Taylor-Aris Dispersion-Assisted Mass Spectrometry for the Analysis of Native Proteins.

As has recently been shown, Taylor-Aris dispersion-assisted mass spectrometry (TADA-MS) can offer direct injection MS determinations in fields where the targets of the analyses are large molecules present in a matrix that would otherwise cause serious interferences. In the present study, we demonstrated the exceptional utility of TADA-MS in native protein analysis: (i) a dramatic improvement in detection sensitivity was found due to its ability to strongly reduce matrix interferences, (ii) more "native-like" conditions can be used during analyses, (iii) the direct injection of non-MS-compatible matrices is allowed into MS, and (iv) a considerable simplification and economization of the workflow is ensured. We investigated the behavior of different types of proteins and protein complexes present under native conditions, demonstrating the unambiguous benefits and simplicity of the method.

Wild Strawberry, Blackberry, and Blueberry Leaf Extracts Alleviate Starch-Induced Hyperglycemia in Prediabetic and Diabetic Mice.

Intestinal α-glucosidase and α-amylase break down nutritional poly- and oligosaccharides to monosaccharides and their activity significantly contributes to postprandial hyperglycemia. Competitive inhibitors of these enzymes, such as acarbose, are effective antidiabetic drugs, but have unpleasant side effects. In our ethnopharmacology inspired investigations, we found that wild strawberry (Fragaria vesca), blackberry (Rubus fruticosus), and European blueberry (Vaccinium myrtillus) leaf extracts inhibit α-glucosidase and α-amylase enzyme activity in vitro and are effective in preventing postprandial hyperglycemia in vivo. Toxicology tests on H9c2 rat embryonic cardiac muscle cells demonstrated that berry leaf extracts have no cytotoxic effects. Oral administration of these leaf extracts alone or as a mixture to normal (control), obese, prediabetic, and streptozotocin-induced diabetic mice attenuated the starch-induced rise of blood glucose levels. The efficiency was similar to that of acarbose on blood glucose. These results highlight berry leaf extracts as candidates for testing in clinical trials in order to assess the clinical significance of their effects on glycemic control.

Synthesis of New Series of 2-C-(ß-D-glucopyranosyl)-Pyrimidines and Their Evaluation as Inhibitors of Some Glycoenzymes.

Despite the substantial interest in C-glycosyl heterocycles as mimetics of biologically active native glycans, the appearance of C-glycopyranosyl derivatives of six-membered heterocycles, both in synthetic and biological contexts, is rather scarce. As part of our ongoing research program aimed at preparing hitherto barely known 2-C-glycopyranosyl pyrimidines, the goal of the present study was to synthesize new 5-mono- and multiply substituted derivatives of this compound class. Thus, 2-C-(ß-D-glucopyranosyl)-5,6-disubstituted-pyrimidin-4(3H)-ones and 4-amino-2-C-(ß-D-glucopyranosyl)-5,6-disubstituted-pyrimidines were prepared by base-mediated cyclocondensations of O-perbenzylated and O-unprotected C-(ß-D-glucopyranosyl) formamidine hydrochlorides with methylenemalonic acid derivatives. The 2-C-(ß-D-glucopyranosyl)-5-substituted-pyrimidines were obtained from the same amidine precursors upon treatment with vinamidinium salts. The deprotected derivatives of these pyrimidines were tested as inhibitors of some glycoenzymes. None of them showed inhibitory activity towards glycogen phosphorylase and α- and ß-glucosidase enzymes, but some members of the sets exhibited moderate inhibition against bovine liver ß-galactosidase.

HPLC method for measurement of human salivary α-amylase inhibition by aqueous plant extracts.

Control of hyperglycemia is an important treatment in metabolic disorders such as type II diabetes and obesity. α-Amylase, as the first enzyme of glucose release from dietary polysaccharides, is a potential target to identify new sources of novel anti-obesity and anti-diabetic drugs. In this work, different herbal extracts as α-amylase inhibitors were studied by measuring the rate of the cleavage of a maltooligomer substrate 2-chloro-4-nitrophenyl-ß-D-maltoheptoside. Measurement of chromophore containing products after reversed phase HPLC separation was used for α-amylase activity measurement. Rates of hydrolysis catalysed by human salivary α-amylase were determined in the presence and absence of lyophilised water extracts of eleven herbs. Remarkable bioactivities were found for extracts of Cinnamomum zeylanicum Blume (bark), Camellia sinensis L. (leaf), Ribes nigrum L. (leaf), Laurus nobilis L. (leaf), Vaccinium macrocarpon Aiton (fruit) and Syzygium aromaticum L. (bud). Determined IC50 values were in 0.017-41 µg/ml range for these six selected plant extracts. Our results confirm the applicability of this HPLC-based method for the quick and reliable comparison of plants as α-amylase inhibitors.

