Nitric oxide exposure of CC531 rat colon carcinoma cells induces gamma-glutamyltransferase which may counteract glutathione depletion and cell death

Gamma-glutamyltransferase (GGT) has a central role in glutathione homeostasis by initiating the breakdown of extracellular GSH. We investigated in the present study whether nitric oxide exposure of CC531 rat colon carcinoma cells modulates GGT and how the activity of the enzyme affects the level of intracellular GSH. The data show that GGT activity was induced in a dose-related manner by two NO-donors (spermineNONOate and nitrosoglutathione) and that antioxidants partly inhibited the induction. SpermineNONOate lowered intracellular GSH and induced apoptosis. Cultivating the cells in cystine-depleted medium also resulted in a 50% lowering of GSH, but this was avoided when GSH was added to the medium. This effect was mediated by the activity of GGT and shown after inhibiting GGT activity with acivicin and cyst(e)ine transporters with alanine and homocysteic acid. This shows that the cells benefit from GGT in maintaining the intracellular GSH level. Cells with induced GGT activity obtained after NO incubation showed a higher uptake rate of cysteine (2-fold), measured by incubating the cells with 5S-radiolabeled GSH. The enzyme was also induced by interferon-gamma and tumor necrosis factor-alpha, but this induction was not connected to activation of the endogenous nitric oxide synthase, as the addition of aminoguanidine, a NO-synthase inhibitor, did not affect the induction. The present study shows that the activity of GGT is upregulated by NO-donors and that the colon carcinoma cells, when cultivated in cystine-depleted medium, benefit from the enzyme in maintaining the intracellular level of GSH. Thus, the enzyme will add to the protective measures of the tumor cells during nitrosative stress.     

Nicotine's attenuation of body weight involves the perifornical hypothalamus

Previously we showed that intermittent administration of nicotine (NIC) in the dark phase decreased food intake and body weight and this could be blocked when the NIC receptor antagonist mecamylamine was infused into the fourth ventricle. Catecholaminergic neurons adjacent to the fourth ventricle contain NIC receptors and directly innervate the perifornical hypothalamus (PFH) which has been shown to be involved in regulation of feeding. This study explored whether NIC regulates feeding behavior by modulating catecholaminergic input to the PFH. Epinephrine and norepinephrine neuronal input was ablated within the PFH by infusion of 6-hydroxydopamine hydrobromide (6-OHDA), while bupropion was infused to protect dopaminergic neurons. After recovery of body weights to pre-surgery levels, food intake, meal size, meal number and body weight were measured after intermittent NIC injections. The results showed the PFH lesioned animals did not exhibit the typical prolonged drop in food intake, meal size and body weight normally associated with NIC administration. High performance liquid chromatography analyses demonstrated that compared to control rats, 6-OHDA administration significantly reduced PFH norepinephrine and epinephrine levels, but not dopamine levels. These results are consistent with NIC reducing food intake in part by acting through catecholaminergic neurons within or extending through the PFH.     

Chronic stress alters dendritic morphology in rat medial prefrontal cortex

Chronic stress produces deficits in cognition accompanied by alterations in neural chemistry and morphology. Medial prefrontal cortex is a target for glucocorticoids involved in the stress response. We have previously demonstrated that 3 weeks of daily corticosterone injections result in dendritic reorganization in pyramidal neurons in layer II-III of medial prefrontal cortex. To determine if similar morphological changes occur in response to chronic stress, we assessed the effects of daily restraint stress on dendritic morphology in medial prefrontal cortex. Male rats were exposed to either 3 h of restraint stress daily for 3 weeks or left unhandled except for weighing during this period. On the last day of restraint, animals were overdosed and brains were stained using a Golgi-Cox procedure. Pyramidal neurons in lamina II-III of medial prefrontal cortex were drawn in three dimensions, and the morphology of apical and basilar arbors was quantified. Sholl analyses demonstrated a significant alteration of apical dendrites in stressed animals: overall, the number and length of apical dendritic branches was reduced by 18 and 32%, respectively. The reduction in apical dendritic arbor was restricted to distal and higher-order branches, and may reflect atrophy of terminal branches: terminal branch number and length were reduced by 19 and 35%. On the other hand, basilar dendrites were not affected. This pattern of dendritic reorganization is similar to that seen after daily corticosterone injections. This reorganization likely reflects functional changes in prefrontal cortex and may contribute to stress-induced changes in cognition.     

