bluuebunny/crossref_metadata_2025 huggingface dataset:
Calpain‐Induced Proteolysis of Normal Human Tau and Tau Associated with Paired Helical Filaments
Authors: Yang Liang‐Sheng , Ksiezak‐Reding Hanna
Published: September 2004

The major components of neurofibrillary tangles (NFT) in Alzheimer's disease are bundles of paired helical filaments (PHF) which are primarily composed of highly phosphorylated tau proteins (PHF‐tau). To further understand the mechanism of PHF accumulation in NFT, we examined the calpain‐induced proteolysis of highly purified and primarily non‐aggregated PHF and normal tau proteins with various contents of phosphate isolated from either fetal (F‐tau) or adult human brain (N‐tau). The extent of proteolysis was determined by decreases in tau immunoreactivity using Western‐blot analysis and a panel of site‐specific tau antibodies (Alz 50, Tau‐2, Tau 14, Tau‐1, AT8, E‐11, AH‐1 and PHF‐1). We found that full‐size polypeptides of N‐tau and F‐tau were similarly and rapidly proteolyzed in vitro by calpain (calpain II, 3.3 units/mg protein) during a 10‐min incubation at 30°C, and that their half lives (t1/2) were 1.5 min and 1.8 min, respectively, Analysis of immunoblots suggests that full‐length polypeptides of tau are first degraded into large fragments similar in size to that generated endogenously, then into smaller fragments. Since both endogenous and in‐vitro‐generated tau fragments retained N‐terminal epitopes, the results suggest that most of the calpain‐sensitive sites may be located in the C‐terminal half of the tau molecule. In contrast, PHF were extremely resistant to degradation and only a fivefold higher concentration of calpain (16.7 units/mg protein) induced partial proteolysis of PHF. A major calpain‐generated fragment was a 45‐kDa polypeptide derived from the C‐terminal region of PHF‐tau, which forms a core of filaments. The results suggest that the inaccessibility of potential calpain‐digestion sites in the filament core could contribute to the resistance of PHF to calpain and subsequently lead to the accumulation of PHF in Alzheimer's disease. The results also suggest that hyperphosphorylation of tau may be mariginally involved in the resistance of PHF to degradation by calpain. Ultrastructural examination revealed that, in contrast to previous studies with trypsin, calpain did not alter the morphologic appearance of filaments; after incubation with calpain, the majority of PHF remained short and disperse and the number of PHF aggregated into NFT‐like clusters was not significantly increased. The results suggest that the role of calpain in promoting the aggregation and clustering of filaments is limited.
Involvement of N‐Linked Carbohydrate Chains of Pig Zona Pellucida in Sperm‐Egg Binding
Authors: Yonezawa Naoto , Aoki Hironori , Hatanaka Yumiko , Nakano Minoru
Published: September 2004

The sperm receptor activity of pig zona pellucida has been previously shown to exist in one of the components, pig zona protein 3α (PZP3α), that can be purified after the removal of sialylated and/or sulfated N‐acetylpoly(lactosamine) by digestion with endo‐β‐galactosidase. In this study, we examined whether N‐linked or O‐linked carbohydrate chains are involved in the sperm receptor activity of pig zona pellucida. The elimination of N‐linked carbohydrate chains from endo‐β‐galactosidase‐digested PZP3α by digestion with N‐glycanase markedly reduced its inhibitory effect on sperm‐egg binding in an in vitro competition assay, whereas the elimination of O‐linked carbohydrate chains by alkali treatment hardly reduced the inhibitory effect. These results indicate that N‐linked carbohydrate chains of PZP3α play a major role in mediating the sperm binding of zona pellucida in pig.
Immunoassay for the Quantification of Intracellular Multi‐Ubiquitin Chains
Authors: Takada Koji , Nasu Hidekazu , Hibi Nozomu , Tsukada Yutaka , Ohkawa Kiyoshi , Fujimuro Masahiro , Sawada Hitoshi , Yokosawa Hideyoshi
Published: September 2004

A sandwich ELISA has been developed to measure intracellular levels of multi‐ubiquitin chains. The mixture of multi‐ubiquitin chains, prepared in vitro by incubation of ubiquitin (plus 125I‐ubiquitin) and lysozyme with ubiquitin‐ligating enzymes and ATP, was partially purified and established as a standard named the multi‐ubiquitin‐chain reference preparation 1 (MUCRP1). The concentration of MUCRP1 was calculated from the recovered radioactivity of 125I‐ubiquitin. All measurements by the ELISA were expressed in terms of MUCRP1. The ELISA showed good sensitivity (98 pg/ml), precision (intra‐assays <6%) and reproducibility (interassay <9%). In addition, there was no substantial cross‐reaction with mono‐, di‐ and tri‐ubiquitin, or mono‐ubiquitinated and di‐ubiquitinated lysozyme in the ELISA, and large multi‐ubiquitin chains (n > approximately 6) may be fully reactive. These results combined with excellent results in the recovery and dilution tests guarantee accurate measurement of multi‐ubiquitin chains in cell extracts prepared with a lysis buffer (water soluble) or the buffer supplemented 8 M urea (urea soluble). The level of the water‐soluble multi‐ubiquitin chains in reticulocytes was lower than that of erythrocytes, but the urea‐soluble chain level was higher in the reticulocytes. Heat‐shock treatment of HeLa cells increased the urea‐soluble multi‐ubiquitin chains. These data indicate that this ELISA provides a useful and reliable approach to the study of intracellular multi‐ubiquitin‐conjugate turnover.
Molecular Cloning of CTP: Phosphocholine Cytidylyltransferase from Plasmodium falciparum
Authors: Yeo Hye‐Jeong , Widada Joannes Sri , Mercereau‐Puijalon Odile , Vial Henri Joseph
Published: September 2004

