RESUMEN

We studied the influence of lipid surface composition on the kinetics of fibrin clot formation and its structure. It was shown that lipid surface affects all phases of fibrin polymerization and chances clot morphology. The magnitude and character of the effect depend on the charge and phase state of lipids that determine the interaction of fibrinogen with the lipid surface and its conformational changes, which modulated the process of fibrinogen conversion into fibrin and, as a result, the formation and morphology of the fibrin clot.

Asunto(s)

RESUMEN

Representatives of Pectobacterium genus are some of the most harmful phytopathogens in the world. In the present study, we have elucidated novel aspects of plant-Pectobacterium atrosepticum interactions. This bacterium was recently demonstrated to form specific 'multicellular' structures - bacterial emboli in the xylem vessels of infected plants. In our work, we showed that the process of formation of these structures includes the pathogen-induced reactions of the plant. The colonisation of the plant by P. atrosepticum is coupled with the release of a pectic polysaccharide, rhamnogalacturonan I, into the vessel lumen from the plant cell wall. This polysaccharide gives rise to a gel that serves as a matrix for bacterial emboli. P. atrosepticum-caused infection involves an increase of reactive oxygen species (ROS) levels in the vessels, creating the conditions for the scission of polysaccharides and modification of plant cell wall composition. Both the release of rhamnogalacturonan I and the increase in ROS precede colonisation of the vessels by bacteria and occur only in the primary xylem vessels, the same as the subsequent formation of bacterial emboli. Since the appearance of rhamnogalacturonan I and increase in ROS levels do not hamper the bacterial cells and form a basis for the assembly of bacterial emboli, these reactions may be regarded as part of the susceptible response of the plant. Bacterial emboli thus represent the products of host-pathogen integration, since the formation of these structures requires the action of both partners.

Asunto(s)

RESUMEN

The type of the Electron String Ion Sources (ESIS) is considered to be the appropriate one to produce pulsed C(4+) and C(6+) ion beams for cancer therapy accelerators. In fact, the new test ESIS Krion-6T already now provides more than 10(10) C(4+) ions per pulse and about 5 × 10(9) C(6+) ions per pulse. Such ion sources could be suitable to apply at synchrotrons. It has also been found that Krion-6T can provide more than 10(11) C(6+) ions per second at the 100 Hz repetition rate, and the repetition rate can be increased at the same or larger ion output per second. This makes ESIS applicable at cyclotrons as well. ESIS can be also a suitable type of ion source to produce the (11)C radioactive ion beams. A specialized cryogenic cell was experimentally tested at the Krion-2M ESIS for pulse injection of gaseous species into the electron string. It has been shown in experiments with stable methane that the total conversion efficiency of methane molecules to C(4+) ions reached 5%÷10%. For cancer therapy with simultaneous irradiation and precise dose control (positron emission tomography) by means of (11)C, transporting to the tumor with the primary accelerated (11)C(4+) beam, this efficiency is preliminarily considered to be large enough to produce the (11)C(4+) beam from radioactive methane and to inject this beam into synchrotrons.

Asunto(s)

RESUMEN

The most recent experimental information on electron string phenomenon, such as two step transition to electron string state, stability of e-strings in condition of electron energy recuperation, are described. The new technology developments of electron string ion sources (ESIS) include pulse injection of gaseous species in e-string and its efficient conversion to ion beams, slow ion extraction, ion-ion cooling of heavy ions with CH(4) coolant, and a progress in the construction of the new Joint Institute for Nuclear Research ESIS with 6 T solenoid are briefly considered.

RESUMEN

Ryanodine receptors (RyRs) are the major ion channels in the sarcoplasmic reticulum responsible for Ca2+ release in muscle cells. Localization of RyRs is therefore critical to our understanding of Ca2+ cycling and Ca2+-dependent processes within ventricular cells. Recently, RyRs were reportedly found in non-classical locations in the middle of the sarcomere, between perinuclear mitochondria and in the inner mitochondrial membrane of cardiac mitochondria. However, for multiple reasons these reports could not be considered conclusive. Therefore, we modified immunogold labeling to visualize the distribution of RyRs in ventricular myocytes. Using antibodies to the voltage-dependent anion channel (i.e. VDAC) or cytochrome c along with our labeling method, we showed that these mitochondrial proteins were appropriately localized to the mitochondrial outer and inner membrane respectively. Immunogold labeling of ultrathin sections of intact and permeabilized ventricular myocytes with antibodies to three types of RyRs confirmed the existence of RyRs between the Z-lines and around the perinuclear mitochondria. However, we did not find any evidence to support localization of RyRs to the mitochondrial inner membrane.

