Interdisk spacing effect on resonant properties of Ge disk lattices on Si substrates.

The light reflection properties of Ge disk lattices on Si substrates are studied as a function of the disk height and the gap width between disks. The interdisk spacing effect is observed even at such large gap widths as 500 nm. The gap width decrease leads to the appearance of the reflection minimum in the short wavelength region relative to one originated from the magnetic and electric dipole resonances in individual Ge disks, thereby essentially widening the antireflection properties. This minimum becomes significantly deeper at small gap widths. The observed behavior is associated with the features of the resonant fields around closely spaced disks according to numerical simulation data. The result shows the importance of using structures with geometrical parameters providing the short-wavelength minimum. This can essentially enhance their other resonant properties, which are widely used for applications, in particular, based on collective lattice resonances.

[Conjugate of podophyllotoxin with chlorambucil: synthesis, biological testing and molecular modeling]. / Kon"iugat podofillotoksina s khlorambutsilom: sintez, biotestirovanie i molekuliarnoe modelirovanie.

In the present work we have studied a novel conjugate of the DNA alkylating agent chlorambucil with podophyllotoxin, a ligand of the colchicine binding site in tubulin. The target compound was obtained by Steglich esterification of podophyllotoxin with the percentage yield of 41%. Results of biotesting carried out on the carcinoma A549 cell line revealed that at a concentration of 2 µM the conjugate caused full depolymerization of microtubules without any other effect on free tubulin. The conjugate inhibited proliferation (IC50=135±30 nM) and growth (EC50=240±30 nM) of A549 cells. The data of computer molecular docking of the novel compound into the 3D model of the colchicine binding site in α,ß-tubulin and molecular dynamics modelling allowed to explain the observed difference in effects of chlorambucil-podophyllotoxin and chlorambucil-colchicine conjugates on microtubules.

[Podophyllotoxin analogue with bicyclo[3.2.1]octane moiety annelated with indole: synthesis, molecular modeling, and biological testing]. / Analog podofillotoksina s bitsiklo[3.2.1]oktanovoi gruppirovkoi, annelirovannoi s indolom: sintez, molekuliarnoe modelirovanie i biotestirovanie.

C4-Ester derivatives of the anticancer agent podophyllotoxin with bridged moieties can either inhibit polymerization of alpha,beta-tubulin with the formation of microtubules (analogously to the parent molecule) or cause an unusual effect of "curling and shortening" of the microtubules (MT). In order to predict the effect of bridged podophyllotoxin derivatives on the MT network using computer molecular modeling it is desirable to enhance the structural diversity of their bridged substituents. In the present work we synthesized novel podophyllotoxin ester with bicyclo[3.2.1]octane moiety annelated with indole core. The target compound was obtained by Steglich esterification of podophyllotoxin by rac-exo-(indolo[2,3-b])bicyclo[3.2.1]oct-2-ene-6-carboxylic acid as diastereomeric (6RS,8SR,9RS) mixture, which could not be separated by thin layer or preparative column chromatography on silica gel. Results of biotesting of 4-O-{(6R,8S,9R)-5,6,7,8,9,10-hexahydro-6,9-methanocyclohepto[b]indol-8-ylcarbonyl}-Lpodophyllotoxin on the carcinoma A549 cells proved its ability to cause full depolymerization of microtubules without curling effect at a concentration 10 µM. Cytotoxicity value of the compound estimated in MTT test was in a high nanomolar concentration interval (EC50=710±30 nM). Computer molecular docking of both isomers of novel compound and earlier synthesized podophyllotoxin esters with bridged moieties into the 3D model of the colchicine domain in alpha,beta-tubulin revealed the difference in positions of the bridge moieties of new compound and MT-curling ligands and allowed to hypothesize that the atypical action on MT might be caused by positioning of their bridge groups near the GTP binding site in alpha-tubulin.

Plasticity of Human THP-1 Cell Phagocytic Activity during Macrophagic Differentiation.

