Metabolic bases for differences in sensitivity of two pea cultivars to sulfur dioxide

An oxidative chain reaction of sulfite initiated by the superoxide ion produced in the Mehler reaction has been implicated in the damage of plants exposed to sulfur dioxide. The toxicity of SO₂ may be alleviated by free radical scavenging systems acting to terminate this chain reaction. Hence, the relative sensitivity of plants to SO₂ toxicity could depend on differences in the responses of the levels of antioxidant metabolites and enzymes. The effect of SO₂ exposure on glutathione and ascorbic acid contents, glutathione reductase, and superoxide dismutase activities was assayed in two cultivars (Progress, Nugget) of pea (Pisum sativum L.) in which apparent photosynthesis showed a differential sensitivity to 0.8 microliter per liter SO₂ (R. Alscher, J. L. Bower, W. Zipfel [1987] J Exp Bot 38:99-108). Total and reduced glutathione increased more rapidly and to a greater extent in the insensitive Progress than in the sensitive Nugget, as did glutathione reductase activities. Superoxide dismutase activities increased significantly in Progress, whereas no such change was observed in Nugget as a result of SO₂ exposure. This increase in superoxide dismutase activity was observed at 210 minutes after 0.8 microliter per liter SO₂ concentration had been reached, in marked contrast to the increases in reduced glutathione content and glutathione reductase activity, which were apparent at the 90 minute time point. These data suggest that one basis for the relative insensitivity of the apparent photosynthesis of the pea cultivar Progress to SO₂ is the enhanced response of glutathione reductase, superoxide dismutase activities, and glutathione content.     

Apoptosis occurs in a new model of thermal brain injury

Apoptosis has been implicated recently as a prominent response of the brain to a variety of insults, such as ischemia and trauma. In this study, we demonstrate that apoptosis is a prominent part of the brain's response to a thermal insult. To examine the brain's response to a thermal insult, a new model of thermal brain injury in the laboratory rat was developed. Water heated to 60 degrees C was passed over an area of thinned calvarium for 1 min. This resulted in an actual brain temperature of 47-48 degrees C. A uniform area of 2,3,5-triphenyl-tetrazolium chloride pallor was demonstrated and pyknotic neurons were seen in the area of injury by hematoxylin-eosin staining. Apoptosis was demonstrated by the characteristic DNA fragmentation seen by agarose gel electrophoresis, ApopTag in situ staining and electron microscopy. The findings of apoptosis were localized to the area of thermal injury and were time dependent, starting 6 h after the insult and peaking approximately 18 h after the insult. This represents one of the first demonstrations that apoptosis occurs in the brain in response to a thermal injury.     

Lamp-1 is upregulated in human glioblastoma cell lines induced to undergo apoptosis

Lysosome-associated membrane protein (LAMP)-1, one of the major protein components of the lysosomal membrane, is upregulated in the human glioblastoma cell lines, U-373 MG and LN-Z308, which undergo cisplatin-induced apoptosis. These human brain tumor cell lines demonstrated apoptosis in response to cisplatin/nifedipine treatment. Both cell lines demonstrated an apoptotic response by more than one criterion. Apoptosis was demonstrated by DNA fragmentation techniques such as DNA laddering, ApopTag in situ labeling, and an ELISA-based method of detecting liberated oligosomes. These cells also had characteristic morphologic changes and upregulation of bax consistent with apoptosis. LAMP-1 expression at the protein and mRNA level was examined and found to increase with cisplatin/nifedipine treatment. LAMP-1 expression was examined using indirect immunofluorescent staining, Northern blot analysis and Western blot analysis. The finding of an augmentation of LAMP-1 in these cells induced to die is enigmatic. These findings raise the possibility of LAMP-1 involvement in the apoptotic process.     

