Electron microscopic study of isolated proteoglycans]

Chemically isolated separate preparations of the non-aggregating protein-chondroitin-keratin sulphate (PCKS) fraction from the hyaline cartilage and hyaluronic acid (HUA) of the vitreous body and of the umbilicus were investigated by electron microscopy. PCKS and HUA in films without cytochrome c were present in the form of granules and differed by structural organization. The proteoglycans of the cytochrome c films were seen as finely-filamentous-cellular network, and were distinctly differentiated by their macromolecular organization. A mixture of both proteoglycans formed complexes as a result of a noncovalent interaction. Uranyl acetate ensured a good contrasting of proteoglycans, especially of PCKS, without cytochrome c.     

The role of different proteoglycan salts as factors in steric exclusion

It has been shown that the capacity of Ca2+ salts of hyaluronic acid (HA) and nonaggregating protein-chondroitin-keratan-sulfate (PCKS) to divide in erythrocyte-saline suspension into liquid and cell phases was stronger than the analogous capacity of K+ salts. It was suggested that this is connected with a tendency to form different three-dimensional structures in solutions, which was more expressed in HA and PCKS Ca2+ salts than in K+ salts of these proteoglycans.     

Study of different proteoglycan salts

The authors have devised the methods for preparing free hyaluronic acid (HA) and non-aggregating fraction of protein-chondroitin-keratan sulfate (PCKS), as well as those for preparing their Na+, K+, Ca2+ and Mg2+ salts (acid and neutral). Infrared spectroscopy has demonstrated the presence of intermolecular hydrogen bonds, formed by hydroxyl groups, in HA and PCKS macrocomplexes and in PCKS acid salts. HA salts appeared not to form macrocomplexes at the expense of intermolecular hydrogen bonds.     

Joint action of protein-chondroitin-4-keratan-sulfate and hyaluronic acid on erythrocyte aggregation and adhesion]

It was shown that the rate and the degree of erythrocytes aggregation brought about by a mixture of protein-chondroitin-4-keratan sulfate (PCKS) and hyaluronic acid (HA) was greater than the sum of the values of the corresponding indices observed during separate independent action of these proteoglycans on the aggregation of the mentioned cells concentrations as in the mixtures. It may be supposed that such phenomenon is connected with formation in the mixture of a hybrid PCKS-HA complex which is more active in respect to the erythrocyte aggregation than its components separately.     

Effect of Dextran 500 on Radial Migration of Erythrocytes in Postcapillary Venules at Low Flow Rates

Recently, we reported that collision efficiency (fraction of total collisions that result in the formation of aggregates) between red blood cells was an important factor in the formation of aggregates in postcapillary venules. In the present study, we focus on how high molecular weight dextran influences the overall radial migration trend of red blood cells in the postcapillary venule along a longitudinal distance of 50 μm from the bifurcation which would in turn affect collision behavior of these cells. A radial migration index, which defines the extent of radial migration of individual cells relative to the vessel center, was found to have a larger magnitude after infusion of dextran (1.9 ± 2.73) compared to that before dextran infusion (1.48 ± 3.89). This implied that dextran-induced aggregation might provide an external force to actively move cells towards the centerline of the vessel, which could contribute to the greater number of red blood cells participating in collision (16% increase) and aggregate formation. Further analysis of the collision behavior of individual red blood cells revealed that collision frequencies of individual cells decreased from a wide range (1 to 14) to a narrow range (1 to 5) after dextran treatment, indicating the alteration of collision behavior of red blood cells by the presence of aggregates along the flow stream.     

A precursor to hemoglobin mRNA in nuclei of immature duck red blood cells

A method is described for sedimenting RNA on a preparative scale in the presence of 85% formamide. It is likely that the RNA is fully denatured, since under the same conditions ribosomal RNA develops full hyperchromicity, and formaldehyde-treated and untreated ribosomal and nuclear RNA sediment in an identical manner.RNA from immature duck red blood cells was fractionated on sucrose gradients. The amount of hemoglobin mRNA and poly(A) in each fraction was measured by hybridization with complementary DNA and poly(U), respectively. We observed that hemoglobin mRNA forms aggregates in the presence of phenol, which are dispersed on formamide gradients. Nuclear RNA became more heavily aggregated, and again the aggregates were dispersed by formamide. A possible nuclear precursor of hemoglobin mRNA was identified. The molecular weight of the precursor is 6–7 × 10⁵, three times as long as hemoglobin mRNA, and it is attached covalently to a poly(A) sequence.     