Studies of the role of macrophages in phagocytosis are of great theoretical and practical importance for understanding how these cells are involved in the organism's defense response and in the development of various pathologies. Here we investigated phagocytic plasticity of THP-1 (acute monocytic human leukemia) cells at different stages (days 1, 3, and 7) of phorbol ester (PMA)-induced macrophage differentiation. Analysis of cytokine profiles showed that PMA at a concentration of 100 nM induced development of the proinflammatory macrophage population. The functional activity of macrophages was assessed on days 3 and 7 of differentiation using unlabeled latex beads and latex beads conjugated with ligands (gelatin, mannan, and IgG Fc fragment) that bind to the corresponding specific receptors. The general phagocytic activity increased significantly (1.5-2.0-fold) in the course of differentiation; phagocytosis occurred mostly through the Fc receptors, as shown previously for M1 macrophages. On day 7, the levels of phagocytosis of gelatin- and Fc-covered beads were high; however, the intensity of ingestion of mannan-conjugated beads via mannose receptors increased 2.5-3.0-fold as well, which indicated formation of cells with an alternative phenotype similar to that of M2 macrophages. Thus, the type and the plasticity of phagocytic activity at certain stages of macrophage differentiation can be associated with the formation of functionally mature morphological phenotype. This allows macrophages to exhibit their phagocytic potential in response to specific ligands. These data are of fundamental importance and can be used to develop therapeutic methods for correcting the M1/M2 macrophage ratio in an organism.

Using Two-Dimensional Distributed Feedback for Synchronization of Radiation from Two Parallel-Sheet Electron Beams in a Free-Electron Maser.

A spatially extended planar 75 GHz free-electron maser with a hybrid two-mirror resonator consisting of two-dimensional upstream and traditional one-dimensional downstream Bragg reflectors and driven by two parallel-sheet electron beams 0.8 MeV/1 kA has been elaborated. For the highly oversized interaction space (cross section 45×2.5 vacuum wavelengths), the two-dimensional distributed feedback allowed realization of stable narrow-band generation that includes synchronization of emission from both electron beams. As a result, spatially coherent radiation with the output power of 30-50 MW and a pulse duration of ∼100 ns was obtained in each channel.

Dynamic phase microscopy reveals periodic oscillations of endoplasmic reticulum during network formation.

Dynamic phase microscopy was used to study the dynamic events of formation of the endoplasmic reticulum (ER) in interphase-arrested Xenopus egg extract. We have shown that the ER periodically oscillated in an ATP-dependent manner in the frequency range of 1.6-2.2 Hz, while the tubular membrane network formed in vitro. The spectral density, i.e. the pattern of a given frequency component in the Fourier spectrum, was strongly correlated with the dynamic events during microtubule-dependent and microtubule-independent ER network formation observed by video-enhanced contrast differential interference contrast and fluorescence microscopy. Because the 1.6-2.2 Hz frequency of oscillation during the network formation was detected both in the presence and absence of microtubules, it appears to be an intrinsic ATP-dependent ER membrane property. Several characteristic active and inactive stages of ER network formation were observed both in the presence and absence of microtubules. However, data analysis of these stages indicated that microtubules and dynein motor activity have a strong influence and a cooperative effect on the kinetics of ER formation by controlled fusion reaction.

Three-dimensional structure of carboxypeptidase T from Thermoactinomyces vulgaris in complex with N-BOC-L-leucine.

The 3D structure of recombinant bacterial carboxypeptidase T (CPT) in complex with N-BOC-L-leucine was determined at 1.38 Å resolution. Crystals for the X-ray study were grown in microgravity using the counter-diffusion technique. N-BOC-L-leucine and SO4(2-) ion bound in the enzyme active site were localized in the electron density map. Location of the leucine side chain in CPT-N-BOC-L-leucine complex allowed identification of the S1 subsite of the enzyme, and its structure was determined. Superposition of the structures of CPT-N-BOC-L-leucine complex and complexes of pancreatic carboxypeptidases A and B with substrate and inhibitors was carried out, and similarity of the S1 subsites in these three carboxypeptidases was revealed. It was found that SO4(2-) ion occupies the same position in the S1' subsite as the C-terminal carboxy group of the substrate.

