Interaction of myelin basic protein and 2',3'-cyclic nucleotide phosphodiesterase with mitochondria.

The content and distribution of myelin basic protein (MBP) isoforms (17 and 21.5 kDa) as well as 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) were determined in mitochondrial fractions (myelin fraction, synaptic and nonsynaptic mitochondria) obtained after separation of brain mitochondria by Percoll density gradient. All the fractions could accumulate calcium, maintain membrane potential, and initiate the opening of the permeability transition pore (mPTP) in response to calcium overloading. Native mitochondria and structural contacts between membranes of myelin and mitochondria were found in the myelin fraction associated with brain mitochondria. Using Western blot, it was shown that addition of myelin fraction associated with brain mitochondria to the suspension of liver mitochondria can lead to binding of CNPase and MBP, present in the fraction with liver mitochondria under the conditions of both closed and opened mPTP. However, induction of mPTP opening in liver mitochondria was prevented in the presence of myelin fraction associated with brain mitochondria (Ca2+ release rate was decreased 1.5-fold, calcium retention time was doubled, and swelling amplitude was 2.8-fold reduced). These results indicate possible protective properties of MBP and CNPase.

[Inclusion of proteins into polyelectrolyte microcapsules by coprecipitation and adsorption].

In present study microcapsules composed of synthetic (PSS and PAA) and biodegradable (DS and PAr) polyelectrolytes on calcium carbonate microparticles were obtained. The ultrastructural organization of biodegradable microcapsules was studied using transmission electron microscopy. The envelope of such capsules consisting of six polyelectrolyte layers is already well-formed, having the average thickness of 44 ± 3.0 nm, and their internal polyelectrolyte matrix is sparser compared to the synthetic microcapsules. Spectroscopy was employed to evaluate the efficiency of incorporation of FITC-labeled BSA into synthetic microcapsules by adsorption, depending on the number of polyelectrolyte layers. It was shown that the maximal amount of protein incorporated into the capsules with 6 or 7 polyelectrolyte layers (4 and 2 pg/capsule, correspondingly). As a result we conclude that, in comparison with co-precipitation, the use of adsorption allows to completely avoid the loss of protein upon encapsulation.

Effect of dopamine on Ehrlich ascites carcinoma cells.

Morphological studies showed that daily intraperitoneal injections of dopamine in doses of 10(-2)and 10(-1)M down-regulates the general number of cells in the Ehrlich ascites carcinoma in 10 and 30 times and decreases their diameter by 27% and 59%, respectively (as compared to the control animals received physiological saline). According to ultrastructural data these injections were followed by the abnormal changes in microvilluses, forming the specific moire fringes in cytosol, thickening of cortical layer, and a significant increase in filament reticulum density (actin fibers) in tumor cells of treatment group specimens. We concluded that the oncocytotoxic effect of dopamine was related to the induced polymerization of cytosol actin.

[Ultrastructure of afferent synapses on the ventral dendrite of mauthner neurons after goldfish adaptation to optokinetic stimulation].

Using the morphometric techniques, the ultrastructural changes of the afferent synapses on the ventral dendrite of the Mauthner neurons (MNs) were studied after the adaptation of goldfish to long-term fatiguing sensory (visual) stimulation, characterized by the growth of MN resistance. It was shown that after the adaptation, the length of active zones (AZs) in the synapses located on the MN ventral dendrite was significantly reduced by 23%. At the same time, the length the AZs of the excitatory visual synapses was reduced by 29% in comparison with the control, while the length of desmosome-like contacts (DLCs) bordering AZs was increased by 71%. It was also found that the length of AZs in the inhibitory synapses was decreased by 19% after the adaptation, which is consistent with the important role of inhibitory processes in the sensory pathways during the memory formation. Taking into account the actin nature of the DLCs, the basis of the adaptation to the visual stimulation is suggested to be in the presynaptic mechanism of neurotransmitter secretion regulation by actin.

[Serotoninergic synapses on the ventral dendrite of the mauthner neuron (ultrastructural study with immunogold labeling)].

Using immunogold labeling, excitatory serotoninergic synapses of both chemical and mixed types, were found on the ventral dendrite (VD) of goldfish Mauthner neuron (MN).They are characterized by the presence of several mitochondria in the bouton and by an obligatory desmosome-like contact (DLC) besides the active zone (AZ). Their AZs were commonly found to make contact with the unlabeled chemical crested synapses, which, in turn, directly interacted with VD. These synapses were practically devoid of mitochondria and had no DLCs, thus allowing to identify them as the inhibitory ones. This "two-level" organization of excitatory serotoninergic and inhibitory synapses appears to be related to the reciprocal mechanism of the regulation of MN functional activity by visual input.

