Vanadate induces apoptosis in epidermal JB6 Pplus cells via hydrogen peroxide-mediated reactions

Apoptosis is a physiological mechanism for the control of DNA integrity in mammalian cells. Vanadium induces both DNA damage and apoptosis. It is suggested that vanadium-induced apoptosis serves to eliminate DNA-damaged cells. This study is designed to clarify a role of reactive oxygen species in the mechanism of apoptosis induced by vanadium. We established apoptosis model with murine epidermal JB6 P⁺ cells in the response to vanadium stimulation. Apoptosis was detected by a cell death ELISA assay and morphological analysis. The result shows that apoptosis induced by vanadate is dose-dependent, reaching its saturation level at a concentration of 100 M vanadate. Vanadyl (IV) can also induce apoptosis albeit with lesser potency. A role of reactive oxygen species was analyzed by multiple reagents including specific scavengers of different reactive oxygen species. The result shows that vanadate-induced apoptosis is enhanced by NADPH, superoxide dismutase and sodium formate, but was inhibited by catalase and deferoxamine. Cells exposed to vanadium consume more molecular oxygen and at the same time, produce more H₂O₂ as measured by the change in fluorescence of scopoletin in the presence of horseradish peroxidase. This change in oxygen consumption and H₂O₂ production is enhanced by NADPH. Taken together, these results show that vanadate induces apoptosis in epidermal cells and H₂O₂ induced by vanadate plays a major role in this process.     

Hydrogen peroxide formation and actin filament reorganization by Cdc42 are essential for ethanol-induced in vitro angiogenesis

This report focuses on the identification of the molecular mechanisms of ethanol-induced in vitro angiogenesis. The manipulation of angiogenesis is an important therapeutic approach for the treatment of cancer, cardiovascular diseases, and chronic inflammation. Our results showed that ethanol stimulation altered the integrity of actin filaments and increased the formation of lamellipodia and filopodia in SVEC4-10 cells. Further experiments demonstrated that ethanol stimulation increased cell migration and invasion and induced in vitro angiogenesis in SVEC4-10 cells. Mechanistically, ethanol stimulation activated Cdc42 and produced H(2)O(2) a reactive oxygen species intermediate in SVEC4-10 cells. Measuring the time course of Cdc42 activation and H(2)O(2) production upon ethanol stimulation revealed that the Cdc42 activation and the increase of H(2)O(2) lasted more than 3 h, which indicates the mechanisms of the long duration effects of ethanol on the cells. Furthermore, either overexpression of a constitutive dominant negative Cdc42 or inhibition of H(2)O(2) production abrogated the effects of ethanol on SVEC4-10 cells, indicating that both the activation of Cdc42 and the production of H(2)O(2) are essential for the actions of ethanol. Interestingly, we also found that overexpression of a constitutive dominant positive Cdc42 itself was sufficient to produce H(2)O(2) and to induce in vitro angiogenesis. Taken together, our results suggest that ethanol stimulation can induce H(2)O(2) production through the activation of Cdc42, which results in reorganizing actin filaments and increasing cell motility and in vitro angiogenesis.     

Nitric oxide production by rat bronchoalveolar macrophages or polymorphonuclear leukocytes following intratracheal instillation of lipopolysaccharide or silica

Exposure of the lung to lipopolysaccharide (LPS) or silica results in an activation of alveolar macrophages (AMs), recruitment of polymorphonuclear leukocytes (PMNs) into bronchoalveolar spaces, and the production of free radicals. Nitric oxide (NO) is one of the free radicals generated by bronchoalveolar lavage (BAL) cell populations following either LPS or silica exposure. The purpose of the present study was to assess the relative contributions of AMs and PMNs to the amounts of NO produced by BAL cells following intratracheal (IT) instillation of either LPS or silica. Male Sprague Dawley rats (265-340 g body wt.) were given LPS (10 mg/100 g body wt.) or silica (5 mg/100 g body wt.). BAL cells were harvested 18-24 h post-IT and enriched for AMs or PMNs using density gradient centrifugation. Media levels of nitrate and nitrite (NOx; the stable decomposition products of NO) were then measured 18 h after ex vivo culture of these cells. Following IT exposure to either LPS or silica, BAL cell populations were approximately 20% AMs and approximately 80% PMNs. After density gradient centrifugation of BAL cells from LPS- or silica-treated rats, cell fractions were obtained which were relatively enriched for AMs (approximately 60%) or PMNs (approximately 90%). The amounts of NOx produced by the AM-enriched fractions from LPS- or silica-treated rats were approximately 2-4-fold greater than that produced by the PMN-enriched fractions. Estimations of the relative contribution of AMs or PMNs to the NOx produced indicated that: (i) following LPS treatment, 75%-89% of the NOx was derived from AMs and 11%-25% from PMNs; and (ii) following silica treatment, 76%-100% of the NOx was derived from AMs and 0-24% from PMNs. Immunohistochemistry for inducible NO synthase on lung tissue sections supported these findings. We conclude that AMs are the major source of the NO produced by BAL cells during acute pulmonary inflammatory responses to LPS or silica.     

