabilising Aβ into small non-toxic oligomers. • Identification of these residues will assist the design of future therapeutic peptides. - Abstract: Self association of the amyloid-β (Aβ{sub 42}) peptide into oligomers, high molecular weight forms, fibrils and ultimately neuritic plaques, has been correlated with progressive cognitive decline in Alzheimer’s disease. Thus, insights into the drivers of the aggregation pathway have the capacity to significantly contribute to our understanding of disease mechanism. Functional assays and a three-dimensional crystal structure of the P3 amyloidogenic region 18–41 of Aβ were used to identify residues important in self-association and to design novel non-aggregating variants of the peptide. Biophysical studies (gel filtration, SDS–PAGE, dynamic light scattering, thioflavin T assay, and electron microscopy) demonstrate that in contrast to wild type Aβ these targeted mutations lose the ability to self-associate. Loss of aggregation also correlates with reduced neuronal toxicity. Our results highlight residues and regions of the Aβ peptide important for future targeting agents aimed at the amelioration of Alzheimer’s disease.

  10. Non-enzymatic palladium recovery on microbial and synthetic surfaces

    DEFF Research Database (Denmark)

    Rotaru, Amelia-Elena; Jiang, Wei; Finster, Kai

    2012-01-01

    in the presence of cells as compared to cell-free controls. We found no difference between native (untreated) and autoclaved cells, and could demonstrate that even a non-enzymatic protein (bovine serum albumin) stimulated Pd(II) reduction as efficiently as bacterial cells. Amine groups readily interact with Pd......(II), and to specifically test their role in surface-assisted Pd(II) reduction by formate, we replaced bacterial cells with polystyrene microparticles functionalized with amine or carboxyl groups. Amine-functionalized microparticles had the same effect on Pd(II) reduction as bacterial cells, and the effect could...... be hampered if the amine groups were blocked by acetylation. The interaction with amine groups was confirmed by infrared spectroscopy on whole cells and amine-functionalized microparticles. In conclusion, bio-supported Pd(II) reduction on microbial surfaces is possibly mediated by a non-enzymatic mechanism...

  11. The specific localization of advanced glycation end-products (AGEs) in rat pancreatic islets.

    Science.gov (United States)

    Morioka, Yuta; Teshigawara, Kiyoshi; Tomono, Yasuko; Wang, Dengli; Izushi, Yasuhisa; Wake, Hidenori; Liu, Keyue; Takahashi, Hideo Kohka; Mori, Shuji; Nishibori, Masahiro

    2017-08-01

    Advanced glycation end-products (AGEs) are produced by non-enzymatic glycation between protein and reducing sugar such as glucose. Although glyceraldehyde-derived AGEs (Glycer-AGEs), one of the AGEs subspecies, have been reported to be involved in the pathogenesis of various age-relating diseases such as diabetes mellitus or arteriosclerosis, little is known about the pathological and physiological mechanism of AGEs in vivo. In present study, we produced 4 kinds of polyclonal antibodies against AGEs subspecies and investigated the localization of AGEs-modified proteins in rat peripheral tissues, making use of these antibodies. We found that Glycer-AGEs and methylglyoxal-derived AGEs (MGO-AGEs) were present in pancreatic islets of healthy rats, distinguished clearly into the pancreatic α and β cells, respectively. Although streptozotocin-induced diabetic rats suffered from remarkable impairment of pancreatic islets, the localization and deposit levels of the Glycer- and MGO-AGEs were not altered in the remaining α and β cells. Remarkably, the MGO-AGEs in pancreatic β cells were localized into the insulin-secretory granules. These results suggest that the cell-specific localization of AGEs-modified proteins are presence generally in healthy peripheral tissues, involved in physiological intracellular roles, such as a post-translational modulator contributing to the secretory and/or maturational functions of insulin. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  12. Glycation of polyclonal IgGs: Effect of sugar excipients during stability studies.

    Science.gov (United States)

    Leblanc, Y; Bihoreau, N; Jube, M; Andre, M-H; Tellier, Z; Chevreux, G

    2016-05-01

    A number of intravenous immunoglobulin preparations are stabilized with sugar additives that may lead over time to undesirable glycation reactions especially in liquid formulation. This study aimed to evaluate the reactivity of sugar excipients on such preparations in condition of temperature, formulation and concentration commonly used for pharmaceutical products. Through an innovative LC-MS method reported to characterize post-translational modifications of IgGs Fc/2 fragments, a stability study of IVIg formulated with reducing and non-reducing sugars has been undertaken. The rate of polyclonal IgGs glycation was investigated during 6months at 5, 25, 30 and 40°C. High levels of glycation were observed with reducing sugars such as glucose and maltose in the first months of the stability study from 25°C. Non-reducing sugars presented a low reactivity even at the highest tested temperature (40°C). Furthermore, a site by site analysis was performed by MS/MS to determine the glycation sites which were mainly identified at Lys246, Lys248 and Lys324. This work points out the high probability of glycation reactions in some commercialized products and describes a useful method to characterize IVIg glycated products issued from reducing sugar excipients. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  13. Skin fluorescence as a clinical tool for non-invasive assessment of advanced glycation and long-term complications of diabetes

    NARCIS (Netherlands)

    Fokkens, Bernardina T.; Smit, Andries J.

    Glycation is important in the development of complications of diabetes mellitus and may have a central role in the well-described glycaemic memory effect in developing these complications. Skin fluorescence has emerged over the last decade as a non-invasive method for assessing accumulation of

  14. A novel strategy for global mapping of O-GlcNAc proteins and peptides using selective enzymatic deglycosylation, HILIC enrichment and mass spectrometry identification.

    Science.gov (United States)

    Shen, Bingquan; Zhang, Wanjun; Shi, Zhaomei; Tian, Fang; Deng, Yulin; Sun, Changqing; Wang, Guangshun; Qin, Weijie; Qian, Xiaohong

    2017-07-01

    O-GlcNAcylation is a kind of dynamic O-linked glycosylation of nucleocytoplasmic and mitochondrial proteins. It serves as a major nutrient sensor to regulate numerous biological processes including transcriptional regulation, cell metabolism, cellular signaling, and protein degradation. Dysregulation of cellular O-GlcNAcylated levels contributes to the etiologies of many diseases such as diabetes, neurodegenerative disease and cancer. However, deeper insight into the biological mechanism of O-GlcNAcylation is hampered by its extremely low stoichiometry and the lack of efficient enrichment approaches for large-scale identification by mass spectrometry. Herein, we developed a novel strategy for the global identification of O-GlcNAc proteins and peptides using selective enzymatic deglycosylation, HILIC enrichment and mass spectrometry analysis. Standard O-GlcNAc peptides can be efficiently enriched even in the presence of 500-fold more abundant non-O-GlcNAc peptides and identified by mass spectrometry with a low nanogram detection sensitivity. This strategy successfully achieved the first large-scale enrichment and characterization of O-GlcNAc proteins and peptides in human urine. A total of 474 O-GlcNAc peptides corresponding to 457 O-GlcNAc proteins were identified by mass spectrometry analysis, which is at least three times more than that obtained by commonly used enrichment methods. A large number of unreported O-GlcNAc proteins related to cell cycle, biological regulation, metabolic and developmental process were found in our data. The above results demonstrated that this novel strategy is highly efficient in the global enrichment and identification of O-GlcNAc peptides. These data provide new insights into the biological function of O-GlcNAcylation in human urine, which is correlated with the physiological states and pathological changes of human body and therefore indicate the potential of this strategy for biomarker discovery from human urine. Copyright

  15. A facile route to glycated albumin detection.

    Science.gov (United States)

    Bohli, Nadra; Meilhac, Olivier; Rondeau, Philippe; Gueffrache, Syrine; Mora, Laurence; Abdelghani, Adnane

    2018-07-01

    In this paper we propose an easy way to detect the glycated form of human serum albumin which is biomarker for several diseases such as diabetes and Alzheimer. The detection platform is a label free impedimetric immunosensor, in which we used a monoclonal human serum albumin antibody as a bioreceptor and electrochemical impedance as a transducing method. The antibody was deposited onto a gold surface by simple physisorption technique. Bovine serum albumin was used as a blocking agent for non-specific binding interactions. Cyclic voltammetry and electrochemical impedance spectroscopy were used for the characterization of each layer. Human serum albumin was glycated at different levels with several concentrations of glucose ranging from 0 mM to 500 mM representing physiological, pathological (diabetic albumin) and suprapathological concentration of glucose. Through the calibration curves, we could clearly distinguish between two different areas related to physiological and pathological albumin glycation levels. The immunosensor displayed a linear range from 7.49% to 15.79% of glycated albumin to total albumin with a good sensitivity. Surface plasmon resonance imaging was also used to characterize the developed immunosensor. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Maillard Proteomics: Opening New Pages

    Directory of Open Access Journals (Sweden)

    Alena Soboleva

    2017-12-01

    Full Text Available Protein glycation is a ubiquitous non-enzymatic post-translational modification, formed by reaction of protein amino and guanidino groups with carbonyl compounds, presumably reducing sugars and α-dicarbonyls. Resulting advanced glycation end products (AGEs represent a highly heterogeneous group of compounds, deleterious in mammals due to their pro-inflammatory effect, and impact in pathogenesis of diabetes mellitus, Alzheimer’s disease and ageing. The body of information on the mechanisms and pathways of AGE formation, acquired during the last decades, clearly indicates a certain site-specificity of glycation. It makes characterization of individual glycation sites a critical pre-requisite for understanding in vivo mechanisms of AGE formation and developing adequate nutritional and therapeutic approaches to reduce it in humans. In this context, proteomics is the methodology of choice to address site-specific molecular changes related to protein glycation. Therefore, here we summarize the methods of Maillard proteomics, specifically focusing on the techniques providing comprehensive structural and quantitative characterization of glycated proteome. Further, we address the novel break-through areas, recently established in the field of Maillard research, i.e., in vitro models based on synthetic peptides, site-based diagnostics of metabolism-related diseases (e.g., diabetes mellitus, proteomics of anti-glycative defense, and dynamics of plant glycated proteome during ageing and response to environmental stress.

  17. Definition of the upper reference limit of glycated albumin in blood donors from Italy.

    Science.gov (United States)

    Bellia, Chiara; Zaninotto, Martina; Cosma, Chiara; Agnello, Luisa; Lo Sasso, Bruna; Bivona, Giulia; Plebani, Mario; Ciaccio, Marcello

    2017-11-27

    Glycated Albumin (GA) has been proposed as a short-term indicator of glycemic homeostasis. The aim of this study is to describe the distribution of GA in a large sample of blood donors from Italy to evaluate whether demographic features, namely age and sex, could influence GA levels and define specific reference limits. The study included 1334 Italian blood donors. GA was measured using an enzymatic method (quantILab Glycated Albumin, IL Werfen, Germany). The upper reference limit (URL) was calculated using the non-parametric percentile method. A modest, although significant, increase of GA was observed in relation to age (psex (12% [11.3-12.8] in males; 12.2% [11.4-13.1] in females; p=0.01). After excluding individuals with fasting plasma glucose ≥7 mmol/L, the calculated GA URL was 14.5% (95% CI: 14.3-14.7). Subjects with GA>14.5% presented a mean age of 48.4±12.2 years, 66.7% were males and the mean glucose was 6.88±2.5 mmol/L. GA in Caucasians shows a similar increasing trend at older ages documented in other ethnicities. The definition of the URL in this population could be useful for both clinical studies, which will clarify the role of GA for diagnosing and monitoring diabetes, and will encourage the introduction of GA in clinical practice.

  18. Serum glycated albumin as a new glycemic marker in pediatric diabetes

    Directory of Open Access Journals (Sweden)

    Ji Woo Lee

    2013-12-01

    Full Text Available PurposeSerum glycated albumin (GA has been recently used as another glycemic marker that reflects shorter term glycemic control than glycated hemoglobin (HbA1c. Insulin secretory function and glycemic fluctuation might be correlated with the ratio of GA to HbA1c (GA/HbA1c in diabetic adult patients. This study investigated the association of GA and GA/HbA1c ratio with the levels of fasting C-peptide, fasting plasma glucose in type 1 and type 2 pediatric diabetes.MethodsTotal 50 cases from 42 patients were included. The subjects were classified into type 1 diabetes mellitus (T1DM (n=30 and type 2 diabetes mellitus (T2DM (n=20 group. The associations among HbA1c, GA, and GA/HbA1c ratio were examined. The relationship between the three glycemic indices and fasting glucose, fasting C-peptide were analyzed.ResultsMean values of GA, the GA/HbA1c ratio were significantly higher in T1DM than T2DM. GA (r=0.532, P=0.001, HbA1c (r=0.519, P=0.002 and the GA/HbA1c ratio (r=0.409, P=0.016 were correlated with the fasting plasma glucose. Fasting C-peptide level arranged 4.22±3.22 ng/mL in T2DM, which was significantly above the values in T1DM (0.26±0.49 ng/mL. There were no significant correlation between HbA1c and fasting C-peptide level. However, GA and the GA/HbA1c ratio exhibited inverse correlations with fasting C-peptide level (r=-0.214, P=0.002; r=-0.516, P<0.001.ConclusionGA seems to more accurately reflects fasting plasma glucose level than HbA1c. GA, GA/HbA1c ratio appear to reflect insulin secretory function.

  19. Dietary Advanced Glycation End Products and Aging

    Directory of Open Access Journals (Sweden)

    Karen Chapman-Novakofski

    2010-12-01

    Full Text Available Advanced glycation end products (AGEs are a heterogeneous, complex group of compounds that are formed when reducing sugar reacts in a non-enzymatic way with amino acids in proteins and other macromolecules. This occurs both exogenously (in food and endogenously (in humans with greater concentrations found in older adults. While higher AGEs occur in both healthy older adults and those with chronic diseases, research is progressing to both quantify AGEs in food and in people, and to identify mechanisms that would explain why some human tissues are damaged, and others are not. In the last twenty years, there has been increased evidence that AGEs could be implicated in the development of chronic degenerative diseases of aging, such as cardiovascular disease, Alzheimer’s disease and with complications of diabetes mellitus. Results of several studies in animal models and humans show that the restriction of dietary AGEs has positive effects on wound healing, insulin resistance and cardiovascular diseases. Recently, the effect of restriction in AGEs intake has been reported to increase the lifespan in animal models. This paper will summarize the work that has been published for both food AGEs and in vivo AGEs and their relation with aging, as well as provide suggestions for future research.

  20. Alzheimer’s disease against peptides products of enzymatic cleavage APP protein: Biological, pathobiological and physico-chemical properties of fibrillating peptides

    Directory of Open Access Journals (Sweden)

    Małgorzata Marszałek

    2017-05-01

    Full Text Available Various peptides products of enzymatic cleavage of key for Alzheimer’s disease Amyloid Precursor Protein (APP are well known, but still are matter of scientific debate. The Aβ type products are especially challenging for experimental and medical research. This paper outlines several, still poorly known, biological and medical processes such as peptides biology, i.e., formation, biodistribution, translocation, transport and finally removal from brain compartments and body fluids like Intracellular Fluid (ICF, Cerebrospinal Fluid (CSF, Interstitial Fluid (ISF, blood serum or urine. In addition, the following studies concerning AD patients might prove challenging and simultaneously promising: peptides translocation through Blood-Brain – Barrier (BBB and Blood–Cerebrospinal Fluid Barrier (BCSFB and their removal from the brain according to a new concept of glymphatic system; – diagnostic difficulties that stem from physico-chemical properties and the nature of proteins or fibrillating peptides itself like low concentration, short half-live and from experimental-technical problems as well like high adsorption or low solubility of Aβ, tau or amylin. The study of diagnostic parameters is very important, as it may better reflect early changes before the disease develops; one such parameter is the Aβ42/Aβ40 ratio, or the ratio with the total tau concentration combination and other new biomarkers like Aβ1-38; other factors include oxidative stress and inflammation process proteins, complement factor H, alpha-2-macroglobulin, or clusterin. The study of various forms of pathological amyloid deposits that emerge in different but specific brain regions AD patients seems to be crucial as well. The composition of the first initial pathological, pre-fibrillating monomers of fibrillating peptides and their role in AD development and disease progression have been described as well. They are even more challenging for science and simultaneously might be

  1. Co-solvent effects on reaction rate and reaction equilibrium of an enzymatic peptide hydrolysis.

    Science.gov (United States)

    Wangler, A; Canales, R; Held, C; Luong, T Q; Winter, R; Zaitsau, D H; Verevkin, S P; Sadowski, G

    2018-04-25

    This work presents an approach that expresses the Michaelis constant KaM and the equilibrium constant Kth of an enzymatic peptide hydrolysis based on thermodynamic activities instead of concentrations. This provides KaM and Kth values that are independent of any co-solvent. To this end, the hydrolysis reaction of N-succinyl-l-phenylalanine-p-nitroanilide catalysed by the enzyme α-chymotrypsin was studied in pure buffer and in the presence of the co-solvents dimethyl sulfoxide, trimethylamine-N-oxide, urea, and two salts. A strong influence of the co-solvents on the measured Michaelis constant (KM) and equilibrium constant (Kx) was observed, which was found to be caused by molecular interactions expressed as activity coefficients. Substrate and product activity coefficients were used to calculate the activity-based values KaM and Kth for the co-solvent free reaction. Based on these constants, the co-solvent effect on KM and Kx was predicted in almost quantitative agreement with the experimental data. The approach presented here does not only reveal the importance of understanding the thermodynamic non-ideality of reactions taking place in biological solutions and in many technological applications, it also provides a framework for interpreting and quantifying the multifaceted co-solvent effects on enzyme-catalysed reactions that are known and have been observed experimentally for a long time.

  2. Chemo-enzymatic peptide synthesis : bioprocess engineering aspects

    NARCIS (Netherlands)

    Vossenberg, P.

    2012-01-01

    Peptides, in particular oligopeptides, play an important role in the fields of health care, nutrition and cosmetics. Chemical synthesis is currently the most mature technique for the synthesis of peptides that range in length from 5 to 80 amino acids. Chemical synthesis is, however,

  3. Arg354 in the catalytic centre of bovine liver catalase is protected from methylglyoxal-mediated glycation.

    Science.gov (United States)

    Scheckhuber, Christian Q

    2015-12-30

    In addition to controlled post-translational modifications proteins can be modified with highly reactive compounds. Usually this leads to a compromised functionality of the protein. Methylglyoxal is one of the most common agents that attack arginine residues. Methylglyoxal is also regarded as a pro-oxidant that affects cellular redox homeostasis by contributing to the formation of reactive oxygen species. Antioxidant enzymes like catalase are required to protect the cell from oxidative damage. These enzymes are also targets for methylglyoxal-mediated modification which could severely affect their catalytic activity in breaking down reactive oxygen species to less reactive or inert compounds. Here, bovine liver catalase was incubated with high levels of methylglyoxal to induce its glycation. This treatment did not lead to a pronounced reduction of enzymatic activity. Subsequently methylglyoxal-mediated arginine modifications (hydroimidazolone and dihydroxyimidazolidine) were quantitatively analysed by sensitive nano high performance liquid chromatography/electron spray ionisation/tandem mass spectrometry. Whereas several arginine residues displayed low to moderate levels of glycation (e.g., Arg93, Arg365, Arg444) Arg354 in the active centre of catalase was never found to be modified. Bovine liver catalase is able to tolerate very high levels of the modifying α-oxoaldehyde methylglyoxal so that its essential enzymatic function is not impaired.

  4. Ex vivo instability of glycated albumin: A role for autoxidative glycation.

    Science.gov (United States)

    Jeffs, Joshua W; Ferdosi, Shadi; Yassine, Hussein N; Borges, Chad R

    2017-09-01

    Ex vivo protein modifications occur within plasma and serum (P/S) samples due to prolonged exposure to the thawed state-which includes temperatures above -30 °C. Herein, the ex vivo glycation of human serum albumin from healthy and diabetic subjects was monitored in P/S samples stored for hours to months at -80 °C, -20 °C, and room temperature, as well as in samples subjected to multiple freeze-thaw cycles, incubated at different surface area-to-volume ratios or under different atmospheric compositions. A simple dilute-and-shoot method utilizing trap-and-elute LC-ESI-MS was employed to determine the relative abundances of the glycated forms of albumin-including forms of albumin bearing more than one glucose molecule. Significant increases in glycated albumin were found to occur within hours at room temperature, and within days at -20 °C. These increases continued over a period of 1-2 weeks at room temperature and over 200 days at -20 °C, ultimately resulting in a doubling of glycated albumin in both healthy and diabetic patients. It was also shown that samples stored at lower surface area-to-volume ratios or incubated under a nitrogen atmosphere experienced less rapid glucose adduction of albumin-suggesting a role for oxidative glycation in the ex vivo glycation of albumin. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. De novo design and engineering of non-ribosomal peptide synthetases

    Science.gov (United States)

    Bozhüyük, Kenan A. J.; Fleischhacker, Florian; Linck, Annabell; Wesche, Frank; Tietze, Andreas; Niesert, Claus-Peter; Bode, Helge B.

    2018-03-01

    Peptides derived from non-ribosomal peptide synthetases (NRPSs) represent an important class of pharmaceutically relevant drugs. Methods to generate novel non-ribosomal peptides or to modify peptide natural products in an easy and predictable way are therefore of great interest. However, although the overall modular structure of NRPSs suggests the possibility of adjusting domain specificity and selectivity, only a few examples have been reported and these usually show a severe drop in production titre. Here we report a new strategy for the modification of NRPSs that uses defined exchange units (XUs) and not modules as functional units. XUs are fused at specific positions that connect the condensation and adenylation domains and respect the original specificity of the downstream module to enable the production of the desired peptides. We also present the use of internal condensation domains as an alternative to other peptide-chain-releasing domains for the production of cyclic peptides.

  6. An Investigation of the Changes in Enzymatic and Non-Enzymatic Salivary Antioxidants Caused by Exhausting Aerobic Activity in Non-Athletic Men

    Directory of Open Access Journals (Sweden)

    Yazgaldi Nazari

    2016-12-01

    Full Text Available Background and Objectives: In the present study, the effect of acute aerobic exercise on enzymatic and non-enzymatic salivary antioxidants variations in non-athlete men, was investigated. Methods: In this experimental study, 25 male non-athlete collegiates (age, 21.2±1.6 years; weight, 68.62±10.1kg; body fat, 16.75±2.9%; and Vo2 max, 37.54±2.4ml/kg/min participated voluntarily in this study. Saliva samples were collected in three phases (before, immediately, and 1 hour after running on treadmill according to Astrand test. The activity of peroxidase and catalase, and concentration of uric acid were measured by laboratory methods. Then, to assess the obtained changes, repeated measures statistical test, and in case of significance, post-hoc Bonferroni test were used for pairwise comparing of the measuring phases at the significance level of p≤0.05 used.  Results: The activity of peroxidase significantly increased immediately and 1 hour after exercise compared to the baseline; Also, the concentration of uric acid significantly increased after aerobic exercise, but catalase enzyme activity significantly decreased after aerobic exercise (p<0.05. No significant change was observed in saliva flow rate after exercise. Conclusion: According to the findings of this study, aerobic exercise causes the production of free radicals, and salivary antioxidant system increases as the body biological response to neutralize and counteract the damaging effects of free radicals.

  7. Calorimetric investigation of diclofenac drug binding to a panel of moderately glycated serum albumins.

    Science.gov (United States)

    Indurthi, Venkata S K; Leclerc, Estelle; Vetter, Stefan W

    2014-08-01

    Glycation alters the drug binding properties of serum proteins and could affect free drug concentrations in diabetic patients with elevated glycation levels. We investigated the effect of bovine serum albumin glycation by eight physiologically relevant glycation reagents (glucose, ribose, carboxymethyllysine, acetoin, methylglyoxal, glyceraldehyde, diacetyl and glycolaldehyde) on diclofenac drug binding. We used this non-steroidal anti-inflammatory drug diclofenac as a paradigm for acidic drugs with high serum binding and because of its potential cardiovascular risks in diabetic patients. Isothermal titration calorimetry showed that glycation reduced the binding affinity Ka of serum albumin and diclofenac 2 to 6-fold by reducing structural rigidity of albumin. Glycation affected the number of drug binding sites in a glycation reagent dependent manner and lead to a 25% decrease for most reagent, expect for ribose, with decreased by 60% and for the CML-modification, increased the number of binding sites by 60%. Using isothermal titration calorimetry and differential scanning calorimetry we derived the complete thermodynamic characterization of diclofenac binding to all glycated BSA samples. Our results suggest that glycation in diabetic patients could significantly alter the pharmacokinetics of the widely used over-the-counter NSDAI drug diclofenac and with possibly negative implications for patients. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Non-ionic Surfactants and Non-Catalytic Protein Treatment on Enzymatic Hydrolysis of Pretreated Creeping Wild Ryegrass

    Science.gov (United States)

    Zheng, Yi; Pan, Zhongli; Zhang, Ruihong; Wang, Donghai; Jenkins, Bryan

    Our previous research has shown that saline Creeping Wild Ryegrass (CWR), Leymus triticoides, has a great potential to be used for bioethanol production because of its high fermentable sugar yield, up to 85% cellulose conversion of pretreated CWR. However, the high cost of enzyme is still one of the obstacles making large-scale lignocellulosic bioethanol production economically difficult. It is desirable to use reduced enzyme loading to produce fermentable sugars with high yield and low cost. To reduce the enzyme loading, the effect of addition of non-ionic surfactants and non-catalytic protein on the enzymatic hydrolysis of pretreated CWR was investigated in this study. Tween 20, Tween 80, and bovine serum albumin (BSA) were used as additives to improve the enzymatic hydrolysis of dilute sulfuric-acid-pretreated CWR. Under the loading of 0.1 g additives/g dry solid, Tween 20 was the most effective additive, followed by Tween 80 and BSA. With the addition of Tween 20 mixed with cellulase loading of 15 FPU/g cellulose, the cellulose conversion increased 14% (from 75 to 89%), which was similar to that with cellulase loading of 30 FPU/g cellulose and without additive addition. The results of cellulase and BSA adsorption on the Avicel PH101, pretreated CWR, and lignaceous residue of pretreated CWR support the theory that the primary mechanism behind the additives is prevention of non-productive adsorption of enzymes on lignaceous material of pretreated CWR. The addition of additives could be a promising technology to improve the enzymatic hydrolysis by reducing the enzyme activity loss caused by non-productive adsorption.

  9. In silico panning for a non-competitive peptide inhibitor

    Directory of Open Access Journals (Sweden)

    Ikebukuro Kazunori

    2007-01-01

    Full Text Available Abstract Background Peptide ligands have tremendous therapeutic potential as efficacious drugs. Currently, more than 40 peptides are available in the market for a drug. However, since costly and time-consuming synthesis procedures represent a problem for high-throughput screening, novel procedures to reduce the time and labor involved in screening peptide ligands are required. We propose the novel approach of 'in silico panning' which consists of a two-stage screening, involving affinity selection by docking simulation and evolution of the peptide ligand using genetic algorithms (GAs. In silico panning was successfully applied to the selection of peptide inhibitor for water-soluble quinoprotein glucose dehydrogenase (PQQGDH. Results The evolution of peptide ligands for a target enzyme was achieved by combining a docking simulation with evolution of the peptide ligand using genetic algorithms (GAs, which mimic Darwinian evolution. Designation of the target area as next to the substrate-binding site of the enzyme in the docking simulation enabled the selection of a non-competitive inhibitor. In all, four rounds of selection were carried out on the computer; the distribution of the docking energy decreased gradually for each generation and improvements in the docking energy were observed over the four rounds of selection. One of the top three selected peptides with the lowest docking energy, 'SERG' showed an inhibitory effect with Ki value of 20 μM. PQQGDH activity, in terms of the Vmax value, was 3-fold lower than that of the wild-type enzyme in the presence of this peptide. The mechanism of the SERG blockage of the enzyme was identified as non-competitive inhibition. We confirmed the specific binding of the peptide, and its equilibrium dissociation constant (KD value was calculated as 60 μM by surface plasmon resonance (SPR analysis. Conclusion We demonstrate an effective methodology of in silico panning for the selection of a non

  10. Effect of Temperature on Tolbutamide Binding to Glycated Serum Albumin

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    Agnieszka Szkudlarek

    2017-03-01

    Full Text Available Glycation process occurs in protein and becomes more pronounced in diabetes when an increased amount of reducing sugar is present in bloodstream. Glycation of protein may cause conformational changes resulting in the alterations of its binding properties even though they occur at a distance from the binding sites. The changes in protein properties could be related to several pathological consequences such as diabetic and nondiabetic cardiovascular diseases, cataract, renal dysfunction and Alzheimer’s disease. The experiment was designed to test the impact of glycation process on sulfonylurea drug tolbutamide-albumin binding under physiological (T = 309 K and inflammatory (T = 311 K and T = 313 K states using fluorescence and UV-VIS spectroscopies. It was found in fluorescence analysis experiments that the modification of serum albumin in tryptophanyl and tyrosyl residues environment may affect the tolbutamide (TB binding to albumin in subdomain IIA and/or IIIA (Sudlow’s site I and/or II, and also in subdomains IB and IIB. We estimated the binding of tolbutamide to albumin described by a mixed nature of interaction (specific and nonspecific. The association constants Ka (L∙mol−1 for tolbutamide at its high affinity sites on non-glycated albumin were in the range of 1.98–7.88 × 104 L∙mol−1 (λex = 275 nm, 1.20–1.64 × 104 L∙mol−1 (λex = 295 nm and decreased to 1.24–0.42 × 104 L∙mol−1 at λex = 275 nm (T = 309 K and T = 311 K and increased to 2.79 × 104 L∙mol−1 at λex = 275 nm (T = 313 K and to 4.43–6.61 × 104 L∙mol−1 at λex = 295 nm due to the glycation process. Temperature dependence suggests the important role of van der Waals forces and hydrogen bonding in hydrophobic interactions between tolbutamide and both glycated and non-glycated albumin. We concluded that the changes in the environment of TB binding of albumin in subdomain IIA and/or IIIA as well as in subdomains IB and IIB influence on

  11. Deamidation of asparagine and glutamine residues in proteins and peptides: structural determinants and analytical methodology

    NARCIS (Netherlands)

    Bischoff, Rainer; Kolbe, H.V.

    1994-01-01

    Non-enzymatic deamidation of asparagine and glutamine residues in proteins and peptides are reviewed by first outlining the well-described reaction mechanism involving cyclic imide intermediates, followed by a discussion of structural features which influence the reaction rate. The second and major

  12. Enzyme-Assisted Discovery of Antioxidant Peptides from Edible Marine Invertebrates: A Review.

    Science.gov (United States)

    Chai, Tsun-Thai; Law, Yew-Chye; Wong, Fai-Chu; Kim, Se-Kwon

    2017-02-16

    Marine invertebrates, such as oysters, mussels, clams, scallop, jellyfishes, squids, prawns, sea cucumbers and sea squirts, are consumed as foods. These edible marine invertebrates are sources of potent bioactive peptides. The last two decades have seen a surge of interest in the discovery of antioxidant peptides from edible marine invertebrates. Enzymatic hydrolysis is an efficient strategy commonly used for releasing antioxidant peptides from food proteins. A growing number of antioxidant peptide sequences have been identified from the enzymatic hydrolysates of edible marine invertebrates. Antioxidant peptides have potential applications in food, pharmaceuticals and cosmetics. In this review, we first give a brief overview of the current state of progress of antioxidant peptide research, with special attention to marine antioxidant peptides. We then focus on 22 investigations which identified 32 antioxidant peptides from enzymatic hydrolysates of edible marine invertebrates. Strategies adopted by various research groups in the purification and identification of the antioxidant peptides will be summarized. Structural characteristic of the peptide sequences in relation to their antioxidant activities will be reviewed. Potential applications of the peptide sequences and future research prospects will also be discussed.

  13. PNA Peptide chimerae

    DEFF Research Database (Denmark)

    Koch, T.; Næsby, M.; Wittung, P.

    1995-01-01

    Radioactive labelling of PNA has been performed try linking a peptide segment to the PNA which is substrate for protein kinase A. The enzymatic phosphorylation proceeds in almost quantitative yields....

  14. Fabrication of Nickel/nanodiamond/boron-doped diamond electrode for non-enzymatic glucose biosensor

    International Nuclear Information System (INIS)

    Dai, Wei; Li, Mingji; Gao, Sumei; Li, Hongji; Li, Cuiping; Xu, Sheng; Wu, Xiaoguo; Yang, Baohe

    2016-01-01

    Highlights: • Nanodiamonds (NDs) were electrophoretically deposited on the BDD film. • The NDs significantly extended the potential window. • Ni/NDs/BDD electrode was prepared by electrodeposition. • The electrode shows good catalytic activity for glucose oxidation. - Abstract: A stable and sensitive non-enzymatic glucose sensor was prepared by modifying a boron-doped diamond (BDD) electrode with nickel (Ni) nanosheets and nanodiamonds (NDs). The NDs were electrophoretically deposited on the BDD surface, and acted as nucleation sites for the subsequent electrodeposition of Ni. The morphology and composition of the modified BDD electrodes were characterized by field-emission scanning electron microscopy and energy-dispersive X-ray spectroscopy, respectively. The Ni nanosheet-ND modified BDD electrode exhibited good current response towards the non-enzymatic oxidation of glucose in alkaline media. The NDs significantly extended the potential window. The response to glucose was linear over the 0.2–1055.4-μM range. The limit of detection was 0.05 μM, at a signal-to-noise ratio of 3. The Ni nanosheet-ND/BDD electrode exhibited good selectivity, reproducibility and stability. Its electrochemical performance, low cost and simple preparation make it a promising non-enzymatic glucose sensor.

  15. Quantification of peptides released during in vitro digestion of cooked meat.

    Science.gov (United States)

    Sayd, T; Chambon, C; Santé-Lhoutellier, V

    2016-04-15

    We aimed to identify and quantify the peptides generated during in vitro digestion of cooked meat by liquid chromatography coupled with high resolution mass spectrometer. A total of 940 non-redundant peptides in the gastric compartment and 989 non-redundant peptides in the intestinal compartment were quantified and identified. Among the 71 different proteins identified, 43 meat proteins were found in the two digestive compartments, 20 proteins were specific to the gastric compartment and 8 proteins to the intestinal compartment. In terms of estimation, the proteins involved in muscle contraction and structure were preferentially enzymatically hydrolyzed in the small intestine. The effect of cooking provided different but less clear patterns of digestion. To the best of our knowledge, this constitutes the highest number of peptides identified in beef meat digests and provides a comprehensive database for meat protein digestion associated with cooking conditions. Such quantitative and qualitative differences may have important nutritional consequences. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Nickel-functionalized reduced graphene oxide with polyaniline for non-enzymatic glucose sensing

    International Nuclear Information System (INIS)

    Zhang, Bing; He, Yu; Liu, Bingqian; Tang, Dianping

    2015-01-01

    We have developed a new class of organic–inorganic hybrid nanostructures based on the use of reduced graphene oxide (rGO), polyaniline, and a nickel metal nanostructure. It was applied to efficient non-enzymatic sensing of glucose based on its electrocatalytic oxidation. Scanning electron microscopy and energy-dispersive X-Ray were employed to characterize the material. It is shown that the doped polyaniline plays an important role in the formation of the hybrid nanostructures. Improved analytical performance is found when the hybrid nanostructures were placed on a glassy carbon electrode and used for non-enzymatic sensing of glucose at a typical working potential of +450 mV and a pH value of 13. Features include a fast response (∼2 s), high sensitivity (6,050 μA mM −1 cm −2 ), a linear range from 0.1 μM to 1.0 mM, and a low detection limit (0.08 μM). The response to glucose follows a Michaelis-Menten kinetic behavior, and the K M value was determined to be 0.241 μM. Reproducibility and specificity are acceptable. Fructose and maltose do not interfere significantly. Importantly, the methodology was validated and evaluated for the analysis of 15 spiked human serum specimens, receiving in a good accordance with the results obtained by the non-enzymatic glucose sensing and the commercialized personal glucose meter. (author)

  17. Correlation of advanced glycation end products to Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Zong-yan MA

    2018-04-01

    Full Text Available Alzheimer's disease (AD is a common retrograde neurodegenerative disease of the central nervous system, as well as the most common type of dementia in the aged, the main manifestations of AD are progressive decline of cognitive function and daily life ability. AD seriously affects the quality of life and physical and mental health of the aged, and increased the burden of family and society. The etiology and pathogenesis of AD remain unclear nowadays, and there is no objective and specific biological marker to help the early diagnosis and effective treatment. Advanced glycation end products (AGEs are stable end products formed by non enzymatic reaction between the free amino groups of proteins, lipids, nucleic acids macromolecules and the carbonyls of glucose or other reduced sugars. Recent years, more and more studies have focused on the correlation between AGEs and its receptors (RAGE in patients with cognitive impairment, however, the role played by AGEs in the pathogenesis of AD remains unclear. The present paper will give an overview from three aspects: the structure and characteristics of AGEs, the relationship between the occurrence and development of AD and AGEs and the relationship between AGEs and prognosis of cognitive impairment which we've known so far. DOI: 10.11855/j.issn.0577-7402.2018.01.16

  18. Therapeutic potential of dairy bioactive peptides: A contemporary perspective.

    Science.gov (United States)

    Sultan, Saira; Huma, Nuzhat; Butt, Masood Sadiq; Aleem, Muhammad; Abbas, Munawar

    2018-01-02

    Dairy products are associated with numerous health benefits. These are a good source of nutrients such as carbohydrates, protein (bioactive peptides), lipids, minerals, and vitamins, which are essential for growth, development, and maintenance of the human body. Accordingly, dairy bioactive peptides are one of the targeted compounds present in different dairy products. Dairy bioactive compounds can be classified as antihypertensive, anti-oxidative, immmunomodulant, anti-mutagenic, antimicrobial, opoid, anti-thrombotic, anti-obesity, and mineral-binding agents, depending upon biological functions. These bioactive peptides can easily be produced by enzymatic hydrolysis, and during fermentation and gastrointestinal digestion. For this reason, fermented dairy products, such as yogurt, cheese, and sour milk, are gaining popularity worldwide, and are considered excellent source of dairy peptides. Furthermore, fermented and non-fermented dairy products are associated with lower risks of hypertension, coagulopathy, stroke, and cancer insurgences. The current review article is an attempt to disseminate general information about dairy peptides and their health claims to scientists, allied stakeholders, and, certainly, readers.

  19. High glucose recovery from direct enzymatic hydrolysis of bisulfite-pretreatment on non-detoxified furfural residues.

    Science.gov (United States)

    Xing, Yang; Bu, Lingxi; Sun, Dafeng; Liu, Zhiping; Liu, Shijie; Jiang, Jianxin

    2015-10-01

    This study reports four schemes to pretreat wet furfural residues (FRs) with sodium bisulfite for production of fermentable sugar. The results showed that non-detoxified FRs (pH 2-3) had great potential to lower the cost of bioconversion. The optimal process was that unwashed FRs were first pretreated with bisulfite, and the whole slurry was then directly used for enzymatic hydrolysis. A maximum glucose yield of 99.4% was achieved from substrates pretreated with 0.1 g NaHSO3/g dry substrate (DS), at a relatively low temperature of 100 °C for 3 h. Compared with raw material, enzymatic hydrolysis at a high-solid of 16.5% (w/w) specifically showed more excellent performance with bisulfite treated FRs. Direct bisulfite pretreatment improved the accessibility of substrates and the total glucose recovery. Lignosulfonate in the non-detoxified slurry decreased the non-productive adsorption of cellulase on the substrate, thus improving enzymatic hydrolysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Glycated albumin as a marker of glycemia in diabetes and its vascular complications

    Directory of Open Access Journals (Sweden)

    Maria Warwas

    2015-05-01

    Full Text Available Effective glycemic control is very important to prevent the onset and the progression of chronic complications in diabetic patients. It is known that glycation of various proteins is increased in diabetic patients compared with non-diabetics. Among these glycated proteins, glycated hemoglobin (HbA1c is commonly used as a gold standard index of glycemic control in the clinical setting. However, it can be unreliable in conditions affecting the lifespan of erythrocytes (120 days as well as in the clinical state in which glycemic control alleviates or deteriorates in a short period. By overcoming the shortcomings of HbA1c, glycated albumin (GA has gained interest as a useful index for an intermediate glycation period (2 weeks and pathogenic protein.After giving a brief overview of the key role of HbA1c as a long-term glycemic marker, this review focuses on (a glycation of human albumin and its main properties, (b methods of GA determination, (c the recent clinical status of GA as a glycemic index in diabetic patients and its association with vascular complications. Finally, conditions with a possible inaccurate GA level are also mentioned.

  1. Non-enzymatic glucose detection using magnetic nanoemulsions

    International Nuclear Information System (INIS)

    Mahendran, V.; Philip, John

    2014-01-01

    We probe the optical properties and intermolecular interactions in magnetically responsive nanoemulsions in the presence of glucose. The equilibrium interdroplet distance between the emulsion droplets in an one-dimensional array increases by several nanometers in the presence of glucose because of intermolecular hydrogen bonding with sodium dodecyl sulphate molecules at the oil-water interface that gives rise to stretched lamellae-like structure. The observed large red shift in the diffracted Bragg peak (∼50–100 nm) and the linear response in the glucose concentration range of 0.25–25 mM offer a simple, fast, and cost effective non-enzymatic approach for glucose detection.

  2. Non-Enzymatic Glucose Sensor Composed of Carbon-Coated Nano-Zinc Oxide

    Directory of Open Access Journals (Sweden)

    Ren-Jei Chung

    2017-02-01

    Full Text Available Nowadays glucose detection is of great importance in the fields of biological, environmental, and clinical analyzes. In this research, we report a zinc oxide (ZnO nanorod powder surface-coated with carbon material for non-enzymatic glucose sensor applications through a hydrothermal process and chemical vapor deposition method. A series of tests, including crystallinity analysis, microstructure observation, and electrochemical property investigations were carried out. For the cyclic voltammetric (CV glucose detection, the low detection limit of 1 mM with a linear range from 0.1 mM to 10 mM was attained. The sensitivity was 2.97 μA/cm2mM, which is the most optimized ever reported. With such good analytical performance from a simple process, it is believed that the nanocomposites composed of ZnO nanorod powder surface-coated with carbon material are promising for the development of cost-effective non-enzymatic electrochemical glucose biosensors with high sensitivity.

  3. The Effect of Irradiation Treatment on the Non-Enzymatic Browning Reaction in Legume Seeds

    International Nuclear Information System (INIS)

    El-Niely, H.F.G.

    2013-01-01

    The present study was conducted to evaluate the effects of gamma irradiation treatment, at room temperature, on the non-enzymatic browning reaction (Millerd reaction products, MRPs) generated in soybeans, broad beans and dried peas seeds at dose levels of 10, 30 and 60 kGy and their effects on the chemical constituents, soluble protein, available lysine and in vitro protein digestibility. The formation of MRPs in the studied legumes was assayed by monitoring the formation of brown pigments (browning intensity) by spectrophotometric method. The results revealed that the chemical composition of irradiated legumes showed non-significant differences relative to the raw one. A dose dependent decrease in soluble proteins and available lysine in the three legumes were observed. The non-enzymatic browning reaction was significantly increased with increasing the radiation dose, which was proved by changes in browning index tests. At the same time, the in vitro protein digestibility was increased after irradiation up to 60 kGy. Irradiation of dried peas with 60 kGy produced higher browning index than the other legumes. A positive correlation was observed between the radiation dose and the browning index for soybeans (R2= 0.96), broad beans (R2 = 0.81) and dried peas (R2 = 0.97) which means that 96%, 81% and 97 of the variation in the incidence of non-enzymatic browning reaction in soybean, broad bean and dried peas, respectively, are due to the effect of irradiation treatments. The present study suggests that the formation of non-enzymatic browning reaction did not impair the nutritional quality of legumes, therefore, the process of irradiation was helpful in increasing the in vitro protein digestibility of studied legumes. These results clearly indicated that gamma irradiation processing at the studied doses can add valuable effects to the studied legumes

  4. The non-peptidic part determines the internalization mechanism and intracellular trafficking of peptide amphiphiles.

    Directory of Open Access Journals (Sweden)

    Dimitris Missirlis

    Full Text Available BACKGROUND: Peptide amphiphiles (PAs are a class of amphiphilic molecules able to self-assemble into nanomaterials that have shown efficient in vivo targeted delivery. Understanding the interactions of PAs with cells and the mechanisms of their internalization and intracellular trafficking is critical in their further development for therapeutic delivery applications. METHODOLOGY/PRINCIPAL FINDINGS: PAs of a novel, cell- and tissue-penetrating peptide were synthesized possessing two different lipophilic tail architectures and their interactions with prostate cancer cells were studied in vitro. Cell uptake of peptides was greatly enhanced post-modification. Internalization occurred via lipid-raft mediated endocytosis and was common for the two analogs studied. On the contrary, we identified the non-peptidic part as the determining factor of differences between intracellular trafficking and retention of PAs. PAs composed of di-stearyl lipid tails linked through poly(ethylene glycol to the peptide exhibited higher exocytosis rates and employed different recycling pathways compared to ones consisting of di-palmitic-coupled peptides. As a result, cell association of the former PAs decreased with time. CONCLUSIONS/SIGNIFICANCE: Control over peptide intracellular localization and retention is possible by appropriate modification with synthetic hydrophobic tails. We propose this as a strategy to design improved peptide-based delivery systems.

  5. Non-enzymatic depolymerization of cotton cellulose by fungal mimicking metabolites

    Science.gov (United States)

    Anne Christine Steenkjaer Hastrup; Caitlin Howell; Bo Jensen; Frederick Green

    2011-01-01

    Small, low molecular weight, non-enzymatic compounds have been linked to the early stages of brown rot decay as the enzymes involved with holocellulose degradation are too large to penetrate the S3 layer of intact wood cells. We investigated the most notable of these compounds, i.e. hydrogen peroxide, iron, and oxalic acid. The former two are involved in the Fenton...

  6. Highly ordered Ni–Ti–O nanotubes for non-enzymatic glucose detection

    Energy Technology Data Exchange (ETDEWEB)

    Hang, Ruiqiang, E-mail: hangruiqiang@tyut.edu.cn [Research Institute of Surface Engineering, Taiyuan University of Technology, Taiyuan 030024 (China); Liu, Yanlian [Research Institute of Surface Engineering, Taiyuan University of Technology, Taiyuan 030024 (China); Gao, Ang [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong (China); Bai, Long; Huang, Xiaobo; Zhang, Xiangyu; Lin, Naiming [Research Institute of Surface Engineering, Taiyuan University of Technology, Taiyuan 030024 (China); Tang, Bin, E-mail: tangbin@tyut.edu.cn [Research Institute of Surface Engineering, Taiyuan University of Technology, Taiyuan 030024 (China); Chu, Paul K. [Department of Physics and Materials Science, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong (China)

    2015-06-01

    Anodization is used to fabricate Ni–Ti–O nanotube (NT) electrodes for non-enzymatic glucose detection. The morphology, microstructure and composition of the materials are characterized by field emission scanning electron microscopy (FE-SEM), high resolution transmission electron microscopy (HR-TEM) and X-ray photoelectron spectroscopy (XPS). Our results show amorphous and highly ordered NTs with diameter of 50 nm, length of 800 nm, and Ni/Ti ratio (at %) of 0.35 can be fabricated in ethylene glycol electrolyte supplemented with 0.2 wt.% NH{sub 4}F and 0.5 vol.% H{sub 2}O at 30 °C and 25 V for 1 h. Electrochemical experiments indicate that at an applied potential of 0.60 V vs. Ag/AgCl, the electrode exhibits a linear response window for glucose concentrations from 0.002 mM to 0.2 mM with a response time of 10 s, detection limit of 0.13 μM (S/N = 3), and sensitivity of 83 μA mM{sup −1} cm{sup −2}. The excellent performance of the electrode is attributed to its large specific area and fast electron transfer between the NT walls. The good electrochemical performance of the Ni–Ti–O NTs as well as their simple and low-cost preparation method make the strategy promising in non-enzymatic glucose detection. - Highlights: • Highly ordered Ni–Ti–O nanotubes have been fabricated by one-step anodization. • We find H{sub 2}O contents in the electrolyte is critical to successful fabrication of the NTs. • The Ni–Ti–O nanotubes are ideal electrode materials for non-enzymatic glucose detection.

  7. Thiomers: potential excipients for non-invasive peptide delivery systems.

    Science.gov (United States)

    Bernkop-Schnürch, Andreas; Krauland, Alexander H; Leitner, Verena M; Palmberger, Thomas

    2004-09-01

    In recent years thiolated polymers or so-called thiomers have appeared as a promising alternative in the arena of non-invasive peptide delivery. Thiomers are generated by the immobilisation of thiol-bearing ligands to mucoadhesive polymeric excipients. By formation of disulfide bonds with mucus glycoproteins, the mucoadhesive properties of these polymers are improved up to 130-fold. Due to formation of inter- and intramolecular disulfide bonds within the thiomer itself, dosage forms such as tablets or microparticles display strong cohesive properties resulting in comparatively higher stability, prolonged disintegration times and a more controlled release of the embedded peptide drug. The permeation of peptide drugs through mucosa can be improved by the use of thiolated polymers. Additionally some thiomers exhibit improved inhibitory properties towards peptidases. The efficacy of thiomers in non-invasive peptide delivery could be demonstrated by various in vivo studies. Tablets comprising a thiomer and pegylated insulin, for instance, resulted in a pharmacological efficacy of 7% after oral application to diabetic mice. Furthermore, a pharmacological efficacy of 1.3% was achieved in rats by oral administration of calcitonin tablets comprising a thiomer. Human growth hormone in a thiomer-gel was applied nasally to rats and led to a bioavailability of 2.75%. In all these studies, formulations comprising the corresponding unmodified polymer had only a marginal or no effect. According to these results drug carrier systems based on thiomers seem to be a promising tool for non-invasive peptide drug delivery.

  8. Carpatizine, a novel bridged oxazine derivative generated by non-enzymatic reactions.

    Science.gov (United States)

    Fu, Peng; MacMillan, John B

    2017-06-27

    Carpatizine (1), a new bridged oxazine derivative, was isolated from a marine-derived Streptomyces strain SNE-011. The structure was fully determined by spectroscopic analysis, ECD calculations and chemical methods. A plausible non-enzymatic reaction mechanism from daryamide D leading to carpatizine was presented, which was confirmed by chemical transformation.

  9. Increased Nepsilon-(carboxymethyl)-lysine levels in cerebral blood vessels of diabetic patients and in a (streptozotocin-treated) rat model of diabetes mellitus

    NARCIS (Netherlands)

    van Deutekom, A.W.; Niessen, H.W.M.; Schalkwijk, C.G.; Heine, R.J.; Simsek, S.

    2008-01-01

    Objective: Non-enzymatic glycation of proteins and their end products (advanced glycation end products, AGE) have been implicated in the pathogenesis of diabetic complications. Our aim was to evaluate the association between diabetes mellitus (DM) and the accumulation of one of the most abundant

  10. Placental peptides metabolism and maternal factors as predictors of risk of gestational diabetes in pregnant women. A case-control study.

    Science.gov (United States)

    Ngala, Robert Amadu; Fondjo, Linda Ahenkorah; Gmagna, Peter; Ghartey, Frank Naku; Awe, Martin Akilla

    2017-01-01

    Gestational diabetes is a risk factor for perinatal complications; include shoulder dystocia, birth injuries such as bone fractures and nerve palsies. It is associated with later development of type 2 diabetes, the risk of macrosomia and other long-term health effects of infants born to diabetic mothers. The study assesses placental peptides and maternal factors as potential predictors of gestational diabetes among pregnant women. A total of 200 pregnant women were recruited for the study, 150 pregnant women without pre gestational diabetes including 50 women with low risk factors of diabetes as controls and 50 other pregnant women with pregestational diabetes as control. Fasting blood glucose and the lipid profile were determined by enzymatic methods using Envoy® 500 reagents (Vital Diagnostics, USA). Glycated haemoglobin was assessed using the Cation Exchange resin method. Leptin and the Human Placenta Lactogen were assayed using the Sandwich-ELISA technique. Beta chorionic gonadotrophin, insulin, progesterone and estradiol were determined using chemilumiscence imunoassay technique on MAGLUMI 600 analyzer. Anthropometry, including BMI and blood pressure were also measured. Fasting plasma glucose (FBG), insulin, insulin resistance, glycated haemoglobin and Human Placenta Lactogen(HPL)were significantly (p0.05) in estradiol, insulin, insulin resistance and HPL between the pregnant women who developed gestational diabetes and those who did not. Leptin, progesterone and FBG were significantly increased in those who developed GDM. The risk of developing gestational diabetes increased with overweight (OR = 1.76, P = 0.370) and family history of diabetes (OR = 2.18, P = 0.282). Leptin, progesterone, estradiol estimated in this study were increased in the gestational diabetes mellitus women and fairly predicted gestational diabetes in the non-diabetics pregnant women. Obesity, aging and family history of diabetes were strongly predictive of gestational diabetes.

  11. Glycation Reactions of Casein Micelles.

    Science.gov (United States)

    Moeckel, Ulrike; Duerasch, Anja; Weiz, Alexander; Ruck, Michael; Henle, Thomas

    2016-04-13

    After suspensions of micellar casein or nonmicellar sodium caseinate had been heated, respectively, in the presence and absence of glucose for 0-4 h at 100 °C, glycation compounds were quantitated. The formation of Amadori products as indicators for the "early" Maillard reaction were in the same range for both micellar and nonmicellar caseins, indicating that reactive amino acid side chains within the micelles are accessible for glucose in a comparable way as in nonmicellar casein. Significant differences, however, were observed concerning the formation of the advanced glycation end products (AGEs), namely, N(ε)-carboxymethyllysine (CML), pyrraline, pentosidine, and glyoxal-lysine dimer (GOLD). CML could be observerd in higher amounts in nonmicellar casein, whereas in the micelles the pyrraline formation was increased. Pentosidine and GOLD were formed in comparable amounts. Furthermore, the extent of protein cross-linking was significantly higher in the glycated casein micelles than in the nonmicellar casein samples. Dynamic light scattering and scanning electron microscopy showed that glycation has no influence on the size of the casein micelles, indicating that cross-linking occurs only in the interior of the micelles, but altered the surface morphology. Studies on glycation and nonenzymatic cross-linking can contribute to the understanding of the structure of casein micelles.

  12. Open tubular capillary electrochromatography: A useful microreactor for collagen I glycation and interaction studies with low-density lipoprotein particles

    International Nuclear Information System (INIS)

    D'Ulivo, Lucia; Witos, Joanna; Ooerni, Katariina; Kovanen, Petri T.; Riekkola, Marja-Liisa

    2010-01-01

    Diabetes, a multifunctional disease and a major cause of morbidity and mortality in the industrialized countries, strongly associates with the development and progression of atherosclerosis. One of the consequences of high level of glucose in the blood circulation is glycation of long-lived proteins, such as collagen I, the most abundant component of the extracellular matrix (ECM) in the arterial wall. Glycation is a long-lasting process that involves the reaction between a carbonyl group of the sugar and an amino group of the protein, usually a lysine residue. This reaction generates an Amadori product that may evolve in advanced glycation end products (AGEs). AGEs, as reactive molecules, can provoke cross-linking of collagen I fibrils. Since binding of low-density lipoproteins (LDLs) to the ECM of the inner layer of the arterial wall, the intima, has been implicated to be involved in the onset of the development of an atherosclerotic plaque, collagen modifications, which can affect the affinity of native and oxidized LDL for collagen I, can promote the entrapment of LDLs in the intima and accelerate the progression of atherosclerosis. In this study, open tubular capillary electrochromatography is proposed as a new microreactor to study in situ glycation of collagen I. The kinetics of glycation was first investigated in a fused silica collagen I-coated capillary. Dimethyl sulphoxide, injected as an electroosmotic flow marker, gave information about the charge of coating. Native and oxidized LDL, and selected peptide fragments from apolipoprotein B-100, the protein covering LDL particles, were injected as marker compounds to clarify the interactions between LDLs and the glycated collagen I coating. The method proposed is simple and inexpensive, since only small amounts of collagen and LDL are required. Atomic force microscopy images complemented our studies, highlighting the difference between unmodified and glycated collagen I surfaces.

  13. Open tubular capillary electrochromatography: A useful microreactor for collagen I glycation and interaction studies with low-density lipoprotein particles

    Energy Technology Data Exchange (ETDEWEB)

    D' Ulivo, Lucia; Witos, Joanna [Laboratory of Analytical Chemistry, Department of Chemistry, P.O. Box 55, FIN-00014 University of Helsinki (Finland); Ooerni, Katariina; Kovanen, Petri T. [Wihuri Research Institute, Kalliolinnantie 4, FIN-00140, Helsinki (Finland); Riekkola, Marja-Liisa, E-mail: marja-liisa.riekkola@helsinki.fi [Laboratory of Analytical Chemistry, Department of Chemistry, P.O. Box 55, FIN-00014 University of Helsinki (Finland)

    2010-04-07

    Diabetes, a multifunctional disease and a major cause of morbidity and mortality in the industrialized countries, strongly associates with the development and progression of atherosclerosis. One of the consequences of high level of glucose in the blood circulation is glycation of long-lived proteins, such as collagen I, the most abundant component of the extracellular matrix (ECM) in the arterial wall. Glycation is a long-lasting process that involves the reaction between a carbonyl group of the sugar and an amino group of the protein, usually a lysine residue. This reaction generates an Amadori product that may evolve in advanced glycation end products (AGEs). AGEs, as reactive molecules, can provoke cross-linking of collagen I fibrils. Since binding of low-density lipoproteins (LDLs) to the ECM of the inner layer of the arterial wall, the intima, has been implicated to be involved in the onset of the development of an atherosclerotic plaque, collagen modifications, which can affect the affinity of native and oxidized LDL for collagen I, can promote the entrapment of LDLs in the intima and accelerate the progression of atherosclerosis. In this study, open tubular capillary electrochromatography is proposed as a new microreactor to study in situ glycation of collagen I. The kinetics of glycation was first investigated in a fused silica collagen I-coated capillary. Dimethyl sulphoxide, injected as an electroosmotic flow marker, gave information about the charge of coating. Native and oxidized LDL, and selected peptide fragments from apolipoprotein B-100, the protein covering LDL particles, were injected as marker compounds to clarify the interactions between LDLs and the glycated collagen I coating. The method proposed is simple and inexpensive, since only small amounts of collagen and LDL are required. Atomic force microscopy images complemented our studies, highlighting the difference between unmodified and glycated collagen I surfaces.

  14. Effect of temperature towards lipid oxidation and non-enzymatic browning reactions in krill oil upon storage

    DEFF Research Database (Denmark)

    Lu, Henna Fung Sieng; Bruheim, I.; Haugsgjerd, B.O.

    2014-01-01

    was assessed by peroxide value and anisidine value, measurement of lipid derived volatiles, lipid classes and antioxidants. The non-enzymatic browning reactions were assessed through the measurement of pyrroles, free amino acids content and Strecker-derived volatiles. The increase of incubation temperature......The main objective of this study was to investigate the effect of temperature towards lipid oxidation and non-enzymatic browning reactions in krill oil upon storage. Krill oil was incubated at two different temperatures (20 and 40°C) for 28 or 42 days. The oxidative stability of krill oil...

  15. Bioinformatic prediction of arthropod/nematode-like peptides in non-arthropod, non-nematode members of the Ecdysozoa.

    Science.gov (United States)

    Christie, Andrew E; Nolan, Daniel H; Garcia, Zachery A; McCoole, Matthew D; Harmon, Sarah M; Congdon-Jones, Benjamin; Ohno, Paul; Hartline, Niko; Congdon, Clare Bates; Baer, Kevin N; Lenz, Petra H

    2011-02-01

    The Onychophora, Priapulida and Tardigrada, along with the Arthropoda, Nematoda and several other small phyla, form the superphylum Ecdysozoa. Numerous peptidomic studies have been undertaken for both the arthropods and nematodes, resulting in the identification of many peptides from each group. In contrast, little is known about the peptides used as paracrines/hormones by species from the other ecdysozoan taxa. Here, transcriptome mining and bioinformatic peptide prediction were used to identify peptides in members of the Onychophora, Priapulida and Tardigrada, the only non-arthropod, non-nematode members of the Ecdysozoa for which there are publicly accessible expressed sequence tags (ESTs). The extant ESTs for each phylum were queried using 106 arthropod/nematode peptide precursors. Transcripts encoding calcitonin-like diuretic hormone and pigment-dispersing hormone (PDH) were identified for the onychophoran Peripatopsis sedgwicki, with transcripts encoding C-type allatostatin (C-AST) and FMRFamide-like peptide identified for the priapulid Priapulus caudatus. For the Tardigrada, transcripts encoding members of the A-type allatostatin, C-AST, insect kinin, orcokinin, PDH and tachykinin-related peptide families were identified, all but one from Hypsibius dujardini (the exception being a Milnesium tardigradum orcokinin-encoding transcript). The proteins deduced from these ESTs resulted in the prediction of 48 novel peptides, six onychophoran, eight priapulid and 34 tardigrade, which are the first described from these phyla. Copyright © 2010 Elsevier Inc. All rights reserved.

  16. Non-enzymatic N-acetylation of Lysine Residues by AcetylCoA Often Occurs via a Proximal S-acetylated Thiol Intermediate Sensitive to Glyoxalase II

    Directory of Open Access Journals (Sweden)

    Andrew M. James

    2017-02-01

    Full Text Available Summary: Acetyl coenzyme A (AcCoA, a key intermediate in mitochondrial metabolism, N-acetylates lysine residues, disrupting and, in some cases, regulating protein function. The mitochondrial lysine deacetylase Sirtuin 3 (Sirt3 reverses this modification with benefits reported in diabetes, obesity, and aging. We show that non-enzymatic lysine N-acetylation by AcCoA is greatly enhanced by initial acetylation of a cysteine residue, followed by SN-transfer of the acetyl moiety to a nearby lysine on mitochondrial proteins and synthetic peptides. The frequent occurrence of an S-acetyl intermediate before lysine N-acetylation suggests that proximity to a thioester is a key determinant of lysine susceptibility to acetylation. The thioesterase glyoxalase II (Glo2 can limit protein S-acetylation, thereby preventing subsequent lysine N-acetylation. This suggests that the hitherto obscure role of Glo2 in mitochondria is to act upstream of Sirt3 in minimizing protein N-acetylation, thus limiting protein dysfunction when AcCoA accumulates. : James et al. show that the non-enzymatic N-acetylation of lysine residues in mitochondrial proteins frequently occurs via a proximal S-acetylated thiol intermediate. Glutathione equilibrates with this intermediate, allowing the thioesterase glyoxalase II to limit protein lysine N-acetylation. These findings expand our understanding of how protein acetylation arises. Keywords: AcetylCoA, lysine acetylation, glyoxalase

  17. Bioactive Peptides in Milk Products. | Tirelli | Journal of Food ...

    African Journals Online (AJOL)

    Some peptides produced in vitro or in vivo by enzymatic hydrolysis of caseins and whey protein can affect some biological functions of the body and therefore they are called bioactive peptides. In this paper the physiological significance of bioactive peptides is reviewed and the analytical methods for their purification and ...

  18. Targeting nanoparticles to M cells with non-peptidic ligands for oral vaccination

    OpenAIRE

    Fievez, Virginie; Plapied, Laurence; des Rieux, Anne; Pourcelle, Vincent; Freichels, Hélène; Wascotte, Valentine; Vanderhaegen, Marie-Lyse; Jérôme, Christine; Vanderplasschen, Alain; Marchand-Brynaert, Jacqueline; Préat, Véronique

    2009-01-01

    The presence of RGD on nanoparticles allows the targeting of β1 integrins at the apical surface of human M cells and the enhancement of an immune response after oral immunization. To check the hypothesis that non-peptidic ligands targeting intestinal M cells or APCs would be more efficient for oral immunization than RGD, novel non-peptidic and peptidic analogs (RGD peptidomimitic (RGDp), LDV derivative (LDVd) and LDV peptidomimetic (LDVp)) as well as mannose were grafted on the PEG chain of P...

  19. Photodissociative Cross-Linking of Non-covalent Peptide-Peptide Ion Complexes in the Gas Phase

    Science.gov (United States)

    Nguyen, Huong T. H.; Andrikopoulos, Prokopis C.; Rulíšek, Lubomír; Shaffer, Christopher J.; Tureček, František

    2018-05-01

    We report a gas-phase UV photodissociation study investigating non-covalent interactions between neutral hydrophobic pentapeptides and peptide ions incorporating a diazirine-tagged photoleucine residue. Phenylalanine (Phe) and proline (Pro) were chosen as the conformation-affecting residues that were incorporated into a small library of neutral pentapeptides. Gas-phase ion-molecule complexes of these peptides with photo-labeled pentapeptides were subjected to photodissociation. Selective photocleavage of the diazirine ring at 355 nm formed short-lived carbene intermediates that underwent cross-linking by insertion into H-X bonds of the target peptide. The cross-link positions were established from collision-induced dissociation tandem mass spectra (CID-MS3) providing sequence information on the covalent adducts. Effects of the amino acid residue (Pro or Phe) and its position in the target peptide sequence were evaluated. For proline-containing peptides, interactions resulting in covalent cross-links in these complexes became more prominent as proline was moved towards the C-terminus of the target peptide sequence. The photocross-linking yields of phenylalanine-containing peptides depended on the position of both phenylalanine and photoleucine. Density functional theory calculations were used to assign structures of low-energy conformers of the (GLPMG + GLL*LK + H)+ complex. Born-Oppenheimer molecular dynamics trajectory calculations were used to capture the thermal motion in the complexes within 100 ps and determine close contacts between the incipient carbene and the H-X bonds in the target peptide. This provided atomic-level resolution of potential cross-links that aided spectra interpretation and was in agreement with experimental data. [Figure not available: see fulltext.

  20. CuO nanoparticles supported on nitrogen and sulfur co-doped graphene nanocomposites for non-enzymatic glucose sensing

    Energy Technology Data Exchange (ETDEWEB)

    Li, Meixia [Hebei University of Engineering, Faculty of Material Science and Engineering (China); Guo, Qingbin, E-mail: guoqingbinhue@163.com [Hebei University of Engineering, Academic Affairs office (China); Xie, Juan; Li, Yongde; Feng, Yapeng [Hebei University of Engineering, Faculty of Material Science and Engineering (China)

    2017-01-15

    Developing highly active catalysts to promote the electrocatalytic glucose oxidation (EGO) is a crucial demand for non-enzymatic glucose sensing. Herein, we reported the use of nitrogen and sulfur co-doped graphene (NSG) as a novel support material for anchoring CuO nanoparticles and obtained CuO/NSG was employed as an efficient EGO catalyst for non-enzymatic glucose sensing. The results showed that the NSG endowed the CuO/NSG with large surface area, increased structural defects, improved conductivity, and strong covalent coupling between NSG and CuO. Owing to the significant contribution of NSG and the synergistic effect of NSG and CuO, the CuO/NSG exhibited a remarkably higher EGO activity than CuO and CuO/reduced graphene oxide. The CuO/NSG-based sensor displayed excellent glucose sensing performances with a considerably low detection limit of 0.07 μM. These findings elucidate that the NSG is a promising support material for non-enzymatic glucose detection.

  1. Vitamin C Degradation Products and Pathways in the Human Lens*

    OpenAIRE

    Nemet, Ina; Monnier, Vincent M.

    2011-01-01

    Vitamin C and its degradation products participate in chemical modifications of proteins in vivo through non-enzymatic glycation (Maillard reaction) and formation of different products called advanced glycation end products. Vitamin C levels are particularly high in selected tissues, such as lens, brain and adrenal gland, and its degradation products can inflict substantial protein damage via formation of advanced glycation end products. However, the pathways of in vivo vitamin C degradation ...

