Assessment of Autophagy in Tumor Cells of Human Skin Melanoma of Different Stages.

High levels of autophagy can increase the viability of tumor cells as well as their resistance to chemotherapy. Evaluation of the dynamics of autophagy processes at different stages of carcinogenesis can extend our understanding of melanoma pathogenesis to develop new therapeutic approaches. We performed a comparative study of tumor cell autophagy in stages II and III human skin melanoma. Tumor cells were characterized by high content of structures associated with autophagy (autophagosomes and autolysosomes). In stage III melanoma characterized by the presence of regional metastases in the lymph nodes, tumor cells showed higher expression of the autophagy marker protein LC3beta in comparison with stage II melanoma cells, which can indicate the involvement of autophagy processes in tumor progression and the formation of metastases in the lymph nodes.

The nucleus acts as a ruler tailoring cell responses to spatial constraints.

The microscopic environment inside a metazoan organism is highly crowded. Whether individual cells can tailor their behavior to the limited space remains unclear. In this study, we found that cells measure the degree of spatial confinement by using their largest and stiffest organelle, the nucleus. Cell confinement below a resting nucleus size deforms the nucleus, which expands and stretches its envelope. This activates signaling to the actomyosin cortex via nuclear envelope stretch-sensitive proteins, up-regulating cell contractility. We established that the tailored contractile response constitutes a nuclear ruler-based signaling pathway involved in migratory cell behaviors. Cells rely on the nuclear ruler to modulate the motive force that enables their passage through restrictive pores in complex three-dimensional environments, a process relevant to cancer cell invasion, immune responses, and embryonic development.

Expression of Molecular Markers of Angiogenesis, Lymphangiogenesis, and Proliferation Depending on the Stage of Skin Melanoma.

The expression of molecular markers characterizing activity of the tumor process and metastases (proliferation marker Ki-67, angiogenesis marker CD34, and lymphangiogenesis markers podoplanin and LYVE-1) was assessed by immunohictochemical method in the primary tumor specimens collected during surgery for cutaneous melanoma (40 patients). Proliferative activity of the tumor tissue and volume density of peritumoral blood and lymph vessels increased with increasing tumor malignancy, which could indicate the risk of metastases.

[The realization of the program of medical care to patients with vascular pathology in the Central Russia: a three year experience of the Saratov Regional Vascular Center]. / Realizatsiia programmy po okazaniiu meditsinskoĭ pomoshchi patsientam s sosudistoĭ patologieĭ v Tsentral'noĭ Rossii: trekhletniĭ opyt regional'nogo sosudistogo tsentra Saratova.

UNLABELLED: Creation of vascular centers in Russian regions is one of the ways of prevention of spreading of vascular diseases. AIM: To analyze three year (2012-2014) activity of the Saratov Regional Vascular Center (RVS). MATERIAL AND METHODS: Several stages of creation and development of RVS as well as the progress achieved in the treatment of acute coronary and cerebrovascular pathology have been analyzed. RESULTS AND CONCLUSION: The realization of protocols, standards, procedures of medical care to patients with cerebrovascular diseases by RVS personnel, wide use of high-technology methods of diagnosis and treatment during 3 years allowed not only to achieve positive results but to find unrealized possibilities of the activity in this direction.

Reaction of dermal structures to subcutaneous injection of gold nanoparticles to CBA mice.

The reactions of dermal structures to subcutaneous injections of gold nanoparticles were studied in CBA mice. Routes of the nanoparticles migration after injection and the mechanisms of their effects on the adjacent tissues were studied. Injected nanoparticles were phagocytosed by macrophages; some of them migrated to lymphoid follicles of the lymph node, while others migrated into blood vessels, where the particles were released from the macrophage cytoplasm into circulating blood. The endothelium was destroyed as a result of the toxic activity of macrophages loaded with nanoparticles. Two mechanisms of angiogenesis inhibition and death of blood vessels in tissues after injection of nanoparticles were distinguished. One mechanism consisted in deactivation of macrophages producing vascular endothelium growth factor inducing the formation of endothelium in the growing blood vessels, but not in direct inhibition of this factor. The other mechanism was realized through direct death of the endothelium in migration of macrophages through the vascular wall.

