The mechanism of nitrogen monoxide (NO)-mediated iron mobilization from cells. NO intercepts iron before incorporation into ferritin and indirectly mobilizes iron from ferritin in a glutathione-dependent manner.

Nitrogen monoxide (NO) is a cytotoxic effector molecule produced by macrophages that results in Fe mobilization from tumour target cells which inhibits DNA synthesis and mitochondrial respiration. It is well known that NO has a high affinity for Fe, and we showed that NO-mediated Fe mobilization is markedly potentiated by glutathione (GSH) generated by the hexose monophosphate shunt [Watts, R.N. & Richardson, D.R. (2001) J. Biol. Chem. 276, 4724-4732]. We hypothesized that GSH completes the coordination shell of an NO[bond]Fe complex that is released from the cell. In this report we have extended our studies to further characterize the mechanism of NO-mediated Fe mobilization. Native PAGE 59Fe-autoradiography shows that NO decreased ferritin-59Fe levels in cells prelabelled with [59Fe]transferrin. In prelabelled cells, ferritin-59Fe levels increased 3.5-fold when cells were reincubated with control media between 30 and 240 min. In contrast, when cells were reincubated with NO, ferritin-59Fe levels decreased 10-fold compared with control cells after a 240-min reincubation. However, NO could not remove Fe from ferritin in cell lysates. Our data suggest that NO intercepts 59Fe on route to ferritin, and indirectly facilitates removal of 59Fe from the protein. Studies using the GSH-depleting agent, L-buthionine-(S,R)-sulphoximine, indicated that the reduction in ferritin-59Fe levels via NO was GSH-dependent. Competition experiments with NO and permeable chelators demonstrated that both bind a similar Fe pool. We suggest that NO requires cellular metabolism in order to effect Fe mobilization and this does not occur via passive diffusion down a concentration gradient. Based on our results, we propose a model of glucose-dependent NO-mediated Fe mobilization.     

Evidence for a junctional feet-ryanodine receptor complex from sarcoplasmic reticulum

Heavy sarcoplasmic reticulum vesicles, labelled with the Ca2+ release channel probe [3H]ryanodine, were solubilized in detergent, then centrifuged through sucrose gradients. A single peak of ryanodine binding activity was observed with an apparent sedimentation coefficient of 30S. Electron microscopy of the peak fraction showed disk structures of 25-28 nm diameter and 10 nm thickness. Proteins specifically enriched in the peak fraction were the Mr 160,000 and 260,000 and junctional feet proteins (Mr 320,000 and 300,000). This suggests that the feet proteins and ryanodine receptor may be specifically associated into a large oligomeric complex comprising subunits of Mr 160,000-320,000.     

Glandular trichome exudate is the critical factor in geranium resistance to foxglove aphid

Resistance to the foxglove-aphid (Acyrthosiphon solani Kaltenbach) has been demonstrated in some inbred geranium lines (Pelargonium Xhortorum Bailey). To establish more definitively the cause/effect relationship between tall glandular trichome exudate and resistance, an intact plant bioassay was performed comparing a resistant plant line, a resistant plant line from which the tall glandular trichome exudate had been removed using a basic buffer solution, a susceptible line and a susceptible line treated with the buffer wash. After 5 days of isolation on the respective surfaces, the number of surviving adult aphids as well as the number of nymphs produced and remaining alive were determined. Aphids on the buffer washed, resistant line exhibited mortality and fecundity which was not significantly different from that produced by the susceptible line. In contrast, the untreated resistance line was clearly resistant with lower adult survival and fewer living nymphs. The tall glandular trichome exudate must therefore be a critical factor in geranium resistance to the foxglove aphid.

