Analysis of dihydroethidium fluorescence for the detection of intracellular and extracellular superoxide produced by NADPH oxidase

All methods used for quantitation of superoxide have limitations when it comes to differentiating between extracellular and intracellular sites of superoxide production. In the present study, we monitored dihydroethidium (DHE)-derived fluorescence at 570 nm, which indicates hydroxyethidium derived from reaction with superoxide produced by human leukemia cells (HL-60) and microvascular endothelial cells (HMEC-1). Phorbol-12-myristate 13-acetate (PMA; 100 ng/ml) caused an increase in fluorescence and lucigenin chemiluminescence in HL-60, which was abolished by superoxide dismutase (SOD; 600 U/ml) indicating that DHE detects extracellular superoxide. Furthermore, both HL-60 cells and HMEC-1 generated a fluorescence signal in the presence of DHE under resting conditions, which was unaffected by SOD, but abolished by polyethylene glycosylated-SOD (PEG-SOD) (100 U/ml) and MnTmPyP (25 μM), indicating that DHE also detects superoxide produced intracellularly. In HMEC-1, silencing of either Nox2 or Nox4 components of NADPH oxidase with small interference RNA (siRNA) resulted in a significant reduction in superoxide detected by both DHE fluorescence (Nox2 siRNA; 71 ± 6% and Nox4 siRNA 83 ± 7% of control) and lucigenin chemiluminescence (Nox2; 54 ± 6% and Nox4 74 ± 4% of control). In conclusion, DHE-derived fluorescence at 570 nm is a convenient method for detection of intracellular and extracellular superoxide produced by phagocytic and vascular NADPH oxidase.     

Thermal inactivation, denaturation and aggregation of mitochondrial aspartate aminotransferase

A comparative study of thermal denaturation and inactivation of aspartate aminotransferase from pig heart mitochondria (mAAT) has been carried out (10 mM Na phosphate buffer, pH 7.5). Analysis of the data on differential scanning calorimetry shows that thermal denaturation of mAAT follows the kinetics of irreversible reaction of the first order. The kinetics of thermal inactivation of mAAT follows the exponential law. It has been shown that the inactivation rate constant (k_in) is higher than the denaturation rate constant (k_den). The k_in/k_den ratio decreases from 28.8 ± 0.1 to 1.30 ± 0.09 as the temperature increases from 57.5 to 77 °C. The kinetic model explaining the discrepancy between the inactivation and denaturation rates has been proposed. The size of the protein aggregates formed at heating of mAAT at a constant rate (1 °C min^{− 1}) has been characterized by dynamic light scattering.     

Characteristics of galactomannanase for degrading konjac gel

Galactomannanase (Glmnase) is an enzyme product derived from Aspergillus niger. The activity of Glmnase degrading (hydrolyzing) the konjac gel were investigated. Significant loss in the enzyme activity was found when the temperature above 60 °C. Similar observations were obtained when the reaction pH above 5. Further increase in the pH value resulted in entirely loss of enzyme activity at the alkaline pH region (pH 8.0 and above). The optimal hydrolyzing temperature and pH were at 60 °C and 5.0, respectively. For the stability test, the purified Glmnase increased its thermostability up to 70 °C at pH 5.0, but it retained only about 60% activity after 60 min incubation at this temperature and its activity became zero after 20 min incubation at 80 °C. The Glmnase was stable at the pH range from 3.0 to 7.0 at room temperature and retained at least 80% activity for 60 min. For the storage temperature test, the lyophilized Glmnase still conserved about 90% activity during 7 days at 30 °C, and was higher than about 80% at 4 °C. The K_m and V_max, were 0.018 mg/ml konjac powder and 0.20 mg/ml reducing sugar per min, respectively.     