A practical guide to culturing mouse and human bone marrow stromal cells.

Bone marrow stromal cells (BMSCs, frequently also called MSCs) represent a cell population within the bone marrow, a subset of which contains multipotent stem cells. Their primary role is to produce and maintain both bone tissue and bone marrow microenvironment necessary for hematopoiesis. The latter is achieved by secreting a wide variety of different cytokines and growth factors, many of which also have a regulatory role in immune processes. BMSCs have recently been introduced into the field of immunobiology after their successful clinical use in GVHD was reported in 2004. Since then, numerous studies confirmed and expanded the knowledge on the immunosuppressive potential of BMSCs in various in vitro and in vivo models. Although the immunomodulatory capacity of BMSCs is well established, there are still many unanswered questions regarding the cytokines, chemokines, receptors, and molecular pathways that play a role in this effect. To study these cells and answer many of the questions, researchers must be able to reliably and reproducibly isolate, culture, and use these cells. Below a practical guide on how to culture and characterize mouse and human BMSCs, which can then be applied in various in vitro and in vivo assays, is provided.

[A novel endogeneous inhibitor from hepatopancreas of Kamchatka crab (Paralithodes camtschaticus)].

A novel endogeneous inhibitor from hepatopancreas of Kamchatka crab (Paralithosed camtschaticus) was isolatyed. The inhibitor was purifeid through fractional affinity chromatography on gramicidin-diasorb followed by gel-filtration at Sephadex G-100. The inhibitor PC is a protein (M, 66 kDa) and active against serine collagenolytic protease PC at temperature optimum 15-20 degrees C, stable at 4-40 degrees C and was completely inactivated after heating to 50 degrees C and higher. 0.9-20% NaCl is necessary for its inhibitor activity. The inhibitor was found to slow down cell spreading in vitro in cell type-dependent manner. Fibroblasts are most prone to inhibitory effect, epithelial tumor derived cells show medium susceptibility, while fibrosarcoma cells were not affected.

Radiosensitizing effect of epothilone B on human epithelial cancer cells.

BACKGROUND: A combined modality treatment employing radiation and chemotherapy plays a central role in the management of solid tumors. In our study, we examined the cytotoxic and radiosensitive effect of the microtubule stabilizer epothilone B on two human epithelial tumor cell lines in vitro and its influence on the microtubule assembly. METHODS: Cancer cells were treated with epothilone B in proliferation assays and in combination with radiation in colony-forming assays. For the analysis of ionizing radiation-induced DNA damage and the influence of the drug on its repair a γH2AX foci assay was used. To determine the effect of epothilone B on the microtubule assembly in cells and on purified tubulin, immunofluorescence staining and tubulin polymerization assay, respectively, were conducted. RESULTS: Epothilone B induced a concentration- and application-dependent antiproliferative effect on the cells, with IC(50) values in the low nanomolar range. Colony forming assays showed a synergistic radiosensitive effect on both cell lines which was dependent on incubation time and applied concentration of epothilone B. The γH2AX assays demonstrated that ionizing radiation combined with the drug resulted in a concentration-dependent increase in the number of double-strand breaks and suggested a reduction in DNA repair capacity. Epothilone B produced enhanced microtubule bundling and abnormal spindle formation as revealed by immunofluorescence microscopy and caused microtubule formation from purified tubulin. CONCLUSION: The results of this study showed that epothilone B displays cytotoxic antitumor activity at low nanomolar concentrations and also enhances the radiation response in the tumor cells tested; this may be induced by a reduced DNA repair capacity triggered by epothilone B. It was also demonstrated that epothilone B in fact targets microtubules in a more effective manner than paclitaxel.

