Lysozyme is an ozone-sensitive component of alveolar type II cell lamellar bodies

Exposure of rats to 3 ppm ozone for up to 8 h results in significant changes in lamellar bodies, the surfactant storing organelles of type II cells. We have previously shown that a 14 kDa lamellar body protein is decreased as early as 4 h after the onset of ozone exposure. We have isolated this ozone-sensitive protein from rat lung lamellar bodies and identified it as lysozyme by immunochemical methods, as well as by its amino acid composition, N-terminal amino acid sequence and bacteriolytic activity. Reduced lysozyme activity in isolated lamellar bodies is detected as early as 4 h after the start of ozone exposure. Following an 8 h ozone exposure, the activity does not return to control levels for at least 48 h. Lamellar body lysozyme is expected to be secreted with surfactant phospholipids, thereby contributing to the antimicrobial defense of the alveolar lining layer. The acute lysozyme deficiency seen in ozone-induced oxidant injury may reduce the resistance of the lung to infection.     

Variations of Some Physiological and Immunological Parameters in Siberian Sturgeon (Acipenser baerii,Brandt, 1869) Subjected to an Acute Stressor

This study was carried out to investigate the effect of an acute stressor on the variation of some physiological and immunological parameters of Siberian sturgeon (Acipenser baerii) juveniles. Fish, reared in 3 tanks for 10 weeks, were used for this study. The acute stress of fish consisted of 2 min of air exposure stress. Plasma levels of cortisol, glucose, and lactate as well as lysozyme activity in plasma were measured before stress and 1 hr, 3 hr, 6 hr, 9 hr, 12 hr, and 24 hr after stress. The plasma cortisol significantly increased in the highest level 1 hr after stress, yet it gradually declined after 3 hr. The glucose significantly increased only 1 hr after stress. There was no significant difference between plasma lactate prestress and poststress. Moreover, lysozyme activity was enhanced by stress, thus reaching the highest level 9 hr after stress. The results of this study indicate that Siberian sturgeon not only have a rapid response to acute stress, but also a great capacity for recovery from stress, thus returning physiological parameters to prestress levels after 6 hr.     

Ozone increases the permeability of isolated pea chloroplasts

The effect of short-term exposure of chloroplasts isolated from the leaves of Pisum sativum to high concentrations of ozone was examined. The inhibitory effect of O₃ on endogenous photophosphorylation was apparently related to an increased permeability of the chloroplast limiting membranes induced by ozone exposure. A 5 min treatment with 50 ppm O₃ reduced the reflection coefficient of meso-erythritol from 0.84 to 0.58 and that of glycerol from 0.26 to 0.03. Such decreases in reflection coefficients indicate that ozone caused a marked increase in the permeability of the limiting membranes of the chloroplasts, which may result from an oxidation of membrane lipids. The decrease in the reflection coefficient of meso-erythritol was proportional both to ozone concentration (up to 30 ppm for 5 min of bubbling) and to time (up to 5 min at 30 ppm). Extrapolating these results to lower concentrations and longer times, ozone injury should be possible for a 2 hr exposure of plants to 0.3 ppm ozone, as is indeed the case.     

Lysozymelike activity in the hemolymph of Biomphalaria glabrata challenged with bacteria.

Levels of lysozyme activity have been determined in the serum and cells of untreated Biomphalaria glabrata and in snails that had been challenged with heat-killed Bacillus megaterium and water at 1, 2, and 4 hr postinjection. Lysozyme activities have also been ascertained in sham-injected snails at 1, 2, and 4 hr postchallenge. Our results indicate significant alterations in the serum lysozyme activity levels at 2 and 4 hr postchallenge with bacteria and at 1 hr postinjection of water. Also, there is a significant increase in cell lysozyme activity at 1 hr postchallenge with B. megaterium. It is concluded that lysozyme is released from phagocytes into serum as a result of challenge with B. megaterium. Although the exact role of the released enzyme is uncertain, it is hypothesized that it may serve as a humoral defense molecule.     