CTP:phosphocholine cytidylyltransferase (CCT) is the rate‐limiting and regulatory enzyme in the synthesis of phosphatidylcholine, the major membrane phospholipid, in Plasmodium. The structural gene encoding CCT was isolated from the human malaria parasite Plasmodium falciparum. This was achieved using the PCR to amplify genomic DNA with degenerate primers constructed on the basis of conserved regions identified within yeast and rat liver CCT molecules, and using the PCR product to screen a genomic library. The P. falciparum CCT gene encodes a protein of 370 amino acids (42. 6 kDa) and displays 41–43% similarity (28–29% identity) to CCT molecules of the other organisms cloned to date. The central domain of CCT, proposed as the catalytic domain of the CTP‐transfer reaction, shows 68–72% similarity and 48–55% identity among P. falciparum, human, rat and yeast enzymes. This gene is present in a single copy, as determined by Southern‐blotting of genomic DNA, and located on chromosome 13 of P. falciparum. Large transcripts were detected by Northern analysis and indicate that this gene is expressed in the asexual intraerythrocytic stages. The coding region of the P. falciparum CCT gene was inserted into an Escherichia coli expression vector to confirm the function of the CCT product. The recombinant CCT expressed in E. coli is catalytically active, as evidenced by the conversion of phosphocholine to CDP‐choline.
The Effect of Cross‐Linking of the Two Heads of Porcine Aorta Smooth Muscle Myosin on Its Conformation and Enzymic Activity
Authors: Katoh Tsuyoshi , Morita Fumi
Published: September 2004

The two heads of porcine aorta smooth muscle myosin can be cross‐linked by a disulfide bridge between the two 17‐kDa essential light chains with 5,5′‐dithiobis(2‐nitrobenzoic acid) [Katoh, T., Tanahashi, K., Hasegawa, Y. & Morita, F. (1995) Eur. J. Biochem. 227, 459–465]. When the cross‐linked myosin sample was visualized by rotary shadowing, the two heads of myosin molecules appeared predominantly to adhere to each other. The cross‐linking of dephosphorylated myosin in the presence of ATP was greatly inhibited by a decrease in the concentration of NaCl from 0.4 M to 0.15 M, suggesting that the cross‐linking of the two heads was suppressed in 10S myosin. However, the fraction of dephosphorylated myosin in a filamentous state at 0.1 M NaCl in the presence of 1 mM ATP was increased from 33% to 83% by the cross‐linking. The cross‐linking of the two heads might inhibit the formation of the 10S conformation, leading to the increase in the fraction of filamentous myosin. The filaments of the cross‐linked myosin sample were visualized by electron microscopy and appeared morphologically similar to those of uncross‐linked myosin. The ATPase activity of the cross‐linked dephosphorylated myosin sample was more than three times as high as that of an uncross‐linked control. The increase in the activity may be related to the increase in the fraction of filamentous myosin caused by the cross‐linking. The ATPase activity of dephosphorylated myosin in the presence of actin was increased more than twofold by the cross‐linking, but the activity of phosphorylated myosin was affected only slightly. The degree of phosphorylation‐dependent regulation of actin‐activated ATPase activity decreased with an increase in the degree of cross‐linking and was extrapolated to zero at 100% cross‐linking. Superprecipitation of acto‐cross‐linked dephosphorylated myosin was activated, while that of acto‐cross‐linked phosphorylated myosin was inhibited only slightly. These results suggest that the freedom of each head in myosin molecules may be required to keep the ATPase activity and Superprecipitation of acto‐dephosphorylated myosin low but not for keeping these activity levels high in acto‐phosphorylated myosin.
Purification and Properties of Codeinone Reductase (NADPH) from Papaver somniferum Cell Cultures and Differentiated Plants
Authors: Lenz Rainer , Zenk Meinhart H.
Published: September 2004

Codeinone reductase (NADPH), which catalyzes the stereospecific reduction of (–)codeinone to (–)codeine, was detected and purified to electrophoretic homogeneity from a cytosolic fraction of Papaver somniferum L. cell cultures. The purification involved ammonium sulfate precipitation (40–80%), affinity chromatography (matrex red A), gel filtration (fractogel TSK HW 55S), affinity chromatography (fractogel TSK AF Blue), ion‐exchange chromatography (DEAE‐Sephacel) and native PAGE. The purified codeinone reductase was found to be a monomeric protein of 35±1 kDa that is highly substrate‐specific, reducing only the C6 oxo group of codeinone and morphinone as well as a few analogues. The physiological forward reaction has a pH optimum at 7.0, the reverse reaction at 9.1. The temperature optimum is at 40°C and the isoelectric point (pl) at 4.4. The apparent Km values (forward reaction) for codeinone and NADPH are 23 μM and 168 μM, respectively. Using capsule tissue of differentiated P. somniferum plants as an enzyme source, two codeinone reductase (NADPH) isoenzymes were detected and purified to homogeneity. These isoenzymes could not be separated for characterization and showed slightly different kinetic features (Km values: codeinone 9 μM; NADPH 81 μM) compared with the cell culture enzyme.
Temperature‐Induced Changes in Fluorescence Properties as a Probe of Porphyrin Microenvironment in Lipid Membranes
Authors: Ricchelli Fernanda , Gobbo Silvano , Jori Giulio , Moreno Giuliana , Salet Christian
Published: September 2004

Temperature‐induced fluorescence changes were studied for hematoporphyrin and protoporphyrin, incorporated into liposomes of dipalmitoylphosphoglycerocholine (Pam2GroPCho) or dimiristoylphosphoglycerocholine (Myr2GroPCho). In some cases, cholesterol or cardiolipin were added to the vesicles for better mimicking the lipid composition of biological membranes. The experimental conditions were appropriately chosen in order to reproduce different possible configurations of the porphyrin molecule in lipid membranes: namely, at the polar water/headgroups, headgroups/lipid and lipid/lipid interfaces.