Molecular arrangement between multivalent glycocluster and Pseudomonas aeruginosa LecA (PA‐IL) by atomic force microscopy: influence of the glycocluster concentration

New therapeutics strategy against cystic fibrosis seeks to prevent the adhesion of the bacterium Pseudomonas aeruginosa (PA) on the epithelial cells in the lungs. One of the factors that induces the adhesion is the interaction between natural glycocluster present on the cells and lectins such as the PA lectin LecA (PA‐IL) present on the bacterium. By introducing synthetic glycoclusters with a great affinity with the lectin PA‐IL, the adhesion can be prevented. In this study, we characterized, by atomic force microscopy, the interaction between a tetra‐galactosylated glycocluster and the PA‐IL lectin for high concentration of lectins (2.5 μM).We showed that the strong lectin/lectin interaction is reduced even for low concentration of glycoclusters (1 for 20 000 lectins). We assumed that it is due to the tensioactive behavior of the glycoclusters. It was shown that the arrangement of the created complexes induced different structures evolving from one‐dimensional elongated aggregates to two‐dimensional compact islands when increasing the glycocluster concentration. This evolution can be interpreted as the predominance of the glycocluster/lectin interaction. Copyright © 2013 John Wiley & Sons, Ltd.     

Phosphatidic acid positively regulates LPS-induced differentiation of RAW264.7 murine macrophage cell line into dendritic-like cells

Phosphatidic acid (PA) is an important second messenger produced by the activation of numerous cell surface receptors. Recent data have suggested that PA regulates multiple cellular processes. In this study, we found that PA positively regulates the lipopolysaccharide (LPS)-induced differentiation of RAW264.7 murine macrophage cells into dendritic-like cells. Co-treatment of PA with LPS further increased dendritic cell surface marker expressions (CD80, CD86, CD40, MHC class I, and class II antigens) and reduced the phagocytic activity of LPS-treated cells. Moreover, PA up regulated allostimulatory activity and the secretion of IL-12 in LPS-treated RAW264.7 cells. Taken together, these data indicate that PA might play a role in the LPS-mediated differentiation of macrophage cells into dendritic-like cells.     

Effects of sedimentation of small red blood cell aggregates on blood flow in narrow horizontal tubes

The flow properties of aggregating red cell suspensions flowing at low flow rates through horizontal tubes are analyzed using a theoretical model. The effects of sedimentation of small aggregates, which will be formed at comparatively high flow rates, on the relative apparent viscosity are considered. In the case in which a large number of small aggregates are formed in a suspension flowing through a horizontal tube, it seems that red cells are transported as a concentrated suspension through the bottom part of the tube because of sedimentation of aggregates. A two-layer flow model is used for the distribution of red cells. It consists of plasma in the upper part and a concentrated red cell suspension in the bottom part of the tube divided by a smooth and horizontal interface. It is assumed that the suspension is a Newtonian fluid whose viscosity increases exponentially with hematocrit. The velocity distribution, the relative apparent viscosity and the flux of red cells are calculated as functions of width of plasma layer for a different discharge hematocrit. The theoretical results are compared with the results obtained from experimental data. The relative apparent viscosity increases rapidly with an increasing degree of sedimentation over a wide range of plasma layer widths.     

