Non-Ionic Contrast Media Induces Oxidative Stress and Apoptosis Through Ca2+ Influx in Human Neutrophils

Non-ionic contrast media (CM) can induce tissue kidney injury via activation of phagocytosis and oxidative stress, although the mechanisms of injury via neutrophils are not clear. We investigated the effects of CM on oxidative stress and Ca²⁺ concentrations in serum and neutrophils of humans. Ten migraine patients were used in the study. Serum and neutrophil samples from patients?? peripheral blood were obtained before (control) and 30?min after non-ionic (iopromide) CM injection. The neutrophils were incubated with non specific transient receptor potential 2 (TRPM2) channel blocker, 2-aminoethoxydiphenyl borate (2-APB), and voltage gated Ca²⁺ channel blockers, verapamil plus diltiazem. Serum and neutrophil lipid peroxidation, apoptosis and intracellular Ca²⁺ concentrations levels were higher in the CM group than in controls. The neutrophilic reduced glutathione (GSH) and glutathione peroxidase (GSH-Px) levels as well as serum vitamin E and ??-carotene concentrations were lower in the CM group than in controls. Neutrophil lipid peroxidation levels were lower in the CM+2-APB and CM+verapamil-diltiazem groups than in the CM group, although GSH, GSH-Px and intracellular Ca²⁺ values increased in the CM+2-APB and CM+verapamil-diltiazem groups. However, caspase-3, caspase-9, vitamin A and vitamin C values were unaltered by CM treatment. In conclusion, we observed that CM induced oxidative stress and Ca²⁺ influx by decreasing vitamin E, ??-carotene and Ca²⁺ release levels in human serum and neutrophils. However, we observed protective effects of Ca²⁺ channel blockers on Ca²⁺ influx in neutrophils.     

The solution structure of the membrane-proximal cytokine receptor domain of the human interleukin-6 receptor

The members of the interleukin-6-type family of cytokines interact with receptors that have a modular structure and are built of several immunoglobulin-like and fibronectin type III-like domains. These receptors have a characteristic cytokine receptor homology region consisting of two fibronectin type III-like domains defined by a set of four conserved cysteines and a tryptophan-serine-X-tryptophan-serine sequence motif. On target cells, interleukin-6 (IL-6) initially binds to its cognate alpha-receptor and subsequently to a homodimer of the signal transducer receptor gp130. The IL-6 receptor (IL-6R) consists of three extracellular domains. The N-terminal immunoglobulin-like domain is not involved in ligand binding, whereas the third membrane-proximal fibronectin-like domain (IL-6R-D3) accounts for more than 90% of the binding energy to IL-6. Here, we present the solution structure of the IL-6R-D3 domain solved by multidimensional heteronuclear NMR spectroscopy.     

Structure/function analysis of spinalin, a spine protein of Hydra nematocysts

The nematocyst capsules of the cnidarians are specialized explosive organelles that withstand high osmotic pressures of approximately 15 MPa (150 bar). A tight disulfide network involving cysteine-rich capsule wall proteins, like minicollagens and nematocyst outer wall antigen, characterizes their molecular composition. Nematocyst discharge leads to the expulsion of a long inverted tubule that was coiled inside the capsule matrix before activation. Spinalin has been characterized as a glycine-rich, histidine-rich protein associated with spine structures on the surface of everted tubules. Here, we show that full-length Hydra spinalin can be expressed recombinantly in HEK293 cells and has the property to form disulfide-linked oligomers, reflecting its state in mature capsules. Furthermore, spinalin showed a high tendency to associate into dimers in vitro and in vivo. Our data, which show incomplete disulfide connectivity in recombinant spinalin, suggest a possible mechanism by which the spine structure may be linked to the overall capsule polymer.     