Effects of Nitrogen Dioxide on Pulmonary Cell Population

Studies from this laboratory have shown that ozone produces changes in the number and function of cells obtained by pulmonary lavage. In similar experiments, rabbits exposed to levels of NO(2) from ambient to 60 ppm demonstrated increased numbers of polymorphonuclear leukocytes in the lung washings. This phenomenon persisted for more than 72 hr after a single 3-hr exposure. When streptococci were instilled in the lungs of NO(2)-exposed anesthetized rabbits 30 min before lavage, a pronounced inhibition of phagocytic activity was observed. With these criteria, NO(2) appeared less effective than ozone as a pulmonary irritant.     

Toxic effects of ozone on murine L929 fibroblasts. Enzyme inactivation and glutathione depletion.

Exposure of L929 murine fibroblasts to ozone resulted in K+ leakage and inhibition of several enzymes. Most sensitive to ozone exposure were glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase. The activities of another cytosolic enzyme, lactate dehydrogenase, the mitochondrial enzymes glutamate dehydrogenase, succinate dehydrogenase, cytochrome c oxidase and the activity of the lysosomal enzymes acid phosphatase and beta-glucuronidase were, initially, not or only slightly affected. The localization of the lysosomal enzymes did not change during ozone exposure. After prolonged exposure complete deterioration of the cells was observed and all enzyme activities declined. The activity of the enzymes was also monitored during ozone exposure of a sonicated cell suspension and it was shown that all these enzymes are in fact susceptible to ozone. These observations clearly demonstrate that, besides the structure and amino acid composition of an enzyme, the localization in the cell plays an important role in its susceptibility to ozone. The intracellular levels of reduced and oxidized glutathione were affected as well. The ATP content, however, proved to be insensitive to ozone exposure.     

Response of photosynthesis and cellular antioxidants to ozone in populus leaves

Atmospheric ozone causes formation of various highly reactive intermediates (e.g. peroxyl and superoxide radicals, H₂O₂, etc.) in plant tissues. A plant's productivity in environments with ozone may be related to its ability to scavenge the free radicals formed. The effects of ozone on photosynthesis and some free radical scavengers were measured in the fifth emergent leaf of poplars. Clonal poplars (Populus deltoides × Populus cv caudina) were fumigated with 180 parts per billion ozone for 3 hours. Photosynthesis was measured before, during, and after fumigation. During the first 90 minutes of ozone exposure, photosynthetic rates were unaffected but glutathione levels and superoxide dismutase activity increased. After 90 minutes of ozone exposure, photosynthetic rates began to decline while glutathione and superoxide dismutase continued to increase. Total glutathione (reduced plus oxidized) increased in fumigated leaves throughout the exposure period. The ratio of GSH/GSSG also decreased from 12.8 to 1.2 in ozone exposed trees. Superoxide dismutase levels increased twofold in fumigated plants. After 4 hours of ozone exposure, the photosynthetic rate was approximately half that of controls while glutathione levels and superoxide dismutase activity remained above that of the controls. The elevated antioxidant levels were maintained 21 hours after ozone exposure while photosynthetic rates recovered to about 75% of that of controls. Electron transport and NADPH levels remained unaffected by the treatment. Hence, elevated antioxidant metabolism may protect the photosynthetic apparatus during exposure to ozone.     

