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Abnormalities in repolarization and its rate dependence are known to related to increased proarrhythmic risk. A number of repolarization-related electrophysiological properties are commonly used as preclinical biomarkers of arrhythmic risk. However, the variability and complexity of repolarization mechanisms make the use of cellular biomarkers to predict arrhythmic risk preclinically challenging. Our goal is to investigate the role of ionic current properties and their variability in modulating cellular biomarkers of arrhythmic risk to improve risk stratification and identification in humans. A systematic investigation into the sensitivity of the main preclinical biomarkers of arrhythmic risk to changes in ionic current conductances and kinetics was performed using computer simulations. Four stimulation protocols were applied to the ten Tusscher and Panfilov human ventricular model to quantify the impact of [+ or -] 15 and [+ or -] 30% variations in key model parameters on action potential (AP) properties, [Ca.sup.2+] and [Na.sup.+] dynamics, and their rate dependence. Simulations show that, in humans, AP duration is moderately sensitive to changes in all repolarization current conductances and in L-type [Ca.sup.2+] current ([I.sub.CaL]) and slow component of the delayed rectifier current ([I.sub.Ks]) inactivation kinetics. AP triangulation, however, is strongly dependent only on inward rectifier [K.sup.+] current ([I.sub.KI]) and delayed rectifier current ([I.sub.Kr]) conductances. Furthermore, AP rate dependence (i.e., AP duration rate adaptation and restitution properties) and intracellular [Ca.sup.2+] and [Na.sup.+] levels are highly sensitive to both [I.sub.CaL] and [Na.sup.+]/[K.sup.+] pump current ([I.sub.NaK]) properties. This study provides quantitative insights into the sensitivity of preclinical biomarkers of arrhythmic risk to variations in ionic current properties in humans. The results show the importance of sensitivity analysis as a powerful method for the in-depth validation of mathematical models in cardiac electrophysiology. biological variability: arrhytbmia: computer simulation doi:10.1152/ajpheart.00263.2009

Malic enzymes (MEs) catalyze the oxidative decarboxylation of malate in the presence of a divalent metal ion. In eukaryotes, well-conserved cytoplasmic, mitochondrial, and plastidic MEs have been characterized. On the other hand, distinct groups can be detected among prokaryotic MEs, which are more diverse in structure and less well characterized than their eukaryotic counterparts. In Escherichia coli, two genes with a high degree of homology to ME can be detected: sfcA and maeB. MaeB possesses a multimodular structure: the N-terminal extension shows homology to ME, while the C-terminal extension shows homology to phosphotransacetylases (PTAs). In the present work, a detailed characterization of the products of E. coli sfcA and maeB was performed. The results indicate that the two MEs exhibit relevant kinetic, regulatory, and structural differences. SfcA is a NAD(P) ME, while MaeB is a NADP-specific ME highly regulated by key metabolites. Characterization of truncated versions of MaeB indicated that the PTA domain is not essential for the ME reaction. Nevertheless, truncated MaeB without the PTA domain loses most of its metabolic ME modulation and its native oligomeric state. Thus, the association of the two structural domains in MaeB seems to facilitate metabolic control of the enzyme. Although the PTA domain in MaeB is highly similar to the domains of proteins with PTA activity, MaeB and its PTA domain do not exhibit PTA activity. Determination of the distinct properties of recombinant products of sfcA and maeB performed in the present work will help to clarify the roles of MEs in prokaryotic metabolism.

The role of [Ca.sup.2} in oxygen evolution is examined by monitoring the [S.sub.1] to [S.sub.2] state transition in photosystem II (PSII) membranes and PSII core complexes upon inhibition of oxygen evolution by [Dy.sup.3}, [Cu.sup.2} and [Cd.sup.2} ions. The results show that [Ca.sup.2} has played a vital structural role in the early S-state transitions and that the functional role of [Ca.sup.2} to activate water in the O-O bond-forming reaction that occurs in the final step of the S state cycle can only be fulfilled by [Ca.sup.2} and [Sr.sup.2}.

