E. S. Snigirevskaya

PROSTAGLANDIN-DEPENDENT OSMOTIC WATER PERMEABILITY OF THE FROG AND TROUT URINARY BLADDER

Washout of autacoids from serosal Ringer solution, using a repeated change of the solution of the frog and trout urinary bladder, was accompanied by a pronounced rise in the osmotic water permeability: the water transport in the frog rose from 0.05 +/- 0.02 to 1.21 +/- 0.26 microliter min-1.cm-2, in the trout, from 0.041 +/- 0.011 to 0.26 +/- 0.034 microliter min-1.cm-2. Such an increase in the osmotic water permeability in the trout and frog urinary bladder occurred in the background of a decrease in the prostaglandin E2 concentration in the serosal Ringer solution. This permeability increase was accompanied by the formation of aggregates of intramembranous particles in the apical plasma membrane of the trout and frog urinary bladder. A decrease in the osmotic water permeability was achieved by the addition to the serosal Ringer solution of 10-8 M prostaglandin. Experiments on the frog urinary bladder have shown that prostaglandins E1, I2 and F2 alpha also decrease the osmotic water permeability. Vasotocin increased the osmotic water permeability in the frog urinary bladder but did not affect the osmotic water permeability of the trout urinary bladder. The data obtained indicates a role of the endogenous prostaglandin production in maintaining the low osmotic water permeability in the frog and trout urinary bladder. A suggestion is made that in the vertebrate evolution, colonisation of the fresh-water was connected with the maintenance of the low osmotic water permeability via participation of prostaglandins, whereas the vasotocin hydroosmotic effect developed in the vertebrate evolution later and provided for the possibility of the water absorption, osmotic homeostasis and animal migration from fresh-water to the land.

Investigation of the kinetics of insulin amyloid fibrils formation

Today, the investigation of the structure of ordered protein aggregates-amyloid fibrils, the influence of the native structure of the protein and the external conditions on the process of fibrillation-is the subject of intense investigations. The aim of the present work is to study the kinetics of formation of insulin amyloid fibrils at low pH values (conditions that are used at many stages of the isolation and purification of the protein) using the fluorescent probe thioflavin T. It is shown that the increase of the fluorescence intensity of ThT during the formation of amyloid fibrils is described by a sigmoidal curve, in which three areas can be distinguished: the lag phase, growth, and a plateau, which characterize the various stages of fibril formation. Despite the variation in the length of the lag phase at the same experimental conditions (pH and temperature), it is seen to drop during solution stirring and seeding. Data obtained by electron microscopy showed that the formed fibrils are long, linear filaments ∼20 nm in diameter. With increasing incubation time, the fibril diameter does not change, while the length increases to 2–3 μm, which is accompanied by a significant increase in the number of fibril aggregates. All the experimental data show that, irrespective of the kinetics of formation of amyloid fibrils, their properties after the completion of the fibrillation process are identical. The results of this work, together with the previous studies of insulin amyloid fibrils, may be important for clarification the mechanism of their formation, as well as for the treatment of amyloidosis associated with the aggregation of insulin.

The role of chromatoid bodies and cytoskeleton in differentiation of rat spermatozoids

An ultrastructural and immunocytochemical study of rat male germ cells at different stages of development has been carried out. Investigation of morphological changes of spermatogenic cells showed the presence of close associations between chromatoid bodies (CBs) and other cell organelles, particularly with the nucleus and Golgi apparatus. In addition, a connection of manchette noncentosomal microtubules (MTs) with spermatid perinuclear ring plasma membrane (PM) in the zone of adhesion intercellular contact, zonula adhaerens (ZA), was revealed. These results, as well as the available literary data, make it possible to analyze expected pathways of noncentrosomal MT nucleation in the late spermatids. It is possible to suggest that noncentorosomal MT are nucleated on the sites of perinuclear ring ZA. The immunocytochemical analysis revealed two novel proteins for these cells: BASP1 and MARCKS. It was shown that these proteins were present in CBs in early spermatids. During spermatozoid differentiation, these proteins are located along the outer dense fibers (ODFs) of the sperm tail. BASP1 and MARCKS are believed to be involved in the processes of calcium accumulation in CBs and ODFs. Calcium ions seem to play a significant role in RNA processing and protein synthesis in spermatids. Calcium is also necessary for sperm mobility defined mainly by ODFs.