A peculiar feature observed in some of the above liposomal systems was the appearence of discontinuities in the Arrhenius plots of the fluorescence quantum yields, with relevant changes of the values of activation energies. These discontinuities were due to an increase of the fluorescence signal in a temperature range corresponding to the transition of the different lipids from the gel‐to‐liquid crystal state. The observed phenomena are consistent with the formation of non‐covalent linear dimers or linear higher aggregates of the porphyrin molecules. The intermolecular contacts required for the formation of these species are favored by at least three situations: disruption of the ordered lipid structure during the gel‐to‐liquid crystal phase transition; competition of other polar groups (e.g., the ‐OH group of cholesterol) with the porphyrin carboxylate groups for the polar phospholipid headgroups; and steric constraints due to overcrowding of porphyrin molecules in a restricted space.
Prevention of Cholesteryl Ester Accumulation in P388D1 Macrophage‐Like Cells by Increased Cellular Vitamin E Depends on Species of Extracellular Cholesterol
Authors: Asmis Reto , Llorente Vicenta C. , Gey K. Fred
Published: September 2004

Since the cellular role of the antioxidative vitamins in the formation of foam cells has not yet been studied in detail, we investigated the effect of α‐tocopherol and ascorbic acid loading of P388D1 macrophage‐like cells on their cholesterol and cholesteryl ester levels and their response to the exposure to different lipoproteins. α‐Tocopherol loading, but not ascorbic acid loading, of P388D1 cells strongly reduced their cellular cholesteryl ester/cholesterol ratio (the crucial indicator of foam cell formation) when fetal calf serum was the only extracellular source of cholesterol. Balance studies suggest that this effect of α‐Tocopherol was mainly due to a reduced uptake of fetal‐calf‐serum‐derived cholesterol. α‐Tocopherol loading, however, did not reduce the cholesteryl ester/cholesterol ratio when human unmodified low‐density lipoprotein (LDL) was added to culture medium containing fetal calf serum. Thus, the uptake of fetal‐calf‐serum‐derived cholesterol was competetively reduced by human LDL, the uptake of which remained unaffected by α‐tocopherol. Similarly, α‐tocopherol loading did not prevent cholesteryl ester formation induced by human LDL either oxidized with Cu2+, ultraviolet light or HOCl, or modified by acetylation, aggregation or by malondialdehyde treatment. The present experimental conditions lacked any pro‐oxidative burden, since (a) ascorbic acid, either alone or combined with α‐tocopherol, did not affect cellular cholesteryl ester levels, (b) foam cell formation was not a linear function of the degree of oxidative LDL modification, and (c) α‐tocopherol lacked specific effects on oxidatively modified LDL. Thus, the reduction of cellular cholesteryl esters by α‐tocopherol in the absence of human unmodified LDL was hardly due to common antioxidative properties of vitamin E.

In conclusion, the present observation that a desirable α‐tocopherol effect on the cholesteryl ester balance in mouse‐tumor‐derived P388D1 cells strongly depended on the species of extracellular cholesterol carrier, cautions against premature generalizations of conventional non‐human cell culture data.
Isolation of a Neuropeptide‐Degrading Endopeptidase from the Leech Theromyzon tessulatum
Authors: Laurent Virginie , Salzet Michel
Published: September 2004

Extracts of head parts prepared from the leech Theromyzon tessulatum hydrolyse the Gly3‐Phe4 bond of synthetic [d‐Ala2, Leu5]enkephalin and the Gly‐His bond of benzoyl‐Gly‐His‐Leu. The metabolism of benzoyl‐Gly‐His‐Leu was completely inhibited by captopril, consistent with an angiotensin‐converting enzyme activity. Such an enzyme has recently been isolated from T. tessulatum. However, the enkephalin hydrolysis by captopril (100 μM) was inhibited to a maximum of 70 %. The residual activity hydrolyzing enkephalin was inhibited by phosphoramidon, consistent with the presence of endopeptidase‐24.11, a mammalian enzyme implicated in the metabolism of neuropeptides. This enzyme was isolated using four steps of purification including gel‐permeation and anion‐exchange chromatographies followed by reverse‐phase HPLC. This neuropeptide endopeptidase (of approximate molecular mass 45 kDa) hydrolyses, at pH 7 and 37°C, both the Gly3‐Phe4 bond of synthetic [d‐Ala2, Leu5]enkephalin and the Phe8‐His9 bond of angiotensin I. Cleavage of [d‐Ala2, Leu5]enkephalin yields, respectively, the Tyr‐d‐Ala‐Gly and PheLeu peptides with a specific activity of 29 nmol Tyr‐d‐Ala‐Gly · min−1· mg protein−1 (Km 95 μM). The hydrolysis of angiotensin I yields angiotensin II and the dipeptide His‐Leu with a specific activity of 1.2 nmol angiotensin min−1· mg protein−1 (Km 330 μM). The metabolism of these peptides was totally inhibited by phosphoramidon. This study therefore provides biochemical evidence for neuropeptide‐degrading endopeptidases in leeches.
Regulation of Rat Mast Cell Protease 1 Activity
Authors: Pejler Gunnar , Berg Lotta
Published: September 2004

Rat mast cell protease 1 (RMCP‐1) is a chymotrypsin‐like serine protease (chymase) that is specifically expressed by connective‐tissue‐type mast cells. It is stored in the secretory granules of the cells in a complex with heparin proteoglycan, and the chymase/heparin proteoglycan complexes are released following mast cell activation. The present study was undertaken to examine if the association with heparin proteoglycan influenced the regulation of RMCP‐1 by various macromolecular protease inhibitors. Endogenous mast cell heparin proteoglycan was shown to significantly block the inhibition of RMCP‐1 by the serpins α1‐protease inhibitor and α1‐antichymotrypsin, as well as the inhibition by α2‐macroglobulin, soybean trypsin inhibitor and plasma. The blocking of protease inhibition showed an optimum at a RMCP‐1/proteoglycan ratio of 5:1 (by mass), corresponding to ≈80 RMCP‐1 molecules bound/proteoglycan molecule. Chymase activity present on intact peritoneal mast cells, i.e. present in its native complex with heparin proteoglycan, was also shown to be largely resistant to inhibition by α1‐antichymotrypsin and α1‐protease inhibitor. Heparin 10‐saccharides and 20‐saccharides were inefficient in preventing the interaction of RMCP‐1 with α1‐antichymotrypsin, whereas pig mucosal heparin (≈50 monosaccharide units) blocked protease inhibition. We have previously shown that heparin potentiates the catalytic activity of RMCP‐1 and, in the present study, we show that the mechanism for chymase activation involves a sixfold reduction of the Km,app value of RMCP‐1 for the chromogenic substrate S‐2586. Thus, the association of mast cell chymase with heparin proteoglycan may serve both to potentiate the catalytic activity of the enzyme and to increase the life‐span of the chymases by preventing their inhibition after exocytosis.
Association of Bile‐Salt‐Dependent Lipase with Membranes of Human Pancreatic Microsomes
Authors: Bruneau Nadine , De La Porte Paulette Lechene , Sbarra Veronique , Lombardo Dominique
Published: September 2004