  2. Advanced glycation end products and sorbitol in blood from differently compensated diabetic dogs.

    Science.gov (United States)

    Comazzi, S; Bertazzolo, W; Bonfanti, U; Spagnolo, V; Sartorelli, P

    2008-06-01

    Canine diabetes mellitus (DM) is a common metabolic disorder with long term complications, most of which are caused by glycosylation of structural proteins, decreases in antioxidant concentrations, altered osmotic balance and hypoxia due to impaired oxygen transport. Previous studies have demonstrated that under hyperglycemic conditions canine erythrocytes undergo swelling, probably due to activation of the polyol pathway. The present work aimed to assess the plasma concentration of advanced glycation end (AGE) products, stable Amadori-products generated by non-enzymatic glycosylation of proteins and the intracellular concentration of sorbitol, produced by the activation of polyol pathway in 34 blood samples from diabetic dogs and in 14 controls. AGE products were significantly higher (pdogs compared with control animals. The sorbitol concentration in erythrocytes was also significantly higher in diabetic dogs and, in particular, in poorly compensated animals and in dogs with ketonuria. In five cases that were analysed before and after clinical improvement, sorbitol concentration was found to correlate with improvement. These results suggest that non-specific glycosylation is increased and that the polyol pathway is activated in diabetic dogs in a manner that is proportionate to the severity of disease. Moreover, the concentration of AGE products and sorbitol may be useful for monitoring the onset of diabetic complications and assessing the most appropriate therapeutic approaches for management of canine DM.

  3. Valorisation of tuna processing waste biomass for recovery of functional and antioxidant peptides using enzymatic hydrolysis and membrane fractionation process.

    Science.gov (United States)

    Saidi, Sami; Ben Amar, Raja

    2016-10-01

    The enzymatic hydrolysis using Prolyve BS coupled to membrane process (Ultrafiltration (UF) and nanofiltration (NF)) is a means of biotransformation of tuna protein waste to Tuna protein hydrolysate (TPH) with higher added values. This method could be an effective solution for the production of bioactive compounds used in various biotechnological applications and minimizing the pollution problems generated by the seafood processing industries. The amino acid composition, functional and antioxidant properties of produced TPH were evaluated. The results show that the glutamic acid, aspartic acid, glycine, alaline, valine and leucine were the major amino acids detected in the TPH profile. After membrane fractionation process, those major amino acids were concentrated in the NF retentate (NFR). The NFR and NF permeate (NFP) have a higher protein solubility (>95 %) when compared to TPH (80 %). Higher oil and water binding capacity were observed in TPH and higher emulsifying and foam stability was found in UF retentate. The NFP showed the highest DPPH radical scavenging activity (65 %). The NFR contained antioxidant amino acid (30.3 %) showed the highest superoxide radical and reducing power activities. The TPH showed the highest iron chelating activity (75 %) compared to other peptide fractions. The effect of the membrane fractionation on the molecular weight distribution of the peptide and their bioactivities was underlined. We concluded that the TPH is a valuable source of bioactive peptides and their peptide fractions may serve as useful ingredients for application in food industry and formulation of nutritional products.

  4. Role of enzymatic and non enzymatic antioxidant in ameliorating salinity induced damage in nostoc muscorum

    International Nuclear Information System (INIS)

    Hend, A.; Abeer, A.; Allah, A.

    2015-01-01

    Presence of high salt concentration in the growth medium adversely affected the plant growth and productivity by altering its metabolic activities. Experiments were conducted on cyanobacteriaum Nostoc muscorum grown in nitrogen free medium supplemented with 250 mM NaCl to evaluate the salt stress induced changes in growth, antioxidants and lipid composition. Salt stress significantly reduced the growth and physio-biochemical attributes. Salt stress increased malonaldehyde content thereby causing alterations in the lipid fraction. Significant reduction in polyunsaturated fatty acids including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI) and phosphatidylserine (PS) was observed. Where as diacylglycerol, sterol ester and non-esterified fatty acids were increased. Activities of antioxidant enzymes and contents of non-enzymatic antioxidants including glutathione enhanced due to salt stress. An increase in accumulation of proline was also observed. Hence increased activity of antioxidants and altered fatty acid composition was observed in salt stressed Nostoc muscorum. (author)

  5. [Non-enzymatic glycosylation of dietary protein in vitro].

    Science.gov (United States)

    Bednykh, B S; Evdokimov, I A; Sokolov, A I

    2015-01-01

    Non-enzymatic glycosylation of proteins, based on discovered by Mayarn reaction of carbohydrate aldehyde group with a free amino group of a protein molecule, is well known to experts in biochemistry of food industry. Generated brown solid in some cases give the product marketable qualities--crackling bread--in others conversely, worsen the product. The biological effects of far-advanced products of non-enzymatic protein glycosylation reaction have not been studied enough, although it was reported previously that they are not split by digestive enzymes and couldn't be absorbed by animals. The objective of this work was to compare the depth of glycosylation of different food proteins of animal and vegetable origin. The objects of the study were proteins of animal (casein, lactoglobulin, albumin) and vegetable (soy isolate, proteins of rice flour, buckwheat, oatmeal) origin, glucose and fructose were selected as glycosylation agents, exposure 15 days at 37 degrees C. Lactoglobulin was glycosylated to a lesser extent among the proteins of animal origin while protein of oatmeal was glycosylated in the least degree among vegetable proteins. Conversely, such proteins as casein and soya isolate protein bound rather large amounts of carbohydrates. Fructose binding with protein was generally higher than the binding of glucose. The only exception was a protein of oatmeal. When of glucose and fructose simultaneously presented in the incubation medium, glucose binding usually increased while binding of fructose, in contrast, reduced. According to the total amount of carbohydrate (mcg), which is able to attach a protein (mg) the studied food proteins located in the following order: albumin (38) > soy protein isolate (23) > casein (15,) > whey protein rice flour protein (6) > protein from buckwheat flour (3) > globulin (2) > protein of oatmeal (0.3). The results obtained are to be used to select the optimal combination of proteins and carbohydrates, in which the glycosylation

  6. BDNF pro-peptide regulates dendritic spines via caspase-3

    OpenAIRE

    Guo, J; Ji, Y; Ding, Y; Jiang, W; Sun, Y; Lu, B; Nagappan, G

    2016-01-01

    The precursor of brain-derived neurotrophic factor (BDNF) (proBDNF) is enzymatically cleaved, by either intracellular (furin/PC1) or extracellular proteases (tPA/plasmin/MMP), to generate mature BDNF (mBDNF) and its pro-peptide (BDNF pro-peptide). Little is known about the function of BDNF pro-peptide. We have developed an antibody that specifically detects cleaved BDNF pro-peptide, but not proBDNF or mBDNF. Neuronal depolarization elicited a marked increase in extracellular BDNF pro-peptide,...

  7. A study on human serum albumin influence on glycation of fibrinogen

    International Nuclear Information System (INIS)

    Kielmas, Martyna; Szewczuk, Zbigniew; Stefanowicz, Piotr

    2013-01-01

    Highlights: •The glycation of fibrinogen was investigated by isotopic labeling method. •The potential glycation sites in fibrinogen were identified. •Human serum albumin (HSA) inhibits the glycation of fibrinogen. •The effect of HSA on fibrinogen glycation is sequence-dependent. -- Abstract: Although in vivo glycation proceeds in complex mixture of proteins, previous studies did not take in consideration the influence of protein–protein interaction on Maillard reaction. The aim of our study was to test the influence of human serum albumin (HSA) on glycation of fibrinogen. The isotopic labeling using [ 13 C 6 ] glucose combined with LC-MS were applied as tool for identification possible glycation sites in fibrinogen and for evaluation the effect of HSA on the glycation level of selected amino acids in fibrinogen. The obtained data indicate that the addition of HSA protects the fibrinogen from glycation. The level of glycation in presence of HSA is reduced by 30–60% and depends on the location of glycated residue in sequence of protein

  8. A study on human serum albumin influence on glycation of fibrinogen

    Energy Technology Data Exchange (ETDEWEB)

    Kielmas, Martyna; Szewczuk, Zbigniew; Stefanowicz, Piotr, E-mail: Piotr.stefanowicz@chem.uni.wroc.pl

    2013-09-13

    Highlights: •The glycation of fibrinogen was investigated by isotopic labeling method. •The potential glycation sites in fibrinogen were identified. •Human serum albumin (HSA) inhibits the glycation of fibrinogen. •The effect of HSA on fibrinogen glycation is sequence-dependent. -- Abstract: Although in vivo glycation proceeds in complex mixture of proteins, previous studies did not take in consideration the influence of protein–protein interaction on Maillard reaction. The aim of our study was to test the influence of human serum albumin (HSA) on glycation of fibrinogen. The isotopic labeling using [{sup 13}C{sub 6}] glucose combined with LC-MS were applied as tool for identification possible glycation sites in fibrinogen and for evaluation the effect of HSA on the glycation level of selected amino acids in fibrinogen. The obtained data indicate that the addition of HSA protects the fibrinogen from glycation. The level of glycation in presence of HSA is reduced by 30–60% and depends on the location of glycated residue in sequence of protein.

  9. Glycated albumin: from biochemistry and laboratory medicine to clinical practice.

    Science.gov (United States)

    Dozio, Elena; Di Gaetano, Nicola; Findeisen, Peter; Corsi Romanelli, Massimiliano Marco

    2017-03-01

    This review summarizes current knowledge about glycated albumin. We review the changes induced by glycation on the properties of albumin, the pathological implications of high glycated albumin levels, glycated albumin quantification methods, and the use of glycated albumin as a complementary biomarker for diabetes mellitus diagnosis and monitoring and for dealing with long-term complications. The advantages and limits of this biomarker in different clinical settings are also discussed.

  10. The mechanism of reduced IgG/IgE-binding of β-lactoglobulin by pulsed electric field pretreatment combined with glycation revealed by ECD/FTICR-MS.

    Science.gov (United States)

    Yang, Wenhua; Tu, Zongcai; Wang, Hui; Zhang, Lu; Kaltashov, Igor A; Zhao, Yunlong; Niu, Chendi; Yao, Honglin; Ye, Wenfeng

    2018-01-24

    Bovine β-lactoglobulin (β-Lg) is a major allergen existing in milk and causes about 90% of IgE-mediated cow's milk allergies. Previous studies showed that pulsed electric field (PEF) treatment could partially unfold the protein, which may contribute to the improvement of protein glycation. In this study, the effect of PEF pretreatment combined with glycation on the IgG/IgE-binding ability and the structure of β-Lg was investigated. The result showed that PEF pretreatment combined with glycation significantly reduced the IgG and IgE binding abilities, which was attributed to the changes of secondary and tertiary structure and the increase in glycation sites and degree of substitution per peptide (DSP) value determined by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD/FTICR-MS). Unexpectedly, glycation sites (K47, K91 and K135) added by two mannose molecules were identified in glycated β-Lg with PEF pretreatment. Moreover, the results indicated that PEF pretreatment at 25 kV cm -1 for 60 μs promoted the reduction of IgG/IgE-binding capacity by increasing the glycation degree of β-Lg, whereas single PEF treatment under the same conditions markedly enhanced the IgG/IgE-binding ability by partially unfolding the structure of β-Lg. The results suggested that ECD/FTICR-MS could help us to understand the mechanism of reduction in the IgG/IgE-binding of β-Lg by structural characterization at the molecular level. Therefore, PEF pretreatment combined with glycation may provide an alternative method for β-Lg desensitization.

  11. Non-enzymatic palladium recovery on microbial and synthetic surfaces.

    Science.gov (United States)

    Rotaru, Amelia-Elena; Jiang, Wei; Finster, Kai; Skrydstrup, Troels; Meyer, Rikke Louise

    2012-08-01

    The use of microorganisms as support for reduction of dissolved Pd(II) to immobilized Pd(0) nanoparticles is an environmentally friendly approach for Pd recovery from waste. To better understand and engineer Pd(0) nanoparticle synthesis, one has to consider the mechanisms by which Pd(II) is reduced on microbial surfaces. Escherichia coli, Shewanella oneidensis, and Pseudomonas putida were used as model organisms in order to elucidate the role of microbial cells in Pd(II) reduction under acidic conditions. Pd(II) was reduced by formate under acidic conditions, and the process occurred substantially faster in the presence of cells as compared to cell-free controls. We found no difference between native (untreated) and autoclaved cells, and could demonstrate that even a non-enzymatic protein (bovine serum albumin) stimulated Pd(II) reduction as efficiently as bacterial cells. Amine groups readily interact with Pd(II), and to specifically test their role in surface-assisted Pd(II) reduction by formate, we replaced bacterial cells with polystyrene microparticles functionalized with amine or carboxyl groups. Amine-functionalized microparticles had the same effect on Pd(II) reduction as bacterial cells, and the effect could be hampered if the amine groups were blocked by acetylation. The interaction with amine groups was confirmed by infrared spectroscopy on whole cells and amine-functionalized microparticles. In conclusion, bio-supported Pd(II) reduction on microbial surfaces is possibly mediated by a non-enzymatic mechanism. We therefore suggest the use of amine-rich biomaterials rather than intact cells for Pd bio-recovery from waste. Copyright © 2012 Wiley Periodicals, Inc.

  12. Characterization and cytological effects of a novel glycated gelatine substrate

    International Nuclear Information System (INIS)

    Boonkaew, Benjawan; Supaphol, Pitt; Tompkins, Kevin; Manokawinchoke, Jeeranan; Pavasant, Prasit

    2014-01-01

    Hyperglycemia in diabetes results in the glycation of long-lived proteins. Protein glycation leads to the formation of advanced glycation end products (AGEs), which are implicated in delayed wound healing and other diabetes-associated pathologies, one of which is periodontal disease. Research into the mechanisms by which glycated long-lived proteins such as collagen exert their effects can allow for the understanding of diabetic pathologies and the development of appropriate treatments. However, the high cost of purified protein can be a limitation for many laboratories around the world. The objective of this study was to develop a low-cost in vitro model of glycated gelatine as an alternative to the glycated collagen model. We investigated the glycation of gelatine type A, a denatured form of collagen, which is low-cost and abundantly available. In this study, gelatine was incubated for 7 days with ribose or methylglyoxal (MG). Cross-linking, autofluorescence and UV–Vis spectrophotometry assays were performed and indicated a dose-dependent linear increase in cross-linking and autofluorescence of gelatine by ribose and MG. MG produced more cross-linking compared to ribose at the same concentrations. The UV–Vis spectra of the glycated gelatines confirmed the presence of AGE fluorophores. Because diabetes is a risk factor for periodontal disease, the effect of the glycated substrates on the basic behaviour of human periodontal ligament (HPDL) cells was evaluated. Glycation dose dependently reduced HPDL attachment and cell spreading, indicating that the novel glycated gelatine substrate affects cell behaviour. These results show that gelatine glycated with ribose or MG can be used as low-cost in vitro models to study the effects of protein glycation on cell behaviour in diabetes and ageing. (paper)

  13. Commercial processed soy-based food product contains glycated and glycoxidated lunasin proteoforms.

    Science.gov (United States)

    Serra, Aida; Gallart-Palau, Xavier; See-Toh, Rachel Su-En; Hemu, Xinya; Tam, James P; Sze, Siu Kwan

    2016-05-18

    Nutraceuticals have been proposed to exert positive effects on human health and confer protection against many chronic diseases. A major bioactive component of soy-based foods is lunasin peptide, which has potential to exert a major impact on the health of human consumers worldwide, but the biochemical features of dietary lunasin still remain poorly characterized. In this study, lunasin was purified from a soy-based food product via strong anion exchange solid phase extraction and then subjected to top-down mass spectrometry analysis that revealed in detail the molecular diversity of lunasin in processed soybean foods. We detected multiple glycated proteoforms together with potentially toxic advanced glycation end products (AGEs) derived from lunasin. In both cases, modification sites were Lys24 and Lys29 located at the helical region that shows structural homology with a conserved region of chromatin-binding proteins. The identified post-translational modifications may have an important repercussion on lunasin epigenetic regulatory capacity. Taking together, our results demonstrate the importance of proper chemical characterization of commercial processed food products to assess their impact on consumer's health and risk of chronic diseases.

  14. Changes in non-enzymatic antioxidant capacity and lipid peroxidation during germination of white, yellow and purple maize seeds

    International Nuclear Information System (INIS)

    Deng, B.; Zhang, Y.; Yang, K.

    2016-01-01

    In this study, the changes in non-enzymatic antioxidant capacity and lipid peroxidation during the germination process of purple, yellow and white maize seeds were compared, under favorable conditions. Results showed that germination can increase non-enzymatic antioxidant capacity (evaluated with ferric reducing power and 2, 2-diphenyl-1-picryl-hydrazyl-hydrate radical scavenging capacity) and lipid peroxidation levels for all these seeds. In addition, non-enzymatic antioxidant capacity observed in the germinating seeds were in the order of purple > yellow > white. However, the highest and lowest levels of lipid peroxidation could be seen during the germination processes of the white and purple seeds, respectively. In addition, the germination rates of the seeds followed the order of white > yellow > purple. Further studies showed that H/sub 2/O/sub 2/ treatment can significantly promote seed germination, especially for purple seeds. In addition, DMTU (dimethylthiourea), a specific scavenger for H/sub 2/O/sub 2/, could slightly but significantly arrest dormancy release. Data analysis showed that a high negative correlation (R/sup 2/ = -0.955) existed between non-enzymatic antioxidant capacity and germination rates. However, a high positive correlation (R/sup 2/ = 0.860) could be detected between lipid peroxidation and germination rates. Finally, lipid peroxidation as a possible novel signaling mechanism for seed germination has been discussed under stress-free conditions. (author)

  15. Unraveling the mechanism responsible for the contrasting tolerance of Synechocystis and Synechococcus to Cr(VI): Enzymatic and non-enzymatic antioxidants

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, Alka [Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India); Ballal, Anand, E-mail: aballal@barc.gov.in [Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India); Homi Bhabha National Institute, Training School Complex, Anushakti Nagar, Mumbai 40085 (India)

    2015-07-15

    Highlights: • Cr(VI) accumulation generates higher ROS in Synechocystis than in Synechococcus. • Synechococcus exhibits better photosynthetic activity in response to Cr(VI). • Synechococcus has higher enzymatic/non-enzymatic antioxidants than Synechocystis. • Synechococcus shows better tolerance to other oxidative stresses than Synechocystis. • Differential detoxification of ROS is responsible for the contrasting tolerance to Cr(VI) - Abstract: Two unicellular cyanobacteria, Synechocystis and Synechococcus, showed contrasting tolerance to Cr(VI); with Synechococcus being 12-fold more tolerant than Synechocystis to potassium dichromate. The mechanism responsible for this differential sensitivity to Cr(VI) was explored in this study. Total content of photosynthetic pigments as well as photosynthetic activity decreased at lower concentration of Cr(VI) in Synechocystis as compared to Synechococcus. Experiments with {sup 51}Cr showed Cr to accumulate intracellularly in both the cyanobacteria. At lower concentrations, Cr(VI) caused excessive ROS generation in Synechocystis as compared to that observed in Synechococcus. Intrinsic levels of enzymatic antioxidants, i.e., superoxide dismutase, catalase and 2-Cys-peroxiredoxin were considerably higher in Synechococcus than Synechocystis. Content of total thiols (both protein as well as non-protein) and reduced glutathione (GSH) was also higher in Synechococcus as compared to Synechocystis. This correlated well with higher content of carbonylated proteins observed in Synechocystis than Synechococcus. Additionally, in contrast to Synechocystis, Synechococcus exhibited better tolerance to other oxidative stresses like high intensity light and H{sub 2}O{sub 2}. The data indicate that the disparity in the ability to detoxify ROS could be the primary mechanism responsible for the differential tolerance of these cyanobacteria to Cr(VI)

  16. Association of grade of non-alcoholic fatty liver disease and glycated albumin to glycated hemoglobin ratio in patients with type 2 diabetes mellitus.

    Science.gov (United States)

    Jung, Chan-Hee; Lee, Bora; Choi, Dug-Hyun; Jung, Sang-Hee; Kim, Bo-Yeon; Kim, Chul-Hee; Kang, Sung-Koo; Mok, Ji-Oh

    2017-03-01

    The aim of this study was to investigate the association between the glycated albumin (GA) to glycated hemoglobin (HbA1c) (GA/HbA1c) ratio and grade of non-alcoholic fatty liver disease (NAFLD) on ultrasonography (US) in patients with type 2 diabetes mellitus (T2DM). This retrospective, cross-sectional study was performed with data obtained from 186 T2DM patients. Participants were assessed for serum GA/HbA1c ratio and fatty liver using US. NAFLD was defined as ultrasonographically detected fatty liver and was graded as normal, mild, moderate, and severe fatty liver. A total of 98 subjects (53%) were diagnosed with NAFLD on US, of which 47 (48%) had moderate-to-severe grade of NAFLD. The mean GA level and GA/HbA1c ratio significantly decreased across increasing NAFLD stages (34% vs. 29% vs. 27% vs. 28%, p=0.023 for trend; 3.1vs. 2.9vs. 2.6vs. 2.7, p=0.001 for trend, respectively), whereas there was no significant difference in HbA1c level among groups (p=0.714 for trend). There was a significant decrease in prevalence of NAFLD across GA/HbA1c ratio tertiles (67% vs. 58% vs. 41%, p for trend=0.007). Multivariate logistic regression analysis showed that individuals with the lowest GA/HbA1c ratio had an odds ratio (OR) of 2.75 (95% CI=1.06-7.13) for having any grade of NAFLD and an OR of 4.48 [1.20-16.74] for moderate-to-severe grade NAFLD compared with the highest GA/HbA1c ratio even after adjustment for confounding factors (p=0.038, p=0.026, respectively). The present study showed that GA/HbA1c ratio was significantly inversely associated with the presence and severity of NAFLD on US. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  17. Synthesis of stable isotopically labeled peptides with filter-assisted enzymatic labeling for the diagnosis of hepatitis B virus infection utilizing mass spectrometry-based proteomics strategy

    International Nuclear Information System (INIS)

    Tsai, Hsing-Fen; Hsiao, He-Hsuan

    2017-01-01

    A facile method for the preparation of stable isotopically labeled peptides was developed by means of filter-assisted tryptic "1"6O/"1"8O water labeling, which could be directly applied to the determination of hepatitis B virus infection from human serum with tandem mass spectrometry. Tryptic peptides of hepatitis B surface antigen or hepatitis B e antigen from different subtypes of hepatitis B virus were synthesized with traditional solid-phase peptide synthesis as potential biomarkers. Trypsin catalyzed oxygen-18 exchange at their amidated c-terminus of arginine or lysine residue. The protease catalyzed oxygen-18 to oxygen-16 back exchange reaction was eliminated due to the complete removal of trypsin by the centrifugal filter containing a thin membrane associated with molecular weight cut-off of 10 KDa. The synthetic isotopic peptides were spiked into trichloroacetic acid/acetone precipitated human serum as internal standards and were selectively detected with multiplexed parallel reaction monitoring on a hybrid quadrupole-orbitrap mass spectrometer. The limit of detection for all synthetic peptides were in the range of 0.09 fmol–1.13 fmol. The results indicated that the peptide YLWEWASVR derived from hepatitis B surface antigen was quantified approximately 200 fmol per μl serum and may serve as a diagnostic biomarker for the detection of hepatitis B virus infected disease. - Highlights: • Facile synthesis of an inexpensive and highly reproducible stable isotopically labeled peptides. • Complete incorporation of two "1"8O atoms into synthesized peptides with filter-assisted enzymatic labeling. • Targeted analysis with parallel reaction monitoring assay for the disease diagnosis.

  18. Synthesis of stable isotopically labeled peptides with filter-assisted enzymatic labeling for the diagnosis of hepatitis B virus infection utilizing mass spectrometry-based proteomics strategy

    Energy Technology Data Exchange (ETDEWEB)

    Tsai, Hsing-Fen; Hsiao, He-Hsuan, E-mail: hhhsiao@dragon.nchu.edu.tw

    2017-03-01

    A facile method for the preparation of stable isotopically labeled peptides was developed by means of filter-assisted tryptic {sup 16}O/{sup 18}O water labeling, which could be directly applied to the determination of hepatitis B virus infection from human serum with tandem mass spectrometry. Tryptic peptides of hepatitis B surface antigen or hepatitis B e antigen from different subtypes of hepatitis B virus were synthesized with traditional solid-phase peptide synthesis as potential biomarkers. Trypsin catalyzed oxygen-18 exchange at their amidated c-terminus of arginine or lysine residue. The protease catalyzed oxygen-18 to oxygen-16 back exchange reaction was eliminated due to the complete removal of trypsin by the centrifugal filter containing a thin membrane associated with molecular weight cut-off of 10 KDa. The synthetic isotopic peptides were spiked into trichloroacetic acid/acetone precipitated human serum as internal standards and were selectively detected with multiplexed parallel reaction monitoring on a hybrid quadrupole-orbitrap mass spectrometer. The limit of detection for all synthetic peptides were in the range of 0.09 fmol–1.13 fmol. The results indicated that the peptide YLWEWASVR derived from hepatitis B surface antigen was quantified approximately 200 fmol per μl serum and may serve as a diagnostic biomarker for the detection of hepatitis B virus infected disease. - Highlights: • Facile synthesis of an inexpensive and highly reproducible stable isotopically labeled peptides. • Complete incorporation of two {sup 18}O atoms into synthesized peptides with filter-assisted enzymatic labeling. • Targeted analysis with parallel reaction monitoring assay for the disease diagnosis.

  19. Glycation inhibits trichloroacetic acid (TCA)-induced whey protein precipitation

    Science.gov (United States)

    Four different WPI saccharide conjugates were successfully prepared to test whether glycation could inhibit WPI precipitation induced by trichloroacetic acid (TCA). Conjugates molecular weights after glycation were analyzed with SDS-PAGE. No significant secondary structure change due to glycation wa...

  20. Electrochemical non-enzymatic glucose sensors

    International Nuclear Information System (INIS)

    Park, Sejin; Boo, Hankil; Chung, Taek Dong

    2006-01-01

    The electrochemical determination of glucose concentration without using enzyme is one of the dreams that many researchers have been trying to make come true. As new materials have been reported and more knowledge on detailed mechanism of glucose oxidation has been unveiled, the non-enzymatic glucose sensor keeps coming closer to practical applications. Recent reports strongly imply that this progress will be accelerated in 'nanoera'. This article reviews the history of unraveling the mechanism of direct electrochemical oxidation of glucose and making attempts to develop successful electrochemical glucose sensors. The electrochemical oxidation of glucose molecules involves complex processes of adsorption, electron transfer, and subsequent chemical rearrangement, which are combined with the surface reactions on the metal surfaces. The information about the direct oxidation of glucose on solid-state surfaces as well as new electrode materials will lead us to possible breakthroughs in designing the enzymeless glucose sensing devices that realize innovative and powerful detection. An example of those is to introduce nanoporous platinum as an electrode, on which glucose is oxidized electrochemically with remarkable sensitivity and selectivity. Better model of such glucose sensors is sought by summarizing and revisiting the previous reports on the electrochemistry of glucose itself and new electrode materials

  1. Reference values for the Chinese population of skin autofluorescence as a marker of advanced glycation end products accumulated in tissue

    NARCIS (Netherlands)

    Yue, X.; Hu, H.; Koetsier, M.; Graaff, R.; Han, C.

    Aim Advanced glycation end products play an important role in the pathophysiology of several chronic and age-related diseases, especially diabetes mellitus. Skin autofluorescence is a non-invasive method for assessing levels of tissue advanced glycation end products. This study aims to establish the

  2. Plant proteases for bioactive peptides release: A review.

    Science.gov (United States)

    Mazorra-Manzano, M A; Ramírez-Suarez, J C; Yada, R Y

    2017-04-10

    Proteins are a potential source of health-promoting biomolecules with medical, nutraceutical, and food applications. Nowadays, bioactive peptides production, its isolation, characterization, and strategies for its delivery to target sites are a matter of intensive research. In vitro and in vivo studies regarding the bioactivity of peptides has generated strong evidence of their health benefits. Dairy proteins are considered the richest source of bioactive peptides, however proteins from animal and vegetable origin also have been shown to be important sources. Enzymatic hydrolysis has been the process most commonly used for bioactive peptide production. Most commercial enzymatic preparations frequently used are from animal (e.g., trypsin and pepsin) and microbial (e.g., Alcalase® and Neutrase®) sources. Although the use of plant proteases is still relatively limited to papain and bromelain from papaya and pineapple, respectively, the application of new plant proteases is increasing. This review presents the latest knowledge in the use and diversity of plant proteases for bioactive peptides release from food proteins including both available commercial plant proteases as well as new potential plant sources. Furthermore, the properties of peptides released by plant proteases and health benefits associated in the control of disorders such as hypertension, diabetes, obesity, and cancer are reviewed.

  3. Open-tubular capillary electrochromatography with bare gold nanoparticles-based stationary phase applied to separation of trypsin digested native and glycated proteins

    Czech Academy of Sciences Publication Activity Database

    Mikšík, Ivan; Lacinová, Kateřina; Zmatlíková, Zdeňka; Sedláková, Pavla; Král, V.; Sýkora, D.; Řezanka, P.; Kašička, Václav

    2012-01-01

    Roč. 35, č. 8 (2012), s. 994-1002 ISSN 1615-9306 R&D Projects: GA ČR(CZ) GA203/09/0675; GA ČR(CZ) GA203/08/1428 Institutional research plan: CEZ:AV0Z50110509; CEZ:AV0Z40550506 Keywords : capillary electrochromatography * gold nanoparticles * glycation * peptide maps * proteins Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 2.591, year: 2012

  4. Beyond genetic factors in familial amyloidotic polyneuropathy: protein glycation and the loss of fibrinogen's chaperone activity.

    Directory of Open Access Journals (Sweden)

    Gonçalo da Costa

    Full Text Available Familial amyloidotic polyneuropathy (FAP is a systemic conformational disease characterized by extracellular amyloid fibril formation from plasma transthyretin (TTR. This is a crippling, fatal disease for which liver transplantation is the only effective therapy. More than 80 TTR point mutations are associated with amyloidotic diseases and the most widely accepted disease model relates TTR tetramer instability with TTR point mutations. However, this model fails to explain two observations. First, native TTR also forms amyloid in systemic senile amyloidosis, a geriatric disease. Second, age at disease onset varies by decades for patients bearing the same mutation and some mutation carrier individuals are asymptomatic throughout their lives. Hence, mutations only accelerate the process and non-genetic factors must play a key role in the molecular mechanisms of disease. One of these factors is protein glycation, previously associated with conformational diseases like Alzheimer's and Parkinson's. The glycation hypothesis in FAP is supported by our previous discovery of methylglyoxal-derived glycation of amyloid fibrils in FAP patients. Here we show that plasma proteins are differentially glycated by methylglyoxal in FAP patients and that fibrinogen is the main glycation target. Moreover, we also found that fibrinogen interacts with TTR in plasma. Fibrinogen has chaperone activity which is compromised upon glycation by methylglyoxal. Hence, we propose that methylglyoxal glycation hampers the chaperone activity of fibrinogen, rendering TTR more prone to aggregation, amyloid formation and ultimately, disease.

  5. Isotope effects in the non enzymic glycation of hemoglobin catalyzed by DPG

    International Nuclear Information System (INIS)

    Gil, Herminia; Uzcategui, Jorge

    1993-01-01

    The paradigmatic reaction of glucose with hemoglobin (Hb A o ) has been studied and is known to occur most rapidly at the N-terminal valine of the β-subunit. An initial, rapid imine formation is succeeded by slower Amadori rearrangement. Non enzymic glycation of Hb A o was studied in vitro in buffer Tris 10 mM in H 2 O and D 2 O, pH 7.3, pD 7.8 at 37 deg C at a fixed concentration of 2,3 diphosphoglycerate (DPG). The reaction exhibits identical rates in protium and deuterium oxides. When D-glucose-2-h is compared with D-glucose-2-d, the kinetic isotope effect for the DPG-dependent rate is 2.1 ± 0.3, while the DPG-independent rate constant shows no isotope effect (1.1 ± 0.1). The absence of a rate in isotopic water solvents shows that proton donation for solvent, lyons or DPG does not limit the rate. The substrate isotope effect of around 2 for the DPG kinetic term indicates that the proton abstraction step of the Amadori rearrangement by DPG is wholly or partially rate-limiting for this reaction. (author)

  6. Effect of topical application of fluoride gel NaF 2% on enzymatic and non-enzymatic antioxidant parameters of saliva.

    Science.gov (United States)

    Leite, Mariana Ferreira; Ferreira, Nayara Ferraz D'Assumpção; Shitsuka, Caleb David Willy Moreira; Lima, Amanda Martins; Masuyama, Mônica Miyuki; Sant'Anna, Giselle Rodrigues; Yamaguti, Paula Mochidome; Polotow, Tatiana G; de Barros, Marcelo Paes

    2012-06-01

    The aim of the study was to evaluate the effect of topical fluoride gel NaF 2% application on antioxidant parameters of whole saliva from children. The saliva mechanically stimulated with parafilm was collected from 25 children (6-12 years) attending the Clinic of Paediatric Dentistry of Universidade Cruzeiro do Sul, São Paulo, Brazil, before (control group) and immediately after application of neutral fluoride gel NaF 2% (fluoride-gel group), according to the Standards for Research Using Human Subjects, Resolution 196/96 of the USA National Health Council of 10/10/1996. Afterwards, pre-post ferric-reducing antioxidant power (FRAP), trolox-equivalent antioxidant capacity (TEAC), uric acid, reduced/oxidised glutathione content (GSH/GSSG) and total peroxidase activity (TPO) were evaluated in whole saliva of both groups. All non-enzymatic antioxidant parameters were augmented by fluoride-gel NaF 2% application, whereas a notable reduction (31%) of peroxidase activity was concomitantly observed in the children's saliva (p ≤ 0.05). Nevertheless, the reducing power of saliva was kept unaltered under these circumstances (p ≤ 0.05). Despite the reduced activity of peroxidase (an important antimicrobial and antioxidant enzyme), the topical fluoride gel NaF 2% favourably stimulated the release of non-enzymatic antioxidant components of saliva, sustaining the reducing power of saliva and the natural defences of the oral cavity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. Nonenzymatic glycation of phosphatidylethanolamine in erythrocyte vesicles

    International Nuclear Information System (INIS)

    Patkowska, M.J.; Horowitz, M.I.

    1986-01-01

    Unsealed inside-out and right-side out vesicles were prepared from human red cells. The vesicles were incubated with D-glucose [ 14 C(U)] and sodium cyanoborohydride in phosphate buffer, pH 7.4. After incubation, lipids were extracted with 1-butanol and non-lipid contaminants removed by Sephadex G-25 chromatography. Phosphatidylethanolamine-sorbitol was purified by chromatography on columns of silicic acid and phenylboronate agarose gel. Phospholipase C (B. cereus) liberated phosphoethanolamine-sorbitol (I) which comigrated on TLC with synthetic I prepared by reductive condensation of phosphoethanolamine and D-glucose and also with the product of phospholipase C (B. cereus) hydrolysis of reference phosphatidylethanolamine-sorbitol. Exposure of I to alkaline phosphatase (E. coli) gave P/sub i/ and ethanolamine-sorbitol (II) which comigrated on TLC with synthetic II prepared by reductive condensation of ethanolamine and D-glucose or by phospholipase D hydrolysis of reference phosphatidylethanolamine-sorbitol. These studies demonstrate that vesicular phosphatidylethanolamine can be reductively glycated and illustrate the applicability of both phospholipase C and phospholipase D in characterizing glycated phosphoglycerides

  8. Inhibition of protein glycation and advanced glycation end products ...

    African Journals Online (AJOL)

    Advanced glycation end products (AGEs) formation is increased in diabetes mellitus, leading to microvascular and macrovascular complications. Recently, much attention has been focused on natural and synthetic inhibitors to delay the onset or progression of diabetes and its comorbidities. Ascorbic acid (AA) can react with ...

  9. Cell-penetrating peptides as tools to enhance non-injectable delivery of biopharmaceuticals

    DEFF Research Database (Denmark)

    Kristensen, Mie; Nielsen, Hanne Mørck

    2016-01-01

    Non-injectable delivery of peptide and protein drugs is hampered by their labile nature, hydrophilicity, and large molecular size; thus limiting their permeation across mucosae, which represent major biochemical and physical barriers to drugs administered via e.g. the oral, nasal, and pulmonary...... routes. However, in recent years cell-penetrating peptides (CPP) have emerged as promising tools to enhance mucosal delivery of co-administered or conjugated peptide and protein cargo and more advanced CPP-cargo formulations are emerging. CPPs act as transepithelial delivery vectors, but the mechanism...... understanding, documentation of CPP-mediated delivery in higher animal species than rodent as well as extensive toxicological studies are necessary for CPP-containing non-injectable DDSs to reach the clinic....

  10. Inhibition of Nonenzymatic Protein Glycation by Pomegranate and Other Fruit Juices

    Science.gov (United States)

    Dorsey, Pamela Garner; Greenspan, Phillip

    2014-01-01

    Abstract The nonenzymatic glycation of proteins and the formation of advanced glycation endproducts in diabetes leads to the crosslinking of proteins and disease complications. Our study sought to demonstrate the effect of commonly consumed juices (pomegranate, cranberry, black cherry, pineapple, apple, and Concord grape) on the fructose-mediated glycation of albumin. Albumin glycation decreased by 98% in the presence of 10 μL of pomegranate juice/mL; other juices inhibited glycation by only 20%. Pomegranate juice produced the greatest inhibition on protein glycation when incubated at both the same phenolic concentration and the same antioxidant potential. Both punicalagin and ellagic acid significantly inhibited the glycation of albumin by ∼90% at 5 μg/mL. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis revealed that pomegranate, but not apple juice, protected albumin from modification. These results demonstrate that pomegranate juice and two of its major constituents are potent inhibitors of fructose-mediated protein glycation. PMID:24433074

  11. Facile synthesis of a silver nanoparticles/polypyrrole nanocomposite for non-enzymatic glucose determination.

    Science.gov (United States)

    Poletti Papi, Maurício A; Caetano, Fabio R; Bergamini, Márcio F; Marcolino-Junior, Luiz H

    2017-06-01

    The present work describes the synthesis of a new conductive nanocomposite based on polypyrrole (PPy) and silver nanoparticles (PPy-AgNP) based on a facile reverse microemulsion method and its application as a non-enzymatic electrochemical sensor for glucose detection. Focusing on the best sensor performance, all experimental parameters used in the synthesis of nanocomposite were optimized based on its electrochemical response for glucose. Characterization of the optimized material by FT-IR, cyclic voltammetry, and DRX measurements and TEM images showed good monodispersion of semispherical Ag nanoparticles capped by PPy structure, with size average of 12±5nm. Under the best analytical conditions, the proposed sensor exhibited glucose response in linear dynamic range of 25 to 2500μmolL -1 , with limit of detection of 3.6μmolL -1 . Recovery studies with human saliva samples varying from 99 to 105% revealed the accuracy and feasibility of a non-enzymatic electrochemical sensor for glucose determination by easy construction and low-cost. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Technical Report for a Study on the Mechanism and Control of Non-Enzymatic Browning Reaction in Gamma-Irradiated Food

    Energy Technology Data Exchange (ETDEWEB)

    Byun, Myung Woo; Lee, Ju Woon; Kim, Jae Hun

    2007-01-15

    Gamma irradiation leads to a non-enzymatic browning reaction (carbonyl -amine reaction) in an aqueous system similar to those induced in a heated one. This reaction may influence the changes of the color in irradiated foods. The intensity of the reaction was dependent on the type of the sugar, if the occurrence is by irradiation or by heating. There was a difference in the browning reaction between irradiation and heating. Although no browning was observed in the heated solution of the non-reducing sugar, the formation of colored products was observed in the irradiated sucrose-lysine solution. It could be explained on the basis that irradiation promotes the breakdown of the glycosidic linkages of the disaccharide, sucrose and the produce of a reducing power. The high molecular weight melanoidin (> MW 12,000-14,000 Da) was produced by gamma irradiation from the non-enzymatic browning reaction between glucose and glycine. The structure of melanoidin was similar to melanodin from heat processing. The results suggested that gamma-irradiation occurred the non-enzymatic browning reaction that is similar the reaction by heat processing. Non-enzymatic browning reaction during gamma-irradiation processing was greatly influenced by pH and medium of reaction system. The brown color development of irradiated sugar solutions with and without glycine is more increased in buffer system especially with alkaline pH than DDW. When food is irradiated, off-color such as browning can be produced due to the non-enzymatic browning reaction and it is influenced by other ions and/or pH of system. This suggests that the browning of irradiated food might be retarded by lowering the pH of the system. Gamma-irradiation produce the free radical and the radiolysis products of sugar and glycine and then they may be condensed to colored products during post-irradiation. However, when the food is irradiated in frozen state, the production of free radical and radiolysis product is inhibited and it

  13. Technical Report for a Study on the Mechanism and Control of Non-Enzymatic Browning Reaction in Gamma-Irradiated Food

    International Nuclear Information System (INIS)

    Byun, Myung Woo; Lee, Ju Woon; Kim, Jae Hun

    2007-01-01

    Gamma irradiation leads to a non-enzymatic browning reaction (carbonyl -amine reaction) in an aqueous system similar to those induced in a heated one. This reaction may influence the changes of the color in irradiated foods. The intensity of the reaction was dependent on the type of the sugar, if the occurrence is by irradiation or by heating. There was a difference in the browning reaction between irradiation and heating. Although no browning was observed in the heated solution of the non-reducing sugar, the formation of colored products was observed in the irradiated sucrose-lysine solution. It could be explained on the basis that irradiation promotes the breakdown of the glycosidic linkages of the disaccharide, sucrose and the produce of a reducing power. The high molecular weight melanoidin (> MW 12,000-14,000 Da) was produced by gamma irradiation from the non-enzymatic browning reaction between glucose and glycine. The structure of melanoidin was similar to melanodin from heat processing. The results suggested that gamma-irradiation occurred the non-enzymatic browning reaction that is similar the reaction by heat processing. Non-enzymatic browning reaction during gamma-irradiation processing was greatly influenced by pH and medium of reaction system. The brown color development of irradiated sugar solutions with and without glycine is more increased in buffer system especially with alkaline pH than DDW. When food is irradiated, off-color such as browning can be produced due to the non-enzymatic browning reaction and it is influenced by other ions and/or pH of system. This suggests that the browning of irradiated food might be retarded by lowering the pH of the system. Gamma-irradiation produce the free radical and the radiolysis products of sugar and glycine and then they may be condensed to colored products during post-irradiation. However, when the food is irradiated in frozen state, the production of free radical and radiolysis product is inhibited and it

  14. Boronate-Modified Interdigitated Electrode Array for Selective Impedance-Based Sensing of Glycated Hemoglobin

    DEFF Research Database (Denmark)

    Boonyasit, Yuwadee; Laiwattanapaisal, Wanida; Chailapakul, Orawon

    2016-01-01

    An impedance-based label-free affinity sensor was developed for the recognition of glycated hemoglobin (HbA1c). Interdigitated gold microelectrode arrays (IDA) were first modified with a self-assembled monolayer of cysteamine followed by cross-linking with glutaraldehyde and subsequent binding of 3......-aminophenylboronic acid (APBA), which selectively binds HbA1c via cis-diol interactions. Impedance sensing was demonstrated to be highly responsive to the clinically relevant HbA1c levels (0.1%-8.36%) with a detection limit of 0.024% (3σ). The specificity of the assay was evaluated with non-glycated hemoglobin (Hb...

  15. Glutamic Acid Selective Chemical Cleavage of Peptide Bonds.

    Science.gov (United States)

    Nalbone, Joseph M; Lahankar, Neelam; Buissereth, Lyssa; Raj, Monika

    2016-03-04

    Site-specific hydrolysis of peptide bonds at glutamic acid under neutral aqueous conditions is reported. The method relies on the activation of the backbone amide chain at glutamic acid by the formation of a pyroglutamyl (pGlu) imide moiety. This activation increases the susceptibility of a peptide bond toward hydrolysis. The method is highly specific and demonstrates broad substrate scope including cleavage of various bioactive peptides with unnatural amino acid residues, which are unsuitable substrates for enzymatic hydrolysis.

  16. Enzymatic vegetable organic extracts as soil biochemical biostimulants and atrazine extenders.

    Science.gov (United States)

    García-Martínez, Ana María; Tejada, Manuel; Díaz, Ana Isabel; Rodríguez-Morgado, Bruno; Bautista, Juan; Parrado, Juan

    2010-09-08

    The purpose of this study was to gather information on the potential effects of organic biostimulants on soil activity and atrazine biodegradation. Carob germ enzymatic extract (CGEE) and wheat condensed distiller solubles enzymatic extract (WCDS-EE) have been obtained using an enzymatic process; their main organic components are soluble carbohydrates and proteins in the form of peptides and free amino acids. Their application to soil results in high biostimulation, rapidly increased dehydrogenase, phosphatase and glucosidase activities, and an observed atrazine extender capacity due to inhibition of its mineralization. The extender capacity of both extracts is proportional to the protein/carbohydrate ratio content. As a result, these enzymatic extracts are highly microbially available, leading to two independent phenomena, fertility and an atrazine persistence that is linked to increased soil activity.

  17. Influence of storage and heating on protein glycation levels of processed lactose-free and regular bovine milk products.

    Science.gov (United States)

    Milkovska-Stamenova, Sanja; Hoffmann, Ralf

    2017-04-15

    Thermal treatment preserves the microbiological safety of milk, but also induces Maillard reactions modifying for example proteins. The purpose of this study was evaluating the influence of consumer behaviors (storage and heating) on protein glycation degrees in bovine milk products. Lactosylation and hexosylation sites were identified in ultra-high temperature (UHT), lactose-free pasteurized, and lactose-free UHT milk (ULF) and infant formula (IF) using tandem mass spectrometry (electron transfer dissociation). Overall, 303 lactosylated and 199 hexosylated peptides were identified corresponding to 170 lactosylation (31 proteins) and 117 hexosylation sites (25 proteins). In quantitative terms, storage increased lactosylation up to fourfold in UHT and IF and hexosylation up to elevenfold in ULF and threefold in IF. These levels increased additionally twofold when the stored samples were heated (40°C). In conclusion, storage and heating appear to influence protein glycation levels in milk at similar or even higher degrees than industrial processing. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. The browning kinetics of the non-enzymatic browning reaction in L-ascorbic acid/basic amino acid systems

    Directory of Open Access Journals (Sweden)

    Ai-Nong YU

    Full Text Available Abstract Under the conditions of weak basis and the reaction temperature range of 110-150 °C, lysine, arginine and histidine were reacted with L-ascorbic acid at equal amount for 30-150 min, respectively and the formation of browning products was monitored with UV–vis spectrometry. The kinetic characteristics of their non-enzymatic browning reaction were investigated. The study results indicated that the non-enzymatic browning reaction of these three amino acids with L-ascorbic acid to form browning products was zero-order reaction. The apparent activation energies for the formation of browning products from L-ascorbic acid/lysine, L-ascorbic acid/arginine and L-ascorbic acid/histidine systems were 54.94, 50.08 and 35.31kJ/mol. The activation energy data indicated the degree of effects of reaction temperature on non-enzymatic browning reaction. Within the temperature range of 110-150 °C, the reaction rate of L-ascorbic acid/lysine system was the fastest one, followed by that of the L-ascorbic acid/arginine system. The reaction rate of L-ascorbic acid/histidine system was the slowest one. Based on the observed kinetic data, the formation mechanisms of browning products were proposed.

  19. Central effects of some peptide and non-peptide opioids and naloxone on thermoregulation in the rabbit

    Science.gov (United States)

    Kandasamy, S. B.; Williams, B. A.

    1983-01-01

    The effects of several peptide and non-peptide opiods and naloxone on induced hyperthermia is studied in rabbits. The effect of tyical mu, kappa, and sigma receptor antagonists (morphine, ketocyclazcine and SKF 10,0 10, 047) and some opioid peptides (Beta-endorphin /BE/, methionine-enkaphalin /ME/, and D-Ala2-methionine-enkaphalin-amide /DAME/ are determined. The role of prostaglandins (PG), cAMP, and norepinephrine (NE) in morphine, BE, and DAME induced hyperthermia is investigated. In addition, the effect of naloxone on pyrogen, arachidonic acid, PGE2, prostacyclin, dibutyryl cAMP, and NE induced hyperthermia is determined. Among other results, it is found that the three receptor antagonists induced hyperthermia in rabbits. BE, ME, and DAME were also found to cause hyperthermia, and it is suggested that they act on the same type of receptor. It is also determined that neither NE nor cAMP is involved in the hyperthermia due to morphine, BE, and DAME. It is suggested that an action of endogenous peptides on naloxone sensitive receptors plays little role in normal thermoregulation or in hyperthermia.

  20. Hepatocyte growth factor protects human endothelial cells against advanced glycation end products-induced apoposis

    International Nuclear Information System (INIS)

    Zhou Yijun; Wang Jiahe; Zhang Jin

    2006-01-01

    Advanced glycation end products (AGEs) form by a non-enzymatic reaction between reducing sugars and biological proteins, which play an important role in the pathogenesis of atherosclerosis. In this study, we assessed AGEs effects on human umbilical vein endothelial cells (HUVECs) growth, proliferation and apoptosis. Additionally, we investigated whether hepatocyte growth factor (HGF), an anti-apoptotic factor for endothelial cells, prevents AGEs-induced apoptosis of HUVECs. HUVECs were treated with AGEs in the presence or absence of HGF. Treatment of HUVECs with AGEs changed cell morphology, decreased cell viability, and induced DNA fragmentation, leading to apoptosis. Apoptosis was induced by AGEs in a dose- and time-dependent fashion. AGEs markedly elevated Bax and decreased NF-κB, but not Bcl-2 expression. Additionally, AGEs significantly inhibited cell growth through a pro-apoptotic action involving caspase-3 and -9 activations in HUVECs. Most importantly, pretreatment with HGF protected against AGEs-induced cytotoxicity in the endothelial cells. HGF significantly promoted the expression of Bcl-2 and NF-κB, while decreasing the activities of caspase-3 and -9 without affecting Bax level. Our data suggest that AGEs induce apoptosis in endothelial cells. HGF effectively attenuate AGEs-induced endothelial cell apoptosis. These findings provide new perspectives in the role of HGF in cardiovascular disease

  1. Isotope effects in the non enzymic glycation of hemoglobin catalyzed by DPG

    Energy Technology Data Exchange (ETDEWEB)

    Gil, Herminia; Uzcategui, Jorge [Universidad de Los Andes, Merida (Venezuela). Dept. de Quimica

    1993-12-31

    The paradigmatic reaction of glucose with hemoglobin (Hb A{sub o}) has been studied and is known to occur most rapidly at the N-terminal valine of the {beta}-subunit. An initial, rapid imine formation is succeeded by slower Amadori rearrangement. Non enzymic glycation of Hb A{sub o} was studied in vitro in buffer Tris 10 mM in H{sub 2} O and D{sub 2} O, pH 7.3, pD 7.8 at 37 deg C at a fixed concentration of 2,3 diphosphoglycerate (DPG). The reaction exhibits identical rates in protium and deuterium oxides. When D-glucose-2-h is compared with D-glucose-2-d, the kinetic isotope effect for the DPG-dependent rate is 2.1 {+-} 0.3, while the DPG-independent rate constant shows no isotope effect (1.1 {+-} 0.1). The absence of a rate in isotopic water solvents shows that proton donation for solvent, lyons or DPG does not limit the rate. The substrate isotope effect of around 2 for the DPG kinetic term indicates that the proton abstraction step of the Amadori rearrangement by DPG is wholly or partially rate-limiting for this reaction. (author) 23 refs., 4 figs.

  2. Archetypal sandwich-structured CuO for high performance non-enzymatic sensing of glucose

    Science.gov (United States)

    Meher, Sumanta Kumar; Rao, G. Ranga

    2013-02-01

    In the quest to enhance the selectivity and sensitivity of novel structured metal oxides for electrochemical non-enzymatic sensing of glucose, we report here a green synthesis of unique sandwich-structured CuO on a large scale under microwave mediated homogeneous precipitation conditions. The physicochemical studies carried out by XRD and BET methods show that the monoclinic CuO formed via thermal decomposition of Cu2(OH)2CO3 possesses monomodal channel-type pores with largely improved surface area (~43 m2 g-1) and pore volume (0.163 cm3 g-1). The fascinating surface morphology and pore structure of CuO is formulated due to homogeneous crystallization and microwave induced self assembly during synthesis. The cyclic voltammetry and chronoamperometry studies show diffusion controlled glucose oxidation at ~0.6 V (vs. Ag/AgCl) with extremely high sensitivity of 5342.8 μA mM-1 cm-2 and respective detection limit and response time of ~1 μM and ~0.7 s, under a wide dynamic concentration range of glucose. The chronoamperometry measurements demonstrate that the sensitivity of CuO to glucose is unaffected by the absence of dissolved oxygen and presence of poisoning chloride ions in the reaction medium, which essentially implies high poison resistance activity of the sandwich-structured CuO. The sandwich-structured CuO also shows insignificant interference/significant selectivity to glucose, even in the presence of high concentrations of other sugars as well as reducing species. In addition, the sandwich-structured CuO shows excellent reproducibility (relative standard deviation of ~2.4% over ten identically fabricated electrodes) and outstanding long term stability (only ~1.3% loss in sensitivity over a period of one month) during non-enzymatic electrochemical sensing of glucose. The unique microstructure and suitable channel-type pore architecture provide structural stability and maximum accessible electroactive surface for unimpeded mobility of glucose as well as the

  3. Inhibition of protein glycation and advanced glycation end products ...

    African Journals Online (AJOL)

    Fatima

    2012-06-26

    Jun 26, 2012 ... acid (AA) can react with proteins, including hemoglobin and possibly interfere with protein glycation process. .... 50 mM) and AA (100, 200, 300 and 400 mM) at 37°C for five weeks ... drops were removed by patting the plate over a paper towel. The .... adults participating in the Beaver Dam Eye Study. In.

  4. Antioxidative capacity and binding affinity of the complex of green tea catechin and beta-lactoglobulin glycated by the Maillard reaction.

    Science.gov (United States)

    Perusko, Marija; Al-Hanish, Ayah; Mihailovic, Jelena; Minic, Simeon; Trifunovic, Sara; Prodic, Ivana; Cirkovic Velickovic, Tanja

    2017-10-01

    Major green tea catechin, epigallocatechin-3-gallate (EGCG), binds non-covalently to numerous dietary proteins, including beta-lactoglobulin of cow's milk. The effects of glycation of proteins via Maillard reaction on the binding capacity for polyphenols and the antiradical properties of the formed complexes have not been studied previously. Binding constant of BLG glycated by milk sugar lactose to EGCG was measured by the method of fluorophore quenching. Binding of EGCG was confirmed by CD and FTIR. The antioxidative properties of the complexes were examined by measuring ABTS radical scavenging capacity, superoxide anion scavenging capacity and total reducing power assay. Glycation of BLG does not significantly influence the binding constant of EGCG for the protein. Conformational changes were observed for both native and glycated BLG upon complexation with EGCG. Masking effect of polyphenol complexation on the antioxidative potential of the protein was of the similar degree for both glycated BLG and native BLG. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Determination of the glycation sites of Bacillus anthracis neoglycoconjugate vaccine by MALDI-TOF/TOF-CID-MS/MS and LC-ESI-QqTOF-tandem mass spectrometry

    Science.gov (United States)

    Jahouh, Farid; Hou, Shu-jie; Kováč, Pavol; Banoub, Joseph H.

    2012-01-01

    We present herein an efficient mass spectrometric method for the localization of the glycation sites of a model neoglycoconjugate vaccine formed by a construct of the tetrasaccharide side chain of the Bacillus anthracis exosporium and the protein carrier bovine serum albumin. The glycoconjugate was digested with both trypsin and GluC V8 endoproteinases, and the digests were then analyzed by MALDI-TOF/TOF-CID-MS/MS and nano-LC-ESI-QqTOF-CID-MS/MS. The sequences of the unknown peptides analyzed by MALDI-TOF/TOF-CID-MS/MS, following digestion with the GluC V8 endoproteinase, allowed us to recognize three glycopeptides whose glycation occupancies were, respectively, on Lys 235, Lys 420, and Lys 498. Similarly, the same analysis was performed on the tryptic digests, which permitted us to recognize two glycation sites on Lys 100 and Lys 374. In addition, we have also used LC-ESI-QqTOF-CID-MS/MS analysis for the identification of the tryptic digests. However, this analysis identified a higher number of glycopeptides than would be expected from a glycoconjugate composed of a carbohydrate–protein ratio of 5.4:1, which would have resulted in glycation occupancies of 18 specific sites. This discrepancy was due to the large number of glycoforms formed during the synthetic carbohydrate–spacer–carrier protein conjugation. Likewise, the LC-ESI-QqTOF-MS/MS analysis of the GluC V8 digest also identified 17 different glycation sites on the synthetic glycoconjugate. PMID:22012665

  6. Collagencin, an antibacterial peptide from fish collagen: Activity, structure and interaction dynamics with membrane

    International Nuclear Information System (INIS)

    Ennaas, Nadia; Hammami, Riadh; Gomaa, Ahmed; Bédard, François; Biron, Éric; Subirade, Muriel; Beaulieu, Lucie; Fliss, Ismail

    2016-01-01

    In this study, we first report characterization of collagencin, an antimicrobial peptide identified from fish collagen hydrolysate. The peptide completely inhibited the growth of Staphylococcus aureus at 1.88 mM. Although non-toxic up to 470 μM, collagencin was hemolytic at higher concentrations. The secondary structure of collagencin was mainly composed by β-sheet and β-turn as determined by CD measurements and molecular dynamics. The peptide is likely to form β-sheet structure under hydrophobic environments and interacts with both anionic (phosphatidylglycerol) and zwitterionic (phosphoethanolamine and phosphatidylcholine) lipids as shown with CD spectroscopy and molecular dynamics. The peptide formed several hydrogen bonds with both POPG and POPE lipids and remained at membrane–water interface, suggesting that collagencin antibacterial action follows a carpet mechanism. Collagenous fish wastes could be processed by enzymatic hydrolysis and transformed into products of high value having functional or biological properties. Marine collagens are a promising source of antimicrobial peptides with new implications in food safety and human health. - Highlights: • Collagencin, an antibacterial (G+ & G-) peptide identified from fish collagen hydrolysate. • The peptide completely inhibited the growth of S. aureus at 1.88 mM and non-toxic at 470 μM. • The secondary structure was mainly composed by β-sheet and turn as determined by CD and MD. • Collagencin interacts with both anionic and zwitterionic lipids as shown with CD and MD. • Collagencin antibacterial action probably follows a carpet mechanism.

  7. Collagencin, an antibacterial peptide from fish collagen: Activity, structure and interaction dynamics with membrane

    Energy Technology Data Exchange (ETDEWEB)

    Ennaas, Nadia [STELA Dairy Research Centre, Institute of Nutrition and Functional Foods, Université Laval, G1V 0A6 Québec, QC (Canada); Hammami, Riadh, E-mail: riadh.hammami@fsaa.ulaval.ca [STELA Dairy Research Centre, Institute of Nutrition and Functional Foods, Université Laval, G1V 0A6 Québec, QC (Canada); Gomaa, Ahmed [STELA Dairy Research Centre, Institute of Nutrition and Functional Foods, Université Laval, G1V 0A6 Québec, QC (Canada); Bédard, François; Biron, Éric [Faculty of Pharmacy, Université Laval and Laboratory of Medicinal Chemistry, CHU de Québec Research Centre, G1V 4G2 Québec, QC (Canada); Subirade, Muriel [STELA Dairy Research Centre, Institute of Nutrition and Functional Foods, Université Laval, G1V 0A6 Québec, QC (Canada); Beaulieu, Lucie, E-mail: lucie.beaulieu@fsaa.ulaval.ca [STELA Dairy Research Centre, Institute of Nutrition and Functional Foods, Université Laval, G1V 0A6 Québec, QC (Canada); Department of Biology, Chemistry and Geography, Université du Québec à Rimouski (UQAR), 300 Allée des Ursulines, Rimouski, QC G5L 3A1 (Canada); Fliss, Ismail, E-mail: ismail.fliss@fsaa.ulaval.ca [STELA Dairy Research Centre, Institute of Nutrition and Functional Foods, Université Laval, G1V 0A6 Québec, QC (Canada)

    2016-04-29

    In this study, we first report characterization of collagencin, an antimicrobial peptide identified from fish collagen hydrolysate. The peptide completely inhibited the growth of Staphylococcus aureus at 1.88 mM. Although non-toxic up to 470 μM, collagencin was hemolytic at higher concentrations. The secondary structure of collagencin was mainly composed by β-sheet and β-turn as determined by CD measurements and molecular dynamics. The peptide is likely to form β-sheet structure under hydrophobic environments and interacts with both anionic (phosphatidylglycerol) and zwitterionic (phosphoethanolamine and phosphatidylcholine) lipids as shown with CD spectroscopy and molecular dynamics. The peptide formed several hydrogen bonds with both POPG and POPE lipids and remained at membrane–water interface, suggesting that collagencin antibacterial action follows a carpet mechanism. Collagenous fish wastes could be processed by enzymatic hydrolysis and transformed into products of high value having functional or biological properties. Marine collagens are a promising source of antimicrobial peptides with new implications in food safety and human health. - Highlights: • Collagencin, an antibacterial (G+ & G-) peptide identified from fish collagen hydrolysate. • The peptide completely inhibited the growth of S. aureus at 1.88 mM and non-toxic at 470 μM. • The secondary structure was mainly composed by β-sheet and turn as determined by CD and MD. • Collagencin interacts with both anionic and zwitterionic lipids as shown with CD and MD. • Collagencin antibacterial action probably follows a carpet mechanism.

  8. Evidence of changes in renal charge selectivity in patients with type 1 (insulin-dependent) diabetes mellitus

    DEFF Research Database (Denmark)

    Kverneland, A; Feldt-Rasmussen, B; Vidal, P

    1986-01-01

    in plasma and urine was determined by a specific, sensitive and highly reproducible chromatographic procedure. In diabetic patients with normal urinary albumin excretion, the selectivity index was increased three-fold compared with that of non-diabetic subjects (2 p less than 0.01). A significant...... clearance of non-glycated plasma albumin/clearance of glycated plasma albumin in 38 patients with Type 1 diabetes mellitus. The two albumin molecules differed slightly in charge, non-enzymatic glycated albumin being more anionic at physiological pH compared with unmodified plasma albumin. Glycated albumin...... patients with increased albumin excretion rate had a significantly lower selectivity index compared with patients with normal albumin excretion (2 p less than 0.01). A significant negative correlation (r = 0.85, 2 p less than 0.001, exponential curve fit) was seen between urinary albumin excretion...

  9. Quantitative Profiling of Peptides from RNAs classified as non-coding

    Science.gov (United States)

    Prabakaran, Sudhakaran; Hemberg, Martin; Chauhan, Ruchi; Winter, Dominic; Tweedie-Cullen, Ry Y.; Dittrich, Christian; Hong, Elizabeth; Gunawardena, Jeremy; Steen, Hanno; Kreiman, Gabriel; Steen, Judith A.

    2014-01-01

    Only a small fraction of the mammalian genome codes for messenger RNAs destined to be translated into proteins, and it is generally assumed that a large portion of transcribed sequences - including introns and several classes of non-coding RNAs (ncRNAs) do not give rise to peptide products. A systematic examination of translation and physiological regulation of ncRNAs has not been conducted. Here, we use computational methods to identify the products of non-canonical translation in mouse neurons by analyzing unannotated transcripts in combination with proteomic data. This study supports the existence of non-canonical translation products from both intragenic and extragenic genomic regions, including peptides derived from anti-sense transcripts and introns. Moreover, the studied novel translation products exhibit temporal regulation similar to that of proteins known to be involved in neuronal activity processes. These observations highlight a potentially large and complex set of biologically regulated translational events from transcripts formerly thought to lack coding potential. PMID:25403355

  10. Biosynthetic Tailoring of Microcin E492m: Post-Translational Modification Affords an Antibacterial Siderophore-Peptide Conjugate

    Science.gov (United States)

    Nolan, Elizabeth M.; Fischbach, Michael A.; Koglin, Alexander; Walsh, Christopher T.

    2008-01-01

    The present work reveals that four proteins, MceCDIJ, encoded by the MccE492 gene cluster are responsible for the remarkable post-translational tailoring of Microcin E492 (MccE492), an 84-residue protein toxin secreted by Klebsiella pneumonaie RYC492 that targets neighboring gram-negative species. This modification results in attachment of a linearized and monoglycosylated derivative of enterobactin, a nonribosomal peptide and iron scavenger (siderophore), to the MccE492m C-terminus. MceC and MceD derivatize enterobactin by C-glycosylation at the C5 position of a N-(2,3-dihydroxybenzoyl) serine (DHB-Ser) moiety and regiospecific hydrolysis of an ester linkage in the trilactone scaffold, respectively. MceI and MceJ form a protein complex that attaches C-glycosylated enterobactins to the C-terminal serine residue of both aC10 model peptide and full-length MccE492. In the enzymatic product, the terminal serine residue is covalently attached to the C4′ oxygen of the glucose moiety. Non-enzymatic and base-catalyzed migration of the peptide to the C6′ position affords the C6′ glycosyl ester linkage observed in the mature toxin, MccE492m, isolated from bacterial cultures. PMID:17973380

  11. Evaluation of a reference material for glycated haemoglobin

    NARCIS (Netherlands)

    Weykamp, CW; Penders, TJ; Muskiet, FAJ; vanderSlik, W

    The use of lyophilized blood as a reference material for glycated haemoglobin was investigated with respect to IFCC criteria for calibrators and control materials. Ninety-two laboratories, using 11 methods, detected no changes in glycated haemoglobin content when the lyophilizate was stored for one

  12. Ketone Body Acetoacetate Buffers Methylglyoxal via a Non-enzymatic Conversion during Diabetic and Dietary Ketosis

    DEFF Research Database (Denmark)

    Salomon, Trine; Sibbersen, Christian; Hansen, Jakob

    2017-01-01

    now demonstrate that during ketosis, another meta- bolic route is operative via direct non-enzymatic aldol reaction between methylglyoxal and the ke- tone body acetoacetate, leading to 3-hydroxyhex- ane-2,5-dione. This novel metabolite is present at a concentration of 10%–20% of the methylglyoxal...

  13. Extracellular matrix glycation and receptor for advanced glycation end-products activation: a missing piece in the puzzle of the association between diabetes and cancer.

    Science.gov (United States)

    Rojas, Armando; Añazco, Carolina; González, Ileana; Araya, Paulina

    2018-04-05

    A growing body of epidemiologic evidence suggests that people with diabetes are at a significantly higher risk of many forms of cancer. However, the molecular mechanisms underlying this association are not fully understood. Cancer cells are surrounded by a complex milieu, also known as tumor microenvironment, which contributes to the development and metastasis of tumors. Of note, one of the major components of this niche is the extracellular matrix (ECM), which becomes highly disorganized during neoplastic progression, thereby stimulating cancer cell transformation, growth and spread. One of the consequences of chronic hyperglycemia, the most frequently observed sign of diabetes and the etiological source of diabetes complications, is the irreversible glycation and oxidation of proteins and lipids leading to the formation of the advanced glycation end-products (AGEs). These compounds may covalently crosslink and biochemically modify structure and functions of many proteins, and AGEs accumulation is particularly high in long-living proteins with low biological turnover, features that are shared by most, if not all, ECM proteins. AGEs-modified proteins are recognized by AGE-binding proteins, and thus glycated ECM components have the potential to trigger Receptor for advanced glycation end-products-dependent mechanisms. The biological consequence of receptor for advanced glycation end-products activation mechanisms seems to be connected, in different ways, to drive some hallmarks of cancer onset and tumor growth. The present review intends to highlight the potential impact of ECM glycation on tumor progression by triggering receptor for advanced glycation end-products-mediated mechanisms.