BASAL BODY REPLICATION AND CILIOGENESIS IN A SUCTORIAN, TOKOPHRYA INFUSIONUM

Basal body replication and ciliogenesis in Tokophrya infusionum were studied in synchronized cultures. Basal body replication occurs during the 1st hr of reproduction, which in Tokophrya is by internal budding. The number of basal bodies increases from about 20 to over 300 within this period. New basal bodies develop in association with mature basal bodies; they are formed at right angles to the mature basal body as short "probasal" bodies, which elongate, slant upward, become parallel to the mature basal body, and elongate to the mature size. Ciliogenesis occurs only during reproduction; the nonreproducing adult is not ciliated, and has only 18–25 barren basal bodies. Cilia first appear as short bulges above the basal body. The axonemal structure is incomplete at first, with one or both central microtubules absent, and occasionally the B fibers of the outer doublets are missing. Several accessory fibers are associated with the basal bodies, both in the adult and during reproduction. One of the fibers appears only after the cilia have sprouted. The scheme of basal body replication and ciliogenesis in Tokophrya is compared to that reported in other organisms, and the role of the accessory fibers is discussed.     

The Ventral Photoreceptor Cells of Limulus : I. The microanatomy

The ventral photoreceptor cells of Limulus polyphemus resemble the retinular cells of the lateral eyes both in electrical behavior and in morphology. Because of the great size of the ventral photoreceptor cells they are easy to impale with glass capillary micropipettes. Their location along the length of the ventral eye nerve makes them easy to dissect out and fix for electron microscopy. Each cell has a large, ellipsoidal soma that tapers into an axon whose length depends upon the distance of the cell from the brain. The cell body contains a rich variety of cytoplasmic organelles with an especially abundant endoplasmic reticulum. The most prominent structural feature is the microvillous rhabdomere, a highly modified infolding of the plasmalemma. The microvilli are tightly packed together within the rhabdomere, and quintuple-layered junctions are encountered wherever microvillar membranes touch each other. Glial cells cover the surface of the photoreceptor cell and send long, sheet-like projections of their cytoplasm into the cell body of the photoreceptor cell. Some of these projections penetrate the rhabdomere deep within the cell and form quintuple-layered junctions with the microvilli. Junctions between glial cells and the photoreceptor cell and between adjacent glial cells are rarely encountered elsewhere, indicating that there is an open pathway between the intermicrovillous space and the extracellular medium. The axon has a normal morphology but it is electrically inexcitable.     

Effect of inhaled crystalline silica in a rat model: time course of pulmonary reactions

