bluuebunny/crossref_metadata_2025 huggingface dataset:
Complete Ammo Acid Sequence of the Aa6 Subunit of the Scorpion Androctonus australis Hemocyanin Determined by Edman Degradation and Mass Spectrometry
Authors: Buzy Armelle , Gagnon Jean , Lamy Josette , Thibault Pierre , Forest Eric , Hudry‐Clergeon Gilbert
Published: September 2004

The primary structure of the hemocyanin Aa6 subunit from the scorpion Androctonus australis was resolved by using protein sequencing and mass spectrometry for analysis of the polypeptide chain and of fragments obtained by CNBr, trypsin, and chymotrypsin cleavage. Due to the high sensitivity of the methodologies used, only a small amount of material, less than 1 mg, was consumed. The complete sequence is composed of 626 amino acid residues and the protein is not glycosylated but probably phosphorylated at Ser374. Its molecular mass measured by mass spectrometry (71 890 ± 7 Da) is about 30 Da higher than the mass calculated from the sequence data (71 860.1 Da). The origin of this difference is not clear but could result from minor molecular heterogeneities. Within the chelicerates, the Aa6 subunit of the arachnid A. australis shares 405 identical residues with chain e of another arachnid, Eurypelma californicum, and 399 with chain α of the merostom Tachypleus tridentatus. The degrees of identity between these three subunits, which are known to occupy the same location in the native hemocyanin oligomers, are significantly higher than those existing between the subunits a, d, and e of E. californicum. This favors the hypothesis that gene duplications, leading to separate chains in one species, have occurred before the divergence between arachnids and merostoms.
Cloning and Properties of a Novel Natriuretic Peptide Receptor, NPR‐D
Authors: Kashiwagi Masahide , Katafuchi Takeshi , Kato Akira , Inuyama Hiromi , Ito Teizo , Hagiwara Hiroki , Takei Yoshio , Hirose Shigehisa
Published: September 2004

A novel natriuretic peptide receptor, which we have termed natriuretic peptide receptor D (NPR‐D), has been cloned and characterized. cDNAs related to the natriuretic peptide receptor (NPR) were amplified by PCR from a template of poly(A)‐rich RNA isolated from the eel gill. Sequencing of the PCR products revealed the presence of a new clone that showed about 70% sequence identity to the eel type‐C receptor, NPR‐C. The PCR fragment was used to determine the tissue distribution of the new NPR‐D message by an RNase protection assay, which gave the strongest signal in brain samples, and then used to screen a brain library to obtain a full‐length cDNA clone. The cDNA clone predicted a protein of 500 amino acids containing a signal sequence and a hydrophobic transmembrane segment. The predicted sequence also contained the NPR motif which is essential for the binding of natriuretic peptides. The protein NPR‐D was expressed in COS cells and shown to have high affinities for eel and rat natriuretic peptides. The newly cloned NPR‐D has a short cytoplasmic tail; in this respect, NPR‐C and NPR‐D are very similar and form a subfamily of the NPR family. Affinity labeling indicated that NPR‐D exists as a disulfide‐linked tetramer. This is a marked contrast to the homodimeric structure of NPR‐C. HS‐142‐1, a non‐peptide natriuretic peptide receptor antagonist of microbial origin previously shown to be selective for the guanylate‐cyclase‐coupled receptors NPR‐A and NPR‐B, competitively inhibited the binding of 125I‐labeled eel natriuretic peptide to eel NPR‐D, whereas it did not affect the binding activity of eel NPR‐C, suggesting that HS‐142‐1 is an antagonist that recognizes the tetrameric structures of NPR since the guanylate‐cyclase‐coupled receptors have also been demonstrated to exist as tetramers.
Expression, Purification and Characterization of the Enzyme II Mannitol‐Specific Domain from Staphylococcus carnosus and Determination of the Active‐Site Cysteine Residue
Authors: Von Strandmann Rembert Pogge , Weigt Christiane , Fischer Roland , Meyer Helmut E. , Kalbitzer Hans Robert , Hengstenberg Wolfgang
Published: September 2004