Effect of red pigment on insulin fibril formation in vitro

The influence of red pigment isolated from yeast Saccharomyces cerevisiae and its low molecular weight derivate on insulin amyloid fibril formation in vitro was studied. The red pigment derivative, which presumably lacked phosphoribosyl moiety due to acid hydrolysis, retained the ability to inhibit fluorescence intensity (FI) of amyloid bound Thioflavine T. It was found that FI inhibition depended on the concentration of both pigment forms. Both forms were also able to compete with Thioflavine T for amyloid fibril binding. Electron microscopy revealed that fibrils reduced in size in the presence of red pigment.

Unusual Golgi Apparatus at the Proliferative Stage of Microsporidian Life Cycle

The Golgi apparatus is a multifunctional organelle of the eukaryotic cell [1, 2]. It is of great importance as a central link in the whole cytoplasmic secretory system responsible for effective processing, modification, and sorting of proteins and lipids. In the higher eukaryotes, a typical Golgi apparatus contains a stack of flattened cisternae with dilated rims. In ultrathin sections, arrays of vesicles can be found near these cisternae. The vesicles are commonly considered as elements of a complex tubulo-vesicular network. Each Golgi stack shows polarization: the cis-pole is associated with the endoplasmic reticulum (ER), while the condensed (secretory) vacuoles and lysosomes are situated near the trans-pole. A so-called medial-Golgi compartment is localized between the cisand trans-poles. The space between the ER and cis-Golgi is referred to as the intermediate compartment [1]. However, in some obligatory intracellular protozoan parasites ( Giardia, Plasmodium , and Toxoplasma ), the Golgi apparatus may be either incomplete in structure or contain an unusual set of constituents. Such a peculiar organization of their Golgi complexes may be associated with a small cellbody size of these eukaryotes and with a general decrease of their enzymatic and secretory systems as a result of adaptation to parasitism. Thus, minute unicellular parasitic eukaryotes may represent promising models for analyzing both the structure of the Golgi compartment itself and the simpliest cell machinery for secretion so far known for eukaryotes [3].

In situ electron microscopic detection of proteasomes in apoptotic U937 cells

75 Continuous protein synthesis and degradation in cells underlie the regulation of important cell func tions, such as cell cycle, transcription, and elimination of proteins damaged or incorrectly folded. According to current notions, there are two ways of degradation of cell proteins: (1) the lysosomal pathway, which pro vides hydrolysis of most membrane and extracellular proteins in the endomembrane vacuolar system con taining many acid proteases and (2) the ubiquitin– proteasome pathway implementing ATP dependent proteolysis of ubiquitinated proteins using protea somes [1–3]. Recently, the ubiquitin–proteasome sys tem has been thought to be of particular importance in cell functioning. The interest toward it is largely deter mined by its key role in neoplastic growth [1]. For example, a known substrate for proteasomes is the suppressor of cancer cell growth p53, which is involved in various cell processes, in particular, apoptosis induction in a number of hematopoietic lineages.

Localization of division protein FtsZ in Mycoplasma hominis

The localization of FtsZ protein in M. hominis cells was studied by immunoelectron microscopy with polyclonal antibodies to this protein. Cell polymorphism typical for mycoplasmas was seen on electron microscopic pictures. Among the diversity of cell shapes, we distinguished dumbbell-shaped dividing cells and cells connected with each other by membrane tubules (former constrictions). The label was predominantly observed in the constriction area of dividing M. hominis cells and on thin membrane tubules. A septum and the gold labeling of this structure have not been described before in mycoplasma cells. For the first time, in some rounded and oval cells, colloidal gold particles labeled the entire plasma membrane, probably marking a submembranous contractile ring (Z ring). These observations confirm the implication of FtsZ protein in M. hominis cytokinesis. In some cells, the spiral-like distribution of gold particles was observed. Most likely, FtsZ protofilaments in M. hominis cells form spiral structures similar to Z spirals in Bacillus subtilis and Escherichia coli. Their presence in mycoplasma cells may be considered to be an important argument in favor of Z ring assembly through the reorganization of Z spirals. FtsZ as a bacterial cytoskeleton protein binding with membrane directly or through intermediates may be engaged in maintenance of M. hominis cell shape.

