β1-Integrin is up-regulated via Rac1-dependent reactive oxygen species as part of the hypertrophic cardiomyocyte response

β₁-Integrin mediates cardiomyocyte growth and survival and its proper regulation is essential for the structural and functional integrity of the heart. β₁-Integrin expression is enhanced in hypertrophy, but the mechanism and significance of its up-regulation are unknown. Because reactive oxygen species (ROS) are important mediators of myocardial remodeling we examined their role in regulated β₁-integrin expression. Hypertrophy was induced in neonatal cardiomyocytes by endothelin-1 (ET-1), which activated the regulatory NADPH oxidase subunit Rac1, evoked ROS, and enhanced fetal gene expression and cardiomyocyte size. ET-1 also enhanced cell adhesion and FAK phosphorylation and inhibited oxidative stress-induced cardiomyocyte apoptosis. Further, ET-1 increased β₁-integrin mRNA and protein expression via Rac1-ROS-dependent MEK/ERK and EGF receptor-PI3K/Akt activation as shown by adenoviral dominant-negative Rac1 or overexpression of copper/zinc-superoxide dismutase. The relevance of regulated β₁-integrin expression was examined in cardiomyocytes, in which targeting siRNA impeded the ET-1-induced β₁-integrin up-regulation. In these cells, ET-1-induced cell adhesion, FAK phosphorylation, and hypertrophic response were significantly blunted, whereas its antiapoptotic effect was predominantly unchanged, suggesting at least partial dissociation of prohypertrophic and prosurvival signaling elicited by ET-1. In conclusion, β₁-integrin up-regulation in response to ET-1 is mediated via Rac1-ROS-dependent activation of prohypertrophic pathways and is mandatory for ET-1-induced FAK activation, cell adhesion, and hypertrophic response.     

Characterization of Receptors for Substance P in Human Astrocytoma Cells: Radioligand Binding and Inositol Phosphate Formation

UC11 cells, derived from a human astrocytoma, have a high density of functional substance P receptors. Radioligand binding studies were conducted with the highly selective neurokinin-1 receptor ligand [3H][Sar9,Met(O2)11]-substance P. Kinetic binding experiments conducted at 4 degrees C yielded an association rate constant k1 of 1.86 x 10(7) M-1 min-1, a dissociation rate constant k-1 of 0.00478 min-1, and a calculated kinetic KD of 257 pM. Saturation binding experiments yielded average values of KD = 447 +/- 103 pM, Bmax = 862 +/- 93 fmol/mg of protein. This Bmax corresponds to more than 150,000 binding sites/cell. Competition binding experiments with unlabeled [Sar9,Met(O2)11]-substance P yielded average values of KD = 491 +/- 48 pM and Bmax = 912 +/- 67 fmol/mg of protein. In [3H]inositol-labeled cells, substance P induced a robust inositol phosphate formation. Inositol trisphosphate levels increased as much as 20-fold within approximately 15 s of addition of substance P. This inositol trisphosphate formation was transient and had returned to baseline within the first 60-120 s. Inositol monophosphate formation, however, was linear for at least 2 h. Structure activity data on binding and inositol monophosphate formation confirmed the presence of a neurokinin-1 receptor subtype in these cells. Thus, the UC11 cell should be a useful model cell for delineating the physiological role of substance P receptors in astrocytes.     

Growth responses of mycorrhizal and non-mycorrhizal tropical forage species to different levels of soil phosphate

Three tropical forage legumes, Stylosanthes capitata, Pueraria phaseoloides and Centrosema macrocarpum, and one grass, Brachiaria dictyoneura, were grown in a sterile phosphate deficient soil amended with soluble or rock phosphate at rates ranging from 0 to 400 mg kg^-1 soil. The effects of inoculation with Glomus manihotis on mycorrhizal infection and plant growth were assessed. Early growth and nodulation of P. phaseoloides in soil with and without rock phosphate fertilizer were also determined. In the legumes, mycorrhizal infection was high at all P levels and sources, except for a significant decrease of infection in S. capitata at high levels of superphosphate. Plant growth was significantly increased by phosphate fertilizer and mycorrhizal inoculation. Mycorrhizal responses were more pronounced at low P levels with both P sources. In B. dictyoneura mycorrhizal infection was decreased with increasing additions of P. No effects of mycorrhizal inoculation (except with no added P) were observed. Growth and nodulation of P. phaseoloides were greatly stimulated by mycorrhizal inoculation.     