The C‐terminal B domain of mannitol‐specific enzyme II (enzyme IIB) of the phosphoenolpyruvate‐dependent phosphotransferase system for mannitol from Staphylococcus carnosus was subcloned, purified and characterized. In Staphylococcal cells, mannitol‐specific enzyme II is composed of a soluble A domain (EIIA) and a transmembrane C domain transporter with a fused enzyme II B (IIB) domain. We purified large amounts of the IIB domain as an in‐frame fusion with six histidine residues. Here, we show that the domain is stable and can be phosphorylated by phosphoenolpyruvate and the phosphotransferase components. It is a dimer over a wide range of pH values and salt conditions. Differences between the published nucleotide sequence data and the mass‐spectroscopic data obtained with the purified protein lead to anewed nucleotide sequencing of the gene. Two errors in the original proposed sequence were found, the correction of the second error leading to a frame shift that adds 10 amino acids to the deduced amino acid sequence. The mass of the phosphorylated domain is 20068 Da, 80 Da more than the mass of the unphosphorylated domain, therefore, no other residues, such as COOH side chains, are directly involved in an additional phosphate linkage concerning the IIB domain. 31P‐NMR experiments as well as chemical modification proved that Cys429 is the phosphoamino acid. Titration of the phosphorylated domain during 31P‐NMR did not lead to the typical shift for the protonation of the thiophosphate in the resonance spectrum. Thus, the thiophosphate remains in the twofold negatively charged state.
Binding Studies on the Combining Site of a GalNAcα1→‐Specific Lectin with Thomsen‐Friedenreich Activity Prepared from Green Marine Algae Codium fragile Subspecies Tomentosoides
Authors: Wu Albert M. , Song Shuh‐Chyung , Hwang Pei‐Yi , Wu June H. , Chang Kenneth S. S.
Published: September 2004

The combining site of a GalNAα1→‐specific lectin (CFT) with Thomsen‐Friedenreich (T, Galβ1→‐3‐GalNAcα1→Ser/Thr) activity, purified from the subspecies tomentosoides of green marine algae Codium fragile was studied by quantitative precipitin and precipitin‐inhibition assays. Of 27 glycoforms tested, Tn (GalNAcα1→Ser/Thr) glycoprotein from armadillo submandibular glands, and asialo porcine submandibular glycoprotein, which contains T, Tn and GalNAcα1→3Gal(A) sequences, completely precipitated the lectin added, and less than 1 μg glycoprotein was required to precipitate 50% 4.7 μg lectin nitrogen. However, CFT precipitated negligibly with Pneumococcus type‐XIV polysaccharide and asialo human α1‐acid glycoprotein, that contain exclusively the human blood‐type‐II precursor sequence (II, Galβ1→4GlcNAc) at the nonreducing ends. Among the sugar inhibitors tested, the human blood A‐active trisaccharide [Ah, GalNAcα1→3(lFucα1→2)Gal] was the best inhibitor; it was about twice as active as the T disaccharide.

Oligosaccharides without GalNAcα1→ as part of their sequences were inactive, indicating that the acetamido group at C2 of galactose is essential for binding and that GalNAc is the main contributor in the T sequence for binding. From the data provided, it is clear that the combining site of CFT requires an α‐anomer of GalNAc and recognizes Ah, internal GalNAcα1→ of T and Tn determinants of glycans, but not the blood group I/II (Galβ1→3/4GlcNAc) sequences. Consequently, CFT is a useful reagent for detecting GalNAcα1→‐containing glycoconjugates.
The Structure of the Lipid A‐Core Region of the Lipopolysaccharides from Vibrio cholerae O1 Smooth Strain 569B (Inaba) and Rough Mutant Strain 95R (Ogawa)
Authors: Vinogradov Evgeny V. , Bock Klaus , Holst Otto , Brade Helmut
Published: September 2004

The lipopolysaccharides (LPS) from Vibrio cholerae 95R, a rough mutant strain of O1 V. cholerae 162 (Ogawa), and from smooth O1 V cholerae 569B (Inaba) were de‐O‐acylated. In each case, one part of the products was treated with 48% aqueous HF which removed the phosphoryl and fructose residues, then reduced, de‐N‐acylated, and N‐acetylated. Another part was de‐N‐acylated by treatment with hot KOH. The products of both degradation pathways were separated by high‐performance anion‐exchange chromatography. The major dephosphorylated and defructosylated product 1 was obtained in pure form, whereas the minor products 2 and 3 were eluted as a mixture, as were, from the second degradation, the phosphorylated oligosaccharides 4 (major product) and 5 (minor product). No phosphorylated component corresponding to oligosaccharide 3 could be identified by NMR spectroscopy in the latter mixture. The following structures of oligosaccharides 1–5 were established on the basis of monosaccharide and methylation analyses, Smith degradation, and 1H‐ and 13C‐NMR investigations (correlated, total correlated, NOE and heteronuclear correlation spectroscopy; all sugars are present as α‐d‐pyranoses except where indicated otherwise; Hep, l‐glycero‐d‐manno‐hsptose; Kdo, 3‐deoxy‐d‐manno‐2‐octulosonic acid).