Role of the neuroglia of human ocular transparent structures in the visual perception concepts.

Physically transparent cornea, lens, and vitreous body are not transparent from histophysiological viewpoint and hence, cannot directly transmit light to the retina. The lens forms from two primordia in the course of the eye development: ectodermal (for capsular epithelium) and neuroglia (for lenticular stroma). These data suggest that the neuroglia migrating from the internal leaflet of the ocular goblet is a source of stromal fibroblasts of the corneal proper substance and also presents the fibroblast differons in the human ocular vitreous body and in the lenticular posterior pole. The common source in the development of the stroma of the transparent structures of the eye is explained and confirmed by the fact that the cornea, lens, vitreous body, and the retinal and brain neuroglia contain special proteins common for all these structures, crystallins. The structural relationship and origin of fibroblasts of the transparent ocular media underlie the identical functions of crystallin production; the physical and chemical characteristics of crystallins prevent the diffusion of light and cumulate it in one direction. In addition, our data on the neuroglial origin of the lenticular stroma suggest that the lenticular cells can act as Muller's glia, regulating the transformed energy flow. Hence, the fibers and stromal cells of the cornea, lens, vitreous body, and retinal glia can serve as components of the universal conduction system perceiving light, transforming it into another type of energy (presumably into electromagnetic waves or some motor pulse), and only then sending it to the photosensor cells. As a result, we have one-way light conduction due to stromal cells of transparent eye structures and inability of the retina to identify these cells. We therefore conclude that the cells located in front of the retina are invisible for the photoreceptors, because they conduct stimulation being not transparent in the physical sense, but just physiologically. We see only what this unique conduction system allows us to see.

[Age-related changes in the nervous apparatus of the blood vessels in the brain and spinal cord].

This work was designed to study changes in the nervous apparatus of the blood vessels in the brain and spinal cord with a view to developing methods for the determination of the biological age in man. Pial and intramedullary vascular systems of the brain and spinal cord were examined in different periods of postnatal ontogenesis (between the age of 1 and 90 years) using histological, fluorescent-histochemical, and immunohistochemical methods as well as by ink injections into the blood vessels, scanning and transmission electron microscopy. The data obtained on the age-related rearrangement of the nervous apparatus of the arterial vessels in the brain and spinal cord have not only theoretical significance but can also be used for the solution of practical problems encountered in the practical work of forensic medical experts.

Dynamics of nonmembranous cell components: role of active transport along microtubules.

Here we discuss some common mechanisms of microtubule-dependent active transport of nonmembranous components in animal cells. We summarize data about mRNA, cytoskeletal elements, structural proteins, and signaling complexes transport. We also characterize the series of molecular interactions that connect nonmembranous cargoes and microtubules and describe the regulatory pathways for these interactions.

[The study of patients? Opinion about the quality of medical care in the N.I. Pirogov National Medical Surgical Center].

The study covered the consumer group of relatives of 192 patients receiving treatment in the surgery departments of the Saint George National center of thoracic and cardiovascular surgery in 2005 and 2009, after implementation of industrial techniques of medical care quality management. The relatives of 84 patients were polled in 2005 and of 108 patients--in 2007. The questionnaire proceeded the standard algorithm. The final stage of the study included the analysis of proposals of relatives of patients concerning the enhancement of quality of Center functioning.

Evaluation of erythrocyte shape and status by laser interference microscopy.

The area, thickness, and volume of erythrocytes of different types (discocytes, stomatocytes, and echinocytes) from normal subjects and coronary patients were studied by laser interference microscopy. Increase of pH value leading to the stomatocyte-discocyte-echinocyte transformations resulted in a slight decrease of cell volume. In coronary patients, erythrocyte had larger area and volume and exhibited increased aggregation capacity compared to erythrocytes from controls. The results recommend laser interference microscopy as an adequate method for erythrocyte evaluation in laboratory diagnostic measurements.

Cytoskeleton inhibitors combined with TRAIL induce apoptosis in HeLa carcinoma cells overexpressing antiapoptotic protein Bcl-2.

