The -665 C>T polymorphism in the eNOS gene predicts cardiovascular mortality and morbidity in white Europeans.

We recently identified rs3918226 as a hypertension susceptibility locus (-665 C>T), TT homozygosity being associated with higher hypertension risk. T compared with C allele transfected cells had lower endothelial nitric oxide synthase (eNOS) expression. In the family-based Flemish Study on Environment, Genes and Health Outcomes (50.9% women; mean age 40.3 years), we investigated whether 32 TT homozygotes had worse outcomes than 2787 C allele carriers. Over 15 years (median), total and cardiovascular mortality and cardiovascular and coronary events amounted to 269 (9.5%), 98 (3.5%), 247 (8.8%) and 120 (4.3%), respectively. While accounting for family clusters, the hazard ratios associated with TT homozygosity were 4.11 (P=0.0052) for cardiovascular mortality (4 deaths), 2.75 (P=0.0067) for cardiovascular events (7 endpoints) and 3.10 (P=0.022) for coronary events (4 endpoints). With adjustment for cardiovascular risk factors, these hazard ratios were 6.01 (P=0.0003), 2.64 (P=0.0091) and 2.89 (P=0.010), respectively. Analyses unadjusted for blood pressure and antihypertensive treatment produced consistent results. For all fatal plus nonfatal cardiovascular events, the positive predictive value, attributable risk and population-attributable risk associated with TT homozygosity were 21.9, 61.5 and 2.0%, respectively. In conclusion, TT homozygosity at the position -665 in the eNOS promoter predicts adverse outcomes, independent of blood pressure and other risk factors.

Determination of local atomic displacements in CeO(1-x)F(x)BiS2 system.

We have used Bi and Ce L3-edges extended x-ray absorption fine structure measurements to study local structure of CeO(1-x)F(x)BiS2 system as a function of F-substitution. The local structure of both BiS2 active layer and CeO1-xFx spacer layer changes systematically. The in-plane Bi-S1 distance decreases (ΔRmax ∼ 0.08 Å) and the out-of-plane Bi-S2 distance increases (ΔRmax ∼ 0.12 Å) with increasing F-content. On the other hand, the Ce-O/F distance increases (ΔRmax ∼ 0.2 Å) with a concomitant decrease of the Ce-S2 distance (ΔRmax ∼ 0.15 Å). Interestingly, the Bi-S1 distance is characterized by a large disorder that increases with F-content. The results provide useful information on the local atomic displacements in CeO(1-x)F(x)BiS2, that should be important for the understanding of the coexistence of superconductivity and low temperature ferromagnetism in this system.

Interplay between microstructure and magnetism in NiO nanoparticles: breakdown of the antiferromagnetic order.

The possibility of tuning the magnetic behaviour of nanostructured 3d transition metal oxides has opened up the path for extensive research activity in the nanoscale world. In this work we report on how the antiferromagnetism of a bulk material can be broken when reducing its size under a given threshold. We combined X-ray diffraction, high-resolution transmission electron microscopy, extended X-ray absorption fine structure and magnetic measurements in order to describe the influence of the microstructure and morphology on the magnetic behaviour of NiO nanoparticles (NPs) with sizes ranging from 2.5 to 9 nm. The present findings reveal that size effects induce surface spin frustration which competes with the expected antiferromagnetic (AFM) order, typical of bulk NiO, giving rise to a threshold size for the AFM phase to nucleate. Ni(2+) magnetic moments in 2.5 nm NPs seem to be in a spin glass (SG) state, whereas larger NPs are formed by an uncompensated AFM core with a net magnetic moment surrounded by a SG shell. The coupling at the core-shell interface leads to an exchange bias effect manifested at low temperature as horizontal shifts of the hysteresis loop (~1 kOe) and a coercivity enhancement (~0.2 kOe).

Local vibrational properties of GaAs studied by extended X-ray absorption fine structure.