Protein kinase activity of phosphoinositide 3-kinase regulates beta-adrenergic receptor endocytosis

Phosphoinositide 3-kinase (PI(3)K) is a unique enzyme characterized by both lipid and protein kinase activities. Here, we demonstrate a requirement for the protein kinase activity of PI(3)K in agonist-dependent beta-adrenergic receptor (betaAR) internalization. Using PI(3)K mutants with either protein or lipid phosphorylation activity, we identify the cytoskeletal protein non-muscle tropomyosin as a substrate of PI(3)K, which is phosphorylated in a wortmannin-sensitive manner on residue Ser 61. A constitutively dephosphorylated (S61A) tropomyosin mutant blocks agonist-dependent betaAR internalization, whereas a tropomyosin mutant that mimics constitutive phosphorylation (S61D) complements the PI(3)K mutant, with only lipid phosphorylation activity reversing the defective betaAR internalization. Notably, knocking down endogenous tropomyosin expression using siRNAs that target different regions if tropomyosin resulted in complete inhibition of betaAR endocytosis, showing that non-muscle tropomyosin is essential for agonist-mediated receptor internalization. These studies demonstrate a previously unknown role for the protein phosphorylation activity of PI(3)K in betaAR internalization and identify non-muscle tropomyosin as a cellular substrate for protein kinase activity of PI(3)K.     

Aging, oxidative responses, and proliferative capacity in cultured mouse aortic smooth muscle cells

The cellular mechanisms that contribute to the acceleration of atherosclerosis in aging populations are poorly understood, although it is hypothesized that changes in the proliferative capacity of vascular smooth muscle cells is contributory. We addressed the relationship among aging, generation of reactive oxygen species (ROS), and proliferation in primary culture smooth muscle cells (SMC) derived from the aortas of young (4 mo old) and aged (16 mo old) mice to understand the phenotypic modulation of these cells as aging occurs. SMC from aged mice had decreased proliferative capacity in response to alpha-thrombin stimulation, yet generated higher levels of ROS and had constitutively increased mitogen-activated protein kinase activity, in comparison with cells from younger mice. These effects may be explained by dysregulation of cell cycle-associated proteins such as cyclin D1 and p27Kip1 in SMC from aged mice. Increased ROS generation was associated with decreased endogenous antioxidant activity, increased lipid peroxidation, and mitochondrial DNA damage. Accrual of oxidant-induced damage and decreased proliferative capacity in SMC may explain, in part, the age-associated transition to plaque instability in humans with atherosclerosis.     

Reduced spontaneous relaxation in immature guinea pig airway smooth muscle is associated with increased prostanoid release

Airway smooth muscle (ASM) from infant guinea pigs has less spontaneous relaxation during stimulation than ASM from adults. Inhibition of cyclooxygenase (COX), which catalyzes the production of prostanoids, increases this relaxation in infant ASM and abolishes age differences, thus suggesting that prostanoids reduce relaxation in infant ASM. In this study, we investigated whether leukotrienes are also involved in reducing spontaneous relaxation; whether the two COX isoforms, COX-1 and COX-2, differentially regulate spontaneous relaxation; and whether prostanoid release is developmentally regulated in guinea pig ASM. In different age groups, we measured relaxation during and after electrical stimulation in tracheal strips as well as prostanoid release from tracheal segments. Relaxation was studied in the absence and in the presence of a lipoxygenase inhibitor, a cysteinyl leukotriene receptor-1 antagonist, a COX-1 inhibitor, or a COX-2 inhibitor. We found that inhibition of lipoxygenase or cysteinyl leukotriene receptor-1 antagonism did not increase spontaneous relaxation at any age, thus excluding a role for leukotrienes in this phenomenon. Inhibition of COX-2, but not COX-1, promoted spontaneous relaxation. The basal release of prostanoids was more abundant in tissue from infant animals and decreased significantly with age. Thromboxane B2 was the most abundant metabolite released at all ages. Electrical stimulation and epithelium removal did not affect the age difference in prostanoid release. We conclude that increased basal prostanoid release contributes to the reduced spontaneous relaxation in immature guinea pig ASM compared with older animals. By regulating ASM relaxation, prostanoids may play a role in the airway hyperresponsiveness at a young age.