bluuebunny/crossref_metadata_2025 huggingface dataset:
The calcium‐induced switch in the troponin complex probed by fluorescent mutants of troponin I
Authors: Oliveira Deodoro C. S. G. , Reinach Fernando C.
Published: July 2003

The Ca2+‐induced transition in the troponin complex (Tn) regulates vertebrate striated muscle contraction. Tn was reconstituted with recombinant forms of troponin I (TnI) containing a single intrinsic 5‐hydroxytryptophan (5HW). Fluorescence analysis of these mutants of TnI demonstrate that the regions in TnI that respond to Ca2+ binding to the regulatory N‐domain of TnC are the inhibitory region (residues 96–116) and a neighboring region that includes position 121. Our data confirms the role of TnI as a modulator of the Ca2+ affinity of TnC; we show that point mutations and incorporation of 5HW in TnI can affect both the affinity and the cooperativity of Ca2+ binding to TnC. We also discuss the possibility that the regulatory sites in the N‐terminal domain of TnC might be the high affinity Ca2+‐binding sites in the troponin complex.
Photochemical cross‐linking of Escherichia coli Fpg protein to DNA duplexes containing phenyl(trifluoromethyl)diazirine groups
Authors: Taranenko Maria , Rykhlevskaya Anna , Mtchedlidze Manana , Laval Jacques , Kuznetsova Svetlana
Published: July 2003

Formamidopyrimidine‐DNA glycosylase (Fpg protein) of Escherichia coli is a DNA repair enzyme that excises oxidized purine bases, most notably the mutagenic 7‐hydro‐8‐oxoguanine, from damaged DNA. In order to identify specific contacts between nucleobases of DNA and amino acids from the E. coli Fpg protein, photochemical cross‐linking was employed using new reactive DNA duplexes containing 5‐[4‐[3‐(trifluoromethyl)‐3H‐diazirin‐3‐yl]phenyl]‐2′‐deoxyuridine dU* residues near the 7‐hydro‐8‐oxoguanosine (oxoG) lesion. The Fpg protein was found to bind specifically and tightly to the modified DNA duplexes and to incise them. The nicking efficiency of the DNA duplex containing a dU* residue 5′ to the oxoG was higher as compared to oxidized native DNA. The conditions for the photochemical cross‐linking of the reactive DNA duplexes and the Fpg protein have been optimized to yield as high as 10% of the cross‐linked product. Our results suggest that the Fpg protein forms contacts with two nucleosides, one 5′ adjacent to oxoG and the other 5′ adjacent to the cytidine residue pairing with oxoG in the other strand. The approaches developed may be applicable to pro‐ and eukaryotic homologues of the E. coli Fpg protein as well as to other repair enzymes.
Phenylalanine‐independent biosynthesis of 1,3,5,8‐tetrahydroxyxanthone
Authors: Wang Chang‐Zeng , Maier Ulrich H. , Keil Michael , Zenk Meinhart H. , Bacher Adelbert , Rohdich Felix , Eisenreich Wolfgang
Published: July 2003

Root cultures of Swertia chirata (Gentianaceae) were grown with supplements of [1‐13C]glucose, [U‐13C6]glucose or [carboxy‐13C]shikimic acid. 1,3,5,8‐Tetrahydroxyxanthone was isolated and analysed by quantitative NMR analysis. The observed isotopomer distribution shows that 1,3,5,8‐tetrahydroxyxanthone is biosynthesized via a polyketide‐type pathway. The starter unit, 3‐hydroxybenzoyl‐CoA, is obtained from an early shikimate pathway intermediate. Phenylalanine, cinnamic acid and benzoic acid were ruled out as intermediates.
Glycoprotein Ib‐mediated platelet activation
Authors: Adam Frédéric , Guillin Marie‐Claude , Jandrot‐Perrus Martine
Published: July 2003

Platelet activation by thrombin plays a major role in the development of haemostasis and thrombosis. Thrombin activates human platelets by cleaving the N‐terminal region of G‐protein‐coupled protease‐activated receptors (PARs). On the other hand, the platelet membrane glycoprotein GPIb acts as a thrombin‐binding site and promotes platelet activation by low thrombin concentrations. We present here new evidence in favour of a thrombin receptor function for GPIb. We have selected conditions in which thrombin–GPIb interactions were enhanced by thrombin immobilization. Activation was studied independently of PAR cleavage by using active‐site‐blocked thrombin. We show that immobilized, proteolytically inactive thrombin induces platelet adhesion and spreading, dense granule secretion and integrin αIIbβ3‐dependent platelet–platelet interactions. The pathway must be dependent on GPIb because it is deficient in platelets from a patient with Bernard Soulier syndrome and inhibited by a monoclonal antibody to GPIb (SZ2) or by an excess of glycocalicin. Secreted ADP plays a major role in GPIb‐dependent thrombin‐induced platelet activation which is, in addition, regulated by cAMP concentration. Thrombin‐induced GPIb‐dependent platelet activation leads to tyrosyl phosphorylation of several proteins. Inhibition of platelet–platelet interactions and protein tyrosine phosphorylations by inhibitors of phosphatidylinositol 3‐kinases and protein kinase C implies that activation of the latter are important steps of the GPIb‐coupled signalling pathway triggered by thrombin.
Hydrolysis of diadenosine polyphosphates by nucleotide pyrophosphatases/phosphodiesterases
Authors: Vollmayer Petra , Clair Timothy , Goding James W. , Sano Kimihiko , Servos Jörg , Zimmermann Herbert
Published: July 2003