Extended X-ray absorption fine structure (EXAFS) has been measured at both the K edges of gallium and arsenic in GaAs, from 14 to 300 K, to investigate the local vibrational and thermodynamic behaviour in terms of bond expansion, parallel, and perpendicular mean square relative displacements and third cumulant. The separate analysis of the two edges allows a self-consistent check of the results and suggests that a residual influence of Ga EXAFS at the As edge cannot be excluded. The relation between bond expansion, lattice expansion, and expansion due to anharmonicity of the effective potential is quantitatively clarified. The comparison with previous EXAFS results on other crystals with the diamond or zincblende structure shows that the values of a number of parameters determined from EXAFS are clearly correlated with the fractional ionicity and with the strength and temperature interval of the lattice negative expansion.

An x-ray absorption spectroscopy study of Ni-Mn-Ga shape memory alloys.

The austenite to martensite phase transition in Ni-Mn-Ga ferromagnetic shape memory alloys was studied by extended x-ray absorption fine structure (EXAFS) and x-ray absorption near-edge structure (XANES) spectroscopy. The spectra at all the three elements', namely, Mn, Ga and Ni, K-edges in several Ni-Mn-Ga samples (with both Ni and Mn excess) were analyzed at room temperature and low temperatures. The EXAFS analysis suggested a displacement of Mn and Ga atoms in opposite direction with respect to the Ni atoms when the compound transforms from the austenite phase to the martensite phase. The first coordination distances around the Mn and Ga atoms remained undisturbed on transition, while the second and subsequent shells showed dramatic changes indicating the presence of a modulated structure. The Mn rich compounds showed the presence of antisite disorder of Mn and Ga. The XANES results showed remarkable changes in the unoccupied partial density of states corresponding to Mn and Ni, while the electronic structure of Ga remained unperturbed across the martensite transition. The post-edge features in the Mn K-edge XANES spectra changed from a double peak like structure to a flat peak like structure upon phase transition. The study establishes strong correlation between the crystal structure and the unoccupied electronic structure in these shape memory alloys.

Local structure of LiCoO2 nanoparticles studied by Co K-edge x-ray absorption spectroscopy.

We have studied the local structure of LiCoO(2) nanoparticles by Co K-edge x-ray absorption spectroscopy as a function of particle size. Extended x-ray absorption fine structure data reveal substantial changes in the near neighbor distances and the associated mean square relative displacements with decreasing particle size. X-ray absorption near edge structure spectra show clear local geometrical changes with decreasing particle size, similar to those that appear in the charging (delithiation) process. The results suggest that the LiCoO(2) nanoparticles are characterized by a large atomic disorder confined to the Co-O octahedra, similar to the distortions generated during the delithiation, and this disorder should be the primary limiting factor for a reversible diffusion of Li ions when nanoparticles of LiCoO(2) are used as cathode material in rechargeable Li ion batteries.

Synthesis of Ge-imogolite: influence of the hydrolysis ratio on the structure of the nanotubes.

The synthesis protocol for Ge-imogolite (aluminogermanate nanotubes) consists of 3 main steps: base hydrolysis of a solution of aluminum and germanium monomers, stabilization of the suspension and heating at 95 °C. The successful synthesis of these nanotubes was found to be sensitive to the hydrolysis step. The impact of the hydrolysis ratio (from n(OH)/n(Al) = 0.5 to 3) on the final product structure was examined using a combination of characterization tools. Thus, key hydrolysis ratios were identified: n(OH)/n(Al) = 1.5 for the formation of nanotubes with structural defects, n(OH)/n(Al) = 2 for the synthesis of a well crystallized Ge imogolite and n(OH)/n(Al) > 2.5 where nanotube formation is hindered. The capability of controlling the degree of the nanotube's crystallinity opens up interesting opportunities in regard to new potential applications.

Unsuccessful application of taurolidine in the treatment of fungal peritonitis in peritoneal dialysis.

Fungal peritonitis (FP) is a serious complication for peritoneal dialysis (PD) patients, determining hospitalization, technique failure, catheter loss and death. In the 2005 update, treatment recommendations for FP from the International Society of Peritoneal Dialysis (ISPD) advocate catheter removal immediately after fungi are identified by microscopy or culture. The availability of more effective medical treatments could therefore be of great importance. The aim of this report is to describe a case of a 43-year-old, diabetic, HIV positive PD patient with fluconazole resistant Candida peritonitis, who was treated with an i.p. taurolidine solution. Taurolidine is a non-antibiotic antimicrobial, with broad bactericidal and fungicidal properties. It has been used during surgery for lavage of the peritoneum in cases of peritonitis. Its mechanism of action is related to direct toxic action on micro-organisms, through a chemical reaction between active taurolidine derivatives and structures on the cell wall. Treatment failed because the patient had severe burning pain during i.p. administration of the drug, limiting its dose. PD catheter removal allowed complete recovery. It remains undetermined if, with different doses and methodology, taurolidine could be more effective in treating bacterial and/or fungal peritonitis. Currently, catheter removal remains the most effective therapy of fungal peritonitis.

