Resolving near-ultraviolet circular dichroism spectra of single trp mutants in tear lipocalin

Near-ultraviolet circular dichroism (near-UV CD) spectra of tryptophan residues in proteins are complicated because the line shapes are derived from the overlap of both the 1L(a) and the 1L(b) electronic bands that vary independently. Contributing to this complexity, tryptophan near-UV CD spectra differ in the relative amplitude of the 0-0 vibronic band compared to the rest of the 1L(b) spectrum, an inherent feature that may result in poor fitting. To resolve this problem, a computer program that incorporated the separation of the 0-0 transition of 1L(b) component from the rest of the 1L(b) was written in LabVIEW and its amplitude was allowed to vary independently. This method showed dramatically improved fitting of 1L(a) and 1L(b) components in the near-UV CD tryptophan spectra in tear lipocalin mutants featuring low intensity of the 0-0 1L(b) component. Side chain dynamic characteristics (mobility and accessibility to the solvent) identified from different spectroscopic techniques were related to differences in Trp near-UV CD spectra. This method is broadly applicable to different types of Trp near-UV CD spectra.     

Determination of degradation of radioactivity and its kinetics in aerobic composting

In this study, the kinetics of disappearance of radioactivity in aerobic composting was investigated. For this purpose, compost materials were prepared by mixing sugar beet wastes, wine factory wastes (grape wastes), straw and biological treatment sludge in different amounts. While alpha-radioactivity was not initially detected in all composting materials, the composting materials had some beta-radioactivity. In the mixtures of sugar beet wastes--straw-biological treatment sludge (1), sugar beet wastes-wine factory wastes (grape wastes)-biological treatment sludge (II) and wine factory wastes (grape wastes)-biological treatment sludge (III), the beta-radioactivity reduced by 82%, 58%, 85% respectively of initial values after 52 d. The beta-radioactivity degradation in the composting process could be represented by first-order kinetics and reaction rate constants of mixtures of I, II and III were k = 0.0693 d(-1) (R2 - 0.84), k = 0.0453 d(-1) (R2 = 0.98), k = 0.0234 d(-1) (R2 = 0.97), respectively.     

Kinetics and effects of trace elements and electron complexes on 2-keto-4-methylthiobutyric acid-dependent biosynthesis of ethylene in soil

AIMS: 2-Keto-4-methylthiobutyric acid (KMBA) is an established intermediate in microbial biosynthesis of ethylene from methionine. This study demonstrates the kinetics and effects of trace elements and electron complexes on substrate (KMBA)-derived C2H4 biosynthesis in soil. METHODS AND RESULTS: We have previously reported KMBA-dependent C2H4 production in soil. We studied the kinetics and effects of various trace elements and electron complexes on KMBA-derived C2H4 biosynthesis in soil by gas chromatography. Kinetic analysis revealed that ethylene forming enzyme (EFE) reaction was linear (R2 = 0.9448) when velocity of reaction (V) was plotted against substrate [S] over the range from 2.5 to 10 mmol l(-1) and thus followed a first order reaction. Application of three linear transformations of the Michaelis-Menten equation indicated high affinity of EFE for the substrate because Km values ranged between 5.4 and 6.67 mmol l(-1) and Vmax of reaction was between 22.4 and 35.7 nmol kg(-1) soil 120 cm(-1). Most of the trace elements exhibited positive effects on KMBA-dependent C2H4 production in soil. Maximum stimulatory effect on C2H4 biosynthesis was observed in response to Co(II) application, while Fe(III) inhibited the biotransformation of KMBA into C2H4. Contrarily, most of the tested electron complexes inhibited KMBA-derived C2H4 biosynthesis in the soil. However, lower concentrations (1.0 mmol l(-1)) of mannitol and hydroquinone were stimulatory to C2H4 production in soil compared with controls (substrate only). Conclusions: The results revealed that both kind and concentration of trace elements and electron complexes affected the substrate-dependent production of C2H4 in soil with different degrees of efficacy. SIGNIFICANCE AND IMPACT OF THE STUDY: The C2H4 in the root environment could be physiologically active even at low concentrations, so knowledge regarding various factors which regulate C2H4 biosynthesis in soil could be of significance for plant growth and development.     

Effect of flagella on initial attachment of Listeria monocytogenes to stainless steel

At 22 degrees C a flagellin mutant of Listeria monocytogenes was found to attach to stainless steel at levels 10-fold lower than wild-type cells, even under conditions preventing active motility. At 37 degrees C, when flagella are not produced, attachment of both strains was identical. Therefore, flagella per se facilitate the early stage of attachment.     

Carbonic anhydrase inhibitors: inhibition of the tumor-associated isozymes IX and XII with a library of aromatic and heteroaromatic sulfonamides

The inhibition of the two transmembrane, tumor-associated isozymes of carbonic anhydrase (CA, EC 4.2.1.1) of human origin, hCA IX and XII, with a library of aromatic and heteroaromatic sulfonamides has been investigated. Most of them were sulfanilamide, homosulfanilamide, and 4-aminoethyl-benzenesulfonamide derivatives, to which tails that should induce diverse physico-chemical properties have been attached at the amino moiety, whereas several of these compounds were derived from metanilamide, benzene-1,3-disulfonamide or the 1,3,4-thiadiazole/thiadiazoline-2-sulfonamides. The tails were of the alkyl/aryl-carboxamido/sulfonamido-, ureido or thioureido type. Against hCA IX the investigated compounds showed inhibition constants in the range of 3-294 nM, whereas against hCA XII in the range of 1.9-348 nM, respectively. The best hCA IX inhibitors were ureas/thioureas incorporating 4-aminoethyl-benzenesulfonamide and metanilamide moieties. The best hCA XII inhibitors were 1,3,4-thiadiazole/thiadiazoline-2-sulfonamides incorporating 5-acylamido or 5-arylsulfonylamido moieties. These compounds also inhibited appreciably the cytosolic isozymes hCA I and II, but some selectivity for the transmembrane, tumor-associated isozymes was observed for some of them, which is an encouraging result for the design of novel therapies targeting hypoxic tumors, in which these carbonic anhydrases are highly overexpressed.     

