An evaluation of the cryosectioning of fixed and cryoprotected rat liver.

The influence of temperature and speed on cryosectioning was studied by replication of the surfaces of both the sections and the specimen blocks. At 90 mm/s and at temperatures lower than -70 degrees C, specimen block surfaces displayed fracture images similar to those encountered with standard freeze-fracturing procedures; but at a speed of 0.1 mm/s, fracture images were found only at temperatures lower than -120 degrees C. Replicas of both sides of cryosections never displayed fracture images. The discrepancy between the surface structure of cryosections and specimen blocks is discussed from the aspect of the preservation of ultrastructure of cells.

The site of action of lithium ions in morphogenesis of Lymnaea stagnalis analyzed by secondary ion mass spectroscopy.

Exogastrulation as a disturbance of development in eggs of Lymnaea stagnalis is caused by the action of LiCl at the second cleavage stage and not at the first or third. The percentage of exogastrulae formed is strongly concentration dependent. To determine the site of action of lithium ions, the cellular contents of Li, C, Na, Mg, P, K, and Ca were analyzed by secondary ion mass spectroscopy (SIMS). The mean elemental concentrations of Na, Mg, K, and Ca are close to those found earlier by electron probe microanalysis and atomic absorption spectroscopy. Lymnaea eggs at the first, second, and third cleavage stage were treated with LiCl in a series of concentrations ranging from 50 to 0.1 mM. In all cases the cells contained a few mM lithium after treatment. After treatment at the insensitive first cleavage stage the lithium content is carried over by the cells through the sensitive second cleavage to the insensitive third cleavage stage. These data allow the conclusion that it is the external lithium concentration which is responsible for the specific effect. This presents direct analytical evidence that the primary action of lithium ions is located at the cell membrane.

Modification of the Reichert FC-2 cryoattachment to improve ultrathin sectioning reproducibility.

Modifications of the Reichert FC-2 cryomicrotome attachment which facilitate sectioning of frozen material are described. Most valuable was the construction of a specimen holder chuck made of brass and connected to the specimen arm with a glass rod. Other changes include improved shielding from heat influx, a constant nitrogen pressure cooling system and a stable cryochamber mount. These modifications considerably improve reproducibility.

Localization of acid phosphatase in Saccharomyces cerevisiae: a clue to cell wall formation.

Acid phosphatase is present in two layers of the cell envelope of Saccharomyces cerevisiae. These are separated by another layer, which is free of acid phosphatase. We have evidence that the cell wall is built up in two stages, which are independent. In the first stage, the cell wall is built up during the formation of the bud. Glucanase vesicles are involved in this process. In the second stage, a thick layer is deposited at the inside against the new cell wall. This results in the thick, rigid wall of the mature yeast cell. This latter layer is probably assembled on the outer surface of the plasmalemma.

Relation of mitochondrial phospholipase A activity to mitochondrial swelling.

Assays of mitochondrial phospholipase A activity and mitochondrial swelling demonstrated that the phospholipase A activity is related to the swelling under the experimental conditions used. Both were stimulated by added free fatty acid and CaCl(2), not affected greatly by the addition of monoacyl phosphoglycerides, and inhibited by EDTA. The amount of fatty acid hydrolyzed from endogenous phosphatidyl ethanolamine and phosphatidyl choline during swelling was calculated to be 20-30 times less than the amount of added free fatty acid that gave comparable swelling. Under the experimental conditions about 4% of the phospholipid was hydrolyzed. Mitochondrial swelling was studied by electron microscopy and turbidity measurements. The results found were in agreement, whether oleic acid was present or not, except for those values obtained after very brief incubation (1 min) and after incubation for longer than 35 min. The lack of direct proportion between swelling and the concentration of lysosomes present indicated that the swelling is related mainly to mitochondrial phospholipase A, although swelling due to contaminating lysosomes cannot be excluded entirely. The temperature dependence of spontaneous, fatty acid-induced, or CaCl(2)-induced swelling suggested that enzymatic activities are responsible for swelling.