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In the untreated lipopolysaccharide, the amino group of the non‐reducing terminal glucosamine residue is not substituted.
Temperature‐Induced Changes in Fluorescence Properties as a Probe of Porphyrin Microenvironment in Lipid Membranes
Authors: Ricchelli Fernanda , Gobbo Silvano , Jori Giulio , Salet Christian , Moreno Giuliana
Published: September 2004

The temperature dependence of hematoporphyrin and protoporphyrin fluorescence quantum yields (ΦF) was studied after delivery to whole mitochondria or isolated inner (IMM) and outer (OMM) mitochondrial membranes, obtained from liver of Wistar rats. These studies are very sensitive to variations of the porphyrin lipid environment. Before incorporation, the porphyrins were dissolved in 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4) NaCl/Pi (only hematoporphyrin) or dispersed into liposomes of dipalmitoylphosphogiycerocholine (Pam2GroPCho), sometimes enriched with cholesterol or cardiolipin. Whole mitochondria show higher incorporation capacity of hematoporphyrin and protoporphyrin than isolated IMM and OMM, probably because additional, energy‐sensitive transport mechanisms for the porphyrin uptake occur in intact organelles. A small decrease in protoporphyrin uptake is observed in OMM in comparison with IMM; in contrast, the decrease in hematoporphyrin uptake by OMM is rather significant.

A comparison between the results obtained with IMM, OMM and whole mitochondria show that both porphyrins, when released to the intact organelles, preferentially localize in the IMM, irrespective of the lipid carrier used. NaCl/Pi‐dissolved hematoporphyrin probably interacts with some membrane proteins, due to the similarity of the Arrhenius plots with those obtained for liposome‐entrapped human serum albumin/hematoporphyrin complexes which were used as models to mimic hematoporphyrin‐membrane protein binding sites. Liposomal hematoporphyrin and protoporphyrin bind to lipid domains. Hematoporphyrin accumulates in specific, localized lipid regions, perhaps in the boundary lipids area surrounding some inner‐mitochondrial carriers; protoporphyrin accomodates in more rigid, lipid areas. On these bases, the higher photoactivity of hematoporphyrin, previously observed in mitochondria, in comparison with protoporphyrin, can be easily explained.

Formation of linear dimers/aggregates, endowed with higher ΦF than that of the monomers, are postulated to occur for both porphyrins only in the inner mitochondrial membrane.
The Amino Acid Sequence and Interaction with the Nucleosome Core DNA of Transition Protein 4 from Boar Late Spermatid Nuclei
Authors: Akama Kuniko , Ichimura Hirokazu , Sato Hiroki , Kojima Shuichi , Miura Kin‐ichiro , Hayashi Hiroaki , Komatsu Yasuhiko , Nakano Minoru
Published: September 2004

The primary structure of transition protein 4 (TP4) from boar late spermatid nuclei was determined by automated Edman degradation of S‐pyridylethylated protein and of peptides generated by cleavage with Staphylococcus aureus V8 protease, lysyl endopeptidase and CNBr. Boar TP4 is a basic protein consisting of a highly basic amino‐terminal half (residues 1–73) and a less basic carboxy‐terminal half (residues 74–138). The latter half includes a highly hydrophobic segment, a four‐times tandemly repeated sequence, N(G)QNKR(K)X, and a carboxy‐terminal segment containing Trpl26. Ultraviolet absorption and CD spectra of TP4–rat‐liver‐nucleosome‐core‐DNA (double‐stranded DNA) complexes suggest a TP4‐induced local melting of DNA. Although at 1 mM NaCl TP4 brought about a slight stabilization of the DNA against thermal melting, a destabilization of the DNA was observed at 50 mM NaCl. From the results of quenching of tryptophan (Trpl26) fluorescence of TP4 upon its binding to double‐stranded and single‐stranded boar liver nucleosome‐core DNA at 50 mM NaCl, the apparent association constants for the binding of TP4 to double‐stranded and single‐stranded DNA were calculated to be 7.3×103 M−1 and 4.1×103M−1, respectively. These results suggest that TP4, having different domain structures from TP1–3 and a higher affinity for double‐stranded DNA, induces a local destabilization of DNA probably through the stacking of Trp126 with nucleic acid bases.
Regulation of Mitochondrial Biogenesis in Saccharomyces cerevisiae
Authors: De Winde Johannes H. , Grivell Leslie A.
Published: September 2004