Neuroprotective Effect of Etomidate in the Central Nervous System of Streptozotocin-Induced Diabetic Rats

It is well known that hyperglycaemia due to diabetes mellitus leads to oxidative stress in the central nervous system. Oxidative stress plays important role in the pathogenesis of neurodegenerative changes. In the present study we investigated the possible neuroprotective effect of etomidate against streptozotocin-induced (STZ-induced) hyperglycaemia in the rat brain and spinal cord. A total of 40 rats were used in this study. Rats were divided into four groups: sham-control, diabetic, diabetic-etomidate treated and vehicle for etomidate treatment group. Diabetes mellitus was induced by a single injection of streptozotocin (60 mg/kg body weight). Three days after streptoztocin injection, etomidate (2 mg/kg) was injected intraperitoneally for etomidate group and lipid emulsion (10%) for vehicle group was injected with corresponding amount intraperitoneally every day for 6 weeks. Six weeks after streptozotocin injection, seven rats from each group were killed and brain, brain stem and cervical spinal cord were removed. The hippocampus, cortex, cerebellum, brain stem and spinal cord were dissected for the biochemical analysis (the level of malondialdehyde [MDA], total nitrite, reduced glutathione [GSH], and xanthine oxidase [XO] activity). STZ-induced diabetes resulted in significantly elevation of MDA, XO and nitrite levels in the hippocampus, cortex, cerebellum, brain stem and spinal cord of the rats (P < 0.05) while etomidate treatment provided significantly lower values (P < 0.05). This study demonstrated that etomidate have neuroprotective effect on the neuronal tissue against the diabetic oxidative damage.     

Attachment and growth of human embryonic stem cells on microcarriers

The use of human embryonic stem cells (hESCs) for cell-based therapies will require large quantities of genetically stable pluripotent cells and their differentiated progeny. Traditional hESC propagation entails adherent culture and is sensitive to enzymatic dissociation. These constraints hamper modifying method from 2-dimensional flat-bed culture, which is expensive and impractical for bulk cell production. Large-scale culture for clinical use will require innovations such as suspension culture for bioprocessing. Here we describe the attachment and growth kinetics of both murine embryonic stem cells (mESCs) and hESCs on trimethyl ammonium-coated polystyrene microcarriers for feeder-free, 3-dimensional suspension culture. mESCs adhered and expanded according to standard growth kinetics. For hESC studies, we tested aggregate (collagenase-dissociated) and single-cell (TrypLE-dissociated) culture. Cells attached rapidly to beads followed by proliferation. Single-cell cultures expanded 3-fold over approximately 5 days, slightly exceeding that of hESC aggregates. Importantly, single-cell cultures were maintained through 6 passages with a 14-fold increase in cell number while still expressing the undifferentiated markers Oct-4 and Tra 1-81. Finally, hESCs retained their capacity to differentiate towards pancreatic, neuronal, and cardiomyocyte lineages. Our studies provide proof-of-principle of suspension-based expansion of hESCs on microcarriers, as a novel, economical and practical feeder-free means of bulk hESC production.     

Influence of trifluoroethanol on membrane interfacial anchoring interactions of transmembrane alpha-helical peptides

Interfacial anchoring interactions between aromatic amino acid residues and the lipid-water interface are believed to be important determinants for membrane protein structure and function. Thus, it is possible that molecules that partition into the lipid-water interface can influence membrane protein activity simply by interfering with these anchoring interactions. Here we tested this hypothesis by investigating the effects of 2,2,2-trifluoroethanol (TFE) on the interaction of a Trp-flanked synthetic transmembrane peptide (acetyl-GW₂(LA)₈LW₂A-NH₂) with model membranes of dimyristoylphosphatidylcholine. Two striking observations were made. First, using ²H nuclear magnetic resonance on acyl chain deuterated lipids, we found that addition of 4 or 8 vol % of TFE completely abolishes the ability of the peptide to order and stretch the lipid acyl chains in these relatively thin bilayers. Second, we observed that addition of 8 vol % TFE reduces the tilt angle of the peptide from 5.3° to 2.5°, as measured by ²H NMR on Ala-d₄ labeled peptides. The “straightening” of the peptide was accompanied by an increased exposure of Trp to the aqueous phase, as shown by Trp-fluorescence quenching experiments using acrylamide. The observation of a reduced tilt angle was surprising because we also found that TFE partioning results in a significant thinning of the membrane, which would increase the extent of hydrophobic mismatch. In contrast to the Trp-flanked peptide, no effect of TFE was observed on the interaction of a Lys-flanked analog (acetyl-GK₂(LA)₈LK₂A-NH₂) with the lipid bilayer. These results emphasize the importance of interfacial anchoring interactions for membrane organization and provide new insights into how molecules such as TFE that can act as anesthetics may affect the behavior of membrane proteins that are enriched in aromatic amino acids at the lipid-water interface.     