TRAIL (Apo2L), a cytokine from the family of tumor necrosis factors (TNF), causes apoptosis in various types of tumor cells but is not toxic for normal cells. Recombinant TRAIL obtained using an original method stimulates the release of cytochrome c from mitochondria into the cytoplasm and apoptosis in HeLa carcinoma cells. Expression of oncoprotein Bcl-2 in these cells blocks both processes. The microtubule inhibitors taxol, nocodazole, and colcemid, as well as an inhibitor of actin microfilaments cytochalasin D, enhance the action of TRAIL and allow it to overcome protection caused by overexpression of Bcl-2. This effect is not associated with enhancement of early steps of TRAIL-dependent apoptosis leading to activation of caspase-8 and Bid protein. The inactivation of Bcl-2 also does not define the effect of cytoskeleton inhibitors. It is supposed that destruction of cytoskeleton alters the mechanism of the TRAIL- (or TNF)-dependent cytochrome c release from mitochondria by making it resistant to Bcl-2. The combined use of cytoskeleton inhibitors, which are antitumor drugs, with the recombinant TRAIL preparations may be efficient in therapy of tumors resistant to traditional chemotherapy.

Modified DNA fragments specifically and irreversibly bind transcription factor NF-kappaB in lysates of human tumor cells.

Covalent binding of a synthetic DNA fragment with eukaryotic transcription factor NF-kappaB has been studied in lysates of human colon carcinoma HCT-116 cells. For binding we used 32P-labeled 17/19 bp nucleotide DNA duplex containing an NF-kappaB recognition site (kappaB-site) in which one of internucleotide phosphate groups was replaced by a chemically active trisubstituted pyrophosphate group. Using gel electrophoresis under denaturing conditions (Laemmli electrophoresis) followed by immunoblotting revealed selective irreversible binding of 32P-labeled DNA duplex with NF-kappaB in lysates of tumor cells in the presence of other cell components. Experiment on delivery of this DNA duplex containing rhodamine at 3 -end of the modified chain in an intact cell revealed that rhodamine-labeled DNA penetrated through the plasma membrane of tumor cells without any additional delivery systems. Using fluorescent microscopy, we found that the rhodamine-labeled DNA is initially localized in the cytoplasm. Confocal laser scanning microscopy revealed that subsequent treatment of the cells with TNF-alpha promoted partial translocation of the DNA reagent into the nucleus.

Application of universal primers for identification of Foot-and-mouth disease virus and Swine vesicular disease virus by PCR and PCR-ELISA.

Two approaches for simultaneous identification of both Foot-and-mouth disease virus (FMDV) and Swine vesicular disease virus (SVDV) are described: (1) a single-step reverse transcription-PCR with three primers and (2) a PCR-ELISA assay with two universal primers for genome amplification and two virus-specific probes for identification. These methods are based on the use of 3D gene universal PCR primers, the structure of which was optimized and refined due to the close relationship between the two viruses belonging to different genera of the Picornaviridae family. In procedure (1), a three-primer PCR containing one universal antisense primer and two virus-specific primers was shown to differentiate between FMDV and SVDV in one reaction, due to the different length of the amplified DNA fragments (600 and 340 base pairs, respectively). In procedure (2), the two viruses were identified by PCR-ELISA, i.e. PCR for the 3D gene followed by two parallel hybridizations with FMDV and SVDV-specific probes in microplate wells and ELISA detection. The application of universal primers could halve the number of PCR experiments in both cases, as compared to the usual virus-specific PCR procedures. Also, we investigated the 3D gene structure of several SVDV strains isolated at different times. No essential changes were detected in the regions coding for conserved motifs of the RNA-dependent RNA polymerase recognized by our universal primers. The multi-primer PCR was successfully tested on 38 FMDV and 15 SVDV strains, and the PCR-ELISA on 32 FMDV and 16 SVDV strains including clinical material from disease cases.

Enhanced crystallization of the Cys18 to Ser mutant of bovine gammaB crystallin.

