Endothelial Akt activation by hyperoxia: role in cell survival

High oxygen concentrations (hyperoxia), often required in the treatment of preterm infants and critically ill patients, cause lung injury, targeting especially the endothelium. Exposure of primary human lung microvascular endothelial cells (HLMVEC) to hyperoxia caused transient Akt activation after 60 min, as determined by Western blot analysis of phosphorylated Ser 473 of Akt. Akt phosphorylation was also increased after 24 h of hyperoxic exposure, which declined at 48 h. Adenoviral (Ad)-mediated expression of constitutively active myrAkt protected HLMVEC against hyperoxic injury. Cell death due to hyperoxia (95% O2, 8 days), which was primarily necrotic, was substantial in control and Ad-LacZ-transduced cells, but was diminished by almost half in myrAkt-transduced cells. Hyperoxia caused increased cellular glucose consumption, an effect that was amplified in cells transduced with myrAkt compared to the LacZ-transduced or the nontransduced controls. Increased glucose consumption in myrAkt-expressing cells was accompanied by increased phosphorylation of mTOR and p70 S6-kinase. Rapamycin treatment decreased glucose consumption in myrAkt-transduced cells to levels comparable to those in control and LacZ-transduced cells exposed to hyperoxia. Ultrastructural morphometric analyses demonstrated that mitochondria and endoplasmic reticulum were less swollen in myrAkt cells relative to controls exposed to hyperoxia. These studies demonstrate that early activation of Akt occurs in hyperoxia in HLMVEC. That this event is a beneficial response is suggested by the finding that constitutive activation of Akt protects against hyperoxic stress, at least in part, by maintaining mitochondrial integrity.     

Glutathione peroxidase-1 expression enhances recovery of human breast carcinoma cells from hyperoxic cell cycle arrest

We previously reported that hyperoxia (95% O(2)) induces an S-phase cell cycle arrest in glutathione peroxidase-deficient human carcinoma cells T47D-H3 (Exp. Cell Res. 256:347-357; 2000). Here, we investigated whether increasing the peroxide scavenging capacity via glutathione peroxidase-1 (GPx1) expression can prevent cell cycle alterations induced by oxidative stress. We show that GPx1-proficient T47D-GPx-2 transfectant cells, in which GPx1 concentration is most elevated in mitochondria (Biochem. Biophys. Res. Commun. 272:416-422; 2000), are partially resistant to cell cycle inhibition induced by hyperoxia or menadione exposure. Transient cell growth resistance was observed at the level of cell cycle phase distribution, Cdk2 activity, and DNA synthesis after 40 h hyperoxia. This differential resistance was associated with an inhibition of ROS production and lipid peroxidation induced by hyperoxia. After 64 h hyperoxic exposure, cell growth was completely abolished in both cell lines, despite elevated glutathione levels. However, in contrast to the GPx1-deficient cells, T47D-GPx-2 cells showed an increased capacity to recover from a cell cycle arrest mediated by a 64 h hyperoxic stress. Differential recovery was also observed at the ultrastructural level between Gpx1-proficient and -deficient cells. These data indicate that GPx1 played an important role in the cell capacity to recover from hyperoxic insults. The limited protection conferred by GPx1 during hyperoxia suggests that the deleterious effects were partially mediated by peroxide-derived free radicals, but also involved the action of nonperoxide-derived reactive species.     

SPF级新生大鼠高氧肺损伤模型的建立

目的建立一种操作简便、性能稳定的新生大鼠高氧肺损伤动物模型。方法①设计制造能自动控制氧浓度的高氧动物饲养舱。②将孕期满21 d出生的SPF级新生大鼠随机分成4组,即：高氧组Ⅰ（入高氧舱中饲养1～7 d）,高氧组Ⅱ（入高氧舱中饲养14 d）,以及相应的空气对照组Ⅰ、Ⅱ,每组14只。舱内氧浓度≥90%,每天23 h。计算肺系数,HE染色与病理学观察。结果高氧组与相应的对照组比较：高氧组大鼠体重轻,肺系数大,HE染色显示部分肺泡萎缩、肺间质及肺泡腔有水肿、出血,中性粒细胞浸润;肺泡发育明显滞后,辐射状肺泡计数明显少。结论本动物饲养舱性能稳定,操作简便;复制的新生大鼠的肺病理变化符合高氧肺损伤的改变。     