A GAAA tetraloop domain and receptor domain are connected by a single-strander A7 linker to investigate the metal ion dependence, kinetics and thermodynamics for the isolated tertiary interaction in RNA of the GAAA-tetraloop motif. Studies of docking induced using a variety of metal ions show that the single-stranded region functions primarily as a flexible tether and inhibition of docking by oligonucleotides complementary to the linker is investigated.

How does iron enter enterocytes? Ablating SLC11A2, the gene for the divalent metal ion transporter DMT1, supports evidence from the Belgrade rat and mk mouse models establishing DMT1 as the primary mechanism serving apical uptake of nonheme iron. DMT1 harnesses the energy from the proton electrochemical potential gradient to drive active transport of [Fe.sup.2+] (and perhaps [Mn.sup.2+] and other metal ions) into enterocytes. Fe(III) must first be reduced by ascorbic acid and surface ferrireductases. Among these is duodenal cytochrome B (DcytB), but lack of an obvious phenotype in DcytB (Cybrd1) knockout mice suggests ferrireductase redundancy. Our understanding of heine absorption has lagged, but the time is ripe for gains. anemia: duodenal cytochrome B; divalent metal ion transporter; ferroportin; hemochromatosis; iron absorption; Nramp2; SLC11A2

Electron paramagnetic resonance (EPR) spectroscopy is used to examine changes in internal structure and dynamics of the hammerhead ribozyme upon metal ion induced folding, changes in pH, and the presence and absence of ribozyme inhibitors. Results reveal that different U7 dynamics are observed in the presence of catalytically efficient divalent metal ion concentrations than in the presence of high concentrations of monovalent metal ions.

Two bis(calix[4]diquinone) compounds and one double (calix[4]diquinone) are synthesized from their corresponding double calix[4]arenas. It is demonstrated that double calix[4]arenas can be synthesized in reasonable yields under high pressure.

Four crystal structures of EcoRV endonuclease mutants K92A and K38A gives new insight into the mechanism of DNA bending and the structural basis for metal-dependent phosphodiester bond cleavage. The studies suggest a novel mechanism in which a single initially bound metal ion in a third distinct site undergoes a shift in position together with movement of the scissile phosphate deeper into the active site cleft, which reconfigures the local environment to permit binding of second metal ion.

Research has been conducted on the metal ion-dependent heterodimer factor VIII, a plasma protein participating in the blood coagulation cascade. The differential effect of the metal ions on the factor VIII subunit association and cofactor activity generation has been investigated.

Research has been conducted on the metal-ion-chelating lipids that bear a reactive diacetylene group. The design of the first metal-ion-chelating lipid that can be used in the fluids is described.

Research has been conducted on the series of diazatrithia-15-crown-5 and diazatrithia-16-crown-5 ligands that contain two 8-hydroxyquinoline sidearms. The synthesis and photophysical properties of these ligands have been investigated.

Research has been conducted on the divalent metal ions. The role of these ions in the 2-phosphoglycerate's enolase-catalyzed dehydration has been investigated by spectroscopic and kinetic methods.

Research has been conducted on the Rhodobacter sphaeroides reaction center which protonates the quinone molecule Q (sub)B to form quinol. The importance of residues Asp-M17 and Asp-L210 for Q (sub)B reduction in the absence of metal ions has been investigated.

Research has been conducted on the N-acetylglucosamine-1-PO (sub)4 uridyltransferase (GlmU). The X-ray crystal structure of the Escherichia coli GlmU has been investigated in complex with UDP-GlcNAc and CoA revealing a two-domain atchitecture responsible for these reactions.

Research has been conducted on the metal ion selective conformational changes of recoverin. The relationship between these recoverin changes and its metal-bound coordination structures has been investigated.

Research has been conducted on the catalytic RNA, hammerhead ribozyme. The use of the (super)31 P NMR spectroscopy in investigating the putative metal sites binding metal ions in this ribozyme is discussed.

Research has been conducted on the kynurenine pathway catabolites. The reaction of these catabolites with iron and copper and interactions with alpha-crystallins have been investigated.

Research has been conducted on the copper chaperone for superoxide dismutaes gene. The encoding of the protein that delivers copper ions to copper-zinc superoxide dismutase is described.

