bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Combination of Coordination Polyhedra in Mg2Co3Sn10+x ‐ The First Phase in the Mg/Co/Sn System
Authors: Schreyer Martin , Kraus Günther , Fässler Thomas F.
Published: October 2004

Mg2Co3Sn10+x is the first compound in the ternary system Mg/Co/Sn. Single crystals for structure analysis were grown from the elements by a tin flux method. The structure (P63/mmc, a = 9.490(1) Å and c = 33.555(7) Å) was determined by X‐ray single crystal analysis (R1 = 0.037). A small phase width of 0 ≤ x ≤ 0.15, due to the partial occupancy of one out of ten tin positions is observed. There are two crystallographically different cobalt sites each with a unique tin coordination sphere. The Co1 site has a square antiprismatic coordination with tin atoms comparable to the ones observed in the tin rich binary stannides. The Co2 site is characterized by a trigonal prismatic coordination with tin similar to previously known ternary stannides. The different polyhedra are arranged in alternating layers in an intricate way which gives rise to tin‐tin distances shorter than 3Å. Magnesium is only found in those layers which have cobalt trigonal prismatically coordinated to tin atoms. Thus, the structure shows an unprecedented arrangement of different coordination spheres, and furthermore provides evidence for homoatomic tin bonding within a metallic framework. Magnetic susceptibility measurements show temperature independent Pauli‐paramagnetism down to 50 K.
The First Reduced Rare Earth Halide with a Group 11 Element as Interstitial: La3I3Au
Authors: Mattausch Hansjürgen , Zheng Chong , Kienle Lorenz , Simon Arndt
Published: October 2004

The title compound was synthesized from La, LaI3 and Au under Ar atmosphere in the temperature range 850‐950 °C. It crystallizes in the cubic space group I4132 with lattice constant a = 12.661(2) Å. The structure features Au‐centered helical octahedron chains arranged in all three directions with 90 degree turns. It is the first reduced rare earth halide with a group 11 interstitial. It is very air and moisture sensitive, and produces crimson‐colored Au precipitate similar to Gold Purple of Cassius upon reaction with water or ethanol.
New Oxovanadium(IV) Complexes with Mixed Ligands ‐ Synthesis, Thermal Stability, and Crystal Structure of (VO)2(acac)2(μ‐OEt)2 and (VO)2(thd)2(μ‐OEt)2
Authors: Ahmed M. A. K. , Fjellvåg H. , Kjekshus A. , Klewe B.
Published: October 2004

Synthesis, crystal structure, thermal stability, and magnetic properties of the new oxovanadium(IV) complexes [(VO)2L2(μ‐OEt)2] with mixed β‐diketonato [L = acac = C5H7O2‐ or L = thd = C11H19O2‐] and ethanolato (OEt) ligands are reported. The structure determinations of (VO)2(acac)2(μ‐OEt)2 [P21/c, with a = 14.775(2), b = 8.788(1), c = 15.742(2) Å, β = 112.43(1)°] and (VO)2(thd)2(μ‐OEt)2 [$P{\bar 1}$, with a = 8.871(3), b = 9.988(4), c = 10.299(4) Å, α = 73.51(1), β = 68.70(1), γ = 65.85(1)°] are based on single‐crystal X‐ray diffraction data collected at 105 K. Structure data for VO(acac)2 and VO(thd)2 are redetermined at 105 and 295 K. (VO)2(acac)2(μ‐OEt)2 and (VO)2(thd)2(μ‐OEt)2 comprise binuclear five‐coordinated molecular units. The configuration of five oxygen atoms around a central vanadium atom is approximately square pyramidal in all the four studied complexes. (VO)2(thd)2(μ‐OEt)2 exhibits conspicuous changes in unit‐cell dimensions (expansion in a and c, shrinkage in b, and appreciable variation in α, β, and γ) on going from 105 to 295 K, which appear to reflect adjustments in the configuration of its (anti‐arranged) ligands. The interatomic distances in the crystal structures are analyzed in terms of the bond‐valence concept. The four‐valent state of vanadium is verified by magnetic susceptibility measurements. All complexes are subject to sublimation.
Automatic measurement of particulate contaminants (dirts) in cotton linters
Authors: Smith J. W. , Ferguson G. N. , Mills J. D.
Published: October 2004

An electronic image scanning measurement method was developed to obtain objective measurements of the area of resistant dark contaminants, broadly referred to as dirts, contained in cottonseed linters and hull fibers. The method is based upon the use of the AOCS standard cellulose yield residue. The yield residue (20 g) was bleached and formed into a 10 × 12 in. test sheet, and the projected area of visible dirts in 42 fields on each side of the sheet was meausred. Areas as small as 0.1 mm2 in a field 30 × 41 mm can be measured. The results were converted to area, summed, and compared with the visual measurements obtained using the method of Jurbergs and Dowling. Statistical analysis gave standard deviations of ca. 8% and 25%, respectively, for new and the earlier procedures. The instrument standard deviation was shown to be ca. 1% relative. A comparison of the two methods using samples of 1099 different lots of linters gave a 0.94 correlation coefficient.
Lubricants and lubricant additives: I. Performance characteristics of N‐mono and N,N‐Disubstituted fatty amides and modified fatty amides
Authors: Magne F. C. , Mod R. R. , Sumrell G. , Parker W. E. , Koos R. E.
Published: October 2004