Mechanism of apoptosis induced by a lysosomotropic agent, L-Leucyl-L-Leucine methyl ester

Lysosomes are fundamental for cell growth, and thus inhibition of the lysosomal function often leads to cell death. L-Leucyl-L-leucine methyl ester (LeuLeuOMe) is a lysosomotropic agent that induces apoptosis of certain immune cells. LeuLeuOMe is taken up through receptor-mediated endocytosis, and then converted into (LeuLeu)_n-OMe (n>3) by dipeptidyl peptidase I (DPPI) in lysosomes, which reportedly causes rupture of the lysosomes and DNA fragmentation. In this study we examined how lysosomal damage causes DNA fragmentation in LeuLeuOMe-treated HL-60 cells. When acridine orange was employed to monitor lysosomal membrane integrity, orange or red granular fluorescence was seen in normal cells. In contrast, LeuLeuOMe-treated cells showed orange, yellow or green cellular fluorescence all over the cytoplasm, suggesting that LeuLeuOMe induced a loss of lysosomal membrane integrity. The loss was inhibited by a DPPI inhibitor, GlyPheCHN₂ (GFCHN₂), but not by a caspase-3 inhibitor, Ac-DEVD-CHO, indicating that a condensation product was responsible for the loss. LeuLeuOMe also induced the activation of caspase-3-like protease and DNA fragmentation, both of which were inhibited by either GFCHN₂ or Ac-DEVD-CHO. Therefore, the activation of caspase-3-like protease links the loss of lysosomal membrane integrity to DNA fragmentation during apoptosis induced by LeuLeuOMe.     

Effects of continuous and episodic hyperoxia on stress and hepatic glutathione levels in one-summer-old rainbow trout (Oncorhynchus mykiss)

One-summer-old rainbow trout ( Oncorhynchus mykiss ) were exposed to continuous hyperoxia (173 ± 24%) and three hyperoxic/normoxic treatments for 14 days. Hepatic glutathione status as the indicator of oxidative stress, as well as classical stress indicators such as hemoglobin, hematocrit and plasma cortisol levels, were measured during normoxic, constantly hyperoxic and the following episodically hyperoxic oxygen treatment regimes: 12 h hyperoxia:12 h normoxia (12 HYP:12 NOR), 24 HYP:24 NOR and 48 HYP:24 NOR. Constant hyperoxia tended to shrink erythrocytes, but the 12 HYP:12 NOR treatment increased the number of erythrocytes and thus enhanced the oxygen carrying capacity of blood. Similarly, a trend toward an elevation in plasma cortisol concentrations was detected in 12 HYP:12 NOR treatment group. The finding that elevated hepatic glutathione (GSH) levels (P < 0.01), indicative of enhanced potential of the liver tissue to resist oxidative stress, coincided with elevated cortisol levels might suggest that in the 12 HYP:12 NOR treatment physiological processes were recruited to increase oxygen carrying capacity in blood and to elevate protection against oxyradicals. However, none of the episodic hyperoxia treatments or continuous hyperoxia caused mortality or resulted in better growth. These data indicate that continuous hyperoxia (173 ± 24%) and hyperoxic-normoxic treatments may be applied in intensive culture of rainbow trout provided that fish have at least 24 h in normoxia prior to the next bout of hyperoxia. Shorter recovery periods, like in a 12 HYP:12 NOR treatment, may result in the increased need of oxygen in tissues followed by an activation of glutathione dependent defence system against an increased oxygen load.     

Characteristics of the intensity of glycolytic and oxidative processes in rat brain tissue under hypoxia and hyperoxia

Effect of hypoxia and hyperoxia on some indexes of energy metabolism was studied in the brain of intact rats and those with preliminarily administered hydrocortisone. So hypoxia (8 and 5.5% of oxygen in the medium) increases considerably the lactate and pyruvate content and has no effect on the oxidation and photophosphorylation in the brain mitochondria. The hyperoxic medium (1 at. abs. for 3-5h) inhibits consumption of oxygen and inorganic phosphate by the brain mitochondria, does not effect the lactate content and lowers the pyruvate level. The preliminary administration of hydrocortisone (1 mg per 100 g) under conditions of hypoxia and hyperoxia leads to the further intensification of the glycolysis independently of the initial level of the process. At the same time hormone administration favours normalization of the oxidative processes in the brain tissue under conditions of hyperoxia.     