[Prevention and treatment of secondary hyperparathyroidism in non-dialyzed patients with stage 3-5 chronic kidney diease]. / Prevenzione e trattamento dell'iperparatiroidismo secondario nel paziente con malattia renale cronica allo stadio 3-5 non in dialisi.

Deficiencies in vitamin D and vitamin D receptor (VDR) activation adversely affect cardiovascular health in the general population and in people at high risk of cardiovascular disease, as well as contributing to secondary hyperparathyroidism in patients with chronic kidney disease (CKD). Furthermore, epidemiological and observational data indicate that there is a close interrelationship between progressive renal dysfunction in CKD, cardiovascular disease, and mortality. The causes of death in patients even with only moderate kidney dysfunction are commonly associated with cardiovascular events. Modulation of vitamin D levels results in correlative regulatory effects on mineral homeostasis, hypertension, vascular disease, and calcification, as well as a number of other endpoints in cardiac and renal disease. The use of VDR activators to treat these and other parameters outside of cardiovascular and renal disease not only results in enhanced patient health but significantly lowers the risk of mortality in CKD and non-CKD patients with low systemic activity of vitamin D. The cardiovascular and renal systems continue to demonstrate their interrelated effects on each other, particularly when vitamin D and VDR signaling are considered.

Metabolic consequences of peritoneal dialysis treatment.

Energy and protein metabolism are both altered in chronic kidney disease (CKD) from its early stages. Patients undergoing peritoneal dialysis (PD) use peritoneal solutions with glucose as osmotic agent, which exposes them to an increased glucose load (40-80 g/day) and during PD there is a net loss of proteins through the peritoneum (4-8 g/day). Insulin resistance may lead to a reduction of the anabolic effects of insulin, while its proliferative effects on adipose tissue are potentially enhanced. Insulin resistance is also an important factor in the development of hypertriglyceridemia in PD patients: it increases free fatty acid availability, which then stimulates the release of large triglyceride-rich VLDL. Moreover, inhibitors of lipolytic enzymes (apoC-III, inflammation, oxidative modification and carbamoylation of apolipoproteins) may reduce lipid clearance, contributing to the development of dyslipidemia. Inflammatory molecules also play an important role in regulating glucose metabolism, and the excessive activation of inflammatory pathways may represent a fundamental step in the development of insulin resistance, including an over-expression of cytokines. Frequently, protein intake is reduced in PD because of under-dialysis, glucose load, abdominal discomfort and abnormal hormones levels, leading to a complex "protein-energy malnutrition". Optimization of dialysis dose, correction of acidosis and anemia and nutritional counseling, together with "non-traditional" management strategies, such as the use of PD solutions without glucose, like icodextrin and amino acid based solutions, represent the best strategies to prevent and correct malnutrition in PD patients. The mainstay of therapy is a reduction of glucose-based PD solutions and a correct dietary prescription.

[Pathogenesis and treatment of vascular calcification in CKD]. / Invecchiamento cardiovascolare nel soggetto uremico: nuove acquisizioni.

Increased vascular calcification is a major cause of cardiovascular events in patients with chronic kidney disease (CKD). It is the result of an active ossification process counteracted by ''bone'' proteins such as osteopontin, alkaline phosphatase, osteoprotegerin, and osteocalcin. Chronic kidney disease - mineral and bone disorder (CKD-MBD) is a systemic disorder of mineral and bone metabolism that occurs in CKD. In addition to abnormalities in the serum calcium and phosphate profile, CKD-MBD is characterized by abnormalities of bone turnover, mineralization, volume and growth as well as vascular calcification. Considering that the presence and extent of vascular calcification in CKD portend a poor prognosis, many efforts have been made to shed light on this complicated phenomenon to prevent vascular calcium deposition and its progression. Indeed, careful control of calcium load, serum phosphate and parathyroid hormone along with the use of calcium-free phosphate binders and vitamin D receptor activators represent a new therapeutic armamentarium to improve quality of life and reduce mortality in CKD.