Immunolocalization studies indicated that, in contrast to other enzyme markers of human pancreatic secretion, bile‐salt‐dependent lipase (BSDL) was partly but specifically associated with endoplasmic reticulum membranes. In microsomes, temperature‐induced phase separation using Triton X‐114 elucidated the partition of BSDL between the aqueous phase and the detergent‐rich phase containing hydrophilic and membrane proteins, respectively. The size of the membrane‐associated BSDL (approx. 100 kDa) is compatible with that of the fully processed enzyme. Fucosylated O‐ and N‐linked oligosaccharide structures were detected by means of specific lectins. The membrane‐associated BSDL might therefore be released from membranes between the trans‐Golgi compartment (where terminal fucose residues were added) and the zymogen granules where BSDL was mainly found in the soluble fraction. Even though BSDL associated with membranes was enzymically active, it appeared less efficient than the soluble form. The association of BSDL with membranes was pH‐dependent and optimal association occurred between pH 5–6. The membrane‐associated BSDL was released by KBr which suggests that the association of BSDL with microsomal membranes involves ionic interactions. Lipid–protein interactions are probably not involved in this association as BSDL did not associate with liver microsome membranes. We attempted to characterize the putative ligand and showed that BSDL and a 94‐kDa protein, immunologically related to a glucose‐regulated protein of 94 kDa (Grp94), were co‐immunoprecipitated by specific antibodies directed against each individual species. It is suggested that the biogenesis of the human pancreatic BSDL involves an association with intracellular membranes and that its folding may be assisted by molecular chaperones.
Dimeric 3‐Phosphoglycerate Kinases from Hyperthermophilic Archaea
Authors: Hess Daniel , Krüger Kerstin , Knappik Achim , Palm Peter , Hensel Reinhard
Published: September 2004

The gene coding for the 3‐phosphoglycerate kinase (EC 2.7.2.3) of Pyrococcus woesei was cloned and sequenced. The gene sequence comprises 1230 bp coding for a polypeptide with the theoretical Mr of 46195. The deduced protein sequence exhibits a high similarity (46.1% and 46.6% identity) to the other known archaeal 3‐phosphoglycerate kinases of Methanobacterium bryantii and Methanothermus fervidus [Fabry, S., Heppner, P., Dietmaier, W. & Hensel, R. (1990) Gene 91, 19–25]. By comparing the 3‐phosphoglycerate kinase sequences of the mesophilic and the two thermophilic Archaea, trends in thermoadaptation were confirmed that could be deduced from comparisons of glyceraldehyde‐3‐phosphate dehydrogenase sequences from the same organisms [Zwickl, P., Fabry, S., Bogedain, C., Haas, A. & Hensel, R. (1990) J. Bacteriol. 172, 4329–4338]. With increasing temperature the average hydrophobicity and the portion of aromatic residues increases, whereas the chain flexibility as well as the content in chemically labile residues (Asn, Cys) decreases.

To study the phenotypic properties of the 3‐phosphoglycerate kinases from thermophilic Archaea in more detail, the 3‐phosphoglycerate kinase genes from P. woesei and M. fervidus were expressed in Escherichia coli. Comparisons of kinetic and molecular properties of the enzymes from the original organisms and from E. coli indicate that the proteins expressed in the mesophilic host are folded correctly. Besides their higher thermostability according to their origin from hyperthermophilic organisms, both enzymes differ from their bacterial and eucaryotic homologues mainly in two respects, (a) The 3‐phosphoglycerate kinases from P. woesei and M. fervidus are homomeric dimers in their native state contrary to all other known 3‐phosphoglycerate kinases, which are monomers including the enzyme from the mesophilic Archaeum M. bryantii. (b) Monovalent cations are essential for the activity of both archaeal enzymes with K+ being significantly more efficient than Na+. For the P. woesei enzyme, non‐cooperative K+ binding with an apparent Kd (K+) of 88 mM could be determined by kinetic analysis, whereas for the M. fervidus 3‐phosphoglycerate kinase the K+ binding is rather complex: from the fitting of the saturation data, non‐cooperative binding sites with low selectivity for K+ and Na+ (apparent Kd= 270 mM) and at least three cooperative and highly specific K+ binding sites/subunit are deduced.

At the optimum growth temperature of P. woesei (100°C) and M. fervidus (83°C), the 3‐phosphoglycerate kinases show half‐lives of inactivation of only 28 min and 44 min, respectively. Potassium salts of polyvalent anions stabilize both 3‐phosphoglycerate kinases, like other enzymes from these organisms. In the case of M. fervidus 3‐phosphoglycerate kinase, the dominant low‐molecular‐mass compound cyclic d‐glycerate 2,3‐bisphosphate found in vivo [Hensel, R. & König, H. (1988) FEMS Microbiol. Lett. 19, 325–333] is the most effective.
Molecular Cloning, Functional Expression in Escherichia coli, and Characterization of Multiple Mitogen‐Activated‐Protein Kinases from Tobacco
Authors: Wilson Cathal , Anglmayer Roswitha , Vicente Oscar , Heberle‐Bors Erwin
Published: September 2004

