Pitfalls in a Method for Assessment of Total Antioxidant Capacity

A relatively simple and widely applied method for quantitating the total antioxidant capacity of body fluids and drug solutions based on the absorbance of the ABTS radical cation was evaluated. In this assay, the end-point is an antioxidant-induced decrease in absorbance at a fixed time. This decrease is used as an index of total antioxidant capacity. It is shown that Trolox, potassium cyanide and quercetin all decrease the absorbance of ABTS radical cations at a fixed time, but by different mechanisms. Trolox scavenges the ABTS radical, potassium cyanide inhibits radical formation, while quercetin acts by both mechanisms. Using this method antioxidant capacity may be overestimated, due to both a scavenger effect and an effect on the rate of ABTS oxidation. To distinguish between these effects, a post-addition assay was used in which the sample is added when the formation of radicals is stable. Using post- addition assay conditions enables discrimination between effects on radical scavenging and on the radical formation, two major mechanisms for antioxidant action. In extrapolating the results to an ill vivo situation it should be questioned: (i) whether the peroxidase process does indeed mimic the process of radical formation in vivo, and (ii) whether the ABTS radicals do resemble the radical species involved in an in vivo situation. Results obtained in the ABTS radical-based methods should therefore be reviewed critically before the antioxidant capacity can be assessed.     

螺旋藻氢酶的纯化与生化特性

本研究用DE－52、SephadexG－75、SephadexG－100柱层析从螺旋藻分离纯化得到比活性提高200倍的氢酶,回收率为14％。凝胶柱层析和SDS－PAGE显示一条带,其分子量为56kd。氨基酸分析结果表明酸性氨基酸比例较大,等电聚焦测定结果证明其等电点为pH4．2。吸收光谱结果显示氢酶是铁硫蛋白。甲基紫晶（MV）是氢酶催化放氢的最佳电子供体,其Km（MV）为0．31mmol／L,最适pH值为7．5－8．0。     

Stetteria hydrogenophila, gen. nov. and sp. nov., a novel mixotrophic sulfur-dependent crenarchaeote isolated from Milos, Greece

A new hyperthermophilic, strictly anaerobic crenarchaeote, Stetteria hydrogenophila DSM11227 representing a new genus within the family of Desulfurococcaceae, was isolated from the sediment of a marine hydrothermal system at Paleohori Bay in Milos, Greece. Cells are gram-negative irregular and disc-shaped cocci, 0.5–1.5 μm in diameter, which are flagellate and can form cytoplasmatic protrusions up to 2 μm in length. The strain grew optimally at 95°C at pH 6.0 and at a NaCl concentration of 3%. The organism grew mixotrophically on peptide substrates. It required elemental sulfur as an external electron acceptor, and in addition, its growth was completely dependent on the presence of molecular hydrogen. Sulfur could be replaced by thiosulfate. H₂S, CO₂, acetate, and ethanol were identified as products of metabolism. The G + C content of DNA was 65 mol%. Analysis of its phylogenetic position by sequence analysis of 16S rRNA placed this organism in the family of Desulfurococcaceae. The dependence of this organism on both hydrogen and sulfur during growth on peptide substrates distinguishes Stetteria from all previously described species of Crenarchaeota. Received: September 4, 1996 / Accepted: November 12, 1996     

Stability of metal ion complexes formed with methyl phosphate and hydrogen phosphate

 The acidity constants of methyl phosphoric acid, CH₃OPO(OH)₂, and orthophosphoric acid, HOPO(OH)₂, and the stability constants of the 1 : 1 complexes formed between Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, or Cd²⁺ and methyl phosphate, CH₃OPO₃ ^2–, or hydrogen phosphate, HOPO₃ ^2–, were determined by potentiometric pH titration in aqueous solution (25  °C;I = 0.1 M, NaNO₃). On the basis of previously established log K versus pK _a straight-line plots for the complexes of simple phosphate monoesters and phosphonate derivatives, R-PO₃ ^2–, where R is a noncoordinating residue, it is shown that the stability of the M(CH₃OPO₃) complexes is solely determined (as one might expect) by the basicity of the –PO₃ ^2– residue. It is emphasized that the mentioned reference lines may also be used to reveal increased complex stabilities, for example, for certain complexes formed with 8-quinolyl phosphate the occurrence of 7-membered chelates can be proven in this way; the same procedure is also applicable to complexes of nucleotides, etc. The M(HOPO₃) complexes are slightly more stable (on average by 0.08 log unit) than it is expected from the basicity of HPO₄ ^2–; this observation is attributed to a more effective solvation, including hydrogen bonding, than is possible with CH₃OPO₃ ^2– species. Received: 9 November 1995 / Accepted: 5 February 1996     

Role of the testa epidermis in the leakage of intracellular substances from imbibing soybean seeds and its implications for seedling survival