Diadenosine polyphosphates (ApnAs) act as extracellular signaling molecules in a broad variety of tissues. They were shown to be hydrolyzed by surface‐located enzymes in an asymmetric manner, generating AMP and Apn‐1 from ApnA. The molecular identity of the enzymes responsible remains unclear. We analyzed the potential of NPP1, NPP2, and NPP3, the three members of the ecto‐nucleotide pyrophosphatase/phosphodiesterase family, to hydrolyze the diadenosine polyphosphates diadenosine 5′,5′′′‐P1,P3‐triphosphate (Ap3A), diadenosine 5′,5′′′‐P1,P4‐tetraphosphate (Ap4A), and diadenosine 5′,5′′′‐P1,P5‐pentaphosphate, (Ap5A), and the diguanosine polyphosphate, diguanosine 5′,5′′′‐P1,P4‐tetraphosphate (Gp4G). Each of the three enzymes hydrolyzed Ap3A, Ap4A, and Ap5A at comparable rates. Gp4G was hydrolyzed by NPP1 and NPP2 at rates similar to Ap4A, but only at half this rate by NPP3. Hydrolysis was asymmetric, involving the α,β‐pyrophosphate bond. ApnA hydrolysis had a very alkaline pH optimum and was inhibited by EDTA. Michaelis constant (Km) values for Ap3A were 5.1 µm, 8.0 µm, and 49.5 µm for NPP1, NPP2, and NPP3, respectively. Our results suggest that NPP1, NPP2, and NPP3 are major enzyme candidates for the hydrolysis of extracellular diadenosine polyphosphates in vertebrate tissues.
Structural characterization of a novel branching pattern in the lipopolysaccharide from nontypeable Haemophilus influenzae
Authors: Månsson Martin , Hood Derek W. , Moxon E. Richard , Schweda Elke K. H.
Published: July 2003

Structural analysis of the lipopolysaccharide (LPS) from nontypeable Haemophilus influenzae strain 981 has been achieved using NMR spectroscopy and ESI‐MS on O‐deacylated LPS and core oligosaccharide (OS) material as well as by ESI‐MSn on permethylated dephosphorylated OS. A heterogeneous glycoform population was identified, resulting from the variable length of the OS branches attached to the glucose residue in the common structural element of H. influenzae LPS, l‐α‐d‐Hepp‐(1→2)‐[PEtn→6]‐l‐α‐d‐Hepp‐(1→3)‐[β‐d‐Glcxp‐(1→4)]‐l‐α‐d‐Hepp‐(1→5)‐[PPEtn→4]‐α‐Kdop‐(2→6)‐Lipid A. Notably, the O‐6 position of the β‐d‐Glcp residue was either substituted by PCho or the disaccharide branch β‐d‐Galp‐(1→4)‐d‐α‐d‐Hepp, while the O‐4 position was substituted by the globotetraose unit, β‐d‐GalpNAc‐(1→3)‐α‐d‐Galp‐(1→4)‐β‐d‐Galp‐(1→4)‐β‐d‐Glcp, or sequentially truncated versions thereof. This is the first time a branching sugar residue has been reported in the outer‐core region of H. influenzae LPS. Additionally, a PEtn group was identified at O‐3 of the distal heptose residue in the inner‐core.
Acetyl‐CoA:1‐O‐alkyl‐sn‐glycero‐3‐phosphocholine acetyltransferase (lyso‐PAF AT) activity in cortical and medullary human renal tissue
Authors: Nomikos Tzortzis N , Iatrou Christos , Demopoulos Constantine A
Published: July 2003

Platelet‐activating factor (PAF) is one of the most potent inflammatory mediators. It is biosynthesized by either the de novo biosynthesis of glyceryl ether lipids or by remodeling of membrane phospholipids. PAF is synthesized and catabolized by various renal cells and tissues and exerts a wide range of biological activities on renal tissue suggesting a potential role during renal injury. The aim of this study was to identify whether cortex and medulla of human kidney contain the acetyl‐CoA:1‐O‐alkyl‐sn‐glycero‐3‐phosphocholine acetyltransferase (lyso‐PAF AT) activity which catalyses the last step of the remodeling biosynthetic route of PAF and is activated in inflammatory conditions. Cortex and medulla were obtained from nephrectomized patients with adenocarcinoma and the enzymatic activity was determined by a trichloroacetic acid precipitation method. Lyso‐PAF AT activity was detected in both cortex and medulla and distributed among the membrane subcellular fractions. No statistical differences between the specific activity of cortical and medullary lyso‐PAF AT was found. Both cortical and medullary microsomal lyso‐PAF ATs share similar biochemical properties indicating common cellular sources.
Glucagon‐like peptide‐2 stimulates the proliferation of cultured rat astrocytes
Authors: Velázquez Esther , Ruiz‐Albusac Juan M. , Blázquez Enrique
Published: July 2003

Glucagon‐like peptide‐2 (GLP‐2) is a potent intestinotrophic/satiety hormone that acts through a G protein‐coupled receptor. To determine whether or not GLP‐2 has any effect on cellular proliferation on neural cells, we examined the effects of this peptide on cultured astrocytes from rat cerebral cortex. The expression of the GLP‐2 receptor gene in both cerebral cortex and astrocytes was determined by RT‐PCR and Southern blotting. Also, cells responded to GLP‐2, producing cAMP in a dose‐dependent manner (EC50 = 0.86 nm). GLP‐2 also stimulated the DNA synthesis rate in rat astrocytes. When proliferation was assessed by measuring [3H]thymidine incorporation into DNA or staining cells with crystal violet, GLP‐2 produced a dose‐dependent increase in both parameters. Similarly, when the numbers of cells in different phases of the cell cycle were measured by flow cytometry, a dose‐dependent decrease in those in the G0‐G1 phase and an increase in those in the S and G2‐M phases were observed after 24 h incubation with GLP‐2. By contrast, the number of hypodiploid cells was not affected during the experimental time. Also, GLP‐2 produced a significant increase in the mRNAs of c‐fos and c‐jun when gene expression was determined by Northern blotting. These results suggest that GLP‐2 directly stimulates the proliferation of rat astrocytes; this may open new insights in the physiological role of this novel neuropeptide.
Isolation, characterization and expression analysis of a hypoxia‐responsive glucose transporter gene from the grass carp, Ctenopharyngodon idellus
Authors: Zhang Ziping , Wu Rudolf S. S. , Mok Helen O. L. , Wang Yilei , Poon Winnie W. L. , Cheng Shuk H. , Kong Richard Y. C.
Published: July 2003

