Ya. Yu. Komissarchik

Comparative analysis of SGLT1 and GLUT2 transporters distribution in rat small-intestine enterocytes and Caco-2 cells during hexose absorption.

The distribution of SGLT1 and GLUT2 hexose transporters has been evaluated in enterocytes of an isolated loop of the small intestine and Caco-2 cell culture after absorption of hexoses at their high and low concentrations. The SGLT1 transporter was found to be located in enterocytes along the edge of the intestinal villus. The GLUT2 transporter after loading with high hexose concentrations is located in the apical part of enterocytes. In culture, Caco-2 cells form a characteristic of enterocytes microvilli and the cell junction complex. During the incubation of the culture in solutions of glucose and galactose, the absorption of these sugars from the incubation medium was observed. The SGLT1 transporter in the Caco-2 cells is located in the apical and perinuclear enterocyte parts and is organized in globules. After loading with hexoses at low concentrations, the GLUT2 transporter is in the basal cell area. The Caco-2 cell culture can serve a model for studying the transport of sugar in the intestinal epithelium.

Probing for Actinase Activity of Protealysin

The ability of protealysin, a thermolysin-like metallopeptidase from Serratia proteamaculans 94, to cleave actin and matrix metalloprotease MMP2 is reported. In globular actin, protealysin and S. proteamaculans 94 cell extracts are shown to hydrolyze the Gly42-Val43 peptide bond within the DNase-binding loop and the Gly63-Ile64 and Thr66-Ile67 peptide bonds within the nucleotide cleft of the molecule. At enzyme/substrate mass ratio of 1: 50 and below, a 36 kDa-fragment produced by the cleavage between Gly42 and Val43 was virtually resistant to further breakdown. Judging from the results of zymography, protealysin transforms proMMP2 into a 66 kDa polypeptide characteristic of mature MMP2, indicating that protealysin can activate MMP2. Upon incubation of S. proteamaculans 94 with human larynx carcinoma Hep-2 cells intracellular bacteria were detected in about 10% of Hep-2 cells, this being the first evidence for invasion of eukaryotic cells with bacteria of this species. Thus, S. proteamaculans 94 turned out to be one more bacterial strain in which synthesis of actin-specific metalloprotease is coupled with bacterial invasion. These results are consistent with the idea of the actinase activity of bacterial metalloproteases being a factor that may promote bacterial invasion of eukaryotic cells.

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.

Caco2 cell culture as an intestinal epithelium model to study hexose transport

The distribution of SGLT1 and GLUT2 hexose transporters, fibrillar actin, and tight junction proteins, as well as glucose absorption, have been considered in Caco2 cell cultures incubated in solutions with different hexose concentrations. Fibrillar actin is concentrated on microvilli closely to a tight junction. The actin distribution does not depend on glucose concentration. There is no SGLT1 association with brush border actin, and the transporter localization does not depend on hexose concentration. GLUT2 is localized in the basal part of Caco2 cells loaded with a low hexose concentration (2.5 mM). The transporter is colocalized with microvilli actin in the apical part of the cells loaded with a high hexose concentration (25 mM). The tight junction proteins occludin and claudin 1, 3, and 4 do not depend on glucose concentration. Claudin 2 protein was not revealed in Caco2 cells. Caco2 cell culture is a suitable model for studying hexose transport in small intestine epithelium.

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.

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.