bluuebunny/crossref_metadata_2025_split huggingface dataset:
Postoperative cognitive dysfunction: true deterioration versus random variation
Authors: Rasmussen L. S. , Siersma V. D. ,   the ispocd group
Published: September 2004

Background:  Postoperative cognitive dysfunction (POCD) is a common complication, especially in the elderly. The aim of this study was to describe how variability in neuropsychological testing could lead to the detection of cognitive improvement and poor consistency of POCD between postoperative test sessions.Methods:  In four published studies performed by the ISPOCD group, we included 2536 patients and 359 healthy controls. Cognitive function was assessed using neuropsychological tests preoperatively and at 7 days and 3 months thereafter, comparing the changes between those at baseline with those after surgery. Postoperative cognitive dysfunction was defined as a Z score greater than 2, and we also defined a corresponding improvement as a Z score less than −2. Consistency of POCD between postoperative test sessions was analyzed and we also assessed test–retest variability using data from healthy control subjects.Results:  Improvement in cognitive function was found in 4.2–8.7% of patients after 1 week and in 5.0–7.8% after 3 months. The ratio between incidence of dysfunction and improvement varied in patients between 3.3 and 6.2 early after major surgery. Of those patients who displayed POCD at the 3‐month test, 30–48% also had POCD at the previous test at 1 week. The test–retest reliability was between 0.56 and 0.90, except for the error score in Concept Shifting Test, where the values were 0.20 and 0.37.Conclusion:  Variability in neuropsychological test data contributes to a low consistency between postoperative test sessions but it does not explain the detection of cognitive dysfunction after major surgery.
Identification of Two Sites in Gelsolin with Different Sensitivities to Adenine Nucleotides
Authors: Laham Lorraine E. , Way Michael , Yin Helen L. , Janmey Paul A. 
Published: September 2004

The affinity of monomeric actin for several actin‐binding proteins, including gelsolin, depends on adenine nucleotides. Gelsolin binds faster and with higher affinity to ADP‐actin than to ATP‐actin. Here, we show that the C‐terminal actin‐binding domain of gelsolin, which is required for filament nucleating activity but not for filament severing activity, contains the site that distinguishes between ATP‐actin and ADP‐actin monomers.In contrast, actin binding to the N‐terminal half of gelsolin depends on solution ATP concentrations, but not on the nucleotide (ATP or ADP) tightly bound in the cleft of the actin monomer. Binding is stronger in the absence of free nucleotide or in the presence of 0.5 mM ADP than in solutions containing 0.5 mM ATP. Complexes formed using different nucleotide concentrations differ in their filament‐severing activities as well as in their abilities to increase the fluorescence of 4‐chloro‐7‐nitrobenzeno‐2‐oxa‐1,3‐diazole‐labeled actin monomers. These results suggest that, at physiologic concentrations of nucleotides, both free and actin‐bound ATP may affect the binding of actin to its accessory proteins and that gelsolin, actin, or the gelsolin‐actin complex, contains a low‐affinity nucleotide‐binding site.
Activation of two Isoforms of Mitogen‐Activated Protein Kinase Kinase in Response to Epidermal Growth Factor and Nerve Growth Factor
Authors: Moriguchi Tetsuo , Gotoh Yukiko , Nishida Eisuke 
Published: September 2004