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Zusammenfassung Widerstandsfähigkeit dem Fingerhut-Blattlaus (Acyrthosiphon solani Kaltenbach) gegenüber wurde in einigen durch Inzucht erzeugten Pelargonie-Linien (Pelargonium Xhortorum Bailey) gezeigt. Um das Verhältnis von Ursache und Wirkung zwischen dem hochgewachsenen glandulären Trichom-Exudat und Widerstandsfähigkeit genauer zu bestimmen, wurde eine Bio-Untersuchung an intakten Pflanzen unternommen. Dabei wurden eine widerstandsfähige Pflanzenlinie, eine widerstandsfähige Pflanzenlinie, von der das hochgewachsene glanduläre Trichom-Exudat durch eine basische Pufferlösung entfernt worden war, eine anfällige Linie und eine mit Pufferlösung behandelte Linie verglichen. Zwei erwachsene weibliche Blattläuse wurden fünf Tage durch ein engmaschiges Netz auf den zu untersuchenden dritten und vierten Knotenblättern eingesperrt. Bei jeder Pflanze wurde die Untersuchung an einem nichtbehandelten Blatt und an einem Blat, von dem das Exudat durch Waschen mit der Pufferlösung entfernt worden war, durchgeführt. Für jede Linie wurden fünf Pflanzen gebraucht, und der ganze Versuch wurde sechsmal wiederholt. Nach einer fünftägigen Isolierung auf den jeweiligen Oberflächen wurden die Blätter von der Pflanze entfernt, und sowohl die Zahl der überlebenden erwachsenen Blattläuse wie auch die der produzierten und noch am Leben gebliebenen Nymphen festgestellt. Mit einer niedrigeren Überlebensrate der Erwachsenen und weniger noch lebenden Nymphen war die nichtbehandelte widerstandsfähige Linie deutlich widerstandsfähig. Im Gegensatz dazu zeigten die Blattläuse auf der mit Puffer gewaschenen widerstandsfähigen Linie eine Sterblichkeit und Fruchtbarkeit, die nicht erheblich höher waren, als die auf der anfälligen Linie, was beweist, daß das Waschen mit der Pufferlösung den Widerstandsfaktor entfernt hatte. Das hochgewachsene glanduläre Trichom-Exudat muß deshalb ein kritischer Faktor in der Widerstandsfähigkeit gegen Pelargonie-Schädlinge sein.

     

Heterotrophic activity and bacterial productivity in assemblages of microbes from sea ice in the high Arctic

Summary Heterotrophic activity in the bottom few cm of annual sea ice in the Canadian Arctic was measured throughout the spring bloom of ice algae, using tritium-labelled thymidine and glucose. Experiments with chloramphenicol and cyclohexamide indicated that thymidine assimilation was due to procaryotic microbes but that about half of the glucose assimilation was due to eucaryotic organisms. Glucose and thymidine assimilation rates increased with salinity, from 10 ppt to 30 ppt. Thymidine assimilation rates increased from 1.16 to 4.94·10^–21mol·cell^–1·h^–1 during the latter half of the algal bloom, while the exponential growth rate of the in situ populations decreased from 0.058 to 0.025 d^–1. Bacterial production and specific growth rates calculated from thymidine assimilation were 149mgC·m^–2 and 0.25 d^–1 or less respectively over the 50 day observation period, compared with net primary production of 5,500 mgC·m^–2. Thymidine assimilation rates suggested that about half of the bacterial production may be consumed or lost from the ice during the bloom.     

Physical identification of branched intron side-products of splicing in Trypanosoma brucei.

Every mRNA in trypanosomes consists of two exons, a common 5' capped mini-exon or spliced leader and a coding-exon. All evidence suggests that the exons are joined by trans-splicing of two individual precursor RNAs, the mini-exon donor RNA or spliced leader precursor RNA (medRNA) and the pre-mRNA. We studied intermediates of the splicing reaction using denaturing two-dimensional PAGE and structurally identified a group of small (approximately 180-300 nt) non-polyadenylated, Y-shaped branched RNAs. The branched Y-shaped RNAs contain the 105 nt medRNA derived intron, joined in a 2'-5' phosphodiester bond to small heterogeneously sized RNAs. These non-polyadenylated branched Y-shaped RNA molecules are analogous to the lariat shaped introns of higher eukaryotes and presumably represent the released intron-like by-products of a trans-splicing reaction which joins the mini-exon and the major coding-exon.