Transcription of the QCR8 gene, encoding subunit VIIl of the Saccharomyces cerevisiae mitochondrial ubiquinol‐cytochrome c oxidoreductase (QCR), is controlled by the carbon‐source‐dependent heme‐activator protein complex HAP2/3/4 and the general transcriptional regulators autonomous replication‐site‐binding factor ABF1 and centromere‐binding and promoter‐binding factor CPF1. In this study, we investigate and dissect the relative contributions and mutual interactions of these regulators in transcriptional control. Transcription was analyzed both under steady‐state conditions and during nutritional shifts, in hapΔ mutants and after site‐specific mutagenesis of the various binding sites in the chromosomal context of the QCR8 gene. We present evidence for both direct and indirect interactions between ABF1 and HAP2/3/4, and show that HAP2/3/4 is essential for a rapid transcriptional induction during transition from repressed to derepressed conditions. However, the activator is not the only determinant for carbon‐source‐dependent regulation, and we observe a functional difference between HAP2/3/4 and the HAP2/3 subcomplex. ABF1 is required for maintainance of basal repressed and derepressed transcription in the steady state of growth. The repressive action of the negative modulator CPFl during escape from glucose repression is overcome through the cooperative action of ABF1 and HAP2/3/4. The implications of the intricate interactions of these DNA‐binding regulators for control of expression of mitochondrial protein genes are discussed.
Molecular Biological Analysis of a Bidirectional Hydrogenase from Cyanobacteria
Authors: Schmitz Oliver , Boison Gudrun , Hilscher Ralf , Hundeshagen Barbara , Zimmer Wolfgang , Lottspeich Fnedrich , Bothe Hermann
Published: September 2004

An 8.9‐kb segment with hydrogenase genes from the cyanobacterium Anabaena variabilis has been cloned and sequenced. The sequences show homology to the methyl‐viologen‐reducing hydrogenases from archaebacteria and, even more striking, to the NAD+‐reducing enzymes from Alcaligenes eutrophus and Nocardia opaca as well as to the NADP+‐dependent protein from Desulfovibrio fructosovorans. The cluster from A. variabilis contains genes coding for both the hydrogenase heterodimer (hoxH and hoxY) and for the diaphorase moiety (hoxU and hoxF) described for the A. eutrophus enzyme. In A. variabilis the gene cluster is split by two open reading frames (between hoxY and hoxH and between hoxU and hoxY, respectively), and a probably non‐coding 0.9‐kb segment in an unusual way. The hoxH partial sequence from Anabaena 7119 and Anacystis nidulans was amplified by PCR. Using the labeled segment from A. 7119 as probe, Southern analysis revealed homologous gene segments in the cyanobacteria A. 7119, Anabaena cylindrica, Anacystis nidulans and A. variabilis. The bidirectional hydrogenase from A. nidulans was purified and digests were sequenced. The amino acid sequences obtained showed partial identities to the amino acid sequences deduced from the DNA data of the 8.9‐kb segment from A. variabilis. Therefore the 8.9‐kb segment contains the genes coding for the bidirectional, reversible hydrogenase from cyanobacteria. Crude extracts from A. nidulans perform NAD(P)H‐dependent H2 evolution corroborating the molecular biological demonstration of the NAD(P)+‐dependent hydrogenase in cyanobacteria.
Valyl‐tRNA Synthetase from Artemia
Authors: Brandsma Margreet , Kerjan Pierre , Dijk Jan , Janssen George M. C. , Möller Wim
Published: September 2004