A screening of four tobacco cDNA libraries by PCR, using degenerate oligonucleotides corresponding to motifs conserved in mitogen‐activated‐protein kinases from animals and yeasts, resulted in the isolation of five different PCR fragments that showed high sequence similarity to mitogen‐activated‐protein kinases from other organisms. Full‐length cDNAs were obtained for two of these, ntf4 and ntf6, and we have previously reported the isolation of one of the other cDNAs, ntf3 [Wilson, C., Eller, N., Gartner, A., Vicente, O. & Heberle‐Bors, E. (1993) Plant Mol. Biol. 23, 543–551], The three cDNAs, ntf3, ntf4 and ntf6, as well as a mutated form of ntf3, were fused to the glutathione‐S‐transferase gene and expressed as fusion proteins in Escherichia coli. All three wild‐type recombinant proteins, with or without the glutathione‐5‐transferase fragment, are capable of autophosphorylation and phosphorylate myelin basic protein, in a reaction that is more strongly supported by Mn2+ than by Mg2+, while the kinase‐negative Ntf3 mutant did not show any activity. Western‐blot analysis showed that the recombinant proteins autophosphorylate on tyrosine residues and are recognized by antibodies prepared against mammalian mitogen‐activated‐protein kinases.
Molecular Cloning of the cDNA Encoding Human Skeletal Muscle Triadin and Its Localisation to Chromosome 6q22–6q23
Authors: Taske Nichole L. , Eyre Helen J. , O'Brien Robert O. , Sutherland Grant R. , Denborough Michael A. , Foster Paul S.
Published: September 2004

We have cloned and sequenced the cDNA encoding triadin, a junctional terminal cisternae protein from human skeletal muscle. The cDNA, 2941 base pairs in length, encodes a protein of 729 amino acids with a predicted molecular mass of 81 545 Da. Hydropathy analysis indicates that triadin of human skeletal muscle has the same topology in the myoplasmic, transmembrane and sarcoplasmic reticulum luminal domains as that of triadin from rabbit skeletal muscle. The number and relative position of potential modulation sites are also conserved between the human and rabbit proteins. The cDNA sequence of the predicted sarcoplasmic reticulum luminal domain of human triadin diverged from that of rabbit, with an observed similarity of 82%, translating to an identity of 77% in amino acid sequence. Two insertions of 9 and 12 residues in the amino acid sequence were observed in the predicted luminal domain of triadin, although the structural and functional consequences of such insertions are expected to be minimal. Using fluorescence in situ hybridisation, we have assigned the gene encoding human triadin to the long arm of chromosome 6 in the region 6q22–6q23. Our structural analysis of human triadin supports a central role for this protein in the mechanism of skeletal muscle excitation/contraction coupling.
Mutations at Positions 11 and 60 of Insulin‐Like Growth Factor 1 Reveal Differences between its Interactions with the Type I Insulin‐Like‐Growth‐Factor Receptor and the Insulin Receptor
Authors: Hodgson Darren R. , May Felicity E. B. , Westley Bruce R.
Published: September 2004

Insulin‐like growth factor 1 (IGF‐1) and three analogues ([V11I]IGF‐1, [V11T]IGF‐1, and [Y60F]IGF‐1), constructed by site‐directed mutagenesis, were expressed as fusion proteins and secreted into the periplasmic space ofEscherichia coli.Purified IGF were obtained following IgG Sepharose affinity and cation‐exchange chromatographies of the products of hydroxylamine cleavage of fusion proteins. The properties of the mutants were assessed using (a) quantification of affinities for the human insulin receptor overexpressed on NIH 3T3 cells, (b) quantification of affinities for the type I IGF receptor via competition for binding to a monolayer of MDA‐MB‐231 cells, (c) promotion of thein vitromitogenesis of growth‐arrested MCF‐7 cells in the presence of 17‐β‐oestradiol, and (d) a competition assay for binding to IGF‐binding proteins secreted by MCF‐7 cells.

The mutants exhibited decreases in affinity for the insulin receptor, relative to IGF‐1, of 2.6‐, 3.8‐and, 8.8‐fold for [Y60F]IGF‐1, [V11I]IGF‐1, and [V11T]IGF‐1, respectively. IGF‐1, [V11I]IGF‐1, and [Y60F]IGF‐1 were of equal potency in the growth assay and in affinity for the type I IGF receptor. [V11 T]IGF‐1 exhibited a three fold loss of potency in the type I IGF receptor‐binding and growth assays. The mutants did not differ significantly from IGF‐1 in their affinities for the IGF‐binding proteins.

The full‐activity of [Y60F]IGF‐1 at the type I IGF receptor, in contrast to the weakened receptor affinity of IGF‐1 with a Leu substitution at this position, indicates a requirement for an aromatic ring, rather than a hydroxyl group, in the interaction of IGF‐1 with the type I IGF receptor. The decrease in affinity for the insulin receptor of all the mutants indicates that, as in insulin, the residues Val11 and Tyr60 are important for the interaction of IGF‐1 with the insulin receptor. The unchanged or minor changes in the affinities of the mutants for the type I IGF receptor contrast with the more deleterious effects of the mutations on insulin receptor binding and with the properties of analogues of insulin mutated at equivalent sites: 3‐fold and 5–10‐fold reductions in biological activity for [VB121]insulin and [YA19F]insulin, respectively. Thus, the results obtained using the mutants indicate important differences between the IGF‐1/type I IGF receptor and insulin/insulin receptor interactions.
Spectroscopic Characterisation of an Aconitase (AcnA) of Escherichia coli
Authors: Bennett Brian , Gruer Megan J. , Guest John R. , Thomson Andrew J.
Published: September 2004

