The caveolae in rabbit sinus node and atrium

Summary Caveolae or membrane vesicles are commonly observed in smooth and skeletal muscle as well as in working heart muscle. Using sections of fixed tissue and replicas of freeze-cleaved material, we show in this study that caveolae are also very numerous in sinus node cells of the rabbit, and to a lesser degree, in the atrial cells.Caveolae increase the plasma membrane surface area by 115% in the leading sinus node, and by 56% in the atrial cells. In these two cell types, the membrane of the caveolae contains four times fewer intramembranous particles than the rest of the plasma membrane, and this difference applies to both PF and EF faces. The role of the caveolae is still unclear, but it does not seem that they have a pinocytotic function.     

Accumulation et excrétion de substances organiques par les cellules à chlorure de la branchie d'Anguilla anguilla L. adaptée à l'eau de mer

Résumé Les sites branchiaux d'accumulation de certaines substances (bleu de méthylène, urée, paraaminohippurate et inuline) injectées dans la circulation générale ont été localisés sur des anguilles adaptées à l'eau de mer. Le bleu de méthylène est accumulé et excrété par les cellules à chlorure. Les autoradiographies des corps marqués au ¹⁴C: inuline, PAH et urée, faites après cryodessication montrent que les cellules à chlorure accumulent ces substances. La signification de ces résultats est discutée.

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Accumulation and excretion of organic substances by the branchial chloride cells in sea-water adapted eel (anguilla anguilla L.)

Summary The branchial site of accumulation of various organic substances (methylene blue, urea, inulin and paraaminohippurate) following intracardiac injection has been localised in sea-water adapted eels. Methylene blue is concentrated and excreted by chloride cells. By combining freeze-drying and radioautographic technique it was possible to demonstrate that chloride cells concentrate urea, inulin and paraaminohippurate. The signification of these results is discussed in relation with the branchial excretion of organic substances.

     

High pressure freezing and freeze substitution improve immunolabeling of S-locus specific glycoproteins in the stigma papillae ofBrassica

Summary High pressure freezing and freeze substitution methods significantly improve the antigenic preservation of S-locus specific glycoproteins (SLSG). The SLSG, which are implicated in the incompatibility response, are localized over the cell wall and cytoplasm. Labeling in the cytoplasm is mainly associated with dictyosomes and rough endoplasmic reticulum. Quantitative analysis show that in cryofixed papillae the labeling was enhanced by approximately 45% over the cell wall and approximately 90% over the dictyosomes compared to chemically fixed papillae.     

Effect of cooling rate on the survival of frozen wood frogs,Rana sylvatica

Summary Wood frogs (Rana sylvatica) were frozen to-2.5°C under five distinct cooling regimes to investigate the effect of cooling rate on survival. Frogs survived freezing when cooled at -0.16°C · h^-1 or -0.18°C · h^-1, but mortality resulted at higher rates (-0.30°C · h^-1,-1.03°C · h^-1, and -1.17°C · h^-1). Surviving frogs in the latter groups required longer periods to recover, and transient injury to the neuromuscular system was evident. Some of the frogs that died had patches of discolored, apparently necrotic skin; vascular damage, as indicated by hematoma, also occurred. It is concluded that slow cooling may be critical to the freeze tolerance of wood frogs. Additional studies examined the effect of cooling rate on physiological responses promoting freeze tolerance. Mean glucose concentrations measured in plasma (15–16 mol · ml^-1) and liver (42–45 mol · g^-1) following a 2-h thaw did not differ between slowly- and rapidly-cooled frogs but in both groups were elevated relative to unfrozen controls. Thus freezing injury to rapidly-cooled frogs apparently was not mitigated by the presence of elevated glucose. Water contents of liver tissue, measured 2 h post-thawing, did not differ between slowly-cooled (mean = 77.6%) and rapidly-cooled (mean = 78.5%) frogs. However, the mean hematocrit of slowly-cooled frogs (48%) was significantly higher than that (37%) of frogs cooled rapidly, possibly owing to differences in the dynamics of tissue water during freezing.     

