No abstract

Metastatic cancer cells seed the lung via blood vessels. Because endothelial cells generate nitric oxide (NO) in response to shear stress, we postulated that the arrest of cancer cells in the pulmonary microcirculation causes the release of NO in the lung. After intravenous injection of B16F1 melanoma cells, pulmonary NO increased sevenfold throughout 20 minutes and approached basal levels by 4 hours. NO induction was blocked by N G -nitro-L-arginine methyl ester (L-NAME) and was not observed in endothelial nitric oxide synthase (eNOS)-deficient mice. NO production, visualized ex vivo with the fluorescent NO probe diaminofluorescein diacetate, increased rapidly at the site of tumor cell arrest, and continued to increase throughout 20 minutes. Arrested tumor cells underwent apoptosis with apoptotic counts more than threefold over baseline at 8 and 48 hours. Neither the NO signals nor increased apoptosis were seen in eNOS knockout mice or mice pretreated with L-NAME. At 48 hours, 83% of the arrested cells had cleared from the lungs of wild-type mice but only ϳ55% of the cells cleared from eNOS-deficient or L-NAME pretreated mice. eNOS knockout and L-NAME-treated mice had twofold to fivefold more metastases than wild-type mice, measured by the number of surface nodules or by histomorphometry. We conclude that tumor cell arrest in the pulmonary microcirculation induces eNOS-dependent NO release by the endothelium adjacent to the arrested tumor cells and that NO is one factor that causes tumor cell apoptosis, clearance from the lung, and inhibition of metastasis. In the lung, metastatic neoplasms are the most common malignant tumors. Their formation usually involves hematogenous seeding of metastatic cancer cells into the lung from remote primary tumors. Interactions between intravascular cancer cells and the endothelium are important determinants of metastatic outcome. 1,2 For example, the expression of constitutive and inducible microvascular adhesion molecules, and the release of reactive oxygen species (NO, O 2 Ϫ , and H 2 O 2 ) by endothelial cells or cancer cells can regulate the mechanisms that govern the metastatic process, including cancer cell adhesion and arrest, 3 the production of endothelial matrix metalloproteinases, 4 and cancer cell apoptosis. 5 Evidence from in vitro and in vivo studies has shown that reactive oxygen and nitrogen species can be cytotoxic to neoplastic cells 6 -9 and reduced their adhesion to postcapillary venules. 10 In vivo, we have recently demonstrated that the arrest of intravascular B16F1 melanoma cells in the liver induces the rapid local release of nitric oxide (NO) that causes apoptosis of the melanoma cells and inhibits their subsequent development into hepatic metastases. 5 Because pulmonary endothelial cells generate NO in response to shear stress, 11 we have postulated that there is a comparable cytotoxic mechanism in the lung.Here we provide data showing that the arrest of B16F1 melanoma cells in the pulmonary circulation of wild-type C57B1/6 mice (WT mice) induces the ...

Chloroplast ultrastructure, pigment content, and composition were investigated during growth of 'Puma' rye at warm and cold-hardening temperatures. Cytological characteristics of leaf mesophyll cells were altered upon growth at cold-hardening temperatures as indicated by an apparent increase in the amount of cytoplasm. Univacuolate and multivacuolate mesophyll cells were found in leaves from cold-hardened plants, but only univacuolate mesophyll cells were found in leaves from warm-grown plants. Chloroplast ultrastructure was affected by growth at low temperature as indicated by a higher frequency of smaller granal stacks. However, there was no significant difference in the number of grana per chloroplast in mesophyll cells from either warm- or cold-grown plants. Chlorophyll per plastid increased by a factor of 1.67 upon growth at cold-hardening temperatures, as did β-carotene and the xanthophylls. However, there was no significant difference in photosynthetic unit size based on P700 measurements between chloroplasts from warm- or cold-grown tissue. Analysis of freeze-fractured thylakoid membranes indicated a loss in the bimodal nature of particle size distribution on the exoplasmic fracture face and a general increase in particle size on the protoplasmic fracture face upon growth and development at cold temperatures. However, particle densities on the exoplasmic and protoplasmic fracture faces were not significantly different in thylakoids from plastids developed at either warm or cold temperatures. It is concluded that chloroplast ultrastructure and pigment content but not composition are altered upon growth and development at cold-hardening temperatures.

We studied the effect of rHuKGF on acute, lethal graft- vs.-host disease (GVHD) in the C57BL/6-->(C57BL/6 X DBA/2)F(1)-hybrid model. rHuKGF-treated recipients did not develop intestinal GVHD despite elevated levels of intestinal NO and TNF alpha, did not develop endotoxemia, and did not die. LPS augmented serum TNF alpha release and intestinal NO production, but did not induce intestinal epithelial cell apoptosis, a phenomenon associated with acute GVHD. These data suggest that KGF prevents the development of acute lethal GVHD by protecting epithelial cell injury mediated by TNF-alpha, NO, and other potential cytotoxic factors. We noted a moderate reduction in intestinal KGFR mRNA expression in untreated GVH mice on day 8, when IFN-gamma mRNA levels were highest. This reduction in KGFR mRNA levels was not seen in recipients of IFN-gamma gene knockout grafts, suggesting that IFN-gamma may be involved in reducing KGFR mRNA expression in the intestine. A similar reduction in intestinal KGFR mRNA expression was also seen in rHuKGF-treated recipients, suggesting that rHuKGF does not mediate its protective effect by maintaining KGFR at control levels. KGF-treatment also redirected the cytokine response in acute GVH mice from Th1 to a mixed pattern of both Th1 and Th2 cytokines. This was associated with histopathologic changes resembling chronic GVHD.

Oxidative damage may be one of the manifestations of cellular damage in the toxicity of ochratoxin A (OA). OA; its three natural analogs, OB, OC and O alpha; and three synthetic analogs, the ethyl amide of OA (OE-OA), O-methylated OA (OM-OA), and the lactone-opened OA (OP-OA) were used to study free radical generation in hepatocytes, mitochondria and microsomes from rats. Electron paramagnetic resonance spectroscopy (EPR) using alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (4-POBN) as a spin trapping agent showed an enhanced free radical generation due to the addition of NADPH to the microsomes. An EPR signal was not observed in the mitochondria and hepatocyte samples when they were treated with a variety of agents. Addition of OM-OA together with NADPH and Fe3+ to the microsomes resulted in a strong EPR signal compared with the other analogs, whereas the signal could be quenched by the addition of catalase. OM-OA does not have a dissociable phenolate group and does not chelate Fe3+. The spin adduct hyperfine splitting constants indicated the presence of alpha-hydroxyethyl radicals resulting from generated hydroxyl radicals, which were trapped by 4-POBN. The results also suggested that the production of hydroxyl radicals by OA does not require a dissociable phenolate group or the prior formation of an OA-Fe complex.