A number of N‐mono and N,N‐disubstituted amides of substituted and unsubstituted C18 fatty acids have been prepared and evaluated as base lubricants and lubricant additives. The neat epithioamides possess extreme pressure lubricant characteristics and are noncorrosive at normal temperatures. The epithioamides also frequently function as extreme pressure additives and antiwear agents for paraffinic or diester base oils, sometimes in both capacities for both base oils. The intensiveness of these properties correlated directly with the degree of thiirane substitution in the compound. Performance in both these capacities at the same levels of epithioation was dependent upon the N‐ or N,N‐substituent groups present.
Rapid and nondestructive determination of seed oil by pulsed nuclear magnetic resonance technique
Authors: Tiwari P. N. , Gambhir P. N. , Rajan T. S.
Published: October 2004

The pulsed NMR technique for rapid and nondestructive determination of oil in oilseeds has been developed. The effects of spin‐lattice relaxation time, spin‐spin relaxation time, seed moisture, angular position of the seeds, sample tube thickness, and sample height upon the magnitude and reproducibility of the NMR signal were studied. Based upon these studies, various parameters for seed oil analysis have been fixed. The oil content of Brassica, peanut, and sunflower seeds was determined. The reproducibility of the measurement is ± 1 %. The technique was tested by measuring the oil content of the same seeds by the cold percolation method (CCl4 extraction). It was further tested by determining the oil content of 60 Brassica seed samples independently at three laboratories. The results of these tests are given.
Excitotoxicity and bioenergetics in glutaryl‐CoA dehydrogenase deficiency
Authors: KÖlker S. , Koeller D. M. , Sauer S. , HÖrster F. , Schwab M. A. , Hoffmann G. F. , Ullrich K. , Okun J. G.
Published: October 2004

Summary: Glutaryl‐CoA dehydrogenase deficiency is an inherited organic acid disorder with predominantly neurological presentation. The biochemical hallmark of this disease is an accumulation and enhanced urinary excretion of two key organic acids, glutaric acid and 3‐hydroxyglutaric acid. If untreated, acute striatal damage is often precipitated by febrile illnesses during a vulnerable period of brain development in infancy or early childhood, resulting in a dystonic dyskinetic movement disorder. 3‐Hydroxyglutaric and glutaric acids are structurally similar to glutamate, the main excitatory amino acid of the human brain, and are considered to play an important role in the pathophysiology of this disease. 3‐Hydroxyglutaric acid induces excitotoxic cell damage specifically via activation of N‐methyl‐D‐aspartate receptors. It has also been suggested that secondary amplification loops potentiate the neurotoxic properties of these organic acids. Probable mechanisms for this effect include cytokine‐stimulated NO production, a decrease in energy metabolism, and reduction of cellular creatine phosphate levels. Finally, maturation‐dependent changes in the expression of neuronal glutamate receptors may affect the vulnerability of the immature brain to excitotoxic cell damage in this disease.
Maintenance treatment of glutaryl‐CoA dehydrogenase deficiency
Authors: Mühlhausen C. , Hoffmann G. F. , Strauss K. A. , KÖlker S. , Okun J. G. , Greenberg C. R. , Naughten E. R. , Ullrich K.
Published: October 2004

Summary: This paper summarizes the published experience as well as results of the 3rd International Workshop on Glutaryl‐CoA Dehydrogenase Deficiency held in October 2003 in Heidelberg, Germany, on the topic treatment of patients with glutaryl‐CoA dehydrogenase (GCDH) deficiency. So far no international recommendation for treatment of GCDH deficiency exists. Such an approach is hampered by several facts, namely the lack of an in‐depth understanding of the pathophysiology of the disease, the lack of prospective studies, including the evaluation of drug monotherapy, and lack of objective documentation of clinical changes (e.g. video documentation) during pharmacotherapy.
Looking forward—An evidence‐based approach to glutaryl‐CoA dehydrogenase deficiency
Authors: KÖlker S. , Burgard P. , Okun J. G. , Schulze‐Bergkamen A. , Assmann B. , Greenberg C. R. , Hoffmann G. F.
Published: October 2004

Summary: Three decades after the first description of glutaryl‐CoA dehydrogenase deficiency, major progress has been achieved in the prevention of acute striatal necrosis and neurological sequelae in affected children, if diagnosis is made early and treatment is started before manifestation of acute encephalopathic crises. However, all concepts for diagnostic work‐up, monitoring, and treatment are solely experience‐based, and 10–35% of early‐diagnosed children do not or only incompletely benefit from the current management. They still develop neurological deterioriation and sequelae despite early implementation of dietary treatment, carnitine supplementation and emergency treatment during acute intercurrent illnesses. International efforts should be made to move management of affected children from experience‐based to evidence‐based medicine. Major tools for this optimization are the establishment of an international patients' database, the implementation of an international prospective clinical study, and the development of international guidelines for diagnostic work‐up, monitoring and therapy.
Isobutyryl‐CoA dehydrogenase deficiency: Isobutyrylglycinuria and ACAD8 gene mutations in two infants
Authors: Sass J. O. , Sander S. , Zschocke J.
Published: October 2004

Summary: Isobutyryl‐CoA dehydrogenase (IBD) is an enzyme involved in the catabolism of the branched‐chain amino acid valine. We report a third and a fourth child with IBD deficiency who were both detected during newborn screening with tandem mass spectrometry and so far do not receive any treatment. The diagnosis was confirmed by biochemical and molecular studies. One of the children is homozygous for the mutation M128I in the ACAD8 gene, which is predicted to affect the substrate binding cavity. The other child is compound heterozygous for a frameshift mutation F33fsins and a missense mutation V203I. It is as yet uncertain whether IBD deficiency may cause significant morbidity in affected children and whether treatment is necessary. In view of the limited experience worldwide, careful monitoring of the children is recommended.
Molecular basis of hypoxanthine‐guanine phosphoribosyltransferase deficiency in Italian Lesch‐Nyhan patients: Identification of nine novel mutations
Authors: Bertelli M. , Randi D. , Micheli V. , Gallo S. , Andrighetto G. , Parmigiani P. , Jacomelli G. , Carella M. , Lievore C. , Pandolfo M.
Published: October 2004