  14. Influences of conformations of peptides on stereoinversions and/or isomerizations of aspartic acid residues.

    Science.gov (United States)

    Oda, Akifumi; Nakayoshi, Tomoki; Fukuyoshi, Shuichi; Kurimoto, Eiji; Takahashi, Ohgi

    2018-07-01

    Recently, non-enzymatic stereoinversions of aspartic acid (Asp) residues in proteins and peptides have been reported. Here, we performed replica exchange molecular dynamics (REMD) simulations of model peptides (exon 6, 26A-1, and 26A-2) extracted from elastin to investigate their structural features, thereby revealing the factor that influences stereoinversions. For REMD trajectories, we calculated distances between carboxyl carbon in Asp and amide nitrogen in the (n + 1) residue (CN distances). Because bond formation between carbon and nitrogen is indispensable to the formation of a succinimide intermediate the distance between them seems to play an important role in stereoinversion. Moreover, we calculated polar surface areas (PSAs) for the trajectories, finding that CN distances and PSA were different for each peptide, with the longest CN distance and smallest PSA observed for exon 6 peptide, where stereoinversion of Asp is the slowest. Although the average CN distance was shorter for exon 26A-1 peptide than for exon 26A-2 peptide, the number of conformations with CN distances acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. An enzymatic method for the rapid measurement of the hemoglobin A1c by a flow-injection system comprised of an electrochemical detector with a specific enzyme-reactor and a spectrophotometer

    International Nuclear Information System (INIS)

    Nanjo, Yoko; Hayashi, Ryuzo; Yao, Toshio

    2007-01-01

    A flow-injection analytical (FIA) system, comprised of an electrochemical detector with a fructosyl-peptide oxidase (FPOX-CET) reactor and a flow-type spectrophotometer, was proposed for the simultaneous measurement of glycohemoglobin and total hemoglobin in blood cell. The blood cell samples were hemolyzed with a surfactant and then treated with protease. In the first stage of operation, total hemoglobin in digested sample was determined spectrophotometrically. In the second stage, fructosyl valyl histidine (FVH) released from glycohemoglobin by the selective proteolysis was determined specifically using the electrochemical detector with the FPOX-CET reactor. The FIA system could be automatically processed at an analytical speed of 40 samples per hour. The proposed assay method could determine selectively only the glycated N-terminal residue of β-chain in glycohemoglobin and total hemoglobin in blood cell. The enzymatic hemoglobin A 1c (HbA 1c ) value calculated by the concentration ratio of the FVH to total hemoglobin, was closely correlated with the HbA 1c values certified by the Japan Diabetic Society (JDS) and the International Federation of Clinical Chemistry (IFCC)

  16. Antioxidant Activity of Purified Active Peptide Derived from Spirulina platensis Enzymatic Hydrolysates

    OpenAIRE

    Nur Maulida Safitri; Endang Yuli Herawati; Jue Liang Hsu

    2017-01-01

    The aim of this study is to isolate the antioxidative peptide from Spirulina platensis. Peptide was obtained by proteolytic digestion, ultrafiltration, fractionation by RP-HPLC, identified by LC-MS/MS—MASCOT Distiller and measured its antioxidant activity by DPPH (2.2-Diphenyl-1-picrylhydrazyl) assay. Results showed that thermolysin was the most effective enzyme to digest this algae. The active peptide Phe-Ser-Glu-Ser-Ser-Ala-Pro-Glu-Gln-His-Tyr (m/z 1281.51) was identified and synthetized, w...

  17. Bioactive peptides: production, health effects and application as natural supplements for functional foods production

    Directory of Open Access Journals (Sweden)

    S. Mirdamadi

    2017-05-01

    Full Text Available Bioactive peptides, are inactive components within the structure of the protein and when they are released by enzymatic hydrolysis, show different physiological functions. Recently, the identification and characterization of bioactive peptides derived from plant and animal sources and different microorganisms is highly regarded. They are produced during enzymatic hydrolysis by gastrointestinal enzymes or enzymes extracted from microorganisms and plants or by proteolytic starter cultures during fermentation process and exhibit different activities including: opioid, mineral binding, immunomodulatory, antioxidant, antimicrobial, anti-inflammatory, chlosterol lowering and so on. Take advantage of bioactive peptides as components of health is related to bio stability assurance, bioavailability and safety of them. The use of computer-based techniques and the use of various databases completed in laboratory studies,  have provided the possibility of studying the mechanisms of action of different peptides.

  18. Towards Tuning the Mechanical Properties of Three-Dimensional Collagen Scaffolds Using a Coupled Fiber-Matrix Model

    Directory of Open Access Journals (Sweden)

    Shengmao Lin

    2015-08-01

    Full Text Available Scaffold mechanical properties are essential in regulating the microenvironment of three-dimensional cell culture. A coupled fiber-matrix numerical model was developed in this work for predicting the mechanical response of collagen scaffolds subjected to various levels of non-enzymatic glycation and collagen concentrations. The scaffold was simulated by a Voronoi network embedded in a matrix. The computational model was validated using published experimental data. Results indicate that both non-enzymatic glycation-induced matrix stiffening and fiber network density, as regulated by collagen concentration, influence scaffold behavior. The heterogeneous stress patterns of the scaffold were induced by the interfacial mechanics between the collagen fiber network and the matrix. The knowledge obtained in this work could help to fine-tune the mechanical properties of collagen scaffolds for improved tissue regeneration applications.

  19. Non-enzymatic glucose detection based on phenylboronic acid modified optical fibers

    Science.gov (United States)

    Sun, Xiaolan; Li, Nana; Zhou, Bin; Zhao, Wei; Liu, Liyuan; Huang, Chao; Ma, Longfei; Kost, Alan R.

    2018-06-01

    A non-enzymatic, sensitive glucose sensor was fabricated based on an evanescent wave absorbing optical fiber probe. The optical fiber sensor was functionalized by fixing a poly (phenylboronic acid) (polyPBA) film onto the conical region of the single mode fiber. The reflected light intensity of the polyPBA-functionalized fiber sensor increased proportionally with glucose concentration in the range of 0-60 mM, and the sensor showed good reproducibility and stability. The developed sensor possessed a high sensitivity of 0.1787%/mM and good linearity. The measurement of glucose concentration in human serum was also demonstrated.

  20. Enzymatic cross-linking of soy proteins within non-fat set yogurt gel.

    Science.gov (United States)

    Soleymanpuori, Rana; Madadlou, Ashkan; Zeynali, Fariba; Khosrowshahi, Asghar

    2014-08-01

    Soy proteins as the health-promoting ingredients and candidate fat substitutes in dairy products are good substrates for the cross-linking action of the enzyme transglutaminase. Non-fat set yogurt samples were prepared from the milks enriched with soy protein isolate (SPI) and/or treated with the enzyme transglutaminase. The highest titrable acidity was recorded for the yogurt enriched with SPI and treated with the enzyme throughout the cold storage for 21 d. SPI-enrichment of yogurt milk increased the water holding capacity. Although enrichment with SPI did not influence the count of Streptococcus themophilus, increased that of Lactobacillus bulgaricus ∼3 log cycles. The enzymatic treatment of SPI-enriched milk however, suppressed the bacteria growth-promoting influence of SPI due probably to making the soy proteins inaccessible for Lactobacillus. SPI-enrichment and enzymatic treatment of milk decreased the various organic acids content in yoghurt samples; influence of the former was more significant. The cross-linking of milk proteins to soy proteins was confirmed with the gel electrophoresis results.

  1. Non-Enzymatic Wearable Sensor for Electrochemical Analysis of Perspiration Glucose.

    Science.gov (United States)

    Zhu, Xiaofei; Ju, Yinhui; Chen, Jian; Liu, Deye; Liu, Hong

    2018-05-16

    We report a non-enzymatic wearable sensor for electrochemical analysis of perspiration glucose. Multi-potential steps are applied on a Au electrode, including a high negative pretreatment potential step for proton reduction which produc-es a localized alkaline condition, a moderate potential step for electrocatalytic oxidation of glucose under the alkaline condi-tion, and a positive potential step to clean and reactivate the electrode surface for the next detection. Fluorocarbon-based materials were coated on the Au electrode for improving the selectivity and robustness of the sensor. A fully integrated wrist-band is developed for continuous real-time monitoring of perspiration glucose during physical activities, and uploading the test result to a Smartphone App via Bluetooth.

  2. Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass.

    Science.gov (United States)

    Su, Xiaoyun; Zhang, Jing; Mackie, Roderick I; Cann, Isaac K O

    2012-01-01

    The glycoside hydrolases (GH) of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry.

  3. Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass.

    Directory of Open Access Journals (Sweden)

    Xiaoyun Su

    Full Text Available The glycoside hydrolases (GH of Caldicellulosiruptor bescii are thermophilic enzymes, and therefore they can hydrolyze plant cell wall polysaccharides at high temperatures. Analyses of two C. bescii glycoside hydrolases, CbCelA-TM1 and CbXyn10A with cellulase and endoxylanase activity, respectively, demonstrated that each enzyme is highly thermostable under static incubation at 70°C. Both enzymes, however, rapidly lost their enzymatic activities when incubated at 70°C with end-over-end shaking. Since crowding conditions, even at low protein concentrations, seem to influence enzymatic properties, three non-glycoside hydrolase proteins were tested for their capacity to stabilize the thermophilic proteins at high temperatures. The three proteins investigated were a small heat shock protein CbHsp18 from C. bescii, a histone MkHistone1 from Methanopyrus kandleri, and bovine RNase A, from a commercial source. Fascinatingly, each of these proteins increased the thermostability of the glycoside hydrolases at 70°C during end-over-end shaking incubation, and this property translated into increases in hydrolysis of several substrates including the bioenergy feedstock Miscanthus. Furthermore, MkHistone1 and RNase A also altered the initial products released from the cello-oligosaccharide cellopentaose during hydrolysis with the cellodextrinase CbCdx1A, which further demonstrated the capacity of the three non-GH proteins to influence hydrolysis of substrates by the thermophilic glycoside hydrolases. The non-GH proteins used in the present report were small proteins derived from each of the three lineages of life, and therefore expand the space from which different polypeptides can be tested for their influence on plant cell wall hydrolysis, a critical step in the emerging biofuel industry.

  4. Assessment of nonenzymatic glycation in protein by FTIR spectroscopy

    Science.gov (United States)

    Otero de Joshi, Virginia; Joshi, Narahari V.; Gil, Herminia; Velasquez, William; Contreras, Silvia; Marquez, Glevis

    1999-04-01

    Detection of nonenzymatic glycated proteins is a very significant feature in diabetes, aging and related diseases, therefore we have carried out an FTIR spectroscopic study for glycated and native proteins such as (gamma) -globulin, human serum albumin. For this purpose, commercially available proteins were glycated by a usual procedure and their FTIR spectra were recorded together with that of the native ones. In order to follow the changes in time, (gamma) -globulin was glycated during 1, 2, 3, 5 and 8 weeks and their spectra were recorded. Direct verification was obtained by examining a model unit where the -NH2 group was attached to glucose. The spectrum shows a strong peak at 3500 cm-1 confirming the observed variation in time dependent spectra. The general features of the spectra are very similar and there was no additional structure or change in the peaks. This is understandable as not all the lysine residues are glycated, only a small fraction. Glucose is attached to the (epsilon) -amino group of lysine to form Amadori products, and therefore, the vibrational modes corresponding to the (epsilon) -NH2 unit of lysine are expected to be altered. This region exactly lies in the Amide I region of protein structure. Careful investigation of this part, indeed, shows a complex structure originated from alternations of -NH2 group. Thus, the present investigation indicates that an optical approach could be a rapid and effective method to identify the nonenzymatic glycation process.

  5. Plasma Atrial Natriuretic Peptide as a non-invasive biochemical ...

    African Journals Online (AJOL)

    Plasma Atrial Natriuretic Peptide as a non-invasive biochemical marker of dyspnoea in congestive heart failure patients. ... University of Mauritius Research Journal ... score assessed by a 10 graded visual analogue scale in the control group (mean score = 1) and an increased from 1.6 to 6.4 in the heart failure patients.

  6. Effect of Enzymatic Digestion of Protein Derivatives Obtained from Mucuna pruriens L. on Production of Proinflammatory Mediators by BALB/c Mouse Macrophages.

    Science.gov (United States)

    Martínez Leo, Edwin E; Arana Argáez, Victor E; Acevedo Fernández, Juan J; Puc, Rosa Moo; Segura Campos, Maira R

    2018-04-25

    Inflammation is considered to be a major risk factor for the pathogenesis of chronic non-communicable diseases. Macrophages are important immune cells, which regulate inflammation and host defense by secretion of proinflammatory mediators. Obtaining biopeptides by enzymatic hydrolysis adds value to proteins of vegetative origin, such as Mucuna pruriens L. The present study evaluated the effect of enzymatic digestion of protein derivatives obtained from M. pruriens L. on the production of proinflammatory mediators by BALB/c mouse macrophages. Five different molecular weight peptide fractions were obtained (F > 10, 5-10, 3-5, 1-3, and < 1 kDa, respectively). At 300 μg/mL, F5-10 kDa inhibited 50.26 and 61.00% NO and H 2 O 2 production, respectively. Moreover, F5-10 kDa reduced the IL-6 and TNFα levels to 60.25 and 69.54%, respectively. After enzymatic digestive simulation, F5-10 kDa decreased the inflammatory mediators.

  7. Enzymatic activities produced by mixed Saccharomyces and non-Saccharomyces cultures: relationship with wine volatile composition.

    Science.gov (United States)

    Maturano, Yolanda Paola; Assof, Mariela; Fabani, María Paula; Nally, María Cristina; Jofré, Viviana; Rodríguez Assaf, Leticia Anahí; Toro, María Eugenia; Castellanos de Figueroa, Lucía Inés; Vazquez, Fabio

    2015-11-01

    During certain wine fermentation processes, yeasts, and mainly non-Saccharomyces strains, produce and secrete enzymes such as β-glucosidases, proteases, pectinases, xylanases and amylases. The effects of enzyme activity on the aromatic quality of wines during grape juice fermentation, using different co-inoculation strategies of non-Saccharomyces and Saccharomyces cerevisiae yeasts, were assessed in the current study. Three strains with appropriate enological performance and high enzymatic activities, BSc562 (S. cerevisiae), BDv566 (Debaryomyces vanrijiae) and BCs403 (Candida sake), were assayed in pure and mixed Saccharomyces/non-Saccharomyces cultures. β-Glucosidase, pectinase, protease, xylanase and amylase activities were quantified during fermentations. The aromatic profile of pure and mixed cultures was determined at the end of each fermentation. In mixed cultures, non-Saccharomyces species were detected until day 4-5 of the fermentation process, and highest populations were observed in MSD2 (10% S. cerevisiae/90% D. vanrijiae) and MSC1 (1% S. cerevisiae/99% C. sake). According to correlation and multivariate analysis, MSD2 presented the highest concentrations of terpenes and higher alcohols which were associated with pectinase, amylase and xylanase activities. On the other hand, MSC1 high levels of β-glucosidase, proteolytic and xylanolytic activities were correlated to esters and fatty acids. Our study contributes to a better understanding of the effect of enzymatic activities by yeasts on compound transformations that occur during wine fermentation.

  8. Implementation of communication-mediating domains for non-ribosomal peptide production in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Siewers, Verena; San-Bento, Rita; Nielsen, Jens

    2010-01-01

    Saccharomyces cerevisiae has in several cases been proven to be a suitable host for the production of natural products and was recently exploited for the production of non-ribosomal peptides. Synthesis of non-ribosomal peptides (NRPs) is mediated by NRP synthetases (NRPSs), modular enzymes, which...... are often organized in enzyme complexes. In these complexes, partner NRPSs interact via communication-mediating domains (COM domains). In order to test whether functional interaction between separate NRPS modules is possible in yeast we constructed a yeast strain expressing two modules with compatible COM...

  9. Raman spectroscopy provides a powerful diagnostic tool for accurate determination of albumin glycation.

    Science.gov (United States)

    Dingari, Narahara Chari; Horowitz, Gary L; Kang, Jeon Woong; Dasari, Ramachandra R; Barman, Ishan

    2012-01-01

    We present the first demonstration of glycated albumin detection and quantification using Raman spectroscopy without the addition of reagents. Glycated albumin is an important marker for monitoring the long-term glycemic history of diabetics, especially as its concentrations, in contrast to glycated hemoglobin levels, are unaffected by changes in erythrocyte life times. Clinically, glycated albumin concentrations show a strong correlation with the development of serious diabetes complications including nephropathy and retinopathy. In this article, we propose and evaluate the efficacy of Raman spectroscopy for determination of this important analyte. By utilizing the pre-concentration obtained through drop-coating deposition, we show that glycation of albumin leads to subtle, but consistent, changes in vibrational features, which with the help of multivariate classification techniques can be used to discriminate glycated albumin from the unglycated variant with 100% accuracy. Moreover, we demonstrate that the calibration model developed on the glycated albumin spectral dataset shows high predictive power, even at substantially lower concentrations than those typically encountered in clinical practice. In fact, the limit of detection for glycated albumin measurements is calculated to be approximately four times lower than its minimum physiological concentration. Importantly, in relation to the existing detection methods for glycated albumin, the proposed method is also completely reagent-free, requires barely any sample preparation and has the potential for simultaneous determination of glycated hemoglobin levels as well. Given these key advantages, we believe that the proposed approach can provide a uniquely powerful tool for quantification of glycation status of proteins in biopharmaceutical development as well as for glycemic marker determination in routine clinical diagnostics in the future.

  10. Raman Spectroscopy Provides a Powerful Diagnostic Tool for Accurate Determination of Albumin Glycation

    Science.gov (United States)

    Dingari, Narahara Chari; Horowitz, Gary L.; Kang, Jeon Woong; Dasari, Ramachandra R.; Barman, Ishan

    2012-01-01

    We present the first demonstration of glycated albumin detection and quantification using Raman spectroscopy without the addition of reagents. Glycated albumin is an important marker for monitoring the long-term glycemic history of diabetics, especially as its concentrations, in contrast to glycated hemoglobin levels, are unaffected by changes in erythrocyte life times. Clinically, glycated albumin concentrations show a strong correlation with the development of serious diabetes complications including nephropathy and retinopathy. In this article, we propose and evaluate the efficacy of Raman spectroscopy for determination of this important analyte. By utilizing the pre-concentration obtained through drop-coating deposition, we show that glycation of albumin leads to subtle, but consistent, changes in vibrational features, which with the help of multivariate classification techniques can be used to discriminate glycated albumin from the unglycated variant with 100% accuracy. Moreover, we demonstrate that the calibration model developed on the glycated albumin spectral dataset shows high predictive power, even at substantially lower concentrations than those typically encountered in clinical practice. In fact, the limit of detection for glycated albumin measurements is calculated to be approximately four times lower than its minimum physiological concentration. Importantly, in relation to the existing detection methods for glycated albumin, the proposed method is also completely reagent-free, requires barely any sample preparation and has the potential for simultaneous determination of glycated hemoglobin levels as well. Given these key advantages, we believe that the proposed approach can provide a uniquely powerful tool for quantification of glycation status of proteins in biopharmaceutical development as well as for glycemic marker determination in routine clinical diagnostics in the future. PMID:22393405

  11. Oncocalyxone A functions as an anti-glycation agent in vitro.

    Directory of Open Access Journals (Sweden)

    Ingrid Sofia Vieira de Melo

    Full Text Available Advanced glycation endproducts (AGE are the result of post-translational changes to proteins, which ultimately compromise their structure and/or function. The identification of methods to prevent the formation of these compounds holds great promise in the development of alternative therapies for diseases such as diabetes. Plants used in traditional medicine are often rich sources of anti-glycation agents. Therefore, in this study, we investigated the anti-glycation activity of one such compound, Oncocalyxone A (Onco A. Using spectrofluorimetric techniques, we determined that Onco A inhibits AGE formation in a concentration-dependent manner. Its IC50 value (87.88 ± 3.08 μM was almost two times lower than the standard anti-glycation compound aminoguanidine (184.68 ± 4.85 μM. The excellent anti-glycation activity of Onco A makes it an exciting candidate for the treatment of diseases associated with excessive accumulation of AGE. However, additional studies are necessary to identify its mechanism of action, as well as the in vivo response in suitable model organisms.

  12. Synthesis of CdS quantum dots decorated graphene nanosheets and non-enzymatic photoelectrochemical detection of glucose

    International Nuclear Information System (INIS)

    Zhang, Xuyan; Xu, Fang; Zhao, Bingqing; Ji, Xin; Yao, Yanwen; Wu, Dapeng; Gao, Zhiyong; Jiang, Kai

    2014-01-01

    Graphical abstract: - Highlights: • Graphene-CdS hybrid materials were prepared via one-step hydrothermal method. • Graphene-CdS was used as non-enzymatic photoelectrochemical sensor to detect glucose. • Glucose in real sample was detected and showed good specificity and sensitivity. - ABSTRACT: Graphene-CdS quantum dots (QDs) hybrid materials were successfully prepared via one-step hydrothermal method. CdS QDs with average size of ∼6 nm were dispersed on graphene sheets with high coverage through non-covalent bonding. Photocurrent and electrochemical impedance spectroscopy (EIS) results suggested that the best dosage of graphene oxide for graphene-CdS hybrid materials is 0.5% (G0.5-CdS). When G0.5-CdS QDs was used as photoanode materials in non-enzymatic sensor, and the sensor was used to detect glucose and displayed satisfactory analytical performance with good linear range from 0.1∼4 mmol dm −3 with a detection limit of 7 μmol dm −3 at a signal-to-noise ratio of 3. The sensor also possessed high selectivity and durability in trace detection of glucose

  13. Glycation induces formation of amyloid cross-beta structure in albumin.

    Science.gov (United States)

    Bouma, Barend; Kroon-Batenburg, Loes M J; Wu, Ya-Ping; Brünjes, Bettina; Posthuma, George; Kranenburg, Onno; de Groot, Philip G; Voest, Emile E; Gebbink, Martijn F B G

    2003-10-24

    Amyloid fibrils are components of proteinaceous plaques that are associated with conformational diseases such as Alzheimer's disease, transmissible spongiform encephalopathies, and familial amyloidosis. Amyloid polypeptides share a specific quarternary structure element known as cross-beta structure. Commonly, fibrillar aggregates are modified by advanced glycation end products (AGE). In addition, AGE formation itself induces protein aggregation. Both amyloid proteins and protein-AGE adducts bind multiligand receptors, such as receptor for AGE, CD36, and scavenger receptors A and B type I, and the serine protease tissue-type plasminogen activator (tPA). Based on these observations, we hypothesized that glycation induces refolding of globular proteins, accompanied by formation of cross-beta structure. Using transmission electron microscopy, we demonstrate here that glycated albumin condensates into fibrous or amorphous aggregates. These aggregates bind to amyloid-specific dyes Congo red and thioflavin T and to tPA. In contrast to globular albumin, glycated albumin contains amino acid residues in beta-sheet conformation, as measured with circular dichroism spectropolarimetry. Moreover, it displays cross-beta structure, as determined with x-ray fiber diffraction. We conclude that glycation induces refolding of initially globular albumin into amyloid fibrils comprising cross-beta structure. This would explain how glycated ligands and amyloid ligands can bind to the same multiligand "cross-beta structure" receptors and to tPA.

  14. Raman Spectroscopy Provides a Powerful Diagnostic Tool for Accurate Determination of Albumin Glycation

    OpenAIRE

    Dingari, Narahara Chari; Kang, Jeon Woong; Dasari, Ramachandra R.; Barman, Ishan; Horowitz, Gary Leigh

    2012-01-01

    We present the first demonstration of glycated albumin detection and quantification using Raman spectroscopy without the addition of reagents. Glycated albumin is an important marker for monitoring the long-term glycemic history of diabetics, especially as its concentrations, in contrast to glycated hemoglobin levels, are unaffected by changes in erythrocyte life times. Clinically, glycated albumin concentrations show a strong correlation with the development of serious diabetes complications...

  15. Antioxidant Activity of Purified Active Peptide Derived from Spirulina platensis Enzymatic Hydrolysates

    Directory of Open Access Journals (Sweden)

    Nur Maulida Safitri

    2017-08-01

    Full Text Available The aim of this study is to isolate the antioxidative peptide from Spirulina platensis. Peptide was obtained by proteolytic digestion, ultrafiltration, fractionation by RP-HPLC, identified by LC-MS/MS—MASCOT Distiller and measured its antioxidant activity by DPPH (2.2-Diphenyl-1-picrylhydrazyl assay. Results showed that thermolysin was the most effective enzyme to digest this algae. The active peptide Phe-Ser-Glu-Ser-Ser-Ala-Pro-Glu-Gln-His-Tyr (m/z 1281.51 was identified and synthetized, which exhibited 45.98 ± 1.7% at concentration 128.15 µg/mL. Therefore, S. platensis is indicated as a potential therapeutic source for combating oxidative stress.

  16. PENGARUH SORPSI AIR DAN SUHU TRANSISI GELAS TERHADAP LAJU PENCOKLATAN NON-ENZIMATIS PADA PANGAN MODEL [The Effect of Water Sorption and Glass Transition Temperature on Non-Enzymatic Browning Reaction of Food Models

    Directory of Open Access Journals (Sweden)

    Dede R Adawiyah1

    2005-12-01

    Full Text Available This research was aimer/ to study the extend of non enzymatic browning reaction in food models containing the mixture of tapioca starch, casein, sucrose and oh at different moisture contents (2.55%, 5.26%, 7.54%, 15.20%. 15.93% and 23.99% and storage temperatures (30, 55 and 700C. The non-enzymatic browning reaction was detected from brown color intensity measured by spechtrophotometer and colorimetric methods. The non-enzymatic browning reaction or food model follow pseudo-zero order reaction, suggesting that browning reaction occurred at moisture content above monolayer zone. T-Tg (T storage - Tg prediction and reaction rate constant (k plots showed that browning reaction occurred at temperature around glass transition and increased significantly at 150 above Tg of casein. Tapioca starch in the food model was under glassy condition. The mobility of substrate increased and diffused at amorphous matrix.

  17. Enzymatic Activity Detection via Electrochemistry for Enceladus

    Science.gov (United States)

    Studemeister, Lucy; Koehne, Jessica; Quinn, Richard

    2017-01-01

    Electrochemical detection of biological molecules is a pertinent topic and application in many fields such as medicine, environmental spills, and life detection in space. Proteases, a class of molecules of interest in the search for life, catalyze the hydrolysis of peptides. Trypsin, a specific protease, was chosen to investigate an optimized enzyme detection system using electrochemistry. This study aims at providing the ideal functionalization of an electrode that can reliably detect a signal indicative of an enzymatic reaction from an Enceladus sample.

  18. Targeting nanoparticles to M cells with non-peptidic ligands for oral vaccination.

    Science.gov (United States)

    Fievez, Virginie; Plapied, Laurence; des Rieux, Anne; Pourcelle, Vincent; Freichels, Hélène; Wascotte, Valentine; Vanderhaeghen, Marie-Lyse; Jerôme, Christine; Vanderplasschen, Alain; Marchand-Brynaert, Jacqueline; Schneider, Yves-Jacques; Préat, Véronique

    2009-09-01

    The presence of RGD on nanoparticles allows the targeting of beta1 integrins at the apical surface of human M cells and the enhancement of an immune response after oral immunization. To check the hypothesis that non-peptidic ligands targeting intestinal M cells or APCs would be more efficient for oral immunization than RGD, novel non-peptidic and peptidic analogs (RGD peptidomimitic (RGDp), LDV derivative (LDVd) and LDV peptidomimetic (LDVp)) as well as mannose were grafted on the PEG chain of PCL-PEG and incorporated in PLGA-based nanoparticles. RGD and RGDp significantly increased the transport of nanoparticles across an in vitro model of human M cells as compared to enterocytes. RGD, LDVp, LDVd and mannose enhanced nanoparticle uptake by macrophages in vitro. The intraduodenal immunization with RGDp-, LDVd- or mannose-labeled nanoparticles elicited a higher production of IgG antibodies than the intramuscular injection of free ovalbumin or intraduodenal administration of either non-targeted or RGD-nanoparticles. Targeted formulations were also able to induce a cellular immune response. In conclusion, the in vitro transport of nanoparticles, uptake by macrophages and the immune response were positively influenced by the presence of ligands at the surface of nanoparticles. These targeted-nanoparticles could thus represent a promising delivery system for oral immunization.

  19. Insulin and C-peptide secretion in non-obese patients with polycystic ovarian disease.

    Science.gov (United States)

    Mahabeer, S; Jialal, I; Norman, R J; Naidoo, C; Reddi, K; Joubert, S M

    1989-09-01

    Plasma glucose, immunoreactive insulin (IRI) and C-peptide responses during an oral glucose tolerance test (oGTT) were assessed in 11 non-obese patients with polycystic ovarian disease (PCOD) and 11 reference subjects matched for age, height and weight. Also, 6 patients with PCOD and 6 normal women were subjected to intravenous glucose tolerance testing (ivGTT) On oGTT, all subjects exhibited normal glucose tolerance; however, PCOD patients had significantly higher mean plasma glucose levels at 30, 60, 90 and 120 min and higher mean incremental glucose areas. In addition the patients with polycystic ovaries showed higher mean basal IRI and C-peptide levels, higher mean glucose stimulated IRI and C-peptide levels and higher mean incremental IRI and C-peptide values. The molar ratios of C-peptide/IRI were significantly lower in the PCOD group at all time intervals after glucose stimulation when compared to the normal women. During ivGTT, there were significantly higher mean glucose levels at 5, 40, 50 and 60 min in the PCOD group when compared to the reference group. The IRI response to intravenous glucose in the PCOD women was similar to the reference group. The findings on oGTT suggest that non-obese patients with PCOD have increased pancreatic IRI secretion as well as impaired hepatic extraction of the hormone.

  20. Non-enzymatic antioxidant accumulations in BR-deficient and BR-insensitive barley mutants under control and drought conditions.

    Science.gov (United States)

    Gruszka, Damian; Janeczko, Anna; Dziurka, Michal; Pociecha, Ewa; Fodor, Jozsef

    2017-12-07

    Drought is one of the most adverse stresses that affect plant growth and yield. Disturbances in metabolic activity resulting from drought cause overproduction of reactive oxygen species. It is postulated that brassinosteroids (BRs) regulate plant tolerance to the stress conditions, but the underlying mechanisms remain largely unknown. An involvement of endogenous BRs in regulation of the antioxidant homeostasis is not fully clarified either. Therefore, the aim of this study was to elucidate the role of endogenous BRs in regulation of non-enzymatic antioxidants in barley (Hordeum vulgare) under control and drought conditions. The plant material included the 'Bowman' cultivar and a group of semi-dwarf near-isogenic lines (NILs), representing mutants deficient in BR biosynthesis or signaling. In general, accumulations of 11 compounds representing various types of non-enzymatic antioxidants were analyzed under both conditions. The analyses of accumulations of reduced and oxidized forms of ascorbate indicated that the BR mutants contain significantly higher contents of dehydroascorbic acid under drought conditions when compared with the 'Bowman' cultivar. The analysis of glutathione accumulation indicated that under the control conditions the BR-insensitive NILs contained significantly lower concentrations of this antioxidant when compared with the rest of genotypes. Therefore, we postulate that BR sensitivity is required for normal accumulation of glutathione. A complete accumulation profile of various tocopherols indicated that functional BR biosynthesis and signaling are required for their normal accumulation under both conditions. Results of this study provided an insight into the role of endogenous BRs in regulation of the non-enzymatic antioxidant homeostasis. © 2017 Scandinavian Plant Physiology Society.

  1. A reagentless non-enzymatic hydrogen peroxide sensor presented using electrochemically reduced graphene oxide modified glassy carbon electrode

    Energy Technology Data Exchange (ETDEWEB)

    Mutyala, Sankararao; Mathiyarasu, Jayaraman, E-mail: al_mathi@yahoo.com

    2016-12-01

    Herein, we report a simple, facile and reproducible non-enzymatic hydrogen peroxide (H{sub 2}O{sub 2}) sensor using electrochemically reduced graphene oxide (ERGO) modified glassy carbon electrode (GCE). The modified electrode was characterized by Fourier transform infrared (FT-IR), UV–Visible, scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques. Cyclic voltammetric (CV) analysis revealed that ERGO/GCE exhibited virtuous charge transfer properties for a standard redox systems and showed excellent performance towards electroreduction of H{sub 2}O{sub 2}. Amperometric study using ERGO/GCE showed high sensitivity (0.3 μA/μM) and faster response upon the addition of H{sub 2}O{sub 2} at an applied potential of − 0.25 V vs. Ag/AgCl. The detection limit is assessed to be 0.7 μM (S/N = 3) and the time to reach a stable study state current is < 3 s for a linear range of H{sub 2}O{sub 2} concentration (1–16 μM). In addition, the modified electrode exhibited good reproducibility and long-term stability. - Graphical abstract: We presented a reagentless non-enzymatic hydrogen peroxide sensor using electrochemically reduced graphene oxide material. - Highlights: • A facile green procedure proposed for high quality graphene synthesis using electrochemical reduction of graphene oxide • A simple, facile and reagentless non-enzymatic hydrogen peroxide sensor developed using ERGO/GCE. • ERGO/GCE exhibited high sensitivity, selectivity and finite limit of detection for H{sub 2}O{sub 2} sensing at low overpotential. • ERGO/GCE exhibited long term stability and good reproducibility.

  2. A reagentless non-enzymatic hydrogen peroxide sensor presented using electrochemically reduced graphene oxide modified glassy carbon electrode

    International Nuclear Information System (INIS)

    Mutyala, Sankararao; Mathiyarasu, Jayaraman

    2016-01-01

    Herein, we report a simple, facile and reproducible non-enzymatic hydrogen peroxide (H 2 O 2 ) sensor using electrochemically reduced graphene oxide (ERGO) modified glassy carbon electrode (GCE). The modified electrode was characterized by Fourier transform infrared (FT-IR), UV–Visible, scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques. Cyclic voltammetric (CV) analysis revealed that ERGO/GCE exhibited virtuous charge transfer properties for a standard redox systems and showed excellent performance towards electroreduction of H 2 O 2 . Amperometric study using ERGO/GCE showed high sensitivity (0.3 μA/μM) and faster response upon the addition of H 2 O 2 at an applied potential of − 0.25 V vs. Ag/AgCl. The detection limit is assessed to be 0.7 μM (S/N = 3) and the time to reach a stable study state current is < 3 s for a linear range of H 2 O 2 concentration (1–16 μM). In addition, the modified electrode exhibited good reproducibility and long-term stability. - Graphical abstract: We presented a reagentless non-enzymatic hydrogen peroxide sensor using electrochemically reduced graphene oxide material. - Highlights: • A facile green procedure proposed for high quality graphene synthesis using electrochemical reduction of graphene oxide • A simple, facile and reagentless non-enzymatic hydrogen peroxide sensor developed using ERGO/GCE. • ERGO/GCE exhibited high sensitivity, selectivity and finite limit of detection for H 2 O 2 sensing at low overpotential. • ERGO/GCE exhibited long term stability and good reproducibility.

  3. Aerobic bacterial microbiota of the conjunctiva in diabetic patients with normal and altered glycated hemoglobin levels in two regions in Brazil

    Directory of Open Access Journals (Sweden)

    Natalia Pimentel Moreno

    2014-12-01

    Full Text Available Purpose: To study the aerobic bacterial microbiota of the conjunctiva in diabetic patients with regard to the management of diabetes, assessed using glycated hemoglobin levels. Methods: A cross-sectional study was conducted using conjunctival smears of diabetic patients from both sexes and with different ages, residing in two different Brazilian cities (Sorocaba and Rio Branco. A control group of non-diabetic patients was also included. The diabetic patients were considered to have controlled diabetes when their glycated hemoglobin level was ≤7% and blood glucose level was ≤126 mg/dL. Patients with non-controlled diabetes were those with glycated hemoglobin levels >7% and blood glucose levels >126 mg/dL. The samples obtained were inoculated in Brain-Heart Infusion broth and in culture media for aerobic bacteria (blood and chocolate agars; bacterial growth was evaluated in a microbiology laboratory. Results: A total of 120 eyes of 120 patients were included in the present study. The percentage of cultures in which bacterial growth was observed was greater in diabetic patients, although the difference was not statistically significant (p=0.103. There was a greater trend toward bacterial growth in the conjunctiva of diabetic patients with altered fasting blood glucose. There was no difference in the frequency of bacterial growth on the conjunctiva between diabetic patients with normal or altered glycated hemoglobin levels. In Sorocaba, conjunctival bacterial growth was similar to that observed in Rio Branco. The microorganism most frequently detected in the present study was Staphylococcus epidermidis, followed by Staphylococcus aureus, Proteus mirabilis, and Escherichia coli. Conclusion: There was no difference between diabetic patients with normal or altered glycated hemoglobin levels. The microorganisms found were similar to those found in studies investigating the conjunctival bacterial flora of diabetic and non-diabetic patients.

  4. Computational Studies of Difference in Binding Modes of Peptide and Non-Peptide Inhibitors to MDM2/MDMX Based on Molecular Dynamics Simulations

    Directory of Open Access Journals (Sweden)

    Yuxin Zhang

    2012-02-01

    Full Text Available Inhibition of p53-MDM2/MDMX interaction is considered to be a promising strategy for anticancer drug design to activate wild-type p53 in tumors. We carry out molecular dynamics (MD simulations to study the binding mechanisms of peptide and non-peptide inhibitors to MDM2/MDMX. The rank of binding free energies calculated by molecular mechanics generalized Born surface area (MM-GBSA method agrees with one of the experimental values. The results suggest that van der Waals energy drives two kinds of inhibitors to MDM2/MDMX. We also find that the peptide inhibitors can produce more interaction contacts with MDM2/MDMX than the non-peptide inhibitors. Binding mode predictions based on the inhibitor-residue interactions show that the π–π, CH–π and CH–CH interactions dominated by shape complimentarity, govern the binding of the inhibitors in the hydrophobic cleft of MDM2/MDMX. Our studies confirm the residue Tyr99 in MDMX can generate a steric clash with the inhibitors due to energy and structure. This finding may theoretically provide help to develop potent dual-specific or MDMX inhibitors.

  5. Relationship between advanced glycation end-products with the severity of chronic heart failure in 85 patients

    Directory of Open Access Journals (Sweden)

    Amir Farhang Zand Parsa

    2013-12-01

    Full Text Available Background: Advanced glycation end-products (AGEs came up with the recent researches regarding new biomarkers for the diagnosis of heart failure. AGEs are the end products of non-enzymatic glycation and oxidation of proteins, lipids and nucleotides during Maillard biochemical reaction. Although it has been known that AGEs have a role in the pathogenesis of chronic heart failure (CHF, information regarding its role and its pathogenetic mechanism is very limited. The aim of this study was to find any relationship between AGEs with the etiology and severity of chronic heart failure.Methods: This study is a prospective cross sectional study that enrolled 85 patients with chronic heart failure. Measurement of left ventricle ejection fraction (LVEF was done by echocardiography. Blood samples were collected for measuring AGEs just before or after echocardiography assessment (in the same session. Measurement of AGEs was done by the enzyme-linked immunosorbent assay (ELISA method. The relationship between AGEs with the severity of CHF and as well as the etiology of CHF were evaluated via SPSS-15.Results: Of 85 patients 48 (56.5% patients were male and 37 (43.5% were female; Mean±SD of their ages was 55.8±13.4 years old (ranges from 27 to 84 years. Correlation coefficient between LVEF and AGEs was 0.269 (P=0.013. Mean of AGEs in patients with and without ischemic etiology of their heart failure were 16.8±9.8µg/ml and 11.6±7.3 µg/ml, respectively. Although trend was in favor of ischemic heart failure, the difference between two groups was not statistically significant (P= 0.141.Conclusion: According to this study the rate of AGES could be helpful in the diagnosis and assessment of severity of CHF. Based on our findings, higher blood levels of AGEs in the ischemic CHF cases, also it could be concluded that in the future this marker may be used for etiologic differentiation of heart failure syndrome.

  6. ENZYMATIC AND NON-ENZYMATIC ANTIOXIDANT DEFENSE WITH ALZHEIMER DISEASE1

    Directory of Open Access Journals (Sweden)

    A. Vaisi-Raygani

    2007-07-01

    Full Text Available The etiopathogenesis of Alzheimer's disease (AD is still unclear.  However, long-term oxidative stress is believed to be one of the major contributing factors in progression of neuronal degeneration and decline of cognitive function in AD. In order to assess the presence of oxidative stress in AD, we examined the enzymatic activities of the erythrocyte Cu-Zn superoxide dismutase (Cu-Zn SOD, glutathione peroxidase (GSH-Px, catalase (CAT, and plasma level of total antioxidant status (TAS in AD and control groups (age and sex-matched. The results showed that the Cu-Zn SOD activity was significantly higher and the level of GSH-Px and TAS activities were significantly lower in AD subjects than that in the control group (2111 ± 324 U/grHb, 43.7 ± 11.6 U/grHb, and 1.17 ± 0.23 mmol/l compared with 1371 ± 211 U/grHb; t= -2.17, P = 0.036, 56.3 ± 9.5 U/grHb; t=3.8, P = 0.014, and 1.54±0.2 mmol/l; t=11.18, P < 0.001, respectively.  While, the erythrocyte CAT activity was lower in AD subjects compared to the control group, the difference was not statistically significant (t = 1.3, P = 0.15. These findings support the idea that the oxidative stress plays an important role in the pathogenesis underlying AD neurodegeneration. In addition, the enzymatic activity of the erythrocyte Cu-Zn SOD and GSH-Px and the plasma level of TAS can be used as a measure of the oxidative stress and a marker for pathological changes in the brain of patients with AD. 

  7. The narrow therapeutic window of glycated hemoglobin and assay variability.

    Science.gov (United States)

    Hosseini, S S; Bibler, I; Charles, M A

    1999-12-01

    Glycated hemoglobin is measured by a variety of assays, each of which has a unique normal level. Our purpose is to show that among the different assays available in the United States, using the same patient's blood sample, assay results may vary widely and may more or less easily achieve a glycated hemoglobin value within the normal range. The following assays were compared using the same patient's blood sample for each pair of assays: glycohemoglobin affinity assay (GHB Reader; Isolab, Akron, OH) versus gel electrophoresis assay (n = 76); Isolab versus ion capture assay (IMX; Abbott Laboratories, Irving, TX) (n = 57); monoclonal antibody assay (DCA2000; Bayer Diagnostics, Pittsburgh, PA) versus IMX (n = 100); and high-performance liquid chromatography (HPLC) assay (Bio-Rad Variant A1c; Bio-Rad Laboratories, Richmond, CA) versus IMX assay (n = 55). Our analyses indicate that a relative ranking can be established for the ease of achieving a normal glycated hemoglobin level. The ranking indicates that the most stringent or difficult assays for achieving a normal level are the Isolab and DCA2000 assays. The intermediate assays are the IMX and Bio-Rad Variant, and the easiest method for achieving a normal value is the gel electrophoresis assay. Our results indicate that various glycated hemoglobin assays vary widely and are associated with more or less difficulty for an individual patient to achieve a glycated hemoglobin level within the normal range. These results are especially significant with respect to (1) the clinically narrow therapeutic window of glycated hemoglobin values in type 1 diabetes to avoid rapidly advancing severe hypoglycemia rates and chronic microvascular complication rates, and (2) the glycated hemoglobin threshold for rapidly advancing macrovascular disease in both type 1 and type 2 patients.

  8. Dual functional rhodium oxide nanocorals enabled sensor for both non-enzymatic glucose and solid-state pH sensing.

    Science.gov (United States)

    Dong, Qiuchen; Huang, Yikun; Song, Donghui; Wu, Huixiang; Cao, Fei; Lei, Yu

    2018-07-30

    Both pH-sensitive and glucose-responsive rhodium oxide nanocorals (Rh 2 O 3 NCs) were synthesized through electrospinning followed by high-temperature calcination. The as-prepared Rh 2 O 3 NCs were systematically characterized using various advanced techniques including scanning electron microscopy, X-ray powder diffraction and Raman spectroscopy, and then employed as a dual functional nanomaterial to fabricate a dual sensor for both non-enzymatic glucose sensing and solid-state pH monitoring. The sensing performance of the Rh 2 O 3 NCs based dual sensor toward pH and glucose was evaluated using open circuit potential, cyclic voltammetry and amperometric techniques, respectively. The results show that the as-prepared Rh 2 O 3 NCs not only maintain accurate and reversible pH sensitivity of Rh 2 O 3 , but also demonstrate a good electrocatalytic activity toward glucose oxidation in alkaline medium with a sensitivity of 11.46 μA mM -1 cm -2 , a limit of detection of 3.1 μM (S/N = 3), and a reasonable selectivity against various interferents in non-enzymatic glucose detection. Its accuracy in determining glucose in human serum samples was further demonstrated. These features indicate that the as-prepared Rh 2 O 3 NCs hold great promise as a dual-functional sensing material in the development of a high-performance sensor forManjakkal both solid-state pH and non-enzymatic glucose sensing. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Minimalistic peptide supramolecular co-assembly: expanding the conformational space for nanotechnology.

    Science.gov (United States)

    Makam, Pandeeswar; Gazit, Ehud

    2018-05-21

    Molecular self-assembly is a ubiquitous process in nature and central to bottom-up nanotechnology. In particular, the organization of peptide building blocks into ordered supramolecular structures has gained much interest due to the unique properties of the products, including biocompatibility, chemical and structural diversity, robustness and ease of large-scale synthesis. In addition, peptides, as short as dipeptides, contain all the molecular information needed to spontaneously form well-ordered structures at both the nano- and the micro-scale. Therefore, peptide supramolecular assembly has been effectively utilized to produce novel materials with tailored properties for various applications in the fields of material science, engineering, medicine, and biology. To further expand the conformational space of peptide assemblies in terms of structural and functional complexity, multicomponent (two or more) peptide supramolecular co-assembly has recently evolved as a promising extended approach, similar to the structural diversity of natural sequence-defined biopolymers (proteins) as well as of synthetic covalent co-polymers. The use of this methodology was recently demonstrated in various applications, such as nanostructure physical dimension control, the creation of non-canonical complex topologies, mechanical strength modulation, the design of light harvesting soft materials, fabrication of electrically conducting devices, induced fluorescence, enzymatic catalysis and tissue engineering. In light of these significant advancements in the field of peptide supramolecular co-assembly in the last few years, in this tutorial review, we provide an updated overview and future prospects of this emerging subject.

  10. A singular enzymatic megacomplex from Bacillus subtilis.

    Science.gov (United States)

    Straight, Paul D; Fischbach, Michael A; Walsh, Christopher T; Rudner, David Z; Kolter, Roberto

    2007-01-02

    Nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS), and hybrid NRPS/PKS are of particular interest, because they produce numerous therapeutic agents, have great potential for engineering novel compounds, and are the largest enzymes known. The predicted masses of known enzymatic assembly lines can reach almost 5 megadaltons, dwarfing even the ribosome (approximately 2.6 megadaltons). Despite their uniqueness and importance, little is known about the organization of these enzymes within the native producer cells. Here we report that an 80-kb gene cluster, which occupies approximately 2% of the Bacillus subtilis genome, encodes the subunits of approximately 2.5 megadalton active hybrid NRPS/PKS. Many copies of the NRPS/PKS assemble into a single organelle-like membrane-associated complex of tens to hundreds of megadaltons. Such an enzymatic megacomplex is unprecedented in bacterial subcellular organization and has important implications for engineering novel NRPS/PKSs.

  11. Why bacteria derived R-M nucleic enzymatic peptides are likely ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-06-17

    Jun 17, 2008 ... (3) Human genome contains several palindromes recognizable by R-M peptides, making ..... mutation and likely has affected genome evolution. .... Microbicide to prevent sexual transmission of HIV among high risk.

  12. Products by Glycation Process

    Directory of Open Access Journals (Sweden)

    Liliana Ortega

    2015-01-01

    Full Text Available The antioxidant properties of sweet and acid whey products were incremented by polymerization of their proteins by glycation of whey protein concentrates (WPC and their hydrolyzates (WPCH with ribose and glucose in individual experiments under similar concentration. Heating at 50°C during 20 h maximum and pH 7 and pH 9 were used in all tests. The higher activity was found in WPC glycosylates products with ribose at pH 7 and heating during 10–15 h. In comparable form, antioxidant activity in WPCH was incremented by prior hydrolysis to glycation with 25–45% of hydrolysis degree. Further functional properties of whey proteins (solubility, emulsion, and foam were also improved by the polymerization with ribose. The color of polymerized products due to Maillard reactions was associated with antioxidant activity of each compound; however comparative color in glycosylates products with glucose and ribose did not show this effect.

  13. Enhanced sensitivity in non-enzymatic glucose detection by improved growth kinetics of Ni-based nanostructures

    Science.gov (United States)

    Urso, M.; Pellegrino, G.; Strano, V.; Bruno, E.; Priolo, F.; Mirabella, S.

    2018-04-01

    Ni-based nanostructures are attractive catalytic materials for many electrochemical applications, among which are non-enzymatic sensing, charge storage, and water splitting. In this work, we clarify the synthesis kinetics of Ni(OH)2/NiOOH nanowalls grown by chemical bath deposition at room temperature and at 50 °C. We applied the results to non-enzymatic glucose sensing, reaching a highest sensitivity of 31 mA cm-2mM-1. Using scanning electron microscopy, x-ray diffraction analysis and Rutherford backscattering spectrometry we found that the growth occurs through two regimes: first, a quick random growth leading to disordered sheets of Ni oxy-hydroxide, followed by a slower growth of well-aligned sheets of Ni hydroxide. A high growth temperature (50 °C), leading mainly to well-aligned sheets, offers superior electrochemical properties in terms of charge storage, charge carrier transport and catalytic action, as confirmed by cyclic voltammetry and electrochemical impedance spectroscopy analyses. The reported results on the optimization and application of low-cost synthesis of these Ni-based nanostructures have a large potential for application in catalysis, (bio)sensing, and supercapacitors areas.

  14. A high performance non-enzymatic glucose sensor based on nickel hydroxide modified nitrogen-incorporated nanodiamonds.

    Science.gov (United States)

    Ko, Chih-Yu; Huang, Jin-Hua; Raina, Supil; Kang, Weng P

    2013-06-07

    A highly selective, sensitive, and stable non-enzymatic glucose sensor based on Ni hydroxide modified nitrogen-incorporated nanodiamonds (Ni(OH)2-NND) was developed. The sensor was fabricated by e-beam evaporation of a thin Ni film on NND followed by the growth of Ni(OH)2 using an electrochemical process. It was found that the Ni film thickness greatly affects the morphology and electro-catalytic activity of the as-synthesized electrode for non-enzymatic glucose oxidation. Owing to its nanostructure characteristics, the best sensor fabricated by 150 nm Ni deposition showed two wide response ranges, namely, 0.02-1 mM and 1-9 mM, with sensitivities of 3.20 and 1.41 mA mM(-1) cm(-2), respectively, and a detection limit of 1.2 μM (S/N = 3). The sensor also showed good long-term stability as well as high selectivity in the presence of interferences such as ascorbic acid, acetaminophen, and uric acid. This finding reveals the possibility of exploiting the NND as an electrochemical biosensor platform where high performance addressable sensor arrays could be built.

  15. Cyclic peptides as potential therapeutic agents for skin disorders.

    Science.gov (United States)

    Namjoshi, Sarika; Benson, Heather A E

    2010-01-01

    There is an increasing understanding of the role of peptides in normal skin function and skin disease. With this knowledge, there is significant interest in the application of peptides as therapeutics in skin disease or as cosmeceuticals to enhance skin appearance. In particular, antimicrobial peptides and those involved in inflammatory processes provide options for the development of new therapeutic directions in chronic skin conditions such as psoriasis and dermatitis. To exploit their potential, it is essential that these peptides are delivered to their site of action in active form and in sufficient quantity to provide the desired effect. Many polymers permeate the skin poorly and are vulnerable to enzymatic degradation. Synthesis of cyclic peptide derivatives can substantially alter the physicochemical characteristics of the peptide with the potential to improve its skin permeation. In addition, cyclization can stabilize the peptide structure and thereby increase its stability. This review describes the role of cyclic peptides in the skin, examples of current cyclic peptide therapeutic products, and the potential for cyclic peptides as dermatological therapeutics and cosmeceuticals.

  16. Superior Antifouling Performance of a Zwitterionic Peptide Compared to an Amphiphilic, Non-Ionic Peptide.

    Science.gov (United States)

    Ye, Huijun; Wang, Libing; Huang, Renliang; Su, Rongxin; Liu, Boshi; Qi, Wei; He, Zhimin

    2015-10-14

    The aim of this study was to explore the influence of amphiphilic and zwitterionic structures on the resistance of protein adsorption to peptide self-assembled monolayers (SAMs) and gain insight into the associated antifouling mechanism. Two kinds of cysteine-terminated heptapeptides were studied. One peptide had alternating hydrophobic and hydrophilic residues with an amphiphilic sequence of CYSYSYS. The other peptide (CRERERE) was zwitterionic. Both peptides were covalently attached onto gold substrates via gold-thiol bond formation. Surface plasmon resonance analysis results showed that both peptide SAMs had ultralow or low protein adsorption amounts of 1.97-11.78 ng/cm2 in the presence of single proteins. The zwitterionic peptide showed relatively higher antifouling ability with single proteins and natural complex protein media. We performed molecular dynamics simulations to understand their respective antifouling behaviors. The results indicated that strong surface hydration of peptide SAMs contributes to fouling resistance by impeding interactions with proteins. Compared to the CYSYSYS peptide, more water molecules were predicted to form hydrogen-bonding interactions with the zwitterionic CRERERE peptide, which is in agreement with the antifouling test results. These findings reveal a clear relation between peptide structures and resistance to protein adsorption, facilitating the development of novel peptide-containing antifouling materials.

  17. Prediction of monomer isomery in Florine: a workflow dedicated to nonribosomal peptide discovery.

    Directory of Open Access Journals (Sweden)

    Thibault Caradec

    Full Text Available Nonribosomal peptides represent a large variety of natural active compounds produced by microorganisms. Due to their specific biosynthesis pathway through large assembly lines called NonRibosomal Peptide Synthetases (NRPSs, they often display complex structures with cycles and branches. Moreover they often contain non proteogenic or modified monomers, such as the D-monomers produced by epimerization. We investigate here some sequence specificities of the condensation (C and epimerization (E domains of NRPS that can be used to predict the possible isomeric state (D or L of each monomer in a putative peptide. We show that C- and E- domains can be divided into 2 sub-regions called Up-Seq and Down-Seq. The Up-Seq region corresponds to an InterPro domain (IPR001242 and is shared by C- and E-domains. The Down-Seq region is specific to the enzymatic activity of the domain. Amino-acid signatures (represented as sequence logos previously described for complete C-and E-domains have been restricted to the Down-Seq region and amplified thanks to additional sequences. Moreover a new Down-Seq signature has been found for Ct-domains found in fungi and responsible for terminal cyclization of the peptides. The identification of these signatures has been included in a workflow named Florine, aimed to predict nonribosomal peptides from NRPS sequence analyses. In some cases, the prediction of isomery is guided by genus-specific rules. Florine was used on a Pseudomonas genome to allow the determination of the type of pyoverdin produced, the update of syringafactin structure and the identification of novel putative products.

  18. Non-enzymatic N-acetylation of Lysine Residues by AcetylCoA Often Occurs via a Proximal S-acetylated Thiol Intermediate Sensitive to Glyoxalase II.

    Science.gov (United States)

    James, Andrew M; Hoogewijs, Kurt; Logan, Angela; Hall, Andrew R; Ding, Shujing; Fearnley, Ian M; Murphy, Michael P

    2017-02-28

    Acetyl coenzyme A (AcCoA), a key intermediate in mitochondrial metabolism, N-acetylates lysine residues, disrupting and, in some cases, regulating protein function. The mitochondrial lysine deacetylase Sirtuin 3 (Sirt3) reverses this modification with benefits reported in diabetes, obesity, and aging. We show that non-enzymatic lysine N-acetylation by AcCoA is greatly enhanced by initial acetylation of a cysteine residue, followed by SN-transfer of the acetyl moiety to a nearby lysine on mitochondrial proteins and synthetic peptides. The frequent occurrence of an S-acetyl intermediate before lysine N-acetylation suggests that proximity to a thioester is a key determinant of lysine susceptibility to acetylation. The thioesterase glyoxalase II (Glo2) can limit protein S-acetylation, thereby preventing subsequent lysine N-acetylation. This suggests that the hitherto obscure role of Glo2 in mitochondria is to act upstream of Sirt3 in minimizing protein N-acetylation, thus limiting protein dysfunction when AcCoA accumulates. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Using Serum Advanced Glycation End Products-Peptides to Improve the Efficacy of World Health Organization Fasting Plasma Glucose Criterion in Screening for Diabetes in High-Risk Chinese Subjects.

    Directory of Open Access Journals (Sweden)

    Zilin Sun

    Full Text Available The efficacy of using fasting plasma glucose (FPG alone as a preferred screening test for diabetes has been questioned. This study was aimed to evaluate whether the use of serum advanced glycation end products-peptides (sAGEP would help to improve the efficacy of FPG in diabetes screening among high-risk Chinese subjects with FPG <7.0 mmol/L. FPG, 2-h plasma glucose (2h-PG, serum glycated haemoglobin A1c (HbA1c, and sAGEP were measured in 857 Chinese subjects with risk factors for diabetes. The areas under receiver operating characteristic (ROC curves generated by logistic regression models were assessed and compared to find the best model for diabetes screening in subjects with FPG <7.0 mmol/L. The optimal critical line was determined by maximizing the sum of sensitivity and specificity. Among the enrolled subjects, 730 of them had FPG <7.0 mmol/L, and only 41.7% new diabetes cases were identified using the 1999 World Health Organization FPG criterion (FPG ≥7.0 mmol/L. The area under ROC curves generated by the model on FPG-sAGEP was the largest compared with that on FPG-HbA1c, sAGEP, HbA1c or FPG in subjects with FPG <7.0 mmol/L. By maximizing the sum of sensitivity and specificity, the optimal critical line was determined as 0.69×FPG + 0.14×sAGEP = 7.03, giving a critical sensitivity of 91.2% in detecting 2h-PG ≥11.1 mmol/L, which was significantly higher than that of FPG-HbA1c or HbA1c. The model on FPG-sAGEP improves the efficacy of using FPG alone in detecting diabetes among high-risk Chinese subjects with FPG <7.0 mmol/L, and is worth being promoted for future diabetes screening.

  20. 99m Tc-HYNIC-(Ser)3 -J18 peptide: A radiotracer for non-small-cell lung cancer targeting.

    Science.gov (United States)

    Shaghaghi, Zahra; Abedi, Seyed Mohammad; Hosseinimehr, Seyed Jalal

    2018-02-14

    Radiolabeled peptide could be a useful tool for the diagnosis of non-small-cell lung cancer (NSCLC). In this study, HYNIC-(Ser) 3 -J18 peptide was labeled with 99m Tc using EDDA/tricine as coligands. The in vitro and in vivo studies of this radiolabeled peptide were performed for cellular-specific binding and tumor targeting in A-549 cells and tumor-bearing mice, respectively. The high radiochemical purity was obtained and this radiolabeled peptide exhibited high stability in buffer and serum. The radiolabeled peptide showed high affinity for the A-549 cells with a dissociation constant value (K D ) of 4.4 ± 0.8 nm. The tumor-muscles ratios were 2.7 and 4.4 at 1 and 2 hr after injection of 99m Tc-(EDDA/tricine)-HYNIC-(Ser) 3 -J18 in tumor-bearing mice. The tumor uptake was decreased after preinjection with non-labeled peptide for this radiolabeled peptide in blocking experiment. The results of this study showed the 99m Tc-(EDDA/tricine)-(Ser) 3 -HYNIC-J18 peptide might be a promising radiolabeled peptide for NSCLC targeting. © 2018 John Wiley & Sons A/S.

  1. Production of a carob enzymatic extract: potential use as a biofertilizer.

    Science.gov (United States)

    Parrado, J; Bautista, J; Romero, E J; García-Martínez, A M; Friaza, V; Tejada, M

    2008-05-01

    In this paper, we describe a biological process that converts carob germ (CG), a proteinic vegetable by-product, into a water-soluble enzymatic hydrolyzate extract (CGHE). The chemical and physical properties are also described. The conversion is done using a proteolytic enzyme mixture. The main component of CGHE extracted by the enzymatic process is protein (68%), in the form of peptides and free amino acids, having a high content of glutamine and arginine, and a minor component of phytohormones, which are also extracted and solubilized from the CG. We have also compared its potential fertilizer/biostimulant capacity on growth, flowering, and fruiting of tomato plants (Licopericon pimpinellifolium cv. Momotaro) with that of an animal enzymatic protein hydrolyzate. CGHE had a significantly beneficial impact, most notably regarding the greater plant height, number of flowers per plant, and number of fruits per plant. This could be due primarily to its phytohormonal action.

  2. Inhibitory effect of alliin from Allium sativum on the glycation of superoxide dismutase.

    Science.gov (United States)

    Anwar, Shehwaz; Younus, Hina

    2017-10-01

    Inhibition of glycation is an important approach for alleviating diabetic complications. Alliin, the most abundant sulphur compound in garlic has been demonstrated to possess antidiabetic activity. However, there is no scientific evidence supporting its antiglycating activity. The objective of this study was to determine the inhibitory effect of alliin on glucose and methyglyoxal (MG)-induced glycation of an important antioxidant enzyme, superoxide dismutase (SOD). Glycation of SOD resulted in a decrease in enzyme activity, fragmentation/cross-linking, reduced cross-reactivity with anti-SOD antibodies, both tertiary and secondary structural changes, and formation of AGEs and fibrils. Alliin offered protection against glucose or MG induced glycation of SOD. The antiglycating potential of alliin appears to be comparable with that of quercetin which is reported to be a potent natural inhibitor of glycation. Alliin has a good antiglycating effect and hence is expected to have therapeutic potential in the prevention of glycation-mediated diabetic complications. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Quantitative kinetics of proteolytic enzymes determined by a surface concentration-based assay using peptide arrays.

    Science.gov (United States)

    Jung, Se-Hui; Kong, Deok-Hoon; Park, Seoung-Woo; Kim, Young-Myeong; Ha, Kwon-Soo

    2012-08-21

    Peptide arrays have emerged as a key technology for drug discovery, diagnosis, and cell biology. Despite the promise of these arrays, applications of peptide arrays to quantitative analysis of enzyme kinetics have been limited due to the difficulty in obtaining quantitative information of enzymatic reaction products. In this study, we developed a new approach for the quantitative kinetics analysis of proteases using fluorescence-conjugated peptide arrays, a surface concentration-based assay with solid-phase peptide standards using dry-off measurements, and compared it with an applied concentration-based assay. For fabrication of the peptide arrays, substrate peptides of cMMP-3, caspase-3, caspase-9, and calpain-1 were functionalized with TAMRA and cysteine, and were immobilized onto amine-functionalized arrays using a heterobifunctional linker, N-[γ-maleimidobutyloxy]succinimide ester. The proteolytic activities of the four enzymes were quantitatively analyzed by calculating changes induced by enzymatic reactions in the concentrations of peptides bound to array surfaces. In addition, this assay was successfully applied for calculating the Michaelis constant (K(m,surf)) for the four enzymes. Thus, this new assay has a strong potential for use in the quantitative evaluation of proteases, and for drug discovery through kinetics studies including the determination of K(m) and V(max).

  4. Machine Learning Reveals a Non-Canonical Mode of Peptide Binding to MHC class II Molecules

    DEFF Research Database (Denmark)

    Andreatta, Massimo; Jurtz, Vanessa Isabell; Kaever, Thomas

    2017-01-01

    binding motif with a non-canonical binding core of length different from nine. This previously undescribed mode of peptide binding to MHCII molecules gives a more complete picture of peptide presentation by MHCII and allows us to model more accurately this event. This article is protected by copyright...

  5. A new peptide (Ruviprase) purified from the venom of Daboia russelii russelii shows potent anticoagulant activity via non-enzymatic inhibition of thrombin and factor Xa.

    Science.gov (United States)

    Thakur, Rupamoni; Kumar, Ashok; Bose, Biplab; Panda, Dulal; Saikia, Debashree; Chattopadhyay, Pronobesh; Mukherjee, Ashis K

    2014-10-01

    Compounds showing dual inhibition of thrombin and factor Xa (FXa) are the subject of great interest owing to their broader specificity for effective anticoagulation therapy against cardiovascular disorders. This is the first report on the functional characterization and assessment of therapeutic potential of a 4423.6 Da inhibitory peptide (Ruviprase) purified from Daboia russelii russelii venom. The secondary structure of Ruviprase is composed of α-helices (61.9%) and random coils (38.1%). The partial N-terminal sequence (E(1)-V(2)-X(3)-W(4)-W(5)-W(6)-A(7)-Q(8)-L(9)-S(10)) of Ruviprase demonstrated significant similarity (80.0%) with an internal sequence of apoptosis-stimulating protein reported from the venom of Ophiophagus hannah and Python bivittatus; albeit Ruviprase did not show sequence similarity with existing thrombin/FXa inhibitors, suggesting its uniqueness. Ruviprase demonstrated a potent in vitro anticoagulant property and inhibited both thrombin and FXa following slow binding kinetics. Ruviprase inhibited thrombin by binding to its active site via an uncompetitive mechanism with a Ki value and dissociation constant (KD) of 0.42 μM and 0.46 μM, respectively. Conversely, Ruviprase demonstrated mixed inhibition (Ki = 0.16 μM) of FXa towards its physiological substrate prothrombin. Furthermore, the biological properties of Ruviprase could not be neutralized by commercial polyvalent or monovalent antivenom. Ruviprase at a dose of 2.0 mg/kg was non-toxic and showed potent in vivo anticoagulant activity after 6 h of intraperitoneal treatment in mice. Because of the potent anticoagulant property as well as non-toxic nature of Ruviprase, the possible application of the peptide as an antithrombotic agent for combating thrombosis-associated ailments appears promising. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  6. Antioxidant properties of a radical-scavenging peptide purified from enzymatically prepared fish skin gelatin hydrolysate.

    Science.gov (United States)

    Mendis, Eresha; Rajapakse, Niranjan; Kim, Se-Kwon

    2005-02-09

    Hoki (Johnius belengerii) skin gelatin was hydrolyzed with three commercial enzymes to identify radical-scavenging potencies of derived peptides. Peptides derived from tryptic hydrolysate exhibited the highest scavenging activities on superoxide, carbon-centered 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals assessed by ESR spectroscopy. Following consecutive chromatographic separations of tryptic hydroolysate, the peptide sequence His-Gly-Pro-Leu-Gly-Pro-Leu (797 Da) acted as a strong radical scavenger under studied conditions. Further, this peptide could act as an antioxidant against linoleic acid peroxidation and the activity was closer to the highly active synthetic antioxidant butylated hydroxytoluene (BHT). In addition, antioxidative enzyme levels in cultured human hepatoma cells were increased in the presence of this peptide and it was presumed to be the peptide involved in maintaining the redox balance in the cell environment. Present data indicate that free-radical-scavenging activities of hoki skin gelatin peptides substantially contribute to their antioxidant properties measured in different oxidative systems.

  7. Computational discovery of specificity-conferring sites in non-ribosomal peptide synthetases

    DEFF Research Database (Denmark)

    Knudsen, Michael; Søndergaard, Dan Ariel; Tofting-Olesen, Claus

    2016-01-01

    Motivation: By using a class of large modular enzymes known as Non-Ribosomal Peptide Synthetases (NRPS), bacteria and fungi are capable of synthesizing a large variety of secondary metabolites, many of which are bioactive and have potential, pharmaceutical applications as e.g.~antibiotics. There ...

  8. A functional mimic of natural peroxidases : synthesis and catalytic activity of a non-heme iron peptide hydroperoxide complex

    NARCIS (Netherlands)

    Choma, CT; Schudde, EP; Kellogg, RM; Robillard, GT; Feringa, BL

    1998-01-01

    Site-selective attachment of unprotected peptides to a non-heme iron complex is achieved by displacing two halides on the catalyst by peptide caesium thiolates, This coupling approach should be compatible with any peptide sequence provided there is only a single reduced cysteine. The oxidation

  9. Expression of advanced glycation end-products on sun-exposed and non-exposed cutaneous sites during the ageing process in humans.

    Science.gov (United States)

    Crisan, Maria; Taulescu, Marian; Crisan, Diana; Cosgarea, Rodica; Parvu, Alina; Cãtoi, Cornel; Drugan, Tudor

    2013-01-01

    The glycation process is involved in both the intrinsic (individual, genetic) and extrinsic (ultraviolet light, polution and lifestyle) aging processes, and can be quantified at the epidermal or dermal level by histological, immunohistochemical (IHC), or imagistic methods. Our study is focused on a histological and immunohistological comparison of sun-protected regions versus sun-exposed regions from different age groups of skin phototype III subjects, related to the aging process. Skin samples collected from non-protected and UV protected regions of four experimental groups with different ages, were studied using histology and IHC methods for AGE-CML [N(epsilon)-(carboxymethyl)lysine]. A semi-quantitative assessment of the CML expression in the microvascular endothelium and dermal fibroblasts was performed. The Pearson one-way ANOVA was used to compare data between the groups. In the dermis of sun-exposed skin, the number and the intensity of CML positive cells in both fibroblasts and endothelial cells (p<0.05) was higher compared to sun-protected skin, and was significantly increased in older patients. The sun-exposed areas had a more than 10% higher AGE-CML score than the protected areas. No statistically significant correlation was observed between the histological score and the IHC expression of CML. We concluded that in healthy integument, the accumulation of final glycation products increases with age and is amplified by ultraviolet exposure. The study provides new knowledge on differences of AGE-CML between age groups and protected and unprotected areas and emphasizes that endothelium and perivascular area are most affected, justifying combined topical and systemic therapies.