臭氧急性暴露对大鼠血管的损伤及其机制

目的:探索不同浓度臭氧(O3)急性暴露对雄性Wistar大鼠血管的损伤效应和可能的机制。方法:120只雄性Wistar大鼠随机分为6组,每组20只;实验动物置于气体染毒柜中,对照组暴露于过滤后空气,处理组分别暴露于浓度为0.12ppm,0.5ppm,1.0ppm,2.0ppm和4.0ppm的臭氧,持续暴露4h。利用PC-lab医学生理信号采集系统获得动脉血压数据;血流变指标和血生化指标由天津迪安诊断实验室检测;血清中内皮素(ET-1)、同型半胱氨酸(HCY)、血管性血友病因子(vWF)、8-羟基脱氧鸟苷(8-OhdG)、白介素(IL-6)和肿瘤坏死因子α(TNF-α)采用酶联免疫(ELISA)微孔板法检测;氧化应激指标超氧化物歧化酶(SOD)活力和丙二醛(MDA)分别采用黄嘌呤氧化酶法、硫代巴比妥酸(TBA)法测定,还原型谷胱甘肽(GSH)和一氧化氮(NO)采用微孔板比色法;取胸主动脉组织制备石蜡切片,经HE染色后观察血管结构改变。结果:0.12ppm臭氧急性暴露可导致动脉收缩血压(SBP)显著升高;不同浓度臭氧暴露均可导致血浆粘度显著升高,1.0ppm臭氧暴露组血沉(ESR)方程K值显著升高,全血高切相对指数和还原粘度均在臭氧浓度为0.5ppm和4.0ppm时显著降低,而红细胞变形指数在臭氧浓度为0.12ppm、0.5ppm、1.0ppm和2.0ppm时显著升高;急性臭氧暴露可导致总胆固醇含量降低,高密度脂蛋白胆固醇(HDL-C)在0.12ppm臭氧暴露组显著降低;当臭氧浓度高于1.0ppm时还可导致机体出现炎症反应(TNF-α升高)和氧化应激反应(MDA升高、GSH降低);臭氧急性暴露可导致血液中ET-1含量升高,在4.0ppm浓度组具有显著性差异,而HCY水平呈现先降低后升高的趋势,在1.0ppm浓度组达到最高值,胸主动脉未见明显的病理改变。结论:臭氧急性暴露可影响大鼠的动脉血压、血流变及胆固醇代谢,可能的机制是臭氧暴露导致炎症反应和氧化应激反应,引起血管内皮功能损伤,并且随着臭氧暴露浓度升高血管内皮细胞功能损伤越显著。     

OZONE DAMAGE TO PONDEROSA PINE: A HISTOLOGICAL AND HISTOCHEMICAL APPRAISAL

Histological and histochemical changes occurred in current-year needles of sensitive clonal selections of Pinus ponderosa Laws under natural summer growing conditions in the San Bernardino National Forest near Los Angeles, California due to fumigations with 0.45 ppm ozone for 12 hr/day. Within five days after the start of fumigation, chloroplasts and carbohydrate stain accumulated in the peripheral portions of mesophyll cells. Concurrently, the homogenous distribution of proteins and nucleic acids was disrupted. Succinate dehydrogenase was localized mostly in guard cells, resin duct epithelial cells, albuminous cells, and differentiating vascular tissues within unfumigated and fumigated leaves. Acid phosphatase activity increased within mesophyll cells during ozone exposure, but there was no association of acid phosphatase or ozone injury with stomata. Wall destruction occurred in mesophyll cells after appreciable intracellular damage. These histological and histochemical changes occurred within 5–7 days, but visual symptoms were not evident until 2–3 weeks after fumigation. It is thus possible to assay ozone damage very soon after exposure if no other external agents cause similar results.     

Increasing tolerance to ozone by elevating foliar ascorbic acid confers greater protection against ozone than increasing avoidance