GIANT VACUOLES ARISING DURING ADH-INDUCED TRANSCELLULAR BULK WATER FLOW ACROSS THE EPITHELIUM OF THE FROG URINARY BLADDER

Structural changes of the cytoplasm of urinary bladder granular cells after an antidiuretic hormone (ADH) stimulation of water transport were studied using standard and cryogenic methods of electron microscopy. Numerous changes occurred in these cells, the cytoplasm of the granular cells becoming swollen, and the intercellular spaces enlarged. Most granules become fused with the apical membrane. Under maximal ADH action, giant vacuoles appear in the cytoplasm of granular cells, in association with microfilaments and microtubules. Analysis of ultrastructure of the granular cells has established the origin of giant vacuoles from the cis ‐cisterna of the Golgi complex. A hypothesis based on the morphofunctional homology of giant vacuoles in granular cells with the contractile vacuoles of Protozoa is proposed in which the giant vacuoles (‘contractile‐like’ vacuoles) are seen as operating a osmoregulatory role in these cells. It is also proposed that microtubules and microfilaments participate in giant vacuole migration through the cytoplasm.

Ultrastructural and X-ray microanalysis of U-937 cells in hypertonia-induced apoptosis

The results of this study of the ultrastructural changes in U-937 cells in the apoptotic state are largely consistent with analogous data available in the literature. However, we also obtained original data on the ultrastructural changes in cellular organelles and immunocytochemical localization and distribution of proteasomes. It was shown that, when apoptosis was induced by incubation of cells in a hypertonic sucrose solution (200–400 mM), the Golgi apparatus (GA) was localized close to the plasma membrane. This fact is indirect evidence that cytoskeletal elements (particularly microtubules, which hold GA in the center of the cell) are depolymerized already at the early stages of apoptosis. At the late stages of apoptosis, the distance between individual GA cisterns significantly increases, which is apparently due to the cleavage of their binding proteins, golgins. Mitochondria in apoptotic cells do not change significantly. They have regularly spaced cristae and a fairly dense matrix containing single vacuoles. At all stages of apoptosis, the nucleus and cytoplasm of U-937 cells contained proteasomes, which were represented by small rod-shaped osmiophilic particles approximately 12 × 30 nm in size. These particles formed aggregates of varying density and size, not covered with a membrane. We assumed that they are similar to the “processing bodies,” or aggresomes, which were described in the literature. The latter are detected in cells under conditions of suppressed transcription in the nucleus and, according to some researchers, are involved in the storage and degradation of various mRNAs, RNPs, and proteins. X-ray spectral analysis revealed changes in the intracellular content of Na+ and K+ at the level of single cells during apoptosis induced by osmotic shock. An increased ratio of intracellular Na+/K+ compared to the control for the majority of cells in apoptosis was shown.

Functional linkage between the transport characteristics of the MDCK1 cell monolayer and their actin cytoskeleton organization

The dynamics of the actin cytoskeleton spatial organization and transepithelial electric resistance (TEER) in the MDCK1 cell monolayer exposed to arginine–vasopressin (AVP) and forskolin, a protein kinase A (PKA) activator, have been studied. These physiologically active substances are shown to depolymerize filamentous actin in MDCK1 cells (in both the apical and basal cytoplasm) and, concurrently, to considerably decrease the TEER of the cell monolayer. A decrease in TEER suggests an increase in the ion current through the cell monolayer. Correspondingly, the created ion gradient stimulates AVP-sensitive water flow. To clarify the routes of ions and water in MDCK monolayer, the localization of claudin-1 and -2 in tight junctions of ATCC (American Type Culture Collection) MDCK (a low TEER) and MDCK1 (a high TEER) cells was studied by immunofluorescence assay. Claudin-1 and -2 are detectable in the tight junctions of ATCC MDCK cells; however, the tight junctions of MDCK1 cells contain only claudin-1, whereas poreforming claudin-2 is absent. The exposure of MDCK1 cells to forskolin fails to change the distribution of the studied claudins, thereby suggesting that a decrease in TEER caused by forskolin is associated with a change in transcellular, rather than paracellular, permeability of the monolayer