Differences in defence responses of Pinus contorta and Pinus banksiana to the mountain pine beetle fungal associate Grosmannia clavigera are affected by water deficit

We tested the hypotheses that responses to the mountain pine beetle fungal associate Grosmannia clavigera will differ between the evolutionarily co‐evolved host lodgepole pine (Pinus contorta var. latifolia) and the naïve host jack pine (Pinus banksiana) and that these responses will be influenced by water availability. G. clavigera inoculation resulted in more rapid stem lesion development in lodgepole than in jack pine; water deficit delayed lesion development in both species. Decreased hydraulic conductivity was observed in inoculated lodgepole pine seedlings, likely because of tracheid occlusion by fungal hyphae and/or metabolite accumulation. Drought but not inoculation significantly impacted bark abscisic acid levels. Jasmonic and salicylic acid were implicated in local and systemic responses of both species to G. clavigera, with salicylic acid appearing to play a greater role in jack pine response to G. clavigera than lodgepole pine. Water deficit increased constitutive levels and/or attenuated induced responses to G. clavigera for several monoterpenes in lodgepole but not jack pine. Instead, inoculation of well‐watered but not water deficit jack pine resulted in a greater number of xylem resin ducts. These findings reveal mechanisms underlying differences in G. clavigera‐induced responses between lodgepole and jack pine hosts, and how water availability modulates these responses.     

The postnatal age of rat lung fibroblasts influences G1/S phase transition in vitro

Summary In the neonatal rat lung, alveolar development occurs from postnatal Days 4–13, during which time there is a fourfold increase in interstitial fibroblasts. Factors influencing emergence of new septa and cell proliferation associated with septal elongation have yet to be identified, in part because of difficulties inherent in studying this process in vivo. Using flow cytometric analysis of the DNA content of freshly isolated lung fibroblasts, we found that proliferation, as indicated by the percentage of cells in S plus G₂/M phases, peaked on postnatal Day 4 (P<0.04). By Days 9–10 the proliferation rate was lower than on Days 3, 4, 5, or 6 (P<0.005). We then evaluated rates of in vitro proliferation as a function of postnatal age in first passage fibroblasts and found that the proliferative phenotype expressed in vivo persists in vitro. Fibroblasts from 4–5-d-old pups increased in number and incorporated ³H-thymidine at a faster rate than did fibroblasts obtained from pups at other postnatal ages (P<0.0001). Age-dependent differences in cell cycle transit time were compared in fibroblasts synchronized by serum starvation and analyzed by flow cytometry at 2-h intervals from 13–21 h after release from serum starvation. A greater percentage of cells from 5-d-old pups entered S phase during this period than was seen for cells obtained from 2-, 9-, 13-, or 23-d-old rat pups (P=0.0001). Cells from 5-, 9-, and 13-d-old pups reentered G₀/G₁ by 21 h after release from serum starvation, in contrast to fibroblasts from 2- and 23-d-old rats which did not. Throughout the 15-h period after release from serum starvation, levels of cyclin E, which peaks at the G₁/S border, were highest in the 5-d-old cells (P<0.025). Synchronization with 2.5 mM hydroxyurea which inhibits DNA synthesis completely abolished age-related differences in cell cycle transit time, implying that age-dependent differences in lung fibroblast proliferation rates are the result of events occurring before S-phase entry.     

Prion protein protects against zinc-mediated cytotoxicity by modifying intracellular exchangeable zinc and inducing metallothionein expression

PrP^C contains several octapeptide repeats sequences toward the N-terminus which have binding affinity for divalent metals such as copper, zinc, nickel and manganese. However, the link between PrP^C expression and zinc metabolism remains elusive. Here we studied the relationship between PrP^C and zinc ions intracellular homeostasis using a cell line expressing a doxycycline-inducible PrP^C gene. No significant difference in ⁶⁵Zn²⁺ uptake was observed in cells expressing PrP^C when compared with control cells. However, PrP^C-expressing cells were more resistant to zinc-induced toxicity, suggesting an adaptative mechanism induced by PrP^C. Using zinquin-ethyl-ester, a specific fluorophore for vesicular free zinc, we observed a significant re-localization of intracellular exchangeable zinc in vesicles after PrP^C expression. Finally, we demonstrated that PrP^C expression induces metallothionein (MT) expression, a zinc-upregulated zinc-binding protein. Taken together, these results suggest that PrP^C modifies the intracellular localization of zinc rather than the cellular content and induces MT upregulation. These findings are of major importance since zinc deregulation is implicated in several neurodegenerative disorders. It is postulated that in prion diseases the conversion of PrP^C to PrP^Sc may deregulate zinc homeostasis mediated by metallothionein.