A spectroscopic study of an aconitase, AcnA, from Escherichia coli is presented. The amino acid sequence of AcnA has 53% identity with mammalian cytosolic aconitase (c‐aconitase) which is the translational regulator known as iron regulatory factor (IRF). In the [3Fe‐4S]+‐containing, inactive state, AcnA displays an EPR signal which is not unlike the corresponding signal from mammalian mitochondrial aconitase (m‐aconitase) but is even more similar to the signal from c‐aconitase. This is perhaps related to the greater similarity of the AcnA amino acid sequence with c‐aconitase. Magnetic circular dichroism (MCD) spectroscopy has revealed that the electronic structure of the [3Fe‐4S] cluster of AcnA must be similar to, but not identical to that of m‐aconitase. Whilst the [3Fe‐4S] clusters from both of these enzymes display some features in their MCD spectra common to [3Fe‐4S] clusters in general, their spectra overall are unique and indicate that the Fea atom of the [4Fe‐4S] form is not the only unusual feature of the [Fe‐S] clusters of aconitases. Active [4Fe‐4S]‐containing AcnA can be reduced to yield an EPR signal due to a [4Fe‐4S]+ cluster which is indistinguishable from the signals from the [4Fe‐4S]+ cluster in the mammalian enzymes. However, in contrast to the mammalian enzymes, the EPR signals of the cluster in AcnA are not significantly perturbed upon the addition of substrate. Furthermore, the catalytic activity of [4Fe‐4S]2+‐containing AcnA is fivefold higher than that of m‐aconitase. The mechanistic implications of these data are discussed. A novel S = 1/2 EPR signal with g≈2 was observed in AcnA upon treatment with EDTA. The species giving rise to this signal is proposed to be an intermediate in cluster deconstruction.
Cyclic Voltammetry and 1H‐NMR of Rhodopseudomonas palustris Cytochrome c2
Authors: Battistuzzi Gianantonio , Borsari Marco , Dallari Daniela , Ferretti Silvia , Sola Marco
Published: September 2004

The effects of increasing concentrations of Cl−, ClO4−, and HCO3− on the redox potential of Rhodopseudomonas palustris cytochrome c2 indicate that the two polyatomic anions bind specifically to the protein at one site, while chloride simply exerts an ionic atmosphere effect. The change in E° upon specific anion binding allows us to probe for the influence of surface charges on the redox potential of cytochromes c. The decrease in redox potential at null ionic strength (ΔEI=0°) due to anion neutralization of one positive surface charge was found to be 23 mV with perchlorate and 33 mV with bicarbonate. These values compare reasonably well with previous theoretical predictions and estimates of the effect of charge alteration on the E° values in cytochromes c chemically modified or mutated at surface lysines. These ΔE° values, determined on the unmodified protein, are unprecedented for c‐type cytochromes. The anion‐induced chemical shift changes of the hyperfine‐shifted heme 1H‐NMR resonances of the oxidized protein yield lower limit values of 53 M−1 and 18 M−1 for the affinity constant for specific HCO3− and ClO− binding, respectively.
Enzymic and Chemical Reduction of the Iron Center of the Escherichia coli Ribonucleotide Reductase Protein R2
Authors: Covès Jacques , Delon Betty , Climent Isabel , Sjöberg Britt‐Marie , Fontecave Marc
Published: September 2004

The active form of protein R2, the small subunit of ribonucleotide reductase, contains a diferric center and a free radical localized at Tyr122. Hydroxyurea scavenges this radical but leaves the iron center intact. The resulting metR2 protein is inactive. The introduction of a radical into metR2 is dependent on the reduction of the iron center. In Escherichia coli, this is achieved by an enzyme system consisting of a NAD(P)H:flavin oxidoreductase and a poorly defined protein fraction, fraction b. Assuming that the iron center is deeply buried within the protein, electron transfer is suggested to occur over long distances. Site‐directed mutagenesis allowed us to identify two invariant residues, Tyr356 at the C‐terminal part of the protein and Tyr122 located 0.5 nm away from the closest iron atom, as mediators of this electron transfer. We also found that deazaflavins were excellent catalysts in the photoreduction of the iron center of metR2 and generation of the tyrosyl radical, providing the simplest and most efficient model for the physiological flavin reductase/fraction b activating system. The properties of the model reaction are described.
Angiotensin converting enzyme inhibition for cardiac hypertrophy in patients with end‐stage renal disease: What is the evidence?
Authors: WANG ANGELA YEE‐MOON , LI PHILIP KAM‐TAO , LUI SIU‐FAI , SANDERSON JOHN E
Published: September 2004

SUMMARY:  Dialysis patients show a high prevalence of cardiovascular complications among which left ventricular hypertrophy is one of the most frequent and is independently predictive of mortality. A recent study indicates that partial regression of left ventricular hypertrophy improves mortality and reduces cardiovascular events in end‐stage renal disease (ESRD) patients, suggesting the importance of targeting therapeutic strategies to reduce cardiac hypertrophy and improve the outcome in these patients. The pathogenesis of left ventricular hypertrophy in ESRD patients is multifactorial and includes hypertension, activation of the renin‐angiotensin system, increased sympathetic activity, chronic volume overload, chronic anaemia and hyperparathyroidism. In this paper, we review the available experimental and clinical evidence showing the important contribution of the renin‐angiotensin system as well as its interaction with the sympathetic nervous system in the pathogenesis of left ventricular hypertrophy in ESRD patients. Furthermore, we summarize the results of currently available clinical studies that examined the effects of angiotensin‐converting enzyme inhibition or angiotensin receptor antagonism on left ventricular hypertrophy in ESRD patients, and review evidences that support the use of angiotensin‐converting enzyme inhibitors or angiotensin receptor antagonists in the ESRD population.
What is the practical conversion dose when changing from epoetin alfa to darbepoetin outside of clinical trials?
Authors: ROGER SIMON D , COOPER BRUCE
Published: September 2004

Background:  The recommended conversion dose for changing from epoetin alfa to darbepoetin alfa is 200 units to 1 µg. However, this may result in the over treatment of uraemic anaemia.

Methods:  All in‐centre haemodialysis patients (n = 60) were converted from an existing subcutaneous epoetin alfa regimen to weekly intravenous darbepoetin alfa. The protocol for anaemia management included a target haemoglobin (Hb) concentration of 120–130 g/L and a ferritin of 300–600 µg/L. Patient treatments were converted from subcutaneous epoetin alfa to weekly, intravenous darbepoetin alfa at month 0, at a conversion dose of 200 units epoetin alfa to 1 µg darbepoetin.

