Mitochondrial phospholipid hydroperoxide glutathione peroxidase suppresses apoptosis mediated by a mitochondrial death pathway.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a key enzyme in the protection of biomembranes exposed to oxidative stress. We investigated the role of mitochondrial PHGPx in apoptosis using RBL2H3 cells that overexpressed mitochondrial PHGPx (M15 cells), cells that overexpressed non-mitochondrial PHGPx (L9 cells), and control cells (S1 cells). The morphological changes and fragmentation of DNA associated with apoptosis occurred within 15 h in S1 and L9 cells upon exposure of cells to 2-deoxyglucose (2DG). The release of cytochrome c from mitochondria was observed in S1 cells after 4 h and was followed by the activation of caspase-3 within 6 h. Overexpression of mitochondrial PHGPx prevented the release of cytochrome c, the activation of caspase-3, and apoptosis, but non-mitochondrial PHGPx lacked the ability to prevent the induction of apoptosis by 2DG. An ability to protect cells from 2DG-induced apoptosis was abolished when the PHGPx activity of M15 cells was inhibited by diethylmalate, indicating that the resistance of M15 cells to apoptosis was indeed due to the overexpression of PHGPx in the mitochondria. The expression of members of the Bcl-2 family of proteins, such as Bcl-2, Bcl-xL, Bax, and Bad, was unchanged by the overexpression of PHGPx in cells. The levels of hydroperoxides, including hydrogen and lipid peroxide, in mitochondria isolated from S1 and L9 cells were significantly increased after the exposure to 2DG for 2 h, while the level of hydroperoxide in mitochondria isolated from M15 cells was lower than that in S1 and L9 cells. M15 cells were also resistant to apoptosis induced by etoposide, staurosporine, UV irradiation, cycloheximide, and actinomycin D, but not to apoptosis induced by Fas-specific antibodies, which induces apoptosis via a pathway distinct from the pathway initiated by 2DG. Our results suggest that hydroperoxide, produced in mitochondria, is a major factor in apoptosis and that mitochondrial PHGPx might play a critical role as an anti-apoptotic agent in mitochondrial death pathways.     

Sex pheromones in maleMelittobia parasitic wasps: Female response to conspecific and congeneric males of 3 species

Presented a choice between conspecific males and 2 congeneric males, virgin females ofMelittobia australica andM. digitata chose conspecific males disproportionately more often, whereasM. femorata females distributed themselves evenly among the choices. Empty tubes, provided as the fourth choice in the test apparatus, were entered much less often than tubes containing live males. Females of all species chose “wrong” males about equally frequently. These observations suggest that even non-conspecific males possess some degree of attractiveness to virgin females. Chemicals in the sex pheromone of the males are presumed to be the source of the males' attractancy. The incomplete species specificity is interpreted in light of the life history of this genus, and it is suggested that specific recognition cues operate primarily after the sexes come together. Supported by a grant from Kagoshima Prefecture in Japan under the exchange program of faculty members between Kagoshima University and the University of Georgia.     

Meal feeding schedule and ventromedial hypothalamic lesions do not affect rhythm of pineal serotonin N-acetyltransferase activity in rats

Effects of meal feeding schedule and bilateral lesions of the ventromedial hypothalamus (VMH) on the circadian rhythm of pineal serotonin N-acetyltransferase (SNAT) activity were examined in rats, under LD (12:12) condition. Neither meal feeding nor VMH lesions affected the phase of the circadian rhythm of pineal SNAT activity, but the VMH lesions reduced the level. Meal feeding caused a shift of the phases of the daily rhythms of phosphoenolpyruvate carboxykinase and tyrosine aminotransferase activities in the liver. These findings suggest that the circadian rhythm of pineal SNAT activity is not entrained by the food intake, and that the VMH does not function as a master oscillator of the rhythm.     

Serodiagnosis of Streptococcus pneumoniae infection in guinea pigs by an enzyme-linked immunosorbent assay

Guineapig antibodies to Streptococcus pneumoniae (SPN) serotype 19F were detected by an enzyme-linked immunosorbent assay using a simple procedure. In experimentally infected hosts, antibody was detectable as early as 2 to 3 weeks after infection, and high titres were maintained for a long period. Antibodies higher than 1:64 were regarded as specific. In a field study, high antibody titres were shown in SPN enzootic colonies in contrast to negative or low antibody titres in a majority of the animals from non-enzootic and SPF colonies.     