  10. Expression of advanced glycation end-products on sun-exposed and non-exposed cutaneous sites during the ageing process in humans.

    Directory of Open Access Journals (Sweden)

    Maria Crisan

    Full Text Available The glycation process is involved in both the intrinsic (individual, genetic and extrinsic (ultraviolet light, polution and lifestyle aging processes, and can be quantified at the epidermal or dermal level by histological, immunohistochemical (IHC, or imagistic methods. Our study is focused on a histological and immunohistological comparison of sun-protected regions versus sun-exposed regions from different age groups of skin phototype III subjects, related to the aging process. Skin samples collected from non-protected and UV protected regions of four experimental groups with different ages, were studied using histology and IHC methods for AGE-CML [N(epsilon-(carboxymethyllysine]. A semi-quantitative assessment of the CML expression in the microvascular endothelium and dermal fibroblasts was performed. The Pearson one-way ANOVA was used to compare data between the groups. In the dermis of sun-exposed skin, the number and the intensity of CML positive cells in both fibroblasts and endothelial cells (p<0.05 was higher compared to sun-protected skin, and was significantly increased in older patients. The sun-exposed areas had a more than 10% higher AGE-CML score than the protected areas. No statistically significant correlation was observed between the histological score and the IHC expression of CML. We concluded that in healthy integument, the accumulation of final glycation products increases with age and is amplified by ultraviolet exposure. The study provides new knowledge on differences of AGE-CML between age groups and protected and unprotected areas and emphasizes that endothelium and perivascular area are most affected, justifying combined topical and systemic therapies.

  11. Novel anti-oxidative peptides from enzymatic digestion of human milk

    DEFF Research Database (Denmark)

    Tsopmo, Apollinaire; Romanowski, Andrea; Banda, Lyness

    2011-01-01

    Humanmilk pepsin and pancreatin digests were separated using molecular membrane and reverse phase chromatography. Chemical screening of the resulting fractions using the ORAC antioxidant assay yielded a peptide fraction (PF-23) with high antioxidant activity (5207 μM Trolox Equivalents (TE...

  12. A Highly Sensitive Electrochemical Glucose Sensor By Nickel-Epoxy Electrode With Non-Enzymatic Sensor

    Directory of Open Access Journals (Sweden)

    Riyanto Riyanto

    2016-03-01

    Full Text Available The preparation of new sensor for glucose was based on the fact that glucose can be determined by non-enzymatic glucose oxidase. The Ni metals (99.98% purity, 0.5 mm thick, Aldrich Chemical Company was used to prepare Ni-Epoxy electrode. The Ni-epoxy electrodes were prepared in square cut of 1 cm and 1 mm by length and wide respectively. The Ni metal electrodes were connected to silver wire with silver conducting paint prior covered with epoxy gum. The prepared of nickel-epoxy modified electrode showed outstanding electro catalytic activity toward the oxidation of glucose in alkaline solution. The result from this research are correlation of determination using Nickel-Epoxyelectrode for electroanalysis of glucose in NaOH was R2 = 0.9984. LOQ, LOD and recovery of the Nickel-Epoxy electrode towards glucose were found to be 4.4 μM, 1.48 μM and 98.19%, respectively. The Nickel-Epoxy wire based electrochemical glucose sensor demonstrates good sensitivity, wide linear range, outstanding detection limit, attractive selectivity, good reproducibility, high stability as well as prominent feasibility use of non-enzymatic sensor for monitoring glucose in human urine owing to its advantages of low cost, simple preparation and excellent properties for glucose detection.

  13. Coptis chinensis Polysaccharides Inhibit Advanced Glycation End Product Formation.

    Science.gov (United States)

    Yang, Ye; Li, Yun; Yin, Dengke; Chen, Song; Gao, Xiangdong

    2016-06-01

    Coptis chinensis Franch (Huanglian) is commonly used to treat diabetes in China. In this study, the effects of the C. chinensis Franch polysaccharides (CCP) on advanced glycation end product (AGE) formation in vitro and in streptozotocin-induced diabetic mice were investigated. CCP significantly inhibited all the three periods of nonenzymatic protein glycation in vitro, including Amadori product, dicarbonyl compound, and AGE formation (P < .01). In diabetic mice, the administration of CCP not only improved both bodyweight and serum insulin and decreased fasting blood glucose and glycated serum protein concentrations but also decreased the AGE accumulations and morphological abnormalities in pancreas and liver. The inhibitory effects of CCP on AGE formation afford a potential therapeutic use in the prevention and treatment of diabetes.

  14. ASSOCIATION BETWEEN ENZYMATIC AND NON-ENZYMATIC ANTIOXIDANT DEFENSE WITH ALZHEIMER DISEASE

    Directory of Open Access Journals (Sweden)

    A. Vaisi-Raygani

    2008-04-01

    Full Text Available The etiopathogenesis of dementia in Alzheimer's disease (AD is still unclear. However, long-term oxidative stress is believed to be one of the major contributing factors in progression of neuronal degeneration and decline of cognitive function in AD. In order to assess the presence of oxidative stress in AD, we examined the enzymatic activities of the erythrocyte Cu-Zn superoxide dismutase (Cu-Zn SOD, glutathione peroxidase (GSH-Px, catalase (CAT, and plasma level of total antioxidant status (TAS in AD and control groups (age and sex-matched. The results showed that the Cu-Zn SOD activity was significantly higher and the level of GSH-Px and TAS activities were significantly lower in AD subjects than that in the control group (2111±324 U/grHb, 43.7±11.6 U/grHb, and 1.17 ±0.23 mmol/L compared with 1371±211 U/gHb; t= -2.17, p=0.036, 56.3±9.5 U/gHb; t=3.8, p=0.014, and 1.54±0.2 mmol/L; t=11.18, P<0.001, respectively. While, the erythrocyte CAT activity was lower in AD subjects compared to the control group, the difference was not statistically significant (t=1.3, P=0.15. These findings support the idea that the oxidative stress plays an important role in the pathogenesis underlying AD neurodegeneration. In addition, the enzymatic activity of the erythrocyte Cu-Zn SOD and GSH-Px and the plasma level of TAS can be used as a measure of the oxidative stress and a marker for pathological changes in the brain of patients with AD.

  15. Standardization and quality control in quantifying non-enzymatic oxidative protein modifications in relation to ageing and disease: Why is it important and why is it hard?

    DEFF Research Database (Denmark)

    Nedić, Olgica; Rogowska-Wrzesinska, Adelina; Rattan, Suresh

    2015-01-01

    Post-translational modifications (PTM) of proteins determine the activity, stability, specificity, transportability and lifespan of a protein. Some PTM are highly specific and regulated involving various enzymatic pathways, but there are other non-enzymatic PTM (nePTM), which occur stochastically...

  16. Electrodeposition of flower-like platinum on electrophoretically grown nitrogen-doped graphene as a highly sensitive electrochemical non-enzymatic biosensor for hydrogen peroxide detection

    International Nuclear Information System (INIS)

    Tajabadi, M.T.; Sookhakian, M.; Zalnezhad, E.; Yoon, G.H.; Hamouda, A.M.S.; Azarang, Majid; Basirun, W.J.; Alias, Y.

    2016-01-01

    Highlights: • Nitrogen doped graphene with different thickness by electrophoretic deposition. • The conductivity of N-graphene layer depends on the tickness. • Support of platinum shows efficient electrocatalytic performance for biosensor. • CV curves and amperometric responses improved and optimized in the presence of N-graphene. - Abstract: An efficient non-enzymatic biosensor electrode consisting of nitrogen-doped graphene (N-graphene) and platinum nanoflower (Pt NF) with different N-graphene loadings were fabricated on indium tin oxide (ITO) glass using a simple layer-by-layer electrophoretic and electrochemical sequential deposition approach. N-graphene was synthesized by annealing graphene oxide with urea at 900 °C. The structure and morphology of the as-fabricated non-enzymatic biosensor electrodes were determined using X-ray diffraction, field emission electron microscopy, transmission electron microscopy, Raman and X-ray photoelectron spectra. The as-fabricated Pt NF-N-graphene-modified ITO electrodes with different N-graphene loadings were utilized as a non-enzymatic biosensor electrode for the detection of hydrogen peroxide (H_2O_2). The behaviors of the hybrid electrodes towards H_2O_2 reduction were assessed using chronoamperometry, cyclic voltammetry and electrochemical impedance spectroscopy analysis. The Pt NF-N-graphene-modified ITO electrode with a 0.05 mg ml"−"1 N-graphene loading exhibited the lowest detection limit, fastest amperometric sensing, a wide linear response range, excellent stability and reproducibility for the non-enzymatic H_2O_2 detection, due to the synergistic effect between the electrocatalytic activity of the Pt NF and the high conductivity and large surface area of N-graphene.

  17. Upregulation of α7 Nicotinic Receptors by Acetylcholinesterase C-Terminal Peptides

    Science.gov (United States)

    Bond, Cherie E.; Zimmermann, Martina; Greenfield, Susan A.

    2009-01-01

    Background The alpha-7 nicotinic acetylcholine receptor (α7-nAChR) is well known as a potent calcium ionophore that, in the brain, has been implicated in excitotoxicity and hence in the underlying mechanisms of neurodegenerative disorders such as Alzheimer's disease. Previous research implied that the activity of this receptor may be modified by exposure to a peptide fragment derived from the C-terminal region of the enzyme acetylcholinesterase. This investigation was undertaken to determine if the functional changes observed could be attributed to peptide binding interaction with the α7-nAChR, or peptide modulation of receptor expression. Methodology/Principal Findings This study provides evidence that two peptides derived from the C-terminus of acetylcholinesterase, not only selectively displace specific bungarotoxin binding at the α7-nAChR, but also alter receptor binding properties for its familiar ligands, including the alternative endogenous agonist choline. Of more long-term significance, these peptides also induce upregulation of α7-nAChR mRNA and protein expression, as well as enhancing receptor trafficking to the plasma membrane. Conclusions/Significance The results reported here demonstrate a hitherto unknown relationship between the α7-nAChR and the non-enzymatic functions of acetylcholinesterase, mediated independently by its C-terminal domain. Such an interaction may prove valuable as a pharmacological tool, prompting new approaches for understanding, and combating, the process of neurodegeneration. PMID:19287501

  18. Non-enzymatic lipid oxidation products in biological systems: assessment of the metabolites from polyunsaturated fatty acids.

    Science.gov (United States)

    Vigor, Claire; Bertrand-Michel, Justine; Pinot, Edith; Oger, Camille; Vercauteren, Joseph; Le Faouder, Pauline; Galano, Jean-Marie; Lee, Jetty Chung-Yung; Durand, Thierry

    2014-08-01

    Metabolites of non-enzymatic lipid peroxidation of polyunsaturated fatty acids notably omega-3 and omega-6 fatty acids have become important biomarkers of lipid products. Especially the arachidonic acid-derived F2-isoprostanes are the classic in vivo biomarker for oxidative stress in biological systems. In recent years other isoprostanes from eicosapentaenoic, docosahexaenoic, adrenic and α-linolenic acids have been evaluated, namely F3-isoprostanes, F4-neuroprostanes, F2-dihomo-isoprostanes and F1-phytoprostanes, respectively. These have been gaining interest as complementary specific biomarkers in human diseases. Refined extraction methods, robust analysis and elucidation of chemical structures have improved the sensitivity of detection in biological tissues and fluids. Previously the main reliable instrumentation for measurement was gas chromatography-mass spectrometry (GC-MS), but now the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunological techniques is gaining much attention. In this review, the types of prostanoids generated from non-enzymatic lipid peroxidation of some important omega-3 and omega-6 fatty acids and biological samples that have been determined by GC-MS and LC-MS/MS are discussed. Copyright © 2014. Published by Elsevier B.V.

  19. Identification of Maillard reaction induced chemical modifications on Ara h 1

    Science.gov (United States)

    The Maillard reaction is a non-enzymatic glycation reaction between proteins and reducing sugars that can modify nut allergens during thermal processing. These modifications can alter the structural and immunological properties of these allergens, and may result in increased IgE binding. Here, we ...

  20. [Therapeutically active dressings--biomaterials in a study of collagen glycation].

    Science.gov (United States)

    Pielesz, Anna; Paluch, Jadwiga

    2012-01-01

    Active dressings (biomaterials, hydrogels) are cross-linked three-dimensional macromolecular networks. One of the most important properties of active dressings, is their ability for controlled uptake, release and retention of molecules. The formation of advanced glycation end products AGEs progressively increases with normal aging. However, AGE products are formed at accelerated rates in age and stress-related diseases (burns, in wound healing) and also in vitro. The aim will be also to develop a series of gels showing ability of controlled uptake, release and retention of molecules. The hydrogels can be used as biologically and therapeutically (antibacterial and anticancer) active biomaterials. The following materials and reagents were used in the examination: dried plants: Equisetum arvense L., Pulmonaria officinalis L., Agropyron repens. Non-defatted films were extracted from the dried plants. The suspension was stirred and extracted. Temperature was controlled using a water bath. The filtrate was vacuum condensed. The gelling precipitate was poured onto Petri plates and dried. The swelling ratio and the percent loading were calculated. The released amount of CaCl2 at different time intervals was determined by measuring the conductivity. The extent of glycation in collagen was measured. Novel types of swelling hydrogels have been prepared from dried plants and alginic acid. The active dressings showed swelling in aqueous medium, swelling characteristics were dependent on the chemical composition of hydrogel. The hydrogels were also loaded with CaCl2 and their potential for release was judged by measuring conductivity. The activity of hydrogels--active dressings on collagen incubated with glucose showed an decrease in glycation. So, hydrogels--active dressings, a known antiglycating agent which have therapeutic role in wound healing.

  1. Advanced glycation end products in the skin are enhanced in COPD

    NARCIS (Netherlands)

    Hoonhorst, Susan J. M.; Loi, Adele T. Lo Tam; Hartman, Jorine E.; Telenga, Eef D.; van den Berge, Maarten; Koenderman, Leo; Lammers, Jan Willem J.; Boezen, H. Marike; Postma, Dirkje S.; ten Hacken, Nick H. T.

    Background. Cigarette smoking is the main cause of chronic obstructive pulmonary disease (COPD) inducing oxidative stress and local tissue injury, resulting in pulmonary inflammation. Advanced glycation end products (AGEs) are produced by glycation and oxidation processes and their formation is

  2. Antioxidant, ACE-Inhibitory and antibacterial activities of Kluyveromyces marxianus protein hydrolysates and their peptide fractions

    Directory of Open Access Journals (Sweden)

    Mahta Mirzaeia

    2016-07-01

    Full Text Available Background: There has been evidence that proteins are potentially excellent source of antioxidants, antihypertensive and antimicrobial peptides, and that enzymatic hydrolysis is an effective method to release these peptides from protein molecules. The functional properties of protein hydrolysates depends on the protein substrate, the specificity of the enzymes, the conditions used during proteolysis, degree of hydrolysis, and the nature of peptides released including molecular weight, amino acid composition, and hydrophobicity. Context and purpose of this study: The biomass of Kluyveromyces marxianus was considered as a source of ACE inhibitory, antioxidant and antimicrobial peptides. Results: Autolysis and enzymatic hydrolysis were completed respectively, after 96 h and 5 h. Overall, trypsin (18.52% DH and chymotrypsin (21.59% DH treatments were successful in releasing antioxidant and ACE inhibitory peptides. Autolysate sample (39.51% DH demonstrated poor antioxidant and ACE inhibitory activity compared to trypsin and chymotrypsin hydrolysates. The chymotrypsin 3-5 kDa (301.6±22.81 μM TE/mg protein and trypsin < 3 kDa (280.16±39.16 μM TE/mg protein permeate peptide fractions showed the highest DPPH radical scavenging activity. The trypsin <3 kDa permeate peptide fraction showed the highest ABTS radical scavenging (1691.1±48.68 μM TE/mg protein and ACE inhibitory (IC50=0.03±0.001 mg/mL activities. The fraction (MW=5-10 kD obtained after autolysis treatment showed antibacterial activity against St. aureus and Lis. monocytogenes in well diffusion screening. The minimum inhibitory concentration (MIC value was 13.3 mg/mLagainst St. aureus and Lis. monocytogenes calculated by turbidimetric assay and it showed bactericidal activity against St. aureus at 21.3 mg/mL protein concentration. Conclusions: Altogether, the results of this study reveal that K. marxianus proteins contain specific peptides in their sequences which can be released by

  3. Non-enzymatic glucose sensing platform using self assembled cobalt oxide/graphene nanocomposites immobilized graphite modified electrode

    DEFF Research Database (Denmark)

    Vivekananth, R.; Babu, R. Suresh; Prasanna, K.

    2018-01-01

    A new strategy to prepare the densely packed cobalt oxide (Co3O4)/graphene nanocomposites by a self-assembly method were adopted in this work. A new non-enzymatic glucose determination has been fabricated by using Co3O4/graphene nanocomposites modified electrode as a sensing material. The nanocom...... of the modified electrode for glucose determination has been evaluated in urine samples....

  4. Non-Enzymatic Glucose Biosensor Based on CuO-Decorated CeO2 Nanoparticles

    Directory of Open Access Journals (Sweden)

    Panpan Guan

    2016-08-01

    Full Text Available Copper oxide (CuO-decorated cerium oxide (CeO2 nanoparticles were synthesized and used to detect glucose non-enzymatically. The morphological characteristics and structure of the nanoparticles were characterized through transmission electron microscopy, X-ray photoelectron spectroscopy, and X-ray diffraction. The sensor responses of electrodes to glucose were investigated via an electrochemical method. The CuO/CeO2 nanocomposite exhibited a reasonably good sensitivity of 2.77 μA mM−1cm−2, an estimated detection limit of 10 μA, and a good anti-interference ability. The sensor was also fairly stable under ambient conditions.

  5. Characterisation of advanced glycation endproducts in saliva from patients with diabetes mellitus

    International Nuclear Information System (INIS)

    Yoon, Min-Sung; Jankowski, Vera; Montag, Susanne; Zidek, Walter; Henning, Lars; Schlueter, Hartmut; Tepel, Martin; Jankowski, Joachim

    2004-01-01

    Patients with diabetes mellitus are prone to develop increased advanced glycation endproducts causing local complications and increased overall morbidity and mortality. Nuclear magnetic resonance spectra were determined in saliva of 52 consecutive patients with diabetes mellitus and 47 age-matched healthy control subjects. Resonance spectra showed specific peaks at 2.3, 7.3, and 8.4 ppm in saliva from patients with diabetes mellitus. These peaks could be generated by incubation of saliva from healthy control subjects with hypochloric acid in vitro, indicating the presence of advanced glycation endproducts. The presence of advanced glycation endproducts in patients with diabetes mellitus was associated with approximal plaque index, indicating increased periodontal damage. The study indicates that increased advanced glycation endproducts are involved in the pathogenesis of diabetic complications

  6. Effect of high hydrostatic pressure on the enzymatic hydrolysis of bovine serum albumin.

    Science.gov (United States)

    De Maria, Serena; Ferrari, Giovanna; Maresca, Paola

    2017-08-01

    The extent of enzymatic proteolysis mainly depends on accessibility of the peptide bonds, which stabilize the protein structure. The high hydrostatic pressure (HHP) process is able to induce, at certain operating conditions, protein displacement, thus suggesting that this technology can be used to modify protein resistance to the enzymatic attack. This work aims at investigating the mechanism of enzymatic hydrolysis assisted by HHP performed under different processing conditions (pressure level, treatment time). Bovine serum albumin was selected for the experiments, solubilized in sodium phosphate buffer (25 mg mL -1 , pH 7.5) with α-chymotrypsin or trypsin (E/S ratio = 1/10) and HPP treatment (100-500 MPa, 15-25 min). HHP treatment enhanced the extent of the hydrolysis reaction of globular proteins, being more effective than conventional hydrolysis. At HHP treatment conditions maximizing the protein unfolding, the hydrolysis degree of proteins was increased as a consequence of the increased exposure of peptide bonds to the attack of proteolytic enzymes. The maximum hydrolysis degree (10% and 7% respectively for the samples hydrolyzed with α-chymotrypsin and trypsin) was observed for the samples processed at 400 MPa for 25 min. At pressure levels higher than 400 MPa the formation of aggregates was likely to occur; thus the degree of hydrolysis decreased. Protein unfolding represents the key factor controlling the efficiency of HHP-assisted hydrolysis treatments. The peptide produced under high pressure showed lower dimensions and a different structure with respect to those of the hydrolysates obtained when the hydrolysis was carried out at atmospheric pressure, thus opening new frontiers of application in food science and nutrition. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  7. Induction of human immunodeficiency virus (HIV-1 envelope specific cell-mediated immunity by a non-homologous synthetic peptide.

    Directory of Open Access Journals (Sweden)

    Ammar Achour

    2007-11-01

    Full Text Available Cell mediated immunity, including efficient CTL response, is required to prevent HIV-1 from cell-to-cell transmission. In previous investigations, we have shown that B1 peptide derived by Fourier transformation of HIV-1 primary structures and sharing no sequence homology with the parent proteins was able to generate antiserum which recognizes envelope and Tat proteins. Here we have investigated cellular immune response towards a novel non-homologous peptide, referred to as cA1 peptide.The 20 amino acid sequence of cA1 peptide was predicted using the notion of peptide hydropathic properties; the peptide is encoded by the complementary anti-sense DNA strand to the sense strand of previously described non-homologous A1 peptide. In this report we demonstrate that the cA1 peptide can be a target for major histocompatibility complex (MHC class I-restricted cytotoxic T lymphocytes in HIV-1-infected or envelope-immunized individuals. The cA1 peptide is recognized in association with different MHC class I allotypes and could prime in vitro CTLs, derived from gp160-immunized individuals capable to recognize virus variants.For the first time a theoretically designed immunogen involved in broad-based cell-immune memory activation is described. Our findings may thus contribute to the advance in vaccine research by describing a novel strategy to develop a synthetic AIDS vaccine.

  8. "STUDY ON THE EFFECT OF GARLIC ON THE IN VITRO ALBUMIN GLYCATION REACTION"

    Directory of Open Access Journals (Sweden)

    N. Sheikh

    2004-05-01

    Full Text Available Garlic, an antioxidant plant, can react with amino groups of proteins to form Schiff bases. As diabetes leads to glycation of various proteins and this in turn has some effects on the structure of proteins and biochemical activity of them, the inhibition of this process seems very vital. For several years researchers in this field have done their best to recognize the antidiabetic compounds. The aim of this study is to determine the effects of garlic on albumin glycation in vitro.In the presence of various concentrations of garlic, albumin was glycated and evaluated using TBA (thio-barbituric acid method. The results showed that garlic has a statistically significant (P<0.05 effect in inhibiting or decreasing the reaction of albumin glycation. The findings of this research shows that garlic probably inhibits the reaction of glycation and decreases complications occurring in diabetes.

  9. Human Achilles tendon glycation and function in diabetes

    DEFF Research Database (Denmark)

    Couppe, Christian; Svensson, Rene Brüggebusch; Kongsgaard, Mads

    2016-01-01

    Diabetic patients have an increased risk of foot ulcers, and glycation of collagen may increase tissue stiffness. We hypothesized that the level of glycemic control (glycation) may affect Achilles tendon stiffness, which can influence gait pattern. We therefore investigated the relationship between...... tissue cross-linking were greater in diabetic patients compared to controls. The higher foot pressure indicates that material stiffness of tendon and other tissue (e.g skin and joint capsule) may influence on foot gait. The difference in foot pressure distribution may contribute to the development...... of foot ulcers in diabetic patients....

  10. Changes in Biochemical Markers of Kidney Function and ...

    African Journals Online (AJOL)

    Cases of diabetic kidney disease continue to increase worldwide despite advances in knowledge of the disease. Oxidative stress has been shown to play major role in the pathogenesis of diabetes mellitus, since free radicals are formed disproportionately in diabetes by glucose oxidation, non-enzymatic glycation of ...

  11. Effect of glycation of albumin on its renal clearance in normal and diabetic rats

    International Nuclear Information System (INIS)

    Layton, G.J.; Jerums, G.

    1988-01-01

    Two independent techniques have been used to study the renal clearances of nonenzymatically glycated albumin and nonglycated albumin in normal and streptozotocin-induced diabetic rats, 16 to 24 weeks after the onset of diabetes. In the first technique, serum and urinary endogenous glycated and nonglycated albumin were separated using m-aminophenylboronate affinity chromatography and subsequently quantified by radioimmunoassay. Endogenous glycated albumin was cleared approximately twofold faster than nonglycated albumin in normal and diabetic rats. However, no difference was observed in the glycated albumin/nonglycated albumin clearance ratios (Cga/Calb) in normal and diabetic rats, respectively (2.18 +/- 0.39 vs 1.83 +/- 0.22, P greater than 0.05). The second technique measured the renal clearance of injected 125I-labelled glycated albumin and 125I-labelled albumin. The endogenous results were supported by the finding that 125I-labelled glycated albumin was cleared more rapidly than 125I-labelled albumin in normal (P less than 0.01) and diabetic (P less than 0.05) rats. The Cga/Calb ratio calculated for the radiolabelled albumins was 1.4 and 2.0 in normal and diabetic rats, respectively. This evidence suggests that nonenzymatic glycation of albumin increases its renal clearance to a similar degree in normal and diabetic rats

  12. Electrodeposition of flower-like platinum on electrophoretically grown nitrogen-doped graphene as a highly sensitive electrochemical non-enzymatic biosensor for hydrogen peroxide detection

    Energy Technology Data Exchange (ETDEWEB)

    Tajabadi, M.T. [University Malaya Centre for Ionic Liquids, Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603 (Malaysia); Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603 (Malaysia); Sookhakian, M., E-mail: m.sokhakian@gmail.com [University Malaya Centre for Ionic Liquids, Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603 (Malaysia); Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603 (Malaysia); Department of Mechanical Convergence Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 133-791, Korea (Korea, Republic of); Zalnezhad, E., E-mail: erfan@hanyang.ac.kr [Department of Mechanical Convergence Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 133-791, Korea (Korea, Republic of); Yoon, G.H. [Department of Mechanical Convergence Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul, 133-791, Korea (Korea, Republic of); Hamouda, A.M.S. [Mechanical and Industrial Engineering Department, College of Engineering, Qatar University, 2713, Doha (Qatar); Azarang, Majid [Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603 (Malaysia); Basirun, W.J. [Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603 (Malaysia); Institute of Nanotechnology & Catalysis Research, Institute of Postgraduate Studies, University Malaya, 50603 Kuala Lumpur (Malaysia); Alias, Y., E-mail: yatimah70@um.edu.my [University Malaya Centre for Ionic Liquids, Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603 (Malaysia); Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur 50603 (Malaysia)

    2016-11-15

    Highlights: • Nitrogen doped graphene with different thickness by electrophoretic deposition. • The conductivity of N-graphene layer depends on the tickness. • Support of platinum shows efficient electrocatalytic performance for biosensor. • CV curves and amperometric responses improved and optimized in the presence of N-graphene. - Abstract: An efficient non-enzymatic biosensor electrode consisting of nitrogen-doped graphene (N-graphene) and platinum nanoflower (Pt NF) with different N-graphene loadings were fabricated on indium tin oxide (ITO) glass using a simple layer-by-layer electrophoretic and electrochemical sequential deposition approach. N-graphene was synthesized by annealing graphene oxide with urea at 900 °C. The structure and morphology of the as-fabricated non-enzymatic biosensor electrodes were determined using X-ray diffraction, field emission electron microscopy, transmission electron microscopy, Raman and X-ray photoelectron spectra. The as-fabricated Pt NF-N-graphene-modified ITO electrodes with different N-graphene loadings were utilized as a non-enzymatic biosensor electrode for the detection of hydrogen peroxide (H{sub 2}O{sub 2}). The behaviors of the hybrid electrodes towards H{sub 2}O{sub 2} reduction were assessed using chronoamperometry, cyclic voltammetry and electrochemical impedance spectroscopy analysis. The Pt NF-N-graphene-modified ITO electrode with a 0.05 mg ml{sup −1} N-graphene loading exhibited the lowest detection limit, fastest amperometric sensing, a wide linear response range, excellent stability and reproducibility for the non-enzymatic H{sub 2}O{sub 2} detection, due to the synergistic effect between the electrocatalytic activity of the Pt NF and the high conductivity and large surface area of N-graphene.

  13. Enzymatic approaches to rare sugar production.

    Science.gov (United States)

    Zhang, Wenli; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    Rare sugars have recently attracted much attention because of their potential applications in the food, nutraceutical, and pharmaceutical industries. A systematic strategy for enzymatic production of rare sugars, named Izumoring, was developed >10years ago. The strategy consists of aldose-ketose isomerization, ketose C-3 epimerization, and monosaccharide oxidation-reduction. Recent development of the Izumoring strategy is reviewed herein, especially the genetic approaches to the improvement of rare sugar-producing enzymes and the applications of target-oriented bioconversion. In addition, novel non-Izumoring enzymatic approaches are also summarized, including enzymatic condensation, phosphorylation-dephosphorylation cascade reaction, aldose epimerization, ulosonic acid decarboxylation, and biosynthesis of rare disaccharides. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. The hydrophobic substituent in aminophospholipids affects the formation kinetics of their Schiff bases.

    Science.gov (United States)

    Caldés, Catalina; Vilanova, Bartolomé; Adrover, Miquel; Donoso, Josefa; Muñoz, Francisco

    2013-04-01

    Schiff bases (SBs) are the initial products of non-enzymatic glycation reactions, which are associated to some diabetes-related diseases. In this work, we used physiological pH and temperature conditions to study the formation kinetics of the SBs of 1,2-dihexanoyl-sn-glycero-3-phosphoethanolamine (DPHE) and 1,2-dihexanoyl-sn-glycero-3-phospho-l-serine (DHPS) with various glycating compounds and with pyridoxal 5'-phosphate (an effective glycation inhibitor). Based on the obtained results, the hydrophobic environment simultaneously decreases the nucleophilic character of the amino group (k1) and increases its pKa, thereby increasing the formation rate of SB (kobs). Therefore, the presence of hydrophobic chains in aminophospholipids facilitates the formation and stabilization of SBs, and also, in a biological environment, their glycation. Additionally, the results confirm the inhibitory action of B6 vitamers on aminophospholipid glycation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Biomimetic and Aggregation-Driven Crystallization Route for Room-Temperature Material Synthesis: Growth of β-Ga2O3 Nanoparticles Using Peptide Assemblies as Nanoreactors

    Science.gov (United States)

    Lee, Sang-Yup; Gao, Xueyun; Matsui, Hiroshi

    2008-01-01

    The room temperature synthesis of β-Ga2O3 nanocrystal was examined by coupling two biomimetic crystallization techniques, the enzymatic peptide nano-assembly templating and the aggregation-driven crystallization. The catalytic template of peptide assembly nucleated and mineralized primary β-Ga2O3 crystals, and then fused them to grow single-crystalline and monodisperse nanoparticles in the cavity of the peptide assembly at room temperature. In this work, the peptide assembly was exploited as a nano-reactor with an enzymatic functionality catalyzing the hydrolysis of gallium precursors. In addition, the characteristic ring-structure of peptide assembly is expected to provide an efficient dehydration pathway and the crystallization control over the surface tension, which are advantageous for the β-Ga2O3 crystal growth. This multifunctional peptide assembly could be applied for syntheses of a variety of nanomaterials that are kinetically difficult to grow at room temperature. PMID:17302413

  16. Enhancement of fermentable sugar yield by competitive adsorption of non-enzymatic substances from yeast and cellulase on lignin.

    Science.gov (United States)

    Tang, Yong; Lei, Fuhou; Cristhian, Carrasco; Liu, Zuguang; Yu, Hailong; Jiang, Jianxin

    2014-03-20

    Enhancement of enzymatic digestibility by some supplementations could reduce enzyme loading and cost, which is still too high to realize economical production of lignocellulosic biofuels. A recent study indicates that yeast hydrolysates (YH) have improved the efficiency of cellulases on digestibility of furfural residues (FR). In the current work, the components of YH were separated by centrifugation and size exclusion chromatography and finally characterized in order to better understand this positive effect. A 60.8% of nitrogen of yeast cells was remained in the slurry (YHS) after hydrothermal treatment. In the supernatant of YH (YHL), substances of high molecular weight were identified as proteins and other UV-absorbing compounds, which showed close molecular weight to components of cellulases. Those substances attributed to a synergetic positive effect on enzymatic hydrolysis of FR. The fraction of YHL ranged from 1.19 to 2.19 mL (elution volume) contained over 50% of proteins in YHL and had the best performance in stimulating the release of glucose. Experiment results proved the adsorption of proteins in YHL on lignin. Supplementation of cellulases with YH enhances enzymatic digestibility of FR mainly by a competitive adsorption of non-enzymatic substances on lignin. The molecular weight of these substances has a significant impact on their performance. Different strategies can be used for a good utilization of yeast cells in terms of biorefinery concept.

  17. Acute insulin resistance mediated by advanced glycation endproducts in severely burned rats.

    Science.gov (United States)

    Zhang, Xing; Xu, Jie; Cai, Xiaoqing; Ji, Lele; Li, Jia; Cao, Bing; Li, Jun; Hu, Dahai; Li, Yan; Wang, Haichang; Xiong, Lize; Xiao, Ruiping; Gao, Feng

    2014-06-01

    Hyperglycemia often occurs in severe burns; however, the underlying mechanisms and importance of managing postburn hyperglycemia are not well recognized. This study was designed to investigate the dynamic changes of postburn hyperglycemia and the underlying mechanisms and to evaluate whether early glycemic control is beneficial in severe burns. Prospective, randomized experimental study. Animal research laboratory. Sprague-Dawley rats. Anesthetized rats were subjected to a full-thickness burn injury comprising 40% of the total body surface area and were randomized to receive vehicle, insulin, and a soluble form of receptor for advanced glycation endproducts treatments. An in vitro study was performed on cultured H9C2 cells subjected to vehicle or carboxymethyllysine treatment. We found that blood glucose change presented a distinct pattern with two occurrences of hyperglycemia at 0.5- and 3-hour postburn, respectively. Acute insulin resistance evidenced by impaired insulin signaling and glucose uptake occurred at 3-hour postburn, which was associated with the second hyperglycemia and positively correlated with mortality. Mechanistically, we found that serum carboxymethyllysine, a dominant species of advanced glycation endproducts, increased within 1-hour postburn, preceding the occurrence of insulin resistance. More importantly, treatment of animals with soluble form of receptor for advanced glycation endproducts, blockade of advanced glycation endproducts signaling, alleviated severe burn-induced insulin resistance. In addition, early hyperglycemic control with insulin not only reduced serum carboxymethyllysine but also blunted postburn insulin resistance and reduced mortality. These findings suggest that severe burn-induced insulin resistance is partly at least mediated by serum advanced glycation endproducts and positively correlated with mortality. Early glycemic control with insulin or inhibition of advanced glycation endproducts with soluble form of receptor

  18. The Effect of Turmeric , Cardamom and Ginger on in vitro Albumin Glycation

    Directory of Open Access Journals (Sweden)

    N. Sheikh

    2004-01-01

    Full Text Available Diabetes mellitus is one of the most common disease in the world that imposes a tremendous health and societal burden whether that burden is measured in terms of sickness , use of health systems resources or costs. Hyperglycemia is the most important clinical sign of diabetes leading to glycation of the various proteins in the body that leads to change in their nature , structure and biochemical activity. One of the probable methods in the treatment of diabetes mellitus is decrease or inhibition of this reaction. It seems that Turmeric , Cardamom and Ginger are useful for this purpose. The main goal of this research is to determine the effect of above agents on in vitro albumin glycation. In the presence of various concentration of these agents , albumin was glycated and evaluated using TBA method. Results showed that these food additives have inhibitory effects on albumin glycation reaction with the concentraction of 1 g/dl , 0.2 g/dl and 0.1 g/dl. Among these agents , Ginger had the most inhibitory effect (78% with the concentration of 1 g/dl. The sequence of effect is : Ginger > Cardamom > Turmeric These findings showed that these agents decrease albumin glycation reaction.

  19. Effect of some high consumption spices on hemoglobin glycation.

    Science.gov (United States)

    Naderi, G H; Dinani, Narges J; Asgary, S; Taher, M; Nikkhoo, N; Boshtam, M

    2014-01-01

    Formation of glycation products is major factor responsible in complications of diabetes. Worldwide trend is toward the use of natural additives in reducing the complications of diseases. Therefore, there is a growing interest in natural antiglycation found in plants. Herbs and spices are one of the most important targets to search for natural antiglycation from the point of view of safety. This study investigated the ability of some of the spices to inhibit glycation process in a hemoglobin/glucose model system and compared their potency with each other. For this subject the best concentration and time to incubate glucose with hemoglobin was investigated. Then the glycosylation degree of hemoglobin in the presence of extracts by the three concentrations 0.25, 0.5 and 1 μg/ml was measured colorimetrically at 520 nm. Results represent that some of extracts such as wild caraway, turmeric, cardamom and black pepper have inhibitory effects on hemoglobin glycation. But some of the extracts such as anise and saffron have not only inhibitory effects but also aggravated this event and have proglycation properties. In accordance with the results obtained we can conclude that wild caraway, turmeric, cardamom and black pepper especially wild caraway extracts are potent antiglycation agents, which can be of great value in the preventive glycation-associated complications in diabetes.

  20. Glucagon-Like Peptide-1 Secreting Cell Function as well as Production of Inflammatory Reactive Oxygen Species Is Differently Regulated by Glycated Serum and High Levels of Glucose

    Directory of Open Access Journals (Sweden)

    Alessandra Puddu

    2014-01-01

    Full Text Available Glucagon-like peptide-1 (GLP-1, an intestinal hormone contributing to glucose homeostasis, is synthesized by proglucagon and secreted from intestinal neuroendocrine cells in response to nutrients. GLP-1 secretion is impaired in type 2 diabetes patients. Here, we aimed at investigating whether diabetic toxic products (glycated serum (GS or high levels of glucose (HG may affect viability, function, and insulin sensitivity of the GLP-1 secreting cell line GLUTag. Cells were cultured for 5 days in presence or absence of different dilutions of GS or HG. GS and HG (alone or in combination increased reactive oxygen species (ROS production and upregulated proglucagon mRNA expression as compared to control medium. Only HG increased total production and release of active GLP-1, while GS alone abrogated secretion of active GLP-1. HG-mediated effects were associated with the increased cell content of the prohormone convertase 1/3 (PC 1/3, while GS alone downregulated this enzyme. HG upregulated Glucokinase (GK and downregulated SYNTHAXIN-1. GS abrogated SYNTHAXIN-1 and SNAP-25. Finally, high doses of GS alone or in combination with HG reduced insulin-mediated IRS-1 phosphorylation. In conclusion, we showed that GS and HG might regulate different pathways of GLP-1 production in diabetes, directly altering the function of neuroendocrine cells secreting this hormone.

  1. Antibody constant region peptides can display immunomodulatory activity through activation of the Dectin-1 signalling pathway.

    Directory of Open Access Journals (Sweden)

    Elena Gabrielli

    Full Text Available We previously reported that a synthetic peptide with sequence identical to a CDR of a mouse monoclonal antibody specific for difucosyl human blood group A exerted an immunomodulatory activity on murine macrophages. It was therapeutic against systemic candidiasis without possessing direct candidacidal properties. Here we demonstrate that a selected peptide, N10K, putatively deriving from the enzymatic cleavage of the constant region (Fc of human IgG(1, is able to induce IL-6 secretion and pIkB-α activation. More importantly, it causes an up-regulation of Dectin-1 expression. This leads to an increased activation of β-glucan-induced pSyk, CARD9 and pIkB-α, and an increase in the production of pro-inflammatory cytokines such as IL-6, IL-12, IL-1β and TNF-α. The increased activation of this pathway coincides with an augmented phagocytosis of non opsonized Candida albicans cells by monocytes. The findings suggest that some Fc-peptides, potentially deriving from the proteolysis of immunoglobulins, may cause an unexpected immunoregulation in a way reminiscent of innate immunity molecules.

  2. Dynamic peptide libraries for the discovery of supramolecular nanomaterials

    Science.gov (United States)

    Pappas, Charalampos G.; Shafi, Ramim; Sasselli, Ivan R.; Siccardi, Henry; Wang, Tong; Narang, Vishal; Abzalimov, Rinat; Wijerathne, Nadeesha; Ulijn, Rein V.

    2016-11-01

    Sequence-specific polymers, such as oligonucleotides and peptides, can be used as building blocks for functional supramolecular nanomaterials. The design and selection of suitable self-assembling sequences is, however, challenging because of the vast combinatorial space available. Here we report a methodology that allows the peptide sequence space to be searched for self-assembling structures. In this approach, unprotected homo- and heterodipeptides (including aromatic, aliphatic, polar and charged amino acids) are subjected to continuous enzymatic condensation, hydrolysis and sequence exchange to create a dynamic combinatorial peptide library. The free-energy change associated with the assembly process itself gives rise to selective amplification of self-assembling candidates. By changing the environmental conditions during the selection process, different sequences and consequent nanoscale morphologies are selected.

  3. The role of chitosan on oral delivery of peptide-loaded nanoparticle formulation.

    Science.gov (United States)

    Wong, Chun Y; Al-Salami, Hani; Dass, Crispin R

    2017-12-01

    Therapeutic peptides are conventionally administered via subcutaneous injection. Chitosan-based nanoparticles are gaining increased attention for their ability to serve as a carrier for oral delivery of peptides and vaccination. They offered superior biocompatibiltiy, controlled drug release profile and facilitated gastrointestinal (GI) absorption. The encapsulated peptides can withstand enzymatic degradation and various pH. Chitosan-based nanoparticles can also be modified by ligand conjugation to the surface of nanoparticle for transcellular absorption and specific-targeted delivery of macromolecules to the tissue of interest. Current research suggests that chitosan-based nanoparticles can deliver therapeutic peptide for the treatment of several medical conditions such as diabetes, bacterial infection and cancer. This review summarises the role of chitosan in oral nanoparticle delivery and identifies the clinical application of peptide-loaded chitosan-based nanoparticles.

  4. Non-enzymatic hydrogen peroxide biosensor based on rose-shaped FeMoO4 nanostructures produced by convenient microwave-hydrothermal method

    International Nuclear Information System (INIS)

    Liu, Hongying; Gu, Chunchuan; Li, Dujuan; Zhang, Mingzhen

    2015-01-01

    Graphical abstract: A non-enzymatic H 2 O 2 sensor with high selectivity and sensitivity based on rose-shaped FeMoO 4 synthesized by the convenient microwave-assisted hydrothermal method, was fabricated. - Highlights: • Rose-shaped FeMoO 4 is synthesized within 10 min via microwave-assisted hydrothermal approach. • Non-enzymatic hydrogen peroxide biosensor based on FeMoO 4 nanomaterials is fabricated. • The biosensor exhibits good performance. - Abstract: In this work, we demonstrated a simple, rapid and reliable microwave-assisted hydrothermal approach to synthesize the uniform rose-shaped FeMoO 4 within 10 min. The morphologies of the synthesized materials were characterized by X-ray powder diffraction and scanning electron microscopy. Moreover, a non-enzymatic amperometric sensor for the detection of hydrogen peroxide (H 2 O 2 ) was fabricated on the basis of the FeMoO 4 as electrocatalysis. The resulting FeMoO 4 exhibited high sensitivity and good stability for the detection of H 2 O 2 , which may be attributed to the rose-shaped structure of the material and the catalytic property of FeMoO 4 . Amperometric response showed that the modified electrode had a good response for H 2 O 2 with a linear range from 1 μM to 1.6 mM, a detection limit of 0.5 μM (S/N = 3), high selectivity and short response time. Additionally, good recoveries of analytes in real milk samples confirm the reliability of the prepared sensor in practical applications

  5. Advanced glycation End-products (AGEs): an emerging concern for processed food industries.

    Science.gov (United States)

    Sharma, Chetan; Kaur, Amarjeet; Thind, S S; Singh, Baljit; Raina, Shiveta

    2015-12-01

    The global food industry is expected to increase more than US $ 7 trillion by 2014. This rise in processed food sector shows that more and more people are diverging towards modern processed foods. As modern diets are largely heat processed, they are more prone to contain high levels of advanced glycation end products (AGEs). AGEs are a group of complex and heterogeneous compounds which are known as brown and fluorescent cross-linking substances such as pentosidine, non-fluorescent cross-linking products such as methylglyoxal-lysine dimers (MOLD), or non-fluorescent, non-cross linking adducts such as carboxymethyllysine (CML) and pyrraline (a pyrrole aldehyde). The chemistry of the AGEs formation, absorption and bioavailability and their patho-biochemistry particularly in relation to different complications like diabetes and ageing discussed. The concept of AGEs receptor - RAGE is mentioned. AGEs contribute to a variety of microvascular and macrovascular complications through the formation of cross-links between molecules in the basement membrane of the extracellular matrix and by engaging the receptor for advanced glycation end products (RAGE). Different methods of detection and quantification along with types of agents used for the treatment of AGEs are reviewed. Generally, ELISA or LC-MS methods are used for analysis of foods and body fluids, however lack of universally established method highlighted. The inhibitory effect of bioactive components on AGEs by trapping variety of chemical moieties discussed. The emerging evidence about the adverse effects of AGEs makes it necessary to investigate the different therapies to inhibit AGEs.

  6. Kinetic study of solid phase demineralization by weak acids in one-step enzymatic bio-refinery of shrimp cuticles

    OpenAIRE

    Baron, Regis; Socol, Marius; Arhaliass, A.; Bruzac, Sandrine; Le Roux, Karine; Del Pino, J. Rodriguez; Berge, Jean-pascal; Kaas, Raymond

    2015-01-01

    We describe a one-step bio-refinery process for shrimp composites by-products. Its originality lies in a simple rapid (6 h) biotechnological cuticle fragmentation process that recovers all major compounds (chitins, peptides and minerals in particular calcium). The process consists of a controlled exogenous enzymatic proteolysis in a food-grade acidic medium allowing chitin purification (solid phase), and recovery of peptides and minerals (liquid phase). At a pH of between 3.5 and 4, protease ...

  7. Neurite regeneration in adult rat retinas exposed to advanced glycation end-products and regenerative effects of neurotrophin-4.

    Science.gov (United States)

    Bikbova, Guzel; Oshitari, Toshiyuki; Yamamoto, Shuichi

    2013-10-09

    The purpose of this study was to determine the effect of low concentrations of advanced glycation end-products on neurite regeneration in isolated rat retinas, and to determine the effects of neurotrophin-4 on regeneration in advanced glycation end-products exposed retinas. Retinal explants of 4 adult Sprague-Dawley rats were cultured on collagen gel and were incubated in; (1) serum-free control culture media, (2) glucose-advanced glycation end-products-bovine serum albumin media, (3) glycolaldehyde-advanced glycation end-products-bovine serum albumin media, (4) glyceraldehyde-advanced glycation end-products-bovine serum albumin media, (5) glucose-advanced glycation end-products+neurotrophin-4 media, (6) glycolaldehyde-advanced glycation end-products+neurotrophin-4 media, or (7) glyceraldehyde-advanced glycation end-products+neurotrophin-4 supplemented culture media. After 7 days, the number of regenerating neurites from the explants was counted. Then, explants were fixed, cryosectioned, and stained for TUNEL. The ratio of TUNEL-positive cells to all cells in the ganglion cell layer was determined. Immunohistochemical examinations for the active-form of caspase-9 and apoptosis-inducing factor were performed. In retinas incubated with advanced glycation end-products containing media, the number of regenerating neurites were fewer than in retinas without advanced glycation end-products, and the number of TUNEL-positive cells and caspase-9- and apoptosis-inducing factor-immunopositive cells was significantly higher than in control media. Neurotrophin-4 supplementation increased the numbers of regenerating neuritis, and the number of TUNEL-positives, caspase-9-, and apoptosis-inducing factor-immunopositive cells were significantly fewer than that in advanced glycation end-products without neurotrophin-4 media. Low doses of advanced glycation end-products impede neurite regeneration in the rat retinas. Neurotrophin-4 significantly enhances neurite regeneration in

  8. Use of Fructosyl Peptide Oxidase for HbA1c Assay

    Science.gov (United States)

    Yonehara, Satoshi; Inamura, Norio; Fukuda, Miho; Sugiyama, Koji

    2015-01-01

    ARKRAY, Inc developed the world’s first automatic glycohemoglobin analyzer based on HPLC (1981). After that, ARKRAY developed enzymatic HbA1c assay “CinQ HbA1c” with the spread and diversification of HbA1c measurement (2007). CinQ HbA1c is the kit of Clinical Chemistry Analyzer, which uses fructosyl peptide oxidase (FPOX) for a measurement reaction. This report mainly indicates the developmental background, measurement principle, and future of the enzymatic method HbA1c reagent. PMID:25633966

  9. Binderless solution processed Zn doped Co3O4 film on FTO for rapid and selective non-enzymatic glucose detection

    CSIR Research Space (South Africa)

    Chowdhury, M

    2016-09-01

    Full Text Available A simple solution based deposition process has been used to fabricate Zn doped Co(sub3)O(sub4) electrode as an electrocatalyst for non-enzymatic oxidation of glucose. XRD, HRTEM, SEM, EELS, AFM, EIS was used to characterise the electrode...

  10. Determination of advanced glycation endproducts by LC-MS/MS in raw and roasted almonds (Prunus dulcis).

    Science.gov (United States)

    Zhang, Gong; Huang, Guangwei; Xiao, Lu; Mitchell, Alyson E

    2011-11-23

    A sensitive and reliable LC-(ESI)MS/MS method was developed and validated for the simultaneous analysis of five common advanced glycation endproducts (AGEs) after enzymatic digestion in raw and roasted almonds. AGEs included carboxymethyl-lysine (CML), carboxyethyl-lysine (CEL), pyralline (Pyr), argpyrimidine (Arg-p), and pentosidine (Pento-s). This method allows accurate quantitation of free and AGE-protein adducts of target AGEs. Results indicate that CML and CEL are found in both raw and roasted almonds. Pyr was identified for the first time in roasted almonds and accounted for 64.4% of free plus bound measured AGEs. Arg-p and Pento-s were below the limit of detection in all almond samples tested. Free AGEs accounted for 1.3-26.8% of free plus bound measured AGEs, indicating that protein-bound forms predominate. The roasting process significantly increased CML, CEL, and Pyr formation, but no significant correlation was observed between these AGEs and roasting temperature.

  11. Advanced glycation end products, measured in skin, vs. HbA1c in children with type 1 diabetes mellitus

    NARCIS (Netherlands)

    Banser, Alena; Naafs, Jolanda C.; Hoorweg-Nijman, Jantine J. G.; van de Garde, Ewoudt M. W.; van der Vorst, Marja M. J.

    2016-01-01

    Background and objectiveAdvanced glycation end products (AGEs) are considered major contributors to microvascular and macrovascular complications in adult patients with diabetes mellitus. AGEs can be measured non-invasively with skin autofluorescence (sAF). The primary aim was to determine sAF

  12. A non-covalent peptide-based strategy for ex vivo and in vivo oligonucleotide delivery.

    Science.gov (United States)

    Crombez, Laurence; Morris, May C; Heitz, Frederic; Divita, Gilles

    2011-01-01

    The dramatic acceleration in identification of new nucleic acid-based therapeutic molecules such as short interfering RNA (siRNA) and peptide-nucleic acid (PNA) analogues has provided new perspectives for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake of nucleic acids together with the low permeability of the cell membrane to negatively charged molecules remain major obstacles to their clinical development. Several non-viral strategies have been proposed to improve the delivery of synthetic short oligonucleotides both in cultured cells and in vivo. Cell-penetrating peptides constitute very promising tools for non-invasive cellular import of oligonucleotides and analogs. We recently described a non-covalent strategy based on short amphiphatic peptides (MPG8/PEP3) that have been successfully applied ex vivo and in vivo for the delivery of therapeutic siRNA and PNA molecules. PEP3 and MPG8 form stable nanoparticles with PNA analogues and siRNA, respectively, and promote their efficient cellular uptake, independently of the endosomal pathway, into a wide variety of cell lines, including primary and suspension lines, without any associated cytotoxicity. This chapter describes easy-to-handle protocols for the use of MPG-8 or PEP-3-nanoparticle technologies for PNA and siRNA delivery into adherent and suspension cell lines as well as in vivo into cancer mouse models.

  13. Amadori albumin in diabetic nephropathy

    Directory of Open Access Journals (Sweden)

    Km. Neelofar

    2015-01-01

    Full Text Available Nonenzymatic glycation of macromolecules in diabetes mellitus (DM is accelerated due to persistent hyperglycemia. Reducing sugar such as glucose reacts non enzymatically with free €- amino groups of proteins through series of reactions forming Schiff bases. These bases are converted into Amadori product and further into AGEs. Non enzymatic glycation has the potential to alter the biological, structural and functional properties of macromolecules both in vitro and in vivo. Studies have suggested that amadori as well as AGEs are involved in the micro-macro vascular complications in DM, but most studies have focused on the role of AGEs in vascular complications of diabetes. Recently putative AGE-induced patho-physiology has shifted attention from the possible role of amadori-modified proteins, the predominant form of the glycated proteins in the development of the diabetic complications. Human serum albumin (HSA, the most abundant protein in circulation contains 59 lysine and 23 arginine residues that could, in theory be involved in glycation. Albumin has dual nature, first as a marker of intermediate glycation and second as a causative agent of the damage of tissues. Among the blood proteins, hemoglobin and albumin are the most common proteins that are glycated. HSA with a shorter half life than RBC, appears to be an alternative marker of glycemic control as it can indicate blood glucose status over a short period (2-3 weeks and being unaffected by RBCs life span and variant haemoglobin, anemia etc which however, affect HbA1c. On the other hand, Amadori albumin may accumulate in the body tissues of the diabetic patients and participate in secondary complications. Amadori-albumin has potential role in diabetic glomerulosclerosis due to long term hyperglycaemia and plays an important role in the pathogenesis of diabetic nephropathy. This review is an approach to compile both the nature of glycated albumin as a damaging agent of tissues and as an

  14. Cross-linking in collagen by nonenzymatic glycation increases the matrix stiffness in rabbit achilles tendon.

    Science.gov (United States)

    Reddy, G Kesava

    2004-01-01

    Nonenzymatic glycation of connective tissue matrix proteins is a major contributor to the pathology of diabetes and aging. Previously the author and colleagues have shown that nonenzymatic glycation significantly enhances the matrix stability in the Achilles tendon (Reddy et al., 2002, Arch. Biochem. Biophys., 399, 174-180). The present study was designed to gain further insight into glycation-induced collagen cross-linking and its relationship to matrix stiffness in the rabbit Achilles tendon. The glycation process was initiated by incubating the Achilles tendons (n = 6) in phosphate-buffered saline containing ribose, whereas control tendons (n = 6) were incubated in phosphate-buffered saline without ribose. Eight weeks following glycation, the biomechanical attributes as well as the degree of collagen cross-linking were determined to examine the potential associations between matrix stiffness and molecular properties of collagen. Compared to nonglycated tendons, the glycated tendons showed increased maximum load, stress, strain, Young's modulus of elasticity, and toughness indicating that glycation increases the matrix stiffness in the tendons. Glycation of tendons resulted in a considerable decrease in soluble collagen content and a significant increase in insoluble collagen and pentosidine. Analysis of potential associations between the matrix stiffness and degree of collagen cross-linking showed that both insoluble collagen and pentosidine exhibited a significant positive correlation with the maximum load, stress, and strain, Young's modulus of elasticity, and toughness (r values ranging from.61 to.94) in the Achilles tendons. However, the soluble collagen content present in neutral salt buffer, acetate buffer, and acetate buffer containing pepsin showed an inverse relation with the various biomechanical attributes tested (r values ranging from.22 to.84) in the Achilles tendons. The results of the study demonstrate that glycation-induced collagen cross

  15. Novel Inhibitor Cystine Knot Peptides from Momordica charantia

    Science.gov (United States)

    Clark, Richard J.; Tang, Jun; Zeng, Guang-Zhi; Franco, Octavio L.; Cantacessi, Cinzia; Craik, David J.; Daly, Norelle L.; Tan, Ning-Hua

    2013-01-01

    Two new peptides, MCh-1 and MCh-2, along with three known trypsin inhibitors (MCTI-I, MCTI-II and MCTI-III), were isolated from the seeds of the tropical vine Momordica charantia. The sequences of the peptides were determined using mass spectrometry and NMR spectroscopy. Using a strategy involving partial reduction and stepwise alkylation of the peptides, followed by enzymatic digestion and tandem mass spectrometry sequencing, the disulfide connectivity of MCh-1 was elucidated to be CysI-CysIV, CysII-CysV and CysIII-CysVI. The three-dimensional structures of MCh-1 and MCh-2 were determined using NMR spectroscopy and found to contain the inhibitor cystine knot (ICK) motif. The sequences of the novel peptides differ significantly from peptides previously isolated from this plant. Therefore, this study expands the known peptide diversity in M. charantia and the range of sequences that can be accommodated by the ICK motif. Furthermore, we show that a stable two-disulfide intermediate is involved in the oxidative folding of MCh-1. This disulfide intermediate is structurally homologous to the proposed ancestral fold of ICK peptides, and provides a possible pathway for the evolution of this structural motif, which is highly prevalent in nature. PMID:24116036

  16. Novel inhibitor cystine knot peptides from Momordica charantia.

    Directory of Open Access Journals (Sweden)

    Wen-Jun He

    Full Text Available Two new peptides, MCh-1 and MCh-2, along with three known trypsin inhibitors (MCTI-I, MCTI-II and MCTI-III, were isolated from the seeds of the tropical vine Momordica charantia. The sequences of the peptides were determined using mass spectrometry and NMR spectroscopy. Using a strategy involving partial reduction and stepwise alkylation of the peptides, followed by enzymatic digestion and tandem mass spectrometry sequencing, the disulfide connectivity of MCh-1 was elucidated to be CysI-CysIV, CysII-CysV and CysIII-CysVI. The three-dimensional structures of MCh-1 and MCh-2 were determined using NMR spectroscopy and found to contain the inhibitor cystine knot (ICK motif. The sequences of the novel peptides differ significantly from peptides previously isolated from this plant. Therefore, this study expands the known peptide diversity in M. charantia and the range of sequences that can be accommodated by the ICK motif. Furthermore, we show that a stable two-disulfide intermediate is involved in the oxidative folding of MCh-1. This disulfide intermediate is structurally homologous to the proposed ancestral fold of ICK peptides, and provides a possible pathway for the evolution of this structural motif, which is highly prevalent in nature.

  17. Monotopic modifications derived from in vitro glycation of albumin with ribose

    Czech Academy of Sciences Publication Activity Database

    Pataridis, Statis; Šťastná, Zdeňka; Sedláková, Pavla; Mikšík, Ivan

    2013-01-01

    Roč. 34, č. 12 (2013), s. 1757-1763 ISSN 0173-0835 R&D Projects: GA ČR(CZ) GAP206/12/0453; GA ČR(CZ) GA203/08/1428 Institutional support: RVO:67985823 Keywords : advanced glycation end product (AGE) * albumin * CE-MS * glycation * LC-MS/MS Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.161, year: 2013

  18. Enzymatic biodiesel production: Technical and economical considerations

    DEFF Research Database (Denmark)

    Munk Nielsen, Per; Brask, Jesper; Fjerbæk, Lene

    2008-01-01

    It is well documented in the literature that enzymatic processing of oils and fats for biodiesel is technically feasible. However, with very few exceptions, enzyme technology is not currently used in commercial-scale biodiesel production. This is mainly due to non-optimized process design...... and a lack of available costeffective enzymes. The technology to re-use enzymes has typically proven insufficient for the processes to be competitive. However, literature data documenting the productivity of enzymatic biodiesel together with the development of new immobilization technology indicates...... that enzyme catalysts can become cost effective compared to chemical processing. This work reviews the enzymatic processing of oils and fats into biodiesel with focus on process design and economy....

  19. Peptide-based subunit vaccine against hookworm infection.

    Directory of Open Access Journals (Sweden)

    Mariusz Skwarczynski

    Full Text Available Hookworms infect more people than HIV and malaria combined, predominantly in third world countries. Treatment of infection with chemotherapy can have limited efficacy and re-infections after treatment are common. Heavy infection often leads to debilitating diseases. All these factors suggest an urgent need for development of vaccine. In an attempt to develop a vaccine targeting the major human hookworm, Necator americanus, a B-cell peptide epitope was chosen from the apical enzyme in the hemoglobin digestion cascade, the aspartic protease Na-APR-1. The A(291Y alpha helical epitope is known to induce neutralizing antibodies that inhibit the enzymatic activity of Na-APR-1, thus reducing the capacity for hookworms to digest hemoglobin and obtain nutrients. A(291Y was engineered such that it was flanked on both termini by a coil-promoting sequence to maintain native conformation, and subsequently incorporated into a Lipid Core Peptide (LCP self-adjuvanting system. While A(291Y alone or the chimeric epitope with or without Freund's adjuvants induced negligible IgG responses, the LCP construct incorporating the chimeric peptide induced a strong IgG response in mice. Antibodies produced were able to bind to and completely inhibit the enzymatic activity of Na-APR-1. The results presented show that the new chimeric LCP construct can induce effective enzyme-neutralising antibodies in mice, without the help of any additional toxic adjuvants. This approach offers promise for the development of vaccines against helminth parasites of humans and their livestock and companion animals.

  20. Relationship between glycated haemoglobin and fasting plasma ...

    African Journals Online (AJOL)

    Emmanuel Musenge

    2016-07-30

    Jul 30, 2016 ... Relationship between glycated haemoglobin and fasting plasma glucose ... major stakeholders in the management of diabetes mellitus to consider FPG as an ..... HbA1c among customers of health examination services.

  1. Cell growth and proteolytic activity of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus in milk as affected by supplementation with peptide fractions.

    Science.gov (United States)

    Gandhi, Akanksha; Shah, Nagendra P

    2014-12-01

    The present investigation examined the effects of supplementation of milk peptide fractions produced by enzymatic hydrolysis on the fermentation of reconstituted skim milk (RSM). Changes in pH, cell growth, proteolytic activity, and angiotensin-converting enzyme (ACE)-inhibitory activity were monitored during fermentation of RSM by pure cultures of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus. The study showed that supplementation with peptide fractions of different molecular weights did not significantly affect the bacterial growth in RSM. All bacteria showed an increased proteolytic activity in RSM supplemented with large peptides (>10 kDa), and L. helveticus in general exhibited the highest proteolytic activity among the bacteria studied. The ACE-inhibitory activity was observed to be the maximum in RSM supplemented with larger peptides (>10 kDa) for all bacteria. The results suggest that proteolysis by bacteria leads to increased production of ACE-inhibitory peptides compared to the supplemented peptides produced by enzymatic hydrolysis.

  2. Bio-optic signatures for advanced glycation end products in the skin in streptozotocin (STZ) Induced Diabetes (Conference Presentation)

    Science.gov (United States)

    Saidian, Mayer; Ponticorvo, Adrien; Rowland, Rebecca A.; Balbado, Melisa L.; Lentsch, Griffin; Balu, Mihaela; Alexander, Micheal; Shiri, Li; Lakey, Jonathan R. T.; Durkin, Anthony J.; Kohen, Roni; Tromberg, Bruce J.

    2017-02-01

    Type 1diabetes (T1D) is an autoimmune disorder that occurs due to the rapid destruction of insulin-producing beta cells, leading to insulin deficiency and the inability to regulate blood glucose levels and leads to destructive secondary complications. Advanced glycation end (AGEs) products, the result of the cross-linking of reducing sugars and proteins within the tissues, are one of the key causes of major complications associated with diabetes such as renal failure, blindness, nerve damage and vascular changes. Non-invasive techniques to detect AGEs are important for preventing the harmful effects of AGEs during diabetes mellitus. In this study, we utilized multiphoton microscopy to image biopsies taken from control rats and compared them to biopsies taken from streptozotocin (STZ) induced adult male diabetic rats. This was done at two and four weeks after the induction of hyperglycemia (>400 mg/dL) specifically to evaluate the effects of glycation on collagen. We chose to use an in-situ multiphoton microscopy method that combines multiphoton auto-florescence (AF) and second harmonic generation (SHG) to detect the microscopic influence of glycation. Initial results show high auto-florescence levels were present on the collagen, as a result of the accumulation of AGEs only two weeks after the STZ injection and considerably higher levels were present four weeks after the STZ injection. Future projects could involve evaluating advanced glycation end products in a clinical trial of diabetic patients.

  3. Characterisation of Maillard reaction products derived from LEKFD--a pentapeptide found in β-lactoglobulin sequence, glycated with glucose--by tandem mass spectrometry, molecular orbital calculations and gel filtration chromatography coupled with continuous photodiode array.

    Science.gov (United States)

    Yamaguchi, Keiko; Homma, Takeshi; Nomi, Yuri; Otsuka, Yuzuru

    2014-02-15

    Maillard reaction peptides (MRPs) contribute to taste, aroma, colour, texture and biological activity. However, peptide degradation or the cross-linking of MRPs in the Maillard reaction has not been investigated clearly. A peptide of LEKFD, a part of β-lactoglobulin, was heated at 110 °C for 24h with glucose and the reaction products were analysed by HPLC with ODS, ESI-MS, ESI-MS/MS and HPLC with gel-filtration column and DAD detector. In the HPLC fractions, an imminium ion of LEK*FD, a pyrylium ion or a hydroxymethyl furylium ion of LEK*FD, and KFD and EK were detected by ESI-MS. Therefore, those products may be produced by the Maillard reaction. The molecular orbital of glycated LEKFD at the lysine epsilon-amino residue with Schiff base form was calculated by MOPAC. HPLC with gel-filtration column showed cross-linking and degradation of peptides. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Iodinated derivatives of vasoactive intestinal peptide (VIP), PHI and PHM: purification, chemical characterization and biological activity

    International Nuclear Information System (INIS)

    McMaster, D.; Suzuki, Y.; Rorstad, O.; Lederis, K.

    1987-01-01

    The iodination of vasoactive intestinal peptide (VIP) was studied, using a variety of enzymatic and chemical iodination methods. Reversed phase high performance liquid chromatography (HPLC) was used to purify the reaction products. The lactoperoxidase-glucose oxidase method gave excellent results in terms of reproducibility, iodine incorporation, and yield of the non-oxidized products [Tyr(I)10]VIP and [Tyr(I)22]VIP, and was used to prepare both 125 I and 127 I labelled derivatives. In both cases, direct application to HPLC and a single column system were used. Although the oxidized peptides [Tyr(I)10,Met(O)17]VIP and [Tyr(I)22,Met(O)17]VIP could be generated to varying degrees directly by iodination of VIP, these were most conveniently prepared by iodination of [Met(O)17]VIP. Iodinated derivatives of the homologous peptides PHI and PHM were likewise prepared by rapid, one-step HPLC procedures. The site and degree of iodination were determined by HPLC peptide mapping of tryptic digests and amino acid analyses, and in the case of [Tyr(I)10]VIP also by sequencing. The vasorelaxant activities of the iodinated peptides in bovine cerebral artery preparations did not differ significantly from those of the corresponding noniodinated peptides, with the exception of [Tyr(I)10,Met(O)17]VIP and [Tyr(I)22,Met(O)17]VIP which, unlike [Met(O)17]VIP itself, had slightly lower potency than VIP

  5. Isotopic and enzymatic IgE assays in non-allergic subjects

    International Nuclear Information System (INIS)

    Stein, R.; Evans, S.; Milner, R.; Rand, C.; Dolovich, J.