Role of oxidative stress in lysosomal membrane permeabilization and apoptosis induced by gentamicin, an aminoglycoside antibiotic

Gentamicin, an aminoglycoside antibiotic used to treat severe bacterial infections, may cause acute renal failure. At therapeutic concentrations, gentamicin accumulates in lysosomes and induces apoptosis in kidney proximal tubular cells. In gentamicin-treated renal LLC-PK1 cells, acridine orange release from lysosomes, previously interpreted as lysosomal membrane permeabilization, precedes the apoptotic cascade that develops during incubation with gentamicin. However, the link between gentamicin lysosomal accumulation and apoptosis remains unclear. We here examined if reactive oxygen species (ROS) production could account for gentamicin-induced acridine orange release and apoptosis, and the implication of iron in these events. We found that gentamicin induced ROS production prior to, and at lower drug concentrations than required for, acridine orange release and apoptosis. ROS antioxidant or scavenger, catalase, and N-acetylcysteine largely prevented these events. Vital confocal imaging revealed that gentamicin-induced ROS production occurs in lysosomes. Deferoxamine, an iron chelator, which is endocytosed and accumulates in lysosomes, largely prevented gentamicin-induced ROS production as well as apoptosis. Direct evidence for gentamicin-induced permeabilization of lysosomal membrane was provided by showing the release into the cytosol of Lucifer yellow, a membrane-impermeant endocytic tracer with a comparable molecular weight as gentamicin. Altogether, our data demonstrate a key role of lysosomal iron and early ROS production in gentamicin-induced lysosomal membrane permeabilization and apoptosis.     

Activation of the G2 cell cycle checkpoint enhances survival of epithelial cells exposed to hyperoxia

Reactive oxygen species produced during hyperoxia damage DNA, inhibit proliferation in G1- through p53-dependent activation of p21(Cip1/WAF1/Sdi1), and kill cells. Because checkpoint activation protects cells from genotoxic stress, we investigated cell proliferation and survival of the murine type II epithelial cell line MLE15 during hyperoxia. These cells were chosen for study because they express Simian large and small-T antigens, which transform cells in part by disrupting the p53-dependent G1 checkpoint. Cell counts, 5-bromo-2'-deoxyuridine labeling, and flow cytometry revealed that hyperoxia slowed cell cycle progression after one replication, resulting in a pronounced G2 arrest by 72 h. Addition of caffeine, which inactivates the G2 checkpoint, diminished the percentage of hyperoxic cells in G2 and increased the percentage in sub-G1 and G1. Abrogation of the G2 checkpoint was associated with enhanced oxygen-induced DNA strand breaks and cell death. Caffeine did not affect DNA integrity or viability of cells exposed to room air. Similarly, caffeine abrogated the G2 checkpoint in hyperoxic A549 epithelial cells and enhanced oxygen-induced toxicity. These data indicate that hyperoxia rapidly inhibits proliferation after one cell cycle and that the G2 checkpoint is critical for limiting DNA damage and cell death.     

rIL‐10 enhances IL‐10 signalling proteins in foetal alveolar type II cells exposed to hyperoxia

Although the mechanisms by which hyperoxia promotes bronchopulmonary dysplasia are not fully defined, the inability to maintain optimal interleukin (IL)‐10 levels in response to injury secondary to hyperoxia seems to play an important role. We previously defined that hyperoxia decreased IL‐10 production and pre‐treatment with recombinant IL‐10 (rIL‐10) protected these cells from injury. The objectives of these studies were to investigate the responses of IL‐10 receptors (IL‐10Rs) and IL‐10 signalling proteins (IL‐10SPs) in hyperoxic foetal alveolar type II cells (FATIICs) with and without rIL‐10. FATIICs were isolated on embryonic day 19 and exposed to 65%‐oxygen for 24 hrs. Cells in room air were used as controls. IL‐10Rs protein and mRNA were analysed by ELISA and qRT‐PCR, respectively. IL‐10SPs were assessed by Western blot using phospho‐specific antibodies. IL‐10Rs protein and mRNA increased significantly in FATIICs during hyperoxia, but JAK1 and TYK2 phosphorylation showed the opposite pattern. To evaluate the impact of IL‐8 (shown previously to be increased) and the role of IL‐10Rs, IL‐10SPs were reanalysed in IL‐8‐added normoxic cells and in the IL‐10Rs’ siRNA‐treated hyperoxic cells. The IL‐10Rs’ siRNA‐treated hyperoxic cells and IL‐8‐added normoxic cells showed the same pattern in IL10SPs with the hyproxic cells. And pre‐treatment with rIL‐10 prior to hyperoxia exposure increased phosphorylated IL‐10SPs, compared to the rIL‐10‐untreated hyperoxic cells. These studies suggest that JAK1 and TYK2 were significantly suppressed during hyperoxia, where IL‐8 may play a role, and rIL‐10 may have an effect on reverting the suppressed JAK1 and TYK2 in FATIICs exposed to hyperoxia.     