Mitogen‐activated protein kinase kinase (MAPKK) is a dual‐specificity protein kinase which phosphorylates and activates mitogen‐activated protein kinase (MAPK). cDNAs encoding two isoforms of MAPKK, MAPKK1 and MAPKK2 (also known as MEK1 and MEK2), have been cloned in mammalian cells. To analyze the characteristics of MAPKK1 and MAPKK2 individually, we have produced specific anti‐MAPKK serum against each isoform. MAPKK1 and MAPKK2 have apparent molecular masses of 45 kDa and 47 kDa, respectively, on SDS/polyacrylamide gel electrophoresis. In mouse tissues, MAPKK1 was highly enriched in brain, while MAPKK2 was present relatively evenly. In rat fibroblastic 3Y1 cells, epidermal growth factor (EOF) treatment induced activation of both MAPKK1 and MAPKK2.Immunoprecipitation experiments have shown that the time courses of activation and deactivation of both isoforms of MAPKK were superimposed. In PC12 cells, both MAPKK1 and MAPKK2 were activated in response to nerve growth factor (NGF) as well as EOF, and the time courses of activation and deactivation of both isoforms were indistinguishable from each other in the NGF‐stimulated cells and also in the EGF‐stimulated cells. Furthermore, localization of both MAPKK1 and MAPKK2 in the cytoplasm was unchanged in response to EGF and NGF. Thus, the same or quite similar mechanisms may operate in the regulation of the activation and deactivation of two isoforms of MAPKK, and both kinases might have redundant functions when expressed in the same cell.
Reversible Dissociation and Unfolding of Dimeric Creatine Kinase Isoenzyme MM in Guanidine Hydrochloride and Urea
Authors: Couthon Fabienne , Clottes Eric , Ebel Christine , Vial Christian 
Published: September 2004

The unfolding of dimeric cytoplasmic creatine kinase (MM) by guanidine hydrochloride and by urea has been investigated using activity measurements, far‐ultraviolet circular dichroism, sedimentation velocity and fluorescence energy transfer experiments to monitor global structural changes. Intrinsic (cysteine and tryptophan residues) and extrinsic probes (1‐anilinonaphthalene‐8‐sulfonate) were also used. The reversibility of the unfolding was checked by monitoring activity and tryptophan fluorescence. The unfolding of creatine kinase in guanidine hydrochloride is a reversible multistep process, as suggested by the non‐coincidence of denaturation curves at equilibrium. Inactivation of the dimer precedes its dissociation into two monomers and an intermediate state was identified during the unfolding of the monomer. This intermediate state is characterized by a relatively high degree of secondary structure (as shown by far‐ultraviolet circular dichroism), of compactness (as shown by fluorescence energy transfer measurements and sedimentation experiments), a fluctuating tertiary structure (as shown by near‐ultraviolet circular dichroism) and a strong affinity for anilinonaphthalene sulfonate (as demonstrated by fluorescence). These results clearly indicate that the intermediate state detected possesses some of the properties of a molten globule. In urea, the unfolding pathway is reversible but differs from that observed in guanidine hydrochloride. Indeed inactivation, dissociation and loss of tertiary structure are coincident but the ellipticity curve is slightly shifted to a higher urea concentration. The dimer is dissociated into two expanded monomers possessing some secondary structure which is progressively lost at a higher urea concentration (6.4 M).These results show that guanidine hydrochloride is approximately six times more effective than urea for inactivation and dissociation, underlining the fact that electrostatic interactions are very important in the stabilization of the active site and of the dimeric state.
Peroxynitrite Formed by Simultaneous Nitric Oxide and Superoxide Generation Causes Cyclosporin‐A‐Sensitive Mitochondrial Calcium Efflux and Depolarisation
Authors: Packer Michael A. , Murphy Michael P. 
Published: September 2004