    1983-01-01

    In order to establish normal values in an adult population for serum IgE concentration, sera were obtaned from 446 ambulatory Canadian caucasian subjects with negative allergy histories. A standard isotopic procedure, the Phadebas paper radioimmunosorbent test (PRIST), was compared with the new enzymatic counterpart, the Phadezyme PRIST. By the isotopic method, serum IgE concentrations in women and men were comparable from one age group to another with no age-related trend in the seven age groups examined (15-20, 21-30, 31-40, 41-50, 51-60, 61-70, above 70). The median and 95th percentile units (U)/ml respectively were 17.5 and 145 for 224 women and 25.5 and 275 for 222 men. Mean values +- 1 SD for women were 43 +- 102 and for men, 58 +- 137. Levels were significantly higher in men as a group. Sera with IgE concentrations above 100 U and a sampling of additional sera were tested for specific IgE antibodies to 13 common allergens by the radioallergosorbent test (RAST). After exclusion of RAST-positive sera, the mean U/ml values +- ISD were 22 +- 29 for 204 women and 37 +- 54 for 196 men. Geometric mean U/ml values for these sera were 14.6 for women and 22.3 for men and the median and 95th percentile U/ml respectively for the women were 15 and 66, for the men, 24 and 135. These 95th percentile values are considered the upper limits of normal in this population. The RAST identifiction of antibodies to allergens to which sensitization was demonstrated provided a potential explanation for serum IgE concentrations above 100 U/ml in less than 30% of the sera in this population with negative allergy histories. The isotopic method and the counterpart enzymatic method (Phadezyme PRIST) were highly comparable; the correlation coefficient (r) for all the sera was +- 0.93(P<0.001). IgE levels were significantly higher in male smokers than non-smokers by both methods but these differences were not significant in women. (author)

  6. Enzymatic hydrolysis of Grass Carp fish skin hydrolysates able to promote the proliferation of Streptococcus thermophilus.

    Science.gov (United States)

    Wang, Xiao-Nan; Qin, Mei; Feng, Yu-Ying; Chen, Jian-Kang; Song, Yi-Shan

    2017-09-01

    The promotion effect on proliferation of Streptococcus thermophilus by enzymatic hydrolysates of aquatic products was firstly studied. The effect of influencing factors of the hydrolysis on the growth of S. thermophilus was investigated. Grass Carp fish skin was hydrolysed to peptides by enzymatic hydrolysis using protease ProteAX, and for the S. thermophilus growth, the optimal enzymatic hydrolysis conditions were temperature of 60 °C, initial pH of 9.0, enzyme concentration of 10 g kg -1 , hydrolysis time of 80 min, and ratio of material to liquid of 1:2. The Grass Carp fish skin hydrolysate (GCFSH) prepared under the optimum conditions was fractionated to five fragments (GCFSH 1, GCFSH 2, GCFSH 3, GCFSH 4, GCFSH 5) according to molecular weight sizes, in which the fragments GCFSH 4 and GCFSH 5, with molecular weights of less than 1000 Da, significantly promoted the growth of S. thermophilus. The hydrolysis process of Grass Carp fish skin can be simplified, and the peptides with molecular weights below 1000 Da in the hydrolysates are the best nitrogen source for proliferation of S. thermophilus. This work can provide a fundamental theoretical basis for the production of multi-component functional foods, especially in milk drinks or yogurt. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  7. A 4'-phosphopantetheinyl transferase mediates non-ribosomal peptide synthetase activation in Aspergillus fumigatus.

    Science.gov (United States)

    Neville, Claire; Murphy, Alan; Kavanagh, Kevin; Doyle, Sean

    2005-04-01

    Aspergillus fumigatus is a significant human pathogen. Non-ribosomal peptide (NRP) synthesis is thought to be responsible for a significant proportion of toxin and siderophore production in the organism. Furthermore, it has been shown that 4'-phosphopantetheinylation is required for the activation of key enzymes involved in non-ribosomal peptide synthesis in other species. Here we report the cloning, recombinant expression and functional characterisation of a 4'-phosphopantetheinyl transferase from A. fumigatus and the identification of an atypical NRP synthetase (Afpes1), spanning 14.3 kb. Phylogenetic analysis has shown that the NRP synthetase exhibits greatest identity to NRP synthetases from Metarhizium anisolpiae (PesA) and Alternaria brassicae (AbrePsy1). Northern hybridisation and RT-PCR analysis have confirmed that both genes are expressed in A. fumigatus. A 120 kDa fragment of the A. fumigatus NRP synthetase, containing a putative thiolation domain, was cloned and expressed in the baculovirus expression system. Detection of a 4'-phosphopantetheinylated peptide (SFSAMK) from this protein, by MALDI-TOF mass spectrometric analysis after coincubation of the 4'-phosphopantetheinyl transferase with the recombinant NRP synthetase fragment and acetyl CoA, confirms that it is competent to play a role in NRP synthetase activation in A. fumigatus. The 4'-phosphopantetheinyl transferase also activates, by 4'-phosphopantetheinylation, recombinant alpha-aminoadipate reductase (Lys2p) from Candida albicans, a key enzyme involved in lysine biosynthesis.

  8. Investigation of bi-enzymatic reactor based on hybrid monolith with nanoparticles embedded and its proteolytic characteristics.

    Science.gov (United States)

    Shangguan, Lulu; Zhang, Lingyi; Xiong, Zhichao; Ren, Jun; Zhang, Runsheng; Gao, Fangyuan; Zhang, Weibing

    2015-04-03

    The bottom-up strategy of proteomic profiling study based on mass spectrometer (MS) has drawn high attention. However, conventional solution-based digestion could not satisfy the demands of highly efficient and complete high throughput proteolysis of complex samples. We proposed a novel bi-enzymatic reactor by immobilizing two different enzymes (trypsin/chymotrypsin) onto a mixed support of hybrid organic-inorganic monolith with SBA-15 nanoparticles embedded. Typsin and chymotrypsin were crossly immobilized onto the mixed support by covalent bonding onto the monolith with glutaraldehyde as bridge reagent and chelation via copper ion onto the nanoparticles, respectively. Compared with single enzymatic reactors, the bi-enzymatic reactor improved the overall functional analysis of membrane proteins of rat liver by doubling the number of identified peptides (from 1184/1010 with trypsin/chymotrypsin enzymatic reactors to 2891 with bi-enzymatic reactor), which led to more proteins identified with deep coverage (from 452/336 to 620); the efficiency of the bi-enzymatic reactor is also better than that of solution-based tandem digestion, greatly shorting the digestion time from 24h to 50s. Moreover, more transmembrane proteins were identified by bi-enzymatic reactor (106) compared with solution-based tandem digestion (95) with the same two enzymes and enzymatic reactors with single enzyme immobilized (75 with trypsin and 66 with chymotrypsin). The proteolytic characteristics of the bi-enzymatic reactors were evaluated by applying them to digestion of rat liver proteins. The reactors showed good digestion capability for proteins with different hydrophobicity and molecular weight. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Inhibitory effect of Piper betle Linn. leaf extract on protein glycation--quantification and characterization of the antiglycation components.

    Science.gov (United States)

    Bhattacherjee, Abhishek; Chakraborti, Abhay Sankar

    2013-12-01

    Piper betle Linn. is a Pan-Asiatic plant having several beneficial properties. Protein glycation and advanced glycation end products (AGEs) formation are associated with different pathophysiological conditions, including diabetes mellitus. Our study aims to find the effect of methanolic extract of P. betle leaves on in vitro protein glycation in bovine serum albumin (BSA)-glucose model. The extract inhibits glucose-induced glycation, thiol group modification and carbonyl formation in BSA in dose-dependent manner. It inhibits different stages of protein glycation, as demonstrated by using glycation models: hemoglobin-delta-gluconolactone (for early stage, Amadori product formation), BSA-methylglyoxal (for middle stage, formation of oxidative cleavage products) and BSA-glucose (for last stage, formation of AGEs) systems. Several phenolic compounds are isolated from the extract. Considering their relative amounts present in the extract, rutin appears to be the most active antiglycating agent. The extract of P. betle leaf may thus have beneficial effect in preventing protein glycation and associated complications in pathological conditions.

  10. Vitreous advanced glycation endproducts and alpha-dicarbonyls in retinal detachment patients with type 2 diabetes mellitus and non-diabetic controls

    NARCIS (Netherlands)

    Fokkens, Bernardina T.; Mulder, Douwe J.; Schalkwijk, Casper G.; Scheijen, Jean L.; Smit, Andries J.; Los, Leonoor I.

    2017-01-01

    Purpose Advanced glycation endproducts (AGEs) and their precursors alpha-dicarbonyls are implicated in the progression of diabetic retinopathy. The purpose of this study was to assess AGEs and a-dicarbonyls in the vitreous of patients with type 2 diabetes mellitus (T2DM) with early stages or absence

  11. Genome-wide association study identifies common loci influencing circulating glycated hemoglobin (HbA1c) levels in non-diabetic subjects

    DEFF Research Database (Denmark)

    An, Ping; Miljkovic, Iva; Thyagarajan, Bharat

    2014-01-01

    Glycated hemoglobin (HbA1c) is a stable index of chronic glycemic status and hyperglycemia associated with progressive development of insulin resistance and frank diabetes. It is also associated with premature aging and increased mortality. To uncover novel loci for HbA1c that are associated with...

  12. PChopper: high throughput peptide prediction for MRM/SRM transition design

    Directory of Open Access Journals (Sweden)

    Huang Jeffrey T-J

    2011-08-01

    Full Text Available Abstract Background The use of selective reaction monitoring (SRM based LC-MS/MS analysis for the quantification of phosphorylation stoichiometry has been rapidly increasing. At the same time, the number of sites that can be monitored in a single LC-MS/MS experiment is also increasing. The manual processes associated with running these experiments have highlighted the need for computational assistance to quickly design MRM/SRM candidates. Results PChopper has been developed to predict peptides that can be produced via enzymatic protein digest; this includes single enzyme digests, and combinations of enzymes. It also allows digests to be simulated in 'batch' mode and can combine information from these simulated digests to suggest the most appropriate enzyme(s to use. PChopper also allows users to define the characteristic of their target peptides, and can automatically identify phosphorylation sites that may be of interest. Two application end points are available for interacting with the system; the first is a web based graphical tool, and the second is an API endpoint based on HTTP REST. Conclusions Service oriented architecture was used to rapidly develop a system that can consume and expose several services. A graphical tool was built to provide an easy to follow workflow that allows scientists to quickly and easily identify the enzymes required to produce multiple peptides in parallel via enzymatic digests in a high throughput manner.

  13. Meat and fermented meat products as a source of bioactive peptides.

    Science.gov (United States)

    Stadnik, Joanna; Kęska, Paulina

    2015-01-01

    Bioactive peptides are short amino acid sequences, that upon release from the parent protein may play different physiological roles, including antioxidant, antihypertensive, antimicrobial, and other bioactivities. They have been identified from a range of foods, including those of animal origin, e.g., milk and muscle sources (with pork, beef, or chicken and various species of fish and marine organism). Bioactive peptides are encrypted within the sequence of the parent protein molecule and latent until released and activated by enzymatic proteolysis, e.g. during gastrointestinal digestion or food processing. Bioactive peptides derived from food sources have the potential for incorporation into functional foods and nutraceuticals. The aim of this paper is to present an overview of the muscle-derived bioactive peptides, especially those of fermented meats and the potential benefits of these bioactive compounds to human health.

  14. Oxidative degradation and non-enzymatic browning due to the interaction between oxidised lipids and primary amine groups in different marine PL emulsions

    DEFF Research Database (Denmark)

    Lu, Henna Fung Sieng; Nielsen, Nina Skall; Baron, Caroline P.

    2012-01-01

    investigated through the measurement of secondary volatile compounds by solid-phase microextraction (SPME) and dynamic headspace (DHS) connected to gas chromatography (GC–MS). Non-enzymatic browning reactions were investigated through the measurement of Strecker derived volatiles, colour changes and pyrrole...

  15. Fisetin lowers methylglyoxal dependent protein glycation and limits the complications of diabetes.

    Directory of Open Access Journals (Sweden)

    Pamela Maher

    Full Text Available The elevated glycation of macromolecules by the reactive dicarbonyl and α-oxoaldehyde methylglyoxal (MG has been associated with diabetes and its complications. We have identified a rare flavone, fisetin, which increases the level and activity of glyoxalase 1, the enzyme required for the removal of MG, as well as the synthesis of its essential co-factor, glutathione. It is shown that fisetin reduces two major complications of diabetes in Akita mice, a model of type 1 diabetes. Although fisetin had no effect on the elevation of blood sugar, it reduced kidney hypertrophy and albuminuria and maintained normal levels of locomotion in the open field test. This correlated with a reduction in proteins glycated by MG in the blood, kidney and brain of fisetin-treated animals along with an increase in glyoxalase 1 enzyme activity and an elevation in the expression of the rate-limiting enzyme for the synthesis of glutathione, a co-factor for glyoxalase 1. The expression of the receptor for advanced glycation end products (RAGE, serum amyloid A and serum C-reactive protein, markers of protein oxidation, glycation and inflammation, were also increased in diabetic Akita mice and reduced by fisetin. It is concluded that fisetin lowers the elevation of MG-protein glycation that is associated with diabetes and ameliorates multiple complications of the disease. Therefore, fisetin or a synthetic derivative may have potential therapeutic use for the treatment of diabetic complications.

  16. Fisetin Lowers Methylglyoxal Dependent Protein Glycation and Limits the Complications of Diabetes

    Science.gov (United States)

    Maher, Pamela; Dargusch, Richard; Ehren, Jennifer L.; Okada, Shinichi; Sharma, Kumar; Schubert, David

    2011-01-01

    The elevated glycation of macromolecules by the reactive dicarbonyl and α-oxoaldehyde methylglyoxal (MG) has been associated with diabetes and its complications. We have identified a rare flavone, fisetin, which increases the level and activity of glyoxalase 1, the enzyme required for the removal of MG, as well as the synthesis of its essential co-factor, glutathione. It is shown that fisetin reduces two major complications of diabetes in Akita mice, a model of type 1 diabetes. Although fisetin had no effect on the elevation of blood sugar, it reduced kidney hypertrophy and albuminuria and maintained normal levels of locomotion in the open field test. This correlated with a reduction in proteins glycated by MG in the blood, kidney and brain of fisetin-treated animals along with an increase in glyoxalase 1 enzyme activity and an elevation in the expression of the rate-limiting enzyme for the synthesis of glutathione, a co-factor for glyoxalase 1. The expression of the receptor for advanced glycation end products (RAGE), serum amyloid A and serum C-reactive protein, markers of protein oxidation, glycation and inflammation, were also increased in diabetic Akita mice and reduced by fisetin. It is concluded that fisetin lowers the elevation of MG-protein glycation that is associated with diabetes and ameliorates multiple complications of the disease. Therefore, fisetin or a synthetic derivative may have potential therapeutic use for the treatment of diabetic complications. PMID:21738623

  17. Antioxidant activity of a novel synthetic hexa-peptide derived from an enzymatic hydrolysate of duck skin by-products.

    Science.gov (United States)

    Lee, Seung-Jae; Cheong, Sun Hee; Kim, Yon-Suk; Hwang, Jin-Woo; Kwon, Hyuck-Ju; Kang, Seo-Hee; Moon, Sang-Ho; Jeon, Byong-Tae; Park, Pyo-Jam

    2013-12-01

    A peptide was synthesized on the basis of our previous study from solid phase peptide synthesis using ASP48S (Peptron Inc.) and identified by the reverse phase high-performance liquid chromatography (HPLC) using a Vydac Everest C18 column. The molecular mass of the peptide found to be 693.90 Da, and the amino acid sequences of the peptide was Trp-Tyr-Pro-Ala-Ala-Pro. The purpose of this study was to evaluate antioxidant effects of the peptide by electron spin resonance (ESR) spectrometer, and on t-BHP-induced liver cells damage in Chang cells. The antioxidative activity of the peptide was evaluated by measuring 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, alkyl and superoxide radical scavenging activity using an ESR spectrometer. The half maximal inhibitory concentration (IC50) value of the peptide for hydroxyl, DPPH, alkyl, and superoxide radical scavenging activity were 45.2, 18.5, 31.5, and 33.4 μM, respectively. In addition, the peptide inhibited productions of cell death against t-BHP-induced liver cell damage in Chang cells. It was presumed to be peptide involved in regulating the apoptosis-related gene expression in the cell environment. The present results indicate that the peptide substantially contributes to antioxidative properties in liver cells. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Membrane-Active Epithelial Keratin 6A Fragments (KAMPs) Are Unique Human Antimicrobial Peptides with a Non-αβ Structure

    Science.gov (United States)

    Lee, Judy T. Y.; Wang, Guangshun; Tam, Yu Tong; Tam, Connie

    2016-01-01

    Antibiotic resistance is a pressing global health problem that threatens millions of lives each year. Natural antimicrobial peptides and their synthetic derivatives, including peptoids and peptidomimetics, are promising candidates as novel antibiotics. Recently, the C-terminal glycine-rich fragments of human epithelial keratin 6A were found to have bactericidal and cytoprotective activities. Here, we used an improved 2-dimensional NMR method coupled with a new protocol for structural refinement by low temperature simulated annealing to characterize the solution structure of these kerain-derived antimicrobial peptides (KAMPs). Two specific KAMPs in complex with membrane mimicking sodium dodecyl sulfate (SDS) micelles displayed amphipathic conformations with only local bends and turns, and a central 10-residue glycine-rich hydrophobic strip that is central to bactericidal activity. To our knowledge, this is the first report of non-αβ structure for human antimicrobial peptides. Direct observation of Staphylococcus aureus and Pseudomonas aeruginosa by scanning and transmission electron microscopy showed that KAMPs deformed bacterial cell envelopes and induced pore formation. Notably, in competitive binding experiments, KAMPs demonstrated binding affinities to LPS and LTA that did not correlate with their bactericidal activities, suggesting peptide-LPS and peptide-LTA interactions are less important in their mechanisms of action. Moreover, immunoprecipitation of KAMPs-bacterial factor complexes indicated that membrane surface lipoprotein SlyB and intracellular machineries NQR sodium pump and ribosomes are potential molecular targets for the peptides. Results of this study improve our understanding of the bactericidal function of epithelial cytokeratin fragments, and highlight an unexplored class of human antimicrobial peptides, which may serve as non-αβ peptide scaffolds for the design of novel peptide-based antibiotics. PMID:27891122

  19. Membrane-Active Epithelial Keratin 6A Fragments (KAMPs Are Unique Human Antimicrobial Peptides with a Non-αβ Structure

    Directory of Open Access Journals (Sweden)

    Judy Tsz Ying Lee

    2016-11-01

    Full Text Available Antibiotic resistance is a pressing global health problem that threatens millions of lives each year. Natural antimicrobial peptides and their synthetic derivatives, including peptoids and peptidomimetics, are promising candidates as novel antibiotics. Recently, the C-terminal glycine-rich fragments of human epithelial keratin 6A were found to have bactericidal and cytoprotective activities. Here, we used an improved 2-dimensional NMR method coupled with a new protocol for structural refinement by low temperature simulated annealing to characterize the solution structure of these kerain-derived antimicrobial peptides (KAMPs. Two specific KAMPs in complex with membrane mimicking sodium dodecyl sulfate (SDS micelles displayed amphipathic conformations with only local bends and turns, and a central 10-residue glycine-rich hydrophobic strip that is central to bactericidal activity. To our knowledge, this is the first report of non-αβ structure for human antimicrobial peptides. Direct observation of Staphylococcus aureus and Pseudomonas aeruginosa by scanning and transmission electron microscopy showed that KAMPs deformed bacterial cell envelopes and induced pore formation. Notably, in competitive binding experiments, KAMPs demonstrated binding affinities to LPS and LTA that did not correlate with their bactericidal activities, suggesting peptide-LPS and peptide-LTA interactions are less important in their mechanisms of action. Moreover, immunoprecipitation of KAMPs-bacterial factor complexes indicated that membrane surface lipoprotein SlyB and intracellular machineries NQR sodium pump and ribosomes are potential molecular targets for the peptides. Results of this study improve our understanding of the bactericidal function of epithelial cytokeratin fragments, and highlight an unexplored class of human antimicrobial peptides, which may serve as non-αβ peptide scaffolds for the design of novel peptide-based antibiotics.

  20. Cross-Linking in Collagen by Nonenzymatic Glycation Increases the Matrix Stiffness in Rabbit Achilles Tendon

    OpenAIRE

    Reddy, G. Kesava

    2004-01-01

    Nonenzymatic glycation of connective tissue matrix proteins is a major contributor to the pathology of diabetes and aging. Previously the author and colleagues have shown that nonenzymatic glycation significantly enhances the matrix stability in the Achilles tendon (Reddy et al., 2002, Arch. Biochem. Biophys., 399, 174–180). The present study was designed to gain further insight into glycation-induced collagen cross-linking and its relationship to matrix stiffness in the rabbit Achilles tendo...

  1. Fluorescence lifetime measurements of native and glycated human serum albumin and bovine serum albumin

    Science.gov (United States)

    Joshi, Narahari V.; Joshi, Virgina O. d.; Contreras, Silvia; Gil, Herminia; Medina, Honorio; Siemiarczuk, Aleksander

    1999-05-01

    Nonenzymatic glycation, also known as Maillard reaction, plays an important role in the secondary complications of the diabetic pathology and aging, therefore, human serum albumin (HSA) and bovine serum albumin (BSA) were glycated by a conventional method in our laboratory using glucose as the glycating agent. Fluorescence lifetime measurements were carried out with a laser strobe fluorometer equipped with a nitrogen/dye laser and a frequency doubler as a pulsed excitation source. The samples were excited at 295 nm and the emission spectra were recorded at 345 nm. The obtained decay curves were tried for double and triple exponential functions. It has been found that the shorter lifetime increases for glycated proteins as compared with that of the native ones. For example, in the case of glycated BSA the lifetime increased from 1.36 ns to 2.30 ns. Similarly, for HSA, the lifetime increases from 1.58 ns to 2.26 ns. Meanwhile, the longer lifetime changed very slightly for both proteins (from 6.52 ns to 6.72 ns). The increase in the lifetime can be associated with the environmental effect; originated from the attachment of glucose to some lysine residues. A good example is Trp 214 which is in the cage of Lys 225, Lys 212, Lys 233, Lys 205, Lys 500, Lys 199 and Lys 195. If fluorescence lifetime technique is calibrated and properly used it could be employed for assessing glycation of proteins.

  2. Immune Response of Multiparous Hyper-Immunized Sows against Peptides from Non-Structural and Structural Proteins of PRRSV

    Directory of Open Access Journals (Sweden)

    Edgar Rascón-Castelo

    2015-11-01

    Full Text Available The purpose of this study was to evaluate the humoral and cellular responses of commercial multiparous and hyper-immunized sows against peptides from non-structural (nsp and structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV. We selected sows with different numbers of parities from a commercial farm. Management practices on this farm include the use of the MLV commercial vaccine four times per year, plus two vaccinations during the acclimation period. The humoral response was evaluated via the antibody recognition of peptides from nsp and structural proteins, and the cellular response was assessed by measuring the frequency of peptide and PRRSV-specific IFN-gamma-secreting cells (IFNγ-SC. Our results show that sows with six parities have more antibodies against peptides from structural proteins than against peptides from nsp. The analysis of the cellular response revealed that the number of immunizations did not affect the frequency of IFNγ-SC and that the response was stronger against peptides from structural proteins (M protein than against nsp (nsp2. In summary, these results demonstrate that multiparous, hyper-immunized sows have a stronger immune humoral response to PRRSV structural peptides than nsp, but no differences in IFNγ-SC against the same peptides were observed.

  3. The Preventive Effect of L-Lysine on Lysozyme Glycation in Type 2 Diabetes

    Directory of Open Access Journals (Sweden)

    Hossein Mirmiranpour

    2016-01-01

    Full Text Available Lysozyme is a bactericidal enzyme whose structure and functions change in diabetes. Chemical chaperones are small molecules including polyamines (e.g. spermine, amino acids (e.g. L-lysine and polyols (e.g. glycerol. They can improve protein conformation in several stressful conditions such as glycation. In this study, the authors aimed to observe the effect of L-lysine as a chemical chaperone on structure and function of glycated lysozyme. In this study, in vitro and in vivo effects of L-lysine on lysozyme glycation were investigated. Lysozyme was incubated with glucose and/or L-lysine, followed by an investigation of its structure by electrophoresis, fluorescence spectroscopy, and circular dichroism spectroscopy and also assessment of its bactericidal activity against M. lysodeikticus. In the clinical trial, patients with type 2 diabetes mellitus (T2DM were randomly divided into two groups of 25 (test and control. All patients received metformin and glibenclamide for a three months period. The test group was supplemented with 3 g/day of L-lysine. The quantity and activity of lysozyme and other parameters were then measured. Among the test group, L-lysine was found to reduce the advanced glycation end products (AGEs in the sera of patients with T2DM and in vitro condition. This chemical chaperone reversed the alteration in lysozyme structure and function due to glycation and resulted in increased lysozyme activity. Structure and function of glycated lysozyme are significantly improved by l-lysine; therefore it can be considered an effective therapeutic supplementation in T2DM, decreasing the risk of infection in these patients.

  4. Toward the assessment of food toxicity for celiac patients: characterization of monoclonal antibodies to a main immunogenic gluten peptide.

    Directory of Open Access Journals (Sweden)

    Belén Morón

    Full Text Available BACKGROUND AND AIMS: Celiac disease is a permanent intolerance to gluten prolamins from wheat, barley, rye and, in some patients, oats. Partially digested gluten peptides produced in the digestive tract cause inflammation of the small intestine. High throughput, immune-based assays using monoclonal antibodies specific for these immunotoxic peptides would facilitate their detection in food and enable monitoring of their enzymatic detoxification. Two monoclonal antibodies, G12 and A1, were developed against a highly immunotoxic 33-mer peptide. The potential of each antibody for quantifying food toxicity for celiac patients was studied. METHODS: Epitope preferences of G12 and A1 antibodies were determined by ELISA with gluten-derived peptide variants of recombinant, synthetic or enzymatic origin. RESULTS: The recognition sequences of G12 and A1 antibodies were hexameric and heptameric epitopes, respectively. Although G12 affinity for the 33-mer was superior to A1, the sensitivity for gluten detection was higher for A1. This observation correlated to the higher number of A1 epitopes found in prolamins than G12 epitopes. Activation of T cell from gluten digested by glutenases decreased equivalently to the detection of intact peptides by A1 antibody. Peptide recognition of A1 included gliadin peptides involved in the both the adaptive and innate immunological response in celiac disease. CONCLUSIONS: The sensitivity and epitope preferences of the A1 antibody resulted to be useful to detect gluten relevant peptides to infer the potential toxicity of food for celiac patients as well as to monitor peptide modifications by transglutaminase 2 or glutenases.

  5. Enteral Tube Feeding Nutritional Protein Hydrolysate Production Under Different Factors By Enzymatic Hydrolysis

    Directory of Open Access Journals (Sweden)

    Nguyen ThiQuynhHoa

    2015-01-01

    Full Text Available Abstract Hydrolysis of proteins involves the cleavage of peptide bonds to give peptides of varying sizes and amino acid composition. There are a number of types of hydrolysis enzymatic acid or alkali hydrolysis. Chemical hydrolysis is difficult to control and reduces the nutritional quality of products destroying L-form amino acids and producing toxic substances such as lysino-alanine. Enzymatic hydrolysis works without destructing amino acids and by avoiding the extreme temperatures and pH levels required for chemical hydrolysis the nutritional properties of the protein hydrolysates remain largely unaffected. In this research we investigate the fat removal and protein hydrolysis from pork meat to produce the enteral tube feeding nutritional protein hydrolysate for patient. Our results are as follows meat moisture 75.1 protein 22.6 lipid 1.71 ash 0.5 vitamin B1 1.384mg100g n hexantreatment at 80oCin 45 minutes and drying 30 minutes in 90oC.Viscosity of the hydrolysate is very low 2.240 0.092 cPand high degree of hydrolysis 31.390 0.138 . The final protein powder has balance nutritional components and acid amines low microorganisms which are safety for human consumption.

  6. Construction of a non-enzymatic sensor based on the poly(o-phenylenediamine)/Ag-NPs composites for detecting glucose in blood

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jinxiang; Wang, Meirong [College of Chemistry and Chemical Engineering, Jiangsu Key Laboratory of Environmental Engineering and Monitoring, Yangzhou University, 180 Si–Wang–Ting Road, Yangzhou 225002 (China); Guan, Jun [Clinical Medical College of Yangzhou University, Subei People' s Hospital of Jiangsu Province, Yangzhou 225002 (China); Wang, Chengyin, E-mail: wangcy@yzu.edu.cn [College of Chemistry and Chemical Engineering, Jiangsu Key Laboratory of Environmental Engineering and Monitoring, Yangzhou University, 180 Si–Wang–Ting Road, Yangzhou 225002 (China); Wang, Guoxiu [School of Mathematical and Physical Sciences, University of Technology Sydney, City Campus, Broadway, Sydney, NSW 2007 (Australia)

    2017-02-01

    A non-enzymatic glucose sensor, based on the silver nanoparticles (Ag-NPs)/poly (o-phenylenediamine) (PoPD) composites, is developed by the electrochemical polymerization of o-phenylenediamine and electrodeposition of silver nanoparticles on an indium tin oxide electrode. The Ag-NPs/PoPD composites are characterized by atomic force microscopy, scanning electronic microscopy and energy dispersive spectrometer. Under the optimized experimental conditions, the proposed glucose sensor demonstrates a wide linear range from 0.15 to 13 mmol L{sup −1} with a correlation coefficient of 0.998. The proposed glucose sensor can be used to detect glucose in blood sample with a satisfactory result. In addition, the proposed sensor presents the advantages, such as facile preparation, low cost, high sensitivity and fast response time. It also exhibits good anti-interference performance and stability. - Highlights: • A facile AgNPs/PoPD/ITO modified sensor was developed for the first time. • The non-enzymatic sensor can detect glucose in human blood directly with a wide detection range. • This sensor is of rapid response, low cost, high sensitivity, and long-time stability.

  7. Non-enzymatic hydrogen peroxide biosensor based on rose-shaped FeMoO{sub 4} nanostructures produced by convenient microwave-hydrothermal method

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Hongying, E-mail: liuhongying@hdu.edu.cn [College of Life Information Science & Instrument Engineering, Hangzhou Dianzi University, Zhejiang, Hangzhou 310018 (China); Gu, Chunchuan [Department of Clinical Laboratory, Hangzhou Cancer Hospital, Zhejiang, Hangzhou 310002 (China); Li, Dujuan; Zhang, Mingzhen [College of Life Information Science & Instrument Engineering, Hangzhou Dianzi University, Zhejiang, Hangzhou 310018 (China)

    2015-04-15

    Graphical abstract: A non-enzymatic H{sub 2}O{sub 2} sensor with high selectivity and sensitivity based on rose-shaped FeMoO{sub 4} synthesized by the convenient microwave-assisted hydrothermal method, was fabricated. - Highlights: • Rose-shaped FeMoO{sub 4} is synthesized within 10 min via microwave-assisted hydrothermal approach. • Non-enzymatic hydrogen peroxide biosensor based on FeMoO{sub 4} nanomaterials is fabricated. • The biosensor exhibits good performance. - Abstract: In this work, we demonstrated a simple, rapid and reliable microwave-assisted hydrothermal approach to synthesize the uniform rose-shaped FeMoO{sub 4} within 10 min. The morphologies of the synthesized materials were characterized by X-ray powder diffraction and scanning electron microscopy. Moreover, a non-enzymatic amperometric sensor for the detection of hydrogen peroxide (H{sub 2}O{sub 2}) was fabricated on the basis of the FeMoO{sub 4} as electrocatalysis. The resulting FeMoO{sub 4} exhibited high sensitivity and good stability for the detection of H{sub 2}O{sub 2}, which may be attributed to the rose-shaped structure of the material and the catalytic property of FeMoO{sub 4}. Amperometric response showed that the modified electrode had a good response for H{sub 2}O{sub 2} with a linear range from 1 μM to 1.6 mM, a detection limit of 0.5 μM (S/N = 3), high selectivity and short response time. Additionally, good recoveries of analytes in real milk samples confirm the reliability of the prepared sensor in practical applications.

  8. Enzymatic synthesis of tasty oligopeptide in organic solvent. Yuki yobaichu deno teimisei origo pepuchido no koso gosei

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, T.; Tomita, K.; Nakanishi, K. (Okayama Univ., Okayama (Japan). Faculty of Engineering)

    1991-10-01

    An introduction is made to examples of the latest researches on methods to manufacture oligopeptides by enzymatic synthesizing methods in regards to to various processes using reactions in organic solvents as a means to increase the yield, particularly on synthesis of one that have taste effects. To raise the yield of peptides that are produced by condensing amino acid and amino acid derivatives, it is necessary that an equilibrium control reaction is operated in such a way that the product concentration and product moisture concentration are reduced, and the concentration of substrates of non-dissociation type is raised. In the case ofamino acid reacting with amino acid ester or amide, a velocity control is carried out, in which the velocity to produce the peptides from the acyl intermediates is raised and the velocity of the ester hydrolysis is slowed down. Examples of synthesis of tasty oligopeptides include, for example, the precursor in manufacturing artificial sweetener, aspartyl-phenylalanine methyl, as well as synthesis in an aqueous solution using special enzymes or in an organic solvent, and synthesis of other acidic amino acid oligomers. 24 refs., 2 figs.

  9. Operation and Control of Enzymatic Biodiesel Production

    DEFF Research Database (Denmark)

    Price, Jason Anthony; Huusom, Jakob Kjøbsted; Nordblad, Mathias

    This work explores the control of biodiesel production via an enzymatic catalyst. The process involves the transesterification of oils/fats with an alcohol (usually methanol or ethanol), using enzymatic catalysts to generate mono-alkyl esters (the basis of biodiesel) and glycerol as by......-product. Current literature indicates that enzymatic processing of oils and fats to produce biodiesel is technically feasible and developments in immobilization technology indicate that enzyme catalysts can become cost effective compared to chemical processing. However, with very few exceptions, enzyme technology...... is not currently used in commercial-scale biodiesel production. This is mainly due to non-optimized process designs, which do not use the full potential of the catalysts in a cost-efficient way. Furthermore is it unclear what process variables need to be monitored and controlled to ensure optimal economics...

  10. Construction of tunable peptide nucleic acid junctions.

    Science.gov (United States)

    Duan, Tanghui; He, Liu; Tokura, Yu; Liu, Xin; Wu, Yuzhou; Shi, Zhengshuang

    2018-03-15

    We report here the construction of 3-way and 4-way peptide nucleic acid (PNA) junctions as basic structural units for PNA nanostructuring. The incorporation of amino acid residues into PNA chains makes PNA nanostructures with more structural complexity and architectural flexibility possible, as exemplified by building 3-way PNA junctions with tunable nanopores. Given that PNA nanostructures have good thermal and enzymatic stabilities, they are expected to have broad potential applications in biosensing, drug delivery and bioengineering.

  11. A review of advanced oral drug delivery technologies facilitating the protection and absorption of protein and peptide molecules.

    Science.gov (United States)

    Choonara, Bibi F; Choonara, Yahya E; Kumar, Pradeep; Bijukumar, Divya; du Toit, Lisa C; Pillay, Viness

    2014-11-15

    The oral delivery of proteins and peptides is a dynamic research field despite the numerous challenges limiting their effective delivery. Successful oral delivery of proteins and peptides requires the accomplishment of three key tasks: protection of the macromolecules from degradation in the gastrointestinal tract (GIT), permeation through the intestinal barrier and absorption of molecules into the systemic circulation. Currently, no clinically useful oral formulations have been developed but several attempts have been made to overcome the challenges of low oral bioavailability resulting from poor absorption, poor permeation and enzymatic degradation of the proteins and peptides in the GIT. Present strategies attempt to provide structural protection of the proteins and peptides and improved absorption through the use of enzyme inhibitors, absorption enhancers, novel polymeric delivery systems and chemical modification. However, each of these technologies has their limitations despite showing positive results. This review attempts to discuss the physical and chemical barriers of the GIT with particular emphasis on the current approaches employed to overcome these barriers, including the evaluation of other non-parenteral routes of protein and peptide delivery. In addition, this review assimilates oral formulation strategies under development and within the clinical trial stage in relation to their benefits and drawbacks with regard to facilitating optimal protection and absorption of proteins and peptides, as well as pertinent future challenges and opportunities governing oral drug delivery. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Residual DNA-bound proteins are a source of in vitro transcription inhibitor peptides

    International Nuclear Information System (INIS)

    Venanzi, F.M.

    1989-01-01

    Enzymatic breakdown of residual proteins occurs at mild alkaline pH (pH optimum 8.5) as monitored by using radioiodinated, purified genomic DNA from calf thymus. These DNA fibers also possess a differential ability to hydrolyze added exogenous small and linker histones. The results described argue strongly that a putative protease activity, co-purified with DNA, is the source of short chain peptides which inhibit transcription in vitro. Therefore, we propose that RNA repressor peptides must be of higher molecular weight than previously reported

  13. The Role Of Milk Peptide As Antimicrobial Agent In Supporting Health Status

    Directory of Open Access Journals (Sweden)

    Eni Kusumaningtyas

    2013-06-01

    Full Text Available Antimicrobial peptide is commonly present in all species as a component of their innate immune defense against infection. Antimicrobial peptides derived from milk such as isracidin, casocidin, casecidin and other fragments with variety of amino acid sequence are released upon enzymatic hydrolysis from milk protein К-casein, α-casein, β-casein, α-lactalbumin and β- lactoglobulin. These peptides were produced by the activity of digestive or microbial protease such as trypsin, pepsin, chymosin or alcalase. The mode of action of these peptides is by interaction of their positive with negative charge of target cell membrane leading to disruption of membrane associated with physiological event such as cell division or translocation of peptide across the membrane to interact with cytoplasmic target. Modification of charged or nonpolar aliphatic residues within peptides can enhance or reduce the activities of the peptides against a number of microbial strains and it seems to be strain dependent. Several peptides act not only as an antimicrobial but also as an angiotensin-converting enzyme inhibitor, antioxidant, immunomodulator, antiinflamation, food and feed preservative. Although the commercial production of these peptides is still limited due to lack of suitable large-scale technologies, fast development of some methods for peptide production will hopefully increase the possibility for mass production.

  14. Glycation of human cortical and cancellous bone captures differences in the formation of Maillard reaction products between glucose and ribose.

    Directory of Open Access Journals (Sweden)

    Grażyna E Sroga

    Full Text Available To better understand some aspects of bone matrix glycation, we used an in vitro glycation approach. Within two weeks, our glycation procedures led to the formation of advanced glycation end products (AGEs at the levels that corresponded to approx. 25-30 years of the natural in vivo glycation. Cortical and cancellous bones from human tibias were glycated in vitro using either glucose (glucosylation or ribose (ribosylation. Both glucosylation and ribosylation led to the formation of higher levels of AGEs and pentosidine (PEN in cancellous than cortical bone dissected from all tested donors (young, middle-age and elderly men and women. More efficient glycation of bone matrix proteins in cancellous bone most likely depended on the higher porosity of this tissue, which facilitated better accessibility of the sugars to the matrix proteins. Notably, glycation of cortical bone from older donors led to much higher AGEs levels as compared to young donors. Such efficient in vitro glycation of older cortical bone could result from aging-related increase in porosity caused by the loss of mineral content. In addition, more pronounced glycation in vivo would be driven by elevated oxidation processes. Interestingly, the levels of PEN formation differed pronouncedly between glucosylation and ribosylation. Ribosylation generated very high levels of PEN (approx. 6- vs. 2.5-fold higher PEN level than in glucosylated samples. Kinetic studies of AGEs and PEN formation in human cortical and cancellous bone matrix confirmed higher accumulation of fluorescent crosslinks for ribosylation. Our results suggest that in vitro glycation of bone using glucose leads to the formation of lower levels of AGEs including PEN, whereas ribosylation appears to support a pathway toward PEN formation. Our studies may help to understand differences in the progression of bone pathologies related to protein glycation by different sugars, and raise awareness for excessive sugar

  15. Peptide-based biosensors: From self-assembled interfaces to molecular probes in electrochemical assays.

    Science.gov (United States)

    Puiu, Mihaela; Bala, Camelia

    2018-04-01

    Redox-tagged peptides have emerged as functional materials with multiple applications in the area of sensing and biosensing applications due to their high stability, excellent redox properties and versatility of biomolecular interactions. They allow direct observation of molecular interactions in a wide range of affinity and enzymatic assays and act as electron mediators. Short helical peptides possess the ability to self-assemble in specific configurations with the possibility to develop in highly-ordered, stable 1D, 2D and 3D architectures in a hierarchical controlled manner. We provide here a brief overview of the electrochemical techniques available to study the electron transfer in peptide films with particular interest in developing biosensors with immobilized peptide motifs, for biological and clinical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. SERUM MAGNESIUM, LIPID PROFILE AND GLYCATED HAEMOGLOBIN IN DIABETIC RETINOPATHY

    Directory of Open Access Journals (Sweden)

    Sunanda Vusikala

    2016-07-01

    Full Text Available BACKGROUND Diabetic retinopathy is one of the important microvascular complications of diabetes mellitus of long duration. Alterations in trace metals like magnesium and lipid profile was observed in diabetic retinopathy with hyperglycaemic status. AIM The study was taken up to assess the role of magnesium, lipid profile and glycated haemoglobin in diabetic retinopathy. MATERIALS AND METHODS A total of 80 subjects between 40-65 years were included in the study. Group 1 includes 20 age and sex matched healthy controls. Group 2 includes 30 cases of Diabetes mellitus without retinopathy. Group 3 includes 30 cases of Diabetes mellitus with retinopathy. RESULTS Magnesium was found to be significantly low in the diabetic group with retinopathy. Serum cholesterol and triglycerides were significantly elevated in the diabetic group with retinopathy. Fasting and Postprandial plasma glucose and glycated haemoglobin (HbA1c levels confirmed the glycaemic status of each of the groups. CONCLUSIONS Hypomagnesemia, hypercholesterolaemia, hypertriglyceridemia was observed in diabetic retinopathy along with increased levels of glycated haemoglobin in our study.

  17. Current therapeutic interventions in the glycation pathway: evidence from clinical studies.

    Science.gov (United States)

    Engelen, L; Stehouwer, C D A; Schalkwijk, C G

    2013-08-01

    The increased formation of advanced glycation endproducts (AGEs) constitutes a potential mechanism of hyperglycaemia-induced micro- and macrovascular disease in diabetes. In vitro and animal experiments have shown that various interventions can inhibit formation and/or actions of AGEs, in particular the specific AGE inhibitor aminoguanidine and the AGEs crosslink breaker alagebrium, and the B vitamins pyridoxamine and thiamine, and the latter's synthetic derivative, benfotiamine. The potential clinical value of these interventions, however, remains to be established. The present review provides, from the clinical point of view, an overview of current evidence on interventions in the glycation pathway relating to (i) the clinical benefits of specific AGE inhibitors and AGE breakers and (ii) the potential AGE-inhibiting effects of therapies developed for purposes unrelated to the glycation pathway. We found that safety and/or efficacy in clinical studies with the specific AGE inhibitor, aminoguanidine and the AGE breaker, alagebrium, appeared to be a concern. The clinical evidence on the potential AGE-inhibiting effects of B vitamins is still limited. Finally, current evidence for AGE inhibition by therapies developed for purposes unrelated to glycation is limited due to a large heterogeneity in study designs and/or measurement techniques, which have often been sub-optimal. We conclude that, clinical evidence on interventions to inhibit formation and/or action of AGEs is currently weak and unconvincing. © 2012 Blackwell Publishing Ltd.

  18. Determination of supplier-to-supplier and lot-to-lot variability in glycation of recombinant human serum albumin expressed in Oryza sativa.

    Directory of Open Access Journals (Sweden)

    Grant E Frahm

    Full Text Available The use of different expression systems to produce the same recombinant human protein can result in expression-dependent chemical modifications (CMs leading to variability of structure, stability and immunogenicity. Of particular interest are recombinant human proteins expressed in plant-based systems, which have shown particularly high CM variability. In studies presented here, recombinant human serum albumins (rHSA produced in Oryza sativa (Asian rice (OsrHSA from a number of suppliers have been extensively characterized and compared to plasma-derived HSA (pHSA and rHSA expressed in yeast (Pichia pastoris and Saccharomyces cerevisiae. The heterogeneity of each sample was evaluated using size exclusion chromatography (SEC, reversed-phase high-performance liquid chromatography (RP-HPLC and capillary electrophoresis (CE. Modifications of the samples were identified by liquid chromatography-mass spectrometry (LC-MS. The secondary and tertiary structure of the albumin samples were assessed with far U/V circular dichroism spectropolarimetry (far U/V CD and fluorescence spectroscopy, respectively. Far U/V CD and fluorescence analyses were also used to assess thermal stability and drug binding. High molecular weight aggregates in OsrHSA samples were detected with SEC and supplier-to-supplier variability and, more critically, lot-to-lot variability in one manufactures supplied products were identified. LC-MS analysis identified a greater number of hexose-glycated arginine and lysine residues on OsrHSA compared to pHSA or rHSA expressed in yeast. This analysis also showed supplier-to-supplier and lot-to-lot variability in the degree of glycation at specific lysine and arginine residues for OsrHSA. Both the number of glycated residues and the degree of glycation correlated positively with the quantity of non-monomeric species and the chromatographic profiles of the samples. Tertiary structural changes were observed for most OsrHSA samples which

  19. Phosphomolybdic acid functionalized graphene loading copper nanoparticles modified electrodes for non-enzymatic electrochemical sensing of glucose

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Jiaoyan; Cao, Xiyue [College of Chemistry and Chemical Engineering, Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, Collaborative Innovation Center for Marine Biomass Fiber Materials and Textiles, Laboratory of Fiber Materials and Modern Textile, The Growing Base for State Key Laboratory, Qingdao University, Qingdao 266071 (China); Xia, Jianfei, E-mail: xiajianfei@126.com [College of Chemistry and Chemical Engineering, Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, Collaborative Innovation Center for Marine Biomass Fiber Materials and Textiles, Laboratory of Fiber Materials and Modern Textile, The Growing Base for State Key Laboratory, Qingdao University, Qingdao 266071 (China); Gong, Shida [College of Chemistry and Chemical Engineering, Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, Collaborative Innovation Center for Marine Biomass Fiber Materials and Textiles, Laboratory of Fiber Materials and Modern Textile, The Growing Base for State Key Laboratory, Qingdao University, Qingdao 266071 (China); Wang, Zonghua, E-mail: wangzonghua@qdu.edu.cn [College of Chemistry and Chemical Engineering, Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, Collaborative Innovation Center for Marine Biomass Fiber Materials and Textiles, Laboratory of Fiber Materials and Modern Textile, The Growing Base for State Key Laboratory, Qingdao University, Qingdao 266071 (China); Lu, Lin [College of Chemistry and Chemical Engineering, Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, Collaborative Innovation Center for Marine Biomass Fiber Materials and Textiles, Laboratory of Fiber Materials and Modern Textile, The Growing Base for State Key Laboratory, Qingdao University, Qingdao 266071 (China); Zibo Normal College, Zibo, Shandong 255100 (China)

    2016-08-31

    A sensitive non-enzymatic glucose electrochemical biosensor (Cu/PMo{sub 12}-GR/GCE) was developed based on the combination of copper nanoparticles (CuNPs) and phosphomolybdic acid functionalized graphene (PMo{sub 12}-GR). PMo{sub 12}-GR films were modified on the surface of glassy carbon electrode (GCE) through electrostatic self-assembly with the aid of poly diallyl dimethyl ammonium chloride (PDDA). Then CuNPs were successfully decorated onto the PMo{sub 12}-GR modified GCE through electrodeposition. The morphology of Cu/PMo{sub 12}-GR/GCE was characterized by scanning electron microscope (SEM). Cyclic voltammetry (CV) and chronoamperometry were used to investigate the electrochemical performances of the biosensor. The results indicated that the modified electrode displayed a synergistic effect of PMo{sub 12}-GR sheets and CuNPs towards the electro-oxidation of glucose in the alkaline solution. At the optimal detection potential of 0.50 V, the response towards glucose presented a linear response ranging from 0.10 μM to 1.0 mM with a detection limit of 3.0 × 10{sup −2} μM (S/N = 3). In addition, Cu/PMo{sub 12}-GR/GCE possessed a high selectivity, good reproducibility, excellent stability and acceptable recovery, which indicating the potential application in clinical field. - Highlights: • Cu/PMo{sub 12}-GR/GCE as a non-enzymatic glucose electrochemical sensor. • PMo{sub 12} is efficient for the uniform growth of Cu-NPs and electron transport. • The sensor exhibits good sensitivity and specificity towards glucose.

  20. Protective Effect of Slforafin on the Non-Enzymatic Antioxidants and Chromosomal Aberrations When Injected with Tc "9"9"m Tin colloid in Mice

    International Nuclear Information System (INIS)

    Alwan, I.F.; Abd-Karim, H.M.; Ahmood, A.M.; Mohamad, H.A.

    2015-01-01

    Study aims to evaluated the preventive effect of (Slforafin ) compound extracted from Broccoli plant to effect on Technetium"9"9"m irradiated isotope user to labeled Tin colloid and used in prevent several Tin colloid changes , antioxidant Non-Enzymatic ( vitamin A,E,C ) and some of the basic elements in serum , such as (Zn, Mn, Se, Mg and Cu) and (Chromosomal Aberrations ) in the bone marrow genes of laboratory animals .Slforafin compound was analyzed by High-performance liquid chromatography technique HPLC. Treated the laboratory animals daily with, concentration (200 mg / kg) of Slforafin Broccoli extract material through mouth for one week ,then were injected with (500 μci / 0.1 ml) doses of preparation Tc"9"9"m Tin colloidal.The results indicated that there are significant differences (p< 0.05) at the deviation level of Non- enzymatic changes and chromosomal genes and some of the basic elements in serum of the Non –treated laboratory animals with Slforafin compound compared with the group of animals treated with (Slforafin) and injected with the same radiation dose compared with the control laboratory animal groups .

  1. The effect of glycation on bovine serum albumin conformation and ligand binding properties with regard to gliclazide

    Science.gov (United States)

    Żurawska-Płaksej, Ewa; Rorbach-Dolata, Anna; Wiglusz, Katarzyna; Piwowar, Agnieszka

    2018-01-01

    Albumin, the major serum protein, plays a variety of functions, including binding and transporting endogenous and exogenous ligands. Its molecular structure is sensitive to different environmental modifiers, among which glucose is one of the most significant. In vivo albumin glycation occurs under physiological conditions, but it is increased in diabetes. Since bovine serum albumin (BSA) may serve as a model protein in in vitro experiments, we aimed to investigate the impact of glucose-mediated BSA glycation on the binding capacity towards gliclazide, as well as the ability of this drug to prevent glycation of the BSA molecule. To reflect normo- and hyperglycemia, the conditions of the glycation process were established. Structural changes of albumin after interaction with gliclazide (0-14 μM) were determined using fluorescence quenching and circular dichroism spectroscopy. Moreover, thermodynamic parameters as well as energy transfer parameters were determined. Calculated Stern-Volmer quenching constants, as well as binding constants for the BSA-gliclazide complex, were lower for the glycated form of albumin than for the unmodified protein. The largest, over 2-fold, decrease in values of binding parameters was observed for the sample with 30 mM of glucose, reflecting the poorly controlled diabetic state, which indicates that the degree of glycation had a critical influence on binding with gliclazide. In contrast to significant changes in the tertiary structure of BSA upon binding with gliclazide, only slight changes in the secondary structure were observed, which was reflected by about a 3% decrease of the α-helix content of glycated BSA (regardless of glucose concentration) in comparison to unmodified BSA. The presence of gliclazide during glycation did not affect its progress. The results of this study indicate that glycation significantly changed the binding ability of BSA towards gliclazide and the scale of these changes depended on glucose concentration. It

  2. Liraglutide attenuates the migration of retinal pericytes induced by advanced glycation end products.

    Science.gov (United States)

    Lin, Wen-Jian; Ma, Xue-Fei; Hao, Ming; Zhou, Huan-Ran; Yu, Xin-Yang; Shao, Ning; Gao, Xin-Yuan; Kuang, Hong-Yu

    2018-07-01

    Retinal pericyte migration represents a novel mechanism of pericyte loss in diabetic retinopathy (DR), which plays a crucial role in the early impairment of the blood-retinal barrier (BRB). Glucagon-like peptide-1 (GLP-1) has been shown to protect the diabetic retina in the early stage of DR; however, the relationship between GLP-1 and retinal pericytes has not been discussed. In this study, advanced glycation end products (AGEs) significantly increased the migration of primary bovine retinal pericytes without influencing cell viability. AGEs also significantly enhanced phosphatidylinositol 3-kinase (PI3K)/Akt activation, and changed the expressions of migration-related proteins, including phosphorylated focal adhesion kinase (p-FAK), matrix metalloproteinase (MMP)-2 and vinculin. PI3K inhibition significantly attenuated the AGEs-induced migration of retinal pericytes and reversed the overexpression of MMP-2. Glucagon-like peptide-1 receptor (Glp1r) was expressed in retinal pericytes, and liraglutide, a GLP-1 analog, significantly attenuated the migration of pericytes by Glp1r and reversed the changes in p-Akt/Akt, p-FAK/FAK, vinculin and MMP-2 levels induced by AGEs, indicating that the protective effect of liraglutide was associated with the PI3K/Akt pathway. These results provided new insights into the mechanism underlying retinal pericyte migration. The early use of liraglutide exerts a potential bebefical effect on regulating pericyte migration, which might contribute to mechanisms that maintain the integrity of vascular barrier and delay the development of DR. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. Non-Enzymatic Detection of Bacterial Genomic DNA Using the Bio-Barcode Assay

    Science.gov (United States)

    Hill, Haley D.; Vega, Rafael A.; Mirkin, Chad A.

    2011-01-01

    The detection of bacterial genomic DNA through a non-enzymatic nanomaterials based amplification method, the bio-barcode assay, is reported. The assay utilizes oligonucleotide functionalized magnetic microparticles to capture the target of interest from the sample. A critical step in the new assay involves the use of blocking oligonucleotides during heat denaturation of the double stranded DNA. These blockers bind to specific regions of the target DNA upon cooling, and prevent the duplex DNA from re-hybridizing, which allows the particle probes to bind. Following target isolation using the magnetic particles, oligonucleotide functionalized gold nanoparticles act as target recognition agents. The oligonucleotides on the nanoparticle (barcodes) act as amplification surrogates. The barcodes are then detected using the Scanometric method. The limit of detection for this assay was determined to be 2.5 femtomolar, and this is the first demonstration of a barcode type assay for the detection of double stranded, genomic DNA. PMID:17927207

  4. A non-equilibrium thermodynamic model for tumor extracellular matrix with enzymatic degradation

    Science.gov (United States)

    Xue, Shi-Lei; Li, Bo; Feng, Xi-Qiao; Gao, Huajian

    2017-07-01

    The extracellular matrix (ECM) of a solid tumor not only affords scaffolding to support tumor architecture and integrity but also plays an essential role in tumor growth, invasion, metastasis, and therapeutics. In this paper, a non-equilibrium thermodynamic theory is established to study the chemo-mechanical behaviors of tumor ECM, which is modeled as a poroelastic polyelectrolyte consisting of a collagen network and proteoglycans. By using the principle of maximum energy dissipation rate, we deduce a set of governing equations for drug transport and mechanosensitive enzymatic degradation in ECM. The results reveal that osmosis is primarily responsible for the compression resistance of ECM. It is suggested that a well-designed ECM degradation can effectively modify the tumor microenvironment for improved efficiency of cancer therapy. The theoretical predictions show a good agreement with relevant experimental observations. This study aimed to deepen our understanding of tumor ECM may be conducive to novel anticancer strategies.

  5. Vegetable foods: a cheap source of proteins and peptides with antihypertensive, antioxidant, and other less occurrence bioactivities.

    Science.gov (United States)

    García, M C; Puchalska, P; Esteve, C; Marina, M L

    2013-03-15

    Despite less explored than foods from animal origin, plant derived foods also contain biologically active proteins and peptides. Bioactive peptides can be present as an independent entity in the food or, more frequently, can be in a latent state as part of the sequence of a protein. Release from that protein requires protein hydrolysis by enzymatic digestion, fermentation or autolysis. Different methodologies have been used to test proteins and peptides bioactivities. Fractionation, separation, and identification techniques have also been employed for the isolation and identification of bioactive proteins or peptides. In this work, proteins and peptides from plant derived foods exerting antihypertensive, antioxidant, hypocholesterolemic, antithrombotic, and immunostimulating capacities or ability to reduce food intake have been reviewed. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Inhibitory effect of gold nanoparticles on the D-ribose glycation of bovine serum albumin

    Directory of Open Access Journals (Sweden)

    Liu W

    2014-11-01

    Full Text Available Weixi Liu,1 Menashi A Cohenford,1–3 Leslie Frost,3 Champika Seneviratne,4 Joel A Dain1 1Department of Chemistry, University of Rhode Island, Kingston, RI, USA; 2Department of Integrated Science and Technology, 3Department of Chemistry, Marshall University, Huntington, WV, USA; 4Department of Chemistry, College of the North Atlantic, Labrador, NL, Canada Abstract: Formation of advanced glycation end products (AGEs by nonenzymatic glycation of proteins is a major contributory factor to the pathophysiology of diabetic conditions including senile dementia and atherosclerosis. This study describes the inhibitory effect of gold nanoparticles (GNPs on the D-ribose glycation of bovine serum albumin (BSA. A combination of analytical methods including ultraviolet–visible spectrometry, high performance liquid chromatography, circular dichroism, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF mass spectrometry were used to determine the extent of BSA glycation in the presence of citrate reduced spherical GNPs of various sizes and concentrations. GNPs of particle diameters ranging from 2 nm to 20 nm inhibited BSA’s AGE formation. The extent of inhibition correlated with the total surface area of the nanoparticles. GNPs of highest total surface area yielded the most inhibition whereas those with the lowest total surface area inhibited the formation of AGEs the least. Additionally, when GNPs’ total surface areas were set the same, their antiglycation activities were similar. This inhibitory effect of GNPs on BSA’s glycation by D-ribose suggests that colloidal particles may have a therapeutic application for the treatment of diabetes and conditions that promote hyperglycemia. Keywords: gold nanoparticles, glycation, AGEs, GNPs, BSA

  7. Fabrication and Optimization of a Nanoporous Platinum Electrode and a Non-enzymatic Glucose Micro-sensor on Silicon

    Directory of Open Access Journals (Sweden)

    Younghun Kim

    2008-10-01

    Full Text Available In this paper, optimal conditions for fabrication of nanoporous platinum (Pt were investigated in order to use it as a sensitive sensing electrode for silicon CMOS integrable non-enzymatic glucose micro-sensor applications. Applied charges, voltages, and temperatures were varied during the electroplating of Pt into the formed nonionic surfactant C16EO8 nano-scaled molds in order to fabricate nanoporous Pt electrodes with large surface roughness factor (RF, uniformity, and reproducibility. The fabricated nanoporous Pt electrodes were characterized using atomic force microscopy (AFM and electrochemical cyclic voltammograms. Optimal electroplating conditions were determined to be an applied charge of 35 mC/mm2, a voltage of -0.12 V, and a temperature of 25 °C, respectively. The optimized nanoporous Pt electrode had an electrochemical RF of 375 and excellent reproducibility. The optimized nanoporous Pt electrode was applied to fabricate non-enzymatic glucose micro-sensor with three electrode systems. The fabricated sensor had a size of 3 mm x 3 mm, air gap of 10 µm, working electrode (WE area of 4.4 mm2, and sensitivity of 37.5 µA•L/mmol•cm2. In addition, it showed large detection range from 0.05 to 30 mmolL-1 and stable recovery responsive to the step changes in glucose concentration.

  8. Investigation of lipid oxidation and non-enzymatic browning reactions in marine PL emulsions

    DEFF Research Database (Denmark)

    Lu, Henna Fung Sieng; Nielsen, Nina Skall; Baron, Caroline P.

    Marine phospholipids (PL) have received much attention recently due to their numerous advantages. One of these advantages is their better resistance towards oxidation as compared to fish oil. In addition to the antioxidative properties of α-tocopherol and phospholipids, the better oxidative...... stability of marine PL might be attributed to antioxidative properties of pyrroles formed between oxidised lipids with amine groups from phosphatidylethanolamine (PE) or residues amino acids that are present in marine PL. The main objective of this study was to investigate if the presence of amine group...... of amino acids (leucine, methionine and lysine) from 2 authentic standards (PC and PE) and 2 purified marine PL (LC and MPL) through sonication method. Emulsions were incubated at 60 ºC for 0, 2, 4 and 6 days. Non-enzymatic browning reactions were investigated through measurement of i) Strecker aldehydes...

  9. Enhanced non-enzymatic glucose sensing based on copper nanoparticles decorated nitrogen-doped graphene.

    Science.gov (United States)

    Jiang, Ding; Liu, Qian; Wang, Kun; Qian, Jing; Dong, Xiaoya; Yang, Zhenting; Du, Xiaojiao; Qiu, Baijing

    2014-04-15

    Copper nanoparticles (NPs) decorated nitrogen-doped graphene (Cu-N-G) was prepared by a facile thermal treatment, and further employed as a novel sensing material for fabricating the sensitive non-enzymatic glucose sensor. Compared with pure Cu NPs, the Cu-N-G showed enhanced electrocatalytic activity to glucose oxidation due to the integration of N-G, which exhibited the oxidation peak current of glucose ca. 23-fold higher than that of pure Cu NPs. The presented sensor showed excellent performances for glucose detection including wide linear range of 0.004-4.5 mM, low detection limit (1.3 μM, S/N=3), high sensitivity (48.13 μA mM(-1)), fast response time (doped graphene as enhanced materials in fabricating sensors for chemical and biochemical analysis. © 2013 Published by Elsevier B.V.

  10. The ethylene glycol template assisted hydrothermal synthesis of Co3O4 nanowires; structural characterization and their application as glucose non-enzymatic sensor

    International Nuclear Information System (INIS)

    Khun, K.; Ibupoto, Z.H.; Liu, X.; Beni, V.; Willander, M.

    2015-01-01

    Highlights: • Ethylene glycol assisted Co 3 O 4 nanowires were synthesized by hydrothermal method. • The grown Co 3 O 4 nanowires were used for sensitive non-enzymatic glucose sensor. • The proposed glucose sensor shows a wide linear range with fast response. • The Co 3 O 4 modified electrode is a highly specific enzyme-less glucose sensor. - Abstract: In the work reported herein the ethylene glycol template assisted hydrothermal synthesis, onto Au substrate, of thin and highly dense cobalt oxide (Co 3 O 4 ) nanowires and their characterization and their application for non-enzymatic glucose sensing are reported. The structure and composition of Co 3 O 4 nanowires have been fully characterized using scanning electron microscopy, X-ray diffraction, high resolution transmission electron microscopy and X-ray photoelectron spectroscopy. The synthesized Co 3 O 4 nanowires resulted to have high purity and showed diameter of approximately 10 nm. The prepared Co 3 O 4 nanowires coated gold electrodes were applied to the non-enzymatic detection of glucose. The developed sensor showed high sensitivity (4.58 × 10 1 μA mM −1 cm −2 ), a wide linear range of concentration (1.00 × 10 −4 –1.2 × 10 1 mM) and a detection limit of 2.65 × 10 −5 mM. The developed glucose sensor has also shown to be very stable and selective over interferents such as uric acid and ascorbic acid. Furthermore, the proposed fabrication process was shown to be highly reproducible response (over nine electrodes)

  11. A CuNi/C Nanosheet Array Based on a Metal-Organic Framework Derivate as a Supersensitive Non-Enzymatic Glucose Sensor

    Science.gov (United States)

    Zhang, Li; Ye, Chen; Li, Xu; Ding, Yaru; Liang, Hongbo; Zhao, Guangyu; Wang, Yan

    2018-06-01

    Bimetal catalysts are good alternatives for non-enzymatic glucose sensors owing to their low cost, high activity, good conductivity, and ease of fabrication. In the present study, a self-supported CuNi/C electrode prepared by electrodepositing Cu nanoparticles on a Ni-based metal-organic framework (MOF) derivate was used as a non-enzymatic glucose sensor. The porous construction and carbon scaffold inherited from the Ni-MOF guarantee good kinetics of the electrode process in electrochemical glucose detection. Furthermore, Cu nanoparticles disturb the array structure of MOF derived films and evidently enhance their electrochemical performances in glucose detection. Electrochemical measurements indicate that the CuNi/C electrode possesses a high sensitivity of 17.12 mA mM-1 cm-2, a low detection limit of 66.67 nM, and a wider linearity range from 0.20 to 2.72 mM. Additionally, the electrode exhibits good reusability, reproducibility, and stability, thereby catering to the practical use of glucose sensors. Similar values of glucose concentrations in human blood serum samples are detected with our electrode and with the method involving glucose-6-phosphate dehydrogenase; the results further demonstrate the practical feasibility of our electrode.

  12. Fabrication and electrochemistry characteristics of nickel-doped diamond-like carbon film toward applications in non-enzymatic glucose detection

    Science.gov (United States)

    Liu, Chi-Wen; Chen, Wei-En; Sun, Yin Tung Albert; Lin, Chii-Ruey

    2018-04-01

    This research work focused on the fabrication of nickel-doped diamond-like carbon (DLC) films and their characteristics including of surface morphology, microstructure, and electrochemical aiming at applications in non-enzymatic glucose detection. Novel nanodiamond target was employed in unbalanced magnetron radio-frequency co-sputtering process to prepared high quality Ni-doped DLC thin film at room temperature. TEM analysis reveals a highly uniform distribution of Ni crystallites in amorphous carbon matrix with fraction ranged from 3 to 11.5 at.% which is considered as active sites for the glucose detection. Our cyclic voltammetry measurements using 0.1 M H2SO4 solution demonstrated that the as-prepared Ni-doped DLC films possess large electrochemical potential window of 2.12 V, and this was also observed to be significantly reduced at high Ni doping level owing to lower sp3 fraction. The non-enzymatic glucose detection investigation indicates that the Ni-doped DLC thin film electrode prepared under 7 W of DC sputtering power on Ni target possesses good detecting performance, high stability, and high sensitivity to glucose concentration up to 10 mM, even with the existence of uric acid and ascorbic acid. The peak current was observed to be proportional to glucose concentration and scanning rate, demonstrating highly reversibility redox process of the film electrode and glucose.

  13. Bioactive Peptides in Animal Food Products

    Directory of Open Access Journals (Sweden)

    Marzia Albenzio

    2017-05-01

    Full Text Available Proteins of animal origin represent physiologically active components in the human diet; they exert a direct action or constitute a substrate for enzymatic hydrolysis upon food processing and consumption. Bioactive peptides may descend from the hydrolysis by digestive enzymes, enzymes endogenous to raw food materials, and enzymes from microorganisms added during food processing. Milk proteins have different polymorphisms for each dairy species that influence the amount and the biochemical characteristics (e.g., amino acid chain, phosphorylation, and glycosylation of the protein. Milk from other species alternative to cow has been exploited for their role in children with cow milk allergy and in some infant pathologies, such as epilepsy, by monitoring the immune status. Different mechanisms concur for bioactive peptides generation from meat and meat products, and their functionality and application as functional ingredients have proven effects on consumer health. Animal food proteins are currently the main source of a range of biologically-active peptides which have gained special interest because they may also influence numerous physiological responses in the organism. The addition of probiotics to animal food products represent a strategy for the increase of molecules with health and functional properties.

  14. Dynamic PET and SPECT imaging with radioiodinated, amyloid-reactive peptide p5 in mice: a positive role for peptide dehalogenation.

    Science.gov (United States)

    Martin, Emily B; Kennel, Stephen J; Richey, Tina; Wooliver, Craig; Osborne, Dustin; Williams, Angela; Stuckey, Alan; Wall, Jonathan S

    2014-10-01

    Dynamic molecular imaging provides bio-kinetic data that is used to characterize novel radiolabeled tracers for the detection of disease. Amyloidosis is a rare protein misfolding disease that can affect many organs. It is characterized by extracellular deposits composed principally of fibrillar proteins and hypersulfated proteoglycans. We have previously described a peptide, p5, which binds preferentially to amyloid deposits in a murine model of reactive (AA) amyloidosis. We have determined the whole body distribution of amyloid by molecular imaging techniques using radioiodinated p5. The loss of radioiodide from imaging probes due to enzymatic reaction has plagued the use of radioiodinated peptides and antibodies. Therefore, we studied iodine-124-labeled p5 by using dynamic PET imaging of both amyloid-laden and healthy mice to assess the rates of amyloid binding, the relevance of dehalogenation and the fate of the radiolabeled peptide. Rates of blood pool clearance, tissue accumulation and dehalogenation of the peptide were estimated from the images. Comparisons of these properties between the amyloid-laden and healthy mice provided kinetic profiles whose differences may prove to be indicative of the disease state. Additionally, we performed longitudinal SPECT/CT imaging with iodine-125-labeled p5 up to 72h post injection to determine the stability of the radioiodinated peptide when bound to the extracellular amyloid. Our data show that amyloid-associated peptide, in contrast to the unbound peptide, is resistant to dehalogenation resulting in enhanced amyloid-specific imaging. These data further support the utility of this peptide for detecting amyloidosis and monitoring potential therapeutic strategies in patients. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Accumulation of advanced glycation endproducts reduces chondrocyte-mediated extracellular matrix turnover in human articular cartilage

    NARCIS (Netherlands)

    Degroot, J.; Verzijl, N.; Jacobs, K. M.; Budde, M.; Bank, R. A.; Bijlsma, J. W.; TeKoppele, J. M.; Lafeber, F. P.

