Carbonic anhydrase IX reduces E-cadherin-mediated adhesion of MDCK cells via interaction with beta-catenin

Carbonic anhydrase IX (CA IX) is a cancer-associated transmembrane isoform of zinc metalloenzymes that catalyse interconversion between carbon dioxide and bicarbonate. CA IX is strongly induced by tumor hypoxia and has been proposed to participate in acidification of tumor microenvironment and in cell adhesion. To elucidate the cell adhesion-related role of CA IX, we investigated its subcellular localization and relationship to E-cadherin, a key adhesion molecule whose loss or destabilization is linked to tumor invasion. For this purpose, we generated MDCK cells with constitutive expression of human CA IX protein. During the monolayer formation, CA IX was localized to cell-cell contacts and its distribution in lateral membranes overlapped with E-cadherin. Calcium switch-triggered disruption and reconstitution of cell contacts resulted in relocalization of both CA IX and E-cadherin to cytoplasm and back to plasma membrane. A similar phenomenon was observed in hypoxia-treated and reoxygenated cells. Moreover, CA IX-expressing MDCK cells exhibited reduced cell adhesion capacity and lower levels of Triton-insoluble E-cadherin. Finally, CA IX was found to coprecipitate with beta-catenin. We conclude that CA IX has a capacity to modulate E-cadherin-mediated cell adhesion via interaction with beta-catenin, which could be of potential significance in hypoxia-induced tumor progression.     

Photodynamic Damage to Yeast Subcellular Organelles Induced by Elevated Levels of Endogenous Protoporphyrin IX

The 2,2"-dipyridyl-induced accumulation of protoporphyrin IX in Saccharomyces cerevisiae cells was shown to be accompanied by the photoinhibition of cell respiration and the enhancement of the photoinduced permeability of plasma membranes to the fluorescent dye primuline. The visible-light illumination (at 400–600 nm) of the mitochondria and plasma membranes isolated from yeast cells with a high level of endogenous protoporphyrin IX intensified lipid peroxidation in these subcellular organelles. Comparative studies showed that the rad 52 mutant cells, which are deficient in the postreplicative recombinational DNA repair system, are considerably more sensitive to the inactivating action of visible light than are the wild-type cells and the rad 3 mutant cells, which are deficient in the excision DNA repair system. The contribution of photodynamic damage to the yeast subcellular organelles to the lethal photodynamic effect is discussed.     

High-resolution structure and biochemical properties of a recombinant Proteus mirabilis catalase depleted in iron

Heme catalases are homotetrameric enzymes with a highly conserved complex quaternary structure, and their functional role is still not well understood. Proteus mirabilis catalase (PMC), a heme enzyme belonging to the family of NADPH-binding catalases, was efficiently overexpressed in E. coli. The recombinant catalase (rec PMC) was deficient in heme with one-third heme and two-thirds protoporphyrin IX as determined by mass spectrometry and chemical methods. This ratio was influenced by the expression conditions, but the enzyme-specific activity calculated relative to the heme content remained unchanged. The crystal structure of rec PMC was solved to a resolution of 2.0 A, the highest resolution obtained to date with PMC. The overall structure was quite similar to that of wild-type PMC, and it is surprising that the absence of iron had no effect on the structure of the active site. Met 53 close to the essential His 54 was found less oxidized in rec PMC than in the wild-type enzyme. An acetate anion was modeled in an anionic pocket, away from the heme group but important for the enzymatic reaction. An alternate conformation observed for Arg 99 could play a role in the formation of the H-bond network connecting two symmetrical subunits of the tetramer.     

Signalling through the platelet glycoprotein Ib-V-IX complex

The glycoprotein Ib-V–IX is one of the major adhesive receptors expressed on the surface of circulating platelets. It is composed of four different polypeptides—GPIb, GPIbβ, GPIX, and GPV—and represents a multifunctional receptor able to interact with a number of ligands, including the adhesive protein von Willebrand factor, the coagulation factors thrombin, factors XI and XII, and the membrane glycoproteins P-selectin and Mac-1. Interaction of GPIb-V–IX with the subendothelial von Willebrand factor is essential for primary haemostasis, as it initiates platelet adhesion to the subendothelial matrix at the sites of vascular injury even under high flow conditions. Upon interaction with von Willebrand factor, GPIb-V–IX initiates transmembrane signalling events for platelet activation, which eventually result in integrin _IIbβ₃ stimulation and platelet aggregation. The investigation of the biochemical mechanisms for platelet activation by GPIb-V–IX has attracted increasing attention during the last years. This review will describe and discuss recent findings that have provided new insights into the events underlying GPIb-V–IX transmembrane signalling. In particular, it will summarise basic concepts on the structure of this receptor, extracellular ligands, and intracellular interactors potentially involved in transmembrane signalling. The recently suggested role of membrane Fc receptors in GPIb-V–IX-initiated platelet activation will also be discussed, along with the involvement of lipid metabolising enzymes, tyrosine kinases, and the cytoskeleton in the crosstalk between GPIb-V–IX and integrin _IIbβ₃.     