Effect of phosphate on the kinetics and specificity of glycation of protein

The glycation (nonenzymatic glycosylation) of several proteins was studied in various buffers in order to assess the effects of buffering ions on the kinetics and specificity of glycation of protein. Incubation of RNase with glucose in phosphate buffer resulted in inactivation of the enzyme because of preferential modification of lysine residues in or near the active site. In contrast, in the cationic buffers, 3-(N-morpholino)propane-sulfonic acid and 3-(N-tris(hydroxymethyl)methyl-amino)-2-hydroxypropanesulfonic acid, the kinetics of glycation of RNase were decreased 2- to 3-fold, there was a decrease in glycation of active site versus peripheral lysines, and the enzyme was resistant to inactivation by glucose. The extent of Schiff base formation on RNAse was comparable in the three buffers, suggesting that phosphate, bound in the active site of RNase, catalyzed the Amadori rearrangement at active site lysines, leading to the enhanced rate of inactivation of the enzyme. Phosphate catalysis of glycation was concentration-dependent and could be mimicked by arsenate. Phosphate also stimulated the rate of glycation of other proteins, such as lysozyme, cytochrome c, albumin, and hemoglobin. As with RNase, phosphate affected the specificity of glycation of hemoglobin, resulting in increased glycation of amino-terminal valine versus intrachain lysine residues. 2,3-Diphosphoglycerate exerted similar effects on the glycation of hemoglobin, suggesting that inorganic and organic phosphates may play an important role in determining the kinetics and specificity of glycation of hemoglobin in the red cell. Overall, these studies establish that buffering ions or ligands can exert significant effects on the kinetics and specificity of glycation of proteins.     

Thermal stability of Artemia HGPRT: effect of substrates on inactivation kinetics

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT, E.C. 2.4.2.8) from Artemia cysts exhibits maximum activity at 70°C. Its thermal stability has been examined following enzymatic activity as a function of temperature. Cold-induced renaturation experiments of samples heated at increasing temperatures showed that reversibility of thermal inactivation depends on the incubation time and final temperature. Prolonged incubation of the thermoinactivated enzyme at 0°C did not afford any further increase of the catalytic activity at 37°C. The complex substrate PRPP:Mg protects HGPRT from thermal inactivation. However, incubations with hypoxanthine rendered a less thermostable enzyme at any temperature tested. The irreversible inactivation of HGPRT proceeds in two exponential steps. The analysis of the apparent rate constants for the fast and the slow phases, λ₁ and λ₂ as per the Lumry and Eyring model suggests the existence of more than three states in the thermal denaturation pathway of the free enzyme. In the presence of PRPP:Mg the irreversible process follows a single exponential and proceeds very slowly below 70°C. PRPP:Mg also protects the enzyme from inactivation by NEM and pCMB, suggesting that -SH groups may be in the vicinity of the active site     

In vitro non-enzymatic glycosylation of myofibrillar proteins

• 1.1. Glycation is non-enzymatic modification of proteins by sugars in which not only structural but also biological properties of proteins are altered.
• 2.2. Our in vitro experiments show that incubation of myofibrillar proteins with ribose results in sugar attachment to proteins and at the same time myofibrillar ATPase activity is lowered.
• 3.3. DETAPAC, aminoguanidine and 2-mercaptoethanol all partially block myofibrillar protein glycation and ATPase activity is less inactivated.
• 4.4. The dependence of ATPase activity of myofibrils incubated with ribose on the amount of 2-mercaptoethanol present suggests that also modification of SH groups is involved in enzyme inactivation.
• 5.5. Electrophoretic studies revealed that heavy chains of myosin, actin, and tropomyosins are proteins which are mainly glycated in vitro.

     

Baker''s yeast activity in an organic solvent system

The baker's yeast mediated reduction of ethyl 3-oxobutanoate-3-¹³C in hexane was conducted in an NMR tube at 20°C and a ¹³C NMR spectrum recorded each hour. A plot of relative peak intensity against time allowed the progress of the reaction to be monitored. A series of reactions was carried out in which the yeast was pretreated with the organic solvent system for 3, 6, 12 and 24 h prior to the addition of the substrate. From the initial rate of these reactions it was determined that in hexane the enzyme activity remained constant for about 12 h and then rapidly decreased until after 24 h very little activity remained. The reaction was also carried out at 10°C and 30°C. At the lower temperature, the reaction was slower but enzyme activity was maintained for more than 60 h, while at 30°C the enzyme activity had ceased after 8 h.     

Effect of heat exposure on the activity of monoamine oxidase in the rat brain and interscapular brown adipose tissue

The exposure of Wistar male rats (200±20 g) to high ambient temperature (38°C) for 20 and 60 min induced an equal decrease in hypothalamic, brain stem and hippocampal monoamine oxidase activity when compared to controls. The interscapular brown adipose tissue monoamine oxidase activity, as well as oxygen consumption and rectal temperature were increased only after a 60 min heat exposure. The adrenal function, assessed by dopamine-beta-hydroxylase activity and cholesterol concentration, was enhanced both after 20 and 60 min. In conclusion, heat induced the increase in adrenal function and interscapular brown adipose tissue monoamine oxidase activity, but the decrease in that of the brain.     

