[Intracellular localization of XCAP-E protein in XL2 (Xenopus laevis) cells under normal conditions and during inhibition of pRNA transcription and processing]. / Issledovanie vnutrikletochnoi lokalizatsii belka XCAP-E v kletkakh linii XL2 (Xenopus laevis) v norme i pri ingibirovanii transkriptsii i protsessinga pRNK

The interaction of condensin subunit XCAP-E with various nucleolar subcompartments in XL2 cells was studied. In the interphase cells, XCAP-E was associated with a granular component of nucleoli (as shown by double staining with antibodies against B23) and with small nucleolus-like structures in the nucleoplasm. Inhibition of transcription by actinomycin D does not disrupt interaction of XCAP-E with the granular compartment of segregated nucleoli. Treatment with DRB 5,6-dichloro-1 beta-ribofuranozide-benzimidazole causes disintegration of nucleolar fibrillar complexes, but does not affect nucleolar localization of XCAP-E. The data suggest that nucleolar association of XCAP-E is independent on the functional state of the nucleolus, and imply a possible role of this protein in rRNA processing and pre-fibosome assembly.

[Induction of nuclear envelope formation around individual chromosomes under impact of hypotonic shock]. / Induktsiia formirovaniia iadernoi obolochki vokrug individual'nykh khromosom pod deistviem gipotonicheskogo shoka.

In the present work we have studied the distribution of some proteins participating in the nuclear envelope assembly (lamins A/C, B and LAP2 alpha) in mitotic cells and after hypotonic treatment with 15% Hank's solution. In untreated cells, these proteins are localized in the nuclei of interphase cells migrate to the cytoplasm during mitosis. Hypotonic treatment of interphase, prophase and telophase cells does not lead to considerable relocalization of lamins A/C and B. However, unlike normal mitosis, in prometaphase and metaphase cells their chromosomes acquire affinity to lamins and LAP2 alpha. Comparative analysis of lamins and LAP2 alpha distribution have revealed that chromosomes have special sites for binding with different proteins.

[Intracellular localization of XCAP-E and pEg7 condensins in normal mitosis and after the treatment inducing artificial changes in structural organization of mitotic chromosomes]. / Vnutrikletochnaia lokalizatsiia kondensinov XCAP-E i pEg7 v norme i pri vozdeistviiakh, vyzyvaiushchikh iskusstvennoe izmenenie strukturnogo sostoiianiia mitoticheskikh khromosom.

Function of condensin subunits XCAP-E and pEg7 (XCAP-D2) in the formation and maintaining of special organization of mitotic chromosomes has been studied in Xenopus laevis cells (XL-2). The experimental conditions involved blocking chromosomes being in the condensed state in cells treated by cytostatics, or during their reversible artificial decondensation. The latter was induced by incubation of living cells in hypotonic medium. In extensively mollen chromosomes, XCAP-E and pEg7, remained associated with axial regions of chromosomes. In contrast, upon adaptation of cells to hypotonic conditions and recondensation of chromosomes to nearly initial state, both proteins dissociated from chromosomes into the cytoplasm. In K-mitotic cells, after a 3-6 h treatment with nocodazole or taxol, considerable dissociation of XCAP-E and pEg7 from chromosomes was observed without significant changes in overall level of chromosome compactization. Taken together the data suggested that condensins play no important role in maintaining mitotic chromosomes being in condensed state. Rather, it seems probable that mitotic function of condensins may be associated either with the formation of the higher order chromosome structure, and/or segregation of sister chromatids, the processes being tightly linked with chromosome compactization. This paper is in memory of Professor Katherine Le Guellec of Rennes-1 University, who left us in June 2001. Professor Le Guellec initiated this work in Rennes and offered all the possible help that this work be continued in Moscow University. Let the memory of Katherine, a great scientist and sympathetic friend, live for ever in ours hearts.

[Characteristics of Actinomycin D-induced segregation of a "noncanonical" nucleolus of the sea urchin Paracentrotus lividus]. / Osobennosti segregatsii "nekanonicheskogo" iadryshka ootsitov morskogo ezha Paracentrotus lividus, indytsirovannoi deistviem Aktinomitsina D.

The structure of a "noncanonical" nucleolus of vitellogenic oocytes in the sea urchin Paracentrotus lividus was studied using the inhibitor of transcription actinomycin D. In the control cells, the nucleolus consists of two separated structural subdomains: the dense fibrillar-granular peripheral area and the fibrillar central area. The nucleolus did not contain subdomains corresponding to the fibrillar center and dense fibrillar component of "typical" nucleoli. After treatment with actinomycin D, numerous argyrophilic granules appeared in the karyoplasm, the intranucleolar DNA became compact, and the nucleolar material was segregated into two or three separated zones, the residual peripheral area being the densest and largest. Lesser zones had a decreased electron density and contained argyrophilic proteins and, apparently, the nucleolar organizer material. These results suggest that, for normal rRNA expression and processing, the presence of structural subdomains in the nucleolus, such as fibrillar complexes and a dense fibrillar component, is not essential.