Regular and anomalous extraordinary optical transmission at the THz-gap.

In this paper Anomalous Extraordinary Transmission (ET) is reported for s-polarization of low loss doubly periodic subwavelength hole arrays patterned on polypropylene (PP) substrates by conventional contact photolithography at the so-called THz-gap (1-10 THz). The unexpected enhanced transmittance for s-polarization (i.e. without spoof plasmons) was previously numerically demonstrated in subwavelength slits arrays. However, subsequently no experimental work has been devoted to this unexpected Extraordinary Transmission neither in subwavelength slits nor in subwavelength holes. Here, numerical study and experimental results of the Anomalous ET and the symmetric and antisymmetric transmittance modes associated with the already well-known p-polarization ET are shown alongside a systematically analysis of the frequency peaks as a function of hole size for both incident polarizations.

Polypropylene-substrate-based SRR- and CSRR- metasurfaces for submillimeter waves.

In this paper it is presented the fabrication of low loss millimeter wave metamaterials based on patterning on polypropylene substrates by conventional contact photolitography. We study numerically and experimentally the transmission and reflection properties of two dimensional arrays of split ring resonators (SRRs), or metasurfaces, and their complementary structure (CSRRs) for co- and cross-polarization excitations up to submillimeter frequencies under normal incidence conditions. The obtained results suggest the possibility of scaling them at terahertz frequencies based on this substrate where other lossy substrates degrade the resonators quality. Left-handed metamaterials derived from these SRRs and CSRRs metasurfaces could be feasible.

Formation and germination of Phytophthora infestans oospores in different regions of Russia.

On occurrence of oospores (sexual stage), 88 Phytophthora infestans populations were investigated. The populations were collected in 14 regions of Russia. In total, 3677 samples have been checked: 2888--from potato leaflets (55 populations), 344--from tomato leaflets (16 populations), 445--from tomato fruits (17 populations). The oospores have been detected both in potato and tomato leaflets and in fruits with different occurrence--4.4%, 11.9%, and 28.8%. Occurrence of oospores per sample was also maximal in tomato fruits. After overwintering a majority of oospores (50-90%) kept their viability. Sometimes, loss of viability of some oospores was caused by the external negative influence. About 25% of oospores formed at crossing between current Russian P. infestans strains were capable to germinate. Treatment with negative temperature (-12 degrees C) or Trichoderma suspension increased percentage of germinated oospores. In some field trials, the oospores caused affection of quite many of potato plants. However, in other trials only particular plants or no plants were affected. Tomato germlings were affected only under unfavourable conditions.

Three-dimensional reconstruction of the dynactin complex by single-particle image analysis.

Dynactin is a large complex of at least nine distinct proteins that co-complexes with cytoplasmic dynein within cells, where it plays a major role as a regulator of the motor's function. Owing to its large size and complexity, relatively little is known about dynactin's 3D structure or the structural basis of its function. Use of single-particle image analysis techniques has enabled us to produce the first 3D reconstruction of the dynactin complex, to a resolution of 3 nm. The actin-related protein (Arp) backbone of the filament has been clearly visualized. Fitting of models of the Arp backbone showed that it consists of 10 subunits. Additional mass, not part of the Arp backbone, was also seen. A preliminary fitting of the capping protein CapZ structure into our 3D reconstruction of the dynactin complex suggests that it is optimally placed to perform its proposed function as a stabilizer of the Arp1 backbone and gives clues as to likely interaction points between the capping protein and Arp subunits. The results provide the first detailed visualization of the dynactin complex and shed light on the mode of interaction between several of its constituent proteins and their possible functions.

[New non-hydrolyzable substrate analogs for 8-oxoguanine-DNA glycosylases]. / Novye negidrolizuemye analogi substratov 8-oksoguanin-DNK-glikolilaz.

