Effective inhibition by beta-carotene of cellular DNA breaking induced by peroxynitrous acid

Peroxynitrous acid synthesized by reaction of hydrogen peroxide and nitrite and generated from 3-morpholinosydononimine (SIN-1) induced cellular DNA breaking of human promyelocytic leukemia HL-60 cells in phosphate buffer (pH 7.5) as assessed by alkaline single cell gel electrophoresis (comet) assay and quantification of comet types. Ascorbate and Trolox inhibited cellular DNA breaking induced by peroxynitrous acid, but the concentrations of these antioxidants required for effective inhibition was about 50-fold higher than that of peroxynitrous acid. βCarotene protected DNA breaking by peroxynitrous acid in 20% tetrahydrofuran-phosphate buffer (pH 7.5) much more effectively than ascorbate and Trolox. The concentrations of β-carotene required for effective inhibition was lower than the concentration of peroxynitrous acid.     

Rapid scavenging of peroxynitrous acid by monohydroascorbate

The reaction of peroxynitrous acid with monohydroascorbate, over the concentration range of 250 μM to 50 mM of monohydroascorbate at pH 5.8 and at 25°C, was reinvestigated and the rate constant of the reaction found to be much higher than reported earlier (Bartlett, D.; Church, D. F.; Bounds, P. L.; Koppenol, W. H. The kinetics of oxidation of L-ascorbic acid by peroxynitrite. Free Radic. Biol. Med. 18:85–92; 1995; Squadrito, G. L.; Jin, X.; Pryor, W. A. Stopped-flow kinetics of the reaction of ascorbic acid with peroxynitrite. Arch. Biochem. Biophys. 322:53–59; 1995). The new rate constants at pH 5.8 are k₁ = 1 × 10⁶ M⁻¹ s⁻¹ and k₋₁ = 500 s⁻¹ for 25°C and k₁ = 1.5 × 10⁶ M⁻¹ s⁻¹ and k₋₁ = 1 × 10³ s⁻¹ for 37°C. These values indicate that even at low monohydroascorbate concentrations most of peroxynitrous acid forms an adduct with this antioxidant. The mechanism of the reaction involves formation of an intermediate, which decays to a second intermediate with an absorption maximum at 345 nm. At low monohydroascorbate concentrations, the second intermediate decays to nitrate and monohydroascorbate, while at monohydroascorbate concentrations greater than 4 mM, this second intermediate reacts with a second monohydroascorbate to form nitrite, dehydroascorbate, and monohydroascorbate. EPR experiments indicate that the yield of the ascorbyl radical is 0.24% relative to the initial peroxynitrous acid concentration, and that this small amount of ascorbyl radicals is formed concomitantly with the decrease of the absorption at 345 nm. Thus, the ascorbyl radical is not a primary reaction product. Under the conditions of these experiments, no homolysis of peroxynitrous acid to nitrogen dioxide and hydroxyl radical was observed. Aside from monohydroascorbate's ability to “repair” oxidatively modified biomolecules, it may play a role as scavenger of peroxynitrous acid.     

Degradation of extracellular matrix by peroxynitrite/peroxynitrous acid

The extracellular matrix (ECM) provides strength and elasticity to tissues and plays a key role in regulating cell behavior; damage to this material is believed to be a major factor in many inflammatory diseases. Peroxynitrite/peroxynitrous acid, which is generated at elevated levels at sites of inflammation, is believed to play a role in ECM damage; however, the mechanisms involved are poorly understood. Here we examined the reactions of bolus peroxynitrite, and that generated in a time-dependent manner by SIN-1 decomposition, with ECM isolated from a vascular smooth muscle cell line and porcine thoracic aorta. Bolus peroxynitrite caused the release of ECM glycosaminoglycans and proteins, the formation of 3-nitroTyr, and the detection of ECM-derived radicals (by immuno-spin trapping) in a concentration-dependent manner. Release and nitration of ECM components were modulated by the local pH and bicarbonate. SIN-1 caused the release of glycosaminoglycan, but not protein, from vascular smooth muscle cell-derived ECM in a concentration-, time-, and pH-dependent manner. The data presented here suggest that peroxynitrite-mediated damage to ECM occurs via a radical-mediated pathway. These reactions may contribute to ECM damage at sites of inflammation and play a role in disease progression, including rupture of atherosclerotic lesions.     