Hyperoxia mediates acute lung injury and increased lethality in murine Legionella pneumonia: the role of apoptosis

Legionella pneumophila is a major cause of life-threatening pneumonia, which is characterized by a high incidence of acute lung injury and resultant severe hypoxemia. Mechanical ventilation using high oxygen concentrations is often required in the treatment of patients with L. pneumophila pneumonia. Unfortunately, oxygen itself may propagate various forms of tissue damage, including acute lung injury. The effect of hyperoxia as a cofactor in the course of L. pneumophila pneumonia is poorly understood. In this study, we show that exposure to hyperoxic conditions during the evolution of pneumonia results in a marked increase in lethality in mice with Legionella pneumonia. The enhanced lethality was associated with an increase in lung permeability, but not changes in either lung bacterial burden or leukocyte accumulation. Interestingly, accelerated apoptosis as evidenced by assessment of histone-DNA fragments and caspase-3 activity were noted in the infected lungs of mice exposed to hyperoxia. TUNEL staining of infected lung sections demonstrated increased apoptosis in hyperoxic mice, predominantly in macrophages and alveolar epithelial cells. In vitro exposure of primary murine alveolar epithelial cells to Legionella in conjunction with hyperoxia accelerated apoptosis and loss of barrier function. Fas-deficient mice demonstrated partial resistance to the lethal effects of Legionella infection induced by hyperoxia, which was associated with attenuated apoptosis in the lung. These results demonstrate that hyperoxia serves as an important cofactor for the development of acute lung injury and lethality in L. pneumophila pneumonia. Exaggerated apoptosis, in part through Fas-mediated signaling, may accelerate hyperoxia-induced acute lung injury in Legionella pneumonia.     

The contribution of vascular endothelial xanthine dehydrogenase/oxidase to oxygen-mediated cell injury.

The conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) and the reaction of XO-derived partially reduced oxygen species (PROS) have been suggested to be important in diverse mechanisms of tissue pathophysiology, including oxygen toxicity. Bovine aortic endothelial cells expressed variable amounts of XDH and XO activity in culture. Xanthine dehydrogenase plus xanthine oxidase specific activity increased in dividing cells, peaked after achieving confluency, and decreased in postconfluent cells. Exposure of BAEC to hyperoxia (95% O2; 5% CO2) for 0-48 h caused no change in cell protein or DNA when compared to normoxic controls. Cell XDH+XO activity decreased 98% after 48 h of 95% O2 exposure and decreased 68% after 48 h normoxia. During hyperoxia, the percentage of cell XDH+XO in the XO form increased to 100%, but was unchanged in air controls. Cell catalase activity was unaffected by hyperoxia and lactate dehydrogenase activity was minimally elevated. Hyperoxia resulted in enhanced cell detachment from monolayers, which increased 112% compared to controls. Release of DNA and preincorporated [8-14C]adenine was also used to assess hyperoxic cell injury and did not significantly change in exposed cells. Pretreatment of cells with allopurinol for 1 h inhibited XDH+XO activity 100%, which could be reversed after oxidation of cell lysates with potassium ferricyanide (K3Fe(CN)6). After 48 h of culture in air with allopurinol, cell XDH+XO activity was enhanced when assayed after reversal of inhibition with K3Fe(CN)6, and cell detachment was decreased. In contrast, allopurinol treatment of cells 1 h prior to and during 48 h of hyperoxic exposure did not reduce cell damage. After K3Fe(CN)6 oxidation, XDH+XO activity was undetectable in hyperoxic cell lysates. Thus, XO-derived PROS did not contribute to cell injury or inactivation of XDH+XO during hyperoxia. It is concluded that endogenous cell XO was not a significant source of reactive oxygen species during hyperoxia and contributes only minimally to net cell production of O2- and H2O2 during normoxia.     