Fractional extraction and physico-chemical characterization of hemicelluloses and cellulose from sugar beet pulp

Four hemicelluloses and cellulose fractions were extracted with 10% KOH or 7.5% NaOH at 15°C for 16 h and with 24% KOH or 17.5% NaOH at 15°C for 2 h from defatted, protein and pectin free, lignified or delignified sugar beet pulp (SBP). There was no significant difference in the yield and sugar composition of isolated hemicelluloses and cellulose obtained from four different procedures. 7.5% NaOH extraction at 15°C for 16 h from lignified SBP gave a slightly higher yield of hemicelluloses (10.96%), while 24% KOH extraction at 15°C for 2 h from delignified SBP produced the highest yield of cellulose (18.35%). Molecular-average weights ranged from 88 850 to 91 330 Da for the hemicelluloses obtained from lignified SBP, and 21 620–21 990 Da for the hemicelluloses isolated from delignified SBP. The neutral sugar composition of the hemicelluloses consisted of glucose, arabinose, galactose, xylose, and minor quantities of rhamnose and mannose. The infrared spectra showed an absorption band at 900 cm⁻¹, indicating some amounts of β-linked polysaccharides. Besides ferulic and p-coumaric acids, six other phenolics were also identified in the mixture of alkaline nitrobenzene oxidation of associated lignin in the isolated hemicelluloses and cellulose fractions.     

Effect of propranolol and cold exposure on the activities of antioxidant enzymes in the brain of rats adapted to different ambient temperatures

The exposure to cold (6 h; 6°C) induced a significant decrease in both hypothalamic and brain stem CuZn-superoxide dismutase as well as an increase in Mn-superoxide dismutase and catalase activities in Wistar male rats, acclimated to 6±1°C as compared to those acclimated to 22±2°C. If the rats were administered with propranolol (15 mg/kg), which is a β-adrenoceptor blocker, there were no significant differences in the enzyme activities in any of the brain regions of the two groups studied. It was concluded that acute exposure to cold induces changes in the hypothalamic and brain stem antioxidant enzyme activities dependent on the previous acclimation to different ambient temperatures and propranolol administration.     

Simple procedures for purification and stabilization of human serum paraoxonase-1

Human paraoxonases-1 is one of the most important detoxifying enzymes. In this study using simple chromatographic procedures human paraoxonases-1 was purified from human pooled plasma. The enzyme was purified using DEAE Sephadex an anion exchanger and G-200 a gel filtration chromatographic media. Results showed a single band of approximately 43 KD proteins in SDS–PAGE, corresponding to the human PON1. Using paraoxon as the substrate the activity was related to the concentration of calcium and sodium ions (K_m = 1.2 ± 0.2 mM). Phenyl acetate hydrolyzing activity was independent of sodium and calcium ions (K_m = 0.78 ± 0.08 mM). Keeping at 25 °C for 20 days 75% of the enzyme original activity was restored in 20% (v/v) glycerol. EDTA and zinc chloride both inhibited the enzyme activity. In conclusion the applied procedures can be used for large scale purification. It would greatly facilitate their structural and functional characterization and permit examination of their weak, yet potentially most biologically relevant activities, in the complete absence of other serum proteins.     

Capillary electrophoretic analysis of advanced glycation endproducts formed from the reaction of reducing sugars with the amino group of glucosamine

Glucosamine (GlcN) is an amino sugar sold over-the-counter and is widely used as a dietary supplement to relieve symptoms of osteoarthritis. It is not known whether it is the GlcN alone or one of its many possible nonenzymatic glycation products that is responsible for this effect. The current study demonstrates that reducing sugars form advanced glycation endproducts (AGEs) with GlcN and, as a result, decrease GlcN autocondensation by reducing the availability of the GlcN amino group. Capillary electrophoresis (CE) was used to analyze the in vitro Maillard reaction of GlcN with glyceraldehyde (GA), glucose (Glc), and fructose (Fru) as well as their inhibition of GlcN autocondensation under physiological conditions. Formation of AGEs was monitored by UV and fluorescence spectroscopy. Major components were separated by CE using a bare capillary and UV detection at 214 nm. AGE species were separated by HPLC and were complementary to the CE results. The effects of sugar concentration and incubation time on the AGE profile are also reported for each of the GlcN reducing sugar model systems. A simple and rapid CE method was developed to analyze the AGE formation in this initial report of the reaction of reducing sugars with the amino group of GlcN.     