Glucose transporters (GLUTs) have been implicated in adaptive and survival responses to hypoxic stress in mammals. In fish, the expression and regulation of GLUT in relation to hypoxia remains unexplored. Here we describe the identification of a hypoxia‐responsive glucose transporter gene (gcGLUT) and the corresponding full‐length cDNA from the grass carp. The gene spans ≈ 11 kb of genomic sequence and consists of 12 exons and 11 introns, and an open reading frame (ORF) of 1599 bp encoding a polypeptide of 533 amino acids, with a predicted molecular mass of ≈ 57 kDa and a pI of 8.34. blastx analysis showed that the ORF shared high sequence identity with the GLUT1 (57–59%), GLUT3 (59–60%) and GLUT4 (55–59%) proteins from different vertebrates. Comparative analysis of GLUT genomic structures showed that the arrangement of exons and position of split codons are highly conserved amongst members of the class I GLUTs suggesting that these genes share a common ancestor. Phylogenetic analysis indicated that gcGLUT is most closely related to the GLUT3 proteins. Northern blot analysis showed that the 3.1‐kb gcGLUT transcript was most abundantly expressed and responsive to hypoxia in kidney. Up‐regulated expression by hypoxia was also evident in eye and gill, but differential patterns of expression were observed. Low expression levels detected in brain, heart, liver and muscle were not responsive to hypoxic stress.
Identification of a novel binding site for calmodulin in ammodytoxin A, a neurotoxic group IIA phospholipase A2
Authors: Prijatelj Petra , Šribar Jernej , Ivanovski Gabriela , Križaj Igor , Gubenšek Franc , Pungerčar Jože
Published: July 2003

The molecular mechanism of the presynaptic neurotoxicity of snake venom phospholipases A2 (PLA2s) is not yet fully elucidated. Recently, new high‐affinity binding proteins for PLA2 toxins have been discovered, including the important intracellular Ca2+ sensor, calmodulin (CaM). In the present study, the mode of interaction of group IIA PLA2s with the Ca2+‐bound form of CaM was investigated by mutational analysis of ammodytoxin A (AtxA) from the long‐nosed viper (Vipera ammodytes ammodytes). Several residues in the C‐terminal part of AtxA were found to be important in this interaction, particularly those in the region 115–119. In support of this finding, introduction of Y115, I116, R118 and N119, present in AtxA, into a weakly neurotoxic PLA2 from Russell's viper (Daboia russellii russellii) increased by sevenfold its binding affinity for CaM. Furthermore, two out of four peptides deduced from different regions of AtxA were able to compete with the toxin in binding to CaM. The nonapeptide showing the strongest inhibition was that comprising the AtxA region 115–119. This stretch contributes to a distinct hydrophobic patch within the region 107–125 in the C‐terminal part of the molecule. This lacks any substantial helical structure and is surrounded by several basic residues, which may form a novel binding motif for CaM on the molecular surface of the PLA2 toxin.
Procarboxypeptidase A from the insect pest Helicoverpa armigera and its derived enzyme
Authors: Bayés Alex , Sonnenschein Anka , Daura Xavier , Vendrell Josep , Aviles Francesc X.
Published: July 2003

Although there is a significant knowledge about mammalian metallocarboxypeptidases, the data available on this family of enzymes is very poor for invertebrate forms. Here we present the biochemical characterization of a metallocarboxypeptidase from the insect Helicoverpa armigera (Lepidoptera: Noctuidae), a devastating pest spread in subtropical regions of Europe, Asia, Africa and Oceania. The zymogen of this carboxypeptidase (PCPAHa) has been expressed at high levels in a Pichia pastoris system and shown to display the characteristics of the enzyme purified from the insect midgut. The in vitro activation process of the proenzyme differs significantly from the mammalian ones. The lysine‐specific endoprotease LysC activates PCPAHa four times more efficiently than trypsin, the general activating enzyme for all previously studied metalloprocarboxypeptidases. LysC and trypsin independently use two different activation targets and the presence of sugars in the vicinity of the LysC activation point affects the activation process, indicating a possible modulation of the activation mechanism. During the activation with LysC the prodomain is degraded, while the carboxypeptidase moiety remains intact except for a C‐terminal octapeptide that is rapidly released. Interestingly, the sequence at the cleavage point for the release of the octapeptide is also found at the boundary between the activation peptide and the enzyme moieties. The active enzyme (CPAHa) is shown to have a very broad substrate specificity, as it appears to be the only known metallocarboxypeptidase capable of efficiently hydrolysing basic and aliphatic residues and, to a much lower extent, acidic residues. Two carboxypeptidase inhibitors, from potato and leech, were tested against CPAHa. The former, of vegetal origin, is the most efficient metallocarboxypeptidase inhibitor described so far, with a Ki in the pm range.
Structure of the exceptionally large nonrepetitive carbohydrate backbone of the lipopolysaccharide of Pectinatus frisingensis strain VTT E‐82164
Authors: Vinogradov Evgeny , Petersen Bent O. , Sadovskaya Irina , Jabbouri Said , Duus Jens Ø. , Helander Ilkka M.
Published: July 2003

The structures of the oligosaccharides obtained after acetic acid hydrolysis and alkaline deacylation of the rough‐type lipopolysaccharide (LPS) from Pectinatus frisingensis strain VTT E‐82164 were analysed using NMR spectroscopy, MS and chemical methods. The LPS contains two major structural variants, differing by a decasaccharide fragment, and some minor variants lacking the terminal glucose residue. The largest structure of the carbohydrate backbone of the LPS that could be deduced from experimental results consists of 25 monosaccharides (including the previously found Ara4NP residue in lipid A) arranged in a well‐defined nonrepetitive structure:

We presume that the shorter variant with R1 = H represents the core‐lipid A part of the LPS, and the additional fragment is present instead of the O‐specific polysaccharide. Structures of this type have not been previously described. Analysis of the deacylation products obtained from the LPS of the smooth strain, VTT E‐79100T, showed that it contains a very similar core but with one different glycosidic linkage.
Functional genomics by NMR spectroscopy
Authors: Ismail Wael , El‐Said Mohamed Magdy , Wanner Barry L. , Datsenko Kirill A. , Eisenreich Wolfgang , Rohdich Felix , Bacher Adelbert , Fuchs Georg
Published: July 2003

Aerobic metabolism of phenylalanine in most bacteria proceeds via oxidation to phenylacetate. Surprisingly, the further metabolism of phenylacetate has not been elucidated, even in well studied bacteria such as Escherichia coli. The only committed step is the conversion of phenylacetate into phenylacetyl‐CoA. The paa operon of E. coli encodes 14 polypeptides involved in the catabolism of phenylacetate. We have found that E. coli K12 mutants with a deletion of the paaF, paaG, paaH, paaJ or paaZ gene are unable to grow with phenylacetate as carbon source. Incubation of a paaG mutant with [U‐13C8]phenylacetate yielded ring‐1,2‐dihydroxy‐1,2‐dihydrophenylacetyl lactone as shown by NMR spectroscopy. Incubation of the paaF and paaH mutants with phenylacetate yielded Δ3‐dehydroadipate and 3‐hydroxyadipate, respectively. The origin of the carbon atoms of these C6 compounds from the aromatic ring was shown using [ring‐13C6]phenylacetate. The paaG and paaZ mutants also converted phenylacetate into ortho‐hydroxyphenylacetate, which was previously identified as a dead end product of phenylacetate catabolism. These data, in conjunction with protein sequence data, suggest a novel catabolic pathway via CoA thioesters. According to this, phenylacetyl‐CoA is attacked by a ring‐oxygenase/reductase (PaaABCDE proteins), generating a hydroxylated and reduced derivative of phenylacetyl‐CoA, which is not re‐oxidized to a dihydroxylated aromatic intermediate, as in other known aromatic pathways. Rather, it is proposed that this nonaromatic intermediate CoA ester is further metabolized in a complex reaction sequence comprising enoyl‐CoA isomerization/hydration, nonoxygenolytic ring opening, and dehydrogenation catalyzed by the PaaG and PaaZ proteins. The subsequent β‐oxidation‐type degradation of the resulting CoA dicarboxylate via β‐ketoadipyl‐CoA to succinyl‐CoA and acetyl‐CoA appears to be catalyzed by the PaaJ, PaaF and PaaH proteins.
Structure and positioning comparison of two variants of penetratin in two different membrane mimicking systems by NMR
Authors: Lindberg Mattias , Biverståhl Henrik , Gräslund Astrid , Mäler Lena
Published: July 2003

The Antennapedia homeodomain protein of Drosophila has the ability to penetrate biological membranes and the third helix of this protein, residues 43–58, known as penetratin (RQIKIWFQNRRMKWKK‐amide) has the same translocating properties as the entire protein. The variant, RQI KIFFQNRRMKFKK‐amide, here called penetratin (W48F,W56F) does not have the same ability. We have determined a solution structure of penetratin and investigated the position of both peptides in negatively charged bicelles. A helical structure is seen for residues Lys46 through Met54. The secondary structure of the variant penetratin(W48F,W56F) in bicelles appears to be very similar. Paramagnetic spin‐label studies and analysis of NOEs between penetratin and the phospholipids show that penetratin is located within the bicelle surface. Penetratin (W48F,W56F) is also located inside the phospholipid bicelle, however, with its N‐terminus more deeply inserted than that of wild‐type penetratin. The subtle differences in the way the two peptides interact with a membrane in an equilibrium situation could be important for their translocating ability. As a comparison we have also investigated the secondary structure of penetratin(W48F,W56F) in SDS micelles and the results show that the structure is very similar in SDS and bicelles. In contrast, penetratin(W48F,W56F) and penetratin appear to be located differently in SDS micelles. This clearly shows the importance of using realistic membrane mimetics for investigating peptide–membrane interactions.
Thyroid hormone induces the expression of 4‐1BB and activation of caspases in a thyroid hormone receptor‐dependent manner
Authors: Yamada‐Okabe Toshiko , Satoh Yasuo , Yamada‐Okabe Hisafumi
Published: July 2003

Thyroid hormone has various effects on cell proliferation, growth and apoptosis. To gain more insight into the molecular dynamics caused by thyroid hormone, gene expression in HeLaTR cells that constitutively overexpressed the thyroid hormone receptor (TR) was analyzed. Gene expression profiling of the HeLaTR cells with an oligonucleotide microarray yielded 229 genes whose expression was significantly altered by T3. Among these genes, the expression of 4‐1BB, which is known to initiate a signal cascade activating NF‐κB, was significantly up‐regulated by T3. Although treatment of the HeLaTR cells with T3 did not induce expression of NF‐κB reporter luciferase, even in the presence of the 4‐1BB‐Ligand, it increased the caspase activities. An increase in the caspase activities was also observed in the HeLaTR cells transfected with 4‐1BB cDNA, and the 4‐1BB‐Ligand further increased the caspase activities of the HeLaTR cells overexpressing the 4‐1BB. Furthermore, up‐regulation of 4‐1BB and an increase in caspase activities also occurred in the rat FRTL cells that expressed only authentic TR. These results demonstrate that the expression of 4‐1BB serves as the mediator of signals from T3 to activate caspases.
Characterization of a functionally expressed dipeptidyl aminopeptidase III from Drosophila melanogaster
Authors: Mazzocco Claire , Fukasawa Kayoko M. , Auguste Patrick , Puiroux Jacques
Published: July 2003