Freeze-fracture analysis of structural reorganization during meiotic maturation in oocytes of Xenopus laevis

Summary During meiotic maturation, the cortex of oocytes of Xenopus laevis undergoes structural reorganization, visualized in this study by freeze-fracture electron microscopy. In the full-grown but immature oocyte, annulate lamellae are dispersed throughout the subcortex of the egg, 5 to 20 m from the plasma membrane. The annulate lamellae consist of well-organized stacks of membrane with visible pores. Stimulation of meiotic maturation by progesterone leads to disruption of the annulate lamellae and formation of an elaborate cortical endoplasmic reticulum which surrounds the cortical granules and intertwines throughout the cortex of the mature egg. Pore-like structures similar to those previously observed in the subcortical annulate lamellae are observed in the mature cortical endoplasmic reticulum. The cortical endoplasmic reticulum is often in close apposition with the plasma membrane and with membranes of cortical granules, but no junctions are visualized. This study provides further evidence that the cortical endoplasmic reticulum develops during progesterone-stimulated meiotic maturation in vitro, and that the annulate lamellae are precursors to the cortical endoplasmic reticulum.     

Effects of Ga3 and Ca2+ on barley aleurone protoplasts: a freeze-fracture study

Summary Freeze-fracture electron microscopy was used to study changes in the endomembrane system of barley (Hordeum vulgare L. cv. Himalaya) aleurone protoplasts. Protoplasts were used for this study because their response to calcium and the plant hormone gibberellic acid (Ga₃) can be monitored prior to rapid freezing of cells for electron microscopy. Protoplasts incubated in Ga₃ plus Ca²⁺ secrete elevated levels of a-amylase relative to cells incubated in Ga₃ or Ca²⁺ alone. The endoplasmic reticulum (ER) and Golgi apparatus of protoplasts incubated in Ga₃ plus Ca²⁺ undergo changes that are well correlated with the synthesis and secretion of a-amylase. The ER, which appears as short, single sheets of membrane in Ca²⁺-and Ga₃-treated protoplasts, exists as a series of long fenestrated stacks of membranes following incubation in Ga₃ plus Ca²⁺. The Golgi apparatus is also more highly developed in protoplasts treated with Ga₃ plus Ca²⁺. This organelle is larger and has more vesicles associated with its periphery in protoplasts that actively secrete a-amylase. Evidence that the Golgi apparatus participates in a-amylase secretion is also provided by experiments with the ionophore monensin, which causes pronounced swelling of Golgi cisternae and inhibits the secretion of a-amylase. We interpret these observations as showing that the ER and Golgi apparatus of barley aleurone participate in the intracellular transport and secretion of a-amylase. The plasmalemma (PF face) of barley aleurone protoplasts shows a high density of intramembranous particles (IMPs) which, in general, are evenly distributed. Occasionally, ordered arrays of IMPs are observed, possibly resulting fro m osmotic stress. after 48 hours the plasmalemma of some Ga₃-treated protoplasts show particle-free areas considered to be indications of senescence.abbreviations ER endoplasmic reticulum - Ga₃ gibberellic acid - IEF isoelectric focusing - IMP intramembranous particle - PF protoplasmic fracture - PL plasmalemma     

The Plasma Membrane of Myxosporidian Valve Cells: Freeze Fracture Data

Freeze fracturing of Myxosporidian spores reveals the occurrence of a continuous layer of transmembrane particles all over the surface area of the valve cells which form the spore envelope. These particles are densely packed all over the P face membrane. Due to their polygonal outline, their diameter (6-7 nm) and their central core, they resemble the particles forming the connections of gap junctions which metabolically couple the neighboring cells in animal tissues. In the present report, the role of the transmembrane particles is still hypothetical. However, they might represent a membrane structural specialization of the spores which are submitted to osmotic variations of the fluid external medium. Furthermore similar transmembrane particles are observed at the level of the septate junction which seals the valve cells. In this occurrence, they are arranged in a series of 40 double rows parallel to the suture of the spore envelope. These findings support the view that Myxosporidia are Metazoa and raise the problem of their origin.     

