Although calcium carbonate is known to be a common biomineral in plants, very little attention has been given to the biological control of calcium carbonate deposition. In mulberry leaves, a subcellular structure is involved in mineral deposition and is described here by a variety of cytological techniques. Calcium carbonate was deposited in large, rounded idioblast cells located in the upper epidermal layer of mulberry leaves. Next to the outmost region ("cap") of young idioblasts, we found that the inner cell wall layer expanded to form a peculiar outgrowth, named cell wall sac in this report. This sac grew and eventually occupied the entire apoplastic space of the idioblast. Inside the mature cell wall sac, various cellulosic membranes developed and became the major site of Ca carbonate deposition. Concentrated Ca2+ was pooled in the peripheral zone, where small Ca carbonate globules were present in large numbers. Large globules were tightly packed among multiple membranes in the central zone, especially in compartments formed by cellulosic membranes and in their neighboring membranes. The maximum Ca sink capacity of a single cell wall sac was quantified using enzymatically isolated idioblasts as approximately 48 ng. The newly formed outgrowth in idioblasts is not a pure calcareous body but a complex cell wall structure filled with substantial amounts of Ca carbonate crystals.

Blepharismins are polycyclic quinones found in the pigment granules of the ciliated protozoan, Blepharisma. Exposure to purified blepharismins results in lethal damage to several other ciliates. We here report that, at cytotoxic concentrations, blepharismins formed cation-selective channels in planar phospholipid bilayer membranes. The channels formed in a diphytanoylphosphatidylcholine bilayer had a K + /Cl 3 permeability ratio of 6.6:1. Single channel recordings revealed the conductance to be quite heterogeneous, ranging from 0.2 to 2.8 nS in solutions containing 0.1 M KCl, possibly reflecting different states of aggregation of blepharismin. Our observations suggest that channel formation is a cytotoxic mechanism of blepharismin's action against predatory protozoa. ß

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Tolerance of the resting cysts of protozoa against gastric acid and proteases is a strategy for surviving in the digestive tract of animals. The present study aimed to examine the protection mechanism against HCl in resting cysts of Colpoda cucullus, which were surrounded by a cyst wall, which is composed of an outermost layer (ectocyst) and several inner layers (endocyst). In addition to water, paraformaldehyde, hydrogen peroxide (H 2 O 2) and ethanol may diffuse across the cyst wall, because the cysts hardly showed tolerance against these molecules. However, the cysts showed tolerance against HCl (0.01 to 1 M) and pepsin. The tolerance against HCl disappeared soon after the onset of excystment induction. Electron micrographs of the excysting cells showed that digestion of the endocyst began within 30 min after the onset of excystment induction. The encysting cells surrounded by the ectocyst (3 h after encystment induction) did not show tolerance against HCl, but acquired it just after a first layer of the endocyst was formed beneath the ectocyst (6 h after encystment induction). These results suggest that the tolerance of the resting cyst of C. cucullus against HCl may be acquired by preventing its diffusion across the endocyst, although the possibility that cytoplasmic molecules and the plasma membrane may acquire tolerance has not been ruled out.