Evolution of iron speciation during hydration of C4AF.

It is now well accepted and demonstrated that calcium silicate, calcium aluminate and calcium sulfo aluminate (ettringite, AFm) phases exhibit a good capability to fix metals and metalloids. Unfortunately the role of minor phases and especially calcium-ferric aluminate phase, shorthand C4AF is not well defined. In other systems like in soils or sediments iron phases play a key role in the fixation of pollutant. In cement sorption isotherms, indicated that various metals can be retained by the C4AF hydrated products. Therefore the capabilities of those phase to retain heavy metal should not be neglected. Previous investigations have shown that the minerals formed during the hydration of C4AF are similar to those formed from C3A (pure tri-calcium aluminate) under comparable conditions. Nevertheless no investigation was conducted at the molecular level and there is still a controversy whether Fe substitutes for Al in the hydrated minerals in whole or in part, or if it forms FeOOH clusters scattered throughout the matrix. In this context we have conducted XAS experiments using synchrotron radiation. It was found that the hydration of C4AF forms C3AH6 (hydrogarnet) in which Fe randomly substitutes for Al as well as an amorphous FeOOH phase. Intermediate products like AFm (i.e., an ill organized lamellar phase) are also formed but rapidly evolve to C3AH6; iron does not seem to be incorporated in the AFm structure.

Involvement of DMT1 in uptake of Cd in MDCK cells: role of protein kinase C.

The involvement of iron (Fe) transporters in the uptake of cadmium (Cd) was examined in Madin-Darby kidney cells (MDCK). The uptake of Cd displayed properties that are associated with the Fe transporter divalent metal transporter 1 (DMT1). For example, the uptake of Cd and Fe was reduced by altering the cell membrane potential. The uptake of Cd was blocked by Fe, and the uptake of Fe was blocked by Cd. Also, the uptake of Cd and Fe was higher in MDCK cells bathed in a buffer at low pH. Increased uptake of Fe and Cd was observed in the HEK-293 cell line overexpressing DMT1. Overnight treatment of MDCK cells with the protein kinase C activator phorbol 12,13-dibutyrate (PDBu) resulted in increased uptake of Cd and Fe and an increase in DMT1 mRNA. An increase in newly transcribed DMT1 mRNA was not observed, suggesting that PDBu does not increase DMT1 mRNA by activating transcription. Rather, the increase was most likely due to greater stability of DMT1 mRNA, because the rate of degradation of DMT1 mRNA was slower in MDCK cells treated with PDBu. Our results suggest that Fe and Cd are transported in MDCK cells by a transporter with biochemical properties similar to those of DMT1.

Maitotoxin stimulates Cd influx in Madin-Darby kidney cells by activating Ca-permeable cation channels.

This study examined the role of calcium channels for the uptake of cadmium (Cd) into Madin-Darby canine kidney (MDCK) cells. Maitotoxin, an activator of different types of calcium channels, increased accumulation of 109Cd and 45Ca in MDCK cells. We found that maitotoxin increased accumulation by stimulating 109Cd influx because it did not affect efflux. An inhibitor of store-operated Ca channels, SKF96365, partially blocked 45Ca influx but did not affect 109Cd influx. Ni and Mn, and loperamide and proadifen (SKF 525a), inhibited 45Ca and 109Cd influx in cells stimulated with maitotoxin, but La and nifedipine did not. Overnight treatment with phorbol 12, 13-ibutyrate (PDBu) to activate protein kinase C resulted in a decrease in the concentration of maitotoxin needed to stimulate 45Ca and 109Cd influx. The effect of PDBu was blocked by treating cells with the protein kinase C inhibitor GF109203X. Additionally, the effect of PDBu was lost in cells treated with an inhibitor of RNA synthesis actinomycin D. These results suggest that a Ca permeable cation channel different from voltage-dependent and store-operated Ca channels mediates the uptake of Cd in MDCK cells. The expression of this channel is regulated by protein kinase C.