Selective inhibition of free arachidonic acid production in activated alveolar macrophages by calmodulin antagonists

The effect of calmodulin antagonists on the amounts of free fatty acids produced by rabbit alveolar macrophages was determined by fluorometric high-performance liquid chromatography. Opsonized zymosan-induced arachidonic acid production was dramatically suppressed in the presence of W-7 and trifluoperazine without an effect on the production of other fatty acids. Calmodulin antagonists inhibited phospholipase A and abolished the release of arachidonic acid from phospholipids. The present results suggest that a zymosan-sensitive pool of 20:4, which is different from that of other fatty acids, is present in macrophages and that calmodulin antagonists selectively inhibit phospholipase A, which preferentially degrades phospholipids with 20:4.     

Lectin cytochemical evaluation of somatosensory neurons and their peripheral and central processes in rat and man

Summary Paraffin sections of the trigeminal nerve root of the rat, and human spinal nerve root and trigeminal ganglion were stained with a battery of lectin-horseradish peroxidase conjugates to localize and characterize glycoconjugate (GC) in situ. In the rat the myelin sheath of the peripheral segment contained GC with sialic acid most probably linked to the penultinate disaccharide galactose(1 4)-N-acetylglucosamine (Gal(1 )-GlcNAc), and complex type N-glycosidic side chains. The myelin sheath in the central segment differed in containing little if any of the GC named above and in containing GC with terminal -Gal linked to N-acetylgalactosamine (GalNAc), terminal GalNAc and fucose. Schwann cells stained for GC with GlcNAc or mannose whereas oligodendroglia stained for GC with the terminal disaccharide Gal-(1 3)-GalNAc and N-glycosidic side chains, especially in presumed Golgi zones, but also in processes continued as the outer myelin sheath. The human myelin sheath in the central segment differed from that of the rat in not staining with lectins specific for fucose and terminal GalNAc. Sialic acid and terminal -Gal were seen in the human central segment but these sugars appeared to bind to astroglial structures rather than to the myelin sheath as in the rat. Astrocytes in both rat and man were stained by two fucose-binding lectins. Several lectins revealed affinity for GC in the neurilemmal sheath, and staining of this structure was stronger in the human specimens. Neurons in the human trigeminal ganglion ranged from unstained to strongly positive for fucoconjugate in cytoplasmic bodies and plasmalemma. Positive ganglion cells gave rise to unmyelinated fibers which also stained for fucoconjugate. Remak fibers and their extensions into the substantia gelatinosa of the human spinal cord stained strongly for content of fucose.The stronger lectin affinity for N-glycosidic core sugars in the peripheral as compared with the central segment suggests that lectins localize P_o protein in peripheral myelin. The reactivity for several sugars in the central segment can possibly be attributed to myelin-associated glycoprotein (MAG) of central myelin, but lectin staining for GalNAc shows in addition a biochemically unrecognized GC with O-glycosidic linked oligosaccharides in myelin. The lectin cytochemistry indicates that the 170 K Dalton glycoprotein with PNA affinity obtained from rat sciatic nerves occurs in nodes of Ranvier.This research was supported by NIH Grants AM-10956, HL-29775 and United Health and Medical Research Foundation of South Carolina, Inc. Grant No. 79     

3(20)alpha-hydroxysteroid dehydrogenase activity of monkey liver indanol dehydrogenase

Homogeneous indanol dehydrogenase from monkey liver catalyzed the reversible conversion of 3 alpha- or 20 alpha-hydroxy groups of several bile acids and 5 beta-pregnanes to the corresponding 3- or 20-ketosteroids. The kcat values for the steroids determined at pH 7.4 were low, but the kcat/Km values for the 3-ketosteroids were comparable to or exceeded those for 1-indanol and xenobiotic carbonyl substrates. The enzyme transferred the 4-pro-R-hydrogen atom of NADPH to the 3 beta- or 20 beta-face of the ketosteroid substrate. Competitive inhibition of the hydroxysteroid dehydrogenase activity of the enzyme by medroxyprogesterone acetate, hexestrol, and 1,10-phenanthroline suggests that both 1-indanol and hydroxysteroid are oxidized at the same active site on the enzyme. The specific inhibitor of the enzyme, 1,10-phenanthroline, suppressed the 3 alpha-hydroxysteroid dehydrogenase activity in the crude extract of monkey liver by 50%. The results strongly suggest that indanol dehydrogenase acts as a 3(20)alpha-hydroxysteroid dehydrogenase in the metabolism of certain steroid hormones and bile acids.