[Features of interactions of p68 anchorosomal protein with the nuclear envelope in the cell cycle]. / Osobennosti vzaimodeistviia belka ankorosom p68 s iadernoi obolochkoi v kletochnom tsikle.

Chromatin associated with the nuclear envelope appears in the interphase nuclei as a layer of anchorosomes, granules 20-25 nm in diameter. The fraction of chromatin directly associated with the nuclear envelope is resistant to decondensation, shows a low level of DNA methylation, and contains specific acid-soluble proteins. However, mechanisms underlying the interaction of chromatin with the nuclear envelope are not fully understood. Specifically, it is not known whether anchorosomes are permanent structures or if they undergo reversible disassembly during mitosis, when contacts between chromatin and the nuclear envelope are destroyed. We obtained immune serum recognizing a 68 kDa protein from the nuclear envelopes fraction and studied the localization of this protein in interphase and mitotic cells. We show that this protein present in the NE/anchorosomal fraction does not remain bound with chromosomes during mitosis. It dissociates from chromosomes at the beginning of the prophase and then can be identified again at the periphery of the newly forming nuclei in the telophase.

Identification of microtubule-organizing centers in interphase melanophores of Xenopus laevis larvae in vivo.

The morphological characteristics of microtubule-organizing centers (MTOCs) in dermal interphase melanophores of Xenopus laevis larvae in vivo at 51-53 stages of development has been studied using immunostained semi-thick sections by fluorescent microscopy combined with computer image analysis. Computer image analysis of melanophores with aggregated and dispersed pigment granules, stained with the antibodies against the centrosome-specific component (CTR210) and tubulin, has revealed the presence of one main focus of microtubule convergence in the cell body, which coincides with the localization of the centrosome-specific antigen. An electron microscopy of those melanophores has shown that aggregation or dispersion of melanosomes is accompanied by changes in the morphological arrangement of the MTOC/centrosome. The centrosome in melanophores with dispersed pigment exhibits a conventional organization, and their melanosomes are situated in an immediate vicinity of the centrioles. In melanophores with aggregated pigment, MTOC is characterized by a three-zonal organization: the centrosome with centrioles, the centrosphere, and an outlying radial arrangement of microtubules and their associated inclusions. The centrosome in interphase melanophores is presumed to contain a pair of centrioles or numerous centrioles. Because of an inability of detecting additional MTOCs, it has been considered that an active MTOC in interphase melanophores of X. laevis is the centrosome. We assume that remaining intact microtubules in the cytoplasmic processes of mitotic melanophores (Rubina et al., 1999) derive either from the aster or the centrosome active at the interphase.

The use of internalizable derivatives of chlorin E6 for increasing its photosensitizing activity.

Experiments with human hepatoma PLC/PRF/5 cells and human embryo skin fibroblasts involving the use of three different tests (colony formation, Trypan blue exclusion, labeled thymidine incorporation) have demonstrated a significantly higher photosensitizing activity of chlorin e6 conjugates with internalizable ligands as compared to that of chlorin e6 itself. Receptor-mediated internalization of chlorin e6 conjugates ensures a greater photosensitization of cells than binding of those conjugates to cell surface receptors. The suitability of such conjugates that permit the delivery of a photosensitizer to sensitive intracellular targets is discussed.

[Effects of remantadine on fusion of lipid membranes of influenza A virus with plasma and internal membranes of lymphoblastic cells]. / Deistvie remantadina na sliianie lipidnoi obolochki virusa grippa A s plazmaticheskimi i vnutrennimi membranami v limfoblastoidnykh kletkakh.

The method of fluorescence dequenching was used to study the interaction between influenza virus A/Krasnodar/101/59 and Namalwa and Raji lymphoblastoid cells. Experiments with endocytosis inhibitors and fluorescence quenchers have shown that at pH = 5.0 the virus lipid envelopes are fused with the plasma membranes of the cells, and at pH = 7.4 the virus lipid envelopes are fused with the internal, presumably endosomal, membranes of the cells. Remantadine at a concentration of 50-1000 micrograms/ml did not influence the fusion of virus lipid envelopes with intracellular membranes at pH = 7.4 whereas at pH = 5.0 it inhibited, beginning from 25 micrograms/ml concentration, the fusion of virus lipid envelopes with the plasma membranes of cells.

[Photodynamic effects of a concanavalin A-chlorin e6 conjugate on human fibroblasts]. / Fotodinamicheskoe deistvie kon"iugata konkanavalina A s khlorinom e6 na fibroblasty cheloveka.