The role of anion exchange in the uptake of Pb by human erythrocytes and Madin-Darby canine kidney cells.

Anion exchange (AE) plays a critical role in regulating intracellular pH in erythrocytes and epithelial cells and has been suggested to facilitate the transport of lead (Pb) across the erythrocyte cell membrane. In this study we examined the role of AE in the uptake of Pb by human erythrocytes and by Madin-Darby canine kidney (MDCK) cells, the kidney epithelial cell line. Functional AE in MDCK cells was evidenced by: increased uptake of SO(4)(2-) at pH 6.0 over pH 7.0, and inhibition of SO(4)(2-) uptake by the AE inhibitor 4, 4'-diisothiocyanostilbene-2, 2'- disulfonic acid (DIDS) as well as by non-halide anions. Accumulation of Pb into MDCK cells was time and temperature dependent. DIDS inhibited uptake of Pb into human erythrocytes but not MDCK cells. In conclusion, uptake of Pb into erythrocytes but not kidney epithelial cells occurs through AE.

Structure of the trigonal crystal form of bovine annexin IV.

The structure of a trigonal crystal form of N-terminally truncated [des-(1-9)] bovine annexin IV, an annexin variant that exhibits the distinctive property of binding both phospholipids and carbohydrates in a Ca2+-dependent manner, has been determined at 3 A (0.3 nm) resolution -space group: R3; cell parameters: a=b=118.560 (8) A and c=82.233 (6) A-. The overall structure of annexin IV, crystallized in the absence of Ca2+ ions, is highly homologous to that of the other known members of the annexin family. The trimeric assembly in the trigonal crystals of annexin IV is quite similar to that found previously in non-isomorphous crystals of human, chicken and rat annexin V and to the subunit arrangement in half of the hexamer of hydra annexin XII. Moreover, it resembles that found in two-dimensional crystals of human annexin V bound to phospholipid monolayers. The propensity of several annexins to generate similar trimeric arrays supports the hypothesis that trimeric complexes of such annexins, including annexin IV, may represent the functional units that interact with membranes.

The X-ray structure of the DNA-binding domain from the Saccharomyces cerevisiae cell-cycle transcription factor Mbp1 at 2.1 A resolution.

The structure of the DNA-binding domain of the Saccharomyces cerevisiae cell-cycle transcription factor Mbp1 has been solved using the multiwavelength anomalous diffraction (MAD) technique on crystals of selenomethionyl protein and refined at 2.1 A resolution. The molecule is globular, consisting of a twisted, six-stranded beta-barrel that is packed against a loose bundle of four alpha-helices. Two of the beta-strands in combination with two of the helices form a structure characteristic of the DNA-binding motif found in the CAP family of helix-turn-helix transcription factors. In Mbp1, this beta2/alpha2 motif is associated with regions of both positive electrostatic potential and sequence conservation within the Mbp1/Swi4 family, suggesting a role in DNA-binding in these proteins. A combination of structural and biochemical data further indicate a similarity to HNF3gamma/fork head, a member of the family of "winged" helix-turn-helix proteins. We propose a model for DNA-binding involving a recognition helix in the major groove, phosphodiester backbone interactions through the beta-hairpin and further base and/or phosphate interactions mediated by a C-terminal, positively charged loop.

Crystal structures and inhibitor binding in the octameric flavoenzyme vanillyl-alcohol oxidase: the shape of the active-site cavity controls substrate specificity.