Fluid dynamics and cell-bound Psl polysaccharide allows microplastic capture,aggregation and subsequent sedimentation by Pseudomonas aeruginosa in water

Decades after incorporating plastics into consumer markets, research shows that these polymers have spread worldwide. Fragmentation of large debris leads to smaller particles, collectively called microplastics (MPs), which have become ubiquitous in aquatic environments. A fundamental aspect of understanding the implications of MP contamination on ecosystems is resolving the complex interactions of these artificial substrates with microbial cells. Using polystyrene microparticles as model polymers, we conducted an exploratory study where these interactions are quantitatively analyzed using an in vitro system consisting of single-bacterial species capturing and aggregating MPs in water. Here we show that the production of Psl exopolysaccharide by Pseudomonas aeruginosa (PA) does not alter MPs colloidal stability but plays a key role in microspheres adhesion to the cell surface. Further aggregation of MPs by PA cells depends on bacterial mobility and the presence of sufficient flow to prevent rapid sedimentation of early MP-PA assembles. Surprisingly, cells in MP-PA aggregates are not in a sessile state despite the production of Psl, enhancing the motility of the aggregates by an order of magnitude relative to passive diffusion. The generated data could inform the creation of predictive models that accurately describe the dynamics and influence of bacterial growth on plastics debris.     

高尿酸血症与肾脏疾病关系的研究进展

高尿酸血症(HUA)是血尿酸(UA)增高的一种疾病,也是常见的机体代谢紊乱,临床上大多数HUA患者无明显的症状。由于经济水平提高和生活方式的改变,其发病率有增高趋势,且男性高于女性。HUA多发于中老年人群,严重影响其生活质量,因此中老年人群HUA已经成为普遍关注的健康问题。近年来研究发现HUA是痛风的主要病因之一,并且血尿酸(UA)水平可导致肾功能减退。已有多项研究表明HUA与肾脏疾病的发生发展密切相关,HUA是Ig A肾病、糖尿病肾病、急性肾损伤等肾脏疾病的独立危险因素,本文主要从HUA的流行病学、危险因素、发病机制及其与肾脏疾病关系这几个方面展开综述。     

ω‐hydroxyundec‐9‐enoic acid induces apoptosis by ROS mediated JNK and p38 phosphorylation in breast cancer cell lines

ω‐Hydroxyundec‐9‐enoic acid (ω‐HUA), a plant secondary metabolite, exhibits anti‐fungal activity. However, its effect on breast cancer cells is unknown. Here, we investigated the anti‐ breast cancer activity of ω‐HUA and its underlying mechanism. Treatment of human breast cancer cell lines, MDA‐MB‐231 and MDA‐MB‐435, with ω‐HUA induced apoptotic cell death with increased cleaved caspase‐3 and poly (ADP‐ribose) polymerase (PARP) levels, and p38 and JNK phosphorylation. Inhibition of these mitogen‐activated protein kinase (MAPK) pathways using specific inhibitors or siRNA, for p38 and JNK, respectively, blocked the ω‐HUA‐induced apoptosis in a dose‐dependent manner. Moreover, pretreatment of the cells with antioxidant N‐acetyl cysteine (NAC) inhibited ω‐HUA‐induced increased reactive oxygen species (ROS) levels, cleaved caspase‐3 and cleaved PARP, and phosphorylated JNK, phosphorylated p38, and increased cell viability and colony‐forming ability. MDA‐MB‐231 xenograft model showed that the ω‐HUA‐treated group exhibited greater tumor regression and significantly reduced tumor weight compared to that exhibited by the vehicle‐administered group. Collectively, ω‐HUA‐induced intracellular ROS generation induced breast cancer cell apoptosis through JNK and p38 signaling pathway activation, resulting in tumor regression. The results suggested that ω‐HUA is an effective supplement for inhibiting human breast cancer growth.     

Protein Aggregates are Transported to Vacuoles by Macroautophagic Mechanism in Nutrient-Starved Plant Cells

When a fusion protein of cytochrome b5 (Cyt b5) and the red fluorescent protein (RFP) are expressed in tobacco BY-2 cells, the expressed protein forms intracellular aggregates that emit red fluorescence. When such cells are grown to the stationary phase or incubated with nutrient limit medium, RFP fluorescence can be detected in the vacuolar lumen. We investigated this transport mechanism using a limited-nitrogen model. E-64 and 3-methyladenine, which inhibit authophagic processes, inhibited the transport of RFP signal to the vacuole. We next traced the autophagic process in tobacco cells using YFP fused with the tobacco Atg8 homologue (YFP–NtAtg8) and analyzed the contribution of autophagy to the vacuolar transport of the aggregates. Under limited-nitrogen conditions, the aggregates were degraded in preference to otherorganelles, and the autophagosomes colocalized with the aggregates at a higher frequency than with mitochondria. This is the first demonstration that selective macroautophagic degradation occurs in plant cells.     