A Drosophila melanogaster cDNA clone (GH01916) encoding a putative 723‐residue long (82 kDa) protein (CG 7415) and displaying 50% identity with mammalian cytosolic dipeptidyl aminopeptidase (DPP) III was functionally expressed in Schneider S2 cells. Immunocytochemical studies using anti‐(rat liver DPP III) Ig indicated the expression of this putative DPP III at the outer cell membrane and into the cytosol of transfected cells. Two protein bands (82 and 86 kDa) were immunologically detected after PAGE and Western blot of cytosol or membrane prepared from transfected cells. Western blot analysis of partially purified D. melanogaster DPP III confirmed the overexpression of these two protein bands into the cytosol and on the membranes of transfected cells. Despite the identification of six potential glycosylation sites, PAGE showed that these protein bands were not shifted after deglycosylation experiments. The partially purified enzyme hydrolysed the insect myotropic neuropeptide proctolin (Arg‐Tyr‐Leu‐Pro‐Thr) at the Tyr‐Leu bond (Km ≈ 4 µm). In addition, low concentration of the specific DPP III inhibitor tynorphin prevented proctolin degradation (IC50 = 0.62 ± 0.15 µm). These results constitute the first characterization of an evolutionarily conserved insect DPP III that is expressed as a cytosolic and a membrane peptidase involved in proctolin degradation.
Novel ATP‐binding and autophosphorylation activity associated with Arabidopsis and human cryptochrome‐1
Authors: Bouly Jean‐Pierre , Giovani Baldissera , Djamei Armin , Mueller Markus , Zeugner Anke , Dudkin Elizabeth A. , Batschauer Alfred , Ahmad Margaret
Published: July 2003

Cryptochromes are blue‐light photoreceptors sharing sequence similarity to photolyases, a class of flavoenzymes catalyzing repair of UV‐damaged DNA via electron transfer mechanisms. Despite significant amino acid sequence similarity in both catalytic and cofactor‐binding domains, cryptochromes lack DNA repair functions associated with photolyases, and the molecular mechanism involved in cryptochrome signaling remains obscure. Here, we report a novel ATP binding and autophosphorylation activity associated with Arabidopsis cry1 protein purified from a baculovirus expression system. Autophosphorylation occurs on serine residue(s) and is absent in preparations of cryptochrome depleted in flavin and/or misfolded. Autophosphorylation is stimulated by light in vitro and oxidizing agents that act as flavin antagonists prevent this stimulation. Human cry1 expressed in baculovirus likewise shows ATP binding and autophosphorylation activity, suggesting this novel enzymatic activity may be important to the mechanism of action of both plant and animal cryptochromes.
Cloning, expression and characterization of a family‐74 xyloglucanase from Thermobifida fusca
Authors: Irwin Diana C. , Cheng Mark , Xiang Bosong , Rose Jocelyn K. C. , Wilson David B.
Published: July 2003

Thermobifida fusca xyloglucan‐specific endo‐β‐1,4‐glucanase (Xeg)74 and the Xeg74 catalytic domain (CD) were cloned, expressed in Escherichia coli, purified and characterized. This enzyme has a glycohydrolase family‐74 CD that is a specific xyloglucanase followed by a family‐2 carbohydrate binding module at the C terminus. The Michaelis constant (Km) and maximal rate (Vmax) values for hydrolysis of tamarind seed xyloglucan (tamXG) are 2.4 µm and 966 µmol xyloglucan oligosaccharides (XGOs) min−1·µmol protein−1. More than 75% of the activity was retained after a 16‐h incubation at temperatures up to 60 °C. The enzyme was most active at pH 6.0–9.4. NMR analysis showed that its catalytic mechanism is inverting. The oligosaccharide products from hydrolysis of tamXG were determined by MS analysis. Cel9B, an active carboxymethylcellulose (CMC)ase from T. fusca, was also found to have activity on xyloglucan (XG) at 49 µmol·min−1·µmol protein−1, but it could not hydrolyze XG units containing galactose. An XG/cellulose composite was prepared by growing Gluconacetobacterxylinus on glucose with tamXG in the medium. Although a mixture of purified cellulases was unable to degrade this material, the composite material was fully hydrolyzed when Xeg74 was added. T. fusca was not able to grow on tamXG, but Xeg74 was found in the culture supernatant at the same level as was found in cultures grown on Solka Floc. The function of this enzyme appears to be to break down the XG surrounding cellulose fibrils found in biomass so that T. fusca can utilize the cellulose as a carbon source.
Ascorbic acid induces a marked conformational change in long duplex DNA
Authors: Yoshikawa Yuko , Suzuki Mari , Chen Ning , Zinchenko Anatoly A. , Murata Shizuaki , Kanbe Toshio , Nakai Tonau , Oana Hidehiro , Yoshikawa Kenichi
Published: July 2003

Ascorbic acid is often regarded as an antioxidant in vivo, where it protects against cancer by scavenging DNA‐damaging reactive oxygen species. However, the detailed mechanism of the action of ascorbic acid on genetic DNA is still unclear. We examined the effect of ascorbic acid on the higher‐order structure of DNA through real‐time observation by fluorescence microscopy. We found that ascorbic acid generates a pearling structure in single giant DNA molecules, with elongated and compact regions coexisting along a molecular chain. Results from electron microscopy and atomic force microscopy indicate that the compact regions assume a loosely packed conformation. A possible mechanism for the induction of this conformational change is discussed in relation to the interplay between the higher‐order and second‐order structures of DNA.
Calcium‐dependent protein–protein interactions induce changes in proximity relationships of Cys48 and Cys64 in chicken skeletal troponin I
Authors: Liou Ying‐Ming , Chen Ming‐Wei
Published: July 2003