Two components of the protein biosynthetic machinery, valyl‐transfer RNA synthetase (VRS) and elongation factor 1 (EF‐1), have been isolated as a complex from several mammalian tissues. However, yeast VRS, which lacks an amino‐terminal extension, does not associate with EF‐1. We purified VRS from the brine shrimp Artemia and investigated its interaction with EF‐1. Western blotting of crude Artemia extracts revealed the presence of two forms of VRS, differing in size and capacity to associate with EF‐1. About 80% of the total VRS corresponds to a polypeptide of 130 kDa which‐behaves as a monomer upon gel filtration. Only the larger form of 140 kDa coelutes, cosediments and co‐immunoprecipitates with the EF‐1α2βγδ complex. The ratio of the two forms of VRS remains constant throughout early development. The possible origin and mode of expression of the two forms of VRS present in Anemia are discussed.
Interactions Responsible for the pH Dependence of the β‐Hairpin Conformational Population Formed by a Designed Linear Peptide
Authors: de Alba Eva , Blanco Francisco J. , Jiménez M. Angeles , Rico Manuel , Nieto José L.
Published: September 2004

In a previous work [Blanco, F. J., Jiménez, M. A., Herranz, J., Rico, M., Santoro, J. & Nieto, J. L. (1993) J. Am. Chem. Soc. 115, 5887–5888] we showed that a short, designed linear peptide, YQNPDG‐SQA (peptide 1), can form a monomeric β hairpin in aqueous solution. The pH dependence of the β‐hairpin conformation formed by the designed peptide and a series of related peptides has been examined in this work using 1H‐NMR methods. Three pH‐dependent interactions have been identified: a local interaction, unimportant structurally, between the C‐terminal carboxylate group and the side‐chain amide group of Q8; an electrostatic interaction between the main‐chain N‐terminus and C‐terminus; and a hydrogen bond involving the side‐chain amide protons of N3 and the side‐chain carboxylate group of D5. The latter two interactions are particularly relevant as they increase the population of the β‐hairpin conformation. We also observe in the mutant peptide A9H that the interaction between Y1 and H9 (of the type proposed to exist in proteins) does not contribute to β‐hairpin stabilisation in our peptide system. Peptide 1 is, therefore, a very suitable model to examine the different interactions that contribute to β‐hairpin stability.
Structure of Amyloid A4‐(1–40)‐Peptide of Alzheimer's Disease
Authors: Sticht Heinrich , Bayer Peter , Willbold Dieter , Dames Sonja , Hilbich Caroline , Beyreuther Konrad , Frank Rainer W. , Rösch Paul
Published: September 2004

One of the principal peptide components of the amyloid plaque deposits of Alzheimer's disease in humans is the 40‐amino‐acid peptide β‐amyloid A4‐(1–40)‐peptide. The full‐length A4‐(1–40)‐peptide was chemically synthesized and the solution structure determined by two‐dimensional nuclear magnetic resonance spectroscopy and restrained molecular‐dynamics calculations. Synthetic human A4‐(1–40)‐peptide was soluble and non‐aggregating for several days in 40% (by vol.) trifluoroethanol/water. All spin systems could be unambiguously assigned, and a total of 203 sequential and medium‐range cross‐peaks were found in the NOESY (nuclear Overhauser enhancement spectroscopy) spectrum. Long‐range NOE cross‐peaks that would indicate tertiary structure of the peptide were absent. The main secondary‐structure elements found by chemical‐shift analysis, sequential and medium‐range NOESY data, and NOE‐based restrained molecular‐dynamics calculations were two helices, Gln15–Asp23 and IIe31–Met35, whereas the rest of the peptide was in random‐coil conformation. A similar secondary structure is suggested for the aggregation part of prions, the postulated causative agents of the transmissible spongiform encephalopathy. The sequence of the helical part of prion proteins was observed to be remarkably similar to the sequence of the helical part of human A4‐(1–40)‐peptide.
The Fusobacterium nucleatum Major Outer‐Membrane Protein (FomA) Forms Trimeric, Water‐Filled Channels in Lipid Bilayer Membranes
Authors: Kleivdal Hans , Benz Roland , Jensen Harald B.
Published: September 2004