Summary: Lesch‐Nyhan syndrome (LSN, McKusick 300322) is an X‐linked genetic disease due, in its typical form, to the complete absence of hypoxanthine‐guanine phosphoribosyltransferase (HPRT, EC 2.4.2.8) enzyme activity. It is characterized by hyperuricaemia, leading to gout and kidney stones, accompanied by severe neurological dysfunction with self‐injurious behaviour, choreoathetosis and spasticity. Based on a worldwide birth incidence estimate of about 1:380000, one or two new cases are expected every year in Italy. We performed biochemical and molecular genetic studies on 28 Italian patients from 25 families who are likely to represent most living individuals with the syndrome in the country. They all had absent HPRT activity and a typical LNS phenotype. Genetic analysis identified 24 HPRT mutations, 9 of which had not been previously reported: 74C>G (P25R), IVS2+1G>C, 194‐195delTC, 329‐332delCAAC insTCTs, IVS9‐1G>A, 506insC, IVS8‐1G>C, 606G>T (L202F), 418G>C (G140R). No mutation hotspots were identified. Only two mutations were found in more than one family, indicating the lack of any major mutation causing LNS in Italy. Three mutations arose de novo, two in the proband's mother, one in the maternal grandmother. The virtual complete absence of HPRT activity was related to deletions, nonsense, or missense mutations leading to nonconservative amino acid changes.
Carnitine transporter defect due to a novel mutation in the SLC22A5 gene presenting with peripheral neuropathy
Authors: Makhseed N. , Vallance H.D. , Potter M. , Waters P.J. , Wong L.T.K. , Lillquist Y. , Pasquali M. , Filippo C. Amat di San , Longo N.
Published: October 2004

Summary: The carnitine transporter defect (McKusick 212140) is an autosomal recessive disorder caused by mutations in the SLC22A5 gene, which encodes the high‐affinity carnitine transporter OCTN2 (Wang et al 2001). Diagnosis is suspected when plasma carnitine levels are extremely low and secondary causes of carnitine loss are excluded. The disease can present with recurrent Reye‐like episodes of hypoketotic hypoglycaemia or with cardiomyopathy associated with myopathy (Stanley et al 1991). Here we report novel clinical findings in a 3‐year‐old with primary carnitine deficiency.
An assessment of the statistical significance of the total ozone changes simulated with global chemical transport model MEZON
Authors: Karol I. , Kiselev A. , Zubov V. , Egorova T. , Hoyle C. , Rozanov E.
Published: October 2004

This paper is intended to investigate the statistical significance of the ozone recovery amounts computed with the chemical transport model (CTM) MEZON, given errors in the reaction rates and model initial conditions. The CTM is used to investigate the sensitivity of the ozone to Montreal Protocol and its Amendments (MPA) restrictions on the emission of ozone depleting substances in the atmosphere during 1992–2002. The known Student's t‐test is applied for the assessment of the statistical significance of the “saved” total ozone (TO) by the MPA. The results reveal that the uncertainties in the initial fields do not substantially reduce the statistical significance of the results. Substantial reduction of the statistical significance is caused by the uncertainty in the kinetic constants. The model noise level reaches its highest values (up to 10%) in the polar areas during late winter ‐ early spring. The “saved” TO also reaches its maximum there and, therefore, it is significant at the 90–95% confidence level.
Improving total column ozone retrievals by using cloud pressures derived from Raman scattering in the UV
Authors: Vasilkov Alexander P. , Joiner Joanna , Yang Kai , Bhartia Pawan K.
Published: October 2004

The higher spectral resolution, coverage, and sampling of the Aura satellite ozone monitoring instrument (OMI), as compared with the total ozone mapping spectrometer (TOMS) should allow for improved ozone retrievals. By default, the TOMS‐like OMI total column ozone algorithm uses climatological cloud‐top pressures based on infrared (IR) measurements to estimate the column ozone below the clouds. Alternatively, cloud pressure can be retrieved using atmospheric rotational Raman scattering. The retrieved cloud pressures should be more consistent with assumptions made in the total ozone algorithm. Here, we use data from the global ozone monitoring experiment (GOME) to estimate total ozone using both the IR‐climatological and retrieved cloud pressures. The resulting ozone differences can be significant but do not exceed ∼15 DU. Use of the cloud pressure retrievals leads to a smoother distribution of ozone along a satellite track by reducing small spatial irregularities presumably caused by the difference between the retrieved and climatological cloud pressures.
Correlation between particle injections observed at geosynchronous orbit and the Dst index during geomagnetic storms
Authors: Moon Ga‐Hee , Ahn B.‐H. , Kamide Y. , Reeves G. D.
Published: October 2004

To understand the relationship between geomagnetic storms and substorms, we examine the correlation between dispersionless proton injections observed by geosynchronous satellites and the Dst index during geomagnetic storms. It is not possible to deduce the amount of the total energy injection into the inner magnetosphere from measurements only by one or two satellites. Nonetheless, we may obtain a quantity that is proportional to the true injection rate during magnetic storms by estimating the flux increase expressed in terms of the flux ratio (fmax/fpre_ave) and the number of injections, where fpre_ave and fmax represent the average flux of prestorm level and onset level, respectively. Thus we propose to introduce a parameter, “total energy injection parameter” (TEIP), defined by the product of the flux ratio and the number of injections, as an indicator of the energy injected into the inner magnetosphere. Several interesting points are noted, particularly for the main phase of storms. First, the number of particle injections tends to increase with the storm size. Second, the flux ratio (fmax/fpre_ave) also tends to increase with the storm size. Third, there is also a significantly high correlation between TEIP and Dstmin. In particular, the correlation coefficients are very high, above 0.85, for those channels of energy, 50–400 keV, which represent the typical energy range of ring current particles. These results indicate that the substorm expansion activity is higher during the main phase than the recovery phase, suggesting that the substorm expansion activity seems to be closely associated with the development of magnetic storms.
Staphylococcus aureus DNA ligase: characterization of its kinetics of catalysis and development of a high-throughput screening compatible chemiluminescent hybridization protection assay
Authors: GUL Sheraz , BROWN Richard , MAY Earl , MAZZULLA Marie , SMYTH Martin G. , BERRY Colin , MORBY Andrew , POWELL David J.
Published: October 2004