Effects of anoxia, hyperoxia, and salinity on neurons in the leech Nephelopsis obscura (Erpobdellidae): RNA redistribution by fluorescence histochemistry

The histochemical distribution of cytoplasmic RNA in ganglion cells of the freshwater leech Nephelopsis obscura has been studied using the fluorochrome acridine orange as a marker of nucleic acids. Two series of experiments, employing 50 adult animals, involved changes in oxygen tension in the water and changes in salinity. Normal leech neurons exhibit finely granular orange fluorescence uniformly distributed throughout the cytoplasm, with a perinuclear ring of especially strong fluorescence. After exposure to anoxic (0% O2), hypoxic (20% O2), or hyperoxic (200% O2) conditions at 20 degrees C for 1-15 days, the orange cytoplasmic fluorescence is no longer uniformly distributed; the redistribution is generally toward the periphery, leaving the perinuclear zone without RNA fluorescence, but irregular zones of cytoplasm devoid of RNA also occur not as a gradient. Leeches exposed to salinity of, or greater than, 2.5 ppt for 15 days exhibit similar changes. These alterations are confirmed by electron microscopy. Seasonal fluctuations in oxygen tension and salinity of lake water affect the distribution and abundance of organisms. The acridine orange method provides one measure of stress to the nervous system in freshwater invertebrates that might be applicable to ecological studies as well as to metabolic studies of individual animals.     

The use of the tetrazolium salt XTT for the estimation of biological activity of activated sludge cultivated under steady-state and transient regimes

We have shown that the growth, starvation and population heterogeneity of Salmonella typhimurium and its isogenic nuoG and cydA mutants can be monitored by flow cytometry. Bacterial cells were analysed unstained, and after staining with rhodamine 123, propidium iodide and acridine orange. In unstained cultures it was possible to distinguish flagellated and non-flagellated cells. nuoG and cydA mutants were less stained with rhodamine confirming their defects in generating membrane potential. Increase in propidium iodide staining associated with reduced membrane integrity was seen between day 4 and 14 in all the strains. Acridine orange staining showed that there was retarded development in stationary phase in nuoG and cydA mutants. Furthermore, up to day 28, a small portion of cells showed high RNA and DNA levels. To determine whether these cells represent a sub-population better adapted for long term survival, we measured the growth of the population by both OD values and viable counts. Because the OD values increased throughout the whole study in both wild-type and mutant strains, while the viable counts gradually decreased, we propose that even in very old cultures there must be a population of cells undergoing replication.     

Acridine orange stabilization of glycosaminoglycans in beginning endochondral ossification

Summary This study indicated that acridine orange, when combined with the initial fixative stabilized soluble matrix glycosaminoglycan in situ in areas where considerable glycosaminoglycan extraction is known to occur. Acridine orange was able to diffuse through bone into areas of undecalcified mineralizing cartilage and to bind with the glycosaminoglycans in these areas equally well as in growth plate cartilage matrix. Matrix staining was visible by light microscopy without further staining and was seen to vary territorially in intensity; although cellular definition was poor. This deficiency was overcome by the additional application of p phenylenediamine, which stained the cells intensely. At the ultrastructure level, glycosaminoglycan was present as electron dense structures in the cartilage matrix. Preliminary X-ray microanalysis studies confirmed that the acridine orange stained structures contain sulphur; this finding extends the use of acridine orange further to quantitative analysis of glycosaminoglycan.     