Simple ITC method for activity and inhibition studies on human salivary α-amylase.

Isothermal titration calorimetry (ITC) has an increasing significance in enzyme kinetic studies owing to its general applicability and sensitivity. In the present work, we aimed at developing a simple ITC-based screening procedure for the measurement of human salivary α-amylase (HSA) activity. Reaction of two substrates was studied with three independent methods (ITC, HPLC and spectrophotometry). ITC experiments were made using free and chromophore-containing maltooligomers of different length as substrates. Detailed studies revealed that maltoheptaose or longer oligomers could model properly starch and the presence of aromatic chromophore group did not affect the KM values considerably. It is the first time, when ITC was used to investigate of HSA-catalysed hydrolysis of different substrates (2-chloro-4-nitrophenyl-4-O-α-D-galactopyranosyl-maltoside, maltoheptaose and starch) in the presence of acarbose inhibitor. All measured IC50 values are in micromolar range (0.9, 18.6 and 29.0 µM, respectively) and increased in parallel with the degree of polymerisation of substrates.

A comparative study of capillary electrophoresis and isothermal titration calorimetry for the determination of binding constant of human serum albumin to monoclonal antibody.

This paper focuses on the investigation of the interactions between the anti-HSA-mAb and its protein antigen using CZE, ACE, and isothermal titration calorimetry. The CZE revealed the formation of the anti-HSA-mAb·HSA and anti-HSA-mAb·(HSA)2 complexes and the binding constants determined by plotting the amount of the bound anti-HSA-mAb as a function of the concentration of HSA. The ACE provided information on the binding strength from the change in effective electrophoretic mobility of the anti-HSA-mAb. These two separation techniques estimated the presence of two binding sites. The equilibrium dissociation constant values obtained by CZE and ACE were found to be 2.26 × 10(-6) M for anti-HSA-mAb·HSA, 1.22 × 10(-6) M for anti-HSA-mAb·(HSA)2 and 4.45 × 10(-8) M for anti-HSA-mAb·HSA, 1.08 × 10(-7) M for anti-HSA-mAb·(HSA)2 , respectively. The dissociation constant data obtained by ACE were in congruence with the values obtained by isothermal titration calorimetry (2.74 × 10(-8) M, 1.04 × 10(-7) M).

From carbohydrates to drug-like fragments: Rational development of novel α-amylase inhibitors.

Starch catabolism leading to high glucose level in blood is highly problematic in chronic metabolic diseases, such as type II diabetes and obesity. α-Amylase catalyzes the hydrolysis of starch, increasing blood sugar concentration. Its inhibition represents a promising therapeutic approach to control hyperglycaemia. However, only few drug-like molecule inhibitors without sugar moieties have been discovered so far, and little information on the enzymatic mechanism is available. This work aims at the discovery of novel small α-amylase binders using a systematic in silico methodology. 3D-pharmacophore-based high throughput virtual screening of small compounds libraries was performed to identify compounds with high α-amylase affinity. Twenty-seven compounds were selected and biologically tested, revealing IC50 values in the micromolar range and ligand efficiency higher than the one of the bound form of acarbose, which is used as a reference for α-amylase inhibition.

Unexpected mode of action of sweet potato ß-amylase on maltooligomer substrates.