[Distribution of proteins inside polyelectrolyte microcapsules depend on pH of medium].

Distribution of bovine serum albumin and ferritin inside polyelectrolyte microcapsules was studied by transmission electron and confocal microscopy at the pH range 2-5. It was estimate that protein's distribution depends on isoelectric point (pI) and first polyelectrolyte used for preparation of capsule shell. So peptide is placed in the bulk of capsule if pH values of medium are lower isoelectric point of protein and polycation was used as a first polyelectrolyte layer. If the first polyelectrolyte was polyanion, the protein is located near internal surface of the shell. The protein is situated near internal surface of the shell for both polyelectrolytes when pH is equal to pI.

[Dopamine as a possible substance for oncotherapy and for quantitative evaluation of cytosolic G-actin].

Viability, histology and ultrastructure of normal cells and cells of different degrees of malignancy after interaction with dopamine as well as the ability of these cells and isolated G-actin in model experiments to stain by Falck technique were studied. It is shown that dopamine, virtually having no effect on the viability of the "normal" non-tumorigenic transformed cells, noticeably reduces cell viability of slightly tumorigenic cells, causes a significant reduction in viability of attachable cancerous cells and a very significant decrease in cell viability of cancerous cells growing in suspension. The intensity of fluorescence of the cytosole in cells treated with dopamine, has been very high and varied in different cultures, and that of isolated actin directly depended on its concentration. Common to all cell morphological feature of damage from the action of dopamine and the putative substrate of fluorescence was actodopamine filaments network strands (identified on the structure and size), which appears in the cytosole loci, where they were absent in control. The data show that dopamine can be used as an oncotherapeutic remedy and diagnostic tool interacting with G-actin as a cellular target.

[Induction of neuron morphological resistance to beta-amyloid].

The effect of training adaptive vestibular stimulations on goldfish Mauthner neurons (MN) function and three-dimensional morphology was studied in experimental amyloidosis caused by application of aggregated beta-amyloid protein (Abeta25-35). It was found that as compared with control, adapted (trained) MN gained significant resistance to Abeta25-35. Taking into consideration the key role of dopamine in MN adaptation to sensory stimulations, its effect on the development of model amyloidosis was studied. It was shown that the application of dopamine onto MN as well as the increase of its concentration in brain by means of L-dopa protected MN structure and function against pathogenic influence of Abeta25-35. Using electron microscopy it was shown that dopamine protective action on neurons was due to its ability to dissociate polymer amyloid molecules into short inactive fragments.

[Morphological changes of THP-1 tumor cells exposed to dopamine in vitro].

The effect of dopamine (DA) on the viability and morphology of cultured tumor THP-1 cells (human acute monocytic leukemia) was studied. DA in concentration of 10(-5) M had virtually no effect on the culture, while in concentration of 10(-4) M to 10(-3) M it stopped the growth and caused a sharp increase in cell death after 24 and 48 hours. Incubation with DA reduced the cell diameter, progressively increased their vacuolization and intensity of fluorescence after treatment by Falck method. Electron microscopical study has shown that cells exposed for 1 day to DA in the concentrations starting with 10(-4) M, demonstrated smoothing of their surface with the disappearance of microvilli and clasmatosis vesicles, actin filaments perforating the plasma membrane, the emergence of an increasingly dense network of filaments in the cytosole and karyoplasm and, finally, apoptotic cell death. It is suggested that the oncotherapeutic cellular target for DA is a cytosolic G-actin, which at a certain DA concentration, turns into filaments that damage the cells, break the cell cycle and cause cell death.

[Morphological bases of dopamine effect on HEP-2 tumor cells viability].

The purpose of the present investigation was to study the morpho-functional organization of a classical object of cytological research - cultured HEp-2 tumor cells, using dopamine as a penetrating agent, inducing the polymerization of cytosolic actin. It was demonstrated that dopamine introduced into the incubation medium reduced viability and caused morphological disturbances of cultured HEp-2 cells; these effects were proportional to dopamine concentrations (1.0 x 10(-4) M to 1.0 x 10(-3) M) and exposure duration (2 to 3 days). These cells, according to ultrastructural data, underwent fusion and lysis because of the appearance of actin filaments network in the loci of globular actin prevalence in control cells. Dopamine receptors had no effect on cytotoxic effect of dopamine. This was indicated by fluorescent microscopical evidence of dopamine penetration into experimental cells in the presence of haloperidol, as well as destruction of HEp-2 cells under the action of pyrimidinethione, similar to dopamine by characteristics, but lacking its own receptors. It is suggested that cytoplasmic target for dopamine is globular actin and that induced polymerization of this cytoskeletal protein caused injury to tumour cells.