Asunto(s)

RESUMEN

The scheme of an internal injection of Au atoms into the working space of the "Krion-2" electron string ion source (ESIS) was applied and tested. In this scheme Au atoms are evaporated from the thin tungsten wire surface in vicinity of the source electron string. Ion beams with charge states up to Au51+ were produced. Ion-ion cooling with use of C and O coolant ions was studied. It allowed increasing of the Au51+ ion yield by a factor of 2. Ions of Kr up to charge state 28+ were also produced in the source. Electron strings were first formed with injection electron energy up to 6 keV. Methods to increase the ESIS ion output are discussed.

RESUMEN

Ryanodine receptors (RyR2s) are ion channels in the sarcoplasmic reticulum (SR) that are responsible for Ca2+ release in rat ventricular myocytes. Localization of RyR2s is therefore crucial for our understanding of contraction and other Ca2+-dependent intracellular processes. Recent results (e.g. circular waves and Ca2+ sparks in perinuclear area) raised questions about the classical views of RyR2 distribution and organization within ventricular cells. A Ca2+ spark is a fluorescent signal reflecting the activation of a small group of RyR2s. Frequency and spatio-temporal characteristics of Ca2+ sparks depend on the state of cytoplasmic and intraluminal macromolecular complexes regulating cardiac RyR2 function. We employed electron microscopy, confocal imaging of spontaneous Ca2+ sparks and immunofluorescence to visualize the distribution of RyR2s in ventricular myocytes and to evaluate the local involvement of the macromolecular complexes in regulation of functional activity of the RyR2 group. An electron microscopy study revealed that the axial tubules of the transverse-axial tubular system probably do not have junctions with the network SR (nSR). The nSR was found to be wrapped around intermyofibrillar mitochondria and contained structures similar to feet of the junctional cleft. Treatment of ventricular myocytes with antibodies against RyR2 showed that in addition to the junctional SR, a small number of RyR2s can be localized at the middle of the sarcomere and in the zone of perinuclear mitochondria. Recordings of spontaneous Ca2+ sparks showed the existence of functional groups of RyR2s in these intracellular compartments. We found that within the sarcomere about 20% of Ca2+ sparks were not colocalized with the zone of the junctional or corbular SR (Z-line zone). The spatio-temporal characteristics of sparks found in the Z-line and A-band zones were very similar, whereas sparks from the zone of the perinuclear mitochondria were about 25% longer. Analysis of the initiation sites of Ca2+ sparks within the same junctional SR cluster suggested that 18-25 RyR2s are in the functional group producing a spark. Because of the similarity of the spatio-temporal characteristics of sarcomeric sparks and ultrastructural characteristics of nSR, we suggest that the functional groups of RyR2s in the middle of the sarcomere are macromolecular complexes of approximately 20 RyR2s with regulatory proteins. Our data allowed us to conclude that a significant number of functional RyR2s is located in the middle of the sarcomere and in the zone of perinuclear mitochondria. These RyR2s could contribute to excitation-contraction coupling, mitochondrial and nuclear signalling, and Ca2+-dependent gene regulation, but their existence raises many additional questions.

Asunto(s)

RESUMEN

The outer mitochondrial membrane (OMM) is the last barrier between the mitochondrion and the cytoplasm. Breaches of OMM integrity result in the release of cytochrome c oxidase, triggering apoptosis. In this study, we used calibrated gold nanoparticles to probe the OMM in rat permeabilized ventricular cells and in isolated cardiac mitochondria under quasi-physiological ionic conditions and during permeability transition. Our experiments showed that under control conditions, the OMM is not permeable to 6-nm particles. However, 3-nm particles could enter the mitochondrial intermembrane space in mitochondria of permeabilized cells and isolated cardiac mitochondria. Known inhibitors of the voltage-dependent anion channel (VDAC), König polyanion, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid inhibited this entrance. Thus, 3-nm particles must have entered the mitochondrial intermembrane space through the VDAC. The permeation of the isolated cardiac mitochondria OMM for 3-nm particles was approximately 20 times that in permeabilized cells, suggesting low availability of VDAC pores within the cell. Experiments with expressed green fluorescent protein showed the existence of intracellular barriers restricting the VDAC pore availability in vivo. Thus, our data showed that 1), the physical diameter of VDAC pores in cardiac mitochondria is >or=3 nm but

Asunto(s)

RESUMEN

The physical organization of the ventricular myocyte includes barriers for the movement of objects of varying dimensions ranging from ions to solid particles. There are two kinds of diffusion in the cell: lateral (in membranes) and aqueous. Here we examine the size constraints of aqueous diffusion pathways and discuss their impact on cellular physiology. Calibrated gold nanoparticles were used to probe the accessibility of the entire transverse-axial tubular system (TATS), the sarcoplasm, and intracellular structures. The TATS tubules, although up to 300 nm in diameter, permitted only particles 3 nm; 3), the mitochondrial voltage-dependent anion channel and the nuclear pore complex in ventricular cells could not be penetrated by particles >/=6 nm; and 4), there is a difference in size clearance between transversal and longitudinal sarcoplasmic diffusional pathways.