Ultrastructural changes associated with renin secretion from the juxtaglomerular apparatus of mice

Summary Thin sections and freeze-fracture replicas were used to investigate the ultrastructural changes associated with renin secretion from the juxtaglomerular part of the afferent arteriole of male mice. Adrenalectomized animals in which renin secretion was stimulated by furosemide application and bleeding were also studied. Exocytosis of mature electron-dense granules was found in all experimental groups. Before extrusion, the region of granule facing the cell membrane changed, with vesicular and/or stacked membrane-like profiles and a small local protrusion of the granule membrane appearance of. Concomitantly, punctuate sites of fusion between the cell and granule membranes were observed. Later, unaltered amorphous, and altered membrane-like granule content was released from omega-shaped cavities into the extracellular space. In stimulated animals the alteration and extrusion of several closely apposed granules was reminiscent of compound exocytosis. Coated pits were frequently seen, suggesting specific retrieval of the former granule membrane. The collapsing silhouette of a depleted granule very rarely took the form of a saccule whose narrow membrane-bounded neck was continuous with the extracellular space.Observed were two additional events by which active and inactive renin may be released. Small electron-lucent vacuoles of undetermined origin fused with the cell membrane and, in stimulated kidneys, some epithelioid cell processes disintegrated. However, the interpretation of the related ultrastructural phenomena was uncertain.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Glucosylglycerol and glucosylglycerate as enzyme stabilizers

Compatible solutes constitute a diverse class of low-molecular-mass organic molecules that are accumulated in high intracellular concentrations in response to the external stress of hyperosmolality or high temperature. Many of these compounds like α, α-trehalose are well known for their stabilizing effect on protein structure and could lead to development of more stable protein formulations. Negatively charged solutes like mannosylglycerate (R-2-O-α-D -mannopyranosyl-glycerate) are widespread among (hyper)thermophilic microorganisms and are thought to be exceptionally potent stabilizers of proteins under high-temperature denaturation conditions. To further inquire into the role of compound charge for protective function, we have compared two naturally occurring and structurally related solutes, glucosylglycerol (2-O-α-D -glucopyranosyl-sn-glycerol) and glucosylglycerate (R-2-O-α-D -glucopyranosyl-glycerate), as stabilizers of different enzymes undergoing inactivation through elevated temperature or freeze drying, and benchmarked their effects against that of α,α-trehalose. Glucosylglycerate in concentrations of ≥0.1 M was the most effective in preventing thermally induced loss of enzyme activity of lactate dehydrogenase, mannitol dehydrogenase, starch phosphorylase, and xylose reductase. α,α-Trehalose could usually be replaced by glucosylglycerol without compromising enzyme stability. Glucosylglycerol and glucosylglycerate afforded substantial (eightfold) protection to mannitol dehydrogenase during freeze drying.     

Nuclear pore dynamics during pollen development and androgenesis in Brassica napus

Changes in nuclear pore complex (NPC) densities, NPCs/nucleus and NPCs/μm³, are described using freeze-fractured Brassica napus microspores and pollen in vivo and in vitro. Early stages of microspore- and pollen-derived embryogenic cells were also analysed. The results of in vivo and in vitro pollen development indicate an increase in activity of the vegetative nucleus during maturation of the pollen. At the onset of microspore and pollen culture, NPC density decreased from 15 NPCs/μm² at the stage of isolation to 9 NPCs/μm², under both embryogenic and non-embryogenic conditions. This implies that the drop in NPC density might be a result of culturing the microspores and pollen rather than an indication for microspore and pollen embryogenesis in Brassica napus. However, after 1 day in culture under embryogenic conditions, the NPC density increased again and stabilised around 13 NPCs/μm², whereas under non-embryogenic conditions the NPC density remained about 9 NPCs/μm². This low density of 9 NPCs/μm² was also found in the nuclei of sperm cells, in contrast to the 19 NPCs/μm² found in the vegetative nucleus. It means that, although both the vegetative and sperm nuclei are believed to be metabolically rather inactive in mature pollen, the NPC density of vegetative nucleus is twice as high as the NPC density of the sperm nuclei. In a few cases, embryos formed suspensor-like structures with a NPC density of 9 NPCs/μm², indicating a lower nucleocytoplasmic exchange of the nuclei of the suspensor cells than with the nuclei in the embryo proper. In addition, observations on NPCs and other organelles, obtained by high resolution cryo-scanning microscopy, are presented. Received: 29 December 1999 / Revision accepted: 3 March 2000