To minimize the side effect of porphyrin photosensitizers and to reduce their active concentration, chlorine e6 was conjugated with concanavalin A. Photodynamic action of chlorine e6 and concanavalin A-chlorine e6 conjugate has been studied in human skin embryonic fibroblasts. The conjugate appeared to be 5 times more effective as compared to chlorine e6 due to concanavalin A-chlorine e6 endocytosis into intracellular compartments.

Volume-dependent regulation of cation transport and polyphosphoinositide metabolism in human and rat erythrocytes: features revealed in primary hypertension.

The rate of delta mu H+ --induced erythrocyte Na+/H+ exchange is increased in both patients with essential hypertension (EH) and spontaneously hypertensive rats (SHR). The increase of Na+,K(+)-cotransport was revealed in erythrocytes of SHR only. This alteration as well as a decrease of mean cell volume were observed in both young and old erythrocytes of SHR. The moderate shrinkage of rat (but not human) erythrocytes results in an increase of the rate of Na+,K(+)-cotransport. The more pronounced shrinkage of rat (but not human) erythrocytes induces the Na+/H+ exchange. These reactions are accompanied by phosphoinositide response. Activator of protein kinase C (TPA) increases delta mu H+ --induced Na+/H+ exchange both in human and rat erythrocytes but it does not modify phosphoinositide metabolism. No differences were observed in the rate of Na+/H+ exchange between TPA-treated erythrocytes of SHR and WKY. We assume that the activation of protein kinase C increases Na+/H+ exchange in primary hypertension. Increased Na+/H(+)-cotransport revealed in an experimental model of primary hypertension is probably due to the decrease of erythrocyte volume.

Volume-dependent regulation of ion transport and membrane phosphorylation in human and rat erythrocytes.

Osmotic swelling of human and rat erythrocytes does not induce regulatory volume decrease. Regulatory volume increase was observed in shrunken erythrocytes of rats only. This reaction was blocked by the inhibitors of Na+/H+ exchange. Cytoplasmic acidification in erythrocytes of both species increases the amiloride-inhibited component of 22Na influx by five- to eight-fold. Both the osmotic and isosmotic shrinkage of rat erythrocytes results in the 10- to 30-fold increase of amiloride-inhibited 22Na influx and a two-fold increase of furosemide-inhibited 86Rb influx. We failed to indicate any significant changes of these ion transport systems in shrunken human erythrocytes. The shrinking of quin 2-loaded human and rat erythrocytes results in the two- to threefold increase of the rate of 45Ca influx, which is completely blocked by amiloride. The dependence of volume-induced 22Na influx in rat erythrocytes and 45Ca influx in human erythrocytes on amiloride concentration does not differ. The rate of 45Ca influx in resealed ghosts was reduced by one order of magnitude when intravesicular potassium and sodium were replaced by choline. It is assumed that the erythrocyte shrinkage increases the rate of a nonselective Cao2+/(Nai+, Ki+) exchange. Erythrocyte shrinking does not induce significant phosphorylation of membrane protein but increases the 32P incorporation in diphosphoinositides. The effect of shrinkage on the 32P labeling of phosphoinositides is diminished after addition of amiloride. It is assumed that volume-induced phosphoinositide response plays an essential role in the mechanism of the activation of transmembrane ion movements.

[Erythrocyte membrane phosphorylation in rats with spontaneous hypertension as affected by protein kinase C activator]. / Fosforilirovanie membrany éritrotsitov krys so spontannoi gipertenziei pri deistvii aktivatora proteinkinazy C.

A study of orthophosphate incorporation into erythrocyte membrane proteins and lipids of spontaneously-hypertensive rats (SHR) at incubation of erythrocytes with 32PO4 and that of erythrocyte ghosts with 32P-gamma-ATP showed basal phosphorylation of proteins as well as polyphosphoinositides of erythrocyte ghosts to be essentially increased in SHRs as compared to normotensive animals, the differences being eliminated by the addition of protein kinase C activator (4 beta-phorbol-12 beta-myristate-13 alpha-acetate, PMA). After PMA-stimulated protein phosphorylation most of the label was incorporated into proteins of Band 4.1. There was no difference in basal phosphorylation of membrane proteins in case of intact erythrocytes. Under PMA stimulation, most of the label was incorporated into proteins of band 4.1 similarly to labelling of erythrocyte ghosts and a significant increment of radioactivity could only be seen in the control animals.

Investigation of membrane proteins in rat erythrocytes in spontaneous hypertension by means of spin-label technique.

It has been suggested that alterations in cell membrane proteins may play a role in changes of erythrocyte membrane structure and function in hypertension. In order to characterize the structure of membrane proteins of erythrocytes from spontaneously hypertensive rats (SHR) the spin-label technique with a maleimide spin-label was used. A significant difference was observed in the characteristic electron-spin resonance (e.s.r.) spectrum of the label between samples from normotensive rats and SHR. The difference was eliminated and the spectrum significantly changed after treatment of the labelled membrane with EDTA followed by washing out the EDTA extracts, whereas the same treatment with EDTA without the following washing had no effect on the e.s.r. spectrum. It is concluded that the EDTA extracts different substances in the different rat groups. The spin-label technique is a useful method for distinguishing cell membrane properties in SHR and normotensive rats.