    2001-01-01

    The prevalence of osteoarthritis (OAs) increases with age and coincides with the accumulation of advanced glycation endproducts (AGEs) in articular cartilage, suggesting that accumulation of glycation products may be involved in the development of OA. This study was designed to examine the effects

  16. Role of protein-bound carbonyl groups in the formation of advanced glycation endproducts.

    Science.gov (United States)

    Liggins, J; Furth, A J

    1997-08-22

    Several mechanisms have been postulated for the formation of advanced glycation endproducts (AGEs) from glycated proteins; they all feature protein-bound carbonyl intermediates. Using 2,4-dinitrophenylhydrazine (DNPH), we have detected these intermediates on bovine serum albumin, lysozyme and beta-lactoglobulin after in vitro glycation by glucose or fructose. Carbonyls were formed in parallel with AGE-fluorophores, via oxidative Maillard reactions. Neither Amadori nor Heyns products contributed to the DNPH reaction. Fluorophore and carbonyl yields were much enhanced in lipid-associated proteins, but both groups could also be detected in lipid-free proteins. When pre-glycated proteins were incubated in the absence of free sugar, carbonyl groups were rapidly lost in a first-order reaction, while fluorescence continued to develop beyond the 21 days of incubation. Another unexpected finding was that not all carbonyl groups were blocked by aminoguanidine, although there was complete inhibition of reactions leading to AGE-fluorescence. It is suggested that carbonyls acting as fluorophore precursors react readily with aminoguanidine, while others are resistant to this hydrazine, possibly because they are involved in ring closure. Factors influencing the relative rates of acyclisation and hydrazone formation are discussed, together with possible implications for antiglycation therapy.

  17. Non-enzymatic electrochemical immunoassay using noble metal nanoparticles: a review

    International Nuclear Information System (INIS)

    Tang, Juan; Tang, Dianping

    2015-01-01

    Electrochemical immunodetection has attracted considerable attention due to its high sensitivity, low cost and simplicity. Large efforts have recently made in order to design ultrasensitive assays. Noble metal nanoparticles (NM-NPs) offer advantages such as high conductivity and large surface-to-volume ratio. NM-NPs therefore are excellent candidates for developing electrochemical platforms for immunodetection and as signal tags. The use of biofunctionalized NM-NPs often results in amplified recognition via stronger loading of signal tags, and also in enhanced signal. This review (with 87 references) gives an overview on the current state in the use of NM-NPs in Non-enzymatic electrochemical immunosensing. We discuss the application of NM-NPs as electrode matrices and as electroactive labels (either as a carrier or as electrocatalytic labels), and compare the materials (mainly nanoparticles of gold, platinum, or of bimetallic materials) in terms of performance (for example by increasing sensitivity via label amplification or via high densities of capture molecules). A conclusion covers current challenges and gives an outlook. Rather than being exhaustive, the review focuses on representative examples that illustrate novel concepts and promising applications. NM-NPs based immunosensing opens a series of concepts for basic research and offers new tools for determination of trace amounts of protein-related analytes in environment and clinical applications. (author)

  18. Purification and identification of O-GlcNAc-modified peptides using phosphate-based alkyne CLICK chemistry in combination with titanium dioxide chromatography and mass spectrometry

    DEFF Research Database (Denmark)

    Parker, Benjamin L; Gupta, Pankaj; Cordwell, Stuart

    2011-01-01

    A selective method for the enrichment of O-GlcNAcylated peptides using a novel CLICK chemistry reagent is described. Peptides modified by O-GlcNAc were enzymatically labeled with N-azidoacetylgalactosamine. The azide was then reacted with a phospho-alkyne using CLICK chemistry and O-GlcNAcGalNAzPO4...

  19. Antisense Oligonucleotides Internally Labeled with Peptides Show Improved Target Recognition and Stability to Enzymatic Degradation

    DEFF Research Database (Denmark)

    Taskova, Maria; Madsen, Charlotte S.; Jensen, Knud J.

    2017-01-01

    Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide, m...... and makes internally labeled POCs an exciting object of study, i.e., showing high target specificity and simultaneous stability in biological media.......Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide......, most often at the terminal position of the oligonucleotide. Herein, we for the first time systematically investigate the influence of internally attached short peptides on the properties of antisense oligonucleotides. We report the synthesis and internal double labeling of 21-mer oligonucleotides...

  20. Driving engineering of novel antimicrobial peptides from simulations of peptide-micelle interactions

    DEFF Research Database (Denmark)

    Khandelia, Himanshu; Langham, Allison A; Kaznessis, Yiannis N

    2006-01-01

    Simulations of antimicrobial peptides in membrane mimics can provide the high resolution, atomistic picture that is necessary to decipher which sequence and structure components are responsible for activity and toxicity. With such detailed insight, engineering new sequences that are active but non...... peptides and their interaction with membrane mimics. In this article, we discuss the promise and the challenges of widely used models and detail our recent work on peptide-micelle simulations as an attractive alternative to peptide-bilayer simulations. We detail our results with two large structural...... classes of peptides, helical and beta-sheet and demonstrate how simulations can assist in engineering of novel antimicrobials with therapeutic potential....

  1. Accumulation of glycation products in. cap alpha. -H pig lens crystallin and its bearing to diabetic cataract genesis

    Energy Technology Data Exchange (ETDEWEB)

    Vidal, P; Cabezas-Cerrato, J

    1988-01-01

    The incorporation of /sup 11/C-glucose in native pig crystalline by in vitro incubation was found, after subsequent dialysis, to affect all 5 classes of crystallin separated by Sepharose CL-6B column chromatography. Though the radioactivity of the ..cap alpha..-H fraction was three times greater than that of any of the others, autoradiographs of SDS-PAGE gels showed /sup 11/C-glucose adducts to be present in all soluble protein subunits, without there being any evidence of preferential glycation of the ..cap alpha..-H subunits. The concentration of stable glycation products in the ..cap alpha..-H chromatographic fraction of soluble crystallins is suggested to be due the addition of glycated material to this fraction as result of glycation-induced hyperaggregation, and not because the ..cap alpha..-H subunits were especially susceptible to glycation.

  2. A Trojan-Horse Peptide-Carboxymethyl-Cytidine Antibiotic from Bacillus amyloliquefaciens.

    Science.gov (United States)

    Serebryakova, Marina; Tsibulskaya, Darya; Mokina, Olga; Kulikovsky, Alexey; Nautiyal, Manesh; Van Aerschot, Arthur; Severinov, Konstantin; Dubiley, Svetlana

    2016-12-07

    Microcin C and related antibiotics are Trojan-horse peptide-adenylates. The peptide part is responsible for facilitated transport inside the sensitive cell, where it gets processed to release a toxic warhead-a nonhydrolyzable aspartyl-adenylate, which inhibits aspartyl-tRNA synthetase. Adenylation of peptide precursors is carried out by MccB THIF-type NAD/FAD adenylyltransferases. Here, we describe a novel microcin C-like compound from Bacillus amyloliquefaciens. The B. amyloliquefaciens MccB demonstrates an unprecedented ability to attach a terminal cytidine monophosphate to cognate precursor peptide in cellular and cell free systems. The cytosine moiety undergoes an additional modification-carboxymethylation-that is carried out by the C-terminal domain of MccB and the MccS enzyme that produces carboxy-SAM, which serves as a donor of the carboxymethyl group. We show that microcin C-like compounds carrying terminal cytosines are biologically active and target aspartyl-tRNA synthetase, and that the carboxymethyl group prevents resistance that can occur due to modification of the warhead. The results expand the repertoire of known enzymatic modifications of peptides that can be used to obtain new biological activities while avoiding or limiting bacterial resistance.

  3. Angiotensin peptides in the non-gravid uterus: Paracrine actions beyond circulation.

    Science.gov (United States)

    Casalechi, Maíra; Dela Cruz, Cynthia; Lima, Luiza C; Maciel, Luciana P; Pereira, Virgínia M; Reis, Fernando M

    2018-03-01

    The renin-angiotensin system (RAS) involves a complex network of precursors, peptides, enzymes and receptors comprising a systemic (endocrine) and a local (paracrine/autocrine) system. The local RAS plays important roles in tissue modulation and may operate independently of or in close interaction with the circulatory RAS, acting in a complementary fashion. Angiotensin (Ang) II, its receptor AT 1 and Ang-(1-7) expression in the endometrium vary with menstrual cycle, and stromal cell decidualization in vitro is accompanied by local synthesis of angiotensinogen and prorenin. Mas receptor is unlikely to undergo marked changes accompanying the cyclic ovarian steroid hormone fluctuations. Studies investigating the functional relevance of the RAS in the non-gravid uterus show a number of paracrine effects beyond circulation and suggest that RAS peptides may be involved in the pathophysiology of proliferative and fibrotic diseases. Endometrial cancer is associated with increased expression of Ang II, Ang-converting enzyme 1 and AT 1 in the tumoral tissue compared to neighboring non-neoplastic endometrium, and also with a gene polymorphism that enhances AT 1 signal. Ang II induces human endometrial cells to transdifferentiate into cells with myofibroblast phenotype and to synthetize extracellular matrix components that might contribute to endometrial fibrosis. Altogether, these findings point to a fully operating RAS within the uterus, but since many concepts rely on preliminary evidence further studies are needed to clarify the role of the local RAS in uterine physiology and pathophysiology. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. A Review of Antioxidant Peptides Derived from Meat Muscle and By-Products

    Directory of Open Access Journals (Sweden)

    Rui Liu

    2016-09-01

    Full Text Available Antioxidant peptides are gradually being accepted as food ingredients, supplemented in functional food and nutraceuticals, to positively regulate oxidative stress in the human body against lipid and protein oxidation. Meat muscle and meat by-products are rich sources of proteins and can be regarded as good materials for the production of bioactive peptides by use of enzymatic hydrolysis or direct solvent extraction. In recent years, there has been a growing number of studies conducted to characterize antioxidant peptides or hydrolysates derived from meat muscle and by-products as well as processed meat products, including dry-cured hams. Antioxidant peptides obtained from animal sources could exert not only nutritional value but also bioavailability to benefit human health. This paper reviews the antioxidant peptides or protein hydrolysates identified in muscle protein and by-products. We focus on the procedure for the generation of peptides with antioxidant capacity including the acquisition of crude peptides, the assessment of antioxidant activity, and the purification and identification of the active fraction. It remains critical to perform validation experiments with a cell model, animal model or clinical trial to eliminate safety concerns before final application in the food system. In addition, some of the common characteristics on structure-activity relationship are also reviewed based on the identified antioxidant peptides.

  5. A Review of Antioxidant Peptides Derived from Meat Muscle and By-Products.

    Science.gov (United States)

    Liu, Rui; Xing, Lujuan; Fu, Qingquan; Zhou, Guang-Hong; Zhang, Wan-Gang

    2016-09-20

    Antioxidant peptides are gradually being accepted as food ingredients, supplemented in functional food and nutraceuticals, to positively regulate oxidative stress in the human body against lipid and protein oxidation. Meat muscle and meat by-products are rich sources of proteins and can be regarded as good materials for the production of bioactive peptides by use of enzymatic hydrolysis or direct solvent extraction. In recent years, there has been a growing number of studies conducted to characterize antioxidant peptides or hydrolysates derived from meat muscle and by-products as well as processed meat products, including dry-cured hams. Antioxidant peptides obtained from animal sources could exert not only nutritional value but also bioavailability to benefit human health. This paper reviews the antioxidant peptides or protein hydrolysates identified in muscle protein and by-products. We focus on the procedure for the generation of peptides with antioxidant capacity including the acquisition of crude peptides, the assessment of antioxidant activity, and the purification and identification of the active fraction. It remains critical to perform validation experiments with a cell model, animal model or clinical trial to eliminate safety concerns before final application in the food system. In addition, some of the common characteristics on structure-activity relationship are also reviewed based on the identified antioxidant peptides.

  6. Impacts of dissolved organic matter on aqueous behavior of nano/micron-titanium nitride and their induced enzymatic/non-enzymatic antioxidant activities in Scenedesmus obliquus.

    Science.gov (United States)

    Zhang, Xin; Wang, Zhuang; Wang, Se; Fang, Hao; Zhang, Fan; Wang, De-Gao

    2017-01-02

    Freshwater dispersion stability and ecotoxicological effects of titanium nitride (TiN) with particle size of 20 nm, 50 nm, and 2-10 μm in the presence of dissolved organic matter (DOM) at various concentrations were studied. The TiN particles that had a more negative zeta potential and smaller hydrodynamic size showed more stable dispersion in an aqueous medium when DOM was present than when DOM was absent. Biochemical assays indicated that relative to the control, the TiN particles in the presence of DOM alleviated to some extent the antioxidative stress enzyme activity in Scenedesmus obliquus. In addition, it was found that the TiN with a primary size of 50 nm at a high concentration presented a significant impact on non-enzymatic antioxidant defense in algal cells.

  7. Tuning Liposome Membrane Permeability by Competitive Peptide Dimerization and Partitioning-Folding Interactions Regulated by Proteolytic Activity

    Science.gov (United States)

    Lim, Seng Koon; Sandén, Camilla; Selegård, Robert; Liedberg, Bo; Aili, Daniel

    2016-02-01

    Membrane active peptides are of large interest for development of drug delivery vehicles and therapeutics for treatment of multiple drug resistant infections. Lack of specificity can be detrimental and finding routes to tune specificity and activity of membrane active peptides is vital for improving their therapeutic efficacy and minimize harmful side effects. We describe a de novo designed membrane active peptide that partition into lipid membranes only when specifically and covalently anchored to the membrane, resulting in pore-formation. Dimerization with a complementary peptide efficiently inhibits formation of pores. The effect can be regulated by proteolytic digestion of the inhibitory peptide by the matrix metalloproteinase MMP-7, an enzyme upregulated in many malignant tumors. This system thus provides a precise and specific route for tuning the permeability of lipid membranes and a novel strategy for development of recognition based membrane active peptides and indirect enzymatically controlled release of liposomal cargo.

  8. Relationships between Cargo, Cell Penetrating Peptides and Cell Type for Uptake of Non-Covalent Complexes into Live Cells

    Directory of Open Access Journals (Sweden)

    Andrea-Anneliese Keller

    2013-02-01

    Full Text Available Modulating signaling pathways for research and therapy requires either suppression or expression of selected genes or internalization of proteins such as enzymes, antibodies, nucleotide binding proteins or substrates including nucleoside phosphates and enzyme inhibitors. Peptides, proteins and nucleotides are transported by fusing or conjugating them to cell penetrating peptides or by formation of non-covalent complexes. The latter is often preferred because of easy handling, uptake efficiency and auto-release of cargo into the live cell. In our studies complexes are formed with labeled or readily detectable cargoes for qualitative and quantitative estimation of their internalization. Properties and behavior of adhesion and suspension vertebrate cells as well as the protozoa Leishmania tarentolae are investigated with respect to proteolytic activity, uptake efficiency, intracellular localization and cytotoxicity. Our results show that peptide stability to membrane-bound, secreted or intracellular proteases varies between different CPPs and that the suitability of individual CPPs for a particular cargo in complex formation by non-covalent interactions requires detailed studies. Cells vary in their sensitivity to increasing concentrations of CPPs. Thus, most cells can be efficiently transduced with peptides, proteins and nucleotides with intracellular concentrations in the low micromole range. For each cargo, cell type and CPP the optimal conditions must be determined separately.

  9. Ovalbumin with Glycated Carboxyl Groups Shows Membrane-Damaging Activity

    Directory of Open Access Journals (Sweden)

    Ching-Chia Tang

    2017-02-01

    Full Text Available The aim of the present study was to investigate whether glycated ovalbumin (OVA showed novel activity at the lipid-water interface. Mannosylated OVA (Man-OVA was prepared by modification of the carboxyl groups with p-aminophenyl α-dextro (d-mannopyranoside. An increase in the number of modified carboxyl groups increased the membrane-damaging activity of Man-OVA on cell membrane-mimicking vesicles, whereas OVA did not induce membrane permeability in the tested phospholipid vesicles. The glycation of carboxyl groups caused a notable change in the gross conformation of OVA. Moreover, owing to their spatial positions, the Trp residues in Man-OVA were more exposed, unlike those in OVA. Fluorescence quenching studies suggested that the Trp residues in Man-OVA were located on the interface binds with the lipid vesicles, and their microenvironment was abundant in positively charged residues. Although OVA and Man-OVA showed a similar binding affinity for lipid vesicles, the lipid-interacting feature of Man-OVA was distinct from that of OVA. Chemical modification studies revealed that Lys and Arg residues, but not Trp residues, played a crucial role in the membrane-damaging activity of Man-OVA. Taken together, our data suggest that glycation of carboxyl groups causes changes in the structural properties and membrane-interacting features of OVA, generating OVA with membrane-perturbing activities at the lipid-water interface.

  10. [Assays of HbA1c and Amadori products in human biology].

    Science.gov (United States)

    Gillery, P

    2014-09-01

    Different Amadori products, formed during the early steps of the non-enzymatic glycation of proteins, may be assayed in current practice in human biology. The most important marker is HbA1c, resulting from the binding of glucose to the N-terminal extremity of HbA beta chains. HbA1c may be evaluated by various techniques (ion exchange or affinity high performance liquid chromatography, capillary electrophoresis, immunoassay, enzymatic technique) and is considered the best marker of diabetic patient survey. Due to its irreversible and cumulative formation, it provides a retrospective information on the glycemic balance over the four to eight weeks preceding blood collection. It benefits from an international standardization, based on a reference method using liquid chromatography coupled to capillary electrophoresis or mass spectrometry, maintained by an international network of reference laboratories. When HbA1c assay cannot be used (anemia, hemolysis, hemoglobinopathy) or when a shorter period of glycemic equilibrium must be evaluated (child and adolescent, pregnancy, therapeutic changes), other Amadori products may be assayed, like plasma fructosamine (all plasma glycated proteins) or glycated albumin. Nevertheless, these assays are less used in practice, because their semiological value has been less evidenced. Besides, fructosamine assay lacks specificity, and glycated albumin assay has been described recently. An expanding use of HbA1c assay is expected, especially for the diagnosis of diabetes mellitus and the evaluation of other risks, especially cardiovascular ones. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  11. Lack of association of glycated haemoglobin with blood pressure ...

    African Journals Online (AJOL)

    2013-04-12

    Apr 12, 2013 ... Therefore, we examined the relationships of fasting glucose and glycated ... but factors that did associate significantly were age, male gender, rural location, abdominal obesity, alcohol intake .... in continuous variables were.

  12. Serum glycated albumin is inversely influenced by fat mass and visceral adipose tissue in Chinese with normal glucose tolerance.

    Directory of Open Access Journals (Sweden)

    Feifei Wang

    Full Text Available BACKGROUND: Recent studies have revealed that body mass index (BMI inversely influenced serum glycated albumin (GA, which may cause an underestimation of GA-monitored short-term hyperglycemic control. OBJECTIVE: This study was to investigate the association between anthropometric variables (BMI and waist circumference (W and accurate adiposity variables (percentage of body fat (%fat, fat mass, free fat mass (FFM, subcutaneous fat area (SFA, and visceral fat area (VFA with serum GA. DESIGN: A total of 2563 subjects (1037 men, 593 premenopausal women, and 933 postmenopausal women with normal glucose tolerance underwent bioelectrical impedance body fat content measurement and magnetic resonance imaging. Serum GA and absolute value of GA (aGA were measured by enzymatic assay. RESULTS: Compared to the BMI <25.0 kg/m(2 group, the BMI ≥25.0 kg/m(2 group had significantly higher fasting plasma glucose, glycated hemoglobin A1c, and body fat parameters including W, %fat, fat mass, FFM, SFA, and VFA, but significantly lower aGA, and GA in all the three sex- and menopause-stratified groups (all P<0.05. GA decreased with the increment of fat mass for all three groups (all P for trend <0.001. In the same BMI category, men and postmenopausal women with elevated %fat (men, ≥25%; women, ≥35% still had significantly lower GA than those with normal %fat (men, <25%; women, <35% (all P<0.05. Multiple stepwise regression showed that %fat, fat mass, and VFA were independently associated with GA. CONCLUSIONS: Serum GA was inversely influenced by fat mass and visceral adipose tissue in Chinese with normal glucose tolerance.

  13. Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products

    International Nuclear Information System (INIS)

    Hegele, Joerg; Buetler, Timo; Delatour, Thierry

    2008-01-01

    Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N ε -fructoselysine (FL), N ε -carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34 ± 3.81 nmol CML per μmol of free Lys (Lys free ) and 81.5 ± 87.8 nmol Pyr μmol -1 Lys free -1 vs. 3.72 ± 1.29 nmol FL μmol -1 Lys free -1 . In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47 ± 0.08 nmol FL μmol -1 of protein-bound Lys (Lys p-b ), 0.04 ± 0.03 nmol CML μmol -1 Lys p-b -1 and 0.06 ± 0.02 nmol Pyr μmol -1 Lys p-b -1 . It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products

  14. Lacto-ghrestatin, a novel bovine milk-derived peptide, suppresses ghrelin secretion.

    Science.gov (United States)

    Aoki, Hayato; Nakato, Junya; Mizushige, Takafumi; Iwakura, Hiroshi; Sato, Masaru; Suzuki, Hideyuki; Kanamoto, Ryuhei; Ohinata, Kousaku

    2017-07-01

    Ghrelin, an endogenous peptide isolated from the stomach, is known to stimulate food intake after peripheral administration. We found that the enzymatic digest of β-lactoglobulin decreases ghrelin secretion from the ghrelin-producing cell line MGN3-1. The peptides present in the digest were comprehensively analyzed using the nanoLC-OrbitrapMS. Among them, we identified that the nonapeptide LIVTQTMKG, corresponding to β-lactoglobulin(1-9), suppresses ghrelin secretion from MGN3-1 cells. We named LIVTQTMKG 'lacto-ghrestatin'. We found that lacto-ghrestatin decreases intracellular cAMP levels and mRNA expression levels of ghrelin production-related genes in MGN3-1 cells. Orally administered lacto-ghrestatin decreases plasma ghrelin levels and food intake in fasted mice. Lacto-ghrestatin is the first food-derived peptide to suppress ghrelin secretion in vitro and in vivo. © 2017 Federation of European Biochemical Societies.

  15. Na+/K+-ATPase interaction with methylglyoxal as reactive metabolic side product.

    Science.gov (United States)

    Svrckova, Marika; Zatloukalova, Martina; Dvorakova, Petra; Coufalova, Dominika; Novak, David; Hernychova, Lenka; Vacek, Jan

    2017-07-01

    Proteins are subject to oxidative modification and the formation of adducts with a broad spectrum of reactive species via enzymatic and non-enzymatic mechanisms. Here we report that in vitro non-enzymatic methylglyoxal (MGO) binding causes the inhibition and formation of MGO advanced glycation end-products (MAGEs) in Na + /K + -ATPase (NKA). Concretely, MGO adducts with NKA amino acid residues (mainly Arg) and N ε -(carboxymethyl)lysine (CML) formation were found. MGO is not only an inhibitor for solubilized NKA (IC 50 =91±16μM), but also for reconstituted NKA in the lipid bilayer environment, which was clearly demonstrated using a DPPC/DPPE liposome model in the presence or absence of the NKA-selective inhibitor ouabain. High-resolution mass spectrometric analysis of a tryptic digest of NKA isolated from pig (Sus scrofa) kidney indicates that the intracellular α-subunit is naturally (post-translationally) modified by MGO in vivo. In contrast to this, the β-subunit could only be modified by MGO artificially, and the transmembrane part of the protein did not undergo MGO binding under the experimental setup used. As with bovine serum albumin, serving as the water-soluble model, we also demonstrated a high binding capacity of MGO to water-poorly soluble NKA using a multi-spectral methodology based on electroanalytical, immunochemical and fluorimetric tools. In addition, a partial suppression of the MGO-mediated inhibitory effect could be observed in the presence of aminoguanidine (pimagedine), a glycation suppressor and MGO-scavenger. All the results here were obtained with the X-ray structure of NKA in the E1 conformation (3WGV) and could be used in the further interpretation of the functionality of this key enzyme in the presence of highly-reactive metabolic side-products, glycation agents and generally under oxidative stress conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Evaluation of glycated albumin (GA) and GA/HbA1c ratio for diagnosis of diabetes and glycemic control: A comprehensive review.

    Science.gov (United States)

    Yazdanpanah, Sara; Rabiee, Mohammad; Tahriri, Mohammadreza; Abdolrahim, Mojgan; Rajab, Asadollah; Jazayeri, Hossein E; Tayebi, Lobat

    2017-06-01

    Diabetes Mellitus (DM) is a group of metabolic diseases characterized by chronic high blood glucose concentrations (hyperglycemia). When it is left untreated or improperly managed, it can lead to acute complications including diabetic ketoacidosis and non-ketotic hyperosmolar coma. In addition, possible long-term complications include impotence, nerve damage, stroke, chronic kidney failure, cardiovascular disease, foot ulcers, and retinopathy. Historically, universal methods to measure glycemic control for the diagnosis of diabetes included fasting plasma glucose level (FPG), 2-h plasma glucose (2HP), and random plasma glucose. However, these measurements did not provide information about glycemic control over a long period of time. To address this problem, there has been a switch in the past decade to diagnosing diabetes and its severity through measurement of blood glycated proteins such as Hemoglobin A1c (HbA1c) and glycated albumin (GA). Diagnosis and evaluation of diabetes using glycated proteins has many advantages including high accuracy of glycemic control over a period of time. Currently, common laboratory methods used to measure glycated proteins are high-performance liquid chromatography (HPLC), immunoassay, and electrophoresis. HbA1c is one of the most important diagnostic factors for diabetes. However, some reports indicate that HbA1c is not a suitable marker to determine glycemic control in all diabetic patients. GA, which is not influenced by changes in the lifespan of erythrocytes, is thought to be a good alternative indicator of glycemic control in diabetic patients. Here, we review the literature that has investigated the suitability of HbA1c, GA and GA:HbA1c as indicators of long-term glycemic control and demonstrate the importance of selecting the appropriate glycated protein based on the patient's health status in order to provide useful and modern point-of-care monitoring and treatment.

  17. MWCNT-ruthenium oxide composite paste electrode as non-enzymatic glucose sensor.

    Science.gov (United States)

    Tehrani, Ramin M A; Ab Ghani, Sulaiman

    2012-01-01

    A non-enzymatic glucose sensor of multi-walled carbon nanotube-ruthenium oxide/composite paste electrode (MWCNT-RuO(2)/CPE) was developed. The electrode was characterized by using XRD, SEM, TEM and EIS. Meanwhile, cyclic voltammetry and amperometry were used to check on the performances of the MWCNT-RuO(2)/CPE towards glucose. The proposed electrode has displayed a synergistic effect of RuO(2) and MWCNT on the electrocatalytic oxidation of glucose in 3M NaOH. This was possible via the formation of transitions of two redox pairs, viz. Ru(VI)/Ru(IV) and Ru(VII)/Ru(VI). A linear range of 0.5-50mM glucose and a limit of detection of 33 μM glucose (S/N=3) were observed. There was no significant interference observable from the traditional interferences, viz. ascorbic acid and uric acid. Indeed, results so obtained have indicated that the developed MWCNT-RuO(2)/CPE would pave the way for a better future to glucose sensor development as its fabrication was without the use of any enzyme. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Oral delivery of peptides and proteins using lipid-based drug delivery systems.

    Science.gov (United States)

    Li, Ping; Nielsen, Hanne Mørck; Müllertz, Anette

    2012-10-01

    In order to successfully develop lipid-based drug delivery systems (DDS) for oral administration of peptides and proteins, it is important to gain an understanding of the colloid structures formed by these DDS, the mode of peptide and protein incorporation as well as the mechanism by which intestinal absorption of peptides and proteins is promoted. The present paper reviews the literature on lipid-based DDS, employed for oral delivery of peptides and proteins and highlights the mechanisms by which the different lipid-based carriers are expected to overcome the two most important barriers (extensive enzymatic degradation and poor transmucosal permeability). This paper also gives a clear-cut idea about advantages and drawbacks of using different lipidic colloidal carriers ((micro)emulsions, solid lipid core particles and liposomes) for oral delivery of peptides and proteins. Lipid-based DDS are safe and suitable for oral delivery of peptides and proteins. Significant progress has been made in this area with several technologies on clinical trials. However, a better understanding of the mechanism of action in vivo is needed in order to improve the design and development of lipid-based DDS with the desired bioavailability and therapeutic profile.

  19. Electro-activation of sweet defatted whey: Impact on the induced Maillard reaction products and bioactive peptides.

    Science.gov (United States)

    Kareb, Ourdia; Gomaa, Ahmed; Champagne, Claude P; Jean, Julie; Aïder, Mohammed

    2017-04-15

    Electro-activation was used to add value to sweet defatted whey. This study aimed to investigate and to characterize the bioactive compounds formed under different electro-activation conditions by molecular and proteomic approaches. The effects of electric current intensity (400, 500 or 600mA) and whey concentration (7, 14 or 21% (w/v)) as a function of the electro-activation time (0, 15, 30 or 45min) were evaluated. The targeted dependent variables were the formation of Maillard reaction products (MRPs), protein hydrolysates and glycated compounds. It was shown that the MRPs derived from electro-activated whey at a concentration of 14% had the highest potential of biological activity. SDS-PAGE analyses indicated the formation of hydrolysates and glycated compounds with different molecular weight distributions. FTIR indicated the predominance of intermediate MRPs, such as the Schiff base compounds. LC-MS/MS and proteomics analysis showed the production of multi-functional bioactive peptides due to the hydrolysis of whey proteins. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

  20. Morintides: cargo-free chitin-binding peptides from Moringa oleifera.

    Science.gov (United States)

    Kini, Shruthi G; Wong, Ka H; Tan, Wei Liang; Xiao, Tianshu; Tam, James P

    2017-03-31

    Hevein-like peptides are a family of cysteine-rich and chitin-binding peptides consisting of 29-45 amino acids. Their chitin-binding property is essential for plant defense against fungi. Based on the number of cysteine residues in their sequences, they are divided into three sub-families: 6C-, 8C- and 10C-hevein-like peptides. All three subfamilies contain a three-domain precursor comprising a signal peptide, a mature hevein-like peptide and a C-terminal domain comprising a hinge region with protein cargo in 8C- and 10C-hevein-like peptides. Here we report the isolation and characterization of two novel 8C-hevein-like peptides, designated morintides (mO1 and mO2), from the drumstick tree Moringa oleifera, a drought-resistant tree belonging to the Moringaceae family. Proteomic analysis revealed that morintides comprise 44 amino acid residues and are rich in cysteine, glycine and hydrophilic amino acid residues such as asparagine and glutamine. Morintides are resistant to thermal and enzymatic degradation, able to bind to chitin and inhibit the growth of phyto-pathogenic fungi. Transcriptomic analysis showed that they contain a three-domain precursor comprising an endoplasmic reticulum (ER) signal sequence, a mature peptide domain and a C-terminal domain. A striking feature distinguishing morintides from other 8C-hevein-like peptides is a short and protein-cargo-free C-terminal domain. Previously, a similar protein-cargo-free C-terminal domain has been observed only in ginkgotides, the 8C-hevein-like peptides from a gymnosperm Ginkgo biloba. Thus, morintides, with a cargo-free C-terminal domain, are a stand-alone class of 8C-hevein-like peptides from angiosperms. Our results expand the existing library of hevein-like peptides and shed light on molecular diversity within the hevein-like peptide family. Our work also sheds light on the anti-fungal activity and stability of 8C-hevein-like peptides.

  1. Non-enzymatic U(VI) interactions with biogenic mackinawite

    Science.gov (United States)

    Veeramani, H.; Qafoku, N. P.; Kukkadapu, R. K.; Murayama, M.; Hochella, M. F.

    2011-12-01

    Reductive immobilization of hexavalent uranium [U(VI)] by stimulation of dissimilatory metal and/or sulfate reducing bacteria (DMRB or DSRB) has been extensively researched as a remediation strategy for subsurface U(VI) contamination. These bacteria derive energy by reducing oxidized metals as terminal electron acceptors, often utilizing organic substrates as electron donors. Thus, when evaluating the potential for in-situ uranium remediation in heterogeneous subsurface media, it is important to understand how the presence of alternative electron acceptors such as Fe(III) and sulfate affect U(VI) remediation and the long term behavior and reactivity of reduced uranium. Iron, an abundant subsurface element, represents a substantial sink for electrons from DMRB, and the reduction of Fe(III) leads to the formation of dissolved Fe(II) or to reactive biogenic Fe(II)- and mixed Fe(II)/Fe(III)- mineral phases. Consequently, abiotic U(VI) reduction by reactive forms of biogenic Fe(II) minerals could be a potentially important process for uranium immobilization. In our study, the DMRB Shewanella putrefaciens CN32 was used to synthesize a biogenic Fe(II)-bearing sulfide mineral: mackinawite, that has been characterized by XRD, SEM, HRTEM and Mössbauer spectroscopy. Batch experiments involving treated biogenic mackinawite and uranium (50:1 molar ratio) were carried out at room temperature under strict anoxic conditions. Following complete removal of uranium from solution, the biogenic mackinawite was analyzed by a suite of analytical techniques including XAS, HRTEM and Mössbauer spectroscopy to determine the speciation of uranium and investigate concomitant Fe(II)-phase transformation. Determining the speciation of uranium is critical to success of a remediation strategy. The present work elucidates non-enzymatic/abiotic molecular scale redox interactions between biogenic mackinawite and uranium.

  2. Glycated Hemoglobin Measurement and Prediction of Cardiovascular Disease

    DEFF Research Database (Denmark)

    Di Angelantonio, Emanuele; Gao, Pei; Khan, Hassan

    2014-01-01

    IMPORTANCE: The value of measuring levels of glycated hemoglobin (HbA1c) for the prediction of first cardiovascular events is uncertain. OBJECTIVE: To determine whether adding information on HbA1c values to conventional cardiovascular risk factors is associated with improvement in prediction of c...

  3. Cerebral ischemia is exacerbated by extracellular nicotinamide phosphoribosyltransferase via a non-enzymatic mechanism.

    Directory of Open Access Journals (Sweden)

    Bing Zhao

    Full Text Available Intracellular nicotinamide phosphoribosyltransferase (iNAMPT in neuron has been known as a protective factor against cerebral ischemia through its enzymatic activity, but the role of central extracellular NAMPT (eNAMPT is not clear. Here we show that eNAMPT protein level was elevated in the ischemic rat brain after middle-cerebral-artery occlusion (MCAO and reperfusion, which can be traced to at least in part from blood circulation. Administration of recombinant NAMPT protein exacerbated MCAO-induced neuronal injury in rat brain, while exacerbated oxygen-glucose-deprivation (OGD induced neuronal injury only in neuron-glial mixed culture, but not in neuron culture. In the mixed culture, NAMPT protein promoted TNF-α release in a time- and concentration-dependent fashion, while TNF-α neutralizing antibody protected OGD-induced, NAMPT-enhanced neuronal injury. Importantly, H247A mutant of NAMPT with essentially no enzymatic activity exerted similar effects on ischemic neuronal injury and TNF-α release as the wild type protein. Thus, eNAMPT is an injurious and inflammatory factor in cerebral ischemia and aggravates ischemic neuronal injury by triggering TNF-α release from glia cells, via a mechanism not related to NAMPT enzymatic activity.

  4. A green synthetic strategy of nickel hexacyanoferrate nanoparticals supported on the graphene substrate and its non-enzymatic amperometric sensing application

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Zhonghua, E-mail: xzh@nwnu.edu.cn [Key Laboratory of Bioelectrochemistry & Environmental Analysis of Gansu Province, College of Chemistry & Chemical Engineering, Northwest Normal University, Lanzhou 730070 (China); He, Nan [Key Laboratory of Bioelectrochemistry & Environmental Analysis of Gansu Province, College of Chemistry & Chemical Engineering, Northwest Normal University, Lanzhou 730070 (China); Rao, Honghong [College of Chemistry and Chemical Engineering, Lanzhou City University, Lanzhou, 730070 (China); Hu, Chenxian; Wang, Xiaofen; Wang, Hui; Liu, Xiuhui [Key Laboratory of Bioelectrochemistry & Environmental Analysis of Gansu Province, College of Chemistry & Chemical Engineering, Northwest Normal University, Lanzhou 730070 (China); Lu, Xiaoquan, E-mail: luxq@nwnu.edu.cn [Key Laboratory of Bioelectrochemistry & Environmental Analysis of Gansu Province, College of Chemistry & Chemical Engineering, Northwest Normal University, Lanzhou 730070 (China)

    2017-02-28

    Highlights: • A sensitive non-enzymatic glucose sensor was explored by using a facile and green strategy. • Well dispersed and uniform NiHCF nanoparticles can be effectively produced by the introduction of electrochemical reduction graphene oxide films. • Metal hexacyanoferrate as a potential electron mediator was proposed and applied into non-enzymatic sensing. - Abstract: Rapid glucose detection is a key requirement for both diagnosis and treatment of diabetes. A facile and green strategy to achieve spherical-shaped nickel hexacyanoferrate (NiHCF) nanoparticals supported on electrochemical reduction graphene oxide by using electrochemical cyclic voltammetry is explored. As a sensing substrate, electrochemical reduction graphene oxide deposited on a glassy carbon electrode surface exhibited obvious positive effect on the electrodeposition of NiHCF nanoparticals with spherical structure and thus effectively improved the electrical conductivity and electrochemical sensing of the proposed amperometric sensor. Proof-concept experiments demonstrated that the proposed nanocomposites modified electrode exhibited excellent sensitivity toward glucose oxidation as well as with a satisfying detection limit of 0.11 μM. More importantly, we also explore that as a simple, green and facile method, electrochemical technology can be employed and provide a new strategy for developing GO and metal hexacyanoferrate based amperometric sensing platform toward glucose and other biomolecules.

  5. A green synthetic strategy of nickel hexacyanoferrate nanoparticals supported on the graphene substrate and its non-enzymatic amperometric sensing application

    International Nuclear Information System (INIS)

    Xue, Zhonghua; He, Nan; Rao, Honghong; Hu, Chenxian; Wang, Xiaofen; Wang, Hui; Liu, Xiuhui; Lu, Xiaoquan

    2017-01-01

    Highlights: • A sensitive non-enzymatic glucose sensor was explored by using a facile and green strategy. • Well dispersed and uniform NiHCF nanoparticles can be effectively produced by the introduction of electrochemical reduction graphene oxide films. • Metal hexacyanoferrate as a potential electron mediator was proposed and applied into non-enzymatic sensing. - Abstract: Rapid glucose detection is a key requirement for both diagnosis and treatment of diabetes. A facile and green strategy to achieve spherical-shaped nickel hexacyanoferrate (NiHCF) nanoparticals supported on electrochemical reduction graphene oxide by using electrochemical cyclic voltammetry is explored. As a sensing substrate, electrochemical reduction graphene oxide deposited on a glassy carbon electrode surface exhibited obvious positive effect on the electrodeposition of NiHCF nanoparticals with spherical structure and thus effectively improved the electrical conductivity and electrochemical sensing of the proposed amperometric sensor. Proof-concept experiments demonstrated that the proposed nanocomposites modified electrode exhibited excellent sensitivity toward glucose oxidation as well as with a satisfying detection limit of 0.11 μM. More importantly, we also explore that as a simple, green and facile method, electrochemical technology can be employed and provide a new strategy for developing GO and metal hexacyanoferrate based amperometric sensing platform toward glucose and other biomolecules.

  6. 3D-copper oxide and copper oxide/few-layer graphene with screen printed nanosheet assembly for ultrasensitive non-enzymatic glucose sensing

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Zhimei [Tianjin Key Laboratory of Film Electronic and Communicate Devices, School of Electronics Information Engineering, Tianjin University of Technology, Tianjin, 300384 (China); Advanced Materials and Printed Electronics Center, School of Electronics Information Engineering, Tianjin University of Technology, Tianjin, 300384 (China); Pan, Peng, E-mail: panpeny@163.com [Tianjin Key Laboratory of Film Electronic and Communicate Devices, School of Electronics Information Engineering, Tianjin University of Technology, Tianjin, 300384 (China); Advanced Materials and Printed Electronics Center, School of Electronics Information Engineering, Tianjin University of Technology, Tianjin, 300384 (China); Liu, Xuewen [Tianjin Key Laboratory of Film Electronic and Communicate Devices, School of Electronics Information Engineering, Tianjin University of Technology, Tianjin, 300384 (China); Advanced Materials and Printed Electronics Center, School of Electronics Information Engineering, Tianjin University of Technology, Tianjin, 300384 (China); Yang, Zhengchun; Wei, Jun [Advanced Materials and Printed Electronics Center, School of Electronics Information Engineering, Tianjin University of Technology, Tianjin, 300384 (China); Wei, Zhen, E-mail: weizhenxinxi@163.com [Tianjin Key Laboratory of Film Electronic and Communicate Devices, School of Electronics Information Engineering, Tianjin University of Technology, Tianjin, 300384 (China); Advanced Materials and Printed Electronics Center, School of Electronics Information Engineering, Tianjin University of Technology, Tianjin, 300384 (China)

    2017-02-01

    Screen-printed copper oxide (CuO) and CuO/few-layer graphene on graphite electrodes were used to fabricate the ultrasensitive nonenzymatic glucose biosensors. Flower-like CuO and flower-like CuO/few-layer graphene composites were prepared by screen-printing method and characterized by X-ray diffraction (XRD), field-emission scanning electron microscopy (FESEM), and high-resolution transmission electron microscopy (HETEM). On the basis of their cyclic voltammetry (CV) and chronoamperometry results, it was concluded that the addition of graphene to CuO significantly improved the performance of the fabricated glucose sensors, exhibiting high and reproducible sensitivity of 3120 μAmM{sup −1} cm{sup −2} with three linear ranges from 4 μM to 13.5 mM and the detection limit of 4 μM (S/N = 3) in a fast response time of 2 s. In addition, the fabricated sensors could effectively avoid the disturbance by interferents, such as Ascorbic Acid (AA), Uric Acid (UA), and Dopamine (DA). Most importantly, the testing results of real blood serum samples demonstrated that the electrodes were applicable and acceptable for the determination of glucose concentrations in human serum. The efficiencies of two non-enzymatic glucose biosensors for glucose determination were comparable with that of a commercial enzymatic sensor. - Highlights: • The method 2D nanosheet turns to 3D microflower by using screen printing was proposed. • Few-layer graphene added improved the sensor’s performance on base of CuO functional material. • Two ultrasensitive non-enzymatic glucose sensors were successfully fabricated. • The proposed sensor shows a high sensitivity of 3120 μA mM{sup −1} cm{sup −2}.

  7. Photoelectrochemical enzymatic biosensors.

    Science.gov (United States)

    Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

    2017-06-15

    Enzymatic biosensors have been valuable bioanalytical devices for analysis of diverse targets in disease diagnosis, biological and biomedical research, etc. Photoelectrochemical (PEC) bioanalysis is a recently emerged method that promptly becoming a subject of new research interests due to its attractive potential for future bioanalysis with high sensitivity and specificity. PEC enzymatic biosensors integrate the inherent sensitivities of PEC bioanalysis and the selectivity of enzymes and thus share their both advantages. Currently, PEC enzymatic biosensors have become a hot topic of significant research and the recent impetus has grown rapidly as demonstrated by increased research papers. Given the pace of advances in this area, this review will make a thorough discussion and survey on the fundamentals, sensing strategies, applications and the state of the art in PEC enzymatic biosensors, followed by future prospects based on our own opinions. We hope this work could provide an accessible introduction to PEC enzymatic biosensors for any scientist. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Polycyclic Polyprenylated Acylphloroglucinols: An Emerging Class of Non-Peptide-Based MRSA- and VRE-Active Antibiotics.

    Science.gov (United States)

    Guttroff, Claudia; Baykal, Aslihan; Wang, Huanhuan; Popella, Peter; Kraus, Frank; Biber, Nicole; Krauss, Sophia; Götz, Friedrich; Plietker, Bernd

    2017-12-11

    In the past 20 years, peptide-based antibiotics, such as vancomycin, teicoplanin, and daptomycin, have often been considered as second-line antibiotics. However, in recent years, an increasing number of reports on vancomycin resistance in pathogens appeared, which forces researchers to find novel lead structures for potent new antibiotics. Herein, we report the total synthesis of a defined endo-type B PPAP library and their antibiotic activity against multiresistant S. aureus and various vancomycin-resistant Enterococci. Four new compounds that combine high activities and low cytotoxicity were identified, indicating that the PPAP core might become a new non-peptide-based lead structure in antibiotic research. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Agatoxin-like peptides in the neuroendocrine system of the honey bee and other insects.

    Science.gov (United States)

    Sturm, Sebastian; Ramesh, Divya; Brockmann, Axel; Neupert, Susanne; Predel, Reinhard

    2016-01-30

    We investigated the peptide inventory of the corpora cardiaca (CC) of the honey bee, Apis mellifera, by direct tissue profiling using MALDI-TOF MS combined with proteomic approaches focusing on cysteine-containing peptides. An agatoxin-like peptide (ALP) was identified as a component of the glandular part of the CC and was associated with the presence of the adipokinetic hormone in mass spectra. Although abundant in the CC, ALP does not belong to the toxins observed in the venom gland of A. mellifera. Homologs of ALP are highly conserved in major groups of arthropods and in line with this we detected ALP in the CC of non-venomous insects such as cockroaches and silverfish. In the American cockroach, Periplaneta americana, ALP was also identified in the CNS and stomatogastric nervous system. This is the first report that establishes the presence of ALPs in the neuroendocrine tissues of insects and further studies are necessary to reveal common functions of these peptides, e.g. as antimicrobial agents, ion channel modulators or classical neuropeptides. Among the messenger molecules of the nervous system, neuropeptides represent the structurally most diverse class and basically participate in the regulation of all physiological processes. The set of neuropeptides, their functions and spatial distribution are particularly well-studied in insects. Until now, however, several potential neuropeptide receptors remained orphan, which indicates the existence of so far unknown ligands. In our study, we used proteomic methods such as cysteine modification, enzymatic digestion and peptide derivatization, combined with direct tissue profiling by MALDI-TOF mass spectrometry, for the discovery of novel putative messenger molecules in the neuroendocrine system. The described presence of agatoxin-like peptides in the nervous system of the honey bee and other insects was overseen so far and is thus a remarkable addition to the very well studied neuropeptidome of insects. It is not

  10. Aspirin-mediated acetylation of haemoglobin increases in presence of high glucose concentration and decreases protein glycation

    Directory of Open Access Journals (Sweden)

    Francesco Finamore

    2015-09-01

    Full Text Available Glycation represents the first stage in the development of diabetic complications. Aspirin was shown to prevent sugars reacting with proteins, but the exact mechanism of this interaction was not well defined. We performed a quantitative analysis to calculate the levels of acetylation and glycation of haemoglobin, among others red blood cell (RBC proteins, using a label free approach. After glucose incubation, increases in the acetylation levels were seen for several haemoglobin subunits, while a parallel decrease of their glycation levels was observed after aspirin incubation. These results suggest that, a mutual influence between these two modifications, occur at protein level.

  11. Synthesis of novel CuO nanosheets and their non-enzymatic glucose sensing applications.

    Science.gov (United States)

    Ibupoto, Zafar Hussain; Khun, Kimleang; Beni, Valerio; Liu, Xianjie; Willander, Magnus

    2013-06-20

    In this study, we have developed a sensitive and selective glucose sensor using novel CuO nanosheets which were grown on a gold coated glass substrate by a low temperature growth method. X-ray differaction (XRD) and scanning electron microscopy (SEM) techniques were used for the structural characterization of CuO nanostructures. CuO nanosheets are highly dense, uniform, and exhibited good crystalline array structure. X-ray photoelectron spectroscopy (XPS) technique was applied for the study of chemical composition of CuO nanosheets and the obtained information demonstrated pure phase CuO nanosheets. The novel CuO nanosheets were employed for the development of a sensitive and selective non-enzymatic glucose sensor. The measured sensitivity and a correlation coefficient are in order 5.20 × 10² µA/mMcm² and 0.998, respectively. The proposed sensor is associated with several advantages such as low cost, simplicity, high stability, reproducibility and selectivity for the quick detection of glucose.

  12. Synthesis of Novel CuO Nanosheets and Their Non-Enzymatic Glucose Sensing Applications

    Directory of Open Access Journals (Sweden)

    Magnus Willander

    2013-06-01

    Full Text Available In this study, we have developed a sensitive and selective glucose sensor using novel CuO nanosheets which were grown on a gold coated glass substrate by a low temperature growth method. X-ray differaction (XRD and scanning electron microscopy (SEM techniques were used for the structural characterization of CuO nanostructures. CuO nanosheets are highly dense, uniform, and exhibited good crystalline array structure. X-ray photoelectron spectroscopy (XPS technique was applied for the study of chemical composition of CuO nanosheets and the obtained information demonstrated pure phase CuO nanosheets. The novel CuO nanosheets were employed for the development of a sensitive and selective non-enzymatic glucose sensor. The measured sensitivity and a correlation coefficient are in order 5.20 × 102 µA/mMcm2 and 0.998, respectively. The proposed sensor is associated with several advantages such as low cost, simplicity, high stability, reproducibility and selectivity for the quick detection of glucose.

  13. Relationship Between Glycated Haemoglobin and Body Mass Index ...

    African Journals Online (AJOL)

    Blood pressure, Height, Weight were all measured and body mass index (BMI) calculated as weight (in kilograms) divided by height (in meters squared). Glycated haemoglobin was estimated using the ion exchange chromatography method. Result: A total of 100 healthy subjects, 50 males and 50 females, ages ranging ...

  14. Glycated haemoglobin may in future be reported as estimated mean blood glucose concentration--secondary publication

    DEFF Research Database (Denmark)

    Borg, R.; Nerup, J.; Nathan, D.M.

    2009-01-01

    Glycated haemoglobin (HbA 1c ) is widely used to determine levels of chronic glycaemia, to judge the adequacy of diabetes treatment and to adjust therapy. HbA 1c results are expressed as the percentage of HbA that is glycated. Day-to-day management is guided by self-monitoring of capillary glucose...

  15. Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products

    Energy Technology Data Exchange (ETDEWEB)

    Hegele, Joerg [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland)], E-mail: joerg.hegele@rdls.nestle.com; Buetler, Timo; Delatour, Thierry [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland)

    2008-06-09

    Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N{sup {epsilon}}-fructoselysine (FL), N{sup {epsilon}}-carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34 {+-} 3.81 nmol CML per {mu}mol of free Lys (Lys{sub free}) and 81.5 {+-} 87.8 nmol Pyr {mu}mol{sup -1} Lys{sub free}{sup -1} vs. 3.72 {+-} 1.29 nmol FL {mu}mol{sup -1} Lys{sub free}{sup -1}. In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47 {+-} 0.08 nmol FL {mu}mol{sup -1} of protein-bound Lys (Lys{sub p-b}), 0.04 {+-} 0.03 nmol CML {mu}mol{sup -1} Lys{sub p-b}{sup -1} and 0.06 {+-} 0.02 nmol Pyr {mu}mol{sup -1} Lys{sub p-b}{sup -1}. It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products.

  16. Association between Fluorescent Advanced Glycation End-Products and Vascular Complications in Type 2 Diabetic Patients

    Directory of Open Access Journals (Sweden)

    Alexis Guerin-Dubourg

    2017-01-01

    Full Text Available Objectives. Diabetes is a major health problem associated with hyperglycemia and chronically increased oxidative stress and enhanced formation of advanced glycation end-products (AGEs. The aim of this study was to determine whether oxidative plasma biomarkers in diabetic patients could be evidenced and associated with vascular complications. Methods. Oxidative stress biomarkers such as thiols, ischemia-modified albumin (IMA, glycated albumin (GA, fructosamine, and AGEs were measured in 75 patients with poorly controlled type 2 diabetes (HbA1c > 7.5% with (44 or without (31 vascular disease and in 31 nondiabetic controls. Results. Most biomarkers of oxidation and glycation were significantly increased in diabetic patients in comparison with nondiabetics. Fructosamines, GA, IMA, and AGEs were positively correlated and levels of fluorescent AGEs were significantly increased in the plasma from patients presenting vascular complication. Conclusions. These results bring new evidence for the potential interest of glycated albumin, oxidative stress, and glycoxidation parameters in the monitoring of type 2 diabetic patients. Furthermore, it emphasizes fluorescent AGEs as a putative indicator for vascular event prediction in diabetic patients.

  17. Production of peptide antisera specific for mouse and rat proinsulin C-peptide 2

    DEFF Research Database (Denmark)

    Blume, N; Madsen, O D; Kofod, Hans

    1990-01-01

    for antibody binding to the immunizing antigen. Antisera to C-peptide 2, stained islet beta-cells on mouse and rat, but not monkey pancreas sections in immunocytochemical analysis. Preabsorption to the synthetic C-peptide 2, but not the synthetic mouse and rat C-peptide 1 abolished staining. In conclusion we......Mice and rats have two functional non-allelic insulin genes. By using a synthetic peptide representing a common sequence in mouse and rat C-peptide 2 as antigen, we have produced rabbit antisera specific for an epitope which is not present in mouse or rat C-peptide 1. Long-term immunization did...... not seem to increase the end point titre as tested in direct ELISA. The specificity of the antiserum was determined by competitive ELISA and histochemistry on pancreas sections. Only the synthetic C-peptide 2, but not the homologous synthetic C-peptide 1 from mouse and rat competed efficiently in ELISA...

  18. Predicting Three-Dimensional Conformations of Peptides Constructed of Only Glycine, Alanine, Aspartic Acid, and Valine

    Science.gov (United States)

    Oda, Akifumi; Fukuyoshi, Shuichi

    2015-06-01

    The GADV hypothesis is a form of the protein world hypothesis, which suggests that life originated from proteins (Lacey et al. 1999; Ikehara 2002; Andras 2006). In the GADV hypothesis, life is thought to have originated from primitive proteins constructed of only glycine, alanine, aspartic acid, and valine ([GADV]-proteins). In this study, the three-dimensional (3D) conformations of randomly generated short [GADV]-peptides were computationally investigated using replica-exchange molecular dynamics (REMD) simulations (Sugita and Okamoto 1999). Because the peptides used in this study consisted of only 20 residues each, they could not form certain 3D structures. However, the conformational tendencies of the peptides were elucidated by analyzing the conformational ensembles generated by REMD simulations. The results indicate that secondary structures can be formed in several randomly generated [GADV]-peptides. A long helical structure was found in one of the hydrophobic peptides, supporting the conjecture of the GADV hypothesis that many peptides aggregated to form peptide multimers with enzymatic activity in the primordial soup. In addition, these results indicate that REMD simulations can be used for the structural investigation of short peptides.

  19. Isolation of prolyl endopeptidase inhibitory peptides from a sodium caseinate hydrolysate.

    Science.gov (United States)

    Hsieh, Cheng-Hong; Wang, Tzu-Yuan; Hung, Chuan-Chuan; Hsieh, You-Liang; Hsu, Kuo-Chiang

    2016-01-01

    Prolyl endopeptidase (PEP) has been associated with neurodegenerative disorders, and the PEP inhibitors can restore the memory loss caused by amnesic compounds. In this study, we investigated the PEP inhibitory activity of the enzymatic hydrolysates from various food protein sources, and isolated and identified the PEP inhibitory peptides. The hydrolysate obtained from sodium caseinate using bromelain (SC/BML) displayed the highest inhibitory activity of 86.8% at 5 mg mL(-1) in the present study, and its IC50 value against PEP was 0.77 mg mL(-1). The F-5 fraction by RP-HPLC (reversed-phase high performance liquid chromatography) from SC/BML showed the highest PEP inhibition rate of 88.4%, and 9 peptide sequences were identified. The synthetic peptides (1245.63-1787.94 Da) showed dose-dependent inhibition effects on PEP as competitive inhibitors with IC50 values between 29.8 and 650.5 μM. The results suggest that the peptides derived from sodium caseinate have the potential to be PEP inhibitors.

  20. PepArML: A Meta-Search Peptide Identification Platform for Tandem Mass Spectra.

    Science.gov (United States)

    Edwards, Nathan J

    2013-12-01

    The PepArML meta-search peptide identification platform for tandem mass spectra provides a unified search interface to seven search engines; a robust cluster, grid, and cloud computing scheduler for large-scale searches; and an unsupervised, model-free, machine-learning-based result combiner, which selects the best peptide identification for each spectrum, estimates false-discovery rates, and outputs pepXML format identifications. The meta-search platform supports Mascot; Tandem with native, k-score and s-score scoring; OMSSA; MyriMatch; and InsPecT with MS-GF spectral probability scores—reformatting spectral data and constructing search configurations for each search engine on the fly. The combiner selects the best peptide identification for each spectrum based on search engine results and features that model enzymatic digestion, retention time, precursor isotope clusters, mass accuracy, and proteotypic peptide properties, requiring no prior knowledge of feature utility or weighting. The PepArML meta-search peptide identification platform often identifies two to three times more spectra than individual search engines at 10% FDR.

  1. Smart biomaterials: Surfaces functionalized with proteolytically stable osteoblast-adhesive peptides

    Directory of Open Access Journals (Sweden)

    Annj Zamuner

    2017-09-01

    Full Text Available Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion, growth, proliferation and differentiation. Recently, covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide (HVP mapped on [351–359] sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays, and to promote osseointegration in in vivo studies. For the first time to our knowledge, in this study we investigated the resistance of adhesion sequences to proteolytic digestion: HVP was completely cleaved after 5 h. In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence. A retro-inverted peptide D-2HVP, composed of D amino acids, was completely stable in serum-containing medium. In addition, glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium. Interestingly, D-2HVP increased expression of IBSP, VTN and SPP1 genes as compared to HVP functionalized surfaces. Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces. Therefore, the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance.

  2. Smart biomaterials: Surfaces functionalized with proteolytically stable osteoblast-adhesive peptides.

    Science.gov (United States)

    Zamuner, Annj; Brun, Paola; Scorzeto, Michele; Sica, Giuseppe; Castagliuolo, Ignazio; Dettin, Monica

    2017-09-01

    Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion, growth, proliferation and differentiation. Recently, covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide (HVP) mapped on [351-359] sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays, and to promote osseointegration in in vivo studies. For the first time to our knowledge, in this study we investigated the resistance of adhesion sequences to proteolytic digestion: HVP was completely cleaved after 5 h. In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence. A retro-inverted peptide D-2HVP, composed of D amino acids, was completely stable in serum-containing medium. In addition, glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium. Interestingly, D-2HVP increased expression of IBSP, VTN and SPP1 genes as compared to HVP functionalized surfaces. Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces. Therefore, the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance.

  3. Computational and theoretical modeling of pH and flow effects on the early-stage non-equilibrium self-assembly of optoelectronic peptides

    Science.gov (United States)

    Mansbach, Rachael; Ferguson, Andrew

    Self-assembling π-conjugated peptides are attractive candidates for the fabrication of bioelectronic materials possessing optoelectronic properties due to electron delocalization over the conjugated peptide groups. We present a computational and theoretical study of an experimentally-realized optoelectronic peptide that displays triggerable assembly in low pH to resolve the microscopic effects of flow and pH on the non-equilibrium morphology and kinetics of assembly. Using a combination of molecular dynamics simulations and hydrodynamic modeling, we quantify the time and length scales at which convective flows employed in directed assembly compete with microscopic diffusion to influence assembly. We also show that there is a critical pH below which aggregation proceeds irreversibly, and quantify the relationship between pH, charge density, and aggregate size. Our work provides new fundamental understanding of pH and flow of non-equilibrium π-conjugated peptide assembly, and lays the groundwork for the rational manipulation of environmental conditions and peptide chemistry to control assembly and the attendant emergent optoelectronic properties. This work was supported by the U.S. Department of Energy, Office of Science, Basic Energy Sciences, under Award # DE-SC0011847, and by the Computational Science and Engineering Fellowship from the University of Illinois at Urbana-Champaign.

  4. Optimization of protein and peptide drugs based on the mechanisms of kidney clearance.

    Science.gov (United States)

    Huang, Jiaguo; Wu, Huizi

    2018-05-30

    Development of proteins and peptides into drugs has been considered as a promising strategy to target certain diseases. However, only few proteins and peptides has been approved as new drugs into the market each year. One major problem is that proteins and peptides often exhibit short plasma half-life times, which limits the application for their clinical use. In most cases a short half-life time is not effective to deliver sufficient amount of drugs to the target organs and tissues, which is generally caused by fast renal clearance and low plasma stability due to proteolytic degradation during systemic circulation, because the most common clearance pathway of small proteins and peptides is through glomerular filtration by the kidneys. In this review, enzymatic degradation of proteins and peptides were discussed. Furthermore, several approaches to lengthen the half-life of peptides and proteins drugs based on the unique structures of glomerular capillary wall and the mechanisms of glomerular filtration were summarized, such as increasing the size and hydrodynamic diameter; increasing the negative charge to delay the filtration; increasing plasma protein binding to decrease plasma clearance. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non-covalent complexation and intracellular delivery.

    Science.gov (United States)

    Upadhya, Archana; Sangave, Preeti C

    2016-10-01

    Cell penetrating peptides are useful tools for intracellular delivery of nucleic acids. Delivery of plasmid DNA, a large nucleic acid, poses a challenge for peptide mediated transport. The paper investigates and compares efficacy of five novel peptide designs for complexation of plasmid DNA and subsequent delivery into cells. The peptides were designed to contain reported DNA condensing agents and basic cell penetrating sequences, octa-arginine (R 8 ) and CHK 6 HC coupled to cell penetration accelerating peptides such as Bax inhibitory mutant peptide (KLPVM) and a peptide derived from the Kaposi fibroblast growth factor (kFGF) membrane translocating sequence. A tryptophan rich peptide, an analogue of Pep-3, flanked with CH 3 on either ends was also a part of the study. The peptides were analysed for plasmid DNA complexation, protection of peptide-plasmid DNA complexes against DNase I, serum components and competitive ligands by simple agarose gel electrophoresis techniques. Hemolysis of rat red blood corpuscles (RBCs) in the presence of the peptides was used as a measure of peptide cytotoxicity. Plasmid DNA delivery through the designed peptides was evaluated in two cell lines, human cervical cancer cell line (HeLa) and (NIH/3 T3) mouse embryonic fibroblasts via expression of the secreted alkaline phosphatase (SEAP) reporter gene. The importance of hydrophobic sequences in addition to cationic sequences in peptides for non-covalent plasmid DNA complexation and delivery has been illustrated. An alternative to the employment of fatty acid moieties for enhanced gene transfer has been proposed. Comparison of peptides for plasmid DNA complexation and delivery of peptide-plasmid DNA complexes to cells estimated by expression of a reporter gene, SEAP. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

  6. Laboratory scale production of glucose syrup by the enzymatic ...

    African Journals Online (AJOL)

    Jen

    Laboratory scale production of glucose syrup by the enzymatic ... The industrial processing of starch to glucose, maltose and dextrin involves gelatinization, ... due to non-availability of appropriate technology and industry to harness these into.

  7. Skin advanced glycation end products in HIV infection are increased and predictive of development of cardiovascular events

    NARCIS (Netherlands)

    Sprenger, Herman G.; Bierman, Wouter F.; Martes, Melanie I.; Graaff, Reindert; van der Werf, Tjip S.; Smit, Andries J.

    2017-01-01

    Objective: HIV-1 infection is associated with an increased cardiovascular disease (CVD) risk. Advanced glycation end products are formed as stable markers of glycaemic and oxidative stress. Skin autofluorescence (SAF) as marker of accumulated advanced glycation end products is increased and

  8. Cost effective purification of intein based syntetic cationic antimicrobial peptide expressed in cold shock expression system using salt inducible E. coli GJ1158

    Directory of Open Access Journals (Sweden)

    Seetha Ram Kotra

    2014-03-01

    Full Text Available Objective:Synthetic cationic antimicrobial peptide (SC-AMP is an important and upcoming therapeutic molecule against onventional antibiotics. In this study, an attempt was made to purify the SC-AMP without the enzymatic cleavage of the affinity tag, by using an intein-based system. Methods:The intein sequence was amplified from pTYB11 vector using PCR methodologies and the N-terminal of intein was ligated with SC-AMP. The designed construct, intein-SC-AMP was cloned into MCS region of cold shock expression vector, pCOLDI and the recombinant peptide was purified on a chitin affinity column by cleaving intein with 50 mM DTT without applying enzymatic cleavage. Later the peptide was quantified and its antibacterial activity of the purified peptide was studied using well diffusion method. Results: Initially, intein-SC-AMP was expressed as a fusion protein in both IPTG inducible E. coli BL21(DE3 and salt inducible E. coli GJ1158. Single step purification using CBD (chitin binding domain - intein tag in salt inducible E. coli GJ1158, yields the SC-AMP in the soluble form at a oncentration of 208 mg/L. The antibacterial activity and minimal inhibitory concentration (MIC of the purified SC-AMP was studied against both Gram positive and Gram negative microorganisms. Conclusion: For the first time, single step purification of soluble SC-AMP was carried out using chitin-binding domain affinity tag in salt inducible E. coli GJ1158 without an application of enzymatic cleavage. J Microbiol Infect Dis 2014;4(1:13-19

  9. Prediction of post translational modifications in avicennia marina Cu-Zn superoxide dismutase: implication of glycation on the enzyme structure

    International Nuclear Information System (INIS)

    Jabeen, U.; Salim, A.; Abbasi, A.

    2012-01-01

    3D homology model of Cu-Zn superoxide dismutase (SOD) from Avicennia marina (AMSOD) was constructed using the structural coordinates of Spinach SOD (SSOD). Prediction of post translational modification was done by PROSITE. The predicted sites were examined in the 3D model. AMSOD model was glycated using modeling software and changes in the structure was analyzed after glycation. The analysis revealed some potential sites and structural changes after glycation. (author)

  10. FY 1998 annual report on the exploration and production of functional peptide from unutilized protein resources; 1998 nendo miriyo tanpakushitsugen kara no kinosei pepuchido no tansaku to seisan ni kansuru kenkyu hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1999-03-01

    This project is aimed at promotion of effective utilization of unutilized resources by transforming histidine-containing peptide (anserine), enzymatically hydrolyzed muscle protein present in lean salmon swimming off the Sanriku District in the spawning season, into functional peptide. A reactor system, comprising an enzyme-immobilized column and rotary bioreactor, is investigated to efficiently produce peptide by enzymatically hydrolyzing the water-insoluble muscle protein. Anserine is isolated by ethanol extraction, ion-exchanging and partition chromatography. The TPTZ and ABTS methods are developed for speeding up measurement of the antioxidant activity. The salmon muscle protein is hydrolyzed by 3 types of enzymes, to measure the free radical erasing activity by the ABTS method. The enzymatically hydrolyzed protein is fractionated by gel permeation chromatography. The fractions having a molecular weight of 10,000 or less show strong antioxidant activity. The hydrolyzed protein shows improved activity by the iron rhodanide method when it has histidine at the center of tripeptide, and strong radical erasing function when it has thyrosine or tryptophan at the carboxyl terminal. (NEDO)

  11. Exendin-4, but not glucagon-like peptide-1, is cleared exclusively by glomerular filtration in anaesthetised pigs

    DEFF Research Database (Denmark)

    Simonsen, L; Holst, Jens Juul; Deacon, C F

    2006-01-01

    AIMS/HYPOTHESIS: The insulinotropic hormone, glucagon-like peptide-1 (GLP-1), is rapidly degraded in vivo as a result of the combination of extensive enzymatic degradation and renal extraction. The GLP-1 receptor agonist, exendin-4, has a longer duration of action, and has recently been approved ...

