Adsorption and uptake of Cu by Emiliania huxleyi in natural seawater.

Short-term kinetic experiments, carried out in natural coastal seawater (with predetermined background levels of trace metals and organic ligands, L) enriched with nitrate and phosphate, demonstrated that Emiliania huxleyi was able to uptake Cu very quickly. After 10 min of exposure (background Cu level in the inoculated cells: [Cu]total cellular = 9.3 x 10(-17) mol cell-1, [Cu]intracellular = 8.4 x 10(-17) mol cell-1, and [Cu]extracellular = 1.0 x 10(-17) mol cell-1) to a natural seawater which contained 29 nM total initial dissolved Cu concentration ([Cu]d) (29 nM [CuL] and 3.2 x 10(-13) M free Cu concentration, [Cu2+]) the intracellular Cu was already 28 x 10(-17) mol cell-1. This value corresponded to 85% of the intracellular metal observed in pseudoequilibrium conditions (after 24 h of exposure) and to 88% of the total metal sorption (adsorption plus uptake) after 10 min. In contrast, the external adsorption after 10 min was only 3.0 x 10(-17) mol cell-1 which corresponded to 60% of the extracellular metal in pseudoequilibrium conditions. Simultaneously occurred a very fast release of organic ligands (L) by E. huxleyi, the majority being identified by cathodic stripping voltammetry as glutathione. The production of exudates increased with both Cu concentration and exposure time. After 10 min of exposure, the production of exudates in a medium with 129 nM [Cu]d (72 nM [CuL] and 7.9 x 10(-13) M [Cu2+]) was 51 nM, about 42% of that observed in pseudoequilibrium. As the Cu complexes with the organic ligands present in the medium were very stable (logarithm of the conditional stability constant: 12.18 +/- 0.06) and the ligand concentration in the medium was relatively high (e.g. 123 nM CL in the medium with 129 nM initial [Cu]d after 10 min of exposure) most of the metal was organically bound in the medium.

Microwave-stimulated recovery of myosin-V immunoreactivity from formalin-fixed, paraffin-embedded human CNS.

The lability of brain myosin-V (BM-V) to aldehyde-fixation has hindered immunohistochemical (IH) studies of this actin-based motor. We show here that BM-V immunoreactivity (IR) can be retrieved from formalin-fixed, paraffin-embedded human tissue. BM-V IR was optimally retrieved by boiling 5 microm cerebellar tissue sections in 10 mM sodium citrate buffer, pH 6, for 15 min, using a microwave oven set at 900 W and 2.45 GHz. A polyclonal, affinity purified anti-BM-V antibody, raised in rabbits against the tail domain of chicken BM-V, was shown here to recognize a single band in Western blots of human cortical homogenates. The combined use of this monospecific antibody and of the antigen retrieval (AR) method above allowed us to verify that BM-V IR is strongly expressed in human Purkinje cell bodies and dendrites, and in granule cells. The same pattern of BM-V IR expression was consistently and maximally detected in tissues stored in 10% formalin from 1 week to 2.5 months. The AR protocol for BM-V described here permits its IH study in formaldehyde-fixed tissues. It is a valuable tool to study BM-V in well fixed tissues, as occurs with the large collection of human archival tissue available.