8-Oxoguanine-DNA glycosylases play a key role in the repair of oxidatively damaged DNA. The Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and human 8-oxoguanine-DNA glycosylase (hOGG1) are DNA base excision repair enzymes that catalyze the removal of 7,8-dihydro-8-oxoguanine (oxoG) residue, and cleave DNA strand. Specific contacts between DNA phosphate groups and amino acids from active centers of these enzymes play a significant role in DNA-protein interactions. In order to design new non-hydrolyzable substrate analogs of Fpg and hOGG1 for structural studies modified DNA duplexes containing pyrophosphate or OEt-substituted pyrophosphate internucleotide (SPI) groups near the damage were tested. We showed that enzymes recognize and specifically bind to DNA duplexes obtained. The mechanism of incision of oxoG by the Fpg and hOGG1 was determined. We revealed that both enzymes were not able to excise the oxoG residue from DNA containing modified phosphates immediately 3' to the oxoG. In contrast, Fpg and hOGG1 effectively incise DNA duplex carrying analogous phosphate modifications 5' to the oxoG. Non-cleavable oxoG-containing DNA duplexes bearing pyrophosphate or substituted pyrophosphate groups immediately 3' to the oxoG are specific inhibitors for both 8-oxoguanine-DNA glycosylases and can be used for structural studies of complexes comprising a oxoG-containing DNA bound to catalytically active wild-type enzymes as well as their pro- and eucaryotic homologs.

Osteoclastogenesis in fibrous dysplasia of bone: in situ and in vitro analysis of IL-6 expression.

Fibrous dysplasia of bone (FD) is caused by somatic mutations of the GNAS1 gene, which lead to constitutive activation of adenylyl cyclase and overproduction of cAMP in osteogenic cells. Previous in vitro studies using nonclonal, heterogeneous strains of FD-derived cells suggested that IL-6 might play a critical role in promoting excess osteoclastogenesis in FD. In this study, we investigated IL-6 expression in FD in situ and its relationship to the actual patterns of osteoclastogenesis within the abnormal tissue. We found that osteoclastogenesis is not spatially restricted to bone surfaces in FD but occurs to a large extent ectopicly in the fibrous tissue, where stromal cells diffusely express IL-6 mRNA and exhibit a characteristic cell morphology. We also observed specific expression of IL-6 mRNA in a proportion of osteoclasts, suggesting that an autocrine/paracrine loop may contribute to osteoclastogenesis in vivo in FD, as in some other bone diseases, including Paget's disease. We also generated homogeneous, clonally derived strains of wild-type and GNAS1-mutated stromal cells from the same individual, parent FD lesions. In this way, we could show that mutated stromal cells produce IL-6 at a basal magnitude and rate that are significantly higher than in the cognate wild-type cells. Conversely, wild-type cells respond to db-cAMP with a severalfold increase in magnitude and rate of IL-6 production, whereas mutant strains remain essentially unresponsive. Our data establish a direct link between GNAS1 mutations in stromal cells and IL-6 production but also define the complexity of the role of IL-6 in regulating osteoclastogenesis in FD in vivo. Here, patterns of osteoclastogenesis and bone resorption reflect not only the cell-autonomous effects of GNAS1 mutations in osteogenic cells (including IL-6 production) but also the local and systemic context to which non-osteogenic cells, local proportions of wild-type vs mutated cells, and systemic hormones contribute.

Liquid level sensor using ultrasonic Lamb waves.

This paper describes a novel, noninvasive method for measurement of liquid level in closed metal tanks that are under high pressure. It is based on the use of ultrasonic Lamb waves propagating along the tank wall. Contact with liquid substantially changes the characteristics of these waves and this can be used as an indicator of liquid presence. Theoretical analysis shows that the symmetric and antisymmetric Lamb wave modes, both fundamental and higher order, are sensitive to presence of the liquid. The optimal wave frequency depends on the thickness of the tank wall and wall material. A prototype level sensor based on this principle has been developed. It uses two pairs of wedge transducers to generate and detect Lamb waves propagating along the circumference of the gas tank. An operating frequency of 100 kHz is found to be optimal for use with tanks having a wall thickness of 30-50 mm. Prototype sensors developed under this program have been used successfully in oil fields in the far northern region of Russia.