  12. Glycation, oxidation and glycoxidation of IgG: a biophysical, biochemical, immunological and hematological study.

    Science.gov (United States)

    Islam, Sidra; Moinuddin; Mir, Abdul Rouf; Raghav, Alok; Habib, Safia; Alam, Khursheed; Ali, Asif

    2017-09-12

    Glycation and oxidation induce structural alterations in the proteins in an interdependent manner with consequent pathological implications. The published literature presents wide range of modifications in conformational characteristics of proteins by glycation and oxidation; however, there is little data that could elaborate the cumulative effect of both the processes. This study has analysed the modifications in IgG by methylglyoxal (MG) (glycative stress), hydroxyl radical ([Formula: see text]) (oxidative stress) and by their combined action i.e. [Formula: see text] treatment of MG glycated IgG (glycoxidation). It further addresses the implications of the altered structural integrity of IgG on its immunological characteristics and impact on haematological parameters in rabbits. Using circular dichroism, FTIR, SDS-PAGE analysis, thioflavin-T fluorescence assay, congo red absorbance analysis, dynamic light scattering, transmission electron microscopy, ELISA, blood cell counts and rectal temperature studies, we report that the glycoxidative modification caused maximum alteration in the IgG as compared to the glycatively and oxidatively modified protein. Far-UV CD results confirmed the highest decline in the beta-pleated sheet content of the protein by glycoxidation. The damage led to the reduced flexibility and enhanced electronic interactions in IgG as observed by near-UV CD. Modifications caused cross-linking and adduct formation in the serum protein. The electron micrograph confirmed amorphous aggregation in modified IgG. The modifications increased the hydrodynamic radius of IgG by allowing the attachment of [Formula: see text] and MG residues. The glycoxidatively modified IgG induced the maximum antibody titres that showed high specificity towards the altered IgG. The glycoxidation of IgG leads to activation of inflammatory pathways.

  13. N-Acetyl Cysteine Attenuated the Deleterious Effects of Advanced Glycation End-Products on the Kidney of Non-Diabetic Rats

    Directory of Open Access Journals (Sweden)

    Karina Thieme

    2016-11-01

    Full Text Available Aim: To assess the renal effects of chronic exposure to advanced glycation end-products (AGEs in the absence of diabetes and the potential impact of concomitant treatment with the antioxidant N-acetyl cysteine (NAC. Methods: Wistar rats received intraperitoneally 20 mg/kg/day of albumin modified (AlbAGE or not (AlbC by advanced glycation for 12 weeks and oral NAC (600mg/L; AlbAGE+NAC and AlbC+NAC, respectively. Biochemical, urinary and renal morphological analyses; carboxymethyl-lysine (CML, an AGE, CD68 (macrophage infiltration, and 4-hydroxynonenal (4-HNE, marker of oxidative stress immunostaining; intrarenal mRNA expression of genes belonging to pathways related to AGEs (Ager, Ddost, Nfkb1, renin-angiotensin system (Agt, Ren, Ace, fibrosis (Tgfb1, Col4a1, oxidative stress (Nox4, Txnip, and apoptosis (Bax, Bcl2; and reactive oxidative species (ROS content were performed. Results: AlbAGE significantly increased urine protein-to-creatinine ratio; glomerular area; renal CML content and macrophage infiltration; expression of Ager, Nfkb1, Agt, Ren, Tgfb1, Col4a1, Txnip, Bax/Bcl2 ratio; and 4-HNE and ROS contents. Some of these effects were attenuated by NAC concomitant treatment. Conclusion: Because AGEs are highly consumed in modern diets and implicated in the progression of different kidney diseases, NAC could be a therapeutic intervention to decrease renal damage, considering that long-term restriction of dietary AGEs is difficult to achieve in practice.

  14. Reversal of obesity and insulin resistance by a non-peptidic glucagon-like peptide-1 receptor agonist in diet-induced obese mice.

    Directory of Open Access Journals (Sweden)

    Min He

    Full Text Available BACKGROUND: Glucagon-like peptide-1 (GLP-1 is recognized as an important regulator of glucose homeostasis. Efforts to utilize GLP-1 mimetics in the treatment of diabetes have yielded clinical benefits. A major hurdle for an effective oral therapy has been the difficulty of finding a non-peptidic GLP-1 receptor (GLP-1R agonist. While its oral bioavailability still poses significant challenges, Boc5, one of the first such compounds, has demonstrated the attainment of GLP-1R agonism in diabetic mice. The present work was to investigate whether subchronic Boc5 treatment can restore glycemic control and induce sustainable weight loss in diet-induced obese (DIO mice, an animal model of human obesity and insulin resistance. METHODOLOGY/PRINCIPAL FINDINGS: DIO mice were treated three times a week with Boc5 (0.3, 1 and 3 mg for 12 weeks. Body weight, body mass index (BMI, food intake, fasting glucose, intraperitoneal glucose tolerance and insulin induced glucose clearance were monitored regularly throughout the treatment. Glucose-stimulated insulin secretion, β-cell mass, islet size, body composition, serum metabolic profiles, lipogenesis, lipolysis, adipose hypertrophy and lipid deposition in the liver and muscle were also measured after 12 weeks of dosing. Boc5 dose-dependently reduced body weight, BMI and food intake in DIO mice. These changes were associated with significant decreases in fat mass, adipocyte hypertrophy and peripheral tissue lipid accumulation. Boc5 treatment also restored glycemic control through marked improvement of insulin sensitivity and normalization of β-cell mass. Administration of Boc5 (3 mg reduced basal but enhanced insulin-mediated glucose incorporation and noradrenaline-stimulated lipolysis in isolated adipocytes from obese mice. Furthermore, circulating leptin, adiponectin, triglyceride, total cholesterol, nonesterified fatty acid and high-density lipoprotein/low-density lipoprotein ratio were normalized to various

  15. Glucagon-like peptide 1: A potential anti-inflammatory pathway in obesity-related asthma.

    Science.gov (United States)

    Nguyen, Dan-Vinh; Linderholm, Angela; Haczku, Angela; Kenyon, Nicholas

    2017-12-01

    Alterations in arginine metabolism and accelerated formation of advanced glycation end-products (AGEs), crucial mechanisms in obesity-related asthma, can be modulated by glucagon-like peptide 1 (GLP-1). l-arginine dysregulation in obesity promotes inflammation and bronchoconstriction. Prolonged hyperglycemia, dyslipidemia, and oxidative stress leads to production of AGEs, that bind to their receptor (RAGE) further potentiating inflammation. By binding to its widely distributed receptor, GLP-1 blunts the effects of RAGE activation and arginine dysregulation. The GLP-1 pathway, while comprehensively studied in the endocrine and cardiovascular literature, is under-recognized in pulmonary research. Insights into GLP-1 and the lung may lead to novel treatments for obesity-related asthma. Published by Elsevier Inc.

  16. Co-localisation of advanced glycation end products and D-β-aspartic acid-containing proteins in gelatinous drop-like corneal dystrophy.

    Science.gov (United States)

    Kaji, Yuichi; Oshika, Tetsuro; Takazawa, Yutaka; Fukayama, Masashi; Fujii, Noriko

    2012-08-01

    Gelatinous drop-like corneal dystrophy (GDLD), also known as familial subepithelial corneal amyloidosis, is an autosomal recessive disorder that causes progressive corneal opacity due to accumulation of amyloid fibrils in the corneal stroma. Genetic analyses have revealed that a mutation in membrane component chromosome 1 surface marker 1 gene is responsible for GDLD. However, the mechanism of amyloid formation in the corneal stroma remains unclear. The present study attempted to reveal the role of advanced glycation end products (AGE) and d-amino acids in amyloid formation in GDLD. Informed consent was obtained from five patients with GDLD, three patients with bullous keratopathy and three patients with interstitial keratitis and all the specimens were analysed. Localisation of amyloid fibrils was analysed using Congo-red and thioflavin T staining. In addition, the localisation of AGE (N(ε)-carboxy(methyl)-L-lysine, pyrraline and pentosidine) and D-β-aspartic acid-containing proteins, a major form of d-amino acid-containing proteins, was analysed immunohistochemically. In all GDLD specimens, strong immunoreactivity to AGE and D-β-aspartic acid-containing proteins was detected in the subepithelial amyloid-rich region. In contrast, amyloid fibrils, AGE, or D-amino acid-containing proteins were slightly detected in the corneal stroma of patients with bullous keratopathy and interstitial keratitis. Abnormally accumulated proteins rich in AGE and D-β-aspartic acid co-localise in the amyloid lesions in GDLD. These results indicate that non-enzymatic post-translational modifications of proteins, including AGE formation and isomerisation of aspartyl residues, will be the cause as well as the result of amyloid fibril formations in GDLD.

  17. Characterization of particulate drug delivery systems for oral delivery of Peptide and protein drugs

    DEFF Research Database (Denmark)

    Christophersen, Philip Carsten; Fano, Mathias; Saaby, Lasse

    2015-01-01

    Oral drug delivery is a preferred route because of good patient compliance. However, most peptide/ protein drugs are delivered via parenteral routes because of the absorption barriers in the gastrointestinal (GI) tract such as enzymatic degradation by proteases and low permeability acrossthe...... delivery of peptide/protein drugs and to provide an overview of formulationand characterization strategies. For a better understanding of the challenges in oral delivery of peptide/protein drugs, the composition of GI fluids and the digestion processes of different kinds of excipients in the GI tract...... biological membranes. To overcome these barriers, different formulation strategies for oral delivery of biomacromolecules have been proposed, including lipid based formulations and polymer-based particulate drug delivery systems (DDS). The aim of this review is to summarize the existing knowledge about oral...

  18. Multi-structure docking analysis of BACE1 crystal structures and non-peptidic ligands.

    Science.gov (United States)

    Haghighijoo, Zahra; Hemmateenejad, Bahram; Edraki, Najmeh; Miri, Ramin; Emami, Saeed

    2017-09-01

    In order to design novel non-peptidic inhibitors of BACE1, many research groups have attempted using computational studies including docking analyses. Since there are too many 3D structures for BACE1 in the protein database, the selection of suitable crystal structures is a key prerequisite for the successful application of molecular docking. We employed a multi-structure docking protocol. In which 615 ligands' structures were docked into 150 BACE1 structures. The large number of the resultant docking scores were post-processed by different data analysis methods including exploratory data analysis, regression analysis and discriminant analysis. It was found that using one crystal structure for docking did not result in high accuracy for predicting activity of the BACE1 inhibitors. Instead, using of the multi-structural docking scores, post-processed by chemometrics methods arrived to highly accurate predictive models. In this regards, the PDB accession codes of 4B70, 4DVF and 2WEZ could discriminate between active and inactive compounds, with higher accuracy. Clustering of the BACE1 structures based on principal component analysis of the crystallographic structures the revealed that the discriminant structures are in the center of the clusters. Thus, these structures can be selected as predominant crystal structures for docking studies of non-peptidic BACE1 inhibitors. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Identification of Angiotensin I-Converting Enzyme Inhibitory Peptides Derived from Enzymatic Hydrolysates of Razor Clam Sinonovacula constricta

    Directory of Open Access Journals (Sweden)

    Yun Li

    2016-06-01

    Full Text Available Angiotensin I-converting enzyme (ACE inhibitory activity of razor clam hydrolysates produced using five proteases, namely, pepsin, trypsin, alcalase, flavourzyme and proteases from Actinomucor elegans T3 was investigated. Flavourzyme hydrolysate showed the highest level of degree of hydrolysis (DH (45.87% followed by A. elegans T3 proteases hydrolysate (37.84% and alcalase (30.55%. The A. elegans T3 proteases was observed to be more effective in generating small peptides with ACE-inhibitory activity. The 3 kDa membrane permeate of A. elegans T3 proteases hydrolysate showed the highest ACE-inhibitory activity with an IC50 of 0.79 mg/mL. After chromatographic separation by Sephadex G-15 gel filtration and reverse phase-high performance liquid chromatography, the potent fraction was subjected to MALDI/TOF-TOF MS/MS for identification. A novel ACE-inhibitory peptide (VQY was identified exhibiting an IC50 of 9.8 μM. The inhibitory kinetics investigation by Lineweaver-Burk plots demonstrated that the peptide acts as a competitive ACE inhibitor. The razor clam hydrolysate obtained by A. elegans T3 proteases could serve as a source of functional peptides with ACE-inhibitory activity for physiological benefits.

  20. Possible consequences of the sucrose replacement by a fructose-glucose syrup

    OpenAIRE

    Judit Süli; Ingrid Hamarová; Anna Sobeková

    2017-01-01

    The fructose-glucose syrup is currently used instead of sucrose in bakery products for economic and technological reasons. The authors investigated the extent to which this change affects the formation of non-enzymatic browning products (Advanced Glycation End - AGE-Products and melanoidins). Formation of these products in model systems - mixtures of various sugars (sucrose, fructose, glucose - concentration 6%) with glycine (concentration 0.7%) or/and lysine (concentration 0.3%), heat-treate...

  1. Glyoxalase I reduces glycative and oxidative stress and prevents age-related endothelial dysfunction through modulation of endothelial nitric oxide synthase phosphorylation.

    Science.gov (United States)

    Jo-Watanabe, Airi; Ohse, Takamoto; Nishimatsu, Hiroaki; Takahashi, Masao; Ikeda, Yoichiro; Wada, Takehiko; Shirakawa, Jun-ichi; Nagai, Ryoji; Miyata, Toshio; Nagano, Tetsuo; Hirata, Yasunobu; Inagi, Reiko; Nangaku, Masaomi

    2014-06-01

    Endothelial dysfunction is a major contributor to cardiovascular disease (CVD), particularly in elderly people. Studies have demonstrated the role of glycation in endothelial dysfunction in nonphysiological models, but the physiological role of glycation in age-related endothelial dysfunction has been poorly addressed. Here, to investigate how vascular glycation affects age-related endothelial function, we employed rats systemically overexpressing glyoxalase I (GLO1), which detoxifies methylglyoxal (MG), a representative precursor of glycation. Four groups of rats were examined, namely young (13 weeks old), mid-age (53 weeks old) wild-type, and GLO1 transgenic (WT/GLO1 Tg) rats. Age-related acceleration in glycation was attenuated in GLO1 Tg rats, together with lower aortic carboxymethyllysine (CML) and urinary 8-hydroxydeoxyguanosine (8-OHdG) levels. Age-related impairment of endothelium-dependent vasorelaxation was attenuated in GLO1 Tg rats, whereas endothelium-independent vasorelaxation was not different between WT and GLO1 Tg rats. Nitric oxide (NO) production was decreased in mid-age WT rats, but not in mid-age GLO1 Tg rats. Age-related inactivation of endothelial NO synthase (eNOS) due to phosphorylation of eNOS on Thr495 and dephosphorylation on Ser1177 was ameliorated in GLO1 Tg rats. In vitro, MG increased phosphorylation of eNOS (Thr495) in primary human aortic endothelial cells (HAECs), and overexpression of GLO1 decreased glycative stress and phosphorylation of eNOS (Thr495). Together, GLO1 reduced age-related endothelial glycative and oxidative stress, altered phohphorylation of eNOS, and attenuated endothelial dysfunction. As a molecular mechanism, GLO1 lessened inhibitory phosphorylation of eNOS (Thr495) by reducing glycative stress. Our study demonstrates that blunting glycative stress prevents the long-term impact of endothelial dysfunction on vascular aging. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons

  2. Combined effect of glycation and sodium carbonate-bicarbonate buffer concentration on IgG binding, IgE binding and conformation of ovalbumin.

    Science.gov (United States)

    Ma, Xiao-juan; Gao, Jin-yan; Chen, Hong-bing

    2013-10-01

    Ovalbumin (OVA) is a major allergen in hen egg. During thermal processing, reducing sugars contained in the hen egg white might easily undergo glycation with OVA, but few studies have been conducted on its corresponding immunoreactivity changes. The aim of the present study was to assess changes of the antigenicity, potential allergenicity and conformation of OVA after glycation in a wet-thermal processing system under different concentrations of sodium carbonate-bicarbonate buffer. IgE binding of the glycated OVA was increased after glycation, and the higher the sodium carbonate-bicarbonate buffer concentration, the higher the IgE binding capacity. The increase in IgE binding of OVA corresponded well with the disruption of the disulfide bond, which exposed the epitopes initially buried. Antigenicity of the glycated OVA was increased, and the amount of the increase varied among samples treated under different buffer concentrations. Glycation increased the allergenic potential for OVA, with the amount of increase varying with different sodium carbonate-bicarbonate buffer concentrations. © 2013 Society of Chemical Industry.

  3. Customized Peptide Biomaterial Synthesis via an Environment-Reliant Auto-Programmer Stigmergic Approach

    Directory of Open Access Journals (Sweden)

    Ravindra V. Badhe

    2018-04-01

    Full Text Available Stigmergy, a form of self-organization, was employed here to engineer a self-organizing peptide capable of forming a nano- or micro-structure and that can potentially be used in various drug delivery and biomedical applications. These self-assembling peptides exhibit several desirable qualities for drug delivery, tissue engineering, cosmetics, antibiotics, food science, and biomedical surface engineering. In this study, peptide biomaterial synthesis was carried out using an environment-reliant auto-programmer stigmergic approach. A model protein, α-gliadin (31, 36, and 38 kD, was forced to attain a primary structure with free –SH groups and broken down enzymatically into smaller fragments using chymotrypsin. This breakdown was carried out at different environment conditions (37 and 50 °C, and the fragments were allowed to self-organize at these temperatures. The new peptides so formed diverged according to the environmental conditions. Interestingly, two peptides (with molecular weights of 13.8 and 11.8 kD were isolated when the reaction temperature was maintained at 50 °C, while four peptides with molecular weights of 54, 51, 13.8, and 12.8 kD were obtained when the reaction was conducted at 37 °C. Thus, at a higher temperature (50 °C, the peptides formed, compared to the original protein, had lower molecular weights, whereas, at a lower temperature (37 °C, two peptides had higher molecular weights and two had lower molecular weights.

  4. Sintetski glikokonjugati: model za proučavanje biomolekularnih procesa i šećerima potaknutih neenzimskih preobrazbi peptida/proteina

    Directory of Open Access Journals (Sweden)

    Horvat, Š.

    2007-03-01

    Full Text Available Although the biological and medical importance of complex carbohydrates and glycoconjugates is firmly established, many of the molecular details of how these intriguing compounds accomplish their functions are not understood well. Unraveling these molecular mechanisms is very important since our understanding of the role of glycoconjugates in nature has traditionally fallen far behind the accumulated knowledge on other biopolymers such as proteins and nucleic acids.The synthesis of pure, well-defined glycoconjugates of bioactive peptides is essential to the study of the biological processes that they mediate. This paper covers the methods for glycoconjugate assembly and biological activity screening. Within this context, the impact of the carbohydrate moiety on receptor selectivity, chemical and metabolic stability will be addressed.The diversity and complexity of nonenzymatic glycation products found in naturally occurring peptides/proteins hasten the need to clarify the sequence of reactions that carbohydrate adducts undergo and to find biomarkers of specific sugar-peptide structures. Addressing these issues, it is demonstrated how the structure of the sugar moiety controls the formation of glycation products from the endogenous opioid peptide, Leu-enkephalin. It is shown that the information gained from carbohydrate-peptide model systems provides a novel mechanism for the generation of the glycation (Maillard reaction intermediates under physiological conditions and better insights into the damage to endogenous peptides as a result of glycation by reducing sugars in vivo.

  5. Hepatic microvascular dysfunction and increased advanced glycation end products are components of non-alcoholic fatty liver disease.

    Science.gov (United States)

    Pereira, Evelyn Nunes Goulart da Silva; Silvares, Raquel Rangel; Flores, Edgar Eduardo Ilaquita; Rodrigues, Karine Lino; Ramos, Isalira Peroba; da Silva, Igor José; Machado, Marcelo Pelajo; Miranda, Rosiane Aparecida; Pazos-Moura, Carmen Cabanelas; Gonçalves-de-Albuquerque, Cassiano F; Faria-Neto, Hugo Caire de Castro; Tibiriça, Eduardo; Daliry, Anissa

    2017-01-01

    This study aimed to investigate the pathophysiology of hepatic microcirculatory dysfunction in non-alcoholic fatty liver disease (NAFLD). In Wistar rats, NAFLD model was induced by 20 weeks of high-fat diet (HFD) feeding. Rolling and adhesion of leukocytes and tissue perfusion in hepatic microcirculation were examined using in vivo microscopic and laser speckle contrast imaging (LSCI), respectively. Oxidative stress and inflamatory parameters were analysed by TBARs, catalase enzyme activity, RT-PCR and ELISA. The participation of advanced glycation end-products (AGE) and its receptor RAGE was evaluated by the measurement of gene and protein expression of RAGE by RT-PCR and Western-blot, respectively and by liver and serum quantification of fluorescent AGEs. Wistar rats fed high-fat diet (HFD) showed increase in epididymal and abdominal fat content, systolic arterial blood pressure, fasting blood glucose levels, hepatic triglycerides and cholesterol, and impairment of glucose and insulin metabolisms. Liver histology confirmed the presence of steatosis and ultrasound analysis revealed increased liver size and parenchymal echogenicity in HFD-fed rats. HFD causes significant increases in leukocyte rolling and adhesion on hepatic microcirculation and decrease in liver microvascular blood flow. Liver tissue presented increase in oxidative stress and inflammtion. At 20 weeks, there was a significantly increase in AGE content in the liver and serum of HFD-fed rats and an increase in RAGE gene expression in the liver. The increase in liver AGE levels and microcirculatory disturbances could play a role in the pathogenesis of liver injury and are key components of NAFLD.

  6. Improved glucose control and reduced body weight in rodents with dual mechanism of action peptide hybrids.

    Directory of Open Access Journals (Sweden)

    James L Trevaskis

    Full Text Available Combination therapy is being increasingly used as a treatment paradigm for metabolic diseases such as diabetes and obesity. In the peptide therapeutics realm, recent work has highlighted the therapeutic potential of chimeric peptides that act on two distinct receptors, thereby harnessing parallel complementary mechanisms to induce additive or synergistic benefit compared to monotherapy. Here, we extend this hypothesis by linking a known anti-diabetic peptide with an anti-obesity peptide into a novel peptide hybrid, which we termed a phybrid. We report on the synthesis and biological activity of two such phybrids (AC164204 and AC164209, comprised of a glucagon-like peptide-1 receptor (GLP1-R agonist, and exenatide analog, AC3082, covalently linked to a second generation amylin analog, davalintide. Both molecules acted as full agonists at their cognate receptors in vitro, albeit with reduced potency at the calcitonin receptor indicating slightly perturbed amylin agonism. In obese diabetic Lep(ob/Lep (ob mice sustained infusion of AC164204 and AC164209 reduced glucose and glycated haemoglobin (HbA1c equivalently but induced greater weight loss relative to exenatide administration alone. Weight loss was similar to that induced by combined administration of exenatide and davalintide. In diet-induced obese rats, both phybrids dose-dependently reduced food intake and body weight to a greater extent than exenatide or davalintide alone, and equal to co-infusion of exenatide and davalintide. Phybrid-mediated and exenatide + davalintide-mediated weight loss was associated with reduced adiposity and preservation of lean mass. These data are the first to provide in vivo proof-of-concept for multi-pathway targeting in metabolic disease via a peptide hybrid, demonstrating that this approach is as effective as co-administration of individual peptides.

  7. Peptide array-based interaction assay of solid-bound peptides and anchorage-dependant cells and its effectiveness in cell-adhesive peptide design.

    Science.gov (United States)

    Kato, Ryuji; Kaga, Chiaki; Kunimatsu, Mitoshi; Kobayashi, Takeshi; Honda, Hiroyuki

    2006-06-01

    Peptide array, the designable peptide library covalently synthesized on cellulose support, was applied to assay peptide-cell interaction, between solid-bound peptides and anchorage-dependant cells, to study objective peptide design. As a model case, cell-adhesive peptides that could enhance cell growth as tissue engineering scaffold material, was studied. On the peptide array, the relative cell-adhesion ratio of NIH/3T3 cells was 2.5-fold higher on the RGDS (Arg-Gly-Asp-Ser) peptide spot as compared to the spot with no peptide, thus indicating integrin-mediated peptide-cell interaction. Such strong cell adhesion mediated by the RGDS peptide was easily disrupted by single residue substitution on the peptide array, thus indicating that the sequence recognition accuracy of cells was strictly conserved in our optimized scheme. The observed cellular morphological extension with active actin stress-fiber on the RGD motif-containing peptide supported our strategy that peptide array-based interaction assay of solid-bound peptide and anchorage-dependant cells (PIASPAC) could provide quantitative data on biological peptide-cell interaction. The analysis of 180 peptides obtained from fibronectin type III domain (no. 1447-1629) yielded 18 novel cell-adhesive peptides without the RGD motif. Taken together with the novel candidates, representative rules of ineffective amino acid usage were obtained from non-effective candidate sequences for the effective designing of cell-adhesive peptides. On comparing the amino acid usage of the top 20 and last 20 peptides from the 180 peptides, the following four brief design rules were indicated: (i) Arg or Lys of positively charged amino acids (except His) could enhance cell adhesion, (ii) small hydrophilic amino acids are favored in cell-adhesion peptides, (iii) negatively charged amino acids and small amino acids (except Gly) could reduce cell adhesion, and (iv) Cys and Met could be excluded from the sequence combination since they have

  8. Development of a peptide substrate for detection of Sunn pest damage in wheat flour.

    Science.gov (United States)

    Hançerlioğulları, Begüm Zeynep; Köksel, Hamit; Dudak, Fahriye Ceyda

    2018-05-07

    Since the common protease substrates did not give satisfactory results for the determination of Sunn pest protease activity in damaged wheat, different peptide substrates derived from the repeat sequences of high molecular weight glutenin subunits were synthesized. Hydrolysis of peptides by pest protease was determined by HPLC. Among three peptides having the same consensus motifs, peptide1 (PGQGQQGYYPTSPQQ) showed the best catalytic efficiency. A novel assay was described for monitoring the enzymatic activity of protease extracted from damaged wheat flour. The selected peptide was labeled with a fluorophore (EDANS) and quencher (Dabcyl) to display fluorescence resonance energy transfer (FRET). The proteolytic activity was measured by the change in fluorescence intensity that occurred when the protease cleaved the peptide substrate. Furthermore, the developed assay was modified for rapid and easy detection of bug damage in flour. Flour samples were suspended in water and mixed with fluorescence peptide substrate. After centrifugation, the fluorescence intensities of the supernatants were determined which is proportional with the protease content of the flour. The total analysis time for the developed assay is estimated as 15 minutes. The developed assay permits a significant decrease in time and labor, offering sensitive detection of Sunn pest damage in wheat flour. This article is protected by copyright. All rights reserved.

  9. An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins

    DEFF Research Database (Denmark)

    Hägglund, Per; Matthiesen, R.; Elortza, F.

    2007-01-01

    and N-acetyl-β-glucosaminidase) are also included. The two strategies were here applied to identify 103 N-glycosylation sites in the Cohn IV fraction of human plasma. In addition, Endo D/H digestion uniquely enabled identification of 23 fucosylated N-glycosylation sites. Several O-glycosylated peptides......, thereby reducing the complexity and facilitating glycosylation site determinations. Here, we have used two different enzymatic deglycosylation strategies for N-glycosylation site analysis. (1) Removal of entire N-glycan chains by peptide- N-glycosidase (PNGase) digestion, with concomitant deamidation...... of the released asparagine residue. The reaction is carried out in H218O to facilitate identification of the formerly glycosylated peptide by incorporatation of 18O into the formed aspartic acid residue. (2) Digestion with two endo-β- N-acetylglucosaminidases (Endo D and Endo H) that cleave the glycosidic bond...

  10. Computer-Aided Design of Antimicrobial Peptides

    DEFF Research Database (Denmark)

    Fjell, Christopher D.; Hancock, Robert E.W.; Jenssen, Håvard

    2010-01-01

    in antimicrobial activity. Consequently, the majority of peptides put into clinical trials have failed at some point, underlining the importance of a thorough peptide optimization. An important tool in peptide design and optimization is quantitative structure-activity relationship (QSAR) analysis, correlating...... chemical parameters with biological activities of the peptide, using statistical methods. In this review we will discuss two different in silico strategies of computer-aided antibacterial peptide design, a linear correlation model build as an extension of traditional principal component analysis (PCA......) and a non-linear artificial neural network model. Studies on structurally diverse peptides, have concluded that the PCA derived model are able to guide the antibacterial peptide design in a meaningful way, however requiring rather a high homology between the peptides in the test-set and the in silico...

  11. Regulation of the mesolimbic dopamine circuit by feeding peptides.

    Science.gov (United States)

    Liu, S; Borgland, S L

    2015-03-19

    Polypeptides produced in the gastrointestinal tract, stomach, adipocytes, pancreas and brain that influence food intake are referred to as 'feeding-related' peptides. Most peptides that influence feeding exert an inhibitory effect (anorexigenic peptides). In contrast, only a few exert a stimulating effect (orexigenic peptides), such as ghrelin. Homeostatic feeding refers to when food consumed matches energy deficits. However, in western society where access to palatable energy-dense food is nearly unlimited, food is mostly consumed for non-homeostatic reasons. Emerging evidence implicates the mesocorticolimbic circuitry, including dopamine neurons of the ventral tegmental area (VTA), as a key substrate for non-homeostatic feeding. VTA dopamine neurons encode cues that predict rewards and phasic release of dopamine in the ventral striatum motivates animals to forage for food. To elucidate how feeding-related peptides regulate reward pathways is of importance to reveal the mechanisms underlying non-homeostatic or hedonic feeding. Here, we review the current knowledge of how anorexigenic peptides and orexigenic peptides act within the VTA. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  12. Produtos da glicação avançada dietéticos e as complicações crônicas do diabetes Dietetics advanced glycation end-products and chronic complications of diabetes

    Directory of Open Access Journals (Sweden)

    Júnia Helena Porto Barbosa

    2009-02-01

    a conduta terapêutica, concorrendo para a melhoria da qualidade de vida dos portadores dessa enfermidade.The generation of advanced glycation end products is one of the principal mechanisms that lead to the pathologies associated with diabetes mellitus, which include cardiopathy, retinopathy, neuropathy and nephropathy. The objective of this revision is to analyse the role of the advanced glycation end products present in food as intermediaries of diabetic complications, presenting strategies to reduce their ingestion. For this purpose, research was carried out in databases of publications of the area, for the last 15 years, taking into account revision, experimental and clinical studies. Advanced glycation end products are a heterogenous group of molecules coming from non-enzymatic reactions between amino and carbonyl groups, examples being carboxymethyllisine and pentosidine found in food and in vivo. The advanced glycation end products ingested are absorbed and, along with endogenous advanced glycation end-products, promote the progression of the complications of diabetes. There is a direct correlation between advanced glycation end products consumption and blood concentration. Their restriction in food results in the suppression of serum levels of the markers of vascular disease and the intermediaries of inflammation directly involved in the development of diabetic degenerations. The current dietary orientations are concentrated on the proportion of nutrients and on energetic restriction. The risk of ingestion of advanced glycation end products formed during the processing of food should be taken in consideration. It is simply recommended that in the preparation of food, the use of low temperatures for short periods, in the presence of water, has important effects in the prevention of the complications of diabetes. The study of the mechanisms involved in the generation of advanced glycation end products and the antiglycation properties of compounds presented in

  13. De novo peptide design and experimental validation of histone methyltransferase inhibitors.

    Directory of Open Access Journals (Sweden)

    James Smadbeck

    Full Text Available Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA–protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2 maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 mM, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly inhibit EZH2

  14. De novo peptide design and experimental validation of histone methyltransferase inhibitors.

    Directory of Open Access Journals (Sweden)

    James Smadbeck

    Full Text Available Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA-protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2 maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 [Formula: see text]M, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly

  15. The effect of irradiation temperature on the non-enzymatic browning reaction in cooked rice

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ju-Woon [Radiation Application Research Division, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, PO Box 1266, Jeongeup, Jeonbuk 580-185 (Korea, Republic of); Oh, Sang-Hee [Radiation Application Research Division, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, PO Box 1266, Jeongeup, Jeonbuk 580-185 (Korea, Republic of); Kim, Jae-Hun [Radiation Application Research Division, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, PO Box 1266, Jeongeup, Jeonbuk 580-185 (Korea, Republic of); Byun, Eui-Hong [Radiation Application Research Division, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, PO Box 1266, Jeongeup, Jeonbuk 580-185 (Korea, Republic of); Ree Kim, Mee [Department of Food and Nutrition, Chungnam National University, Gung-Dong 220, Yuseong, Daejeon 305-764 (Korea, Republic of); Baek, Min [Atomic Energy Policy Division, Ministry of Science and Technology, Government Complex-Gwacheon, Kyunggi 427-715 (Korea, Republic of); Byun, Myung-Woo [Radiation Application Research Division, Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, PO Box 1266, Jeongeup, Jeonbuk 580-185 (Korea, Republic of)]. E-mail: mwbyun@kaeri.re.kr

    2007-05-15

    The effect of irradiation temperature on the non-enzymatic browning reaction in a sugar-glycine solution and cooked rice generated by gamma irradiation was evaluated in the present study. When the sugar-glycine solution and cooked rice were irradiated at room temperature, the browning reaction was dramatically increased during the post-irradiation period. In the case of irradiation at below the freezing point, the browning by irradiation was retarded during not only irradiation but also a post-irradiation period. The changes of the sugar profile, such as a sugar loss or reducing power of the irradiated sugar-glycine solution and the electron spin resonance signal intensity of the irradiated cooked rice were also decreased with lower irradiation temperature. The present results may suggest that the production of free radicals and a radiolysis product is inhibited during gamma irradiation in the frozen state and it may prevent the browning reaction generated by gamma irradiation from occurring.

  16. The effect of irradiation temperature on the non-enzymatic browning reaction in cooked rice

    International Nuclear Information System (INIS)

    Lee, Ju-Woon; Oh, Sang-Hee; Kim, Jae-Hun; Byun, Eui-Hong; Ree Kim, Mee; Baek, Min; Byun, Myung-Woo

    2007-01-01

    The effect of irradiation temperature on the non-enzymatic browning reaction in a sugar-glycine solution and cooked rice generated by gamma irradiation was evaluated in the present study. When the sugar-glycine solution and cooked rice were irradiated at room temperature, the browning reaction was dramatically increased during the post-irradiation period. In the case of irradiation at below the freezing point, the browning by irradiation was retarded during not only irradiation but also a post-irradiation period. The changes of the sugar profile, such as a sugar loss or reducing power of the irradiated sugar-glycine solution and the electron spin resonance signal intensity of the irradiated cooked rice were also decreased with lower irradiation temperature. The present results may suggest that the production of free radicals and a radiolysis product is inhibited during gamma irradiation in the frozen state and it may prevent the browning reaction generated by gamma irradiation from occurring

  17. Non-Enzymatic Decomposition of Collagen Fibers by a Biglycan Antibody and a Plausible Mechanism for Rheumatoid Arthritis

    Energy Technology Data Exchange (ETDEWEB)

    Antipova, Olga; Orgel, Joseph P.R.O. (IIT)

    2013-04-08

    Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory and destructive joint disorder that affects tens of millions of people worldwide. Normal healthy joints maintain a balance between the synthesis of extracellular matrix (ECM) molecules and the proteolytic degradation of damaged ones. In the case of RA, this balance is shifted toward matrix destruction due to increased production of cleavage enzymes and the presence of (autoimmune) immunoglobulins resulting from an inflammation induced immune response. Herein we demonstrate that a polyclonal antibody against the proteoglycan biglycan (BG) causes tissue destruction that may be analogous to that of RA affected tissues. The effect of the antibody is more potent than harsh chemical and/or enzymatic treatments designed to mimic arthritis-like fibril de-polymerization. In RA cases, the immune response to inflammation causes synovial fibroblasts, monocytes and macrophages to produce cytokines and secrete matrix remodeling enzymes, whereas B cells are stimulated to produce immunoglobulins. The specific antigen that causes the RA immune response has not yet been identified, although possible candidates have been proposed, including collagen types I and II, and proteoglycans (PG's) such as biglycan. We speculate that the initiation of RA associated tissue destruction in vivo may involve a similar non-enzymatic decomposition of collagen fibrils via the immunoglobulins themselves that we observe here ex vivo.

  18. The minimum activation peptide from ilvH can activate the catalytic subunit of AHAS from different species.

    Science.gov (United States)

    Zhao, Yuefang; Niu, Congwei; Wen, Xin; Xi, Zhen

    2013-04-15

    Acetohydroxyacid synthases (AHASs), which catalyze the first step in the biosynthesis of branched-chain amino acids, are composed of a catalytic subunit (CSU) and a regulatory subunit (RSU). The CSU harbors the catalytic site, and the RSU is responsible for the activation and feedback regulation of the CSU. Previous results from Chipman and co-workers and our lab have shown that heterologous activation can be achieved among isozymes of Escherichia coli AHAS. It would be interesting to find the minimum peptide of ilvH (the RSU of E. coli AHAS III) that could activate other E. coli CSUs, or even those of ## species. In this paper, C-terminal, N-terminal, and C- and N-terminal truncation mutants of ilvH were constructed. The minimum peptide to activate ilvI (the CSU of E. coli AHAS III) was found to be ΔN 14-ΔC 89. Moreover, this peptide could not only activate its homologous ilvI and heterologous ilvB (CSU of E. coli AHAS I), but also heterologously activate the CSUs of AHAS from Saccharomyces cerevisiae, Arabidopsis thaliana, and Nicotiana plumbaginifolia. However, this peptide totally lost its ability for feedback regulation by valine, thus suggesting different elements for enzymatic activation and feedback regulation. Additionally, the apparent dissociation constant (Kd ) of ΔN 14-ΔC 89 when binding CSUs of different species was found to be 9.3-66.5 μM by using microscale thermophoresis. The ability of this peptide to activate different CSUs does not correlate well with its binding ability (Kd ) to these CSUs, thus implying that key interactions by specific residues is more important than binding ability in promoting enzymatic reactions. The high sequence similarity of the peptide ΔN 14-ΔC 89 to RSUs across species hints that this peptide represents the minimum activation motif in RSU and that it regulates all AHASs. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. In Vitro Inhibitory Activity of Acca sellowiana Fruit Extract on End Products of Advanced Glycation.

    Science.gov (United States)

    Muñiz, Alethia; Garcia, Abraham H; Pérez, Rosa M; García, Efren V; González, Daphne E

    2018-02-01

    Hyperglycemia plays an important role in the pathogenesis of diabetic complications, as it increases protein glycation, as well as the progressive accumulation of advanced glycation end products (AGEs), which are complex structures that produce fluorescence. The glycation reaction raises the levels of protein carbonyl, N ε -(carboxymethyl)lysine (CML), and fructosamine and decreases the level of thiol groups. In the present study, the antiglycation activity was determined by fluorescence intensity using the bovine serum albumin (BSA)/glucose, CML method, and the level of fructosamine. The oxidation of proteins was determined by the carbonyl protein content and thiol groups. The results show that the hexane extract of Acca sellowiana (FOH) at different concentrations (0.30-5 mg/ml) significantly inhibited the formation of AGEs in the BSA/glucose model during the 4 weeks of the study. FOH reduced the levels of fructosamine and CML. Our results showed a significant effect of FOH in the prevention of oxidative damage of proteins, as well as an effect on the oxidation of thiol groups and carbonyl proteins. The present study indicates that FOH is effective in inhibiting the glycation of proteins in vitro, so it can prevent or ameliorate the chronic conditions of diabetes associated with the formation of AGEs.

  20. Efficacy of antibacterial peptides against peptide-resistant MRSA is restored by permeabilisation of bacteria membranes

    Directory of Open Access Journals (Sweden)

    Joshua Thomas Ravensdale

    2016-11-01

    Full Text Available Clinical application of antimicrobial peptides, as with conventional antibiotics, may be compromised by the development of bacterial resistance. This study investigated antimicrobial peptide resistance in methicillin resistant Staphylococcus aureus, including aspects related to the resilience of the resistant bacteria towards the peptides, the stability of resistance when selection pressures are removed, and whether resistance can be overcome by using the peptides with other membrane-permeabilising agents. Genotypically variant strains of S. aureus became equally resistant to the antibacterial peptides melittin and bac8c when grown in sub-lethal concentrations. Subculture of a melittin-resistant strain without melittin for 8 days lowered the minimal lethal concentration of the peptide from 170 µg ml-1 to 30 g ml-1. Growth for 24 h in 12 g ml-1 melittin restored the MLC to 100 g ml-1. Flow cytometry analysis of cationic fluorophore binding to melittin-naïve and melittin-resistant bacteria revealed that resistance coincided with decreased binding of cationic molecules, suggesting a reduction in nett negative charge on the membrane. Melittin was haemolytic at low concentrations but the truncated analogue of melittin, mel12-26, was confirmed to lack haemolytic activity. Although a previous report found that mel12-26 retained full bactericidal activity, we found it to lack significant activity when added to culture medium. However, electroporation in the presence of 50 µg ml-1 of mel12-26, killed 99.3% of the bacteria. Similarly, using a low concentration of the non-ionic detergent Triton X-100 to permeabilize bacteria to mel12-26 markedly increased its bactericidal activity. The observation that bactericidal activity of the non-membranolytic peptide mel12-26 was enhanced when the bacterial membrane was permeablised by detergents or electroporation, suggests that its principal mechanism in reducing bacterial survival may be through

  1. Advanced glycation end products and RAGE: a common thread in aging, diabetes, neurodegeneration, and inflammation.

    Science.gov (United States)

    Ramasamy, Ravichandran; Vannucci, Susan J; Yan, Shirley Shi Du; Herold, Kevan; Yan, Shi Fang; Schmidt, Ann Marie

    2005-07-01

    The products of nonenzymatic glycation and oxidation of proteins and lipids, the advanced glycation end products (AGEs), accumulate in a wide variety of environments. AGEs may be generated rapidly or over long times stimulated by a range of distinct triggering mechanisms, thereby accounting for their roles in multiple settings and disease states. A critical property of AGEs is their ability to activate receptor for advanced glycation end products (RAGE), a signal transduction receptor of the immunoglobulin superfamily. It is our hypothesis that due to such interaction, AGEs impart a potent impact in tissues, stimulating processes linked to inflammation and its consequences. We hypothesize that AGEs cause perturbation in a diverse group of diseases, such as diabetes, inflammation, neurodegeneration, and aging. Thus, we propose that targeting this pathway may represent a logical step in the prevention/treatment of the sequelae of these disorders.

  2. Purification of labeled cyanogen bromide peptides of the alpha polypeptide from sodium ion and potassium ion activated adenosinetriphosphatase modified with N-[3H]ethylmaleimide

    International Nuclear Information System (INIS)

    Le, D.T.

    1986-01-01

    Sodium ion and potassium ion activated adenosinetriphosphatase, isolated from canine kidney, was reacted with N-[ 3 H]ethylmaleimide while it was poised in three different conformations, ostensibly E2-P, E2, and E1, respectively. These assignments were made from a consideration of the particular concentrations of ligands in the respective alkylation mixtures. After a 30-min reaction, the remaining enzymatic activity was found to vary among these three different samples from 90 to 30% of that of unalkylated controls. In all cases, the alpha polypeptide was purified and subjected to digestion with cyanogen bromide, and in each digest the same two distinct radioactive peptides were identified and purified by gel filtration on a column of Sephadex LH-60. The incorporation of N-[ 3 H]ethylmaleimide into one of these two peptides correlated closely with enzymatic inactivation, while the incorporation into the other was most extensive when the portion of the active site to which ATP binds was unoccupied. Alkylation of the residue within the latter peptide, however, does not result in inactivation of the enzyme. Both peptides were further purified by high-pressure liquid chromatography, and their amino-terminal sequences were determined by manual dansyl Edman or solid-phase techniques. The peptide containing the sulfhydryl protected by ATP has, as its amino terminus, the lysine that reacts exclusively with fluoresceinyl 5'-isothiocyanate

  3. Processed Meat Protein and Heat-Stable Peptide Marker Identification Using Microwave-Assisted Tryptic Digestion

    Directory of Open Access Journals (Sweden)

    Magdalena Montowska

    2016-01-01

    Full Text Available New approaches to rapid examination of proteins and peptides in complex food matrices are of great interest to the community of food scientists. The aim of the study is to examine the influence of microwave irradiation on the acceleration of enzymatic cleavage and enzymatic digestion of denatured proteins in cooked meat of five species (cattle, horse, pig, chicken and turkey and processed meat products (coarsely minced, smoked, cooked and semi-dried sausages. Severe protein aggregation occurred not only in heated meat under harsh treatment at 190 °C but also in processed meat products. All the protein aggregates were thoroughly hydrolyzed aft er 1 h of trypsin treatment with short exposure times of 40 and 20 s to microwave irradiation at 138 and 303 W. There were much more missed cleavage sites observed in all microwave-assisted digestions. Despite the incompleteness of microwave-assisted digestion, six unique peptide markers were detected, which allowed unambiguous identification of processed meat derived from the examined species. Although the microwave-assisted tryptic digestion can serve as a tool for rapid and high-throughput protein identification, great caution and pre-evaluation of individual samples is recommended in protein quantitation.

  4. Enzymatic activities of Azotobacter chroococcum and survival in ...

    African Journals Online (AJOL)

    Enzymatic activities of Azotobacter chroococcum and survival in schloropyrifos amended sterile and non-sterile. M Shukla, V Kumar, RL Thakur, N Narula. Abstract. No Abstract. Cameroon Journal of Experimental Biology Vol. 2 (2) 2006: pp. 88-94. AJOL African Journals Online. HOW TO USE AJOL... for Researchers · for ...

  5. Graphite powder as an alternative or supplement to reversed-phase material for desalting and concentration of peptide mixtures prior to matrix-assisted laser desorption/ionization-mass spectrometry

    DEFF Research Database (Denmark)

    Larsen, Martin Røssel; Cordwell, Stuart J; Roepstorff, Peter

    2002-01-01

    The success attributed to identification and characterization of gel separated proteins by mass spectrometry (MS) is highly dependent on the percentage of an entire sequence covered by matching peptides derived from enzymatic digestion. Desalting and concentration of peptide mixtures on reversed......-phase (RP) microcolumns prior to mass spectrometric analysis have resulted in increased signal-to-noise ratio and sensitivity, and consequently higher sequence coverage. A large proportion of peptides, however, remains undetected by MS presumably because they are lost during sample preparation...

  6. Enzymatic Release and Characterization of Novel Bioactive Peptides from Milk Proteins

    DEFF Research Database (Denmark)

    De Gobba, Cristian

    -inhibitory, antioxidant and antimicrobial peptides) released from milk proteins by mean of enzyme-catalysed hydrolysis. Goat milk fractions (produced using microfiltration membranes) and bovine casein were used as substrates. The goat milk fractions (retentate, permeate and skimmed milk) were hydrolysed with two...... commercial enzymes. The bovine casein was hydrolysed using the supernatant of a Greenlandic bacterium (Arsukibacterium ikkense), produced in the NOVENIA project, which contains cold-active proteolytic enzymes. The hydrolysates were tested for the relevant bioactivities and active fractions were fractionated...... protein hydrolysates made in other studies. Regarding radical scavenging activity, the bovine casein hydrolysates also showed a positive correlation between extent of hydrolysis and activity, although the difference between the unhydrolysed sample and the hydrolysates was less marked. The goat milk...

  7. Committee on Diabetes Mellitus Indices of the Japan Society of Clinical Chemistry-recommended reference measurement procedure and reference materials for glycated albumin determination.

    Science.gov (United States)

    Takei, Izumi; Hoshino, Tadao; Tominaga, Makoto; Ishibashi, Midori; Kuwa, Katsuhiko; Umemoto, Masao; Tani, Wataru; Okahashi, Mikiko; Yasukawa, Keiko; Kohzuma, Takuji; Sato, Asako

    2016-01-01

    Glycated albumin is an intermediate glycaemic control marker for which there are several measurement procedures with entirely different reference intervals. We have developed a reference measurement procedure for the purpose of standardizing glycated albumin measurements. The isotope dilution liquid chromatography/tandem mass spectrometry method was developed as a reference measurement procedure for glycated albumin. The stable isotopes of lysine and fructosyl-lysine, which serve as an internal standard, were added to albumin isolated from serum, followed by hydrogenation. After hydrolysis of albumin with hot hydrochloric acid, the liberated lysine and fructosyl-lysine were measured by liquid chromatography/tandem mass spectrometry, and their concentrations were determined from each isotope ratio. The reference materials (JCCRM611) for determining of glycated albumin were prepared from pooled patient blood samples. The isotope dilution-tandem mass spectrometry calibration curve of fructosyl-lysine and lysine showed good linearity (r = 0.999). The inter-assay and intra-assay coefficient of variation values of glycated albumin measurement were 1.2 and 1.4%, respectively. The glycated albumin values of serum in patients with diabetes assessed through the use of this method showed a good relationship with routine measurement procedures (r = 0.997). The relationship of glycated albumin values of the reference material (JCCRM611) between these two methods was the same as the relationship with the patient serum samples. The Committee on Diabetes Mellitus Indices of the Japan Society of Clinical Chemistry recommends the isotope dilution liquid chromatography/tandem mass spectrometry method as a reference measurement procedure, and JCCRM611 as a certified reference material for glycated albumin measurement. In addition, we recommend the traceability system for glycated albumin measurement. © The Author(s) 2015.

  8. Antimicrobial Peptides from Plants

    Directory of Open Access Journals (Sweden)

    James P. Tam

    2015-11-01

    Full Text Available Plant antimicrobial peptides (AMPs have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic, lipid transfer proteins, α-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms.

  9. The ethylene glycol template assisted hydrothermal synthesis of Co{sub 3}O{sub 4} nanowires; structural characterization and their application as glucose non-enzymatic sensor

    Energy Technology Data Exchange (ETDEWEB)

    Khun, K., E-mail: kimleang.khun@liu.se [Department of Science and Technology, Linköping University, SE-60174 Norrköping (Sweden); Ibupoto, Z.H. [Dr M.A. Kazi Institute of Chemistry, University of Sindh Jamshoro, Sindh Jamshoro (Pakistan); Liu, X. [Department of Physics, Chemistry and Biology, Linköping University, 58183 Linköping (Sweden); Beni, V. [Biosensors and Biolelectronics Centre, Department of Physics, Chemistry and Biology, Linköping University, 58183 Linköping (Sweden); Willander, M. [Department of Science and Technology, Linköping University, SE-60174 Norrköping (Sweden)

    2015-04-15

    Highlights: • Ethylene glycol assisted Co{sub 3}O{sub 4} nanowires were synthesized by hydrothermal method. • The grown Co{sub 3}O{sub 4} nanowires were used for sensitive non-enzymatic glucose sensor. • The proposed glucose sensor shows a wide linear range with fast response. • The Co{sub 3}O{sub 4} modified electrode is a highly specific enzyme-less glucose sensor. - Abstract: In the work reported herein the ethylene glycol template assisted hydrothermal synthesis, onto Au substrate, of thin and highly dense cobalt oxide (Co{sub 3}O{sub 4}) nanowires and their characterization and their application for non-enzymatic glucose sensing are reported. The structure and composition of Co{sub 3}O{sub 4} nanowires have been fully characterized using scanning electron microscopy, X-ray diffraction, high resolution transmission electron microscopy and X-ray photoelectron spectroscopy. The synthesized Co{sub 3}O{sub 4} nanowires resulted to have high purity and showed diameter of approximately 10 nm. The prepared Co{sub 3}O{sub 4} nanowires coated gold electrodes were applied to the non-enzymatic detection of glucose. The developed sensor showed high sensitivity (4.58 × 10{sup 1} μA mM{sup −1} cm{sup −2}), a wide linear range of concentration (1.00 × 10{sup −4}–1.2 × 10{sup 1} mM) and a detection limit of 2.65 × 10{sup −5} mM. The developed glucose sensor has also shown to be very stable and selective over interferents such as uric acid and ascorbic acid. Furthermore, the proposed fabrication process was shown to be highly reproducible response (over nine electrodes)

  10. The Effects of Leucine, Zinc, and Chromium Supplements on Inflammatory Events of the Respiratory System in Type 2 Diabetic Rats

    OpenAIRE

    Kolahian, Saeed; Sadri, Hassan; Shahbazfar, Amir Ali; Amani, Morvarid; Mazadeh, Anis; Mirani, Mehdi

    2015-01-01

    Diabetes mellitus is a major cause of serious micro- and macrovascular diseases that affect nearly every system in the body, including the respiratory system. Non-enzymatic protein glycation due to hyperglycaemic stress has fundamental implications due to the large capillary network and amount of connective tissue in the lung. The current study was designed to determine whether leucine, zinc, and chromium supplementations influence the function and histological structure of the respiratory tr...

  11. d-Ribose as a Contributor to Glycated Haemoglobin

    Directory of Open Access Journals (Sweden)

    Xixi Chen

    2017-11-01

    Full Text Available Glycated haemoglobin (HbA1c is the most important marker of hyperglycaemia in diabetes mellitus. We show that d-ribose reacts with haemoglobin, thus yielding HbA1c. Using mass spectrometry, we detected glycation of haemoglobin with d-ribose produces 10 carboxylmethyllysines (CMLs. The first-order rate constant of fructosamine formation for d-ribose was approximately 60 times higher than that for d-glucose at the initial stage. Zucker Diabetic Fatty (ZDF rat, a common model for type 2 diabetes mellitus (T2DM, had high levels of d-ribose and HbA1c, accompanied by a decrease of transketolase (TK in the liver. The administration of benfotiamine, an activator of TK, significantly decreased d-ribose followed by a decline in HbA1c. In clinical investigation, T2DM patients with high HbA1c had a high level of urine d-ribose, though the level of their urine d-glucose was low. That is, d-ribose contributes to HbA1c, which prompts future studies to further explore whether d-ribose plays a role in the pathophysiological mechanism of T2DM.

  12. Anti-oxidant, anti-inflammatory and immunomodulating properties of an enzymatic protein hydrolysate from yellow field pea seeds.

    Science.gov (United States)

    Ndiaye, Fatou; Vuong, Tri; Duarte, Jairo; Aluko, Rotimi E; Matar, Chantal

    2012-02-01

    Enzymatic protein hydrolysates of yellow pea seed have been shown to possess high anti-oxidant and anti-bacterial activities. The aim of this work was to confirm the anti-oxidant, anti-inflammatory and immunomodulating activities of an enzymatic protein hydrolysate of yellow field pea seeds. The anti-oxidant and anti-inflammatory properties of peptides from yellow field pea proteins (Pisum sativum L.) were investigated in LPS/IFN-γ-activated RAW 264.7 NO⁻ macrophages. The immunomodulating potential of pea protein hydrolysate (PPH) was then studied in a murine model. Pea protein hydrolysate, after a 12 h pre-treatment, showed significant inhibition of NO production by activated macrophages up to 20%. Moreover, PPH significantly inhibited their secretion of pro-inflammatory cytokines, TNF-α- and IL-6, up to 35 and 80%, respectively. Oral administration of PPH in mice enhanced the phagocytic activity of their peritoneal macrophages and stimulated the gut mucosa immune response. The number of IgA+ cells was elevated in the small intestine lamina propria, accompanied by an increase in the number of IL-4+, IL-10+ and IFN-γ+ cells. This was correlated to up-regulation of IL-6 secretion by small intestine epithelial cells (IEC), probably responsible for B-cell terminal differentiation to IgA-secreting cells. Moreover, PPH might have increased IL-6 production in IECs via the stimulation of toll-like receptors (TLRs) family, especially TLR2 and TLR4 since either anti-TLR2 or anti-TLR4 was able to completely abolish PPH-induced IL-6 secretion. Enzymatic protein degradation confers anti-oxidant, anti-inflammatory and immunomodulating potentials to pea proteins, and the resulted peptides could be used as an alternative therapy for the prevention of inflammatory-related diseases.

  13. A qualitative and quantitative evaluation of the peptide characteristics of microwave- and ultrasound-assisted digestion in discovery and targeted proteomic analyses.

    Science.gov (United States)

    Guo, Zhengguang; Cheng, Jie; Sun, Haidan; Sun, Wei

    2017-08-30

    Fast digestion methods can dramatically accelerate enzyme digestion and increase the throughput of proteomic analysis. However, the peptide characteristics of fast digestion methods and their performance in discovery and targeted proteomic analysis must be systematically evaluated. Three digestion methods, including overnight digestion, microwave-assisted protein enzymatic digestion (MAPED), and high-intensity focused ultrasonic-assisted enzymatic digestion (HIFUSAED), in trypsin or in trypsin/Lys-C were comprehensively compared in both discovery and targeted proteomics analysis using the HeLa cell proteome. In discovery proteomic analysis, the highest numbers of peptides and proteins were identified when the sample was digested via the MAPED method with trypsin/Lys-C. The fast digestion methods showed a higher mis-cleavage rate and a lower semi-tryptic rate than the overnight digestion method. In both label-free quantitative analysis and targeted proteomic analysis, both fully cleaved peptides (FCPs) and mis-cleaved peptides (MCPs) from the fast digestion methods and the overnight digestion method showed good reproducibility if they showed good abundance. When both the FCPs and MCPs were included in the analysis, the MAPED with trypsin/Lys-C method showed the best results for both discovery proteomic analysis and relative quantitative targeted proteomic analysis. These results will be beneficial for the application of fast digestion methods to proteomics. Copyright © 2017 John Wiley & Sons, Ltd.

  14. Novel peptide ligand with high binding capacity for antibody purification

    DEFF Research Database (Denmark)

    Lund, L. N.; Gustavsson, P. E.; Michael, R.

    2012-01-01

    Small synthetic ligands for protein purification have become increasingly interesting with the growing need for cheap chromatographic materials for protein purification and especially for the purification of monoclonal antibodies (mAbs). Today, Protein A-based chromatographic resins are the most...... commonly used capture step in mAb down stream processing; however, the use of Protein A chromatography is less attractive due to toxic ligand leakage as well as high cost. Whether used as an alternative to the Protein A chromatographic media or as a subsequent polishing step, small synthetic peptide...... ligands have an advantage over biological ligands; they are cheaper to produce, ligand leakage by enzymatic degradation is either eliminated or significantly reduced, and they can in general better withstand cleaning in place (CIP) conditions such as 0.1 M NaOH. Here, we present a novel synthetic peptide...

  15. Factors of enzymatic biodiesel production from sludge palm oil (SPO ...

    African Journals Online (AJOL)

    ika

    2013-07-31

    Jul 31, 2013 ... Biodiesel is a non-toxic, renewable and environmental friendly fuel. This study ... of biodiesel from sludge palm oil (SPO), a low-cost waste oil via enzymatic catalysis. ... Increasing energy crisis and environmental concerns by.

  16. Purification of labeled cyanogen bromide peptides of the alpha polypeptide from sodium ion and potassium ion activated adenosinetriphosphatase modified with N-(/sup 3/H)ethylmaleimide

    Energy Technology Data Exchange (ETDEWEB)

    Le, D.T.

    1986-05-06

    Sodium ion and potassium ion activated adenosinetriphosphatase, isolated from canine kidney, was reacted with N-(/sup 3/H)ethylmaleimide while it was poised in three different conformations, ostensibly E2-P, E2, and E1, respectively. These assignments were made from a consideration of the particular concentrations of ligands in the respective alkylation mixtures. After a 30-min reaction, the remaining enzymatic activity was found to vary among these three different samples from 90 to 30% of that of unalkylated controls. In all cases, the alpha polypeptide was purified and subjected to digestion with cyanogen bromide, and in each digest the same two distinct radioactive peptides were identified and purified by gel filtration on a column of Sephadex LH-60. The incorporation of N-(/sup 3/H)ethylmaleimide into one of these two peptides correlated closely with enzymatic inactivation, while the incorporation into the other was most extensive when the portion of the active site to which ATP binds was unoccupied. Alkylation of the residue within the latter peptide, however, does not result in inactivation of the enzyme. Both peptides were further purified by high-pressure liquid chromatography, and their amino-terminal sequences were determined by manual dansyl Edman or solid-phase techniques. The peptide containing the sulfhydryl protected by ATP has, as its amino terminus, the lysine that reacts exclusively with fluoresceinyl 5'-isothiocyanate.

  17. Fluorescent eosin probe in investigations of structural changes in glycated proteins

    Science.gov (United States)

    Pravdin, A. B.; Kochubey, V. I.; Mel'Nikov, A. G.

    2010-08-01

    The possibility of using the luminescent-kinetic probe method to investigate structural changes in bovine serum albumin (BSA) upon nonenzymatic thermal glycation is studied. An increase in the glycation time lead to a decrease in the intensity of the probe (eosin) fluorescence and to a long-wavelength shift of its maximum, as well as to an increase in the eosin phosphorescence intensity, which indicates that eosin binds to hydrophobic regions of protein at any times of incubation of BSA with glucose. From a decrease in the rate constant of the triplet-triplet energy transfer between the donor (eosin) and acceptor (anthracene) bound to proteins, it is found that the changes observed in the spectral characteristics of eosin are caused by structural changes in albumin globules as a result of glycosylation.

  18. Beneficial effects of Psidium guajava leaf extract on diabetic myocardium.

    Science.gov (United States)

    Soman, Sowmya; Rajamanickam, Chellam; Rauf, Arun A; Indira, Madambath

    2013-01-01

    Non enzymatic glycosylation (glycation) between reducing sugar and protein results in the formation of advanced glycation end products (AGEs), which is believed to play an important role in diabetes associated cardiovascular complications. Thus agents that inhibit the formation of AGEs are believed to have therapeutic potential against diabetic complications. In the present study we evaluated the antiglycative potential of ethyl acetate fraction of Psidium guajava leaves (PGEt) by administering the extract into streptozotocin induced diabetic rats. Daily administration of the extract for a period of one month significantly decreased the blood glucose, glycated hemoglobin and fructosamine levels in a dose dependent manner. Evaluation of the toxicity markers like SGOT and SGPT revealed the non toxic nature of the extract. Apart from this we evaluated the presence of cardiac isoform of liver alpha 2 macroglobulin, which is a major protein associated with earlier stages of cardiac hypertrophy. SDS-PAGE analysis showed that the level of this protein decreased significantly in extract treated groups compared to diabetic control. These findings support that the administration of PGEt extract may be beneficial for preventing cardiovascular complications associated with diabetes. Copyright © 2011 Elsevier GmbH. All rights reserved.

  19. Analysis of glycation induced protein cross-linking inhibitory effects of some antidiabetic plants and spices.

    Science.gov (United States)

    Perera, Handunge Kumudu Irani; Handuwalage, Charith Sandaruwan

    2015-06-09

    Protein cross-linking which occurs towards the latter part of protein glycation is implicated in the development of chronic diabetic complications. Glycation induced protein cross-linking inhibitory effects of nine antidiabetic plants and three spices were evaluated in this study using a novel, simple, electrophoresis based method. Methanol extracts of thirteen plants including nine antidiabetic plants and three spices were used. Lysozyme and fructose were incubated at 37 °C in the presence or absence of different concentrations of plant extracts up to 31 days. Standard glycation inhibitor aminoguanidine and other appropriate controls were included. A recently established sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) method was used to detect the products of protein cross-linking in the incubation mixtures. High molecular weight protein products representing the dimer, trimer and tetramer of lysozyme were detected in the presence of fructose. Among the nine antidiabetic plants, seven showed glycation induced protein cross-linking inhibitory effects namely Ficus racemosa (FR) stem bark, Gymnema sylvestre (GS) leaves, Musa paradisiaca (MP) yam, Phyllanthus debilis (PD) whole plant, Phyllanthus emblica (PE) fruit, Pterocarpus marsupium (PM) latex and Tinospora cordifolia (TC) leaves. Inhibition observed with Coccinia grandis (CG) leaves and Strychnos potatorum (SP) seeds were much low. Leaves of Gymnema lactiferum (GL), the plant without known antidiabetic effects showed the lowest inhibition. All three spices namely Coriandrum sativum (CS) seeds, Cinnamomum zeylanicum (CZ) bark and Syzygium aromaticum (SA) flower buds showed cross-link inhibitory effects with higher effects in CS and SA. PD, PE, PM, CS and SA showed almost complete inhibition on the formation of cross-linking with 25 μg/ml extracts. Methanol extracts of PD, PE, PM, CS and SA have shown promising inhibitory effects on glycation induced protein cross-linking.

  20. The in vitro effects of advanced glycation end products on basophil functions.

    Science.gov (United States)

    Han, Kaiyu; Suzukawa, Maho; Yamaguchi, Masao; Sugimoto, Naoya; Nakase, Yuko; Toda, Takako; Nagase, Hiroyuki; Ohta, Ken

    2011-01-01

    Basophils are thought to play pivotal roles in the pathogenesis of allergic reactions, but their roles in inflammation associated with systemic abnormalities such as metabolic disorders remain largely unknown. Advanced glycation end products (AGEs) are potentially important substances produced in high-glucose disease conditions. In this in vitro study, we investigated whether the biological functions of human basophils can be influenced by AGEs. We analyzed the effects of AGEs on various functions and markers of human basophils, including CD11b expression, apoptosis, degranulation, and cytokine production. Flow cytometric analysis indicated that the level of the receptor for AGEs (RAGE) on the surface of freshly isolated basophils was very low but was clearly upregulated by IL-3. Apoptosis of basophils was induced by high concentrations of glycated albumin. Although glycated albumin failed to affect the level of surface CD11b expression or to trigger degranulation or production of IL-4 and IL-13 in basophils, it dose-dependently induced IL-6 and IL-8 secretion. AGEs seem to act on human basophils; they suppress the cells' longevity but elicit secretion of inflammatory cytokines. Through these biological changes, basophils might play some roles in inflammatory conditions associated with metabolic disorders presenting elevated levels of AGEs. Copyright © 2011 S. Karger AG, Basel.

  1. Rapid and non-enzymatic in vitro retrieval of tumour cells from surgical specimens.

    Directory of Open Access Journals (Sweden)

    Brigitte Mack

    Full Text Available The study of tumourigenesis commonly involves the use of established cell lines or single cell suspensions of primary tumours. Standard methods for the generation of short-term tumour cell cultures include the disintegration of tissue based on enzymatic and mechanical stress. Here, we describe a simple and rapid method for the preparation of single cells from primary carcinomas, which is independent of enzymatic treatment and feeder cells. Tumour biopsies are processed to 1 mm(3 cubes termed explants, which are cultured 1-3 days on agarose-coated well plates in specified medium. Through incisions generated in the explants, single cells are retrieved and collected from the culture supernatant and can be used for further analysis including in vitro and in vivo studies. Collected cells retain tumour-forming capacity in xenotransplantation assays, mimic the phenotype of the primary tumour, and facilitate the generation of cell lines.

  2. Glutamine reduces postprandial glycemia and augments the glucagon-like peptide-1 response in type 2 diabetes patients

    DEFF Research Database (Denmark)

    Samocha-Bonet, Dorit; Wong, Olivia; Synnott, Emma-Leigh

    2011-01-01

    Impaired glucagon-like peptide (GLP-1) secretion or response may contribute to ineffective insulin release in type 2 diabetes. The conditionally essential amino acid glutamine stimulates GLP-1 secretion in vitro and in vivo. In a randomized, crossover study, we evaluated the effect of oral...... glutamine, with or without sitagliptin (SIT), on postprandial glycemia and GLP-1 concentration in 15 type 2 diabetes patients (glycated hemoglobin 6.5 ± 0.6%). Participants ingested a low-fat meal (5% fat) after receiving either water (control), 30 g l-glutamine (Gln-30), 15 g L-glutamine (Gln-15), 100 mg...... concentration and limiting postprandial glycemia in type 2 diabetes....

  3. Assessment of cardiac risk before non-cardiac surgery: brain natriuretic peptide in 1590 patients.

    Science.gov (United States)

    Dernellis, J; Panaretou, M

    2006-11-01

    To evaluate the predictive value of brain natriuretic peptide (BNP) for assessment of cardiac risk before non-cardiac surgery. Consecutively treated patients (947 men, 643 women) whose BNP was measured before non-cardiac surgery were studied. Clinical and ECG variables were evaluated to identify predictors of postoperative cardiac events. Events occurred in 6% of patients: 21 cardiac deaths, 20 non-fatal myocardial infarctions, 41 episodes of pulmonary oedema and 14 patients with ventricular tachycardia. All of these patients had raised plasma BNP concentrations (best cut-off point 189 pg/ml). The only independent predictor of postoperative events was BNP (odds ratio 34.52, 95% confidence interval (CI) 17.08 to 68.62, p 300 pg/ml); postoperative event rates were 0%, 5%, 12% and 81%, respectively. In this population of patients evaluated before non-cardiac surgery, BNP is an independent predictor of postoperative cardiac events. BNP > 189 pg/ml identified patients at highest risk.