Protective effect of melatonin on contractile activity and oxidative injury induced by ischemia and reperfusion of rat ileum

Free radicals derived from molecular oxygen have been reported to be responsible for changes in motility and mucosal damage observed in intestinal ischemia-reperfusion injury. Melatonin has been considered as an antioxidant that prevents injuries resulted from I/R in various tissues. The present study was designed to determine the effect of melatonin on the contractile responses of acetylcholine (Ach) and KCl, on malondialdehyde (MDA), a product of lipid peroxidation, and reduced glutathione (GSH) levels and to assess histopathological changes in the smooth muscle of terminal ileum subjected to ischemia-reperfusion. The intestinal ischemia-reperfusion was induced by occlusion of superior mesenteric artery of rat for 30 min, followed by a period of reperfusion for 3 h. Melatonin at doses of 10 or 50 mg/kg was administered via the tail vein in 5 min prior to reperfusion. Following reperfusion, segments of terminal ileum were rapidly taken and transferred into isolated organ bath and responses to Ach and KCl were recorded. Samples of terminal ileum were also taken for measuring the MDA and GSH levels. EC50 values of these contracting substances were seriously reduced in the ischemia-reperfusion group compared to that of the sham-operated control group. The decreased contraction response to Ach and KCl was significantly ameliorated by a dosage of 50 mg/kg of melatonin, while not by a dosage of 10 mg/kg. Similar pattern of the effect was observed in the tissue levels of MDA and GSH as well as in histological improvement. Melatonin appeared to be restoring the amounts of tissue MDA and GSH back to about control levels. These results suggest that the high dose of melatonin not only physiologically but also biochemically and morphologically could be useful to normalize contractility injured by oxidative stress in intestinal ischemia-reperfusion.     

Purification and characterization of polyphenol oxidase from nettle (Urtica dioica L.) and inhibitory effects of some chemicals on enzyme activity

Polyphenol oxidase (PPO) of nettle (Urtica dioica L.) was extracted and purified through (NH₄)₂SO₄ precipitation, dialysis, and CM-Sephadex ion-exchange chromatography and was used for its characterization. The PPO showed activity to catechol, 4-methylcatechol, L-3,4-dihydroxyphenylalanine (L-DOPA), L-tyrosine, p-cresol, pyrogallol, catechin and trans-cinnamic acid. For each of these eight substrates, optimum conditions such as pH and temperature were determined and L-tyrosine was found to be one of the most suitable substrates. Optimum pH and temperature were found at pH 4.5 and 30°C respectively and K_m and V_max values were 7.90?×?10^?4?M, and 11290?EU/mL for with L-tyrosine as substrate. The inhibitory effect of several inhibitors, L-cysteine chloride, sodium azide, sodium cyanide, benzoic acid, salicylic acid, L-ascorbic acid, glutathione, thiourea, sodium diethyl dithiocarbamate, β-mercaptoethanol and sodium metabisulfite were tested. The most effective was found to be sodium diethyl dithiocarbamate which acted as a competitive inhibitor with a K_i value of 1.79?×?10^?9?M. In addition one isoenzyme of PPO was detected by native polacrylamide slab gel electrophoresis.     

Age-related regulation of excitation–contraction coupling in rat heart

Hearts from subjects with different ages have different Ca²⁺ signaling. Release of Ca²⁺ from intracellular stores in response to an action potential initiates cardiac contraction. Both depolarization-stimulated and spontaneous Ca²⁺ releases, Ca²⁺ transients and Ca²⁺ sparks, demonstrate the main events of excitation–contraction coupling (ECC). Global increase in free Ca²⁺ concentration ([Ca²⁺] _i ) consists of summation of Ca²⁺ release events in cardiomyocytes. Since the Ca²⁺ flux induced by Ca²⁺ sparks reports a summation of ryanodine-sensitive Ca²⁺ release channels (RyR2s)’s behavior in a spark cluster, evaluation of the properties of Ca²⁺ sparks and Ca²⁺ transients may provide insight into the role of RyR2s on altered heart function between 3-month-old (young adult) and 6-month-old (mature adult) rats. Basal [Ca²⁺] _i and Ca²⁺ sparks frequency were significantly higher in mature adult rats compared to those of young adults. Moreover, amplitudes of Ca²⁺ sparks and Ca²⁺ transients were significantly smaller in mature adults than those of young adults with longer time courses. A smaller L-type Ca²⁺ current density and decreased SR Ca²⁺ load was observed in mature adult rats. In addition, RyR2s were markedly hyperphosphorylated, and phosphorylation levels of PKA and CaMKII were higher in heart from mature adults compared to those of young adults, whereas their SERCA protein levels were similar. Our data demonstrate that hearts from rats with different ages have different Ca²⁺ signaling including hyperphosphorylation of RyR2s and higher basal [Ca²⁺] _i together with increased oxidized protein-thiols in mature adult rats compared to those of young adults, which play important roles in ECC. Finally, we report that ECC efficiency changes with age during maturation, partially related with an increased cellular oxidation level leading to reduced free protein-thiols in cardiomyocytes.