Gnathodiaphyseal dysplasia: a syndrome of fibro-osseous lesions of jawbones, bone fragility, and long bone bowing.

We report an unusual generalized skeletal syndrome characterized by fibro-osseous lesions of the jawbones with a prominent psammomatoid body component, bone fragility, and bowing/sclerosis of tubular bones. The case fits with the emerging profile of a distinct syndrome with similarities to previously reported cases, some with an autosomal dominant inheritance and others sporadic. We suggest that the syndrome be named gnathodiaphyseal dysplasia. The patient had been diagnosed previously with polyostotic fibrous dysplasia (PFD) elsewhere, but further clinical evaluation, histopathological study, and mutation analysis excluded this diagnosis. In addition to providing a novel observation of an as yet poorly characterized syndrome, the case illustrates the need for stringent diagnostic criteria for FD. The jaw lesions showed fibro-osseous features with the histopathological characteristics of cemento-ossifying fibroma, psammomatoid variant. This case emphasizes that the boundaries between genuine GNAS1 mutation-positive FD and other fibro-osseous lesions occurring in the jawbones should be kept sharply defined, contrary to a prevailing tendency in the literature. A detailed pathological study revealed previously unreported features of cemento-ossifying fibroma, including the participation of myofibroblasts and the occurrence of psammomatoid bodies and aberrant mineralization, within the walls of blood vessels. Transplantation of stromal cells grown from the lesion into immunocompromised mice resulted in a close mimicry of the native lesion, including the sporadic formation of psammomatoid bodies, suggesting an intrinsic abnormality of bone-forming cells.

Myosin Va bound to phagosomes binds to F-actin and delays microtubule-dependent motility.

We established a light microscopy-based assay that reconstitutes the binding of phagosomes purified from mouse macrophages to preassembled F-actin in vitro. Both endogenous myosin Va from mouse macrophages and exogenous myosin Va from chicken brain stimulated the phagosome-F-actin interaction. Myosin Va association with phagosomes correlated with their ability to bind F-actin in an ATP-regulated manner and antibodies to myosin Va specifically blocked the ATP-sensitive phagosome binding to F-actin. The uptake and retrograde transport of phagosomes from the periphery to the center of cells in bone marrow macrophages was observed in both normal mice and mice homozygous for the dilute-lethal spontaneous mutation (myosin Va null). However, in dilute-lethal macrophages the accumulation of phagosomes in the perinuclear region occurred twofold faster than in normal macrophages. Motion analysis revealed saltatory phagosome movement with temporarily reversed direction in normal macrophages, whereas almost no reversals in direction were observed in dilute-lethal macrophages. These observations demonstrate that myosin Va mediates phagosome binding to F-actin, resulting in a delay in microtubule-dependent retrograde phagosome movement toward the cell center. We propose an "antagonistic/cooperative mechanism" to explain the saltatory phagosome movement toward the cell center in normal macrophages.

Circulating skeletal stem cells.

We report the isolation of adherent, clonogenic, fibroblast-like cells with osteogenic and adipogenic potential from the blood of four mammalian species. These cells phenotypically resemble but are distinguishable from skeletal stem cells found in bone marrow (stromal stem cells, "mesenchymal stem cells"). The osteogenic potential of the blood-borne cells was proven by an in vivo transplantation assay in which either polyclonal or single colony-derived strains were transplanted into the subcutis of immunocompromised mice, and the donor origin of the fully differentiated bone cells was proven using species-specific probes. This is the first definitive proof of the existence of circulating skeletal stem cells in mammals.