Hemadsorption of Mumps Virus Examined by Light and Electron Microscopy

The hemadsorption (HAD) reaction of chick embryo cells infected with mumps virus was studied by means of light and electron microscopy, with special reference to the plasma membrane of the infected cell. The concomitant observation of membrane-free aggregates of viral nucleocapsid in the cytoplasm and attached red blood cells on the surface of the same cell indicated that only infected cells hemadsorbed and that hemagglutinin is confined within the infected cell. The attachment of red blood cells to morphologically intact cell membrane prior to its differentiation into viral envelope suggested that the HAD phenomenon, dependent on the presence of hemagglutinin, was independent of the viral maturation process. The gap of low electron density normally separating the morphologically intact membrane of the tissue culture cell and that of the red blood cell at the binding site was replaced by newly formed surface projections in HAD involving a segment of differentiated plasma membrane.     

Protein aggregates are transported to vacuoles by a macroautophagic mechanism in nutrient-starved plant cells

When a fusion protein of cytochrome b5 (Cyt b5) and the red fluorescent protein (RFP) are expressed in tobacco BY-2 cells, the expressed protein forms intracellular aggregates that emit red fluorescence. When such cells are grown to the stationary phase or incubated in nutrient limited medium, RFP fluorescence can be detected in the vacuolar lumen. We investigated this transport mechanism using a limited-nitrogen model. E-64 and 3-methyladenine, which inhibit autophagic processes, blocked the transport of the RFP signal to the vacuole. We next traced the autophagic process in tobacco cells using YFP fused with the tobacco Atg8 homologue (YFP-NtAtg8) and analyzed the contribution of autophagy to the vacuolar transport of the aggregates. Under limited-nitrogen conditions, the aggregates were degraded in preference to other organelles, and the autophagosomes colocalized with the aggregates at a higher frequency than with mitochondria. This is the first demonstration that selective macroautophagic degradation can occur in plant cells.     

The Na+/Ca2+ exchanger is active and working in the reverse mode in human umbilical artery smooth muscle cells

The data presented in this work suggest that in human umbilical artery (HUA) smooth muscle cells, the Na(+)/Ca(2+) exchanger (NCX) is active and working in the reverse mode. This supposition is based on the following results: (i) microfluorimetry in HUA smooth muscle cells in situ showed that a Ca(2+)-free extracellular solution diminished intracellular Ca(2+) ([Ca(2+)](i)), and KB-R7943 (5microM), a specific inhibitor of the Ca(2+) entry mode of the exchanger, also decreased [Ca(2+)](i) (40.6+/-4.5% of Ca(2+)-free effect); (ii) KB-R7943 produced the relaxation of HUA rings (-24.7+/-7.3gF/gW, n=8, p<0.05); (iii) stimulation of the NCX by lowering extracellular Na(+) increases basal [Ca(2+)](i) proportionally to Na(+) reduction (Delta fluorescence ratio=0.593+/-0.141 for Na(+)-free solution, n=8) and HUA rings' contraction (peak force=181.5+/-39.7 for 130mM reduction, n=8), both inhibited by KB-R7943 and a Ca(2+)-free extracellular solution. In conclusion, the NCX represents an important Ca(2+) entry route in HUA smooth muscle cells.     

THE MECHANISM OF COMPLEMENT FIXATION

1. Complement fixation is obtained in every antigen-antibody reaction involving the presence or formation of a heterogeneous phase (red cells, bacteria, precipitate). 2. The physical constants of fixation (temperature coefficient, velocity, quantitative relationships between the reactants) are those commonly associated with adsorption processes, and are the same in the three types of fixation studied. 3. All the in vitro immune reactions involve an aggregation of immune-serum globulins upon the surface of the antigen. It has been shown that the "fixation" of complement is an adsorption by the aggregates so formed; whether these aggregates are visible as a flocculent precipitate (e.g., sheep serum vs. anti-serum) or concentrated as a surface film on a cellular antigen (sensitized cells; agglutinated bacteria), the reaction is fundamentally the same. 4. As yet, it is unknown whether this adsorption is determined by the physical state of the precipitate, and thus, differs only quantitatively from that by Kaolin, charcoal, normal bacteria, heat-denatured proteins, etc.; or whether the comparatively enormous avidity of these aggregates for complement is due to a specific chemical affinity.     