DNA ligases are key enzymes involved in the repair and replication of DNA. Prokaryotic DNA ligases uniquely use NAD+ as the adenylate donor during catalysis, whereas eukaryotic enzymes use ATP. This difference in substrate specificity makes the bacterial enzymes potential targets for therapeutic intervention. We have developed a homogeneous chemiluminescence-based hybridization protection assay for Staphylococcus aureus DNA ligase that uses novel acridinium ester technology and demonstrate that it is an alternative to the commonly used radiometric assays for ligases. The assay has been used to determine a number of kinetic constants for S. aureus DNA ligase catalysis. These included the Km values for NAD+ (2.75±0.1 μM) and the acridinium-ester-labelled DNA substrate (2.5±0.2 nM). A study of the pH-dependencies of kcat, Km and kcat/Km has revealed values of kinetically influential ionizations within the enzyme–substrate complexes (kcat) and free enzyme (kcat/Km). In each case, the curves were shown to be composed of one kinetically influential ionization, for kcat, pKa=6.6±0.1 and kcat/Km, pKa=7.1±0.1. Inhibition characteristics of the enzyme against two Escherichia coli DNA ligase inhibitors have also been determined with IC50 values for these being 3.30±0.86 μM for doxorubicin and 1.40±0.07 μM for chloroquine diphosphate. The assay has also been successfully miniaturized to a sufficiently low volume to allow it to be utilized in a high-throughput screen (384-well format; 20 μl reaction volume), enabling the assay to be used in screening campaigns against libraries of compounds to discover leads for further drug development.
Diminished synthesis of subunit a (ATP6) and altered function of ATP synthase and cytochrome c oxidase due to the mtDNA 2 bp microdeletion of TA at positions 9205 and 9206
Authors: JEŠINA Pavel , TESAŘOVÁ Markéta , FORNŮSKOVÁ Daniela , VOJTÍŠKOVÁ Alena , PECINA Petr , KAPLANOVÁ Vilma , HANSÍKOVÁ Hana , ZEMAN Jiří , HOUŠTĚK Josef
Published: October 2004

Dysfunction of mitochondrial ATPase (F1Fo-ATP synthase) due to missense mutations in ATP6 [mtDNA (mitochondrial DNA)-encoded subunit a] is a frequent cause of severe mitochondrial encephalomyopathies. We have investigated a rare mtDNA mutation, i.e. a 2 bp deletion of TA at positions 9205 and 9206 (9205ΔTA), which affects the STOP codon of the ATP6 gene and the cleavage site between the RNAs for ATP6 and COX3 (cytochrome c oxidase 3). The mutation was present at increasing load in a three-generation family (in blood: 16%/82%/>98%). In the affected boy with severe encephalopathy, a homoplasmic mutation was present in blood, fibroblasts and muscle. The fibroblasts from the patient showed normal aurovertin-sensitive ATPase hydrolytic activity, a 70% decrease in ATP synthesis and an 85% decrease in COX activity. ADP-stimulated respiration and the ADP-induced decrease in the mitochondrial membrane potential at state 4 were decreased by 50%. The content of subunit a was decreased 10-fold compared with other ATPase subunits, and [35S]-methionine labelling showed a 9-fold decrease in subunit a biosynthesis. The content of COX subunits 1, 4 and 6c was decreased by 30–60%. Northern Blot and quantitative real-time reverse transcription–PCR analysis further demonstrated that the primary ATP6 – COX3 transcript is cleaved to the ATP6 and COX3 mRNAs 2–3-fold less efficiently. Structural studies by Blue-Native and two-dimensional electrophoresis revealed an altered pattern of COX assembly and instability of the ATPase complex, which dissociated into subcomplexes. The results indicate that the 9205ΔTA mutation prevents the synthesis of ATPase subunit a, and causes the formation of incomplete ATPase complexes that are capable of ATP hydrolysis but not ATP synthesis. The mutation also affects the biogenesis of COX, which is present in a decreased amount in cells from affected individuals.
Glycoprotein VI/Fc receptor γ chain-independent tyrosine phosphorylation and activation of murine platelets by collagen
Authors: JARVIS Gavin E. , BEST Denise , WATSON Steve P.
Published: October 2004