Single-cell analysis demonstrates how nutrient deprivation creates apoptotic and quiescent cell populations in tumor cylindroids

Understanding how quiescent and apoptotic populations form in tumors is necessary because these cell types can considerably diminish therapeutic efficacy. Most cancer therapeutics are ineffective against quiescent cells because they target rapidly proliferating cells. Distinguishing apoptosis is important because apoptotic cells are committed to death and do not require treatment. Regrowth of quiescent cell can lead to tumor re-occurrence and metastasis, which are the leading causes of cancer mortality. We hypothesized that cylindroid cultures and acridine orange staining could be used to determine how nutrient diffusion creates apoptotic and quiescent regions in tumors. To test this hypothesis we developed a microscopy technique to measure cellular DNA and RNA content in single cells using thin cylindroids and acridine orange staining. Cell classification was compared to flow cytometry of cells grown in defined monolayer cultures. The presence of apoptosis was confirmed by morphological nuclear analysis. The effect of diffusion was determined by varying incubation time, cylindroid size, and exposing cylindroids to nutrient-deficient media. Four overlapping regions were identified as a function of cylindroid radius: an outer viable/quiescent region; a second quiescent/apoptotic region; a third late-stage apoptotic region; and an inner dead region. In monolayer cultures the absence of glutamine and growth factors induced apoptosis and hypoxia induced quiescence. Treating with nutrient-deficient media suggested that cells became quiescent near the periphery because of glucose and oxygen limitations, and became apoptotic and died further from the edge because of glutamine and growth factor limitations. These results show that cellular microenvironments can be identified in cylindroids using simple acridine orange staining and that single cell fluorescence can be measured in three-dimensional culture. The developed techniques will be useful for developing cancer therapies and determining how cell death and apoptosis are induced in three-dimensional tumor tissue.     

Über die Vitalfluorochromierung von Mitochondrien in HeLa- un LM-Zellen mit neuen Acridinfarbstoffen

Zusammenfassung Es wird über die Fluorochromierung von Mitochondrien in lebenden Zellen mit neuen Acridinfarbstoffen berichtet. Die verwendeten Fluorochrome sind Derivate von Acridinorange (AO) und von 3-Amino-6-methoxyacridin (AMA) mit verschiedenen Resten in 9- bzw. 10-Stellung (Formelschema 1). Sie sind entweder permanente Farbstoffkationen oder liegen im Kulturmedium als Kationen vor. HeLa-Zellen und Mäusefibroblasten (LM-Zellen) wurden fluorochromiert. Unter günstigen Bedingungen gelang es, die Mitochondrien nicht nur orthochromatisch sondern auch metachromatisch zu färben. Über photodynamische Effekte, die bei der Bestrahlung unter dem Fluoreszenzmikroskop auftreten, wird berichtet. Die Reste in 9- bzw. 10-Stellung begünstigen die Farbstoffakkumulation in den Mitochondrien. vitalfärbung mit den Grundkörpern AO bzw. AMA ergibt demgegenüber metachromatisch gefärbte Lysosomen im orthochromatisch gefärbten Cytoplasma. Der Farbstoff 3-Amino-6-methoxy-9-(2-hydroxyethyl)-acridin fluorochromiert den Kern lebender Zellen.

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The fluorescent staining of mitochondria in living HeLa- and LM-cells with new acridine dyes

Summary The fluorescent staining of mitochondria in living cells with new acridine dyes is reported. The fluorescent dyes used are derivatives of acridine orange (AO) and of 3-amino-6-methoxyacridine (AMA) with various residues in 9- or 10-position (Scheme 1). They are either permanent cationic dyes or cations which are formed by protonation in the culture medium. HeLa cells and mouse fibroblasts (LM cells) have been used for our staining experiments. On favourable conditions we succeeded in staining the mitochondria not only orthochromatically but also metachromatically. Photodynamical effects which have been observed during the exposure of the stained cells in the fluorescence microscope are described. The residues in 9- or 10-position favour the dye accumulation in the mitochondria. Vital staining with the basic compounds AO and AMA however leads to the formation of metachromatically stained lysosomes in the orthochromatically stained cytoplasm. The dye 3-amino-6-methoxy-9-(2-hydroxyethyl)acridine stains the nucleus of living cells.