The pore‐forming activity of the major outer‐membrane protein FomA of the anaerobic Fusobacterium nucleatum was studied in artificial lipid bilayer membranes. FomA was isolated from F. nucleatum strains Fev1, ATCC 10953, and ATCC 25586 by extraction with lithium dodecyl sulfate and lithium chloride and had an apparent molecular mass of about 40 kDa. When solubilized at low temperatures, the protein ran with an apparent molecular mass of about 62 kDa on SDS/PAGE. Cross‐linking experiments and two‐dimensional SDS/PAGE gave evidence that the 62‐kDa protein band represented the trimeric form of FomA. The protein trimers were susceptible to SDS and temperature. The stability of the porin trimers varied among the strains. The properties of the FomA channels were studied in reconstitution experiments with black lipid bilayer membranes. The F. nucleatum porins formed channels with single‐channel conductances in the range 0.66–1.30 nS in 1 M KCl. The single‐channel conductance was a function of the mobilities of the ions present in the aqueous solution bathing the bilayer membrane. This means that FomA forms general diffusion channels since (a) the conductance showed a linear dependence on the salt concentration, (b) the ion selectivity was small and varied for the three strains, and (c) the channels did not exhibit any binding site for maltotriose or triglycine. The water‐filled channel was voltage dependent, and conductance decrements were observed at transmembrane potentials of ± 50 mV. The conductance decrement steps were about one‐third of the total conductance of a functional unit in its fully ‘open’ state. This strongly suggests that the trimer is the functional unit of the porin.
A Casein‐Kinase‐2‐Related Protein Kinase is Tightly Associated with the Large T Antigen of Simian Virus 40
Authors: Götz Claudia , Koenig Michael G. , Issinger Olaf‐Georg , Montenarh Mathias
Published: September 2004

The simian virus 40 (SV40) large T antigen is a multifunctional protein involved in SV40 cell transformation and lytic virus infection. Some of its activities are regulated by interaction with cellular proteins and/or by phosphorylation of T antigen by various protein kinases. In this study, we show that immunopurified T antigen from SV40‐transformed cells and from baculovirus‐infected insect cells is tightly associated with a protein kinase that phosphorylates T antigen in vitro. In the presence of heparin or a peptide resembling a protein kinase CK2 recognition site, the phosphorylation of T antigen by the associated kinase is reduced whereas a p34cdc2‐kinase‐specific peptide has no influence. In addition, the T‐antigen‐associated protein kinase can use GTP and ATP as phosphate donors. These properties together with the observation that immunopurified T antigen can be phosphorylated by the addition of protein kinase CK2 suggest that at least one of the T‐antigen‐associated protein kinases is CK2 or a protein‐kinase‐CK2‐related enzyme. The association of recombinant CK2 with T antigen was strongly confirmed by in vitro binding studies. Experiments with temperature‐sensitive SV40‐transformed cells provide evidence for a close correlation between cell transformation and phosphorylation of T antigen by the associated protein kinase.
Compositional analysis of Glucosaminyl(acyl)phosphatidylinositol Accumulated in HeLa S3 Cells
Authors: Sevlever Daniel , Humphrey Dawn R. , Rosenberry Terrone L.
Published: September 2004

GlcN(acyl)PtdIns, a derivative of phosphatidylinositol (PtdIns) in which glucosamine and a fatty acid are linked to inositol hydroxyl groups, has been proposed to be an intermediate in the mammalian biosynthetic pathway for glycosylphosphatidylinositol (glycosyl‐PtdIns) anchors of membrane proteins. In this report, GlcN(acyl)PtdIns metabolically labeled with [3H]inositol is shown to accumulate in a HeLa S3 cell subline. The amount of GlcN(acyl)PtdIns in these HeLa S3 cells is about 107molecules/cell, a level comparable to those of the most abundant glycosyl‐PtdIns‐containing molecules reported to date. GlcN(acyl)PtdIns was purified by a two‐step procedure involving octyl‐Sepharose and thin‐layer chromatography. Octyl‐Sepharose separated phospholipids according to their number of hydrocarbon chains: one in 2‐lysoPtdIns, two in PtdIns, and three in GlcN(acyl)PtdIns. Purification also was aided by prior treatment of lipid extracts with bee venom phospholipase A2, an enzyme that did not cleave GlcN(acyl)PtdIns. The GlcN‐inositol head group in purified GlcN(acyl)PtdIns was confirmed by a number of procedures, including cation‐exchange chromatography and mass spectrometry; after radiomethylation, an equal molar ratio of GlcN(Me)2/inositol was measured. Fatty acid analysis indicated an overall stoichiometry of 2.3 mol fatty acid/mol inositol with palmitic (16:0), stearic (18:0) and oleic (18:1) acids being predominant. Analysis of GlcN(acyl)inositol produced by HF fragmentation showed that palmitate was the acyl group attached to inositol and indicated that stearic and oleic acids were in the glycerolipid. Base methanolysis revealed that about 15% of the purified GlcN(acyl)PtdIns contained alkylglycerol. A substantial conversion of GlcN(acyl)PtdIns to a slightly more polar lipid occurred after overnight incubation in even mildly alkaline buffers. Although the current data do not allow proposal of a structure for this lipid, its formation from GlcN(acyl)PtdIns may be important because the conversion appeared to occurin vivo.
Immunosuppressive treatments for immunoglobulin A nephropathy: A meta‐analysis of randomized controlled trials
Authors: SAMUELS JOSHUA A , STRIPPOLI GIOVANNI FM , CRAIG JONATHAN C , SCHENA FRANCESCO P , MOLONY DONALD A
Published: September 2004

