Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate

Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [32P] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [³²P] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca²⁺ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC and , the major isotypes of PKC in BPAECs, by TPA ( 100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [³²P] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.     

Platelet-activating factor receptor agonists mediate xeroderma pigmentosum A photosensitivity

To date, oxidized glycerophosphocholines (Ox-GPCs) with platelet-activating factor (PAF) activity produced non-enzymatically have not been definitively demonstrated to mediate any known disease processes. Here we provide evidence that these Ox-GPCs play a pivotal role in the photosensitivity associated with the deficiency of the DNA repair protein xeroderma pigmentosum type A (XPA). It should be noted that XPA-deficient cells are known to have decreased antioxidant defenses. These studies demonstrate that treatment of human XPA-deficient fibroblasts with the pro-oxidative stressor ultraviolet B (UVB) radiation resulted in increased reactive oxygen species and PAF receptor (PAF-R) agonistic activity in comparison with gene-corrected cells. The UVB irradiation-generated PAF-R agonists were inhibited by antioxidants. UVB irradiation of XPA-deficient (Xpa-/-) mice also resulted in increased PAF-R agonistic activity and skin inflammation in comparison with control mice. The increased UVB irradiation-mediated skin inflammation and TNF-α production in Xpa-/- mice were blocked by systemic antioxidants and by PAF-R antagonists. Structural characterization of PAF-R-stimulating activity in UVB-irradiated XPA-deficient fibroblasts using mass spectrometry revealed increased levels of sn-2 short-chain Ox-GPCs along with native PAF. These studies support a critical role for PAF-R agonistic Ox-GPCs in the pathophysiology of XPA photosensitivity.     

Lipid biomarkers, pigments and cyanobacterial diversity of microbial mats across intertidal flats of the arid coast of the Arabian Gulf (Abu Dhabi, UAE)

Variations in morphology, fatty acids, pigments and cyanobacterial community composition were studied in microbial mats across intertidal flats of the arid Arabian Gulf coast. These mats experience combined extreme conditions of salinity, temperature, UV radiation and desiccation depending on their tidal position. Different mat forms were observed depending on the topology of the coast and location. The mats contained 63 fatty acids in different proportions. The increased amounts of unsaturated fatty acids (12–39%) and the trans/cis ratio (0.6–1.6%) of the cyanobacterial fatty acid n- 18:1ω9 in the higher tidal mats suggested an adaptation of the mat microorganisms to environmental stress. Chlorophyll a concentrations suggested lower cyanobacterial abundance in the higher than in the lower intertidal mats. Scytonemin concentrations were dependent on the increase in solar irradiation, salinity and desiccation. The mats showed richness in cyanobacterial species, with Microcoleus chthonoplastes and Lyngbya aestuarii morphotypes as the dominant cyanobacteria. Denaturing gradient gel electrophoresis patterns suggested shifts in the cyanobacterial community dependent on drainage efficiency and salinity from lower to higher tidal zones. We conclude that the topology of the coast and the variable extreme environmental conditions across the tidal flat determine the distribution of microbial mats as well as the presence or absence of different microorganisms.     

Nitric oxide attenuates H(2)O(2)-induced endothelial barrier dysfunction: mechanisms of protection

Nitric oxide (.NO) attenuates hydrogen peroxide (H(2)O(2))-mediated injury in porcine pulmonary artery endothelial cells (PAECs) and modulates intracellular levels of cGMP and cAMP. We hypothesized that.NO attenuates H(2)O(2)-induced PAEC monolayer barrier dysfunction through cyclic nucleotide-dependent signaling mechanisms. To examine this hypothesis, cultured PAEC monolayers were treated with H(2)O(2), and barrier function was measured as transmonolayer albumin clearance. H(2)O(2) caused significant PAEC barrier dysfunction that was attenuated by intracellular as well as extracellular.NO generation.NO increased PAEC cGMP and cAMP levels, but treatment with inhibitors of soluble guanylate cyclase or protein kinase G did not abrogate.NO-mediated barrier protection. In contrast, H(2)O(2) decreased protein kinase A activity, and inhibiting protein kinase A abrogated the protective effect of.NO. H(2)O(2)-induced barrier dysfunction was not associated with decreased levels of cGMP or cAMP. 3-Isobutyl-1-methylxanthine and the cGMP analog 8-bromo-cGMP had little effect on H(2)O(2)-mediated endothelial barrier dysfunction, whereas 8-bromo-cAMP plus 3-isobutyl-1-methylxanthine was protective. These results indicate that.NO modulates vascular endothelial barrier function through cAMP-dependent signaling mechanisms.     

A morphological model of vertebral trabecular bone

In their micro-structures, typical natural cellular materials such as vertebral trabecular bone have a network of doubly tapered struts, thickening near the strut joints. However, past analytical models for vertebral trabecular bone do not take account of the effect of strut taper on the mechanical properties.This paper presents an analytical cell model comprised of doubly tapered struts to predict the global mechanical properties of vertebral trabecular bone. The predicted results for male, female, and both sexes fit the experimental data well. By considering several strut taper geometries, it is shown that the horizontal Young's modulus and the horizontal uniaxial collapse stress are, in some cases, approximately 1.8- and 2.2-fold higher, respectively, than those of the uniform strut model. This finding illustrates the importance of increased trabecular thickening near the strut joints (i) for improving the accuracy of calculating the mechanical properties and (ii) for the effective treatment of aged bone using drug therapy. It also highlights the need to combine trabecular architecture measurements with information about the morphology near the strut joints.