ß-Amylase (EC 3.2.1.2), one of the main protein of the sweet potato, is an exo-working enzyme catalyzing the hydrolysis of α(1,4) glycosidic linkages in polysaccharides and removes successively maltose units from the non-reducing ends. The enzyme belongs to glycoside hydrolase GH14 family and inverts the anomeric configuration of the hydrolysis product. Multiple attack or processivity is an important property of polymer active enzymes and there is still limited information about the processivity of carbohydrate active enzymes. Action pattern and kinetic measurements of sweet potato ß-amylase were made on a series of aromatic chromophor group-containing substrates (degree of polymerization DP 3-13) using HPLC method. Measured catalytic efficiencies increased with increasing DP of the substrates. Processive cleavage was observed on all substrates except the shortest pentamer. The mean number of steps without dissociation of enzyme-product complex increases with DP of substrate and reached 3.3 in case of CNPG11 indicating that processivity on longer substrates was more significant. A unique transglycosylation was observed on those substrates, which suffer processive cleavage and the substrates were re-built by the enzyme. Our results are the first presentation of a transglycosylation during an inverting glycosidase catalyzed hydrolysis. The yield of transglycosylation was remarkable high as shown in the change of the CNPG11 quantity. The CNPG11 concentration was doubled (from 0.24 to 0.54mM) in the early phase of the reaction.

Variability of microcystins and its synthetase gene cluster in Microcystis and Planktothrix waterblooms in shallow lakes of Hungary.

Waterbloom samples of Microcystis aeruginosa and Planktothrix agardhii were collected from a variety of ponds, lakes and reservoirs in Hungary. Samples were tested with matrix-assisted laser desorption/ionization - time-of-flight mass spectrometry (MALDI-TOF MS) to identify the microcystin forms. The concentration of the microcystins was measured with capillary electrophoresis and the toxicity was tested by sinapis test. DNA was extracted from the samples and tested using a range of primers linked to the biosynthesis of microcystin. All of the fourteen collected samples gave positive results for the presence of the mcy genes with PCR products with sizes between of 425 and 955 bp, respectively, indicating the presence of the genes implicated in the production of microcystins. The results showed that a wide range of microcystin (MC) forms were detected in the Microcystis containing samples, among which MC-LR, -RR, and -YR were the most common. The highest MC concentration was 15,701 mg g-1, which was detected in an angling pond. The samples containing Planktothrix agardhii were less toxic, and the most common form in this species was the Asp3-MC-LR.

Determination of phenylethanoid glycosides and iridoid glycosides from therapeutically used Plantago species by CE-MEKC.

CE methods are valuable tools for medicinal plant quality management, screening, and analysis. Therefore, the aim of the current study was to optimize and validate a CE-MEKC method for simultaneous quantification of four chief bioactive metabolites from Plantago species. The two most important secondary metabolite groups were aimed to be separated. Different electrolyte and surfactant types were tested. Surfactant concentration, BGE pH, electrolyte concentration, and buffering capacity were optimized. The final BGE consisted of 15 mM sodium tetraborate, 20 mM TAPS, and 250 mM DOC at pH 8.50. Acceptable precision, good stability, and accuracy were achieved, with high resolution for phenylethanoid glycosides. Analytes were separated within 20 min. The method was shown to be suitable for the quantification of the iridoid glycosides aucubin and catalpol, and the phenylethanoid glycosides acteoside (verbascoside) and plantamajoside from water extracts of different samples. The method was shown to be applicable to leaf extracts of Plantago lanceolata, Plantago major, and Plantago asiatica, the main species with therapeutic applications, and a biotechnological product, plant tissue cultures (calli) of P. lanceolata. Baseline separation of the main constituents from minor peaks was achieved, regardless of the matrix type.

Towards high-siderophore-content foods: optimisation of coprogen production in submerged cultures of Penicillium nalgiovense.

BACKGROUND: Fungal siderophores are likely to possess atheroprotective effects in humans, and therefore studies are needed to develop siderophore-rich food additives or functional foods to increase the siderophore uptake in people prone to cardiovascular diseases. In this study the siderophore contents of mould-ripened cheeses and meat products were analysed and the coprogen production by Penicillium nalgiovense was characterised. RESULTS: High concentrations of hexadentate fungal siderophores were detected in penicillia-ripened Camembert- and Roquefort-type cheeses and also in some sausages. In one sausage fermented by P. nalgiovense, the siderophore content was comparable to those found in cheeses. Penicillium nalgiovense produced high concentrations of coprogen in submerged cultures, which were affected predominantly by the available carbon and nitrogen sources under iron starvation. Considerable coprogen yields were still detectable in the presence of iron when the fermentation medium was supplemented with the iron chelator Na2-EDTA or when P. nalgiovense was co-cultivated with Saccharomyces cerevisiae. CONCLUSION: These data may be exploitable in the future development of high-siderophore-content foods and/or food additives. Nevertheless, the use of P. nalgiovense fermentation broths for these purposes may be limited by the instability of coprogen in fermentation media and by the ß-lactam production by the fungus.