Continued proliferation of mitogen-stimulated human peripheral blood lymphocytes: requirement for the restimulation of progeny

The nature and specificity of the stimuli required for the continued proliferation of lymphocytes has been studied by DNA density transfer experiments in which restimulated cells were incubated in medium containing bromodeoxyuridine and also by double label autoradiography. The progeny of cells stimulated first by concanavalin A require an additional stimulus with concanavalin A to replicate. The majority of cells stimulated with phytohemagglutinin, streptolysin O, or staphylococcal filtrate must be restimulated with mitogen in order to replicate. We attribute the quantitative difference between concanavalin A and other mitogens in our experiments to the availability of the competitor, methyl-α-d-mannoside, which permits complete removal of concanavalin A. The progeny of cells stimulated by streptolysin O or staphylococcal filtrate can be restimulated by other mitogens, although in both cases slightly greater stimulation was obtained with the homologous mitogen.     

Differential Expression of Phospholipase C Specific for Inositol Phospholipids at the Cell Surface of Rat Glial Cells and REF52 Rat Embryo Fibroblasts

Abstract: Phosphatidylinositol(PI)-specific phospholipase C activity was detected on the surface of rat astrocytes, rat C6 glioma cells, and rat embryo (REF52) fibroblasts. The cell surface phospholipase C (ecto-PLC) activity was calcium-dependent, did not result from secreted phopholipase C, and was not released from the cell surface by bacterial PI-specific phospholipase C. Agents known to stimulate intracellular PI turnover, including carbachol, L-glutamic acid, acetylcholine, and orthovanadate, did not induce measurable alterations in the activity of the ecto-PLC. The expression of ecto-PLC activity by REF52 fibroblasts was density-dependent: subconfluent cultures of REF52 exhibited low levels of activity (less than 80 pmol of inositol phosphate formed/min/10⁶ cells), whereas in confluent cultures ecto-PLC activity increased to approximately 300 pmol/min/10⁶ cells. In contrast to this behavior and that exhibited by previously reported ecto-PLC-positive cell types, the ecto-PLC activity exhibited by astrocytes (approximately 1,000 pmol/min/10⁶ cells) and by C6 glioma cells (approximately 100 pmol/min/10⁶ cells) was independent of cell culture density up to confluence. The constitutive expression of ecto-PLC activity of astroglial cells may be related to their function as accessory cells in close association with neurons.     

Oligomeric states of bacteriophage T7 gene 4 primase/helicase

Electron microscopic and crystallographic data have shown that the gene 4 primase/helicase encoded by bacteriophage T7 can form both hexamers and heptamers. After cross-linking with glutaraldehyde to stabilize the oligomeric protein, hexamers and heptamers can be distinguished either by negative stain electron microscopy or electrophoretic analysis using polyacrylamide gels. We find that hexamers predominate in the presence of either dTTP or beta,gamma-methylene dTTP whereas the ratio between hexamers and heptamers is nearly the converse in the presence of dTDP. When formed, heptamers are unable to efficiently bind either single-stranded DNA or double-stranded DNA. We postulate that a switch between heptamer to hexamer may provide a ring-opening mechanism for the single-stranded DNA binding pathway. Accordingly, we observe that in the presence of both nucleoside di- and triphosphates the gene 4 protein exists as a hexamer when bound to single-stranded DNA and as a mixture of heptamer and hexamer when not bound to single-stranded DNA. Furthermore, altering regions of the gene 4 protein postulated to be conformational switches for dTTP-dependent helicase activity leads to modulation of the heptamer to hexamer ratio.     

Differences between calcifuge and acidifuge plants in root exudation of low-molecular organic acids

The nature and quantity of low-molecular organic acids (LOAs) exuded by the roots of nine species of calcifuge and nine species of acidifuge wild plants from northern Europe were determined by ion chromatography. Particular attention was paid to differences between the calcifuge and the acidifuge species in the proportions of different LOAs in their root exudates. Great differences in mol% root exudation between the calcifuge and the acidifuge species were found in some acids. The calcifuge species exuded more acetic acid, the acidifuge species more oxalic acid and much more citric acid. In three calcifuge species, however, root exudation of oxalic acid was appreciable, whereas acetic acid exudation was low in these species. The phosphate- and Fe-solubilizing ability of eight LOAs in a rhizosphere limestone soil was also tested. Oxalic acid was the most efficient phosphate solubilizer and citric acid, by far, the most efficient Fe-solubilizer at the concentration (10 mM) tested. It might be hypothesized that acidifuge species use oxalate to solubilize phosphate and citrate to solubilize Fe, in limestone soil. The inability of calcifuge species to grow in limestone soil might, therefore, be due to low root exudation of these acids and, as a result, inability to solubilize phosphate and Fe in limestone soil.     