The generation of ethylene from 1-aminocyclopropane-1-carboxylic acid (ACC) added to a cell-free preparation from etiolated pea ( Pisum sativum L. cv. Alaska) epicotyls was found not to be due to a specific ACC oxidase or to oxygen radicals. Rather, endogenously produced H₂O and manganese ions are coupled in a reaction sequence which produces ethylene from ACC. In a model system, H₂O and Mn²⁺ converted ACC to ethylene under conditions similar to those in the pea preparation. Ultrafiltration of the pea preparation inhibited ethylene production, but it could be reconstituted either by adding an H₂O₂-generating system to the ultrafiltrate or Mn²⁺ to the retentate. H₂O₂-generating systems could reconstitute ethylene formation in a heat-inactivated cell-free sample while the loss of ability to produce ethylene upon dialysis of the pea preparation correlated with the loss of Mn²⁺ from the sample. Studies using cell-free preparations to investigate ethylene synthesis should take care to exclude the possible involvement of H₂O₂ and Mn^2+.     

From self-assembly of life to present-day bacteria: a possible role for nanocells

A proposed sequence of major events for the self-assembly of life on Earth is examined. This sequence starts with a construction kit of elements and simple compounds from which a primitive membrane and then a nanocell with a minimal genome is self-assembled. The genome and cell increase in size and complexity and become capable of cell division, similar to present-day bacteria. Another factor to understanding this self-assembly of life is identifying the energy source(s) the first self-assembling nanocells were capable of using. This will also be examined from an evolutionary perspective with hydrogen as the postulated universal energy source [Morita, R. (2000) Microb. Ecol. 38, 307-320].     

氧化诱导K_(562)细胞凋亡机制的初步探讨

以过氧化氢（Ｈ２Ｏ２）诱导人慢性髓细胞白血病（Ｋ５６２）细胞为凋亡模型,采用流式细胞仪（ｆｌｏｗｃｙｔｏｍｅｔｒｙ,ＦＣＭ）和激光共聚焦扫描显微镜（ｌａｓｅｒｃｏｎｆｏｃａｌｓｃａｎｎｉｎｇｍｉｃｒｏｓｃｏｐｙ,ＬＣＳＭ）研究细胞凋亡,形态观察出现核固缩,核碎裂及凋亡小体等典型凋亡特征．ＤＮＡ电泳图谱出现“Ｌａｄｄｅｒ”．ＦＣＭ检测在Ｇ０／Ｇ１峰前出现一低ＤＮＡ含量的凋亡峰．ＬＣＳＭ显示凋亡细胞ｃ－Ｆｏｓ．ｃ－Ｊｕｎ和ＮＦκＢ表达量均有不同程度的增加．该结果提示上述三种转录因子可能参与氧化诱导凋亡过程中基因的调控作用．     

The immune response to Lactococcus lactis: Implications for its use as a vaccine delivery vehicle

Abstract The development of hydrogenotrophic bacteria in the rumen of lambs was investigated by culture and labeling experiments. ¹⁴CO₂ and ¹³CO₂ incorporation by the rumen microflora of a 24-h-old lamb showed that while there was no labeled methane, double-labeled acetate was formed indicating the presence of hydrogen-dependent acetogenesis. In vitro counts from rumen fluid of 20-h-old lambs confirmed an extensive colonization of acetogenic bacteria while methanogens were absent. Methanogens appeared in the rumen of 30-h-old lambs, and as they developed there was a proportional decrease in the numbers of acetogens, indicating a competition for hydrogen between these two groups. Hydrogen-utilizing sulfate-reducing bacteria, which were established by the 3rd day after birth, did not seem to be affected by this competition.     

Antioxidant action of sodium diethyldithiocarbamate: Reaction with hydrogen peroxide and superoxide radical

The oxidation of sodium diethyldithiocarbamate (DDC) by hydrogen peroxide or superoxide radicals has been investigated. Hydrogen peroxide oxidizes DDC, leading to the formation of a hydrated form of disulfiram, a dimer of DDC having a disulfide group. In equimolar conditions, the overall process appears as a first-order reaction (k = 0.025±0.005 s⁻¹), the first step being a second-order reaction (k = 5.0±0.1mol⁻¹.1. s⁻¹). No radical intermediate was observed in this process. In the presence of an excess of any of the reagents, the hydrated form of disulfiram transforms into different products corresponding to the fixation of oxygen by sulfur atoms or replacement of C = S group by ketone function, in the presence of an excess of hydrogen peroxide. Superoxide anions (produced by steady-state ⁶⁰Co γ-radiolysis) oxidize DDC, yielding similar products to those obtained with hydrogen peroxide with a maximum oxidation G-value of 0.3 μmol.J⁻¹. The rate constant k(O₂^·− + DDC) is equal to 900 mol⁻¹. 1. s⁻¹.