Research has been conducted on the hammerhead ribozyme with a phosporodithioate linkage. The role of the metal ion in the catalysis has been investigated.

Research has been conducted on L-ribulose-5-phosphate 4-epimerase mutants. The activity of these mutants when activated by Zn (super)2+ has been investigated.

Catalysis of the hydrolytic removal of the N-terminal formyl group from ribosome-synthesized polypeptides by peptide deformylase is discussed. The ability of the metal ion to ionize the water molecule has been analyzed.

Isothermal titration calorimetry, ITC and gel electrophoretic analysis have been used to investigate the role of metal ions in junction binding and cleavage by enzyme Cce1. Results demonstrate the pivotal role of metal ions in binding Cce1 to DNA junction.

The article discusses the influence of the metal identity on the selectivity of the organic binding and the effect of organic guest on metal affinity. A new approach to the tuning of metal selectivity in fluorescent metal sensors is discussed.

Research concerning ion-selective sensor developing to monitor metal ion concentrations in aqueous solutions has been conducted. Proton-ionizable ligating units have been incorporated into ion-selective macrocycles to provide concomitant changes in the photophysical properties of the system.

Pseudomonas aeruginosa porphobilinogen synthases (PBGS) metalloenzymes bind only four functional active site Mg2+ per octamer that stimulate a metal ion independent catalytic activity. These four Mg2+ are identified by atomic absorption spectroscopy with or without substrate/product. Mn2+ was used to examine P. aeruginosa PBGS while porphobilinogen was used to investigate the active sites. Results show that substrate binding does not involve Mg2+ and that P. aeruginosa PBGS is a type V enzyme which is capable of synthesizing porphobilinogen in a metal ion independent fashion.

Research was conducted to examine the significance of metal ions, namely, an essential structural zinc and a nonessential structural iron, in galactose-1-phosphate uridylyltransferase (GalT). Zinc ligand variants C52S-, C55S-, H115N- and H164N-GalT and the iron ligand variants E182Q- and E182A-GalT were constructed to enable the selective removal of metal from the enzyme. Results indicate that of the two structural metals in GalT, only zinc is important to maintain enzyme activity.

A bispyrenyl bishydroxamate calix(4)arene-based receptor is discussed relative to synthesis and solution structure and its use as a fluorescent chemosensor for Ni(super.2+) and Cu(super.2+) metal ions. In a solution of water with 80% methanol, intensity of fluoresence was related to proton concentration and to presence of transition-metal ions.

Research was conducted to examine the identity of the physiologically relevant metal ions for the methionyl aminopeptidase (MetAP) from Escherichia coli. Inductively coupled plasma emission analysis was used to determine the metal content of whole cells in the absence and presence of expression of the type I MetAP from E. coli. Results indicate that the in vivo metal ions for the MetAP enzyme from E. coli are likely Fe(II) ions.

Research was conducted to examine the potassium and sodium binding to the outer mouth of the K+ channel. The molecular dynamics simulations of the K+ channel from Streptomyces lividans were carried out in a membrane-mimetic environment with Na+ and K+ in various initial locations. Results indicate that the hydration state of the metal ions depends substantially on the conformational flexibility of the channel. The different hydration state of the two ions may be a determinant factor in the ion selectivity of the channel.

Research was conducted to examine the molecular details of the interaction between conserved residues within the metal ion-dependent adhesion site (MIDAS) motif and Mg2+ or Mn2+. The objective was to determine the role of cation in I domain domain function. Results demonstrate that the structural stability provided to the alpha-1-I domain by divalent cations may contribute to augmented ligand binding that occurs in the presence of these cations.

A study of the human ABeta peptide reveals that it directly produces hydrogen peroxide through the reduction of metal ions, Fe(III) or Cu(II). The peptide reduces Fe(III) to Fe(II) and Cu(II) to Cu(I). The molecular oxygen is trapped by ABeta and reduced to hydrogen peroxide in a reaction that is induced by substoichiometric amounts of Fe(II) or Cu(I). The peptide produces a positive thiobarbituric-reactive substance assay in the presence of Fe(III) or Cu(II). These results imply that the accumulation of ABeta could be a direct source of oxidative stress in Alzheimer's disease.