Oxidative stress implication in a new phenotype of amyotrophic quadricipital syndrome with cardiac involvement due to lamin A/C mutation

This study aimed at evaluating OS in an amyotrophic quadricipital syndrome with cardiac impairment in a family of 80 members with a mutation in lamin A/C gene. Twelve patients had cardiac involvement (5 cardiac and skeletal muscles impairment). OS was evaluated in blood samples (thiobarbituric acid-reactive substances (TBARS), carbonylated proteins (PCO)) 6 “affected patients” with phenotypic and genotypic abnormalities without heart failure and 3 “healthy carrier” patients. OS was higher in affected patients than in healthy, as shown by the higher TBARS and PCO values. Patients with cardiac and peripheral myopathy exhibited a higher OS than patients with only cardiac disease (TBARS: 1.73 ± 0.05 vs. 1.51 ± 0.04 mmol/l (p = 0.051), PCO: 2.73 ± 0.34 vs. 0.90 ± 0.10 nmol/mg protein (p = 0.47)), and with healthy carriers patients (TBARS: 1.73 ± 0.05 vs. 1.16 ± 0.14 mmol/l (p = 0.05), PCO: 2.73 ± 0.34 vs. 0.90 ± 0.20 nmol/mg protein (p = 0.47)).

OS may thus contribute to the degenerative process of this laminopathy. ROS production occurs, prior to heart failure symptoms. We suggest that the extent activation may also promote the variable phenotypic expression of the disease.     

Thermal stabilization of trypsin with glycol chitosan

Glycol chitosan was evaluated as thermoprotectant additive for trypsin in aqueous solutions. Maximal stabilization was achieved by using a polymer/protein ratio of 2 (w/w). The catalytic properties of trypsin were not affected by the presence of the polysaccharide. The enzyme thermostability was increased from 49 °C to 93 °C in the presence of the additive. Trypsin was also 37-fold more stable against incubation at 55 °C and its activation free energy of thermal inactivation was increased by 9.9 kJ/mol when adding glycol chitosan.     

Stabilization of heat-induced changes in plant peroxidase preparations by ClpX, a bacterial heat shock protein

Peroxidases (PODs) are known to be quite stable at elevated temperatures. Moreover, partially denatured peroxidases are able to regain their catalytic activity during incubation at room temperature. In this paper, we describe the effects of some heat shock proteins on the self-reactivation of plant peroxidase preparations. Horseradish and artichoke peroxidases (HRP and ARP, respectively) were first heated (at 60 °C or 90 °C), then incubated at a slightly elevated temperature (30 °C). The heat-treatment resulted in a considerable loss of activity of both enzymes but the subsequent incubation allowed their reactivation. However, no reactivation could be detected when incubation was carried out in the presence of the molecular chaperone ClpX. Other chaperones that were tested (DnaK, DnaJ and GrpE) did not show the inhibitory effect. Electrophoretic analyses further indicated that the heat-treated horseradish peroxidase, but not the native enzyme, binds to ClpX eliminating the possibility of undesirable protein refolding that would result in aggregation.     

Influence of Several Effectors on the Structure-Activity Relationship of Spleen Phosphodiesterase

The influence of Mg(II) and organic solvents on the structure-activity relationship of spleen phosphodiesterase II was analyzed using UV and fluorescence spectroscopies. An increase in the RNase activity found in the presence of Mg(II) was related to the enzyme-Mg(II) interaction detected by UV spectroscopy. In the fluorescence spectra of phosphodiesterase strong hypochromic and bathochromic effects were observed when RNA was present at a concentration (52 μg ml^-1) of the same magnitude as the concentration that inhibits the activity (Ki = 40 μg ml^-1). The strong quenching observed in the presence of RNA shows the importance of large dynamic and static quenching of the Trp residues of the enzyme. The denaturation temperature, approx. 60°C, was detected by the pattern observed in the intensity of fluorescence versus temperature curve. Organic solvents produced an alteration in the enzyme conformation, detected by fluorescence. This alteration was diminished in the presence of Mg(II), which stabilizes the conformation of the enzyme.     

Enzyme-linked immunosorbent assay for 4-hydroxynonenal-histidine conjugates

Highly reactive aldehyde 4-hydroxynonenal (HNE) is the final product of lipid peroxidation, known as a second messenger of free radicals and a signaling molecule. It forms protein conjugates involved in pathology of various diseases. To determine cellular HNE-protein conjugates we developed indirect ELISA based on well-known, monoclonal antibody against HNE-histidine (HNE-His) adducts. The method was calibrated using HNE-albumin conjugates as standards (R² = 0.999) and validated on human osteosarcoma cell cultures (HOS). The ELISA showed good sensitivity (8.1 pmol HNE-His/mg of protein), precision ( ± 8% intra-assay and ± 12% inter-assay) and spiking recovery ( ± 9%). The assay revealed 60-fold increase of cellular HNE-His adducts upon copper-induced lipid peroxidation of HOS. The ELISA matched HNE-immunocytochemistry of HNE-treated HOS cells and quantified the increase of cellular HNE-His conjugates in parallel to the decrease of free HNE in culture medium. The ELISA was developed as ELISA Stress for severe lipid peroxidation and ELISA Fine for studies on HNE physiology.     

