Physical and catalytic properties of a peroxidase derived from cytochrome c

Except for its redox properties, cytochrome c is an inert protein. However, dissociation of the bond between methionine-80 and the heme iron converts the cytochrome into a peroxidase. Dissociation is accomplished by subjecting the cytochrome to various conditions, including proteolysis and hydrogen peroxide (H₂O₂)-mediated oxidation. In affected cells of various neurological diseases, including Parkinson's disease, cytochrome c is released from the mitochondrial membrane and enters the cytosol. In the cytosol cytochrome c is exposed to cellular proteases and to H₂O₂ produced by dysfunctional mitochondria and activated microglial cells. These could promote the formation of the peroxidase form of cytochrome c. In this study we investigated the catalytic and cytolytic properties of the peroxidase form of cytochrome c. These properties are qualitatively similar to those of other heme-containing peroxidases. Dopamine as well as sulfhydryl group-containing metabolites, including reduced glutathione and coenzyme A, are readily oxidized in the presence of H₂O₂. This peroxidase also has cytolytic properties similar to myeloperoxidase, lactoperoxidase, and horseradish peroxidase. Cytolysis is inhibited by various reducing agents, including dopamine. Our data show that the peroxidase form of cytochrome c has catalytic and cytolytic properties that could account for at least some of the damage that leads to neuronal death in the parkinsonian brain.     

Structural diversity in thallium chemistry, IV. Further examples of solid state bromothallate(III) derivatives obtained by employing certain classes of alkyl diammonium cations

Six new bromothallate(III)-containing salts with different alkyl diammonium cations have been prepared from bromide containing solutions and studied by single-crystal X-ray crystallographic analyses. The N,N′-diethyl-N,N,N′,N′-tetramethyl-1,2-ethylenediammonium, N-methyl-1,3-propanediammonium, N,N,N′,N′-tetramethyl-1,3-propanediammonium and N,N,N′,N′-tetraethyl-1,2-ethylenediammonium cations yield complexes (I, II, III and IV, respectively) with the [TlBr₅]²⁻ anionic stoichiometry. For I and II, both complexes contain the [TlBr₅]²⁻ anion. In complex II, this appears as a distorted octahedron with one long Tl?Br2′ contact of 3.632(4) Å from an adjacent anion, thus completing the hexacoordination about an otherwise distorted square pyramid. On the other hand, for III and IV, both complexes contain a tetrahedral [TlBr₄]⁻ anion together with an isolated, but hydrogen-bonded, Br⁻ anion. The 1,5-hexanediammonium complex (V) contains tetrahedral [TlBr₄]⁻, slightly distorted octahedral [TlBr₆]³⁻ and Br⁻ anions. The asymmetric unit of the N,N-diethyl-1,3-propanediammonium salt (VI) contains one cation and half of each of a [TlBr₄]⁻ and an axially compressed octahedral [TlBr₆]³⁻ anion. Extensive hydrogen-bonded networks exist in complexes II-VI. NH?Br hydrogen bonds generally have a significant influence on the nature of the anions present in species with the formal [TlBr₅] stoichiometry.     

Properties of native and hydrophobic laccases immobilized in the liquid-crystalline cubic phase on electrodes

Both native Trametes hirsuta laccase and the same laccase modified with palmytic chains to turn it more hydrophobic were prepared and studied with cyclic voltammetry and Raman spectroscopy. Native laccase immobilized in the monoolein cubic phase was characterized with resonance Raman spectroscopy, which demonstrated that the structure at the “blue” copper site of the protein remained intact. The diamond-type monoolein cubic phase prevents denaturation of enzymes on the electrode surface and provides contact of the enzyme with the electrode either directly or through the mediation by electroactive probes. Direct electron transfer for both laccases incorporated into a lyotropic liquid crystal was obtained under anaerobic conditions, whereas bioelectrocatalytic activity was shown only for the native enzyme. The differences in electrochemical behavior of native and hydrophobic laccase as well as possible mechanisms of direct and mediated electron transfers are discussed. The Michaelis constant for 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS²⁻), K _M^app, and the maximal current, I _max, for the native enzyme immobilized onto the electrode were estimated to be 0.24 mM, and 5.3 μA, respectively. The maximal current density and the efficiency of the catalysis, I _max/K _M^app, were found to be 73 μA cm⁻² and 208.2 μA cm⁻² mM⁻¹, respectively, and indicated a high efficiency of oxygen electroreduction by the enzyme in the presence of ABTS²⁻ in the cubic-phase environment. Rate constants were calculated to be 7.5 × 10⁴ and 3.6 × 10⁴ M⁻¹ s⁻¹ for native and hydrophobic laccase, respectively.     