New 8-hydroxyquinoline- and 8-aminoquinoline-substituted tetraazacrown ethers were prepared by reductive amination of the proper aldehydes with the crown ethers using sodium triacetoxyborohydride as the reducing agent. The 8-hydroxyquinoline- and 8-aminoquinoline-containing macrocycles were synthesized by reductive amination of 8-acetoxyquinoline-2-carboxaldehyde or 8-nitroquinoline-2-carboxaldehyde and by removing the acetate groups or reducing the nitro groups to amino groups. Designed to selectively bind transition and post-transition metal ions while modulating the absorption and fluorescence spectra of the compounds, the tetraazacrown ethers were found to selectively complex the target group of metal ions.

Calixarenes carrying amino acids on the lower rim were synthesized by means of the reaction of carboxyl-protected amino acids with the acid chlorides of O-carboxymethylcalix(4)arenes. Using solvent extractions, 1H NMR and CD titrations, it was found that these calixarenes exhibited selective extraction capability toward Na+ and Ag+ ions, while complexing ability was affected by the nature of the solvents. 1H NMR data revealed that the complexation of one calixarene with Na+ ion triggers a change of the orientation of the amide groups from a network-like pattern with circular NH- - -O=C intramolecular hydrogen bonding to a pattern wherein carbonyl groups converge on the Na+ ion.

The mechanism underlying RNA cleavage by the influenza virus endonuclease is investigated using several divalent and trivalent metal ions. It is shown that virus-specific cleavage exists with various metal ions, with maximum cleavage activity derived with 100 microM Mn(super 2+) or 100 microM Co(super 2+). There is also synergistic activation of cleavage activity with combinations of a number of metal ions at different concentrations. These findings suggest that RNA cleavage for the influenza virus cap-dependent endonuclease involves a two-metal ion mechanism.

Spectrophotometric and spectrophotofluorometric methods were used to study the protonation and metal ion complexation behavior of silver ion fluoroionophores synthesized from anthracene-linked polythiazaalkane and polythiaalkane derivatives. The stabilities of the silver ion complexes were found to increase with the number of the sulfur atoms in the complexing components of the fluoroionophores. Results also showed that derivatives containing four sulfur atoms form very stable complexes compared to those containing two sulfur atoms.

The mechanism of conformation and folding of the four-way RNA junction of the hairpin ribozyme was investigated using fluorescence resonance energy transfer. It was shown that the junction exhibits a square configuration of coaxially stacked arms based on A on D and B on C stacking in the absence of added metal ions. The junction undergoes an ion-induced transition to an antiparallel conformation with the addition of magnesium ions. These results are in agreement with folding induced by the binding of a single ion.

The metal ion requirements of ribonucleoprotein ribonuclease P (RNase P) from the slime mold Dictyostelium discoideum were determined through transcription reaction and enzyme purification. RNase P contained both RNA and protein and was considered as the endonuclease required for maturation of the 5'-termini of transfer RNA precursors. It was revealed that in the presence of Mg2+, a bimodal action at the kinetic phase of the RNase P reaction was induced by the alkaline earth metals Ca2+, Sr2+ and Ba2+. Also, the enzyme necessitated an absolute requirement for NH4+ and Mg2+ ions.

A study was conducted to examine the influence of metal ions on the activity and global structure of hairpin ribozymes in their natural junction conformation. T7 RNA polymerase was utilized to determine RNA from synthetic DNA templates while phosphoramidite chemistry was carried out to synthesize DNA and mixed DNA-RNA oligonucleotides. Results indicated that natural ribozyme activity can be influenced by the conformation of the four-way junction.

A study was conducted to analyze photoresponsive tweezers for alkali metal ions. 1H nuclear magnetic resonance was carried out to determine the energy barriers between parallel and antiparallel conformations and was recorded at 200 MHz. Experimental results indicated that the extractability of various alkali picrates was altered by dithienylethene receptors supporting a couple of benzo-crown ethers.