The goal of this study was to relate conformational changes in the N‐terminal domain of chicken troponin I (TnI) to Ca2+ activation of the actin–myosin interaction. The two cysteine residues in this region (Cys48 and Cys64) were labeled with two sulfhydryl‐reactive pyrene‐containing fluorophores [N‐(1‐pyrene)maleimide, and N‐(1‐pyrene)iodoacetamide]. The labeled TnI showed a typical fluorescence spectrum: two sharp peaks of monomer fluorescence and a broad peak of excimer fluorescence arising from the formation of an excited dimer (excimer). Results obtained show that forming a binary complex of labeled TnI with skeletal TnC (sTnC) in the absence of Ca2+ decreases the excimer fluorescence, indicating a separation of the two residues. This reduction in excimer fluorescence does not occur when labeled TnI is complexed with cardiac TnC (cTnC). The latter causes only partial activation of the Ca2+‐dependent myofibrillar ATPase. The binding of Ca2+ to the two N‐terminal sites of sTnC causes a significant decrease in excimer fluorescence and an increase in monomer fluorescence in complexes of labeled TnI with skeletal TnC or TnC/TnT, while Ca2+ binding to site II of cTnC only causes an increase in monomer fluorescence but no change in excimer fluorescence. Thus a conformational change in the N‐terminal region of TnI may be necessary for full activation of muscle contraction.
Early Maternal Changes Contributing to the Formation of the Chorioallantoic and Yolk Sac Placentas in Rat: a Morphological Study
Authors: Kaloğlu C. , Gürsoy E. , Onarlioğlu B.
Published: July 2003

In order to determine the maternal changes contributing to the formation of the chorioallantoic and yolk‐sac placentas, rat gestation sites were examined by light and electron microscopy on days 7 through 10 of pregnancy.

On day 7, the implantation chamber showed different compartments and contained the blastocyst in the antimesometrial chamber. The epithelial lining of the implantation chamber disappeared at the antimesometrial chamber, transformed into disintegrated cells in the mesometrial chamber, and showed signs of the programmed cell death in the decidual crypt. On day 8, the mesometrial chamber lumen contained red blood cells and it was continuous with subepithelial sinusoids. The endothelial cells lining the mesometrial sinusoids also showed some characteristics of the sprouting type angiogenesis such as hypertrophy and cell proliferation. While the yolk‐sac placental circulation was more obvious with participation of the giant trophoblasts at the antimesometrial pole of the conceptus on day 9, the antimesometrial cells showed autophagic degeneration after the formation of the chorioallantoic placenta on day 10. The contribution of the regional cell death and angiogenesis to form both of the two placentas are discussed.
Characteristics of trees drawn by patients with paranoid schizophrenia
Authors: Inadomi Hiroyuki , Tanaka Goro , Ohta Yasuyuki
Published: July 2003

Abstract The objective of the present study was to investigate the relationship between schizophrenia subtype and morphological characteristics of trees drawn in the Baum test. Subjects comprised the following three groups: 20 patients diagnosed with paranoid schizophrenia according to International Classification of Diseases (10th revision; ICD‐10) criteria; 26 patients with non‐paranoid schizophrenia according to ICD‐10 criteria; and 53 healthy individuals. Differences in psychiatric symptoms as assessed using the Brief Psychiatric Rating Scale (BPRS) score were compared between patients with paranoid and non‐paranoid schizophrenia. In addition, differences in two morphological characteristics of trees, namely trunk‐to‐crown ratio and trunk end opening, were compared between the three groups. No differences in psychiatric symptoms were identified between patients with paranoid and non‐paranoid schizophrenia. Conversely, mean ± SD trunk‐to‐crown ratio was 13.1 ± 8.0 for patients with non‐paranoid schizophrenia, 8.8 ± 4.6 for patients with paranoid schizophrenia, and 5.4 ± 3.4 for healthy individuals. Significant differences were identified between all three groups. Furthermore, mean trunk end opening was 0.80 ± 0.7 for patients with paranoid schizophrenia, 0.38 ± 0.6 for patients with non‐paranoid schizophrenia, and 0.06 ± 0.3 for healthy individuals. Again, significant differences were apparent between all three groups. These findings suggest that morphological differences in trees drawn in the Baum test can be observed between the two schizophrenia subtypes in terms of not only psychopathological interpretation, but also gestalt formation, as assessed on the basis of trees with collapsed gestalt or with some degree of gestalt. This suggests the possibility of multiple disorders at a physiological level. The present study confirmed that the Baum test can quantitatively assess facets of schizophrenia that existing scales such as BPRS are unable to analyze, and is useful for investigating brain function in patients with schizophrenia.
Symmetry reversal in schizophrenia
Authors: Kalb Roland , Raydt Gesine , Reulbach Udo , Kornhuber Johannes
Published: July 2003

Abstract Schizophrenia is associated with cortical asymmetries concentrated in the left fronto‐temporal hemisphere. In order to look for functional asymmetries between the two hemispheres, the stimulus–response times of patients were split into smaller periods and the interhemispheric and intrahemispheric correlations between these periods were investigated. Three groups were compared to each other: 22 patients with schizophrenia (Diagnostic and Statistical Manual of Mental Disorders, 4th edn; DSM‐IV) treated with neuroleptics; 24 psychiatric neuroleptic‐treated patients without schizophrenia; and 30 healthy subjects. All subjects were investigated by simple (one stimulus–one response) and complex (two stimuli–two responses), auditory and visual, right‐hemispheric and left‐hemispheric stimulus–response tasks. There were no intrahemispheric but significant interhemispheric correlations between the two auditory and between the two visual time fragments in both the healthy and the neuroleptic control group. In contrast there was a significant intrahemispheric correlation between the auditory and visual time fragment in the left hemisphere of patients with schizophrenia and no interhemispheric correlation between the auditory times. The reduction of the interhemispheric auditory correlation is interpreted as an auditory disintegration, the appearance of the left‐hemispheric audiovisual correlation as an audiovisual ‘hyperintegration’ in patients with schizophrenia. It is questionnable as to whether these findings are due to schizophrenia or to the neuroleptic treatment.
Differences between patients with chronic fatigue syndrome and with chronic fatigue at an infectious disease clinic in Stockholm, Sweden
Authors: Evengård Birgitta , Jonzon Eva , Sandberg Anneli , Theorell Töres , Lindh Gudrun
Published: July 2003