Effect of high relative humidity on dried Plantago lanceolata L. leaves during long-term storage: effects on chemical composition, colour and microbiological quality.

INTRODUCTION: Modern phytotherapy and quality assurance requires stability data on bioactive metabolites to identify and minimise decomposing factors during processing and storage. A compound's stability in a complex matrix can be different from the stability of the purified compound. OBJECTIVE: To test the stability of iridoids and acteoside and quantify changes in colour and microbiological quality in a common herbal tea, dried P. lanceolata leaves during exposure to high-humidity air. To test the contribution of fungi to metabolite decomposition. METHODOLOGY: Dried P. lanceolata leaves were exposed to atmospheres of different relative humidity (75, 45 and 0%) for 24 weeks. Changes in aucubin and catalpol concentration were determined by CE-MEKC, and those in acteoside on TLC. Colour and chlorophyll-like pigments were measured by different spectrophotometric methods. The number of fungi was monitored; 10 strains were isolated from the plant drug, and their ability to decompose the analytes of interest was tested. RESULTS: During incubation at 75% relative humidity (RH), aucubin, catalpol and acteoside concentrations decreased by 95.7, 97.0 and 70.5%, respectively. Strong shifts were detected in CIELAB parameters a* and b* (browning) as a result of conversion of chlorophyll to pheophytin. Intensive microbial proliferation was also observed. Changes at 45 or 0% RH were typically insignificant. Seven of the 10 isolated fungal strains could decompose both iridoids, and five could decompose acteoside in vitro. CONCLUSION: It was shown that exposure to water results in loss of bioactive molecules of P. lanceolata dried leaves, and that colonising fungi are the key contributors to this loss.

Observation of sward destruction caused by irrigation with toxic Microcystis morphospecies containing water in Southern Hungary.

In the summer of 2006 bloom-like phenomenon occurred in a garden pond in Szeged, Southern Hungary. After regular watering of a sward with pond water containing the algal mass, destruction of garden grass occurred. Microcystis aeruginosa, Microcystis viridis, Microcystis ichthyoblabe, and Microcystis wesenbergii were identified by light microscopy in the water sample; microcystin-FR, -LR, -RR and -YR were determined by matrix-assisted laser desorption/ionization--time-of-flight analysis. There was an 80% decrease in the green mass (87% in chlorophyll-content) of the grass in a 1 m² area of the garden irrigated with pond water.

Extending the investigation of 4-thiazolidinone derivatives as potential multi-target ligands of enzymes involved in diabetes mellitus and its long-term complications: A study with pancreatic α-amylase.

Diabetes mellitus is a multifactorial disease, which is frequently complicated by the development of hyperglycaemia-induced chronic complications. The therapy of diabetes mellitus often requires combinations of two or more drugs in order both to control glycaemic levels and to prevent hyperglycaemia-induced dangerous affairs. The application of multi-target agents, which are able to control simultaneously several pathogenic mechanisms, represents a useful alternative and, in fact, their discovery is a pursued aim of the research. Some (5-arylidene-4-oxo-2-thioxothiazolidin-3-yl)acetic acids, which we had previously reported as inhibitors of selected enzymes critically implicated in diabetes mellitus, were tested against pancreatic α-amylase and intestinal α-glucosidase. These enzymes catalyse the hydrolysis of dietary oligo- and polysaccharides into monosaccharides and, consequently, are responsible for postprandial hyperglycaemia; therefore, their inhibition is one of the possible strategies to control glycaemic levels in diabetes mellitus. In addition, we investigated the aggregation tendency of the tested compounds, through direct and indirect methods, in order to evaluate the mechanism of their multiple action and discover if aggregation may contribute to the inhibition of the target enzymes. Overall, compounds 1, 3 and 4 exhibited the most favourable profile since they were shown to act as multi-target inhibitors of enzymes involved in pathways related to diabetes mellitus, without producing aggregates even at high micromolar concentrations and, therefore, can be promising agents for further developments.

