Heparin‐integrin interaction in endothelial cells: Downstream signaling and heparan sulfate expression

Endothelial cells (ECs) are a source of physiologically important molecules that are synthesized and released to the blood and/or to the subendothelial extracellular matrix such as a heparan sulfate proteoglycan (HSPG) with antithrombotic properties. Previously, we have shown that heparin stimulates the synthesis and modifies the sulfation pattern of this HSPG. Here the molecular mechanisms involved in the up‐regulation of HSPG synthesis by heparin in endothelial cells were decoded. The cells were stimulated with heparin and the expression of HSPG and intracellular pathways were evaluated by a combination of methods involving confocal microscopy, flow cytometry, Western blotting analyses, and [³⁵S]‐sulfate metabolically labeling of the cells. We observed that the up‐regulation of HSPG synthesis evoked by heparin is dependent on the interaction of heparin with integrin since RGD peptide abolishes the effect. The activation of integrin leads to tyrosine‐phosphorylation of focal adhesion‐associated proteins such as FAK, Src, and paxillin. In addition, heparin induces ERK1/2 phosphorylation and inhibitors of Ras and MEK decreased heparin‐dependent HSPG synthesis. Moreover, heparin also induced intracellular Ca²⁺ release, PLCγ1 (phospholipase Cγ1) and CaMKII (calcium calmodulin kinase II) activation, as well as an increase in nitric oxide (NO) production. Finally, an intracellular Ca²⁺ chelator, Ca²⁺ signaling inhibitors, and an endothelial NO synthase inhibitor were all able to abolish the effect in heparan sulfate synthesis. In conclusion, the heparin‐induced up‐regulation of HSPG expression is associated with the phosphorylation of focal adhesion proteins and Ras/Raf/MEK/ERK MAP and Ca²⁺/NO pathways. J. Cell. Physiol. 227: 2740–2749, 2012. © 2011 Wiley Periodicals, Inc.     

Extramedullary hematopoiesis in a case of benign mixed mammary tumor in a female dog: cytological and histopathological assessment

Backgroud  

Extramedullary hematopoiesis (EMH) is defined as the presence of hematopoietic stem cells such as erythroid and myeloid lineage plus megakaryocytes in extramedullary sites like liver, spleen and lymph nodes and is usually associated with either bone marrow or hematological disorders. Mammary EMH is a rare condition either in human and veterinary medicine and can be associated with benign mixed mammary tumors, similarly to that described in this case.     

Centrocestus formosanus (Opisthorchiida: Heterophyidae) as a cause of death in gray tilapia fry Oreochromis niloticus (Perciforme: Cichlidae) in the dry Pacific of Costa Rica

Centrocestusformosanus is a zoonotic trematode from Asia and has been mainly associated as cause of death of cultured fish. To identify pathogen trematode species in tilapia fry (Oreochromis niloticus) and to determine mollusks hosting these parasites, freshwater mollusks were collected from tilapia cultured ponds and experimental infections were carried out with tilapia fries and different mollusk species. A total of 907 freshwater mollusks were obtained from tilapia ponds and were identified to species level, four gastropods and one bivalve were determined: Melania tuberculata, Melanoides turricula, Pomacea flagellata, Haitia cubensis and Anodontiles luteola. For the first time, the presence of M. turricula and H. cubensis are reported in Costa Rica. Seven morphotypes of cercariae (Xifiodiocercaria, Equinostoma, Oftalmocercaria, Parapleurolofocercus, Cistocerca, Furcocercaria and Leptocercaria) parasitizing all five species of mollusks were found, all of distome type. Experimental exposure of tilapia fry to M. tuberculata demonstrated that the parapleurolofocercus morphotype found in the mollusk is in accordance with the finding of C. formosanus in tilapia fry. An abundance and mean intensity of 1018-1027 digeneans per gill in each exposed fish was determined. Centrocestus formosanus is reported for the first time in Costa Rica, for which the primary and secondary intermediate hosts were also determined.     