We have investigated the ability of collagen to induce signalling and functional responses in suspensions of murine platelets deficient in the FcRγ (Fc receptor γ) chain, which lack the collagen receptor GPVI (glycoprotein VI). In the absence of the FcRγ chain, collagen induced a unique pattern of tyrosine phosphorylation which was potentiated by the thromboxane analogue U46619. Immunoprecipitation studies indicated that neither collagen alone nor the combination of collagen plus U46619 induced phosphorylation of the GPVI-regulated proteins Syk and SLP-76 (Src homology 2-containing leucocyte protein of 76 kDa). A low level of tyrosine phosphorylation of phospholipase Cγ2 was observed, which was increased in the presence of U46619, although the degree of phosphorylation remained well below that observed in wild-type platelets (∼10%). By contrast, collagen-induced phosphorylation of the adapter ADAP (adhesion- and degranulation-promoting adapter protein) was substantially potentiated by U46619 to levels equivalent to those observed in wild-type platelets. Collagen plus U46619 also induced significant phosphorylation of FAK (focal adhesion kinase). The functional significance of collagen-induced non-GPVI signals was highlighted by the ability of U46619 and collagen to induce the secretion of ATP in FcRγ chain-deficient platelets, even though neither agonist was effective alone. Protein tyrosine phosphorylation and the release of ATP were abolished by the anti-(α2 integrin) antibodies Ha1/29 and HMα2, but not by blockade of αIIbβ3. These results illustrate a novel mechanism of platelet activation by collagen which is independent of the GPVI–FcRγ chain complex, and is facilitated by binding of collagen to integrin α2β1.
Disruption of dimerization and substrate phosphorylation inhibit factor inhibiting hypoxia-inducible factor (FIH) activity
Authors: LANCASTER David E. , McNEILL Luke A. , McDONOUGH Michael A. , APLIN Robin T. , HEWITSON Kirsty S. , PUGH Christopher W. , RATCLIFFE Peter J. , SCHOFIELD Christopher J.
Published: October 2004

HIF (hypoxia-inducible factor) is an αβ transcription factor that modulates the hypoxic response in many animals. The cellular abundance and activity of HIF-α are regulated by its post-translational hydroxylation. The hydroxylation of HIF is catalysed by PHD (prolyl hydroxylase domain) enzymes and FIH (factorinhibiting HIF), all of which are 2-oxoglutarate- and Fe(II)-dependent dioxygenases. FIH hydroxylates a conserved asparagine residue in HIF-α (Asn-803), which blocks the binding of HIF to the transcriptional co-activator p300, preventing transcription of hypoxia-regulated genes under normoxic conditions. In the present paper, we report studies on possible mechanisms for the regulation of FIH activity. Recently solved crystal structures of FIH indicate that it is homodimeric. Site-directed mutants of FIH at residues Leu-340 and Ile-344, designed to disrupt dimerization, were generated in order to examine the importance of the dimeric state in determining FIH activity. A single point mutant, L340R (Leu-340→Arg), was shown to be predominantly monomeric and to have lost catalytic activity as measured by assays monitoring 2-oxoglutarate turnover and asparagine hydroxylation. In contrast, the I344R (Ile-344→Arg) mutant was predominantly dimeric and catalytically active. The results imply that the homodimeric form of FIH is required for productive substrate binding. The structural data also revealed a hydrophobic interaction formed between FIH and a conserved leucine residue (Leu-795) on the HIF substrate, which is close to the dimer interface. A recent report has revealed that phosphorylation of Thr-796, which is adjacent to Leu-795, enhances the transcriptional response in hypoxia. Consistent with this, we show that phosphorylation of Thr-796 prevents the hydroxylation of Asn-803 by FIH.
Cathepsin K: a cysteine protease with unique kinin-degrading properties
Authors: GODAT Emmanuel , LECAILLE Fabien , DESMAZES Claire , DUCHÊNE Sophie , WEIDAUER Enrico , SAFTIG Paul , BRÖMME Dieter , VANDIER Christophe , LALMANACH Gilles
Published: October 2004

Taking into account a previous report of an unidentified enzyme from macrophages acting as a kininase, the ability of cysteine proteases to degrade kinins has been investigated. Wild-type fibroblast lysates from mice, by contrast with cathepsin K-deficient lysates, hydrolysed BK (bradykinin), and released two metabolites, BK-(1–4) and BK-(5–9). Cathepsin K, but not cathepsins B, H, L and S, cleaved kinins at the Gly4–Phe5 bond and the bradykinin-mimicking substrate Abz (o-aminobenzoic acid)-RPPGFSPFR-3-NO2-Tyr (3-nitrotyrosine) more efficiently (pH 6.0: kcat/Km=12500 mM−1·s−1; pH 7.4: kcat/Km=6930 mM−1·s−1) than angiotensin-converting enzyme hydrolysed BK. Conversely Abz-RPPGFSPFR-3-NO2-Tyr was not cleaved by the Y67L (Tyr67→Leu)/L205A (Leu205→Ala) cathepsin K mutant, indicating that kinin degradation mostly depends on the S2 substrate specificity. Kininase activity was further evaluated on bronchial smooth muscles. BK, but not its metabolites BK(1-4) and BK(5-9), induced a dose-dependent contraction, which was abolished by Hoe140, a B2-type receptor antagonist. Cathepsin K impaired BK-dependent contraction of normal and chronic hypoxic rats, whereas cathepsins B and L did not. Taking together vasoactive properties of kinins and the potency of cathepsin K to modulate BK-dependent contraction of smooth muscles, the present data support the notion that cathepsin K may act as a kininase, a unique property among mammalian cysteine proteases.
Vif is an auxiliary factor of the HIV-1 reverse transcriptase and facilitates abasic site bypass
Authors: CANCIO Reynel , SPADARI Silvio , MAGA Giovanni
Published: October 2004

The HIV-1 accessory protein Vif was found to modulate the RNA- and DNA-dependent DNA synthesis activity of the viral RT (reverse transcriptase) in two ways: (i) it stimulated the binding of the viral RT to the primer by increasing the association rate kcat/Km and by decreasing the thermodynamic barrier ΔH[ES] for complex fomation, and (ii) it increased the polymerization rate of HIV-1 RT. A Vif mutant lacking the final 56 amino acids at the C-terminus failed to stimulate the viral RT. On the other hand, another Vif mutant lacking the first 43 amino acids at the N-terminus, which are involved in RNA binding and interaction with the viral protease, was able to stimulate RT activity. In addition, Vif was found to promote the bypass of an abasic site by HIV-1 RT.
Sulpho-conjugation of ethanol in humans in vivo and by individual sulphotransferase forms in vitro
Authors: SCHNEIDER Heiko , GLATT Hansruedi
Published: October 2004