Gallotannins are non-specific inhibitors of α-amylase: Aggregates are the active species taking part in inhibition.

The versatile biological activity of gallotannins has been investigated for a long time, including their use as α-amylase inhibitors for the treatment of diabetes and its complications. The effectiveness of gallotannins on a wide range of enzymes refers to promiscuity. We proved that gallotannins are non-specific promiscuous α-amylase inhibitors, which exert their effect through their aggregates. A gallotannin of Aleppo oak origin fulfilled all the criteria for aggregators; significant changes could be observed in the IC50 values in the presence of Triton™ X-100 detergent (from 2.3 to 110 µg/ml) and after enzyme-inhibitor preincubation (from 2.3 to 0.65 µg/ml). Increasing the enzyme concentration also led to the moderation of the inhibition by gallotannin. In addition, we observed that gallotannin molecules are those, which are involved in aggregation, and discrete protein molecules are adsorbed to the aggregates. This was revealed by the increasing particle size of gallotannin, which became three orders of magnitude higher after 150 min, whereas the size of α-amylase remained unchanged. Consequently, gallotannins should be used as anti-diabetic drugs only if the necessity of higher dose due to their promiscuity is taken into account. Aggregation propensity should not be ignored in case of in vivo applications.

Colorado potato beetle alpha-amylase: Purification, action pattern and subsite mapping for exploration of active centre.

Colorado potato beetle is an invasive insect herbivore and one of the most challenging agricultural pests globally. This study is the first characterization of the active centre of Colorado potato beetle (Leptinotarsa decemlineata) α-amylase (LdAmy). Bond cleavage frequency values for LdAmy were determined by HPLC product analysis on a chromophore labelled maltooligomer substrate series. Binding energies between amino acid moieties of subsites and glucose residues of substrate were calculated. Active site contains six subsites in the binding region of LdAmy; four glycone- (-4, -3, -2, -1) and two aglycone-binding sites (+1, +2). Subsite map calculation resulted in apparent binding energies -11.8 and - 11.0 kJ/mol for subsites (+2) and (-3), respectively, which revealed very favorable interactions at these positions. Structures of binding sites of LdAmy and mammalian α-amylases show similarity, but there are variations in the binding energies at subsite (-2) and (-4). Differences were interpreted by comparison of amino acid sequences of human salivary α-amylase (HSA) and porcine pancreatic α-amylase (PPA) and two insect (Leptinotarsa decemlineata and Tenebrio molitor) enzymes. The observed substitution of positively charged His305 in HSA at subsite (-2) with an acidic Asp in LdAmy in the same position may explain the obtained energy reduction.

Evidence for pentagalloyl glucose binding to human salivary alpha-amylase through aromatic amino acid residues.

We demonstrate here that pentagalloyl glucose (PGG), a main component of gallotannins, was an effective inhibitor of HSA and it exerted similar inhibitory potency to Aleppo tannin used in this study. The inhibition of HSA by PGG was found to be non-competitive and inhibitory constants of K(EI)=2.6 microM and K(ESI)=3.9 microM were determined from Lineweaver-Burk secondary plots. PGG as a model compound for gallotannins was selected to study the inhibitory mechanism and to characterize the interaction of HSA with this type of molecules. Surface plasmon resonance (SPR) binding experiments confirmed the direct interaction of HSA and PGG, and it also established similar binding of Aleppo tannin to HSA. Saturation transfer difference (STD) experiment by NMR clearly demonstrated the aromatic rings of PGG may be involved in the interaction suggesting a possible stacking with the aromatic side chains of HSA. The role of aromatic amino acids of HSA in PGG binding was reinforced by kinetic studies with the W58L and Y151M mutants of HSA: the replacement of the active site aromatic amino acids with aliphatic ones decreased the PGG inhibition dramatically, which justified the importance of these residues in the interaction.

Development and characterization of a FIA system for selective assay of L-ascorbic acid in food samples.