AbstractBackground data were collected from patients presenting with fatigue at the clinic of infectious diseases at Huddinge University Hospital, Stockholm. The main purpose was to look for differences as to demographic and functional status for patients fulfilling criteria for chronic fatigue syndrome (CFS) and chronic fatigue (CF). A cross‐sectional questionnaire survey was performed using a variety of instruments. A thorough medical investigation was performed. No difference was found as to social situation, occupation and illness attributions for patients in the two categories. Patients with CFS reported in general a higher degree of ‘sickness’ with more self‐reported somatic symptoms, more self‐reported functional impairment and more absence from work. A higher degree of psychiatric comorbidity was observed in CF than in CFS patients. A majority of CFS patients (80%) had an acute infectious onset compared to 43% in the CF group. Presently used criteria might, according to findings presented here, define two different patient categories in a population characterized by severe, prolonged fatigue. Because CFS patients (compared to patients with CF) have more somatic symptoms, more often report an infectious, sudden onset and have less psychiatric comorbidity, and CF patients seem to have more of an emotional, burn‐out‐like component one could speculate about the existence of different pathogenetic backgrounds behind the two diagnoses.
Two case studies of hypothermia induced by an increased dosage of zotepine in a combination therapy
Authors: Chen Kao Ching , Yang Yen Kuang , Chen Po See , Yeh Tzung Lieh , Yang Ming Jen
Published: July 2003

Abstract The purpose of the present paper was to remind physicians that hypothermia is a possible side‐effect of combining zotepine, valproate,and benzodiazepine. Two cases of hypothermia occurred after combining the use of zotepine, valproate, and benzodiazepine. The valproate was under therapeutic blood level when zotepine dosage was raised to 200 mg/day, and hypothermia occurred. The dosage of zotepine might constitute a positive correlation with hypothermia. The combination of zotepine, valproate or benzodiazepine may cause hypothermia as a side‐effect.
Correlation between performance on the Continuous Performance Test and economic costs in patients with schizophrenia
Authors: Ko Sheng Ying , Chen Po See , Yang Yen Kuang , Liao Yi Cheng , Lee Yih Dar , Yeh Tzung Lieh , Yang Ming Jen
Published: July 2003

Abstract The aim of the present study was to determine whether a relationship existed between the performance on the Continuous Performance Test (CPT) and the economic costs of schizophrenic patients in Taiwan. Forty‐six schizophrenic patients and their caregivers were enrolled. The CPT is a vigilance task requiring the monitoring of rapid information processing and the detection of briefly presented target stimuli. Each patients undertook two CPT sections: the unmasked task and the masked task. Economic Cost Questionnaires were used for assessing the direct and indirect costs of the illness. The patients’ performance on the masked version of the CPT correlated significantly with the indirect costs, although there was no significant relationship between CPT scores and direct costs. Schizophrenic patients with more severely impaired sustained attention, particularly as measured by CPT, may incur higher indirect costs.
Factor structure of the 12‐item General Health Questionnaire in the Japanese general adult population
Authors: Doi Yuriko , Minowa Masumi
Published: July 2003

Abstract The 12‐item General Health Questionnaire (GHQ‐12) has been extensively used in a variety of settings across countries. The main aim of the present study was to assess the factor structure of the GHQ‐12 for the Japanese general adult population. Data came from a sample of 1808 Japanese aged 20 years or older who were randomly selected based on the 1995 census (897 men and 911 women). Cronbach's α coefficients were 0.83 for men and 0.85 for women. Overall, the corrected item‐total correlation coefficients were >0.20 for both genders. The GHQ‐12 yielded a two‐factor solution of psychological distress (items 2, 5, 6, 9, 10 and 11) and social dysfunction (items 1, 3, 4, 7 and 8), which jointly accounted for 49.1% of the total variance, for women. Item 12 on happiness was not discernable. For men, item 12 was separated from a social dysfunction factor and yielded the third factor with item 3 on social role, and the three factors jointly accounted for 57.6%. The results of the present study suggest that the GHQ‐12 can be used as an internally reliable and homogenous scale that produces mainly the factors of psychological distress and social dysfunction. Item 12 may be structurally different in the case of Japanese adults.
Multi‐center study on post‐ictal headache in patients with localization‐related epilepsy
Authors: Ito Masumi , Adachi Naoto , Nakamura Fumihiro , Koyama Tsukasa , Okamura Takehiko , Kato Masaaki , Kanemoto Kousuke , Nakano Takashi , Matsuura Masato , Hara Sumio
Published: July 2003