We studied whether ethanol is sulphonated in humans with the perspective of using the urinary excretion of ethyl sulphate after ethanol consumption as a biomarker for SULT (sulphotransferase) activity. We developed a sensitive and selective HPLC–MS/MS method for determining ethyl sulphate in urine. Ten volunteers received a low dose of ethanol (0.1 g/kg of body mass). In general, excretion of ethyl sulphate was maximal in the first or second hour after dosage. Within 8 h, 2.5–6.8 μmol of ethyl sulphate was excreted. A 5-fold increase in the dose of ethanol led to an increase in the amount of ethyl sulphate excreted within 8 h (28–95 μmol) and the presence of this metabolite in urine for at least 24 h. Since ethyl sulphate was still being excreted for a substantial period after the elimination of ethanol, it might be used as a medium-time biomarker for preceding ethanol consumption. We have expressed previously all human SULT forms identified in Salmonella typhimurium. Ethanol sulphonation was studied in cytosolic preparations of these strains. The highest activities were observed with SULT1A2, 1B1 and 1C2, followed by 1A3. Activities were markedly lower with SULT1E1, 1A1 and 2A1, and were negligible with SULT1C1, 2B1a, 2B1b and 4A1. If the expression levels in tissues are additionally taken into account, SULT1A3 might be the predominant form for the sulphonation of ethanol in vivo, although a robust estimate requires further studies. With this limitation, urinary ethyl sulphate excretion appears very promising as a biomarker for SULT activity in vivo.
The NRF-1/α-PAL transcription factor regulates humanE2F6promoter activity
Authors: KHERROUCHE Zoulika , DE LAUNOIT Yvan , MONTE Didier
Published: October 2004

E2F6 is widely expressed in human tissues and cell lines. Recent studies have demonstrated its involvement in developmental patterning and in the regulation of various genes implicated in chromatin remodelling. Despite a growing number of studies, nothing is really known concerning the E2F6 expression regulation. To understand how cells control E2F6 expression, we analysed the activity of the previously cloned promoter region of the human E2F6 gene. DNase I footprinting, gel electrophoreticmobility shift, transient transfection and site-directed mutagenesis experiments allowed the identification of two functional NRF-1/α-PAL (nuclear respiratory factor-1/α-palindrome-binding protein)-binding sites within the human E2F6 core promoter region, which are conserved in the mouse and rat E2F6 promoter region. Moreover, ChIP (chromatin immunoprecipitation) analysis demonstrated that overexpressed NRF-1/α-PAL is associated in vivo with the E2F6 promoter. Furthermore, overexpression of full-length NRF-1/α-PAL enhanced E2F6 promoter activity, whereas expression of its dominant-negative form reduced the promoter activity. Our results indicate that NRF-1/α-PAL is implicated in the regulation of basal E2F6 gene expression.
Cell-penetrating peptides do not cross mitochondrial membranes even when conjugated to a lipophilic cation: evidence against direct passage through phospholipid bilayers
Authors: ROSS Meredith F. , FILIPOVSKA Aleksandra , SMITH Robin A. J. , GAIT Michael J. , MURPHY Michael P.
Published: October 2004

CPPs (cell-penetrating peptides) facilitate the cellular uptake of covalently attached oligonucleotides, proteins and other macromolecules, but the mechanism of their uptake is disputed. Two models are proposed: direct movement through the phospholipid bilayer and endocytic uptake. Mitochondria are a good model system to distinguish between these possibilities, since they have no vesicular transport systems. Furthermore, CPP-mediated delivery of macromolecules to the mitochondrial matrix would be a significant breakthrough in the study of mitochondrial function and dysfunction, and could also lead to new therapies for diseases caused by mitochondrial damage. Therefore we investigated whether two CPPs, penetratin and Tat, could act as mitochondrial delivery vectors. We also determined whether conjugation of the lipophilic cation TPP (triphenylphosphonium) to penetratin or Tat facilitated their uptake into mitochondria, since TPP leads to uptake of attached molecules into mitochondria driven by the membrane potential. Neither penetratin nor Tat, nor their TPP conjugates, are internalized by isolated mitochondria, indicating that these CPPs cannot cross mitochondrial phospholipid bilayers. Tat and TPP–Tat are taken up by cells, but they accumulate in endosomes and do not reach mitochondria. We conclude that CPPs cannot cross mitochondrial phospholipid bilayers, and therefore cannot deliver macromolecules directly to mitochondria. Our findings shed light on the mechanism of uptake of CPPs by cells. The lack of direct movement of CPPs through mitochondrial phospholipid bilayers, along with the observed endosomal accumulation of Tat and TPP–Tat in cells, makes it unlikely that CPPs enter cells by direct membrane passage, and instead favours cellular uptake via an endocytic pathway.
Dilating the degradome: matrix metalloproteinase 2 (MMP-2) cuts to the heart of the matter
Authors: OVERALL Christopher M.
Published: October 2004