[Ultrastructure of Mauthner neurons after optokinetic stimulation and eye enucleation].

It was previously shown that the contralateral (relative to preferred side of turns) optokinetic stimulation and ipsilateral eye enucleation cause a significant, 2- to 4-fold reduction of the ventral dendrite (VD) volume in one of two goldfish Mauthner neurons (MN) that becomes more active functionally. In this study, we investigated the MN ultrastructure after mentioned unilateral visual effects. In both cases, devastation of the afferent synapses was detected along the full length of the reduced VD, with simultaneous compaction of its cytoskeleton, in contrast to those of VD of the contralateral MN and of lateral dendrites and cell bodies of both neurons. It is suggested that the depleted synapses belong to the excitatory visual afferent input, and both cytoskeletal and synaptic mechanisms are involved in the regulation of MN functional activity through VD.

Effect of dopamine on viability of BHK-21 cells.

We studied the effects of dopamine added to culture medium on survival of floating or adherent BHK-21 cells differing by organization of actin cytoskeleton. The viability of floating cells more drastically decreased with increasing dopamine concentration and duration of exposure than that of adherent cells. The cells worse adhered to the substrate and formed a monolayer. The formed monolayer degrades, cell borders become blurred, cells, polygonal in the control, are rounded. Preliminary blockade of dopamine receptors with haloperidol, inessential for cell survival and morphology, does not prevent the destructive effect of dopamine on the cells. Ultrastructural study revealed increased density of filamentous actin threads in deep compartments of cell cytoplasm after dopamine treatment, this increase being more pronounced in cells grown in suspension. Bearing in mind the polymerizing effect of dopamine on globular actin in vitro and the fact that the content of this protein in floating cells is higher than in adherent cells, we can conclude that the decrease in viability of BHK-21 cells is caused by interaction of dopamine with cytoplasmic globular actin.

[Localization in a cell of a protein forming the ATP-dependent potassium-selective channels in the bilayer lipid membrane. An ultrastructural study].

The localization in the cell of the protein forming the ATP-dependent potassium-selective channels in the bilayer lipid membrane has been studied. The electron microscope investigation of rat liver and heart tissue sections after their incubation with Abs against this protein and the visualization of the protein with secondary Abs conjugated with colloid gold were carried out. Colloid gold particles were observed both in mitochondrial membranes and in membranes of endoplasmic and sarcoplasmic reticulum. In heart mitochondria, these particles were significantly greater than in liver mitochondria. The localization of the channel protein both in mitochondria and reticulum, as well as the structural similarity between the mitochondrial channel and the precursor of calreticulin suggest that the channel protein belongs to the family of calreticulins. The possible function of the protein as a channel subunit of the mitochondrial ATP-dependent potassium channel is discussed.

[Cytoskeletal regulation of the cellular function by dopamine].

The interaction of dopamine with model membranes, isolated G-actin, and living cells, such as Mauthner neurons and fibroblast-like BHK-21 cells has been studied. It was found that in vitro dopamine passes through the phospholipid membrane and directly polymerizes G-actin due to incorporation into threads as their integral part. In in vivo conditions, it penetrates inside the cell and induces the appearance of a network of actin filaments in loci rich in globular actin. The data suggest that there exists a mechanism of dopamine interaction with living cells, which is based on direct polymerization of cytosolic G-actin as its cellular target. The reorganization of the actin cytoskeleton leads to changes in the morphofunctional status of cells.

[Correlation between the sizes of the individual parts of goldfish Mauthner neuron and its integral function after eye enucleation].

Using the method of 3D reconstruction, the structural correlates of significantly increased functional activity of denervated Mauthner neuron (MN) were studied after the unilateral eye enucleation, that resulted in the irreversible shift of the goldfish motor asymmetry to a "blind" side. It was established that in some cases the functional dominance of MN was significantly correlated with the reduction of the volume of its ventral dendrite, while in the other cases it was correlated with the increase in sizes of its soma and the lateral dendrite. Both structural features, probably, were caused by local redistribution of the neurotransmitters due to the stress of sensory inputs that remained undamaged. Thus, it was demonstrated that the prolonged adaptive changes in goldfish behavior could be regulated by means of specific morphological reorganizations of MN at the level of their individual dendrites by the principle of feedback or feedforward mechanisms.

[Cytochemical and ultrastructural characteristic of BHK-21 cells exposed to dopamine].