Five- and six-coordinate ruthenium(II) porphyrin tetiaryphosphine complexes,and their reactions with dioxygen via inner- and outer-sphere mechanisms

The 5-coordinate ruthenium(II) octaethylporphyrin complex Ru(OEP)(PPh₃) has been prepared by reduction of Ru(OEP)(PPh₃)Br using zinc amalgam. Both the Ru(OEP)(PPh₃)₃ complexes (n = 1,2) undergo reaction in toluene with O₂ to generate OPPh₃, RuO₂, and the parent porphyrin H₂(OEP); trace water and the μ-oxo dimer [Ru(OEP)(OH)]₂O are implicated in the oxidation reaction, which is considered to be initiated by coordination of O₂ to Ru(OEP)(PPh₃). In contrast, a catalytic O₂-oxidation of excess PPh₃ to the oxide probably goes via an initial outer-sphere reaction with Ru(OEP)(PPh₃)₂, that generates superoxides and Ru(III), both detectable by ESR; the superoxide is believed to be stabilized via portion addition as HO₂· that subsequently disproportionates to O₂ and H₂O₂. PPh₃ is oxidized by the peroxide, and during a reduction step that regenerates the Ru(II) catalyst from Ru(III).     

Constitutive expression of human coagulating factor IX in HeLa cells by homologous recombination of the promoter

Constitutive expression of hFIX protein in nonhepatocytes was studied. The gene targeting vector was constructed and transferred into HeLa cells. With the detection system of PCR, we demonstrated that the endogenoushFIX promoter was replaced with anhCMV promoter when targeted insertion of the constructor was directed by the sequence homology. The expression ofhFIX in the modified HeLa cells, 11.2 ng/10⁶ cell/24 h, strongly suggested thathFIX gene could be activated by a powerful promoter in nonhepatocytes. The results would make it possible to examine the feasibility of re-regulate gene expression by promoter replacement.     

Gene therapy for hemophilia B mediated by recombinant adeno-associated viral vector with hFIXR338A, a high catalytic activity mutation of human coagulation factor IX

A mutant human factor IX with arginine at 338 residual changed to alanine (hFIXR338A) by site-directed mutagenesis was introduced into AAV vectors, and a recombinant adeno-associ- ated viral vector containing hFIXR338A, prepared by rHSV/AAV hybrid helper virus system, was directly introduced to the hind leg muscle of factor IX knock out mice. The expression and the biological activity of human factor IX mutant, hFIXR338A, and the immune response against it in the treated mice were assayed and detected. The results showed that (i) the high-level expression of human factor IX mutant protein, hFIXR338A, has been detected in rAAV-hFIXR338A treated hemophilia B mice and lasted more than 15 weeks; (ii) the clotting activity of hFIXR338A in plasma is 34.2%± 5.23%, which is remarkably higher than that of (14.27% ± 3.4%) of wild type hFIX treated mice in the activated partial thromboplastin assay; (iii) immune response against factor IX R338A was absent, with no factor IX mutant protein (hFIXR338A) inhibitors development in the treated mice; and (iv) no local or systemic side-effects and toxicity associated with the gene transfer were found. It demonstrated the potential use of treating hemophilia B by recombinant adeno-associated viral vectors with mutant hFIXR338A gene, an alternative strategy for hemophilia B gene therapy to wild-type human factor IX.     

The influence of light and darkness on cutaneous fluorescence in mice.

The present work was carried out to investigate the role of light and darkness on the endogenous biosynthesis of porphyrins in mammalian skin (hairless BALB/c mouse) in vivo. In the skin of mice that were constantly kept in darkness (DD), increased endogenous porphyrin fluorescence was observed, which mainly originated from protoporphyrin IX (PpIX). No significant increase in the porphyrin levels was observed in mice that were kept under a normal day-night cycle (LD 12:12 h). The presence of cutaneous PpIX together with ambient light may comprise a photosensitizing mechanism by which PpIX may be a photomessenger between ambient light and internal rhythms.     

Thermal broadening of the Soret band in heme complexes and in heme-proteins: role of iron dynamics

We report the thermal broadening of the Soret band in heme-CO, heme-OH and protoporphyrin IX in the temperature range 300–20 K. For protoporphyrin IX the temperature dependent Gaussian line broadening follows the behavior predicted by the harmonic approximation in the entire temperature range investigated. In contrast, for heme-CO and heme-OH the harmonic behavior is obeyed only up to about 180 K and an anomalous line broadening increase is observed at higher temperatures. This effect is attributed to the onset of anharmonic motions of the iron atom with respect to the porphyrin plane. Comparison with previously reported analogous data for heme proteins enables us to suggest that the onset of substate interconversions in these latter systems can be reflected in motions of the iron atom with respect to the porphyrin plane. Correspondence to: L. Cordone