Freezing resistance improvement of Lactobacillus reuteri by using cell immobilization

Lactobacillus reuteri shows certain beneficial effects to human health and is recognized as a probiotic. However, its application in frozen foods is still not popular because of its low survival during freezing and frozen storage. Cell immobilization technique could effectively exert protection effects to microbial cells in order to enhance their endurance to unfavorable environmental conditions as well as to improve their viability and cell concentration. Ca-alginate and κ-carrageenan were used to immobilize L. reuteri in this research, and the immobilized cells were exposed to different freezing temperatures, i.e. − 20 °C, − 40 °C, − 60 °C, − 80 °C, and stored at − 40 °C and − 80 °C for 12 weeks. The objectives were to study the protection effects of cell immobilization against the adverse conditions of freezing and frozen storage, and the effects of freezing temperatures to the immobilized cells. Cell immobilization was used to raise the survival of L. reuteri during freezing and frozen storage in order to develop frozen foods with the probiotic effects of L. reuteri. Results indicated that immobilized L. reuteri possessed better survival in both freezing and frozen storage. The survival of immobilized L. reuteri was higher than that of free cells, and the effects of lower freezing temperature were better than higher freezing temperature. The immobilization effects of Ca-alginate were found to be superior to κ-carrageenan. Cell immobilized L. reuteri exhibits potential to be used in frozen foods.     

Nitrosylmyoglobin as antioxidant--kinetics and proposed mechanism for reduction of hydroperoxides

Nitrosylmyoglobin (MbFe^IINO), which is believed to have a protective role during ischemia and reperfusion injury, was oxidized by tert-butyl hydroperoxide (t-BuOOH), and by hydrogen peroxide (H₂O₂) to the nitrite anion and metmyoglobin (MbFe^III). Further characterization of the reaction of MbFe^IINO with excess of t-BuOOH was investigated with respect to reaction stoichiometry, temperature and pH dependence. It was found that the reaction between MbFe^IINO with excess of t-BuOOH followed a simple stoichiometry and had moderate pH and temperature dependence with the activation parameters ΔH^‡ = 57.4 ± 1.4 kJ mol^- 1 and ΔS^‡ = - 112.0 ± 5.1 J mol^- 1 K^- 1, which is consistent with an associative reaction mechanism. Moreover, t-BuOOH-induced oxidation of MbFe^IINO did not result in any detectable formation of the hypervalent myoglobin (Mb) species, i.e. perferrylmyoglobin, (^√MbFe^IV = O) or ferrylmyoglobin (MbFe^IV = O), and hereby differed from H₂O₂-induced oxidation of MbFe^IINO, which results in the formation of MbFe^IV = O. Based on the obtained results and on published data, different mechanisms for the reaction of the MbFe^IINO with t-BuOOH and H₂O₂ are proposed.     

Detection of dideoxyosone intermediates of glycation using a monoclonal antibody: characterization of major epitope structures

Glycation or the Maillard reaction in proteins forms advanced glycation end products (AGEs) that contribute to age- and diabetes-associated changes in tissues. Dideoxyosones, which are formed by the long-range carbonyl shift of the Amadori product, are newly discovered intermediates in the process of AGE formation in proteins. They react with o-phenylenediamine (OPD) to produce quinoxalines. We developed a monoclonal antibody against 2-methylquinoxaline-6-carboxylate coupled to keyhole limpet hemocyanin. The antibody reacted strongly with ribose and fructose (+OPD)-modified RNase A and weakly with glucose and ascorbate (+OPD)-modified RNase A. Reaction with substituted quinoxalines indicated that this antibody favored the 2-methyl group on the quinoxaline ring. We used high performance liquid chromatography to isolate and purify three antibody-reactive products from a reaction mixture of N alpha-hippuryl-L-lysine+ribose+OPD. The two most reactive products were identified as diastereoisomers of N1-benzoylglycyl-N6-(2-hydroxy-3-quinoxalin-2-ylpropyl)lysine and the other less reactive product as N1-benzoylglycyl-N6-[2-hydroxy-2-(3-methylquinoxalin-2-yl)ethyl]lysine. Our study confirms that dideoxyosone intermediates form during glycation and offers a new tool for the study of this important pathway in diabetes and aging.