Action mechanism of PEGylated magainin 2 analogue peptide

PEGylation is frequently used to improve the efficacy of protein and peptide drugs. Recently, we investigated its effects on the action mechanism of the cyclic β-sheet antimicrobial peptide tachyplesin I isolated from Tachypleus tridentatus [Y. Imura, M. Nishida, Y. Ogawa, Y. Takakura, K. Matsuzaki, Action Mechanism of Tachyplesin I and Effects of PEGylation, Biochim. Biophys. Acta 1768 (2007) 1160-1169]. PEGylation did not change the basic mechanism behind the membrane-permeabilizing effect of the peptide on liposomes, however, it decreased the antimicrobial activity and cytotoxicity. To obtain further information on the effects of PEGylation on the activities of antimicrobial peptides, we designed another structurally different PEGylated antimicrobial peptide (PEG-F5W, E19Q-magainin 2-amide) based on the α-helical peptide magainin 2 isolated from the African clawed frog Xenopus laevis. The PEGylated peptide induced the leakage of calcein from egg yolk l-α-phosphatidylglycerol/egg yolk l-α-phosphatidylcholine large unilamellar vesicles, however, the activity was weaker than that of the control peptides. The PEGylated peptide induced lipid flip-flop coupled to the leakage and was translocated into the inner leaflet of the bilayer, indicating that PEGylation did not alter the basic mechanism of membrane permeabilization of the parent peptide. The cytotoxicity of the non-PEGylated peptides was nullified by PEGylation. At the same time, the antimicrobial activity was weakened only by 4 fold. The effects of PEGylation on the activity of magainin were compared with those for tachyplesin.     

Chloride intracellular channel 1 (CLIC1): Sensor and effector during oxidative stress

Oxidative stress, characterized by overproduction of reactive oxygen species (ROS), is a major feature of several pathological states. Indeed, many cancers and neurodegenerative diseases are accompanied by altered redox balance, which results from dysregulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In this review, we consider the role of the intracellular chloride channel 1 (CLIC1) in microglial cells during oxidative stress. Following microglial activation, CLIC1 translocates from the cytosol to the plasma membrane where it promotes a chloride conductance. The resultant anionic current balances the excess charge extruded by the active NADPH oxidase, supporting the generation of superoxide by the enzyme. In this scenario, CLIC1 could be considered to act as both a second messenger and an executor.     

Effects of osmotic pretreatments on oxidative stress, antioxidant profiles and cryopreservation of olive somatic embryos

A three-day pretreatment of olive somatic embryos (SE) with 0.75 M sucrose, combined with cryoprotection (0.5 M DMSO, 1 M sucrose, 0.5 M glycerol and 0.009 M proline) and controlled rate cooling, supported regrowth (as 34.6% fresh weight gain) and resumption of embryo development after cryopreservation. Pretreatment with mannitol or sorbitol did not support regrowth. Profiles of sugars, proline, antioxidant enzymes, Reactive oxygen species (ROS), secondary oxidation products and ethylene were constructed for the most successful (0.75 M) pretreatment series. Sucrose was the optimal pretreatment for supporting recovery, it also elevated glutathione reductase (GR) activity compared to controls, whereas superoxide dismutase (SOD), catalase and guaiacol peroxidase activities remained relatively unchanged. Superoxide dismutase activity was higher in SE pretreated with sucrose, compared with those pretreated with polyols; H₂O₂ was enhanced in SE pretreated with sorbitol and sucrose compared to mannitol. The overall trend for ethylene and OH production revealed their levels were highest in SE pretreated with polyols albeit, for individual treatments this was not always the case. Generally, pretreatments did not significantly change embryo secondary oxidation profiles of ThioBarbituric Acid Reactive Substances (TBARS) and Schiff's bases. In combination these studies suggest oxidative processes may influence regrowth of cryopreserved olive SE and that optimal pretreatments could, in part, increase tolerance by an overall enhancement of endogenous antioxidants (particularly GR), proline and sugars.