[The role of free calmodulin in the regulation of calcium-pump activity in erythrocytes]. / Ob uchastii svobodnoi formy kal'modulina v reguliatsii aktivnosti Ca-nasosa éritrotsitov.

The activity of Ca-pump in inside-out oriented vesicles obtained from erythrocyte membranes after their 30 min treatment with EGTA at 20 degrees C (membranes A) and 37 degrees C (membranes B) was investigated. It was shown that in membranes A placed into an incubation medium containing 0.1 mM EGTA (pH 7.4) the overall effect of exogenous calmodulin is due to the increase in the maximal activity of the enzyme, its affinity for Ca2+ being unaffected thereby. In membranes B placed into the same medium (pH 6.75) the activation of the Ca-pump by calmodulin is due to the increased affinity for Ca2+ at a constant maximal activity of the enzyme. The dependencies of the value of the calmodulin-stimulated component of membranes A and the Ca2+-binding capacity of calmodulin measured by the intensity of N-phenyl-1-naphthylamine fluorescence on the concentration of free Ca2+ are coincident. In the case of membranes B, the stimulation of Ca-pump by calmodulin occurs at much lower Ca2+ concentrations than the Ca2+ binding-induced conformational shifts in calmodulin. The experimental results suggest that the affinity of the Ca-pump for Ca2+ may affect calmodulin existing in a Ca2+-independent state. The hydrophobic interactions between the Ca-calmodulin complex and the Ca-ATPase molecule are apparently essential for the regulation of the maximal enzyme activity.

[Study of the structure of erythrocyte membrane in spontaneous genetic hypertension using the spin-label method]. / Izuchenie struktury membranyéritrotsitov pri spontannoi geneticheskoi gipertenzii metodom spinovykh metok.

An analysis of ESR spectra of maleimid spin-labeled erythrocyte membranes of spontaneously hypertensive rats of SHR line and normotensive rats of the control line WKY showed differences in the structure of membrane proteins in the norm and pathology. These differences were compared with the differences between the erythrocyte membranes of SHR and WKY, found earlier by fluorescent probe method. An important role of membrane peripheric proteins in the appearance of the above differences in suggested.

Microcalorimetry and electrophoresis of the erythrocyte membrane of spontaneously hypertensive rats.

Abnormalities of both structure and function have been described in the erythrocyte membrane of spontaneously hypertensive rats (SHR). In order to elucidate the molecular basis of these abnormalities we have carried out differential scanning microcalorimetry of the erythrocyte membrane and gel electrophoresis of membrane polypeptides. The partial enthalpy of so-called 'C-transition' (at 63 degrees C) was found to be increased. This may be explained by increased content of band 3 protein in SHR erythrocyte membrane.

Calcium transport and protein content in cell plasma membranes of spontaneously hypertensive rats.

In the absence of depolarizing agents, 45Ca uptake by synaptosomes of the brain tissue of spontaneously hypertensive rats (SHR) was 40% greater than in normotensive controls. This difference disappeared after membrane treatment with depolarizing agents or with the addition of the Ca channel blocker verapamil. Increased 45Ca uptake was also observed in isolated platelets of SHR. It can be assumed that in both cases these differences were caused by partial depolarization of the plasma membrane as a result of its increased permeability to Na+. The addition of calmodulin to microsomal fractions of SHR brain tissue resulted in a considerably diminished increase in ATP-dependent calcium accumulation. These data are in accord with previous findings obtained in a study on the erythrocyte membrane Ca pump. Data obtained by means of differential scanning microcalorimetry of erythrocyte membranes and gel electrophoresis of membrane proteins indicated a higher band-3 protein content in the erythrocyte membranes of SHR. These data support the concept of primary hypertension as a type of generalized membrane pathology.

[Physico-chemical properties of the erythrocyte membranes of normotensive and spontaneously hypertensive rats]. / Issledovanie fiziko-khimicheskikh svoistv membrany éritrotsitov normotenzivnykh i spontanno gipertenzivnykh krys.

To detect the substrate of molecular alterations in a plasma membrane, found earlier to be typical of chronic hypertension, microcalorimetric study of erythrocyte membranes of spontaneously hypertensive rats was performed. It was found that characteristics of some irreversible therminal transitions in the membranes differ from the corresponding control values. The supposed predominant role of membrane protein denaturation in these transitions is confirmed by the analysis of temperature-dependent infrared spectra, spectra of membrane protein fluorescence and circular dichroism.