Nitric oxide reacts rapidly with superoxide to form the potent oxidant peroxynitrite. Mitochondria in vivo produce superoxide and in pathological situations the amount of superoxide produced increases; therefore, in the presence of nitric oxide, mitochondria will be a major site of peroxynitrite formation. Oxidative stress induces cyclosporin‐A‐sensitive mitochondrial calcium efflux and depolarisation which may contribute to tissue damage in pathological situations. To determine whether peroxynitrite could induce calcium efflux and depolarisation we exposed mitochondria to both nitric oxide and superoxide simultaneously, thus subjecting the mitochondria to a continual flux of peroxynitrite similar to that found in pathological situations. Our results show that: (a) exposure of mitochondria to nitric oxide plus super‐oxide induces cyclosporin‐A‐sensitive mitochondrial calcium efflux and depolarisation; (b) neither nitric oxide nor superoxide on their own induce calcium efflux or depolarisation under these conditions; (c) calcium efflux and depolarisation occur when peroxynitrite production (from nitric oxide and superoxide) exceeds about 0.99±0.03 nmol peroxynitrite min‐1 mg mitochondrial protein‐1. This rate of production of peroxynitrite is similar in magnitude to superoxide formation by mitochondria in pathological situations, suggesting that mitochondria can produce sufficient peroxynitrite in vivo to cause mitochondrial calcium efflux and depolarisation.Our experiments suggest a plausible model for tissue damage when mitochondrial superoxide production increases, for example in ischaemia‐reperfusion injury or following exposure to neurotoxins. In the presence of nitric oxide the mitochondrial superoxide production will lead to peroxynitrite formation which induces cyclosporin‐A‐sensitive mitochondrial calcium efflux and depolarisation. This disruption to mitochondrial function may in turn contribute to cell damage and death.
Substrate Specificity of Flavin‐Dependent Vanillyl‐Alcohol Oxidase from Penicillium Simplicissimum
Authors: Fraaije Marco W. , Veeger Cees , Van Berkel Willem J. H. 
Published: September 2004

The substrate specificity of the flavoprotein vanillyl‐alcohol oxidase from Penicillium simplicissimum was investigated. Vanillyl‐alcohol oxidase catalyzes besides the oxidation of 4‐hydroxybenzyl alcohols, the oxidative deamination of 4‐hydroxybenzylamines and the oxidative demethylation of 4‐(methoxy‐methyl)phenols. During the conversion of vanillylamine to vanillin, a transient intermediate, most probably vanillylimine, is observed.Vanillyl‐alcohol oxidase weakly interacts with 4‐hydroxyphenylglycols and a series of catechol‐amines. These compounds are converted to the corresponding ketones. Both enantiomers of (nor)epineph‐rine are substrates for vanillyl‐alcohol oxidase, but the R isomer is preferred.Vanillyl‐alcohol oxidase is most active with chavicol and eugenol. These 4‐allylphenols are converted to coumaryl alcohol and coniferyl alcohol, respectively. Isotopic labeling experiments show that the oxygen atom inserted at the Cγ atom of the side chain is derived from water. The 4‐hydroxycinnamyl alcohol products and the substrate analog isoeugenol are competitive inhibitors of vanillyl alcohol oxidation.The binding of isoeugenol to the oxidized enzyme perturbs the optical spectrum of protein‐bound FAD. pH‐dependent binding studies suggest that vanillyl‐alcohol oxidase preferentially binds the phenolate form of isoeugenol (pKa<6, 25°C). From this and the high pH optimum for turnover, a hydride transfer mechanism involving a P ‐quinone methide intermediate is proposed for the vanillyl‐alcohol‐oxidase‐catalyzed conversion of 4‐allylphenols.
Different Susceptibility of Protein Kinases to Staurosporine Inhibition
Authors: Meggio Flavio , Deana Arianna Donella , Ruzzene Maria , Brunati Anna M. , Cesaro Luca , Guerra Barbara , Meyer Thomas , Mett Helmut , Fabbro Doriano , Furet Pascal , Dobrowolska Grazyna , Pinna Lorenzo A. 
Published: September 2004