An amperometric detector and an enzymatic reaction were combined for the measurement of L-ascorbic acid. The enzyme cell (containing immobilized ascorbate oxidase) was connected to a flow injection analyzer (FIA) system with a glassy carbon electrode as an amperometric detector. During optimization and measurements two sample injectors were used, one before and one after the enzyme cell, thus eliminating the background interferences. Subtraction of the signal area given in the presence of enzyme from the one given in the absence of enzyme was applied for measuring analyte concentrations and calibration at 400 mV. Analysis capacity of system is 25 samples/hour. The relative standard deviation (RSD) was below 5% (5 times repeated, 400 µmol/L conc.), linearity up to 400 µmol/L, limit of detection (LOD) 5 µmol/L, fitting of calibration curve in 25-400 µmol/L range was R (2) = 0.99.

Characterization of gfdB, putatively encoding a glycerol 3-phosphate dehydrogenase in Aspergillus nidulans.

The genome of Aspergillus nidulans accommodates two glycerol 3-phosphate dehydrogenase genes, gfdA and gfdB. Previous studies confirmed that GfdA is involved in the osmotic stress defence of the fungus. In this work, the physiological role of GfdB was characterized via the construction and functional characterization of the gene deletion mutant ΔgfdB. Unexpectedly, ΔgfdB strains showed oxidative stress sensitivity in the presence of a series of well-known oxidants including tert-butyl-hydroperoxide (tBOOH), diamide as well as hydrogen peroxide. Moderate sensitivity of the mutant towards the cell wall stress inducing agent CongoRed was also observed. Hence, both Gfd isoenzymes contributed to the environmental stress defence of the fungus but their functions were stress-type-specific. Furthermore, the specific activities of certain antioxidant enzymes, like catalase and glutathione peroxidase, were lower in ΔgfdB hyphae than those recorded in the control strain. As a consequence, mycelia from ΔgfdB cultures accumulated reactive species at higher levels than the control. On the other hand, the specific glutathione reductase activity was higher in the mutant, most likely to compensate for the elevated intracellular oxidative species concentrations. Nevertheless, the efficient control of reactive species failed in ΔgfdB cultures, which resulted in reduced viability and, concomitantly, early onset of programmed cell death in mutant hyphae. Inactivation of gfdB brought about higher mannitol accumulation in mycelia meanwhile the erythritol production was not disturbed in unstressed cultures. After oxidative stress treatment with tBOOH, only mannitol was detected in both mutant and control mycelia and the accumulation of mannitol even intensified in the ΔgfdB strain.

Two secondary carbohydrate binding sites on the surface of barley alpha-amylase 1 have distinct functions and display synergy in hydrolysis of starch granules.

Some polysaccharide processing enzymes possess secondary carbohydrate binding sites situated on the surface far from the active site. In barley alpha-amylase 1 (AMY1), two such sites, SBS1 and SBS2, are found on the catalytic (beta/alpha)(8)-barrel and the noncatalytic C-terminal domain, respectively. Site-directed mutagenesis of Trp(278) and Trp(279), stacking onto adjacent ligand glucosyl residues at SBS1, and of Tyr(380) and His(395), making numerous ligand contacts at SBS2, suggested that SBS1 and SBS2 act synergistically in degradation of starch granules. While SBS1 makes the major contribution to binding and hydrolysis of starch granules, SBS2 exhibits a higher affinity for the starch mimic beta-cyclodextrin. Compared to that of wild-type AMY1, the K(d) of starch granule binding by the SBS1 W278A, W279A, and W278A/W279A mutants thus increased 15-35 times; furthermore, the k(cat)/K(m) of W278A/W279A was 2%, whereas both affinity and activity for Y380A at SBS2 were 10% of the wild-type values. Dual site double and triple SBS1/SBS2 substitutions eliminated binding to starch granules, and the k(cat)/K(m) of W278A/W279A/Y380A AMY1 was only 0.4% of the wild-type value. Surface plasmon resonance analysis of mutants showed that beta-cyclodextrin binds to SBS2 and SBS1 with K(d,1) and K(d,2) values of 0.07 and 1.40 mM, respectively. A model that accounts for the observed synergy in starch hydrolysis, where SBS1 and SBS2 bind ordered and free alpha-glucan chains, respectively, thus targeting the enzyme to single alpha-glucan chains accessible for hydrolysis, is proposed. SBS1 and SBS2 also influence the kinetics of hydrolysis for amylose and maltooligosaccharides, the degree of multiple attack on amylose, and subsite binding energies.