With recent work revealing that MMPs (matrix metalloproteinases) cleave an increasingly large degradome of bioactive and signalling molecules, the dogma that MMPs are extracellular-matrix-remodelling proteases is under challenge. In this issue of the Biochemical Journal, Martínez et al. have reported that AM (adrenomedullin), a potent vasodilator predominantly expressed by blood vessel endothelial and smooth muscle cells, and microvasculature-rich tissues, is another new bioactive substrate for MMPs in vivo. Cleavage by MMP-2, but not MMP-9, generates a series of peptides; two of which retain receptor agonist and vasodilator activity, three are inactive and, excitingly, AM(11–22), a small product containing a canonical disulphide loop, is a vasoconstrictor. In view of the robust vasodilatory and other cardiac protective activities of AM in inhibiting myocardial fibrosis this represents a potent new systemic role for MMP-2 in the cardiovasculature. Hence, the paper by Martínez et al. directly implicates MMP activity in the development of hypertension and paradoxically in stimulating myocardial fibrosis, therefore pointing to exciting new possibilities for utilizing MMP-2-specific inhibitors as a new mode to treat blood pressure and heart disease.
Proteomic analysis for the assessment of diabetic renal damage in humans
Authors: MISCHAK Harald , KAISER Thorsten , WALDEN Michael , HILLMANN Meike , WITTKE Stefan , HERRMANN Alena , KNUEPPEL Stefan , HALLER Hermann , FLISER Danilo
Published: October 2004

Renal disease in patients with Type II diabetes is the leading cause of terminal renal failure and a major healthcare problem. Hence early identification of patients prone to develop this complication is important. Diabetic renal damage should be reflected by a change in urinary polypeptide excretion at a very early stage. To analyse these changes, we used an online combination of CE/MS (capillary electrophoresis coupled with MS), allowing fast and accurate evaluation of up to 2000 polypeptides in urine. Employing this technology, we have examined urine samples from 39 healthy individuals and from 112 patients with Type II diabetes mellitus and different degrees of albumin excretion rate. We established a ‘normal’ polypeptide pattern in the urine of healthy subjects. In patients with Type II diabetes and normal albumin excretion rate, the polypeptide pattern in urine differed significantly from normal, indicating a specific ‘diabetic’ pattern of polypeptide excretion. In patients with higher grade albuminuria, we were able to detect a polypeptide pattern indicative of ‘diabetic renal damage’. We also found this pattern in 35% of those patients who had low-grade albuminuria and in 4% of patients with normal albumin excretion. Moreover, we could identify several of the indicative polypeptides using MS/MS sequencing. We conclude that proteomic analysis with CE/MS permits fast and accurate identification and differentiation of polypeptide patterns in urine. Longitudinal studies should explore the potential of this powerful diagnostic tool for early detection of diabetic renal damage.
B-group vitamin supplementation mitigates oxidative damage after acute ischaemic stroke
Authors: ULLEGADDI Rajesh , POWERS Hilary J. , GARIBALLA Salah E.
Published: October 2004

Evidence shows that there is a rapid increase in the production of markers of oxidative damage immediately following acute stroke and that endogenous antioxidant defences are rapidly depleted, thus permitting further tissue damage. Several studies point to an antioxidant effect of B-group vitamins and a pro-oxidant effect of elevated plasma tHcy (total homocysteine). In the present study, we assessed whether supplementary B-group vitamins during this critical period will enhance antioxidant capacity and mitigate oxidative damage. Forty-eight patients with acute ischaemic stroke within 12 h of symptom onset were assigned to receive daily oral supplements of B-group vitamins comprising 5 mg of folate, 5 mg of vitamin B2, 50 mg of vitamin B6 and 0.4 mg of vitamin B12 (n=24) or no supplements (n=24) for 14 days. The treatment group and controls were matched for stroke subtype and age. Blood samples were obtained before intervention and also at 7 and 14 days post-recruitment for measurement of the following biomarkers: red cell folate (whole blood folate corrected with haematocrit), erythrocyte glutathione reductase activity coefficient (EGRAC; measure of vitamin B2 status), plasma pyridoxal phosphate (vitamin B6 status), plasma vitamin B12, plasma α-tocopherol, plasma ascorbic acid, plasma TAOC (total antioxidant capacity), plasma MDA (malondialdehyde), plasma tHcy and CRP (C-reactive protein). Supplementation for 14 days with B-group vitamins significantly increased the plasma concentrations of pyridoxal phosphate and red blood cell folate and improved a measure of B2 status compared with the control group (P<0.05). Plasma tHcy decreased in both groups albeit less in the control group, but differences in cumulative changes were not significant. There was, however, a decrease in plasma MDA concentration in the treatment group, in contrast with the increase seen in the control group and these differences were significant (P=0.05). CRP concentration, a marker of tissue inflammation, was significantly lower in the treatment group compared with controls (P<0.05). In conclusion, B-group vitamin supplementation immediately post-infarct may have antioxidant and anti-inflammatory effects in stroke disease independent of a homocysteine-lowering effect.
Nitric oxide and the hyperdynamic circulation in cirrhosis: is there a role for selective intestinal decontamination?
Authors: RASARATNAM Brindhesha , CONNELLY Nathan , CHIN-DUSTING Jaye
Published: October 2004

Abnormal vascular tone is responsible for many of the complications seen in cirrhosis making the identification of the pathophysiology of abnormal dilatation a major focus in hepatology research. The study of abnormal vascular tone is complicated by the multiple vascular beds involved (hepatic, splanchnic, peripheral, renal and pulmonary), the differences in the underlying cause of portal hypertension (hepatic versus pre-hepatic) and the slow evolution of the hyperdynamic state. The autonomic nervous system, circulating vasodilators and abnormalities in vascular smooth muscle cells (receptors, ion channels, signalling systems and contraction) have all been implicated. There is overwhelming evidence for an overproduction of NO (nitric oxide) contributing to the peripheral dilatation in both animal models of, and in humans with, cirrhosis and portal hypertension. This review focuses on the proposal that endotoxaemia, possibly from gut-derived bacterial translocation, causes induction of NOS (NO synthase) leading to increased vascular NO production, which is the primary stimulus for the development of vasodilatation in cirrhosis and its accompanying clinical manifestations. The current controversy lies not in whether NO production is elevated, but in which isoform of NOS is responsible. We review the evidence for endotoxaemia in cirrhosis and the factors contributing to gut-derived bacterial translocation, including intestinal motility and permeability, and finally discuss the possible role of selective intestinal decontamination in the management of circulatory abnormalities in cirrhosis.
Inflammatory and redox responses to ischaemia/reperfusion in human skeletal muscle
Authors: HUDA Ruksana , SOLANKI Daneshvari R. , MATHRU Mali
Published: October 2004