Ascorbic acid (Asc) is the most abundant antioxidant in plants and serves as a major contributor to the cell redox state. Exposure to environmental ozone can cause significant damage to plants by imposing conditions of oxidative stress. We examined whether increasing the level of Asc through enhanced Asc recycling would limit the deleterious effects of environmental oxidative stress. Plants overexpressing dehydroascorbate reductase (DHAR), which results in an increase in the endogenous level of Asc, were exposed to acute or chronic levels of ozone. DHAR-overexpressing plants had a lower oxidative load, a lower level of oxidative-related enzyme activities, a higher level of chlorophyll, and a higher level of photosynthetic activity 24 h following an acute exposure (2 h) to 200 ppb ozone than control plants, despite exhibiting a larger stomatal area. Reducing the size of the Asc pool size through suppression of DHAR expression had the opposite effect. Following a chronic exposure (30 d) to 100 ppb ozone, plants with a larger Asc pool size maintained a larger stomatal area and a higher oxidative load, but retained a higher level of photosynthetic activity than control plants, whereas plants suppressed for DHAR had a substantially reduced stomatal area, but also a substantially lower level of photosynthetic activity. Together, these data indicate that, despite a reduced ability to respond to ozone through stomatal closure, increasing the level of Asc through enhanced Asc recycling provided greater protection against oxidative damage than reducing stomatal area.     

Effect of ozone exposure on prostacyclin synthesis in lung

The effect of ozone exposure on prostacyclin (PGI2) synthesis in the rat lung was studied. Male Wistar rats were exposed to 0.2, 0.4, 0.8, 1.2 and 1.8 ppm ozone for 24h. The higher concentration (1.8 ppm) significantly depressed-PGI2 synthesizing activity of lung homogenates. Time-courses (1, 3, 5, 7, 14 and 28 days) of the effect of ozone (0.4 and 0.8 ppm) exposure on the PGI2-synthesizing activity of lung homogenates were studied. The PGI2-synthesizing activity of the lung decreased, reaching a maximum at 5 days and then gradually returning to normal by day 14, and remaining normal at day 28, even though the ozone exposure continued. The formation of lipid peroxides due to ozone exposure may cause the depression of PGI2-synthesizing activity of lung. Induction of anti-oxidative enzymes may relate to the recovery of the PGI2-synthesizing activity.     

Schistosoma mansoni: species-selective response to N-chloroethyl-acetylcholine derivatives

Three new acetylcholine mustard analogs were tested on schistosome and vertebrate neuromuscular preparations. These compounds extend a previously reported structure-activity series. The new compounds investigated include isopropyl-, cyclohexyl-, and benzyl-2-acetoxyethyl-2'-chloroethylamine (PrM, ChM, and BzM). In schistosome motor activity studies, all of the compounds caused irreversible reductions in the activity of Schistosoma mansoni after 1 hr exposure followed by 19 hr in drug-free medium. Under nonlethal conditions of dosage and exposure time, all compounds blocked carbachol-induced paralysis, indicating a possible action at schistosome cholinergic sites. All the compounds also reduced the labeling of schistosomes by dimethylaminonapthalene-5-sulfonamidoethyl dimethylamine hydrochloride (DDNS), a fluorescent ACh analog. In isolated vertebrate tissue experiments. PrM and ChM had slight agonist activity in the guinea pig ileum, and only PrM had agonist activity in the frog rectus abdominis preparation. PrM and BzM were found to antagonize ACh responses in the guinea pig ileum. Exposure of vertebrate tissues for 1 hr to high concentrations of ChM caused no long-lasting inhibition of ACh, pilocarpine, and serotonin (5HT) responses. Histamine responses were slightly reduced from control. Following 1 hr exposure of vertebrate tissues to PrM, initial reductions in all responses were seen, followed by recoveries to near control. BzM treatment, however, reduced all responses far below control; the tissues did not recover even after washing for 2 hr. It is concluded that ChM and, to a lesser extent, PrM, had a greater effect on schistosomes than on vertebrate tissues. BzM did not display species selectivity in this favorable direction.     