A study was conducted to examine the nature of the catalyzed ethanolysis of N-methyl-2,2,2-trifluoroacetanilide. Kinetic solvent isotope effects were also analyzed to investigate the transition-state structure. Results indicated that a metal coordinated ethanol molecule supported catalysis during the presence of metal ions. Findings also showed that the interaction of the metal ion in the transition state complex supported an important stabilizing function.

A study was conducted to examine the interactions of the N-terminal cellulose-binding domain from Cellulomonas fimi beta-1,4-glucanase CenC with calcium by calorimetry and nuclear magnetic resonance (NMR) spectroscopy. A 50 mM sodium chloride was utilized as a buffer for the NMR studies while a line shape analysis was carried out to determine calcium kinetics. Results indicated that metal ion binding was thermodynamically correlated with native-state stability.

Research was conducted to examine the metal sensor properties of porphyrazines and norphthalocyanines possessing nitrogen donor pockets. The preparation of amino-functionalized porphyrazines and norphthalocyanines was done using Linstead macrocyclization reactions of dinitrile precursors and were subsequently subjected to UV-vis titrations in the presence of inorganic ions. Results indicate that porphyrazines and the norphthalocyanines can bind inorganic metal ions to the peripheral ligating functionality with substantial changes in the UV-vis spectra.

Experiments using exchange-inert metal complex were performed to show that the catalytic metal ion in an acyl-transferase ribozyme acts through outer-sphere coordination. Results showed that acyl-transferase ribozyme needed divalent metal ions for catalytic function. Moreover, the formation of folded structure essential in the function of the ribozyme also needs divalent metal ions such as Mg2+. The number of proton transfer reactions occurring in the rate-limiting step also affects the degree of solvent isotype.

A study was conducted to examine the yvsA-yvqA region of the Bacillus subtilis chromosome which supports genes associated with metal ion uptake and a putative sigma factor. DNA fragments were determined by random primed labeling using the Rediprime system while pUC18 clones were prepared by universal forward and reverse primers. Results indicated that the yvrC, yvrB, yvrA and yvqK genes were arranged in an operon of 3.9 kb in size encoding another putative transporter.

Research was conducted to examine the effects of copper on the structure and role of the subunits of factor VIII, a blood coagulation protein defective in persons with hemophilia A. A Jasco-720 spectropolarimeter was utilized to record CD spectra in the range of 200-250 nm while fluorescence measurements were conducted using a SPEX Fluorolog 1681 spectrometer. Results showed that the conformations of both heavy and light chains can be changed by divalent metal ions.

Metal-ion promoted hydrolytic reactions of the S(sub P) and R(sub P) diastereomers of the phosphoromonothioate analogues of uridylyl(3',5')uridine and their cleavage products, diastereomeric uridine 2',3'-cyclic phosphates were studied by high performance liquid chromatography. The rate-accelerations were almost similar with teh S(sub P) and R(sub P) diastereomers. The metal-ion-promoted reaction was first-order in both the hydroxide and metal ion concentration, and it proceeded through the inversion of configuration at phosphorus.

A thermodynamic experiment was conducted to evaluate the complexation of alkali metal ions with N-phenyl-aza-15-crown-5, N-octylaza-15-crown-5, N-benzylaza-15-crown-5, N-benzylaza-18-crown-6, N-benzylaza-21-crown-7, and N-benzylaza-24-crown-8 in methanol at 25 degrees centigrade. Results show that N-substituents are important in the complexation of alkali metal ions with a nondonating side-chain.

The formation by three new (dialkylamino)pyridine (DAAP)-based ligand amphiphiles with several metal ions as well as their esterolytic capacities in cationic micellar media were examined. A comparison of the catalytic behavior and the cleavage efficiency of the micellar amphiphiles reveals that differences in their structures do not substantially influence their reactivities in the presence or in the absence of Cu(II) ions. Results show that all the Cu(II) complexes discussed suggest true catalytic behavior.

Electronic absorption and electron paramagnetic resonance (EPR) spectroscopy were used to study the changes in the environment, particularly the coordination and spin state of ferric iron of lignin peroxidase isozymes (LiP) during reversible inactivation. It was assumed that removal of loosely bound calcium have no contribution to spectroscopic changes and that the distal calcium plays an important role in the coordination and spin state of heme iron in LiP.