Results:  Both Hb and ferritin concentrations remained within the target range, but darbepoetin dosages fell from 50.8 to 42.3 µg/week by month 3 (P = 0.02). The initial conversion factor of 210 units/µg rose to 275 units/µg (P = 0.01) at month 4. No difference in conversion dosage could be determined between patients who were epoetin sensitive (<200 units/kg per week) or resistant (>200 units/kg per week, P = NS). Patients were then switched to fortnightly darbepoetin alfa dosing treatments; the existing weekly dose being doubled and Hb levels fell from 125 to 110 g/L (P < 0.0001), despite an increase in the mean dose from 44.9 to 47.5 µg/week (P = 0.02).

Conclusion:  The original dosage reduction after the switch from epoetin alfa to weekly intravenous darbepoetin alfa may offset the increased relative cost of the latter. When administered weekly and intravenously, darbepoetin alfa maintains Hb at a more favourable conversion rate than is currently recommended.
Oxidative stress from rapid versus slow intravenous iron replacement in haemodialysis patients
Authors: TIRANATHANAGUL KHAJOHN , EIAM‐ONG SOMCHAI , TOSUKHOWONG PIYARATANA , PRADITPORNSILPA KEARKIAT , TUNGSANGA KRIANG
Published: September 2004

Methods and Results:  Oxidative stress was examined in 19 erythropoietin‐treated haemodialysis patients who were receiving 100 mg of iron sucrose every 2 weeks by two intravenous methods, rapid injection and slow infusion. There were no significant differences in incidence of iron oversaturation state between the two methods. Regarding oxidative stress markers, the values of plasma and red blood cell thiobarbituric acid reactive substances (TBARS) expressed in terms of malonyldialdehyde (MDA) equivalents following the two methods did not increase, and the values of area under the curve (AUC) of both markers were not different between both regimens. Also, there were no significant differences in the values of plasma and AUC of anti‐oxidant markers including total anti‐oxidant status, reduced thiols, and vitamin E among both periods treated with two intravenous iron methods.

Conclusion:  As such, both intravenous iron methods could be safely used without enhancing oxidative stress in haemodialysis patients. The rapid injection method would be the preferred method of intravenous iron administration because it is more convenient while still retaining the safety profile.
Status of dialyser reuse in Korea
Authors: CHO HYEON‐KYEONG , SHIN GYU‐TAE , KIM HEUNGSOO
Published: September 2004

Background and Aim:  Dialyser reuse treatments in Korea were first practised at a facility in 1985. Until 1999, there was only one facility practising dialyser reuse treatments, but the reuse practice has gradually increased since 1999. The purpose of this study was to gather and analyse the current (April 2002) dialyser reuse treatment data in Korea.

Method and Results:  Data was collected via a questionnaire sent to the each hospital that reuses the dialyser, and we received a response from 26 out of 29 facilities (89.7%). Twenty‐nine facilities comprised 7.7% (29 of 376) of the total nationwide haemodialysis facilities in Korea. The percentage of patients on dialyser reuse treatments was 6.2% (1234 of 20 010). All facilities used an automated reuse processing technique for dialyser reuse and 22 facilities used a peracetic acid mixture (PAM) without hypochlorite. There was one facility that used the heated citric acid method. Eighty per cent of facilities used only high flux membranes (Kuf ≥ 20 mL/h per mmHg) and 12% of the facilities used both high and low flux membranes. The average number of the reuse treatments was 15‐fold (range 10−22) and the average of the maximum number of reuse treatments was 20‐fold (range 10–50).

Conclusion:  Although dialyser reuse treatments are not a common practice in Korea, the reuse programs are steadily increasing. Strict quality control and further regulations regarding the reuse program  should be promptly enacted to provide a better quality of haemodialysis treatment for patients in Korea.
LAPAROSCOPIC TREATMENT FOR PERFORATED APPENDICITIS WITH PELVIC ABSCESS
Authors: Yano Hiroshi , Murakami Masahiro , Nakano Yoshiaki , Tono Takeshi , Ohnishi Tadashi , Iwazawa Takashi , Kimura Yutaka , Kanoh Toshiyuki , Monden Takushi
Published: September 2004

We performed laparoscopic appendectomy and drainage to treat panperitonitis due to perforated appendicitis that occurred in a 28‐year‐old woman. We believe this is an appropriate procedure to treat perforated appendicitis because it is safe and minimally invasive, and faster recovery can be expected than after conventional open appendectomy.
DIAGNOSIS OF GASTRIC MUCINOUS CARCINOMA BY ENDOSCOPIC ULTRASONOGRAPHY
Authors: Hirasawa Dai , Fujita Naotaka , Yuki Toyohiko , Sato Tadasu , Ishida Kazuhiko , Senoo Shigeharu , Suzuki Takashi , Noda Yutaka , Kobayashi Go , Kimura Katsumi , Matsunaga Atsuo , Nomura Mikiko , Ito Kei , Sugawara Toshiki , Horaguchi Jun , Takasawa Osamu
Published: September 2004

Background:  Biopsy diagnosis of gastric mucinous carcinoma (GMC) is sometimes difﬁcult. Endoscopic ultrasonography (EUS) can visualize the change in the gastric wall in detail. We reviewed the EUS images and histological ﬁndings of cases of GMC and evaluated the correlation between them to identify the characteristic ﬁndings of GMC on EUS.

Methods:  Thirty‐one GMC patients who had undergone both preoperative EUS and surgery were studied. The EUS images of these patients and the histological ﬁndings were compared with those for other histological types of gastric cancer (OGC).

Results:  The tumor echo showed high echoic in GMC cases more frequently than that in OGC. The frequency of visualization of the layer structure above the tumor was higher compared with OGC. Histology veriﬁed the presence of ﬁne septa composed of thin ﬁbrotic tissue and ﬂoating cancer cells in the mucus lake. The mucus lake was often located in the submucosa and the tumor was covered with cancerous mucosa of other histological types with preserved lamina muscularis mucosae.

