PMC

a)PrP(23–28,57–91) w/1 eq copper titrated with zinc b) PrP(23–28,57–91) with and without 300 μM Zn titrated with copper c) recombinant PrP(23–231) w/2 eq copper titrated with zinc d) PrP(23–231) with and without 300 μM Zn titrated with copper. Total copper bound (black), Component 3 (blue), 1+2 (red) and non-octarepeat (green). Copper and zinc (solid), copper only (dotted).

(A) Copper (gold circles) enters enterocytes via two membranes: the apical membrane that faces the intestinal lumen and the basolateral membrane that faces the blood. The high-affinity copper uptake, inhibited by silver, takes place via the basolateral membrane and is likely to be mediated (as in other cells) by CTR1. The low-affinity copper uptake, which is not inhibited by silver, occurs via the apical membrane through an uncharacterized process (black arrow). Copper taken up via the apical pathway appears unavailable in the cytosol in the absence of CTR1 and an additional role of CTR1 could be to release dietary copper from subapical vesicles for further utilization. Once released, copper binds to cytosolic proteins (brown semicircles) and is also delivered to copper chaperones and cytochrome C oxidase in mitochondria, as well as to Cu-ATPases in the secretory pathway. Cu-ATPase ATP7A transports copper to vesicles, which then fuse with the basolateral membrane and copper is released into the blood. (B) Genetic inactivation of CTR1 in the intestine results in accumulation of copper in enterocytes; however, this copper is sequestered (presumably in vesicles) and does not reach cytosolic proteins, mitochondria or secretory pathways, resulting in low activity of copper-dependent enzymes and impaired copper efflux from the enterocytes.

Level of intracellular calcium in E. siliculosus cultivated in seawater without copper addition (empty circles) and with 2.5 μM copper (black circles) for up to 350 min (A), and from 360 to 720 min (B). To assess VDCCs at 3 h (C) and 9 h (D) of copper exposure, treatments were: without copper (control); with 2.5 μM copper (copper); with copper and 250 nM of verapamil (Ver); with copper and 250 nM nifedpine (Nif); and with copper and 250 nM diltiazem (Dil). To address the nature of VDCCs, levels of intracellular calcium in the alga under copper excess at 3 h (E) and 9 h (F) were measured with the treatments: without copper (control); with 2.5 μM copper (copper); with copper and 1 mM EGTA; and with copper and 250 nM thapsigargin (Thap). The level of intracellular calcium is expressed as the difference among initial and final fluorescence intensity of Fluo 3 normalized to initial intensity of Fluo3. Symbols and bars represent mean values of three independent experiments ± SD.

Approximately 5×10⁴ pfu were applied to test surfaces that had been acclimatised to required temperature (copper (•), phosphor bronze (95% copper) (○), copper nickel (▾), cartridge brass (70% copper) (Δ), nickel silver (65% copper) (▪), stainless steel (□)) in 20 µL (‘wet’ inoculum). Virus was removed and assessed for infectivity using plaque assay.