  4. Effects of glucagon-like peptide-1 on glucagon secretion in patients with non-alcoholic fatty liver disease

    DEFF Research Database (Denmark)

    Junker, Anders E; Gluud, Lise L; van Hall, Gerrit

    2016-01-01

    BACKGROUND & AIMS: We evaluated the glucagon-suppressive effect of glucagon-like peptide-1 (GLP-1) and its potential effects on endogenous glucose production and whole body lipolysis in non-diabetic patients with non-alcoholic fatty liver disease (NAFLD). METHODS: On two separate days 10 non-diabetic...... patients with liver biopsy-verified NAFLD (NAFLD activity score 2.5±1.0) and 10 matched controls underwent a 2-hour intravenous infusions of GLP-1 (0.8 pmol × kg(-1) × min(-1)) and placebo. Since GLP-1-mediated glucagon suppression has been shown to be glucose-dependent, plasma glucose was clamped...

  5. Heparanase facilitates cell adhesion and spreading by clustering of cell surface heparan sulfate proteoglycans.

    Directory of Open Access Journals (Sweden)

    Flonia Levy-Adam

    2008-06-01

    Full Text Available Heparanase is a heparan sulfate (HS degrading endoglycosidase participating in extracellular matrix degradation and remodeling. Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions. Non-enzymatic activities of heparanase include enhanced adhesion of tumor-derived cells and primary T-cells. Attempting to identify functional domains of heparanase that would serve as targets for drug development, we have identified heparin binding domains of heparanase. A corresponding peptide (residues Lys(158-Asp(171, termed KKDC was demonstrated to physically associate with heparin and HS, and to inhibit heparanase enzymatic activity. We hypothesized that the pro-adhesive properties of heparanase are mediated by its interaction with cell surface HS proteoglycans, and utilized the KKDC peptide to examine this possibility. We provide evidence that the KKDC peptide interacts with cell membrane HS, resulting in clustering of syndecan-1 and syndecan-4. We applied classical analysis of cell morphology, fluorescent and time-lapse microscopy and demonstrated that the KKDC peptide efficiently stimulates the adhesion and spreading of various cell types, mediated by PKC, Src, and the small GTPase Rac1. These results support, and further substantiate the notion that heparanase function is not limited to its enzymatic activity.

  6. Equilibrium and non-equilibrium conformations of peptides in lipid bilayers.

    Science.gov (United States)

    Boden, N; Cheng, Y; Knowles, P F

    1997-04-22

    A synthetic, hydrophobic, 27-amino-acid-residue peptide 'K27', modelled on the trans-membrane domain of the slow voltage-gated potassium channel, IsK, has been incorporated into a lipid bilayer and its conformational properties studied using FT-IR spectroscopy. The conformation following reconstitution is found to be dependent on the nature of the solvent employed. When the reconstitution is conducted by solvent evaporation from a methanol solution, aggregates comprised of beta-strands are stabilised and their concentration is essentially invariant with time. By contrast, when trifluoroethanol is used, the initial conformation of the peptide is alpha-helical. This then relaxes to an equilibrium state between alpha-helices and beta-strands. The alpha-helix-to beta-strand conversion rate is relatively slow, and this allows the kinetics to be studied by FT-IR spectroscopy. The reverse process is much slower but again can be demonstrated by FT-IR. Thus, it appears that a true equilibrium structure can only be achieved by starting with peptide in the alpha-helical conformation. We believe this result should be of general validity for hydrophobic peptide reconstitution. The implications for conformational changes in membrane proteins are discussed.

  7. N-acetylcysteine Counteracts Adipose Tissue Macrophage Infiltration and Insulin Resistance Elicited by Advanced Glycated Albumin in Healthy Rats

    Directory of Open Access Journals (Sweden)

    Karolline S. da Silva

    2017-09-01

    Full Text Available Background: Advanced glycation endproducts elicit inflammation. However, their role in adipocyte macrophage infiltration and in the development of insulin resistance, especially in the absence of the deleterious biochemical pathways that coexist in diabetes mellitus, remains unknown. We investigated the effect of chronic administration of advanced glycated albumin (AGE-albumin in healthy rats, associated or not with N-acetylcysteine (NAC treatment, on insulin sensitivity, adipose tissue transcriptome and macrophage infiltration and polarization.Methods: Male Wistar rats were intraperitoneally injected with control (C or AGE-albumin alone, or, together with NAC in the drinking water. Biochemical parameters, lipid peroxidation, gene expression and protein contents were, respectively, determined by enzymatic techniques, reactive thiobarbituric acid substances, RT-qPCR and immunohistochemistry or immunoblot. Carboxymethyllysine (CML and pyrraline (PYR were determined by LC/mass spectrometry (LC-MS/MS and ELISA.Results: CML and PYR were higher in AGE-albumin as compared to C. Food consumption, body weight, systolic blood pressure, plasma lipids, glucose, hepatic and renal function, adipose tissue relative weight and adipocyte number were similar among groups. In AGE-treated animals, insulin resistance, adipose macrophage infiltration and Col12a1 mRNA were increased with no changes in M1 and M2 phenotypes as compared to C-albumin-treated rats. Total GLUT4 content was reduced by AGE-albumin as compared to C-albumin. NAC improved insulin sensitivity, reduced urine TBARS, adipose macrophage number and Itgam and Mrc mRNA and increased Slc2a4 and Ppara. CD11b, CD206, Ager, Ddost, Cd36, Nfkb1, Il6, Tnf, Adipoq, Retn, Arg, and Il12 expressions were similar among groups.Conclusions: AGE-albumin sensitizes adipose tissue to inflammation due to macrophage infiltration and reduces GLUT4, contributing to insulin resistance in healthy rats. NAC antagonizes AGE

  8. Y2K+1 state-of-the-art on non-peptide phosphoantigens, a novel category of immunostimulatory molecules.

    Science.gov (United States)

    Espinosa, E; Belmant, C; Sicard, H; Poupot, R; Bonneville, M; Fournié, J J

    2001-07-01

    Some human T cells are activated in vivo and in vitro by small non-peptide antigens, so-called phosphoantigens. Since their discovery in 1994, several reports have continuously documented novel members of this category of immunostimulatory molecules. This article reviews the current knowledge on their biochemical properties.

  9. Effect of enzymatic hydrolysis of pancreatin and alcalase enzyme on some properties of edible bird's nest hydrolysate

    Science.gov (United States)

    Khushairay, Etty Syarmila Ibrahim; Ayub, Mohd Khan; Babji, Abdul Salam

    2014-09-01

    Edible bird nest (EBN) is a dried glutinous secretion from the salivary glands of several different swiftlet species. It is widely consumed as a health food due to its high beneficial effects to human health and has been considered to be one of the most precious food items by the Chinese for thousands of years. The aim of this study was to evaluate the effect of enzymatic hydrolysis on protein solubility (μg/g), the degree of hydrolysis (DH%), and peptide content (μg/g) of edible bird's nest hydrolysate. Initial protein solubility of boiled EBN was 25.5mg/g EBN. Treating the solubilized EBN with pancreatin 4NF for 1.0 - 1.5hours increased EBN protein solubility to 163.9mg/g and produced hydrolysate with DH% of 86.5% and 109.5mg/g peptide. EBN hydrolyzed with alcalase for 1.5 hours produced hydrolysate with protein solubility of 86.7mg/g, 82.7 DH% and had 104.1mg/g peptide content.

  10. Dietary advanced glycation endproducts

    DEFF Research Database (Denmark)

    Poulsen, Malene Wibe

    High heat cooking induces flavor, aroma, and color of food, but leads to formation of advanced glycation endproducts (AGEs) by the Maillard reaction. In addition to the formation in food, AGEs are also formed in vivo, and increased endogenous formation of AGEs has been linked to diabetic...... on postprandial subjective appetite sensations, appetite hormones, and markers of inflammation of two cooking methods that respectively induce or limit AGE formation were investigated in healthy overweight individuals. It was concluded that the meals affected subjective appetite sensations similarly, but the high...... sensitivity of cooking methods that induce or limit AGE formation were investigated in healthy overweight women. It was concluded that insulin sensitivity was improved with use of low heat cooking methods, compared with high heat cooking methods. In a rat study, effects on expression of AGE receptors, insulin...

  11. Antimicrobial peptides design by evolutionary multiobjective optimization.

    Directory of Open Access Journals (Sweden)

    Giuseppe Maccari

    Full Text Available Antimicrobial peptides (AMPs are an abundant and wide class of molecules produced by many tissues and cell types in a variety of mammals, plant and animal species. Linear alpha-helical antimicrobial peptides are among the most widespread membrane-disruptive AMPs in nature, representing a particularly successful structural arrangement in innate defense. Recently, AMPs have received increasing attention as potential therapeutic agents, owing to their broad activity spectrum and their reduced tendency to induce resistance. The introduction of non-natural amino acids will be a key requisite in order to contrast host resistance and increase compound's life. In this work, the possibility to design novel AMP sequences with non-natural amino acids was achieved through a flexible computational approach, based on chemophysical profiles of peptide sequences. Quantitative structure-activity relationship (QSAR descriptors were employed to code each peptide and train two statistical models in order to account for structural and functional properties of alpha-helical amphipathic AMPs. These models were then used as fitness functions for a multi-objective evolutional algorithm, together with a set of constraints for the design of a series of candidate AMPs. Two ab-initio natural peptides were synthesized and experimentally validated for antimicrobial activity, together with a series of control peptides. Furthermore, a well-known Cecropin-Mellitin alpha helical antimicrobial hybrid (CM18 was optimized by shortening its amino acid sequence while maintaining its activity and a peptide with non-natural amino acids was designed and tested, demonstrating the higher activity achievable with artificial residues.

  12. Non-Enzymatic Glucose Sensing Using Carbon Quantum Dots Decorated with Copper Oxide Nanoparticles

    Directory of Open Access Journals (Sweden)

    Houcem Maaoui

    2016-10-01

    Full Text Available Perturbations in glucose homeostasis is critical for human health, as hyperglycemia (defining diabetes leads to premature death caused by macrovascular and microvascular complications. However, the simple and accurate detection of glucose in the blood at low cost remains a challenging task, although it is of great importance for the diagnosis and therapy of diabetic patients. In this work, carbon quantum dots decorated with copper oxide nanostructures (CQDs/Cu2O are prepared by a simple hydrothermal approach, and their potential for electrochemical non-enzymatic glucose sensing is evaluated. The proposed sensor exhibits excellent electrocatalytic activity towards glucose oxidation in alkaline solutions. The glucose sensor is characterized by a wide concentration range from 6 µM to 6 mM, a sensitivity of 2.9 ± 0.2 µA·µM−1·cm−2, and a detection limit of 6 µM at a signal-to-noise ratio S/N = 3. The sensors are successfully applied for glucose determination in human serum samples, demonstrating that the CQDs/Cu2O-based glucose sensor satisfies the requirements of complex sample detection with adapted potential for therapeutic diagnostics.

  13. Peptide chemistry toolbox - Transforming natural peptides into peptide therapeutics.

    Science.gov (United States)

    Erak, Miloš; Bellmann-Sickert, Kathrin; Els-Heindl, Sylvia; Beck-Sickinger, Annette G

    2018-06-01

    The development of solid phase peptide synthesis has released tremendous opportunities for using synthetic peptides in medicinal applications. In the last decades, peptide therapeutics became an emerging market in pharmaceutical industry. The need for synthetic strategies in order to improve peptidic properties, such as longer half-life, higher bioavailability, increased potency and efficiency is accordingly rising. In this mini-review, we present a toolbox of modifications in peptide chemistry for overcoming the main drawbacks during the transition from natural peptides to peptide therapeutics. Modifications at the level of the peptide backbone, amino acid side chains and higher orders of structures are described. Furthermore, we are discussing the future of peptide therapeutics development and their impact on the pharmaceutical market. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. Identification of a preferred substrate peptide for transglutaminase 3 and detection of in situ activity in skin and hair follicles.

    Science.gov (United States)

    Yamane, Asaka; Fukui, Mina; Sugimura, Yoshiaki; Itoh, Miho; Alea, Mileidys Perez; Thomas, Vincent; El Alaoui, Said; Akiyama, Masashi; Hitomi, Kiyotaka

    2010-09-01

    Transglutaminases (TGases) are a family of enzymes that catalyze cross-linking reactions between proteins. During epidermal differentiation, these enzymatic reactions are essential for formation of the cornified envelope, which consists of cross-linked structural proteins. Two main transglutaminases isoforms, epidermal-type (TGase 3) and keratinocyte-type (TGase 1), are cooperatively involved in this process of differentiating keratinocytes. Information regarding their substrate preference is of great importance to determine the functional role of these isozymes and clarify their possible co-operative action. Thus far, we have identified highly reactive peptide sequences specifically recognized by TGases isozymes such as TGase 1, TGase 2 (tissue-type isozyme) and the blood coagulation isozyme, Factor XIII. In this study, several substrate peptide sequences for human TGase 3 were screened from a phage-displayed peptide library. The preferred substrate sequences for TGase 3 were selected and evaluated as fusion proteins with mutated glutathione S-transferase. From these studies, a highly reactive and isozyme-specific sequence (E51) was identified. Furthermore, this sequence was found to be a prominent substrate in the peptide form and was suitable for detection of in situ TGase 3 activity in the mouse epidermis. TGase 3 enzymatic activity was detected in the layers of differentiating keratinocytes and hair follicles with patterns distinct from those of TGase 1. Our findings provide new information on the specific distribution of TGase 3 and constitute a useful tool to clarify its functional role in the epidermis.

  15. Flanking signal and mature peptide residues influence signal peptide cleavage

    Directory of Open Access Journals (Sweden)

    Ranganathan Shoba

    2008-12-01

    Full Text Available Abstract Background Signal peptides (SPs mediate the targeting of secretory precursor proteins to the correct subcellular compartments in prokaryotes and eukaryotes. Identifying these transient peptides is crucial to the medical, food and beverage and biotechnology industries yet our understanding of these peptides remains limited. This paper examines the most common type of signal peptides cleavable by the endoprotease signal peptidase I (SPase I, and the residues flanking the cleavage sites of three groups of signal peptide sequences, namely (i eukaryotes (Euk (ii Gram-positive (Gram+ bacteria, and (iii Gram-negative (Gram- bacteria. Results In this study, 2352 secretory peptide sequences from a variety of organisms with amino-terminal SPs are extracted from the manually curated SPdb database for analysis based on physicochemical properties such as pI, aliphatic index, GRAVY score, hydrophobicity, net charge and position-specific residue preferences. Our findings show that the three groups share several similarities in general, but they display distinctive features upon examination in terms of their amino acid compositions and frequencies, and various physico-chemical properties. Thus, analysis or prediction of their sequences should be separated and treated as distinct groups. Conclusion We conclude that the peptide segment recognized by SPase I extends to the start of the mature protein to a limited extent, upon our survey of the amino acid residues surrounding the cleavage processing site. These flanking residues possibly influence the cleavage processing and contribute to non-canonical cleavage sites. Our findings are applicable in defining more accurate prediction tools for recognition and identification of cleavage site of SPs.

  16. DNA-dispersed graphene/NiO hybrid materials for highly sensitive non-enzymatic glucose sensor

    International Nuclear Information System (INIS)

    Lv Wei; Jin Fengmin; Guo Quangui; Yang Quanhong; Kang Feiyu

    2012-01-01

    Highlights: ► We investigated the potential of GNS/NiO/DNA hybrid used as a nonenzymatic sensor. ► DNA is a highly efficient disperse agent for GNS/NiO hybrid than ionic surfactants. ► GNS/NiO/DNA hybrid shows fast electron transfer in the electrochemical reaction. ► GNS/NiO/DNA hybrid shows good detection performance towards glucose. - Abstract: We demonstrate graphene nanosheet/NiO hybrids (GNS/NiO) as the active material for high-performance non-enzymatic glucose sensors. Such sensors are fabricated by DNA-dispersed GNS/NiO suspension deposited on glassy carbon electrodes. ss-DNA shows strong dispersing ability for the GNS/NiO hybrid materials resulting in stable water-dispersible GNS/NiO/DNA hybrids with fully separated layers. The GNS/NiO/DNA hybrids show enhanced electron transfer in the electrocatalytic reaction process, and accordingly, such hybrids modified electrodes show good sensing performance towards glucose and are characterized by large detection ranges, short response periods, low detection limit and high sensitivity and stability.

  17. Current Status of HbA1c Biosensors

    Science.gov (United States)

    Lin, Hua; Yi, Jun

    2017-01-01

    Glycated hemoglobin (HbA1c) is formed via non-enzymatic glycosylation reactions at the α–amino group of βVal1 residues in the tetrameric Hb, and it can reflect the ambient glycemic level over the past two to three months. A variety of HbA1c detection methods, including chromatography, immunoassay, enzymatic measurement, electrochemical sensor and capillary electrophoresis have been developed and used in research laboratories and in clinics as well. In this review, we summarize the current status of HbA1c biosensors based on the recognition of the sugar moiety on the protein and also their applications in the whole blood sample measurements. PMID:28777351

  18. Yeast Metabolites of Glycated Amino Acids in Beer.

    Science.gov (United States)

    Hellwig, Michael; Beer, Falco; Witte, Sophia; Henle, Thomas

    2018-06-01

    Glycation reactions (Maillard reactions) during the malting and brewing processes are important for the development of the characteristic color and flavor of beer. Recently, free and protein-bound Maillard reaction products (MRPs) such as pyrraline, formyline, and maltosine were found in beer. Furthermore, these amino acid derivatives are metabolized by Saccharomyces cerevisiae via the Ehrlich pathway. In this study, a method was developed for quantitation of individual Ehrlich intermediates derived from pyrraline, formyline, and maltosine. Following synthesis of the corresponding reference material, the MRP-derived new Ehrlich alcohols pyrralinol (up to 207 μg/L), formylinol (up to 50 μg/L), and maltosinol (up to 6.9 μg/L) were quantitated for the first time in commercial beer samples by reverse phase high performance liquid chromatography tandem mass spectrometry in the multiple reaction monitoring mode. This is equivalent to ca. 20-40% of the concentrations of the parent glycated amino acids. The metabolites were almost absent from alcohol-free beers and malt-based beverages. Two previously unknown valine-derived pyrrole derivatives were characterized and qualitatively identified in beer. The metabolites investigated represent new process-induced alkaloids that may influence brewing yeast performance due to structural similarities to quorum sensing and metal-binding molecules.

  19. The clinical relevance of assessing advanced glycation endproducts accumulation in diabetes

    NARCIS (Netherlands)

    Meerwaldt, Robbert; Links, Thera; Zeebregts, Clark; Tio, Rene; Hillebrands, Jan-Luuk; Smit, Andries

    2008-01-01

    Cardiovascular disease is the major cause of morbidity and mortality associated with diabetes. There is increasing evidence that advanced glycation endproducts (AGEs) play a pivotal role in atherosclerosis, in particular in diabetes. AGE accumulation is a measure of cumulative metabolic and

  20. Enzymatic biodiesel production from sludge palm oil (SPO) using ...

    African Journals Online (AJOL)

    Biodiesel is a non-toxic, renewable and environmental friendly fuel. This study involved the production of biodiesel from sludge palm oil (SPO), a low-cost waste oil via enzymatic catalysis. The enzyme catalyst was a Candida cylindracea lipase, locally-produced using palm oil mill effluent as the low cost based medium.

  1. Rapid near infrared spectroscopy for prediction of enzymatic hydrolysis of corn bran after various pretreatments

    DEFF Research Database (Denmark)

    Baum, Andreas; Wittrup Agger, Jane; Meyer, Anne S.

    2012-01-01

    Efficient generation of a fermentable hydrolysate is a primary requirement in the utilization of fibrous plant biomass as feedstocks in bioethanol processes. The first biomass conversion step usually involves a hydrothermal pretreatment before enzymatic hydrolysis. The purpose of the pretreatment...... step is to increase the responsivity of the substrate to enzymatic attack and the type of pretreatment affects the enzymatic conversion efficiency. Destarched corn bran is a fibrous, heteroxylan-rich side-stream from the starch industry which may be used as a feedstock for bioethanol production...... release of different levels of arabinose, xylose and glucose from all the differently pretreated destarched corn bran samples. The present study also demonstrates a generic, non-destructive solution to determine the enzymatic monosaccharide release from polymers in biomass side-streams, thereby...

  2. The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) is enhanced by NPM-ALK

    DEFF Research Database (Denmark)

    Boccalatte, Francesco E; Voena, Claudia; Riganti, Chiara

    2009-01-01

    . A well-defined set of ALK-associated tyrosine phospho-peptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins was identified. Validation studies confirmed that VASP and ATIC associated with NPM-ALK and their phosphorylation required ALK activity. ATIC phosphorylation was also...... documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampering the methotrexate-mediated transformylase activity inhibition...

  3. Ascorbic acid glycation of lens proteins produces UVA sensitizers similar to those in human lens

    International Nuclear Information System (INIS)

    Ortwerth, B.J.; Linetsky, Mikhail; Olesen, P.R.

    1995-01-01

    Soluble calf lens proteins were extensively glycated during a 4 week incubation with ascorbic acid in the presence of oxygen. Amino acids analysis of the dialyzed proteins removed at weekly intervals showed an increasing loss of lysine, arginine and histidine, consistent with the extensive protein cross-linking observed. Irradiation of the dialyzed samples with UVA light (1.0 kJ/cm 2 total illumination through a 338 nm cutoff filter) caused an increasing loss of tryptophan, an additional loss of histidine and the production of micromolar concentrations of hydrogen peroxide. No alteration in amino acid content and no photolytic effects were seen in proteins incubated without ascorbic acid in proteins incubated with glucose for 4 weeks. The rate of hydrogen peroxide formation was linear with each glycated sample with a maximum production of 25 nmol/mg protein illuminated. The possibility that the sensitizer activity was due to an ascorbate-induced oxidation of tryptophan was eliminated by the presence of a heavy metal ion chelator during the incubation and by showing equivalent effects with ascorbate-incubated ribonuclease A, which is devoid of tryptophan. The ascorbate-incubated samples displayed increasing absorbance at wavelengths above 300 nm and increasing fluorescence (340/430) as glycation proceeded. The spectra of the 4 week glycated proteins were identical to those obtained with a solubilized water-insoluble fraction from human lens, which is known to have UVA sensitizer activity. (Author)

  4. Vaccination with lipid core peptides fails to induce epitope-specific T cell responses but confers non-specific protective immunity in a malaria model.

    Directory of Open Access Journals (Sweden)

    Simon H Apte

    Full Text Available Vaccines against many pathogens for which conventional approaches have failed remain an unmet public health priority. Synthetic peptide-based vaccines offer an attractive alternative to whole protein and whole organism vaccines, particularly for complex pathogens that cause chronic infection. Previously, we have reported a promising lipid core peptide (LCP vaccine delivery system that incorporates the antigen, carrier, and adjuvant in a single molecular entity. LCP vaccines have been used to deliver several peptide subunit-based vaccine candidates and induced high titre functional antibodies and protected against Group A streptococcus in mice. Herein, we have evaluated whether LCP constructs incorporating defined CD4(+ and/or CD8(+ T cell epitopes could induce epitope-specific T cell responses and protect against pathogen challenge in a rodent malaria model. We show that LCP vaccines failed to induce an expansion of antigen-specific CD8(+ T cells following primary immunization or by boosting. We further demonstrated that the LCP vaccines induced a non-specific type 2 polarized cytokine response, rather than an epitope-specific canonical CD8(+ T cell type 1 response. Cytotoxic responses of unknown specificity were also induced. These non-specific responses were able to protect against parasite challenge. These data demonstrate that vaccination with lipid core peptides fails to induce canonical epitope-specific T cell responses, at least in our rodent model, but can nonetheless confer non-specific protective immunity against Plasmodium parasite challenge.

  5. Subcritical Water Hydrolysis of Peptides: Amino Acid Side-Chain Modifications

    Science.gov (United States)

    Powell, Thomas; Bowra, Steve; Cooper, Helen J.

    2017-09-01

    Previously we have shown that subcritical water may be used as an alternative to enzymatic digestion in the proteolysis of proteins for bottom-up proteomics. Subcritical water hydrolysis of proteins was shown to result in protein sequence coverages greater than or equal to that obtained following digestion with trypsin; however, the percentage of peptide spectral matches for the samples treated with trypsin were consistently greater than for those treated with subcritical water. This observation suggests that in addition to cleavage of the peptide bond, subcritical water treatment results in other hydrolysis products, possibly due to modifications of amino acid side chains. Here, a model peptide comprising all common amino acid residues (VQSIKCADFLHYMENPTWGR) and two further model peptides (VCFQYMDRGDR and VQSIKADFLHYENPTWGR) were treated with subcritical water with the aim of probing any induced amino acid side-chain modifications. The hydrolysis products were analyzed by direct infusion electrospray tandem mass spectrometry, either collision-induced dissociation or electron transfer dissociation, and liquid chromatography collision-induced dissociation tandem mass spectrometry. The results show preferential oxidation of cysteine to sulfinic and sulfonic acid, and oxidation of methionine. In the absence of cysteine and methionine, oxidation of tryptophan was observed. In addition, water loss from aspartic acid and C-terminal amidation were observed in harsher subcritical water conditions. [Figure not available: see fulltext.

  6. Targeted separation of antibacterial peptide from protein hydrolysate of anchovy cooking wastewater by equilibrium dialysis.

    Science.gov (United States)

    Tang, Wenting; Zhang, Hui; Wang, Li; Qian, Haifeng; Qi, Xiguang

    2015-02-01

    Anchovy (Engraulis japonicus) cooking wastewater (ACWW) is a by-product resulted from the production of boiled-dried anchovies in the seafood processing industry. In this study, the protein hydrolysate of ACWW (ACWWPH) was found to have antimicrobial activity after enzymatic hydrolysis with Protamex. For the targeted screening of antibacterial peptides, liposomes constructed from Staphylococcus aureus membrane lipids were used in an equilibrium dialysis system. The hydrolysate was further purified by liposome equilibrium dialysis combined with high performance liquid chromatography. The purified antimicrobial peptide (ACWWP1) was determined to be GLSRLFTALK, with a molecular weight of 1104.6622Da. The peptide exhibited no haemolytic activity up to a concentration of 512μg/ml. It displayed a dose-dependent bactericidal effect in reconstituted milk. The change in cell surface hydrophobicity and membrane-permeable action of the purified ACWWP1 may have contributed to the antibacterial effect. This study suggests that liposome equilibrium dialysis can be used for the targeted screening of antimicrobial peptides. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Sequencing Cyclic Peptides by Multistage Mass Spectrometry

    Science.gov (United States)

    Mohimani, Hosein; Yang, Yu-Liang; Liu, Wei-Ting; Hsieh, Pei-Wen; Dorrestein, Pieter C.; Pevzner, Pavel A.

    2012-01-01

    Some of the most effective antibiotics (e.g., Vancomycin and Daptomycin) are cyclic peptides produced by non-ribosomal biosynthetic pathways. While hundreds of biomedically important cyclic peptides have been sequenced, the computational techniques for sequencing cyclic peptides are still in their infancy. Previous methods for sequencing peptide antibiotics and other cyclic peptides are based on Nuclear Magnetic Resonance spectroscopy, and require large amount (miligrams) of purified materials that, for most compounds, are not possible to obtain. Recently, development of mass spectrometry based methods has provided some hope for accurate sequencing of cyclic peptides using picograms of materials. In this paper we develop a method for sequencing of cyclic peptides by multistage mass spectrometry, and show its advantages over single stage mass spectrometry. The method is tested on known and new cyclic peptides from Bacillus brevis, Dianthus superbus and Streptomyces griseus, as well as a new family of cyclic peptides produced by marine bacteria. PMID:21751357

  8. Effects of alagebrium, an advanced glycation endproduct breaker, on exercise tolerance and cardiac function in patients with chronic heart failure

    NARCIS (Netherlands)

    Hartog, Jasper W. L.; Willemsen, Suzan; van Veldhuisen, Dirk J.; Posma, Jan L.; van Wijk, Leen M.; Hummel, Yoran M.; Hillege, Hans L.; Voors, Adriaan A.

    Aims Advanced glycation endproducts (AGEs) have been associated with the development and progression of chronic heart failure (CHF). Advanced glycation endproducts-crosslink breakers might be of benefit in HF, but only small-scale and uncontrolled data are available. Our aim was to conduct a

  9. Kinetic modelling of enzymatic starch hydrolysis

    NARCIS (Netherlands)

    Bednarska, K.A.

    2015-01-01

    Kinetic modelling of enzymatic starch hydrolysis – a summary

    K.A. Bednarska

    The dissertation entitled ‘Kinetic modelling of enzymatic starch hydrolysis’ describes the enzymatic hydrolysis and kinetic modelling of liquefaction and saccharification of wheat starch.

  10. Testing isotopic labeling with [¹³C₆]glucose as a method of advanced glycation sites identification.

    Science.gov (United States)

    Kielmas, Martyna; Kijewska, Monika; Stefanowicz, Piotr; Szewczuk, Zbigniew

    2012-12-01

    The Maillard reaction occurring between reducing sugars and reactive amino groups of biomolecules leads to the formation of a heterogeneous mixture of compounds: early, intermediate, and advanced glycation end products (AGEs). These compounds could be markers of certain diseases and of the premature aging process. Detection of Amadori products can be performed by various methods, including MS/MS techniques and affinity chromatography on immobilized boronic acid. However, the diversity of the structures of AGEs makes detection of these compounds more difficult. The aim of this study was to test a new method of AGE identification based on isotope (13)C labeling. The model protein (hen egg lysozyme) was modified with an equimolar mixture of [(12)C(6)]glucose and [(13)C(6)]glucose and then subjected to reduction of the disulfide bridges followed by tryptic hydrolysis. The digest obtained was analyzed by LC-MS. The glycation products were identified on the basis of characteristic isotopic patterns resulting from the use of isotopically labeled glucose. This method allowed identification of 38 early Maillard reaction products and five different structures of the end glycation products. This isotopic labeling technique combined with LC-MS is a sensitive method for identification of advanced glycation end products even if their chemical structure is unknown. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Characteristics and enhanced antioxidant activity of glycated Morchella esculenta protein isolate

    Directory of Open Access Journals (Sweden)

    Qiang ZHANG

    Full Text Available Abstract Morchella esculenta (L Pers. is a highly valued edible and medicinal fungus that remains underutilized. For this study, the effects of glycation treatment on antioxidant activity and characteristics of the M. esculenta protein isolate (MPI were investigated via the Maillard reaction. Conjugation between MPI and xylose was proven via UV-vis, FT-IR, intrinsic fluorescence analysis, and SDS-PAGE. Amino acid analysis revealed involvement of lysine, arginine and tyrosine in MPI, forming a covalent cross-link with xylose. Differential scanning calorimetry (DSC results showed that glycated MPI (MPIG possesses a more favorable thermal stability compared to native MPI (MPIN, heated MPI (MPIH and an unheated mixture of MPI and xylose (MPI-XM. MPIG exhibited significantly enhanced antioxidant activity compared to MPIN, MPIH, and MPI-XM. These results indicate MPIG can serve as a promising novel source of nutraceutical and functional ingredients that exert antioxidant activity.

  12. Determination of glycated hemoglobin in patients with advanced liver disease

    Science.gov (United States)

    Lahousen, Theresa; Hegenbarth, Karin; Ille, Rottraut; Lipp, Rainer W.; Krause, Robert; Little, Randie R.; Schnedl, Wolfgang J.

    2004-01-01

    AIM: To evaluate the glycated hemoglobin (HbA 1c) determination methods and to determine fructosamine in patients with chronic hepatitis, compensated cirrhosis and in patients with chronic hepatitis treated with ribavirin. METHODS: HbA1c values were determined in 15 patients with compensated liver cirrhosis and in 20 patients with chronic hepatitis using the ion-exchange high performance liquid chromatography and the immunoassay methods. Fructosamine was determined using nitroblue tetrazolium. RESULTS: Forty percent of patients with liver cirrhosis had HbA1c results below the non-diabetic reference range by at least one HbA1c method, while fructosamine results were either within the reference range or elevated. Twenty percent of patients with chronic hepatitis (hepatic fibrosis) had HbA1c results below the non -diabetic reference range by at least one HbA1c method. In patients with chronic hepatitis treated with ribavirin, 50% of HbA1c results were below the non-diabetic reference using at least one of the HbA1c methods. CONCLUSION: Only evaluated in context with all liver function parameters as well as a red blood count including reticulocytes, HbA 1c results should be used in patients with advanced liver disease. HbA 1c and fructosamine measurements should be used with caution when evaluating long-term glucose control in patients with hepatic cirrhosis or in patients with chronic hepatitis and ribavirin treatment. PMID:15259084

  13. Characterization of particulate drug delivery systems for oral delivery of Peptide and protein drugs.

    Science.gov (United States)

    Christophersen, Philip Carsten; Fano, Mathias; Saaby, Lasse; Yang, Mingshi; Nielsen, Hanne Mørck; Mu, Huiling

    2015-01-01

    Oral drug delivery is a preferred route because of good patient compliance. However, most peptide/ protein drugs are delivered via parenteral routes because of the absorption barriers in the gastrointestinal (GI) tract such as enzymatic degradation by proteases and low permeability acrossthe biological membranes. To overcome these barriers, different formulation strategies for oral delivery of biomacromolecules have been proposed, including lipid based formulations and polymer-based particulate drug delivery systems (DDS). The aim of this review is to summarize the existing knowledge about oral delivery of peptide/protein drugs and to provide an overview of formulationand characterization strategies. For a better understanding of the challenges in oral delivery of peptide/protein drugs, the composition of GI fluids and the digestion processes of different kinds of excipients in the GI tract are summarized. Additionally, the paper provides an overview of recent studies on characterization of solid drug carriers for peptide/protein drugs, drug distribution in particles, drug release and stability in simulated GI fluids, as well as the absorption of peptide/protein drugs in cell-based models. The use of biorelevant media when applicable can increase the knowledge about the quality of DDS for oral protein delivery. Hopefully, the knowledge provided in this review will aid the establishment of improved biorelevant models capable of forecasting the performance of particulate DDS for oral peptide/protein delivery.

  14. Regenerative, Highly-Sensitive, Non-Enzymatic Dopamine Sensor and Impact of Different Buffer Systems in Dopamine Sensing

    Directory of Open Access Journals (Sweden)

    Saumya Joshi

    2018-01-01

    Full Text Available Carbon nanotube field-effect transistors are used extensively in ultra-sensitive biomolecule sensing applications. Along with high sensitivity, the possibility of regeneration is highly desired in bio-sensors. An important constituent of such bio-sensing systems is the buffer used to maintain pH and provide an ionic conducting medium, among its other properties. In this work, we demonstrate highly-sensitive regenerative dopamine sensors and the impact of varying buffer composition and type on the electrolyte gated field effect sensors. The role of the buffer system is an often ignored condition in the electrical characterization of sensors. Non-enzymatic dopamine sensors are fabricated and regenerated in hydrochloric acid (HCl solution. The sensors are finally measured against four different buffer solutions. The impact of the nature and chemical structure of buffer molecules on the dopamine sensors is shown, and the appropriate buffer systems are demonstrated.

  15. High Hydrostatic Pressure-Assisted Enzymatic Treatment Improves Antioxidant and Anti-inflammatory Properties of Phosvitin.

    Science.gov (United States)

    Yoo, Heejoo; Bamdad, Fatemeh; Gujral, Naiyana; Suh, Joo-Won; Sunwoo, Hoon

    2017-01-01

    Phosvitin (PV) is a highly-phosphorylated metal-binding protein in egg yolk. Phosphoserine clusters make PV resistant to enzymatic digestion, which might be nutritionally undesirable. This study was designed to determine the effects of high hydrostatic pressure and enzymatic hydrolysis (HHP-EH) on the antioxidant and anti-inflammatory properties of PV hydrolysates (PVHs). PV was hydrolyzed by alcalase, elastase, savinase, thermolysin, and trypsin at 0.1, 50, and 100 MPa pressure levels. PVHs were evaluated for degree of hydrolysis, molecular weight distribution patterns, antioxidant and anti-inflammatory properties in chemical and cellular models. The effect of PVH on gene expression of pro-inflammatory cytokines (TNF-α and IL-1β) was also evaluated using real time-PCR. The hydrolysate with most potent antioxidant and anti-inflammatory properties was subjected to LC-MS/MS analysis to identify the peptide sequence. Hydrolysates produced at 100 MPa exhibited higher degree of hydrolysis and greater reducing power and free radical scavenging activity compared to those obtained at atmospheric pressure. After adjusting the phosphate content, alcalase- and trypsin-digested PVHs showed superior iron chelation capacity (69-73%), regardless of pressure. Both alcalase- and trypsin-digested PVHs significantly inhibited nitric oxide production by RAW264.7 macrophage cells. LPS-stimulated up-regulation of proinflammatory cytokines was also suppressed by alcalase-digested PVH. The HHP-EH method could play a promising role in the production of bioactive peptides from hydrolysis-resistant proteins. HHP-assisted PVH may be useful in preparing a potential pharmaceutical with antioxidant and anti-inflammatory properties. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. Hyperthermic responses to central injections of some peptide and non-peptide opioids in the guinea-pig

    Science.gov (United States)

    Kandasamy, S. B.; Williams, B. A.

    1983-01-01

    The intracerebroventricular administration of prototype nonpeptide opioid receptor (mu, kappa, and sigma) agonists, morphine, ketocyclazocine, and N-allyl normetazocine and an agonist at both kappa and sigma receptors, pentazocine, was found to induce hyperthermia in guinea pigs. The similar administration of peptide opioids like beta endorphin, methionine endkephalin, leucine endkephaline, and several of their synthetic analogues was also found to cause hyperthermia. Only the liver-like transport system of the three anion transport systems (iodide, hippurate, and liver-like) present in the choroid plexus was determined to be important to the central inactivation of beta-endorphin and two synthetic analogues. Prostaglandins and norepinephrine (NE) as well as cAMP were not involved in peptide and nonpeptide opioid-induced hyperthermia. Naloxone-sensitive receptors were found to be involved in the induction of hyperthermia by morphine and beta-endorphin, while hyperthermic responses to ketocyclazocine, N-allyl normetazocine, pentazocine, Met-enkephalin, Leu-enkephalin, and two of the synthetic analogues were not antagonized by nalozone. The lack of antagonism of naloxone on pyrogen, arachidonic acid, PGE2, dibutyryl cAMP, and NE-induced hyperthermia shows that endogenous opioid peptides are not likely to be central mediators of the hyperthermia induced by these agents.

  17. Diagnostic value of C-peptide determination

    International Nuclear Information System (INIS)

    Kober, G.; Rainer, O.H.

    1983-01-01

    C-peptide and insulin serum determinations were performed in 94 glucagon-stimulated diabetics and in 15 healthy persons. A minimal increase of 1.5 ng C-peptide/ml serum after glucagon injection (1 mg i.v.) was found to be a useful parameter for the differentiation of insulin dependent and non-insulin dependent diabetics. The maximal response to glucagon occurred during the first 10-minutes after the injection (blood was drawn at 2-minutes intervals). Serum insulin levels and basal C-peptide concentrations were of no value in predicting insulin-dependency. Basal C-peptide levels were significantly different from control in juvenile insulin dependent diabetics (decrease) only. (Author)

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

    DEFF Research Database (Denmark)

    Poulsen, Malene Wibe; Andersen, Jeanette Marker; Hedegaard, Rikke Susanne Vingborg

    2016-01-01

    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)...

  19. B-type natriuretic peptides. A diagnostic breakthrough in heart failure.

    Science.gov (United States)

    McCullough, P A

    2003-04-01

    B-type natriuretic peptide (BNP) is a neurohormone synthesized in the cardiac ventricles, which is released as N-terminal pro-brain natriuretic peptide (NT-proBNP) and then enzymatically cleaved in to the NT fragment and the immunoreactive BNP. Both tests have been used to identify patients with congestive heart failure (CHF). Important considerations for these tests include their half-lives in plasma, dependence on renal function for clearance, and the interpretation of their units of measure. In general, a BNP level below 100 pg/mL has strong negative predictive value in the assessment of patients with dyspnea caused by a disorder other than CHF. In addition, BNP levels can be used to gauge the effect of short-term treatment of acutely decompensated heart failure, and the peptide has been shown to be a reliable independent predictor of sudden cardiac death. In the absence of renal dysfunction NT-proBNP has also been shown to be an independent predictor of sudden death in CHF patients. Because both a large area of myonecrosis or concomitant left ventricular failure are related to prognosis in acute coronary syndromes, B-type natriuretic peptides have also been linked to outcomes in this condition. This article describes the physiology and timing of release of B-type natriuretic peptides and the rationale for their use in the following settings: 1) evaluation of decompensated CHF, 2) screening for chronic CHF, 3) prognosis of CHF and sudden death, and 4) prognosis in acute coronary syndromes with inferred left ventricular dysfunction.

  20. A Cuprous Oxide Thin Film Non-Enzymatic Glucose Sensor Using Differential Pulse Voltammetry and Other Voltammetry Methods and a Comparison to Different Thin Film Electrodes on the Detection of Glucose in an Alkaline Solution

    Directory of Open Access Journals (Sweden)

    Yifan Dai

    2018-01-01

    Full Text Available A cuprous oxide (Cu2O thin layer served as the base for a non-enzymatic glucose sensor in an alkaline medium, 0.1 NaOH solution, with a linear range of 50–200 mg/dL using differential pulse voltammetry (DPV measurement. An X-ray photoelectron spectroscopy (XPS study confirmed the formation of the cuprous oxide layer on the thin gold film sensor prototype. Quantitative detection of glucose in both phosphate-buffered saline (PBS and undiluted human serum was carried out. Neither ascorbic acid nor uric acid, even at a relatively high concentration level (100 mg/dL in serum, interfered with the glucose detection, demonstrating the excellent selectivity of this non-enzymatic cuprous oxide thin layer-based glucose sensor. Chronoamperometry and single potential amperometric voltammetry were used to verify the measurements obtained by DPV, and the positive results validated that the detection of glucose in a 0.1 M NaOH alkaline medium by DPV measurement was effective. Nickel, platinum, and copper are commonly used metals for non-enzymatic glucose detection. The performance of these metal-based sensors for glucose detection using DPV were also evaluated. The cuprous oxide (Cu2O thin layer-based sensor showed the best sensitivity for glucose detection among the sensors evaluated.

  1. Signal Diversity of Receptor for Advanced Glycation End Products.

    Science.gov (United States)

    Sakaguchi, Masakiyo; Kinoshita, Rie; Putranto, Endy Widya; Ruma, I Made Winarsa; Sumardika, I Wayan; Youyi, Chen; Tomonobu, Naoko; Yamamoto, Ken-Ichi; Murata, Hitoshi

    2017-12-01

    The receptor for advanced glycation end products (RAGE) is involved in inflammatory pathogenesis. It functions as a receptor to multiple ligands such as AGEs, HMGB1 and S100 proteins, activating multiple intracellular signaling pathways with each ligand binding. The molecular events by which ligand-activated RAGE controls diverse signaling are not well understood, but some progress was made recently. Accumulating evidence revealed that RAGE has multiple binding partners within the cytoplasm and on the plasma membrane. It was first pointed out in 2008 that RAGE's cytoplasmic tail is able to recruit Diaphanous-1 (Dia-1), resulting in the acquisition of increased cellular motility through Rac1/Cdc42 activation. We also observed that within the cytosol, RAGE's cytoplasmic tail behaves similarly to a Toll-like receptor (TLR4)-TIR domain, interacting with TIRAP and MyD88 adaptor molecules that in turn activate multiple downstream signals. Subsequent studies demonstrated the presence of an alternative adaptor molecule, DAP10, on the plasma membrane. The coupling of RAGE with DAP10 is critical for enhancing the RAGE-mediated survival signal. Interestingly, RAGE interaction on the membrane was not restricted to DAP10 alone. The chemotactic G-protein-coupled receptors (GPCRs) formyl peptide receptors1 and 2 (FPR1 and FPR2) also interacted with RAGE on the plasma membrane. Binding interaction between leukotriene B4 receptor 1 (BLT1) and RAGE was also demonstrated. All of the interactions affected the RAGE signal polarity. These findings indicate that functional interactions between RAGE and various molecules within the cytoplasmic area or on the membrane area coordinately regulate multiple ligand-mediated RAGE responses, leading to typical cellular phenotypes in several pathological settings. Here we review RAGE's signaling diversity, to contribute to the understanding of the elaborate functions of RAGE in physiological and pathological contexts.

  2. Computational study on a puzzle in the biosynthetic pathway of anthocyanin: Why is an enzymatic oxidation/ reduction process required for a simple tautomerization?

    Science.gov (United States)

    Sato, Hajime; Wang, Chao; Yamazaki, Mami; Saito, Kazuki; Uchiyama, Masanobu

    2018-01-01

    In the late stage of anthocyanin biosynthesis, dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS) mediate a formal tautomerization. However, such oxidation/reduction process requires high energy and appears to be unnecessary, as the oxidation state does not change during the transformation. Thus, a non-enzymatic pathway of tautomerization has also been proposed. To resolve the long-standing issue of whether this non-enzymatic pathway is the main contributor for the biosynthesis, we carried out density functional theory (DFT) calculations to examine this non-enzymatic pathway from dihydroflavonol to anthocyanidin. We show here that the activation barriers for the proposed non-enzymatic tautomerization are too high to enable the reaction to proceed under normal aqueous conditions in plants. The calculations also explain the experimentally observed requirement for acidic conditions during the final step of conversion of 2-flaven-3,4-diol to anthocyanidin; a thermodynamically and kinetically favorable concerted pathway can operate under these conditions.

  3. High-performance non-enzymatic catalysts based on 3D hierarchical hollow porous Co3O4 nanododecahedras in situ decorated on carbon nanotubes for glucose detection and biofuel cell application.

    Science.gov (United States)

    Wang, Shiyue; Zhang, Xiaohua; Huang, Junlin; Chen, Jinhua

    2018-03-01

    In this work, high-performance non-enzymatic catalysts based on 3D hierarchical hollow porous Co 3 O 4 nanododecahedras in situ decorated on carbon nanotubes (3D Co 3 O 4 -HPND/CNTs) were successfully prepared via direct carbonizing metal-organic framework-67 in situ grown on carbon nanotubes. The morphology, microstructure, and composite of 3D Co 3 O 4 -HPND/CNTs were characterized by scanning electron microscopy, transmission electron microscopy, micropore and chemisorption analyzer, and X-ray diffraction. The electrochemical characterizations indicated that 3D Co 3 O 4 -HPND/CNTs present considerably catalytic activity toward glucose oxidation and could be promising for constructing high-performance electrochemical non-enzymatic glucose sensors and glucose/O 2 biofuel cell. When used for non-enzymatic glucose detection, the 3D Co 3 O 4 -HPND/CNTs modified glassy carbon electrode (3D Co 3 O 4 -HPND/CNTs/GCE) exhibited excellent analytical performance with high sensitivity (22.21 mA mM -1  cm -2 ), low detection limit of 0.35 μM (S/N = 3), fast response (less than 5 s) and good stability. On the other hand, when the 3D Co 3 O 4 -HPND/CNTs/GCE worked as an anode of a biofuel cell, a maximum power density of 210 μW cm -2 at 0.15 V could be obtained, and the open circuit potential was 0.68 V. The attractive 3D hierarchical porous structural features, the large surface area, and the excellent conductivity based on the continuous and effective electron transport network in 3D Co 3 O 4 -HPND/CNTs endow 3D Co 3 O 4 -HPND/CNTs with the enhanced electrochemical performance and promising applications in electrochemical sensing, biofuel cell, and other energy storage and conversion devices such as supercapacitor. Graphical abstract High-performance non-enzymatic catalysts for enzymeless glucose sensing and biofuel cell based on 3D hierarchical hollow porous Co 3 O 4 nanododecahedras anchored on carbon nanotubes were successfully prepared via direct carbonizing

  4. HEMOGLOBINA GLICOSILADA O HEMOGLOBINA GLICADA, ¿CUÁL DE LAS DOS? | GLYCOSILATED HEMOGLOBIN OR GLYCATED HEMOGLOBIN, WHICH OF THE TWO?

    Directory of Open Access Journals (Sweden)

    Mariela Bracho-Nava

    2015-11-01

    , according to the International Federation of Clinical Chemistry (IFCC, as a generic term referring to a group of substances that are formed from biochemical reactions between hemoglobin A (HbA and some reducing sugars present in the bloodstream, glucose being the most abundant of them. This reaction is known as the Maillard reaction, which is based on a non-enzimatic glycosylation, or more correctly called, in a glycation. Custom, ignorance or confusion among both chemical processes has led to use the term glycosylated hemoglobin instead of glycated hemoglobin. This article provides a review of the process of formation of hemoglobin A1c, defining the reaction of glycosylation and the protein glycation, the chemical species that favor the glycation, the characteristics of the process of glycation of hemoglobin, stages in which it occurs and the effects related to the glycation of proteins in human beings, to finally conclude with a passage of designations which has received the HbA1c to the present; all with the aim of clarifying and giving property to the use of the term glycated hemoglobin.

  5. Generation of Soluble Advanced Glycation End Products Receptor (sRAGE)-Binding Ligands during Extensive Heat Treatment of Whey Protein/Lactose Mixtures Is Dependent on Glycation and Aggregation

    NARCIS (Netherlands)

    Liu, Fahui; Teodorowicz, Gosia; Wichers, Harry J.; Boekel, van Tiny; Hettinga, Kasper A.

    2016-01-01

    Heating of protein- and sugar-containing materials is considered the primary factor affecting the formation of advanced glycation end products (AGEs). This study aimed to investigate the influence of heating conditions, digestion, and aggregation on the binding capacity of AGEs to the soluble AGE

  6. Non-enzymatic hydrogen peroxide detection at NiO nanoporous thin film- electrodes prepared by physical vapor deposition at oblique angles

    International Nuclear Information System (INIS)

    Salazar, Pedro; Rico, Victor; González-Elipe, Agustín R.

    2017-01-01

    Highlights: • A non-enzymatic sensor for H 2 O 2 detection based on nickel thin film is reported. • Nanostructured nickel thin films are prepared by physical vapor deposition at oblique angles. • Main analytical parameters were obtained under optimal operation conditions. • Sensors depict an outstanding selectivity and a high stability. • Sensors are successfully used to determine H 2 O 2 in antiseptic solutions. - Abstract: In this work we report a non-enzymatic sensor for hydrogen peroxide (H 2 O 2 ) detection based on nanostructured nickel thin films prepared by physical vapor deposition at oblique angles. Porous thin films deposited on ITO substrates were characterized by X-ray diffraction analysis, scanning electron microcopy (SEMs), X-ray photoelectron spectroscopy (XPS) and electrochemical techniques such as Cyclic Voltammetry (CV) and Constant Potential Amperometry (CPA). The microstructure of the thin films consisted of inclined and separated Ni nanocolumns forming a porous thin layer of about 500 nm thickness. Prior to their use, the films surface was electrochemically modified and the chemical state studied by CV and XPS analysis. These techniques also showed that Ni 2+ /Ni 3+ species were involved in the electrochemical oxidation and detection of H 2 O 2 in alkaline medium. Main analytical parameters such as sensitivity (807 mA M −1 cm −2 ), limit of detection (3.22 μM) and linear range (0.011–2.4 mM) were obtained under optimal operation conditions. Sensors depicted an outstanding selectivity and a high stability and they were successfully used to determine H 2 O 2 concentration in commercial antiseptic solutions.

  7. Determination of the minimal fusion peptide of bovine leukemia virus gp30

    International Nuclear Information System (INIS)

    Lorin, Aurelien; Lins, Laurence; Stroobant, Vincent; Brasseur, Robert; Charloteaux, Benoit

    2007-01-01

    In this study, we determined the minimal N-terminal fusion peptide of the gp30 of the bovine leukemia virus on the basis of the tilted peptide theory. We first used molecular modelling to predict that the gp30 minimal fusion peptide corresponds to the 15 first residues. Liposome lipid-mixing and leakage assays confirmed that the 15-residue long peptide induces fusion in vitro and that it is the shortest peptide inducing optimal fusion since longer peptides destabilize liposomes to the same extent but not shorter ones. The 15-residue long peptide can thus be considered as the minimal fusion peptide. The effect of mutations reported in the literature was also investigated. Interestingly, mutations related to glycoproteins unable to induce syncytia in cell-cell fusion assays correspond to peptides predicted as non-tilted. The relationship between obliquity and fusogenicity was also confirmed in vitro for one tilted and one non-tilted mutant peptide

  8. Study on the C-peptide radioimmunoassay with synthetized connecting peptide

    Energy Technology Data Exchange (ETDEWEB)

    Nakagawa, S; Sasaki, T; Nakayama, H; Watanabe, T; Aoki, S [Hokkaido Univ., Sapporo (Japan). School of Medicine

    1976-01-01

    A method of C-peptide radioimmunoassay with the synthetized connecting peptide by Yanaihara was tested for the determination of serum C-peptide immunoreactivity (CPR) in normal people and in diabetics with or without insulin treatment. The CPR value obtained by this method was not interfered with by the presence of serum proteins or by the insulin of people with or without insulin treatment judged by the dilution test and the recovery test. The normal fasting CPR was 2.80 +- 0.78 ng/ml with the synthetized C-peptide as a standard. The CPR value increased and reached a maximum 90 minutes after the ingestion of 50 g of glucose. The increase after the glucose loading reduced corresponding to the severity of diabetes, and some juvenile-onset diabetes showed no response. Adult-type diabetics under insulin treatment, however, showed weak but significant CPR response. The increment of CPR and immunoreactive insulin after glucose loading in normal people and non-treated diabetics was well correlated (..gamma..=0.8262). Judged from the above mentioned results, CPR determination in insulin-treated diabetics was thought to be a useful method for the assessment of the insulin-secreting ability of beta-cells of the pancreas.

  9. Soluble Receptor for Advanced Glycation End Product: A Biomarker for Acute Coronary Syndrome

    Directory of Open Access Journals (Sweden)

    Louise J. N. Jensen

    2015-01-01

    Full Text Available The receptor of advanced glycation end products (RAGE and its ligands are linked to the pathogenesis of coronary artery disease (CAD, and circulating soluble receptor of advanced glycation end products (sRAGE, reflecting the RAGE activity, is suggested as a potential biomarker. Elevated sRAGE levels are reported in relation to acute ischemia and this review focuses on the role of sRAGE as a biomarker for the acute coronary syndrome (ACS. The current studies demonstrated that sRAGE levels are elevated in relation to ACS, however during a very narrow time period, indicating that the time of sampling needs attention. Interestingly, activation of RAGE may influence the pathogenesis and reflection in sRAGE levels in acute and stable CAD differently.

  10. One-pot non-enzymatic formation of firefly luciferin in a neutral buffer from p-benzoquinone and cysteine.

    Science.gov (United States)

    Kanie, Shusei; Nishikawa, Toshio; Ojika, Makoto; Oba, Yuichi

    2016-04-21

    Firefly luciferin, the substrate for the bioluminescence reaction of luminous beetles, possesses a benzothiazole ring, which is rare in nature. Here, we demonstrate a novel one-pot reaction to give firefly luciferin in a neutral buffer from p-benzoquinone and cysteine without any synthetic reagents or enzymes. The formation of firefly luciferin was low in yield in various neutral buffers, whereas it was inhibited or completely prevented in acidic or basic buffers, in organic solvents, or under a nitrogen atmosphere. Labelling analysis of the firefly luciferin using stable isotopic cysteines showed that the benzothiazole ring was formed via the decarboxylation and carbon-sulfur bond rearrangement of cysteine. These findings imply that the biosynthesis of firefly luciferin can be developed/evolved from the non-enzymatic production of firefly luciferin using common primary biosynthetic units, p-benzoquinone and cysteine.

  11. Fast and Selective Modification of Thiol Proteins/Peptides by N-(Phenylseleno)phthalimide

    Science.gov (United States)

    Wang, Zhengfang; Zhang, Yun; Zhang, Hao; Harrington, Peter B.; Chen, Hao

    2012-03-01

    We previously reported that selenamide reagents such as ebselen and N-(phenylseleno)phthalimide (NPSP) can be used to selectively derivatize thiols for mass spectrometric analysis, and the introduced selenium tags are useful as they could survive or removed with collision-induced dissociation (CID). Described herein is the further study of the reactivity of various protein/peptide thiols toward NPSP and its application to derivatize thiol peptides in protein digests. With a modified protocol (i.e., dissolving NPSP in acetonitrile instead of aqueous solvent), we found that quantitative conversion of thiols can be obtained in seconds, using NPSP in a slight excess amount (NPSP:thiol of 1.1-2:1). Further investigation shows that the thiol reactivity toward NPSP reflects its chemical environment and accessibility in proteins/peptides. For instance, adjacent basic amino acid residues increase the thiol reactivity, probably because they could stabilize the thiolate form to facilitate the nucleophilic attack of thiol on NPSP. In the case of creatine phosphokinase, the native protein predominately has one thiol reacted with NPSP while all of four thiol groups of the denatured protein can be derivatized, in accordance with the corresponding protein conformation. In addition, thiol peptides in protein/peptide enzymatic digests can be quickly and effectively tagged by NPSP following tri- n-butylphosphine (TBP) reduction. Notably, all three thiols of the peptide QCCASVCSL in the insulin peptic digest can be modified simultaneously by NPSP. These results suggest a novel and selective method for protecting thiols in the bottom-up approach for protein structure analysis.

  12. Effect of taurine on advanced glycation end products-induced hypertrophy in renal tubular epithelial cells

    International Nuclear Information System (INIS)

    Huang, J.-S.; Chuang, L.-Y.; Guh, J.-Y.; Yang, Y.-L.; Hsu, M.-S.

    2008-01-01

    Mounting evidence indicates that advanced glycation end products (AGE) play a major role in the development of diabetic nephropathy (DN). Taurine is a well documented antioxidant agent. To explore whether taurine was linked to altered AGE-mediated renal tubulointerstitial fibrosis in DN, we examined the molecular mechanisms of taurine responsible for inhibition of AGE-induced hypertrophy in renal tubular epithelial cells. We found that AGE (but not non-glycated BSA) caused inhibition of cellular mitogenesis rather than cell death by either necrosis or apoptosis. There were no changes in caspase 3 activity, bcl-2 protein expression, and mitochondrial cytochrome c release in BSA, AGE, or the antioxidant taurine treatments in these cells. AGE-induced the Raf-1/extracellular signal-regulated kinase (ERK) activation was markedly blocked by taurine. Furthermore, taurine, the Raf-1 kinase inhibitor GW5074, and the ERK kinase inhibitor PD98059 may have the ability to induce cellular proliferation and cell cycle progression from AGE-treated cells. The ability of taurine, GW5074, or PD98059 to inhibit AGE-induced hypertrophy was verified by the observation that it significantly decreased cell size, cellular hypertrophy index, and protein levels of RAGE, p27 Kip1 , collagen IV, and fibronectin. The results obtained in this study suggest that taurine may serve as the potential anti-fibrotic activity in DN through mechanism dependent of its Raf-1/ERK inactivation in AGE-induced hypertrophy in renal tubular epithelial cells

  13. Prior lactose glycation of caseinate via the Maillard reaction affects in vitro activities of the pepsin-trypsin digest toward intestinal epithelial cells.

    Science.gov (United States)

    Wang, X P; Zhao, X H

    2017-07-01

    The well-known Maillard reaction in milk occurs between lactose and milk proteins during thermal treatment, and its effects on milk nutrition and safety have been well studied. A lactose-glycated caseinate was prepared via this reaction and digested using 2 digestive proteases, pepsin and trypsin. The glycated caseinate digest was assessed for its in vitro activities on rat intestinal epithelial cells in terms of growth proliferation, anti-apoptotic effect, and differentiation induction using caseinate digest as reference, to verify potential effects of the Maillard reaction on these activities of caseinate digest to the cells. Two digests had proliferative and anti-apoptotic effects, and reached the highest effects at 0.02 g/L of digest concentration with treatment time of 24 h. In comparison with caseinate digest, glycated caseinate digest always showed weaker proliferative (5.3-14.2%) and anti-apoptotic (5.9-39.0%) effects, and was more toxic to the cells at 0.5 g/L of digest concentration with treatment time of 48 h. However, glycated caseinate digest at 2 incubation times of 4 to 7 d showed differentiation induction higher than caseinate digest, as it could confer the cells with increased activities in lactase (16.3-26.6%), sucrase (22.4-31.2%), and alkaline phosphatase (17.4-24.8%). Transmission electron microscopy observation results also confirmed higher differentiation induction of glycated caseinate digest. Amino acid loss and lactose glycation partially contributed to these decreased and enhanced activities of glycated caseinate digest, respectively. The Maillard reaction of caseinate and lactose is thus shown in this study to have effects on the activities of caseinate digest to intestinal epithelial cells. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  14. Cell-cycle regulation of non-enzymatic functions of the Drosophila methyltransferase PR-Set7.

    Science.gov (United States)

    Zouaz, Amel; Fernando, Céline; Perez, Yannick; Sardet, Claude; Julien, Eric; Grimaud, Charlotte

    2018-04-06

    Tight cell-cycle regulation of the histone H4-K20 methyltransferase PR-Set7 is essential for the maintenance of genome integrity. In mammals, this mainly involves the interaction of PR-Set7 with the replication factor PCNA, which triggers the degradation of the enzyme by the CRL4CDT2 E3 ubiquitin ligase. PR-Set7 is also targeted by the SCFβ-TRCP ligase, but the role of this additional regulatory pathway remains unclear. Here, we show that Drosophila PR-Set7 undergoes a cell-cycle proteolytic regulation, independently of its interaction with PCNA. Instead, Slimb, the ortholog of β-TRCP, is specifically required for the degradation of the nuclear pool of PR-Set7 prior to S phase. Consequently, inactivation of Slimb leads to nuclear accumulation of PR-Set7, which triggers aberrant chromatin compaction and G1/S arrest. Strikingly, these phenotypes result from non-enzymatic PR-Set7 functions that prevent proper histone H4 acetylation independently of H4K20 methylation. Altogether, these results identify the Slimb-mediated PR-Set7 proteolysis as a new critical regulatory mechanism required for proper interphase chromatin organization at G1/S transition.

  15. Biomass enzymatic saccharification is determined by the non-KOH-extractable wall polymer features that predominately affect cellulose crystallinity in corn.

    Science.gov (United States)

    Jia, Jun; Yu, Bin; Wu, Leiming; Wang, Hongwu; Wu, Zhiliang; Li, Ming; Huang, Pengyan; Feng, Shengqiu; Chen, Peng; Zheng, Yonglian; Peng, Liangcai

    2014-01-01

    Corn is a major food crop with enormous biomass residues for biofuel production. Due to cell wall recalcitrance, it becomes essential to identify the key factors of lignocellulose on biomass saccharification. In this study, we examined total 40 corn accessions that displayed a diverse cell wall composition. Correlation analysis showed that cellulose and lignin levels negatively affected biomass digestibility after NaOH pretreatments at pcorn samples indicated that cellulose and lignin should not be the major factors on biomass saccharification after pretreatments with NaOH and H2SO4 at three concentrations. Notably, despite that the non-KOH-extractable residues covered 12%-23% hemicelluloses and lignin of total biomass, their wall polymer features exhibited the predominant effects on biomass enzymatic hydrolysis including Ara substitution degree of xylan (reverse Xyl/Ara) and S/G ratio of lignin. Furthermore, the non-KOH-extractable polymer features could significantly affect lignocellulose crystallinity at pcorn.

  16. The Presence of Two Cyclase Thioesterases Expands the Conformational Freedom of the Cyclic Peptide Occidiofungin

    Science.gov (United States)

    Ravichandran, Akshaya; Gu, Ganyu; Escano, Jerome; Lu, Shi-En; Smith, Leif

    2014-01-01

    Occidiofungin is a cyclic nonribosomally synthesized antifungal peptide with submicromolar activity produced by Gram-negative bacterium Burkholderia contaminans. The biosynthetic gene cluster was confirmed to contain two cyclase thioesterases. NMR analysis revealed that the presence of both thioesterases is used to increase the conformational repertoire of the cyclic peptide. The loss of the OcfN cyclic thioesterase by mutagenesis results in a reduction of conformational variants and an appreciable decrease in bioactivity against Candida species. Presumably, the presence of both asparagine and β-hydroxyasparagine variants coordinate the enzymatic function of both of the cyclase thioesterases. OcfN has presumably evolved to be part of the biosynthetic gene cluster due to its ability to produce structural variants that enhance antifungal activity against some fungi. The enhancement of the antifungal activity from the incorporation of an additional cyclase thioesterase into the biosynthetic gene cluster of occidiofungin supports the need to explore new conformational variants of other therapeutic or potentially therapeutic cyclic peptides. PMID:23394257

  17. Non-enzymatic browning in citrus juice: chemical markers, their detection and ways to improve product quality.

    Science.gov (United States)

    Bharate, Sonali S; Bharate, Sandip B

    2014-10-01

    Citrus juices are widely consumed due to their nutritional benefits and variety of pharmacological properties. Non-enzymatic browning (NEB) is one of the most important chemical reactions responsible for quality and color changes during the heating or prolonged storage of citrus products. The present review covers various aspects of NEB in citrus juice viz. chemistry of NEB, identifiable markers of NEB, analytical methods to identify NEB markers and ways to improve the quality of citrus juice. 2,5-Dimethyl-4-hydroxy-3(2H)-furanone (DMHF) is one of the promising marker formed during browning process with number of analytical methods reported for its analysis; therefore it can be used as an indicator for NEB process. Amongst analytical methods reported, RP-HPLC is more sensitive and accurate method, which can be used as analytical tool. NEB can be prevented by removal of amino acids/ proteins (via ion exchange treatment) or by targeting NEB reactions (e.g. blockage of furfural/ HMF by sulphiting agent).

  18. Thermodynamically based solvent design for enzymatic saccharide acylation with hydroxycinnamic acids in non-conventional media

    DEFF Research Database (Denmark)

    Zeuner, Birgitte; Kontogeorgis, Georgios; Riisager, Anders

    2012-01-01

    as well as other enzymatic hydroxycinnamate acylations in ionic liquid systems. The choice of solvent system is highly decisive for enzyme stability, selectivity, and reaction yields in these synthesis reactions. To increase the understanding of the reaction environment and to facilitate solvent screening......-free microemulsions of a hydrocarbon, a polar alcohol, and water are interesting solvent systems because they accommodate different substrate and product solubilities and maintain enzyme stability. Ionic liquids may provide advantages as solvents in terms of increased substrate and product solubility, higher...... of their amphiphilicity and antioxidative potential. Synthetic reactions using mono- or disaccharides as one of the substrates may moreover direct new routes for biomass upgrading in the biorefinery. The paper reviews the available data for enzymatic hydroxycinnamate saccharide ester synthesis in organic solvent systems...

  19. Isolation and characterization of a thermolysin peptide containing acetyllysine from enzymatically acetylated f2al histone

    International Nuclear Information System (INIS)

    Horiuchi, Kentaro; Fujimoto, Daisaburo

    1973-01-01

    Previous studies (vol. 72, 433, '72) in this laboratory showed that histone acetylase in the cytosol of calf thymus introduced acetyl groups primarily into the epsilon-amino groups of lysine residues in a histone fraction, f2al. In an attempt to examine the site of acetylation in f2al by the enzyme, 14 C-acetylated f2al was isolated and digested by thermolysin. A radioactive peptide, which accounted for 50 - 60% of the total radioactivity, was obtained from the thermolysin digest and identified as the fragment containing amino acid residues 10-21. It appears, therefore, that the major sites of acetylation by the enzyme are the lysine 12 or 16 or both, which are known to be acetylated in vivo. It was also shown that the peptide was not deacetylated by histone deacetylase, in contrast with the whole f2al molecule. (author)

  20. Genetic determinants of glycated hemoglobin levels in the Greenlandic Inuit population

    DEFF Research Database (Denmark)

    Appel, Emil V R; Moltke, Ida; Jørgensen, Marit E

    2018-01-01

    We previously showed that a common genetic variant leads to a remarkably increased risk of type 2 diabetes (T2D) in the small and historically isolated Greenlandic population. Motivated by this, we aimed at discovering novel genetic determinants for glycated hemoglobin (HbA1C) and at estimating...