Activated protein C enhances human keratinocyte barrier integrity via sequential activation of epidermal growth factor receptor and Tie2

Keratinocytes play a critical role in maintaining epidermal barrier function. Activated protein C (APC), a natural anticoagulant with anti-inflammatory and endothelial barrier protective properties, significantly increased the barrier impedance of keratinocyte monolayers, measured by electric cell substrate impedance sensing and FITC-dextran flux. In response to APC, Tie2, a tyrosine kinase receptor, was rapidly activated within 30 min, and relocated to cell-cell contacts. APC also increased junction proteins zona occludens, claudin-1 and VE-cadherin. Inhibition of Tie2 by its peptide inhibitor or small interfering RNA abolished the barrier protective effect of APC. Interestingly, APC did not activate Tie2 through its major ligand, angiopoietin-1, but instead acted by binding to endothelial protein C receptor, cleaving protease-activated receptor-1 and transactivating EGF receptor. Furthermore, when activation of Akt, but not ERK, was inhibited, the barrier protective effect of APC on keratinocytes was abolished. Thus, APC activates Tie2, via a mechanism requiring, in sequential order, the receptors, endothelial protein C receptor, protease-activated receptor-1, and EGF receptor, which selectively enhances the PI3K/Akt signaling to enhance junctional complexes and reduce keratinocyte permeability.     

The cnidarian nematocyst: a miniature extracellular matrix within a secretory vesicle

Nematocysts are the taxon-defining features of all cnidarians including jellyfish, sea anemones, and corals. They are highly sophisticated organelles used for the capture of prey and defense. The nematocyst capsule is produced within a giant post-Golgi vesicle, which is continuously fed by proteins from the secretory pathway. Mature nematocysts consist of a hollow capsule body in which a long tubule is coiled up that, upon discharge, is expelled in a harpoon-like fashion. This is accompanied by the release of a toxin cocktail stored in the capsule matrix. Nematocyst discharge, which is one of the fastest processes in biology, is driven by an extreme osmotic pressure of about 150 bar. The molecular analysis of the nematocyst has from the beginning indicated a collagenous nature of the capsule structure. In particular, a large family of unusual minicollagens has been demonstrated to form the highly resistant scaffold of the capsule. Recent findings on the molecular composition of Hydra nematocysts have confirmed the notion of a specialized extracellular matrix, which is assembled during an intracellular secretion process to form the most complex predatory apparatus at the cellular level.     

Spatio-temporal genetic variation in populations of wild emmer wheat, <Emphasis Type="Italic">Triticum turgidum</Emphasis> ssp. <Emphasis Type="Italic">dicoccoides</Emphasis>, as revealed by AFLP analysis

Genetic structure of natural populations of wild crop relatives has been the subject of many studies. Yet, most of them focused on the assessment of spatial genetic diversity, while information on long-term variation, affected by yearly changes, has been considered only in few cases. The present study aimed therefore, to estimate the spatio-temporal genetic variation in populations of wild emmer wheat, the progenitor of domesticated wheat, and to assess the contribution of spatial versus temporal factors to the maintenance of genetic variation in a population. Single spikes were collected in the years 1988 and 2002 from plants that grew in the same sampling points, from six different habitats in the Ammiad conservation site, Eastern Galilee, Israel. Seeds were planted in a nursery and DNA was extracted from each plant and analyzed by the AFLP method. Fourteen primer combinations yielded 1,545 bands of which 50.0 and 48.8% were polymorphic in the years 1988 and 2002, respectively. Genetic diversity was much larger within populations than between populations and the temporal genetic diversity was considerably smaller than the spatial one. Nevertheless, population genetic structure may vary to some degree in different years, mainly due to fluctuations in population size because of yearly rainfall variations. This may lead to predominance of different genotypes in different years. Clustering the plants by their genetic distances grouped them according to their habitats, indicating the existence of genotype-environment affinities. The significance of the results in relation to factors affecting the maintenance of polymorphism in natural populations is discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.