A systematic analysis reveals that out of 20 protein kinases examined, specific for either Ser/Thr or Tyr, the majority are extremely sensitive to staurosporine, with IC50 values in the low nanomolar range. A few of them however, notably protein kinases CK1 and CK2, mitogen‐activated protein (MAP) kinase and protein‐tyrosine kinase CSK, are relatively refractory to staurosporine inhibition, exhibiting IC50 values in the micromolar range. With all protein kinases tested, namely PKA, CK1, CK2, MAP kinase (ERK‐1), c‐Fgr, Lyn, CSK and TPK‐IIB/p38syk, staurosporine inhibition was competitive with respect to ATP, regardless of its inhibitory power. In contrast, either uncompetitive or noncompetitive kinetics of inhibition with respect to the phosphoacceptor substrate were exhibited by Ser/Thr and Tyr‐specific protein kinases, respectively, consistent with a different mechanism of catalysis by these two sub‐families of kinases. Computer modeling based on PKA crystal structure in conjunction with sequence analysis suggest that the low sensitivity to staurosporine of CK2 may be accounted for by the bulky nature of three residues, Val66, Phe113 and Ile174 which are homologous to PKA Ala70, Metl20 and Thr183, respectively. In contrast these PKA residues are either conserved or replaced by smaller ones in protein kinases highly sensitive to staurosporine inhibition. On the other hand, His160 which is homologous to PKA Glul70, appears to be responsible for the unique behaviour of CK2 with respect to a staurosporine derivative (CGP44171A) bearing a negatively charged benzoyl substituent: while CGP44171A is 10– 100‐fold less effective than staurosporine against PKA and most of the other protein kinases tested, it is actually more effective than staurosporine for CK2 inhibition, but it looses part of its efficacy if it is tested on a CK2 mutant (H160D) in which Hisl60 has been replaced by Asp.It can be concluded from these data that the catalytic sites of protein kinases are divergent enough as to allow a competitive inhibitor like staurosporine to be fairly selective, a feature that can be enhanced by suitable modifications designed based on the structure of the catalytic site of the kinase.
Functional Properties of a Recombinant Chimeric Protein with Combined Thrombin Inhibitory and Plasminogen‐Activating Potential
Authors: Lijnen H. Roger , Wnendt Stephan , Schneider Johannes , Janocha Elke , Van Hoef Berthe , Collen Désiré , Steffens Gerd J. 
Published: September 2004

A chimeric protein (rscu‐PA‐40‐kDa/Hir), consisting of the C‐terminal amino acids 53–65 of hirudin (Hir), fused via a 14‐amino‐acid linker sequence to the C‐terminal of a 40–kDa fragment (Ser47‐Leu411) of recombinant (r) single‐chain (sc) urokinase‐type plasminogen activator (rscu‐PA), was produced by expression of the corresponding chimeric cDNA in Escherichia coli cells. The thrombin inhibitory potential of purified rscu‐PA‐40‐kDa/Hir was confirmed by complete inhibition of the coagulant activity of thrombin at 20–30‐fold molar excess of the chimera, and by the resistance of rscu‐PA‐40‐kDa/Hir to proteolytic cleavage by thrombin. rscu‐PA‐40‐kDa/Hir prolonged the thrombin time of normal human plasma in a dose‐dependent way (reduction of the apparent thrombin concentration to 50% with 95 nM chimeric protein as compared to 4.7 nM hirudin), and inhibited thrombin‐mediated platelet aggregation (reduction of the apparent thrombin concentration to 50% with 40 nM chimeric protein).The chimera had a specific activity on fibrin films of 57000 IU/mg as compared to 95000 IU/mg for rscu‐PA. The urokinase‐like amidolytic activity of the single‐chain protein was only 220 ILJ/mg but increased to 169000 ILJ/mg after treatment with plasmin, which resulted in quantitative conversion to a two‐chain (tc) derivative (rtcu‐PA‐40–kDa/Hir). Corresponding values for rscu‐PA were 270 and 226000 IU/mg. The catalytic efficiencies for plasmin‐mediated conversion to two‐chain molecules were comparable for rscu‐PA‐40–kDa/Hir and rscu‐PA (0.63 and 0.65 μM‐1 s‐1, respectively). The plasminogen‐activating potential of the single‐chain chimera was comparable to that of rscu‐PA; the catalytic efficiencies for plasminogen activation by their two‐chain counterparts were also similar (0.55 and 0.73 μM‐1 s‐1, respectively). In 2 h, 50% lysis of 125I‐fibrin‐labeled clots prepared from platelet‐poor human plasma and immersed in normal plasma was obtained with 1.3 μg/ml rscu‐PA‐40‐kDa/Hir and with 0.67 μg/ml rscu‐PA, with corresponding residual fibrinogen levels of 74% and 87%, respectively. In the absence of fibrin, 50% fibrinogenolysis in 2 h in normal human plasma required 2.1 μg/ml rscu‐PA, but 7.9 μg/ml rscu‐PA‐40‐kDa/Hir.Thus, the chimera rscu‐PA‐40‐kDa/Hir has maintained the specific fibrinolytic and plasminogen activating activity of rscu‐PA as well as its fibrinolytic potency in plasma, whereas it displayed a similar or somewhat better fibrin specificity. In addition, the fibrinolytically active concentration in a plasma medium is severalfold lower than the concentration required for thrombin inhibition, which may limit systemic anticoagulant activity. Therefore, further evaluation of the thrombolytic and antithrombotic potential of such chimeric molecules seems to be warranted.
Urokinase‐Type Plasminogen Activator/Type‐2 Plasminogen‐Activator Inhibitor Complexes are not Internalized Upon Binding to the Urokinase‐Type‐Plasminogen‐Activator Receptor in THP‐1 Cells
Authors: Ragno Pia , Montuori Nunzia , Rossi Guido 
Published: September 2004