Free radical yields from the homolysis of peroxynitrous acid.

A recent study reports discordant results for maximal free-radical yields from the decomposition of peroxynitrous acid using different assay reagents and a puzzling decrease (to zero) of the radical yields with increasing pH. An assay method using 2,2,'azino-bis(3-ethyl-benzthiazoline sulphonate) (ABTS) has been tested using stopped-flow kinetic studies, and the results imply that the assay is satisfactory when [HOONO] much greater than [-OONO] but that reactions involving peroxynitrite anion interfere at higher pH. Evidence is presented that peroxynitrite was an interfering species in the earlier studies.     

Effect of ozone and nitrogen dioxide on the agglutination of rat alveolar macrophages by concanavalin A

$\text{In vitro}$ exposure of rat alveolar macrophages to 0.5 ppm ozone for 60 minutes results in a 76% decrease in agglutination by concanavalin A. A decrease in the agglutinability of rat alveolar macrophages by concanavalin A was also observed following inhalation of 0.5 or 1.0 ppm ozone for two hours. In contradistinction, $\text{in vitro}$ exposure of rat alveolar macrophages to 2.4 ppm nitrogen dioxide for 60 minutes produced a 64% increase in agglutination by concanavalin A; and increased agglutinability was also noted following inhalation of 12.1 ppm nitrogen dioxide for two hours. Agglutination was almost completely inhibited by alpha-methyl-mannose. Neither pollutant significantly altered the binding of ³H-concanavalin A to rat alveolar macrophages. These two air pollutants, both of which are known to potentiate respiratory tract infections, appear to affect the response of the alveolar macrophage membrane to concanavalin A in a dissimilar fashion.     

Effect of light and atmospheric carbon dioxide concentration on nitrogen fixation by herbage legumes

Summary Lucerne, red clover and white clover were grown at two atmospheric concentrations of CO₂ (300 and 1000 μl l⁻¹) and the effects on N₂ fixation, nodule mass/number and root/shoot dry matter production determined. Pea plants were similarly evaluated as a comparison with grain legumes. CO₂ enrichment increased N₂ fixation activity in all cases but activity/unit nodule mass was significantly increased only in the pea. The enhancement of N₂ fixation in herbage legumes by CO₂ enrichment reflected an increase in nodule mass which in turn was attributed to increased nodule number, and results show that under the experimental conditions obtaining here photosynthate supply did not limit nodule N₂ fixation in these plants though it was limiting in the case of peas. White clover growing in a 6 and 14 hour photoperiod was studied for response of the N₂ fixing system to light. Long photoperiod (14 hour) plants assayed at constant temperature (20°C) did not show a significant response to light at the end of the dark period either in terms of fixation per plant or per unit nodule mass, in contrast with short photoperiod (6 hour) plants which showed significant responses. Short photoperiod plants compensated for reduced photosynthates by maintaining only half the root nodule mass and fixation activity of 14 hour photoperiod plants though plants in both systems supported similar rates of N₂ fixation per unit mass of nodule during the photoperiod. Comparison of N₂ fixation activities in whole and decapitated plant systems indicates the importance of shoot reserves for sustaining nitrogenase activity in white clover during short-term interruption of photosynthesis. These results support the conclusion of the CO₂ enrichment studies, that herbage legumes have the potential for supplying their nodule photosynthate requirements for sustaining optimum rates of N₂ fixation and excess carbon supply is used solely to promote further nodulation. Nodules of short photoperiod white clover plants were less efficient in N₂ fixation in that they evolved more H₂ relative to N₂ (C₂H₂) reduced than did long photoperiod plants.     

Biosynthesis of gamma-linolenic acid and beta-carotene by Zygomycetes fungi

Due to increasing demand for natural sources of both polyunsaturated fatty acids (PUFAs) and beta-carotene, 28 Zygomycetes fungal soil isolates were screened for their potential to synthesize these biologically active compounds. Although all fungi produced C₁₈ PUFAs, only nine strains also formed beta-carotene. Although Actinomucor elegans CCF 3218 was the best producer of gamma-linolenic acid (GLA) (251 mg/L), Umbelopsis isabellina CCF 2412 was found to be the most valuable fungus because of the dual production of GLA (217 mg/L) and beta-carotene (40.7 mg/L). The calculated ratio of formed PUFAs provided new insight into activities of individual fatty acid desaturases involved in biosynthetic pathways for various types of PUFAs. The maximal activity of delta-9 desaturase was accompanied by high accumulation of storage lipids in fungal cells. On the other hand, maximal activity of delta-15 desaturase was found in strains synthesizing low amounts of oleic acid due to diminished delta-9 desaturase. Activities of delta-6 desaturase showed competition for fatty acids engaged in n3, n6, and n9 biosynthetic pathways. Such knowledge about fatty acid desaturase activities provides new challenges for the regulation of biotechnological production of PUFAs by Zygomycetes fungi.     