Conclusions:  The EUS ﬁndings of GMC are characteristic and reﬂect its histological changes well, and would complement the preoperative diagnosis of this tumor.
ENDOSCOPY IS ONE OF THE VALUABLE DIAGNOSTIC METHODS FOR PRIMARY AORTO‐ENTERIC FISTULA
Authors: Honjo Kie , Okada Hiroyuki , Sumii Kengo , Kobatake Toshitsugu , Fujioka Shinichi , Kumagai Isao , Takeda Sho , Teraoka Akira
Published: September 2004

The present report describes a case of primary aorto‐enteric ﬁstula (PAEF) in an 81‐year‐old man with no previous abdominal surgery. The patient was admitted to Teraoka Memorial Hospital in shock with repeated episodes of melena and hematemesis. We performed emergency endoscopy to establish a diagnosis and identiﬁed a ruptured aortic aneurysm visualized through a ﬁstula in the second portion of the duodenum. As most PAEF cause massive gastrointestinal hemorrhage, prompt diagnosis is critical to ensure a successful outcome. Although the patient and his family refused surgery and the patient died, we were able to make the diagnosis at an early stage by endoscopy. While PAEF is usually very difﬁcult to diagnose by a single modality, endoscopy can be one of the modalities to identify the source of gastrointestinal hemorrhages, even in cases of PAEF.
ENDOSCOPIC INJECTION SCLEROTHERAPY FOR ESOPHAGEAL VARICES IN TWO PATIENTS WITH IDIOPATHIC MYELOFIBROSIS
Authors: Sato Takahiro , Yamazaki Katsu , Toyota Jouji , Karino Yoshiyasu , Ohmura Takumi , Akaike Jun , Kuwata Yasuaki , Suga Toshihiro
Published: September 2004

Case 1: A 75‐year‐old woman was admitted to our department in February 1996 with tarry stool. At 71 years of age, she had been diagnosed with idiopathic myelofibrosis (IMF) based upon laboratory data and a biopsy of iliac bone marrow. Fibergastroscopic examination on admission revealed esophageal varices graded as Ls, F2, Cb, and red color (RC)(+ +). Esophageal varices were considered the most likely cause of the bleeding. Computed tomography (CT) showed a markedly enlarged spleen and large collateral veins (left gastric vein, spleno‐renal shunt). We performed endoscopic injection sclerotherapy (EIS) twice weekly using 5% ethanolamine oleate with iopamidol (5% EOI), the total amount of sclerosant was 13 mL. No further variceal bleeding occurred until she died in June 1996. Case 2: A 67‐year‐old man was admitted to our hospital in February 1992 with anemia and leukocytosis after a health check. He was diagnosed with IMF based upon laboratory data and a biopsy of iliac bone marrow. Screening upper gastrointestinal endoscopy demonstrated esophageal varices graded as Ls, F3, Cb, and red color (RC)(+) and gastric varices graded as Lg–cf, F2, and RC(–). CT showed a markedly enlarged spleen and large collateral veins (left gastric vein, short gastric vein). We performed EIS four times weekly using 5% EOI, the total amount of sclerosant was 32 mL. Two weeks after EIS the varices were graded as F1, RC(–). This patient experienced esophageal varix recurrence up to 1 year later, and EIS was performed twice using 5% EOI.
A CASE OF INDETERMINATE COLITIS UNDERGOING SUBTOTAL COLECTOMY
Authors: Yokoyama Hiroshi , Takagi Sho , Utsunomiya Kyoko , Negoro Kenichi , Takahashi Seiichi , Kinouchi Yoshitaka , Shimosegawa Tooru , Masuda Takayuki
Published: September 2004

The nomenclature inflammatory bowel disease (IBD) generally defines ulcerative colitis (UC) and Crohn's disease (CD). However, we sometimes find patients who cannot be diagnosed as UC or CD, because of the presence of both characteristics. These cases have been reported as indeterminate colitis (IC) since the first report in 1978. In our department, we have experienced IBD since 1993 (366 cases of CD), but only three cases were diagnosed as IC. Of the three, we report here the clinical and pathological features of a patient who was followed up since the diagnosis.

The case was a 19‐year‐old Japanese woman without any particular past history. In December 1997, she was admitted to our hospital complaining of abdominal pain, high fever, diarrhea and fresh blood in the stool. Based on the findings of various tests, we made a diagnosis of IC, and started to treat her with prednisolone intravenous injection (i.v) at a dose of 40 mg per day. Because symptoms and signs of inflammation relapsed frequently, it was judged that there was a relative indication for surgical operation. She underwent subtotal colectomy with end‐to‐end ileorectal anastomosis on 6 January 2002. On the histopathological examination of surgical specimens, several characteristics of both UC and CD were observed. Therefore, we finally diagnosed this case as IC. After operation, she has not experienced a relapse so far and maintained fair physical condition.
ENDOSCOPIC LIGATION BY DETACHABLE SNARE FOR THE HEMOSTASIS OF COLONIC BLEEDING FOLLOWED BY SUCCESSFUL LIVER TRANSPLANTATION
Authors: Lee Yun‐Jung , Myung Seung‐Jae , Byeon Jeong‐Sik , Yang Suk‐Kyun , Kim Kyu‐Jong , Hong Seong Soo , Jung Hwoon‐Yong , Lee Yung‐Sang , Hong Weon‐Seon , Kim Jin‐Ho , Min Young‐Il
Published: September 2004

A 40‐year‐old male patient with liver cirrhosis developed hematochezia resulting in hepatic encephalopathy. Colonoscopic examination revealed a large, approximately 4.0 cm‐sized villous adenoma in the sigmoid colon which showed diffuse blood oozing. We succeeded in the control of bleeding from the lesion only with a detachable snare. The patient recovered from hepatic encephalopathy and may have an opportunity for liver transplantation. Fortunately, the patient regained normal liver function after a successful liver transplantation.