Augmentation of human natural cell-mediated cytotoxicity by recombinant human interleukin 2

Human peripheral blood mononuclear cells (PBMC) demonstrated increased natural cell-mediated cytotoxicity (NCMC) activity after only 5 min of exposure to purified recombinant human IL 2 or interferon (IFN)-gamma. The mechanism of NCMC augmentation by treatment with IL 2 is not entirely dependent on IFN-gamma production because: a) IL 2 was found to augment NCMC activity at levels which did not induce detectable IFN-gamma; b) IL 2 required only 5 min of exposure to PBMC to augment NCMC activity, whereas 3 hr of contact were required to demonstrate detectable IFN-gamma levels; c) the levels of NCMC enhancement by treatment with IL 2 exceeded the amount of NCMC enhancement that could be due to IFN alone; d) anti-recombinant IFN-gamma, which totally eliminated the augmentation of NCMC enhancement by IFN-gamma, only partially reduced the augmentation of NCMC activity by IL 2; and e) combination treatment of PBMC with IL 2 and IFN-gamma resulted in a synergistic enhancement of NCMC. The results strongly support the conclusion that augmentation of NCMC by IL 2 and IFN-gamma involve overlapping mechanisms.     

萝卜块根中两个具溶菌酶活性的几丁质结合蛋白的纯化及其特性

通过亲和层析和羧甲基一纤维素离子交换层析从萝卜的块根中分离到两个具溶菌酶活性的酶组份：CBP1和CBP2。两者经SDS-PAGE均显示单一蛋白染色条带,其对应的分子量分别为26．9kD和24．8kD。两种蛋白除有溶菌酶活性外,还有几丁质酶活性,但无壳聚糖酶活性。各种类型的几丁质对CBP1和CBP2都有较强的吸附作用,而在还原／非还原的单向SDS-PAGE中却观察不到两者分子中存在二硫键。     

Effect of cold exposure on brain 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in some vertebrate species

1. The changes in 5-HT and 5-HIAA levels were studied in the brain regions of Gerbillus pyramidum, Streptopelia senegalensis aegyptiaca and Agama stellio following exposure to cold. 2. In general, the 5-HT levels increased in the Gerbillus brain parts and decreased in those of Streptopelia. 3. Cold exposure in the Agama brain regions caused a transient decrease in the 5-HT levels of the cerebral hemispheres, midbrain and pons plus medulla after 6 hr and a general increase after 12, 24 and 48 hr. 4. It is concluded that cold exposure may be associated with increased activity of 5-HT ergic neurons and the rate of turnover of 5-HT to 5-HIAA.     

Experimental exposure of rainbow trout Oncorhynchus mykiss (Walbaum) to the infective stages of the sea louse Lepeophtheirus salmonis (Krøyer) influences the physiological response to an acute stressor

The influence of infection with the juvenile stages of the sea louse, Lepeophtheirus salmonis (Kr?yer) on the response of rainbow trout Oncorhynchus mykiss (Walbaum) to a net confinement protocol was investigated. The experiment consisted of two groups of seawater-adapted rainbow trout, one which was exposed to a total of 4000 nauplii/copepodid stages of L. salmonis 30, 25 and 14 days prior to confinement. Confinement elicited a greater stress response in the lice-exposed fish, than in the controls, as seen by higher plasma cortisol and glucose levels. A reduced spleen somatic index in exposed fish following 6 h confinement coincided with increased erythrocyte and lymphocyte numbers in the blood. Circulating lymphocyte numbers were significantly reduced in both groups 24 h post-confinement, when a lower alternative complement activity was recorded in control fish. Prior to confinement, lice-exposed fish had an elevated serum lysozyme activity and reduced oxygen radical production by blood leukocytes. Following confinement, lysozyme activity was gradually reduced in lice-exposed trout. During confinement, oxygen radical production decreased in control fish and increased in infested fish. Overall, transient exposure to juvenile lice altered the response to a second stressor, which has implications for management procedures of L. salmonis exposed fish.     

In vivo adaptive control of beta-receptors and adenylate cyclase during short-term heat exposure in rat parotid glands.