Effect of temperature on nitrogen transformation in Hawaiian soils

Summary In a field experiment soil samples buried at the warmer temperature regime nitrified added ammonium faster than soils buried at the cooler temperature regime. Nitrification occurred more rapidly under both regimes in a soil which had developed in a warm climatic zone than in two other soils developed under cooler conditions.The rate of nitrification of added ammonium in soils incubated at 5, 15, 25 and 40°C in the laboratory increased with increase in the temperature up to 25°C in three out of four soils. In the fourth soil nitrification was as active at 40°C as at 25°C. The temperature range for appreciable nitrification to occur in a soil was related to the environmental conditions where the soil was formed.Mineralization of organic nitrogen occurred to a greater extent at 40°C than at three lower incubating temperatures of 5, 15, and 25°C. Rapid and active mineralization was associated with high organic matter and C/N ratio in soils     

Biogenesis of retinoic acid from beta-carotene. Differences between the metabolism of beta-carotene and retinal

The ability of beta-carotene to serve as precursor to retinoic acid was examined in vitro with cytosol prepared from rat tissues. The rate of retinoic acid synthesis from 10 microM beta-carotene ranged from 120 to 224 pmol/h/mg of protein with intestinal cytosol, and from 344 to 488 pmol/h/mg of protein with cytosols prepared from kidney, lung, testes, and liver. Retinol generated during beta-carotene metabolism was not the major substrate for retinoic acid synthesis. At low substrate concentrations (2.5 microM), the rates of retinoic acid synthesis in intestinal cytosol from beta-carotene or retinol were equivalent, and at higher concentrations (10 microM) the rates of retinoic acid synthesis from beta-carotene or retinol in intestine, testes, lung, and kidney were comparable. Thus, beta-carotene metabolism may be an important source of retinoic acid in retinoid target tissues, particularly in species such as humans that are capable of accumulating high concentrations of tissue carotenoids. Retinal, considered an initial retinoid product of beta-carotene metabolism, was not detected as a product of beta-carotene metabolism in vitro. A ratio of retinol and retinoic acid different from that observed during beta-carotene metabolism in vitro was observed with incubations of retinal under identical conditions. These data indicated that beta-carotene metabolism is not merely a simple process of producing retinal and releasing it into solution to be metabolized independently.     

Spin trapping of nitrogen dioxide and of radicals generated from nitrous acid

Spin trapping of nitrogen dioxide radical by several nitrones has been studied. The reaction results in the formation of persistent acyl nitroxides, after the oxidation of the intermediate spin adducts having an -ONO group on C-2 atom. The intermediate is effectively detected when DEPMPO is used as the spin trap. The reaction between PBN or 5,7-di-tert-butyl-3,3-dimethyl indoline N-oxide with nitrous acid gives the corresponding acyl nitroxide only when oxygen is present in the reaction milieu.

On the other hand, nitroso spin traps do not trap ^⋅NO₂ confirming that the unpaired electron of nitrogen dioxide is localized on the oxygen atom.     