BACKGROUND: Lignin degradation leads to the formation of a broad spectrum of aromatic molecules that can be used by various fungal micro-organisms as their sole source of carbon. When grown on phenolic compounds, Penicillium simplicissimum induces the strong impression of a flavin-containing vanillyl-alcohol oxidase (VAO). The enzyme catalyses the oxidation of a vast array of substrates, ranging from aromatic amines to 4-alkyphenols. VAO is a member of a novel class of widely distributed oxidoreductases, which use flavin adenine dinucleotide (FAD) as a cofactor covalently bound to the protein. We have carried out the determination of the structure of VAO in order to shed light on the most interesting features of these novel oxidoreductases, such as the functional significance of covalent flavinylation and the mechanism of catalysis. RESULTS: The crystal structure of VAO has been determined in the native state and in complexes with four inhibitors. The enzyme is an octamer with 42 symmetry; the inhibitors bind in a hydrophobic, elongated cavity on the si side of the flavin molecule. Three residues, Tyr108, Tyr503 and Arg504 form an anion-binding subsite, which stabilises the phenolate form of the substrate. The structure of VAO complexed with the inhibitor 4-(1-heptenyl)phenol shows that the catalytic cavity is completely filled by the inhibitor, explaining why alkylphenols bearing aliphatic substituents longer than seven carbon atoms do not bind to the enzyme. CONCLUSIONS: The shape of the active-site cavity controls substrate specificity by providing a 'size exclusion mechanism'. Inside the cavity, the substrate aromatic ring is positioned at an angle of 18 degrees to the flavin ring. This arrangement is ideally suited for a hydride transfer reaction, which is further facilitated by substrate deprotonation. Burying the substrate beneath the protein surface is a recurrent strategy, common to many flavoenzymes that effect substrate oxidation or reduction via hydride transfer.

Distinct mechanisms of neurotransmitter release from PC 12 cells exposed to lead.

Two enzymes, protein kinase C and microsomal Ca(2+)-ATPase help regulate levels of Ca2+ in many types of cells. Since proteins that regulate Ca2+ often influence sensitivity to Pb2+, we determined the possible roles played by protein kinase C and microsomal Ca(2+)-ATPase for the Pb(2+)-evoked release of norepinephrine (NOR) in PC cells. NOR release was observed at 10 microM Pb2+ when PC 12 cells were stimulated with inhibitors of microsomal Ca(2+)-ATPase such as thapsigargin, cyclopiazonic acid, or 2,5-di-(t-butyl)-hydroquinone. At 5 microM, Pb2+ evoked the release of NOR in PC 12 cells stimulated with activators of protein kinase C such as phorbol 12-myristate 13-acetate (PMA) or (-)-7-octylindolactam. NOR release was observed at 1 microM Pb2+ in the presence of both PMA and thapsigargin. Ni2+ and Cd2+ blocked NOR release stimulated by Pb2+ in the presence of thapsigargin but not by PMA. NOR released by thapsigargin stimulation was not altered in PC 12 cells depleted of protein kinase C. Two proteins found in vesicles, chromogranin B and secretogranin-II were released with NOR. Our results indicate that in PC 12 cells, PB(2+)-evokes the release of neurotransmitters. Furthermore, thapsigargin and PMA increase the cell's sensitivity to Pb2+ by different pathways.

Phosphorylation of membrane proteins in erythrocytes treated with lead.

In immature rat microvessels, endothelial cells and glioma cells, exposure to lead results in an increase in the level of protein kinase C in membranes. In this paper we have extended these studies to human erythrocytes and, in addition, studied the phosphorylation of membrane proteins. A significant increase in the phosphorylation of membrane cytoskeletal proteins of molecular mass 120, 80, 52 and 45 kDa was observed in human erythrocytes treated for 60 min with lead acetate at concentrations greater than 100 nM. These same proteins were phosphorylated when erythrocytes were treated for 10 min with 50 nM phorbol 12-myristate 13-acetate (PMA). Similarly, protein kinase C activity was elevated and an increase in the amount of protein kinase C-alpha was observed in membranes from erythrocytes exposed to concentrations of lead acetate above 100 nM. No changes, however, in the activities of cAMP-dependent protein kinase, protein phosphatases I and IIA or casein kinase were observed. Phosphorylation of these membrane proteins stimulated by lead acetate or by PMA was not observed in erythrocytes depleted of protein kinase C by a 72-h treatment with 500 nM phorbol 12,13-dibutyrate. Finally, no changes in the levels of calcium or diacylglycerol were observed in erythrocytes stimulated with 100 nM lead acetate. These results indicate that, in erythrocytes, lead acetate stimulates the phosphorylation of membrane cytoskeletal proteins by a mechanism dependent on protein kinase C. Since levels of calcium or diacylglycerols did not increase, it appears that lead may activate the enzyme by a direct interaction.