The urokinase‐type plasminogen activator (uPA) and its inhibitor PAI‐2 form a covalent complex that, upon binding to the uPA receptor (uPA‐R), is cleaved into two fragments of molecular masses 70 kDa and 22 kDa. The 70‐kDa fragment results from the interaction of the B chain of uPA and PAI‐2 whereas the 22‐kDa fragment is the A chain of the enzyme [13].We prove that, at 37°C, the 70–kDa fragment is released into the medium, whereas the 22–kDa fragment remains bound to the cell surface. uPA complexed with its other specific inhibitor, PAI‐1, is cleaved into fragments of identical sizes, but the 70–kDa component is internalized via the a2‐macroglobulin receptor.At 4°C, both uPA/PAI‐2 complex degradation products remain bound to the uPA‐R. We propose that the 70–kDa molecule, which lacks the uPA binding region for uPA‐R, is bound to uPA‐R via a new binding site, unmasked only when uPA‐R is occupied by uPA/PAI‐2 complexes.
New Inhibitors of the Ubiquinol Oxidase of Higher Plant Mitochondria
Authors: Hoefnagel Marcel H. N. , Wiskich Joseph T. , Madgwick Sally A. , Patterson Zoe , Oettmeier Walter , Rich Peter R. 
Published: September 2004