The cysteine residues of the gamma crystallins, a family of ocular lens proteins, are involved in the aggregation and phase separation of these proteins. Both these phenomena are implicated in cataract formation. We have used bovine gammaB crystallin as a model system to study the role of the individual cysteine residues in the aggregation and phase separation of the gamma crystallins. Here, we compare the thermodynamic and kinetic behavior of the recombinant wild-type protein (WT) and the Cys18 to Ser (C18S) mutant. We find that the solubilities of the two proteins are similar. The kinetics of crystallization, however, are different. The WT crystallizes slowly enough for the metastable liquid-liquid coexistence to be easily observed. C18S, on the other hand, crystallizes rapidly; the metastable coexisting liquid phases of the pure mutant do not form. Nevertheless, the coexistence curve of C18S can be determined provided that crystallization is kinetically suppressed. In this way we found that the coexistence curve coincides with that of the WT. Despite the difference in the kinetics of crystallization, the two proteins were found to have the same crystal forms and almost identical X-ray structures. Our results demonstrate that even conservative point mutations can bring about dramatic changes in the kinetics of crystallization. The implications of our findings for cataract formation and protein crystallization are discussed.

Amyloid beta-protein oligomerization: prenucleation interactions revealed by photo-induced cross-linking of unmodified proteins.

Assembly of the amyloid beta-protein (Abeta) into neurotoxic oligomers and fibrils is a seminal event in Alzheimer's disease. Understanding the earliest phases of Abeta assembly, including prenucleation and nucleation, is essential for the development of rational therapeutic strategies. We have applied a powerful new method, photoinduced cross-linking of unmodified proteins (PICUP), to the study of Abeta oligomerization. Significant advantages of this method include an extremely short reaction time, enabling the identification and quantification of short lived metastable assemblies, and the fact that no pre facto structural modification of the native peptide is required. Using PICUP, the distribution of Abeta oligomers existing prior to assembly was defined. A rapid equilibrium was observed involving monomer, dimer, trimer, and tetramer. A similar distribution was seen in studies of an unrelated amyloidogenic peptide, whereas nonamyloidogenic peptides yielded distributions indicative of a lack of monomer preassociation. These results suggest that simple nucleation-dependent polymerization models are insufficient to describe the dynamic equilibria associated with prenucleation phases of Abeta assembly.

[Phylogenetic analysis of swine fever virus strains]. / Filogeneticheskii analiz shtammov virusa chumy plotoiadnykh.

Amplification of H-gene fragment in combination with cDNA nucleotide sequencing can be used for indication and strain differentiation of classical swine fever virus.

Crystal cataracts: human genetic cataract caused by protein crystallization.

Several human genetic cataracts have been linked recently to point mutations in the gammaD crystallin gene. Here we provide a molecular basis for lens opacity in two genetic cataracts and suggest that the opacity occurs because of the spontaneous crystallization of the mutant proteins. Such crystallization of endogenous proteins leading to pathology is an unusual event. Measurements of the solubility curves of crystals of the Arg-58 to His and Arg-36 to Ser mutants of gammaD crystallin show that the mutations dramatically lower the solubility of the protein. Furthermore, the crystal nucleation rate of the mutants is enhanced considerably relative to that of the wild-type protein. It should be noted that, although there is a marked difference in phase behavior, there is no significant difference in protein conformation among the three proteins.

Fitting the correlation function.

The whole correlation function of the intensity of scattered light is usually determined from a single realization of the photocurrent. As a result, the values of the correlation function at different delay times are not statistically independent. A standard least-squares fitting procedure is not optimal for an analysis of such data. However, the benefits of mathematically rigorous but highly nonlinear and less stable methods are not known. We consider the test case of a Gaussian signal with a single-exponential correlation function without shot noise. In this case the fitting procedure, which is based on the maximum-likelihood principle for the observed signal, permits an analytical solution. We demonstrate that such a rigorous statistical analysis produces an approximately two times more-accurate result for the relaxation time than does the standard least-squares fit. This gain, however, is greatly reduced by the presence of shot noise, which introduces additional uncorrelated errors into the values of the correlation function.