Cryofixed blood morula cells of Phallusia mammillata (Cuvier), which are considered to be vanadium-accumulating cells, were examined by X-ray microanalysis using STEM (scanning transmission electron microscopy) and SEM (scanning electron microscopy). It is thought that cryopreparation preserves the native distribution of diffusible elements such as sodium, chlorine, and potassium, and prevents the displacement of vanadium, all of which may occur during conventional preparation. The results show that morula cell globules contain a large amount of sulphur and chlorine, and some sodium, magnesium, bromium and potassium, but very little or no vanadium.
Tunicates (Urochorda) concentrate vanadium in their blood cells to a million times the concentration in sea water." -31 Although the vanadium compound in tunicates was discovered 80 years ago, its structure is still not known. A proven fact is that in Phallusia mammillata and Ascidia nigra vanadium is predominantly t r i~a l e n t . Because the peptides of the blood cells are exceptionally unstable and sensitive to oxidation, we have developed a very simple isolation involving few losses for the peptides from P. marnmillata. The tunichromes were extracted with methanol quite selectively from lyophilized cells and purified by HPLC on polystyrene phases with a recovery rate 80-90 % or by capillary electrophoresis, or introduced on-line from HPLC into an ion spray tandem mass spectrometer. In contrast, the separation on reversed phases based on silica gel caused large losses. All operations were performed in an inert atmosphere. Figure 1 depicts the HPLC separation of the methanol extract. Besides three minor components, a major component was obtained, which was studied by ion spray MS, tandem MS, 'H and 13C NMR, UVjVIS, and IR spectroscopy. The separation of the methanol extract by capillary zone electrophoresis also proved valuable, as it also provided a major component.
Some ascidians (sea squirts) accumulate the transitional metal vanadium in their blood cells at concentrations of up to 350 mM, about 10(7) times its concentration found in seawater. There are approximately 10 different types of blood cell in ascidians. The identity of the true vanadium-containing blood cell (vanadocyte) is controversial and little is known about the subcellular distribution of vanadium. A scanning x-ray microscope installed at the ID21 beamline of the European Synchroton Radiation Facility to visualize vanadium in ascidian blood cells. Without fixation, freezing or staining realized the visualization of vanadium localized in living signet ring cells and vacuolated amoebocytes of two vanadium-rich ascidian species, Phallusia mammillata and Ascidia sydneiensis samea. A combination of transmission and fluorescence images of signet ring cells suggested that in both species the vacuoles contain vanadium.
X-ray histospectrographic analysis at the scanning and transmission electron microscope (STEM) are made on the blood cells of Phallusia mamillata Cuvier and Ciona intestinalis, to study the 'direct' intracellular sites of accumulation of vanadium. The results show a clear accumulation of vanadium on the membrane and in the granules of vacuoles of amebocytes, signet ring cell, compartment cell and traces of metal in the 'vanadophores' of vanadocytes.
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