Peroxyl-oxidized erythrocyte membrane band 3 protein with anion transport capacity is degraded by membrane-bound proteinase

Human red blood cells anion exchange protein (band 3) exposed to peroxyl radicals produced by thermolysis of 2,2'-azo-bis(2-amidinopropane) (AAPH) is degraded by proteinases that prevent accumulation of oxidatively damaged proteins. To assess whether this degradation affects anion transport capacity we used the anionic fluorescent probe 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-y) amino] ethanosulfonate (NBD-taurine). A decrease of band 3 function was observed after exposure to peroxyl radicals. In the presence of proteinase inhibitors the decrement of anion transport through band 3 was smaller indicating that removal achieved by proteinases includes oxidized band 3 which still retain transport ability. Proteinases recognize band 3 aggregates produced by peroxyl radicals as was evaluated by immunoblotting. It is concluded that decrease of band 3 transport capacity may result from a direct protein oxidation and from its degradation by proteinases and that band 3 aggregates removal may prevent macrophage recognition of the senescent condition which would lead to cell disposal.     

Peroxyl-oxidized Erythrocyte Membrane Band 3 Protein with Anion Transport Capacity is Degraded by Membrane-bound Proteinase

Human red blood cells anion exchange protein (band 3) exposed to peroxyl radicals produced by thermolysis of 2,2′-azo-bis(2-amidinopropane) (AAPH) is degraded by proteinases that prevent accumulation of oxidatively damaged proteins. To assess whether this degradation affects anion transport capacity we used the anionic fluorescent probe 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-y) amino] ethanosulfonate (NBD-taurine). A decrease of band 3 function was observed after exposure to peroxyl radicals. In the presence of proteinase inhibitors the decrement of anion transport through band 3 was smaller indicating that removal achieved by proteinases includes oxidized band 3 which still retain transport ability. Proteinases recognize band 3 aggregates produced by peroxyl radicals as was evaluated by immunoblotting. It is concluded that decrease of band 3 transport capacity may result from a direct protein oxidation and from its degradation by proteinases and that band 3 aggregates removal may prevent macrophage recognition of the senescent condition which would lead to cell disposal.     

1 alpha,25-dihydroxyvitamin D3 modulation in lipid metabolism in established bone marrow-derived stromal cells, MC3T3-G2/PA6.

MC3T3-G2/PA6 (PA6) cells established from newborn mouse calvaria are preadipocytic stromal cells, which differentiate into adipocytes in response to glucocorticoids. We examined the effects of 1 alpha,25-dihydroxyvitamin D3[1 alpha,25(OH)2D3] on adipogenesis in PA6 cells. When PA6 cells were cultured with 10(-8) M dexamethasone, adipocytes containing oil red O-positive droplets first appeared on day 7 (3 days after confluence was attained) and the maximal synthesis of neutral lipids occurred on day 12. Simultaneous addition of 1 alpha,25(OH)2D3 at 10(-9)M completely blocked this dexamethasone-induced neutral lipid synthesis throughout the 14-day culture period. Dose-response studies of vitamin D3 derivatives showed that 1 alpha,25(OH)2D3 was the most potent in inhibiting neutral lipid synthesis in PA6 cells, followed by 1 alpha-hydroxyvitamin D3, 25-hydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3, in that order. Dexamethasone greatly enhanced incorporation of [14C]-acetic acid into triacylglycerol in PA6 cells. The incorporation was markedly inhibited by the addition of 10(-9) M 1 alpha,25(OH)2D3. Instead, 1 alpha,25(OH)2D3 greatly increased incorporation of [14C]-acetic acid into phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, irrespective of the presence or absence of dexamethasone. These results suggest that 1 alpha,25(OH)2D3 modulation of lipid metabolism in bone marrow stromal cells is receptor mediated.