Ultrastructure of laevigate hilate spores in sporangia and spore masses from the Upper Silurian and Lower Devonian of the Welsh Borderland

Spore masses and isolated sporangia, containing laevigate hilate cryptospores attributable to the dispersed taxon Laevolancis divellomedia sensu lato, have been recovered on bulk maceration of Upper Silurian (Pridoli) and Lower Devonian (Lochkovian) deposits from the Welsh Borderland. Detailed morphological, anatomical and ultrastructural analysis, using light microscope, scanning electron microscope and transmission electron microscope techniques, reveals subtle differences between the specimens and they can be grouped into five distinct types. The different groups are distinguished principally by using sporangia-spore mass characteristics, presence or absence of extra-exosporal material and nature of spore-wall ultrastructure. Of the groups, one has a uniformly homogeneous exospore and the other four groups have a bilayered exospore. In the former the spores lack extra-exosporal material and occur in a discoidal sporangium. Of the bilayered groups, two have exospores of homogeneous composition but with the two layers differing in electron density. They occur in discoidal sporangia and spore masses and are distinguished on the presence or absence of extra-exosporal material and differences in the widths of the two layers. Finally, two bilayered groups possess a lamellate inner layer, but vary in presumed sporangial shape. Elongate sporangia have spores with concentric continuous lamellae, lacking further ultrastructure. In contrast, spores from a discoidal spore mass have white-line-centred, presumably tripartite, lamellae which are laterally discontinuous, overlapping and irregularly spaced. These findings, which suggest that morphologically similar spores were produced by a number of plant taxa, have important implications regarding the assessment of early land-plant diversity. The affinities of hilate cryptospore-producing plants are unknown and problematic, particularly as no extant non-angiosperm plants produce dyads, other than through meiotic irregularity, and spore-sporangial characters have no exact counterpart in coeval plants. Studies of specimens with in situ hilate cryptospores suggest that they derive from rhyniophytoids, i.e. plants that resemble the simplest of vascular plants but lack evidence of vascular tissue, although hilate cryptospore-containing examples show no axial branching. It might be argued, based on evidence from spore wall ultrastructure, that some of the plants have more in common with lycopsids and filicopsids than bryophytes, a surprising finding bearing in mind the stratigraphic distribution of hilate cryptospores-dyads and inferences that the producers were bryophyte-like. Detailed studies of wall structure in the hilate cryptospores permit consideration of spore wall development. It is suggested that extra-exosporal material derives from a tapetum and is thus produced by the diploid sporophyte. The white-line-centred lamellae in a single specimen provide the earliest evidence for the presence of such structures in early land plant spores and provide further evidence that sporopollenin deposition on such structures is the most primitive mode of sporopollenin deposition among land plants.     

Juvenile hormone regulation of structural changes and DNA synthesis in the follicular epithelium of Leucophaea maderae

Juvenile hormone (JH I) stimulates specific morphological and biochemical changes in the follicular epithelium surrounding the terminal oöcytes in Leucophaea maderae. These include extracellular and intracellular structural changes, increased rates of follicle cell DNA synthesis, and elevated follicle cell DNA concentrations.Using females decapitated 24 hr after ecdysis, we have shown that JH I injections stimulate the following structural changes in the follicular epithelium: the appearance of channels between adjacent follicle cells and of spaces between the follicular epithelium and the maturing oöcyte; an increase in follicle cell size; the development of an extensive rough endoplasmic reticulum system; and an enlarged nucleus within each follicle cell. No increase in the number of follicle cells surrounding the developing terminal follicles is found in 7-day JH I-treated females, although the terminal follicles are almost twice as long as those in untreated females.In addition, we have demonstrated that JH stimulates the following biochemical events in the ovary: a 3.5 fold increase in thymidine incorporation into follicle cell DNA, with no subsequent transfer of such DNA to the developing oöcyte, and a 1.4 fold increase in ovarian DNA in 7-day JH-treated females. These data indicated that JH stimulates follicle cell DNA synthesis. The absence of any corresponding division of follicle cells suggests that JH I may induce polyploidy in follicle cells.Extended exposure of decapitated females to JH I does not result in complete ovarian maturation. Although fat bodies in the treated insects continue to display an increasing rate of vitellogenin synthesis, DNA synthesis in the terminal follicles declines rapidly after day 9, and the terminal follicles ultimately degenerate.