1. Adaptation of beta-adrenergic receptors (beta-AR) and adenylate cyclase (AC) in rat parotid glands during short-term heat exposure (33 degrees C) were studied. 2. Heat exposure reduced AC activity in response to isoproterenol (IPR). 3. The number of beta-AR on the cell surface significantly increased after 24 hr but returned to control level after 48 hr. 4. IPR-induced [3H]GDP release was significantly reduced throughout exposure. 5. The data suggest that the major factor which results in the desensitization of AC during short-term heat exposure is a blunted coupling between beta-AR and GTP binding protein(s).     

Transgenic tobacco plants expressing antisense RNA for cytosolic ascorbate peroxidase show increased susceptibility to ozone injury

Ascorbate peroxidase (APX), as a participant in the ascorbate—glutathione cycle, has been suggested to be a particularly important antioxidant enzyme in helping plants survive oxidative stress, but direct evidence for this has not been reported. We demonstrate that expressing antisense RNA comprising 45% of the 3'-coding region of the tobacco cytosolic APX, can reduce significantly both the endogenous APX mRNA levels and the APX catalytic activity in transgenic tobacco plants. Those transgenic plants showing a reduction in both endogenous APX mRNA levels and extractable APX activity display a significant increase in ozone injury following high-level ozone exposure. Lower-level ozone exposure reveals even more drastic differences between the antisense and control plants, suggesting that even a partial loss of APX function in oxidative defence cannot be fully compensated for by other antioxidant measures.     

AMP-activated protein kinase deficiency reduces ozone-induced lung injury and oxidative stress in mice

Background

Acute ozone exposure causes lung oxidative stress and inflammation leading to lung injury. At least one mechanism underlying the lung toxicity of ozone involves excessive production of reactive oxygen and nitrogen intermediates such as peroxynitrite. In addition and beyond its major prooxidant properties, peroxynitrite may nitrate tyrosine residues altering phosphorylation of many protein kinases involved in cell signalling. It was recently proposed that peroxynitrite activates 5''-AMP-activated kinase (AMPK), which regulates metabolic pathways and the response to cell stress. AMPK activation as a consequence of ozone exposure has not been previously evaluated. First, we tested whether acute ozone exposure in mice would impair alveolar fluid clearance, increase lung tissue peroxynitrite production and activate AMPK. Second, we tested whether loss of AMP-activated protein kinase alpha1 subunit in mouse would prevent enhanced oxidative stress and lung injury induced by ozone exposure.

Methods

Control and AMPKα1 deficient mice were exposed to ozone at a concentration of 2.0 ppm for 3 h in glass cages. Evaluation was performed 24 h after ozone exposure. Alveolar fluid clearance (AFC) was evaluated using fluorescein isothiocyanate tagged albumin. Differential cell counts, total protein levels, cytokine concentrations, myeloperoxidase activity and markers of oxidative stress, i.e. malondialdehyde and peroxynitrite, were determined in bronchoalveolar lavage (BAL) and lung homogenates (LH). Levels of AMPK-Thr¹⁷² phosphorylation and basolateral membrane Na(+)-K(+)-ATPase abundance were determined by Western blot.

Results

In control mice, ozone exposure induced lung inflammation as evidence by increased leukocyte count, protein concentration in BAL and myeloperoxidase activity, pro-inflammatory cytokine levels in LH. Increases in peroxynitrite levels (3 vs 4.4 nM, p = 0.02) and malondialdehyde concentrations (110 vs 230 μmole/g wet tissue) were detected in LH obtained from ozone-exposed control mice. Ozone exposure consistently increased phosphorylated AMPK-Thr¹⁷² to total AMPK ratio by 80% in control mice. Ozone exposure causes increases in AFC and basolateral membrane Na(+)-K(+)-ATPase abundance in control mice which did not occur in AMPKα1 deficient mice.

Conclusions

Our results collectively suggest that AMPK activation participates in ozone-induced increases in AFC, inflammation and oxidative stress. Further studies are needed to understand how the AMPK pathway may provide a novel approach for the prevention of ozone-induced lung injury.