Solar‐induced chlorophyll fluorescence is strongly correlated with terrestrial photosynthesis for a wide variety of biomes: First global analysis based on OCO‐2 and flux tower observations

Solar‐induced chlorophyll fluorescence (SIF) has been increasingly used as a proxy for terrestrial gross primary productivity (GPP). Previous work mainly evaluated the relationship between satellite‐observed SIF and gridded GPP products both based on coarse spatial resolutions. Finer resolution SIF (1.3 km × 2.25 km) measured from the Orbiting Carbon Observatory‐2 (OCO‐2) provides the first opportunity to examine the SIF–GPP relationship at the ecosystem scale using flux tower GPP data. However, it remains unclear how strong the relationship is for each biome and whether a robust, universal relationship exists across a variety of biomes. Here we conducted the first global analysis of the relationship between OCO‐2 SIF and tower GPP for a total of 64 flux sites across the globe encompassing eight major biomes. OCO‐2 SIF showed strong correlations with tower GPP at both midday and daily timescales, with the strongest relationship observed for daily SIF at the 757 nm (R² = 0.72, p < 0.0001). Strong linear relationships between SIF and GPP were consistently found for all biomes (R² = 0.57–0.79, p < 0.0001) except evergreen broadleaf forests (R² = 0.16, p < 0.05) at the daily timescale. A higher slope was found for C₄ grasslands and croplands than for C₃ ecosystems. The generally consistent slope of the relationship among biomes suggests a nearly universal rather than biome‐specific SIF–GPP relationship, and this finding is an important distinction and simplification compared to previous results. SIF was mainly driven by absorbed photosynthetically active radiation and was also influenced by environmental stresses (temperature and water stresses) that determine photosynthetic light use efficiency. OCO‐2 SIF generally had a better performance for predicting GPP than satellite‐derived vegetation indices and a light use efficiency model. The universal SIF–GPP relationship can potentially lead to more accurate GPP estimates regionally or globally. Our findings revealed the remarkable ability of finer resolution SIF observations from OCO‐2 and other new or future missions (e.g., TROPOMI, FLEX) for estimating terrestrial photosynthesis across a wide variety of biomes and identified their potential and limitations for ecosystem functioning and carbon cycle studies.     

Spectral components of detergent-solubilized bovine cytochrome oxidase

Cytochrome oxidase is the terminal oxidase of the mitochondrial electron transport chain and pumps 4 protons per oxygen reduced to water. Spectral shifts in the α-band of heme a have been observed in multiple studies and these shifts have the potential to shed light on the proton pumping intermediates. Previously we found that heme a had two spectral components in the α-band during redox titrations in living RAW 264.7 mouse macrophage cells, the classical 605?nm form and a blue-shifted 602?nm form. To confirm these spectral changes were not an artifact due to the complex milieu of the living cell, redox titrations were performed in the isolated detergent-solubilized bovine enzyme from both the Soret- and α-band using precise multiwavelength spectroscopy. This data verified the presence of the 602?nm form in the α-band, revealed a similar shift of heme a in the Soret-band and ruled out the reversal of calcium binding as the origin of the blue shift. The 602?nm form was found to be stabilized at high pH or by binding of azide, which is known to blue shift the α-band of heme a. Azide also stabilized the 602?nm form in the living cells. It is concluded there is a form of cytochrome oxidase in which heme a undergoes a blue shift to a 602?nm form and that redox titrations can be successfully performed in living cells where the oxidase operates in its authentic environment and in the presence of a proton motive force.     

Chronic Administration of Lithium Chloride Increases Immunodetectable Glial Fibrillary Acidic Protein in the Rat Hippocampus

Abstract: We studied the effect of treating rats with lithium salts on the content and in vitro phosphorylation rate of the astrocyte cell marker, glial fibrillary acidic protein (GFAP), in brain slices. Rats were fed a diet incorporating lithium chloride until the concentration of Li⁺ in serum reached 0.6–1.2 m M , a range similar to that achieved in clinical practice. Hippocampal tissue was analyzed for immunoreactive GFAP by a dot assay, and slices of hippocampus and caudate nucleus were labeled with [³²P]-phosphate to determine the in vitro rate of phosphorylation of GFAP. Compared with controls, the level of immunoreactive GFAP in the hippocampus from lithium-treated rats was increased 34%, and GFAP in hippocampal slices incorporated 39% more ³²P. This effect of lithium was apparently not confined to the hippocampus because the in vitro rate of phosphorylation of GFAP in caudate slices was also increased in the treated rats.     