The objective of this study was to identify cellular and plasma marker(s) of post-I/R (ischaemia/reperfusion) in patients undergoing elective knee surgery where a tourniquet was used to facilitate a bloodless surgical field. We evaluated the inflammatory and redox response by measuring the mRNA levels of ICAM-1 (intercellular cell-adhesion molecule-1), MnSOD (manganese superoxide dismutase), GST-μ (glutathione transferase-μ) and Cu/ZnSOD (copper/zinc superoxide dismutase) in the operated muscle and blood cells pre-operatively (pre-tourniquet) and at various times after reperfusion (tourniquet release). We also measured plasma concentrations of IL (interleukin)-6, IL-8, sICAM-1 (soluble ICAM-1), IL-1β and TNF-α (tumour necrosis factor-α) using ELISA. Our results show a strong induction of MnSOD and GST-μ in granulocytes (but not in mononuclear cells or muscle) after reperfusion (2 and 4 h). There was no change in the mRNA level of Cu/ZnSOD after reperfusion. An up-regulation of membrane ICAM-1 in muscle and a decrease in sICAM-1 in plasma were detected after reperfusion. Plasma IL-6 and IL-8 levels (but not TNF-α or IL-1β) increased significantly over baseline at 2 and 4 h after reperfusion. Elevated expression of ICAM-1 in muscle, MnSOD and GST-μ in granulocytes and increased levels of plasma IL-6 and IL-8 may be considered as phase- and cell-specific markers of post-I/R of skeletal muscle in humans.
Genetic and environmental factors modulating serum concentrations and activities of the antioxidant enzyme paraoxonase-1
Authors: DEAKIN Sara P. , JAMES Richard W.
Published: October 2004

PON1 (paraoxonase-1) is an HDL (high-density lipoprotein)-associated enzyme capable of hydrolysing diverse substrates from OP (organophosphate) toxins to oxidized phospholipids. As such, it has been linked with both the prevention of OP poisoning and inhibition of atherosclerosis initiated by oxidatively modified LDL (low-density lipoprotein). Mice deficient in PON1 are more susceptible to OP poisoning and oxidative stress and more prone to develop atherosclerosis than their wild-type siblings. There are a number of polymorphisms in the PON1 gene which affect serum PON1 activity and concentration. Many (but not all) studies in human populations have suggested that these polymorphisms may be a risk factor for atherosclerosis. The serum concentration of PON1 across the general population is highly variable and there is some debate as to whether genotype or phenotype (i.e. the quantity or quality of the enzyme) is most accurately associated with risk of disease development. What is clear is that factors influencing serum levels of PON1, be they genetic or environmental, will, in turn, affect the capacity of HDL to protect LDL from oxidation and, consequently, may be linked to atherosclerosis. This review will focus on mechanisms which determine the serum concentration of PON1, including gene expression and genetic polymorphisms, protein secretion and association with HDL, pharmacological and environmental factors.
Measurement of coronary vasomotor function: getting to the heart of the matter in cardiovascular research
Authors: HIRATA Kumiko , AMUDHA Kadirvelu , ELINA Raja , HOZUMI Takeshi , YOSHIKAWA Junichi , HOMMA Shunichi , LANG Chim C.
Published: October 2004

Measurement of endothelial function in patients has emerged as a useful tool for cardiovascular research. Although no gold standard for the measurement of endothelial function exists, the measurement of flow-mediated dilation in the brachial artery, assessed with Doppler ultrasonography, is the most studied method. However, the assumption that endothelial dysfunction detected in brachial arteries is a manifestation of systemic endothelial dysfunction including the coronary circulation may not be entirely valid. Brachial and myocardial circulations differ in terms of the microvascular architecture, the pattern of blood flow and vascular resistance (e.g. shunt vessels occur in the hand but not in the myocardium), their metabolic regulation, type of receptors that contribute to humoral regulation and the pathways that are activated to induce hyperaemia. In this context, measuring coronary vasomotor function may be more useful than brachial artery measures to predict and assess potential myocardial damage related to limited vascular responsiveness. This review aims to provide an overview of the basic concept of coronary flow reserve and its different modalities of measurement, as well as its utility in cardiovascular research.
Potential energy surfaces and dynamics of Ni2+ ion aqueous solution: Molecular dynamics simulation of the electronic absorption spectrum
Authors: Iuchi Satoru , Morita Akihiro , Kato Shigeki
Published: October 2004

We develop a model effective Hamiltonian for describing the electronic structures of first-row transition metals in aqueous solutions using a quasidegenerate perturbation theory. All the states consisting of 3dn electronic configurations are determined by diagonalizing a small effective Hamiltonian matrix, where various intermolecular interaction terms such as the electrostatic, polarization, exchange, charge transfer, and three-body interactions are effectively incorporated. This model Hamiltonian is applied to constructing the ground and triplet excited states potential energy functions of Ni2+ in aqueous solution, based on the ab initio multiconfiguration quasidegenerate perturbation theory calculations. We perform molecular dynamics simulation calculations for the ground state of Ni2+ aqueous solution to calculate the electronic absorption spectral shape as well as the ground state properties. Agreement between the simulation and experimental spectra is satisfactory, indicating that the present model can well describe the Ni2+ excited state potential surfaces in aqueous solution.