A screen has been performed of possible inhibitors of the ubiquinol oxidase of higher plant mitochondria by assaying their effects on cyanide‐insensitive NADH oxidase of mitochondria of Arum maculatum. A number of compounds which have powerful inhibitory effects have been identified.Potent inhibition was found with compounds related to the previously described N‐propyl gallate, but with the N‐propyl sidechain replaced with alkyl chains of greater hydrophobicity. Titration of a range of partial reactions showed that the inhibitors act specifically on the ubiquinol oxidase. The concentrations of inhibitor required are dependent on the respiratory substrate and on the amount of mitochondria used in the assay. Octyl gallate also proved to be a potent inhibitor of the ubiquinol oxidase in tobacco cell suspensions.A second class of compounds which strongly inhibit cyanide‐insensitive NADH oxidation is aurachin C and its analogues. Compounds related to aurachin D are much less effective. Titrations of a range of partial reactions indicate that inhibition is caused by a direct action on the ubiquinol oxidase. However, both types of aurachins also act strongly at the Qi site of the cytochrome bc1, complex, as already known to be the case in other systems, and so they are of more limited value for studies of the ubiquinol oxidase.Titration of the oxidation of NADH via the ubiquinol oxidase in a purified mitochondrial fraction from the spadices of Arum maculatum with octyl gallate gave a half‐maximal effect at a concentration of around 6 nM when the protein concentration was 14 μg ml−1. A similar litre was obtained with a decyl derivative of aurachin C. This allowed us to estimate an upper limit for the concentration of ubiquinol oxidase in these mitochondria of 0.72 ± 0.15 nmol mg−1 protein, or a ratio of ubiquinol oxidase/cytochrome oxidase of about 15 ± 7:1. The measurements also provide a minimal turnover number for the ubiquinol oxidase of 186 ± 42 electrons s−1.Titration of the ubiquinol oxidase in soybean cotyledon mitochondria with these compounds gave the concentration of inhibitor required to elicit 50% of the maximum observed effect (I50) values about one order of magnitude higher than those found with Arum mitochondria, and again the values depended on the respiratory substrate. An explanation for the variation in I50 values may be found in terms of differences in oxidase concentrations in the different mitochondrial membranes and in the differences in rate‐controlling steps with substrates of different activities.
Compressibility of the Heme Pocket of Substrate Analogue Complexes of CytochromeP‐450cam‐CO
Authors: Jung Christiane , Bon Hoa Gaston Hui , Davydov Dmitri , Gill Edward , Heremans Karel 
Published: September 2004

The effect of hydrostatic pressure on the electronic absorption spectrum of the carbon monoxide complex of cytochrome P‐450cam (CYP101) in the presence of various substrates was studied.With increasing pressure the wavenumber of the Soret band in the cytochromeP‐450–CO complex shifts linearily to lower values (red‐shift) and the half‐width increases (broadening). The microscopic theory of solvent–solute interaction discussed by Laird and Skinner is used to explain the observed pressure effects. According to this theory, the slope of the red‐shift of the Soret band is related to the compressibility of the chromophore environment, that is the heme moiety of the hemoproteins. It was found that the slope of the red‐shift and the slope of the broadening of the Soret band for the CO complex in the presence of various substrate analogues increase with the decrease of the initial high‐spin content at 0.1 MPa in the oxidized state. Variation of the high‐spin content reflects the changes in the number of water molecules and/or changes in the polarity of the heme environment. The higher compressibility of the cytochromeP‐450 complexes with the substrate analogues, which induce a lower degree of the high‐spin content in the oxidized protein, is explained by the ability of the water molecules in the heme moiety to transmit the pressure effect on the protein structure to the heme chromophore. Therefore, a larger pressure‐induced red‐shift of the Soret band in the CO complex of cytochromeP‐450cam might indicate a higher water content in the heme environment.
NMR Studies of the Action of Hypochlorous Acid on Native Pig Articular Cartilage
Authors: Schiller Jürgen , Arnhold Jürgen , Arnold Klaus 
Published: September 2004

The action of sodium hypochlorite on pig articular cartilage was studied by 1H‐NMR spectroscopy to model some aspects of degradation processes of cartilage during rheumatoid arthritis. Two effects of NaOCl on cartilage polysaccharides have been observed. Hypochlorous acid causes an enhanced release of oligomeric polysaccharides from cartilage. The second effect concerns the degradation of N‐acetyl side chains of carbohydrates to acetate via a chlorinated transient product.Signal intensities for N‐acetyl groups (≈2.0 ppm) increase during the first 2 h of incubation of cartilage with NaOCl. Then they decrease again. However, acetate (1.90 ppm) as the final product of degradation of N‐acetyl side chains increases continuously over the period of incubation with NaOCl.In addition to polysaccharides, effects of NaOCl were only observed in cartilage samples on amino acids like alanine. The alanine resonance disappeared already at NaOCl concentrations where only small effects on cartilage polysaccharides have been observed.