Economic evaluation of water loss saving due to the biological control of water hyacinth at New Year’s Dam,Eastern Cape province,South Africa

Water hyacinth Eichhornia crassipes is considered the most damaging aquatic weed in the world. However, few studies have quantified the impact of this weed economically and ecologically, and even fewer studies have quantified the benefits of its control. This paper focuses on water loss saving as the benefit derived from biological control of this plant between 1990 and 2013 at New Year’s Dam, Alicedale, Eastern Cape, South Africa. Estimates of water loss due to evapotranspiration from water hyacinth vary significantly; therefore, the study used three different rates, high, medium and low. A conservative raw agriculture value of R 0.26 per m³ was used to calculate the benefits derived by the water saved. The present benefit and cost values were determined using 10% and 5% discount rates. The benefit/cost ratio at the low evapotranspiration rate was less than one, implying that biological control was not economically viable but, at the higher evapotranspiration rates, the return justified the costs of biological control. However, at the marginal value product of water, the inclusion of the costs of damage to infrastructure, or the adverse effects of water hyacinth on biodiversity, would justify the use of biological control, even at the low transpiration rate.     

Insulin Resistance in PCOS Patients Enhances Oxidative Stress and Leukocyte Adhesion: Role of Myeloperoxidase

Cardiovascular diseases and oxidative stress are related to polycystic ovary syndrome (PCOS) and insulin resistance (IR). We have evaluated the relationship between myeloperoxidase (MPO) and leukocyte activation in PCOS patients according to homeostatic model assessment of IR (HOMA-IR), and have explored a possible correlation between these factors and endocrine and inflammatory parameters. This was a prospective controlled study conducted in an academic medical center. The study population consisted of 101 PCOS subjects and 105 control subjects. We divided PCOS subjects into PCOS non-IR (HOMA-IR<2.5) and PCOS IR (HOMA-IR>2.5). Metabolic and anthropometric parameters, total and mitochondrial reactive oxygen species (ROS) production, MPO levels, interactions between human umbilical vein endothelial cells and leukocytes, adhesion molecules (E-selectin, ICAM-1 and VCAM-1) and proinflammatory cytokines (IL-6 and TNF-α) were evaluated. Oxidative stress was observed in PCOS patients, in whom there was an increase in total and mitochondrial ROS production and MPO levels. Enhanced rolling flux and adhesion, and a decrease in polymorphonuclear cell rolling velocity were also detected in PCOS subjects. Increases in IL-6 and TNF-α and adhesion molecules (E-selectin, ICAM-1 and VCAM-1) were also observed, particularly in the PCOS IR group, providing evidence that inflammation and oxidative stress are related in PCOS patients. HOMA-IR was positively correlated with hsCRP (p<0.001, r = 0.304), ROS production (p<0.01, r = 0.593), leukocyte rolling flux (p<0.05, r = 0.446), E-selectin (p<0.01, r = 0.436) and IL-6 (p<0.001, r = 0.443). The results show an increase in the rate of ROS and MPO levels in PCOS patients in general, and particularly in those with IR. Inflammation in PCOS induces leukocyte-endothelium interactions and a simultaneous increase in IL-6, TNF-α, E-selectin, ICAM-1 and VCAM-1. These conditions are aggravated by the presence of IR.     

Cytotoxicity effect of algal polysaccharides on HL60 cells

The present study shows the cytotoxic effect of three different classes of algal polysaccharides on HL60 cells. Three galactofucans, fucoidan, and glucan were the polysaccharides utilized in this analysis. The parameters used for evaluating cell viability were [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) reduction, protein content, and phosphatase activity. We demonstrated stimulation of phosphatase activity, MTT reduction, and protein content in relation to three types of galactofucans (1, 2, and 3) with different molecular weights (1600, 1200, and 360 kD). However, when HL60 cells were treated with galactofucan type 3, the total protein remained unchanged. Under the same experimental conditions, an expressed increase in the phosphatase activity was detected when galactofucan 3 was utilized. In relation to the mitochondrial function, the stimulation was higher in cells treated with galactofucan type 1. Fucoidan did not have a significant effect on MTT reduction, but protein content was decreased (IC₅₀ around 30 μg/ml). Glucan also activated all the parameters that were analyzed, and this effect was more expressed in the phosphatase activity and in the protein content. This study provides new insights into the cytotoxic action of polysaccharides on HL60 cells and suggests for the first time the possible involvement of phosphatases in this process. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 12, pp. 1613–1617.