Numerous investigations have been conducted to elucidate mechanisms involved in the initiation and progression of silicosis. However, most of these studies involved bolus exposure of rats to silica, i.e. intratracheal instillation or a short duration inhalation exposure to a high dose of silica. Therefore, the question of pulmonary overload has been an issue in these studies. The objective of the current investigation was to monitor the time course of pulmonary reactions of rats exposed by inhalation to a non-overload level of crystalline silica. To accomplish this, rats were exposed to 15 mg/m3 silica, 6 h/day, 5 days/week for up to 116 days of exposure. At various times (5-116 days exposure), animals were sacrificed and silica lung burden, lung damage, inflammation, NF-KB activation, reactive oxygen species and nitric oxide production, cytokine production, alveolar type II epithelial cell activity, and fibrosis were monitored. Activation of NF-KB/DNA binding in BAL cells was evident after 5 days of silica inhalation and increased linearly with continued exposure. Parameters of pulmonary damage, inflammation and alveolar type II epithelial cell activity rapidly increased to a significantly elevated but stable new level through the first 41 days of exposure and increased at a steep rate thereafter. Pulmonary fibrosis was measurable only after this explosive rise in lung damage and inflammation, as was the steep increase in TNF-alpha and IL-1 production from BAL cells and the dramatic rise in lavageable alveolar macrophages. Indicators of oxidant stress and pulmonary production of nitric oxide exhibited a time course which was similar to that for lung damage and inflammation with the steep rise correlating with initiation of pulmonary fibrosis. Staining for iNOS and nitrotyrosine was localized in granulomatous regions of the lung and bronchial associated lymphoid tissue. Therefore, these data demonstrate that the generation of oxidants and nitric oxide, in particular, is temporally and anatomically associated with the development of lung damage, inflammation, granulomas and fibrosis. This suggests an important role for nitric oxide in the initiation of silicosis.     

Enhanced nitric oxide and reactive oxygen species production and damage after inhalation of silica

In previous reports from this study, measurements of pulmonary inflammation, bronchoalveolar lavage cell cytokine production and nuclear factor-kappa B activation, cytotoxic damage, and fibrosis were detailed. In this study, we investigated the temporal relationship between silica inhalation, nitric oxide (NO), and reactive oxygen species (ROS) production, and damage mediated by these radicals in the rat. Rats were exposed to a silica aerosol (15 mg/m(3) silica, 6 h/day, 5 days/wk) for 116 days. We report time-dependent changes in 1) activation of alveolar macrophages and concomitant production of NO and ROS, 2) immunohistochemical localization of inducible NO synthase and the NO-induced damage product nitrotyrosine, 3) bronchoalveolar lavage fluid NO(x) and superoxide dismutase concentrations, and 4) lung lipid peroxidation levels. The major observations made in this study are as follows: 1) NO and ROS production and resultant damage increased during silica exposure, and 2) the sites of inducible NO synthase activation and NO-mediated damage are associated anatomically with pathological lesions in the lungs.     

Ultrastructure of secretion in the atrial gland of a mollusc (Aplysia)

Extracts of the atrial gland of the sea hare Aplysia californiea (Mollusca) induce egg laying when injected into mature individuals. Since egg laying is controlled endogenously by a peptide secreted by neuroendocrine cells in the central nervous system, the relationship between the atrial gland and these central neurons has become an issue of interest. With the particular objective of examining secretory structures we undertook an ultrastructural study of the atrial gland and adjacent tissues. This study revealed that the atrial gland epithelium is composed of two major cell types: ‘goblet-like’ exocrine cells containing large electron-dense granules, and ciliated ‘capping cells’. A non-secretory, and possibly post-secretory, cell containing electron-lucent granules was noted. A region of the large hermaphroditic duct contiguous to the atrial gland, known as the red hemiduct, also displayed capping cells and secretory cells with large granules. The content of these granules is organized into crista-like condensations. The cell also contains iron-rich pigment inclusions.     

The ultrastructure of nuclear division in a suctorian,Tokophrya infusionum

Summary Ultrastructural changes in the micro- and macronucleus throughout division were followed in synchronized cultures of the suctorian, Tokophrya infusionum. After an initial swelling, the micronucleus elongates enormously; microtubules within the micronucleus proliferate and lengthen as the micronucleus elongates. Changes in the macronucleus become visible only after micronuclear division is well underway. The chromatin bodies fuse into long chromatin strands, and the large bundles of microtubules present in the resting macronucleus break up into small groups which parallel the chromatin strands. Colchicine, which prevents reproduction in Tokophrya, seems to block division at a very early stage. The macronucleus appears the same as the resting nucleus of untreated organisms, with numerous microtubules and distinct chromatin bodies. The chromatin in the micronucleus aggregates into large clumps, however, and proliferation of microtubules does not occur.Supported by a Graduate Fellowship at The Rockefeller University.Supported by Grant A1-01407-12 USPHS and Grant A1-08989-01 USPHS.