甘草酸二铵对百草枯中毒致急性肺损伤大鼠TLR-4表达的影响

目的探讨甘草酸二铵(DG)对百草枯(PQ)中毒致急性肺损伤(ALI)大鼠的保护作用及其机制。方法选择健康SD大鼠50只,随机分为百草枯组(PQ组)、甘草酸二铵组(DG组)和正常对照组(NS组),PQ组和DG组予百草枯100mg/kg灌胃1次,DG组于灌胃后立即腹腔注射DG 50mg/kg,每日1次,对照组与PQ组注射等剂量的生理盐水。观察至48h处死大鼠,取肺组织检测肺湿干重比;肺组织HE染色评价肺组织损伤情况;采用RT-PCR法检测肺组织TLR-4mRNA和NF-κB mRNA的表达情况。另选择健康SD大鼠50只,分组及各组处置方法同上,观察其7天内死亡率。结果 PQ组与DG组肺湿干比、肺组织TLR-4mRNA和NF-κB mRNA表达较NS组明显升高(P0.01);DG组各指标明显低于PQ组(P0.01)。HE染色结果,NS组肺部结构正常,PQ组、DG组可见肺组织水肿、出血及炎性细胞浸润等肺损伤表现,DG组病变轻于PQ组。群体死亡率比较,PQ组7天死亡率为90%,DG组为40%,NS组无死亡。结论甘草酸二铵可减轻百草枯中毒致急性肺损伤大鼠肺部炎症,其机制可能与降低TLR-4、NF-κB的表达有关。     

Food consumption of Sarotherodon mossambicus (Trewaves) exposed to sublethal concentration of diammonium phosphate

S. mossambicus was exposed to toxic and sublethal concentrations of the fertilizer diammonium phosphate (0.2 to 1.0 g l^–1). Mortality, food utilization and growth were studied. At a concentration of 0.6 g l^–1 DAP, 100% mortality was observed within 96 h; no mortality occurred at 0.5 g l^–1; LC50 was 0.55 g l^–1. Rearing the fish in increasing sublethal concentrations of DAP, it was found that the feeding rate decreased from 25.4 mg g^–1 fish^–1 d^–1 (fish reared in DAP-free water) to 10.1 mg g^–1 d^–1 at the highest sublethal concentration (0.5 g l^–1). Growth rate was drastically reduced. At high sublethal concentrations of DAP, the fish lost reserve energy, in addition to the energy obtained from food intake for survival, as a result of increased swimming activity and opercular beats.     

Regulation of n-acetylglucosamine uptake in yeast

Various yeasts have been investigated for their ability to grow on N-acetylglucosamine as the sole carbon source and only those which are associated with the disease, candidiasis, gave positive results. The yeasts unable to grow on N-acetylglucosamine lacked the capacity to transport the aminosugar across the cell membrane. In pathogenic yeasts, two systems of different affinity for substrate were found to operate in the uptake of N-acetylglucosamine. In glucose-grown cells a constitutive, low affinity uptake system was present, but upon addition of inducer, a specific high affinity uptake system was synthesized. Experiments with the inhibitors of macromolecule synthesis suggested that the synthesis of RNA and protein is necessary for induction whereas the synthesis of DNA is not.In glucose-grown Candida albicans cells which are devoid of N-acetylglucosamine enters into the cells as phosphorylated form using a constitutive uptake system. Uranyl acetate (0.01 mM) which binds to cell membrane-associated polyphosphates, inhibited completely the inducible uptake of N-acetylglucosamine. Labelling experiments, designed to determine the temporal sequence of appearance of N-acetylglucosamine in intracellular free sugar and sugar-phosphate pools, indicated that N-acetylglucosamine first appeared in the cells as phosphorylated form. Similar results were obtained with Saccharomyces cerevisiae 3059 and some other yeasts which are devoid of N-acetylglucosamine kinase in both uninduced and induced conditions. These results are consistent with the model of van Steveninck that involves phosphorylation during transport. Furthermore, inhibitors of energy metabolism (arsenate, azide and cyanide), proton conductor (m-chlorocarbonylcyanide phenylhydrazine) and dibenzyl diammonium ion (membrane permeable cation) inhibited the inducible N-acetylglucosamine uptake in C. albicans.     

Toxoplasma gondii: an evaluation of diagnostic value of recombinant antigens in a murine model

Laboratory diagnostics of toxoplasmosis depends primarily on serological methods detecting specific antibodies. Since these methods do not always enable specific and sensitive recognition of the infection and phase of toxoplasmosis, the search for new diagnostic tools continues. Recombinant antigens promise a new alternative in diagnostics of Toxoplasma gondii infections. In this work the usefulness of six recombinant T. gondii antigens: GRA1, GRA6, GRA7, p35, SAG1, and SAG2 in the detection of primary murine toxoplasmosis was evaluated. Sera obtained from infected mice differing in their natural susceptibility to T. gondii infection, BALB/c (relatively resistant) and C57BL/6 (relatively susceptible), were tested using ELISA. During acute infection high response to GRA7, GRA6, and p35 antigens was noticed, whereas a strong reactivity with surface antigens SAG1 and SAG2 was characteristic for chronic toxoplasmosis. Our results show that the recombinant antigens are useful in distinguishing between acute and chronic toxoplasmosis regardless of the genetically determined susceptibility of the host.