     

A Bacillus thuringiensis delta-endotoxin induces programmed cell death in mosquito larvae

We present evidence that a delta-endotoxin isolated from Bacillus thuringiensis subsp.israelensis induces programmed cell death in polytene midgut cells of Culex pipiens larvae. After exposure to toxin, polytene nuclei in the anterior region of the larval midgut undergo many of the morphological and physiological changes which are characteristic of apoptosis, including the ability to stain with the vital dye, acridine orange, and fragmentation of nuclear DNA as demonstrated by agarose gel electrophoresis and in situ TUNEL labeling. The temporal sequence of toxin ingestion, acridine orange staining and larval death suggests a cause and effect relationship between programmed cell death and larval death. Amino sugars that interfere with toxicity also interfere with the time course of acridine orange staining of larval polytene nuclei. The toxin first causes programmed cell death of anterior midgut and gastric caeca cells and, subsequently, posterior midgut cells. This pattern is similar to the temporal sequence of larval polytene cell death that occurs during metamorphosis. From the size and distribution of the nuclei that are stained with acridine orange, it appears that only polytene midgut cells are affected by toxin and that the diploid regenerative cell are not affected.     

Mitochondrial metabolism underlies hyperoxic cell damage

Exposure of mammals to hyperoxia causes pulmonary and ocular pathology. Hyperoxic damage and cell death may derive from enhanced intracellular formation of reactive oxygen species (ROS), probably of mitochondrial origin. There is, however, controversy on this point. When wild-type and respiration-deficient (rho(o)) HeLa cells were cultured in 80% O2, wild-type cells stopped growing after 5 days and died thereafter whereas rho(o) cells survived and grew to confluence. This tolerance of rho(o) cells for hyperoxia was not associated with greater resistance to oxidants such as hydrogen peroxide and t-butyl hydroperoxide. Under both 20% and 80% O2, rho(o) cells exhibited substantially decreased ROS production, and, under 80% O2, rho(o) cells showed no suppression of aconitase activity or mitochondrial protein carbonyl formation. Replacement of normal mitochondria in rho(o) cells restored ROS production and susceptibility to hyperoxia. Two other approaches that diminished mitochondrial ROS generation also increased tolerance for hyperoxia. HeLa cells constantly exposed to the protonophoric uncoupler carbonyl cyanide m-chlorophenylhydrazone, which enhances respiration but decreases ROS production, showed preferential survival under 80% O2, as did HeLa cells treated with chloramphenicol, which suppresses both respiration and mitochondrial ROS production. We conclude that interactions between respiring mitochondria and O2 are primarily responsible for hyperoxic cell damage.     

Parathyroid hormone-related protein reduces alveolar epithelial cell proliferation during lung injury in rats

Parathyroid hormone-related protein (PTHrP) is a growth inhibitor for alveolar type II cells and could be a regulatory factor for alveolar epithelial cell proliferation after lung injury. We investigated lung PTHrP expression in rats exposed to 85% oxygen. Lung levels of PTHrP were significantly decreased between 4 and 8 days of hyperoxia, concurrent with increased expression of proliferating cell nuclear antigen and increased incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA in lung corner cells. PTHrP receptor was present in both normal and hyperoxic lung. To test whether the fall in PTHrP was related to cell proliferation, we instilled PTHrP into lungs on the fourth day of hyperoxia. Eight hours later, BrdU labeling in alveolar corner cells was 3.2 +/- 0.4 cells/high-power field in hyperoxic PBS-instilled rats compared with 0.5 +/- 0.3 cells/high-power field in PTHrP-instilled rats (P < 0. 01). Thus PTHrP expression changes in response to lung injury due to 85% oxygen and may regulate cell proliferation.     

苯甲基磺酰氟（PMSF）在犬精子4℃保存中的作用

目的探讨4℃保存的条件下,苯甲基磺酰氟（phenylmethylsulfonyl fluoride,PMSF）对犬精子的保护作用。方法犬精子以含不同浓度PMSF的Tris-卵黄液（Tris-egg yolk,EYT）处理后,于4℃低温保存,在24、48、72、96 h分别以伊红、瑞-吉和吖啶橙染色检测精子活率、顶体膜和DNA完整性。结果低温保存24 h后,吖啶橙染色检测DNA断裂率（%）：对照组（5.93±0.32）,实验组：20μg/mL（4.50±0.38）、40μg/mL（4.20±0.25）、80μg/mL（4.21±0.46）、160μg/mL（3.87±0.67）,P〈0.05;各浓度组之间没有明显差异;72 h后,犬精子活率仍〉30%;96 h内,对照组和实验组犬精子在活率、顶体膜完整性上未见明显差异。结论PMSF于4℃低温保存条件下对犬精子具有保护作用,24 h内能有效保护精子DNA双链的完整性,4℃低温保存72 h后,犬精子仍符合人工授精要求。