Asunto(s)

RESUMEN

This article discusses the importance and implications of regulating carbon partitioning to cellulose synthesis, the characteristics of cells that serve as major sinks for cellulose deposition, and enzymes that participate in the conversion of supplied carbon to cellulose. Cotton fibers, which deposit almost pure cellulose into their secondary cell walls, are referred to as a primary model system. For sucrose synthase, we discuss its proposed role in channeling UDP-Glc to cellulose synthase during secondary wall deposition, its gene family, its manipulation in transgenic plants, and mechanisms that may regulate its association with sites of polysaccharide synthesis. For cellulose synthase, we discuss the organization of the gene family and how protein diversity could relate to control of carbon partitioning to cellulose synthesis. Other enzymes emphasized include UDP-Glc pyrophosphorylase and sucrose phosphate synthase. New data are included on phosphorylation of cotton fiber sucrose synthase, possible regulation by Ca2+ of sucrose synthase localization, electron microscopic immunolocalization of sucrose synthase in cotton fibers, and phylogenetic relationships between cellulose synthase proteins, including three new ones identified in differentiating tracheary elements of Zinnia elegans. We develop a model for metabolism related to cellulose synthesis that implicates the changing intracellular localization of sucrose synthase as a molecular switch between survival metabolism and growth and/or differentiation processes involving cellulose synthesis.

Asunto(s)

RESUMEN

The synthesis of crystalline cellulose microfibrils in plants is a highly coordinated process that occurs at the interface of the cortex, plasma membrane, and cell wall. There is evidence that cellulose biogenesis is facilitated by the interaction of several proteins, but the details are just beginning to be understood. In particular, sucrose synthase, microtubules, and actin have been proposed to possibly associate with cellulose synthases (microfibril terminal complexes) in the plasma membrane. Differentiating tracheary elements of Zinnia elegans L. were used as a model system to determine the localization of sucrose synthase and actin in relation to the plasma membrane and its underlying microtubules during the deposition of patterned, cellulose-rich secondary walls. Cortical actin occurs with similar density both between and under secondary wall thickenings. In contrast, sucrose synthase is highly enriched near the plasma membrane and the microtubules under the secondary wall thickenings. Both actin and sucrose synthase lie closer to the plasma membrane than the microtubules. These results show that the preferential localization of sucrose synthase at sites of high-rate cellulose synthesis can be generalized beyond cotton fibers, and they establish a spatial context for further work on a multi-protein complex that may facilitate secondary wall cellulose synthesis.

Asunto(s)

RESUMEN

Flax (Linum usitatissimum L.) fibers originate from procambial cells of the protophloem and develop in cortical bundles that encircle the vascular cylinder. We determined the polysaccharide composition of the cell walls from various organs of the developing flax plant, from fiber-rich strips peeled from the stem, and from the xylem. Ammonium oxalate-soluble polysaccharides from all tissues contained 5-linked arabinans with low degrees of branching, rhamnogalacturonans, and polygalacturonic acid. The fiber-rich peels contained, in addition, substantial amounts of a buffer-soluble, 4-linked galactan branched at the 0-2 and 0-3 positions with nonreducing terminal-galactosyl units. The cross-linking glycans from all tissues were (fucogalacto)xyloglucan, typical of type-I cell walls, xylans containing (1->)-[beta]-D-xylosyl units branched exclusively at the xylosyl O-2 with t-(4-O-methyl)-glucosyluronic acid units, and (galacto)glucomannans. Tissues containing predominantly primary cell wall contained a larger proportion of xyloglucan. The xylem cells were composed of about 60% 4-xylans, 32% cellulose, and small amounts of pectin and the other cross-linking polysaccharides. The noncellulosic polysaccharides of flax exhibit an uncommonly low degree of branching compared to similar polysaccharides from other flowering plants. Although the relative abundance of the various noncellulosic polysaccharides varies widely among the different cell types, the linkage structure and degree of branching of several of the noncellulosic polysaccharides are invariant.