BHK-21 cells were incubated in a medium containing dopamine (DA) and then their catecholamine content evaluated by using the Falck cytochemical method. The significant intensification of cell fluorescence as compared to that one in control preparations was detected; this effect was proportional to DA concentration and exposure duration and was more pronounced in cells in suspension than in those attached to the substrate. Simultaneous ultrastructural investigation has shown that an increased intensity of the cytoplasm fluorescence correlated with the appearance of the dense network of fibrils that were morphologically identified as F-actin microfilaments. Prior blockade of dopaminergic receptors by haloperidol did not change the following DA effect both on the fluorescence intensity and cell ultrastructure. The data obtained suggest that DA chronically acting on the living cells was able to penetrate into the cytoplasm, causing actin polymerization and incorporating into the newly formed actin cytoskeleton. Structurally, this may be manifested by cytoskeleton and its derivative hypertrophy, that could have a substantial effect on general morphology of the cell.

[The influence of dopamine on the ultrastructure of BHK-21 cells].

The influence of dopamine on the haloperidol of BHK-21 cells being in suspension or attached to substrate was investigated. It was shown that the ultrastructural changes affected mainly the cellular loci enriched by the cytoskeleton actin such as intercellular desmosome-like contacts, microvilli and cortical layer or mesh just beneath the plasmatic membrane. The desmosome-like contacts were hypertrophied, their electron density was increased and fibrilar bridges appeared in specialized contacts. Many microvilli fused with each other and with plasma membrane of the neighboring cells, or, on the contrary, split up. Frequently, the membrane surface between microvilli and particularly their apical parts was seen to be pierced by thin thread, morphologically similar to actin filaments. The cytoplasmatic matrix onto ultrathin sections had blotched appearance and at the ultrastructural level was represented by numerous randomly oriented actin filaments. The effect of dopamine was more pronounced in the BHK-21 cells when being in suspension than in attached to the substrate ones, which presumably occurred due to known lesser differentiation of the cytoskeleton in the formers. Finally, it was established that the preliminary blockade of cellular D2 receptors with haloperidol neither affected the ultrastructure of BHK-21 cells nor prevented the following effect of dopamine. The data obtained suggest the direct interactions of dopamine with the actin cytoskeleton.

[Stabilization of mauthner neuron structure in goldfish adapted to the contralateral optokinetic stimulation].

Previously we have demonstrated, that the contralateral optokinetic (visual) stimulation (COS) induces an inversion of goldfish motor asymmetry and three-fold decrease of the ventral dendrite of ipsilateral Mauthner neuron (MN) volume, whereas repetitive in training mode daily COS sessions induced a motor behavior resistance to this influence in fish. In the present investigation we have examined the effect of the training COS sessions on the structure of MN and their parts. It was found that daily visual training stabilizes the size of the ventral dendrites, significantly increasing their resistance to single prolonged COS. Thus, the adapted morpho-functional state was induced in the individual dendrite and in the whole neuron by repetitive stimulation of that dendrite. These results make it possible to investigate in more detail the role of an individual dendrite in the modification of functional activity of the whole neuron and in adaptation and memory mechanisms at the cellular level.

[Morpho-functional changes in the goldfish Mauthner neurons after beta-amyloid application].

The influence of aggregated beta-amyloid peptide fragment 25-35 application on three-dimensional structure and volume of Mauthner cells (MCs), as determined by reconstruction from serial histological sections, and on goldfish motor asymmetry was studied. It was shown that in intact and control goldfish motor asymmetry was stable and strongly correlated with structural asymmetry of neurons. But under the influence of beta-amyloid, motor asymmetry appeared to be strongly changed or inverted, did not correlate with structural asymmetry and frequently even was opposite to it. This resulted from strong dystrophy or, on the contrary, hypertrophy of individual neurons and their separate dendrites with the change in the proportions between them. It is suggested that injurious effect of beta-amyloid peptide on MCs structure, discordant with ("irregular") fish behavior, could be the result of mechanical deformation, induced by ribbon-like fibrils of amyloid peptide. These findings collectively suggest that MCs are the adequate object for the study of the structural aspects of amyloidosis.

Changes in the ultrastructure and function of goldfish Mauthner neurons in the presence of 3,4-dihydro-2(1H)-pyrimidinethione.

The behavioral effects of 3,4-dihydro-2(1H)-pyrimidinethione (DPT, a pyrimidine derivative), which is used as a test system for detecting tumor growth, on the ultrastructure and function of Mauthner neurons (MN), were studied in goldfish. Application of DPT to MN was found to lead to increased resistance of neurons to exhaustive stimulation, which was accompanied by increases in the sizes of actin-containing membrane desmosome-like contacts, along with the formation of bundles of actin stress fibers; these effects are similar to those previously reported with dopamine. The similarity of the morphofunctional changes in MN on exposure to an artificial chemical substance for which there are no membrane receptors and dopamine itself suggests that they have trophic effects on the stabilization and polymerization of cytoskeletal actin due to direct penetration into postsynaptic neurons.