氨基酸添加对亚热带森林红壤氮素转化的影响

为探究氨基酸氮形态对亚热带土壤氮素含量及转化的影响,选择建瓯市万木林保护区的山地红壤为对象,采用室内培养实验法,通过设计60%和90%WHC两种土壤含水量并添加不同性质氨基酸,测定了土壤中铵态氮、硝态氮、可溶性有机氮的含量和氧化亚氮的释放量,分析了可溶性有机碳、土壤p H值的大小变化及其与氮素的相互关系。结果表明:与对照处理相比,氨基酸添加显著增加了土壤NH_4~+-N含量并使土壤p H值升高,且在一定程度上解除了高含水量(90%WHC)对NH_4~+-N产生的抑制,其中甲硫氨基酸的效果最为明显。酸性、碱性、中性氨基酸对土壤NO_3~--N含量和N_2O释放影响不显著,但甲硫氨基酸可显著抑制土壤硝化从而导致NH_4~+-N的积累,并在培养前期抑制土壤N_2O产生而在培养后期促进N_2O释放,总体上促进N_2O释放。60%WHC的氨基酸添加处理较90%WHC条件下降低土壤可溶性有机氮的幅度更大。氨基酸对土壤氮素转化的影响与带电性关系较小,而可能与其分解产物密切相关。可见,不同性质氨基酸处理对森林土壤氮素含量及转化存在不同程度的影响,且甲硫氨基酸对土壤氮素转化的影响机理值得深入研究。     

Inhibition of arachidonic acid oxidation by beta-carotene, retinol and alpha-tocopherol

Prostaglandin and hydroxyeicosatetraenoic acid (HETE) production from arachidonic acid in bovine seminal vesicles and kidney as influenced by the addition of beta-carotene, retinol or alpha-tocopherol was studied. The major product formed was prostaglandin E2 (approximately 85% prostaglandin E2 of control), and its proportion decreased with increasing concentration of the additives, while the proportion of HETE increased. Prostaglandin and HETE production was considerably inhibited by beta-carotene and retinol, and to a lesser extent by alpha-tocopherol; HETE production was inhibited less than that of prostaglandin. It appears that beta-carotene, retinol and alpha-tocopherol influence both the cyclooxygenase and lipoxygenase pathways; this modulation of arachidonic acid oxidation by physiological compounds may have important in vivo implications.     

Effect of plant growth on transformation of mineral nitrogen in soils

Summary The effects of plants on NH₄- and NO₃-N transformations were studied on a clay loam soil pH 7.7, organic carbon 2.4%, and total N 0.25%. Without plants 80% of the extractable NH₄-N from a 0.3 ppm addition was nitrified in one hour, and 100% in one day. The disappearance of NH₄-N was increased from 20%, where plants were not grown, to 50% where barley plants were grown for two weeks. Only 5% of the NO₃-N added to soil disappeared in the absence of plant growth, but 40% disappeared with plants. N also disappeared when added to soil from which growing plants had recently been removed. Several hypotheses, including transformation to gaseous forms, are advanced to explain the disappearance.     

Effect of dietary beta-carotene on the accumulation of beta-carotene and vitamin A in plasma and tissues of gilts

The absorption of beta-carotene in pigs is limited. Nevertheless beta-carotene might positively affect reproduction. In this study the absorption and tissue distribution of beta-carotene as well as its function as precursor of vitamin A was investigated in gilts that were fed according to one of three dietary treatments: VA (4000 IU vitamin A), VA + VA (4000 IU + 8300 IU) and VA + BC (4000 IU + 100 mg beta-carotene per kg diet) for 14 weeks. Only in the VA + BC group was beta-carotene detected in plasma (1-8 ng x mL(-1)), liver, adrenals and corpora lutea, indicating that pigs absorb intact beta-carotene at low rates. Liver levels of vitamin A were higher (P < 0.01) at comparable levels in the VA + VA and VA + BC group than in the VA group, indicating a conversion rate of beta-carotene to vitamin A of 40 to 1 on the basis of weight for beta-carotene at this level (100 mg x kg(-1)) in the diet. Higher levels of vitamin A in the uterus of the VA + BC group (P < 0.01) as well as the accumulation of beta-carotene in adrenals and corpora lutea might reflect some influence of beta-carotene on local vitamin A metabolism which might be of importance for reproductive performance in gilts.     

双氰胺对碳酸氢铵在土壤中氮素转化的影响

采用室内培养试验方法,研究了双氰胺对碳酸氢铵中的铵态氮在土壤中动态变化的影响。结果表明,无论是碳铵与双氰胺的机械混施,还是含双氰胺的长效碳铵,在抑制铵态氮硝化产生硝态氮的同时对氨挥发也有一定的抑制作用,使土壤在更长的时间内保持更高的铵态氮含量,并对碳铵施入后土壤的酸碱化强度起到了缓冲作用。添加双氰胺不但提高了碳铵的氮肥利用率,还减少了肥料氮素损失.