Abstract The nature of post‐ictal headache (PIH) remains unclear. A multicenter study was conducted in order to evaluate frequency and risk factors for PIH. The subjects consisted of 97 patients with temporal lobe epilepsy (TLE), 65 patients with frontal lobe epilepsy (FLE) and 37 patients with occipital lobe epilepsy (OLE). The subjects were asked about PIH at each institute. Clinical factors of epilepsy were reviewed for each patient and correlated with PIH. Post‐ictal headache occurred in 41% of TLE patients, 40% of FLE patients and 59% of OLE patients. Logistic regression analysis revealed that the risk of PIH was significantly higher in OLE than in FLE. In contrast, there were no significant differences in the incidence of PIH between TLE and either OLE or FLE patients. Post‐ictal headache occurred significantly more frequently in subjects with generalized tonic–clonic seizure (GTCS) than in those without GTCS. Other clinical factors, such as sex, age, age at onset, duration of illness, seizure frequency, family history of headache and interictal headache were found to have no associations with PIH. A difference was found in the incidence of PIH depending on classification of epilepsy, but only to a relatively slight extent. It was also found that GTCS was significantly related to PIH. These results suggest that both the location of epileptogenic focus and the involvement of certain cerebral areas in the spread of seizure discharges may be closely related to PIH.
Auditory discrimination in female adolescents varying in schizotypal features: Preliminary findings
Authors: Li Chiang‐Shan Ray , Yang Yong‐Yi , Chen Mon‐Chu , Chen Wei J. , Liu Jo‐Lan
Published: July 2003

Abstract Earlier experiments have suggested impaired auditory perception in patients with schizophrenia. The purpose of the present study was to investigate auditory discrimination in young adults varying in schizotypal features. Three hundred and forty‐nine female adolescent subjects were rated with the Schizotypal Personality Questionnaire (SPQ) and the Perceptual Aberration Scale (PAS) and with questions from the Chinese Health Questionnaire (CHQ) that address non‐psychotic, psychological status of the subjects. Sixty‐one subjects were selected to participate in an auditory detection and discrimination task, with 20 subjects each with low and high, and 21 subjects with intermediate, SPQ scores. The sensitivity in auditory discrimination and the response bias were computed, using signal detection theory. The results show that the ratings of the subjects in the three questionnaires correlate with one another and that subjects with higher SPQ scores appear to perform worse in auditory discrimination. The impaired performance occurs for most of the signal‐to‐noise ratios. Taken together, these preliminary findings are consistent with earlier studies demonstrating impaired auditory processing in schizophrenia patients. In contrast, the significant correlation between both the SPQ and PAS scores and the CHQ score suggests that further studies are required to rule out the effect of non‐psychotic factors such as anxiety on the current results. Second, the three groups of subjects did not differ in the response bias. This latter finding provides evidence at odds with the ‘self‐monitoring’ model of auditory hallucination.
Tumor necrosis factor‐α gene polymorphism at position −308 and schizophrenia in the Korean population
Authors: Pae Chi‐Un , Chae Jeong‐Ho , Bahk Won‐Myong , Han Hoon , Jun Tae‐Youn , Kim Kwang‐Soo , Kwon Yong‐Sil , Serretti Alessandro
Published: July 2003

Abstract The purpose of the present study was to examine the association between the tumor necrosis factor (TNF)‐α gene (A) polymorphism and schizophrenia in a Korean sample of schizophrenic patients and control subjects. Genotyping for the TNFA polymorphism was performed by polymerase chain reaction–restriction fragment length polymorphism. Genotype and allele distributions of the TNFA polymorphism between schizophrenic patients and controls were not significantly different. In the light of these results, the TNFA polymorphism seems not to confer susceptibility to the pathogenesis of schizophrenia, at least in the Korean population.
Citalopram treatment of children and adolescents with obsessive–compulsive disorder: A preliminary report
Authors: Mukaddes Nahit Motavallý , Abali Osman , Kaynak Nimet
Published: July 2003

Abstract The purpose of the present study was to assess the efficacy and tolerability of citalopram in the acute treatment of children and adolescents with obsessive compulsive disorder (OCD) during an 8‐week, open‐label study. Fifteen patients (six female, nine male) with a mean age of 12.1 ± 3.3 years (range: 6–17 years) were treated with citalopram (range of dose: 20–30 mg/day, mean dose: 24 ± 5.5 mg/day). The children's version of the Yale–Brown Obsessive–Compulsive scale (CY‐BOCS) was rated at the baseline, at the 4th week and at the 8th week of treatment. Fourteen patients had a decrease in total score of CY‐BOCS from baseline to the 4th week of treatment (P < 0.01) and the 8th week of treatment (P < 0.01). Sedation (n = 1) and insomnia (n = 1) were reported in the first weeks of treatment. The primary data suggest the efficacy and tolerability of citalopram in young patients with OCD.
Exploratory eye movements during the Benton Visual Retention Test: Characteristics of visual behavior in schizophrenia
Authors: Obayashi Shigeru , Matsushima Eisuke , Ando Harunobu , Ando Katsumi , Kojima Takuya
Published: July 2003

AbstractIn order to investigate the relationship between the behavioral patterns and clinical symptomatology in schizophrenia, exploratory eye movements of schizophrenic subjects and healthy controls during the Benton Visual Retention Test were examined using an eye‐mark recorder. The results were as follows: (i) with card 1, the number of eye fixations of schizophrenic subjects was fewer, and the total and mean eye scanning lengths of schizophrenic subjects were shorter than those of healthy controls; (ii) with card 3, almost none of the schizophrenic subjects looked at the blank area opposite the peripheral figure on the right; (iii) with cards 3, 5, 6 and 9 there were some schizophrenic subjects who did not look at the peripheral figures; (iv) with card 6, many of the schizophrenic subjects made stereotypical movements; (v) with card 9, schizophrenic subjects used a narrow vertical gaze to look at the large figure on the right. Based on these characteristics among the schizophrenic subjects themselves, factors such as longer eye scanning length, looking at peripheral figures without fail, and not making stereotypical movements were reflected directly in the results of the Benton test, while there was no relationship between the width of the vertical gaze and the Benton results. Correlations between visual behavior and some psychiatric symptoms were observed. The visual behavioral patterns of schizophrenic subjects were various according to the characteristics of the Benton figures, while those of normal subjects were always almost the same. It was suggested that these results were caused by disturbances of the mental attitude of schizophrenic subjects toward objects or environments.