SUMMARY:  Immunoglobulin A (IgA) nephropathy is a worldwide disease that causes end‐stage kidney disease (ESRD) in up to 15–20% of affected patients within 10 years from the apparent onset of disease and in up to 30–40% of individuals within 20 years from diagnosis. No specific treatment has been established and there is wide variation in current practice. This systematic review evaluates the use of immunosuppressive agents to treat patients with IgA nephropathy. The Cochrane Renal Group Specialized Register, Cochrane Controlled Trial Registry, MEDLINE, EMBASE and article reference lists were searched for randomized or quasi randomized trials. Two independent reviewers assessed studies for inclusion criteria (biopsy proven IgA nephropathy, randomized trial, use of immunosuppressive agents) and extracted data regarding the effects of immunosuppressive agents on ESRD, doubling of serum creatinine, glomerular filtration rate, urinary protein excretion and side‐effects. Data were analysed with a random effects model. The published trials were few (13 trials, 623 patients) and were generally of poor quality. Compared with placebo, steroids were associated with a lower risk of progression to ESRD (six trials, 341 patients, RR 0.44, 95% CI 0.25–0.80) and lower end‐of‐trial proteinuria (six trials, 263 patients, weighted mean difference (WMD) −0.49 g/day, 95% CI −0.25 to −0.72). Treatment with alkylating agents significantly reduced end of treatment proteinuria (two trials, 122 patients, WMD −0.94, 95% CI −0.46 to −1.43). Although the optimal management of patients with IgA nephropathy remains uncertain because of limitations with the existing published data, immunosuppressive agents are a promising strategy and should be investigated further.
Incidence of end‐stage renal disease in overseas‐born, compared with Australian‐born, non‐indigenous Australians
Authors: STEWART JOHN H , MCCREDIE MARGARET RE , MCDONALD STEPHEN P
Published: September 2004

Background:   Barriers to immigration from non‐European sources were relaxed in the 1970s. As a result, more Australians are now of Middle Eastern, Asian or Pacific Islander origin, rather than British or European. Currently, overseas‐born persons comprise one‐third of non‐indigenous Australians with end‐stage renal disease (ESRD).

Methods:  Using data recorded by the Australia and New Zealand Dialysis and Transplant (ANZDATA) Registry, age‐standardized incidence rates were calculated for ESRD due to all causes and to certain primary renal diseases for all non‐indigenous Australians who were aged over 15 years when first treated for ESRD between 1993 and 2001. Truncated age‐standardized incidence rates were calculated for ESRD due to glomerulonephritis by type.

Results:  Immigrants from the British Isles and ‘rest of Europe’ had less, and those from the Pacific Island nations, East/South‐East Asia, Indian subcontinent, Middle East and Southern Europe more ESRD from all causes than the Australian‐born. Two diseases accounted for most of the excess: Type 2 diabetic nephropathy and glomerulonephritis (the latter not significant for the Indian‐born). There was a small excess (not always significant) of hypertensive/arteriopathic renal disease in Asian‐ and Middle Eastern‐born persons. The East/South‐East Asian‐born had the highest rates  of  ESRD  due  to mesangial  immunoglobulin  A  (IgA)  disease  and  lupus  nephritis,  and  the  Middle  Eastern‐born the highest rates from focal sclerosing glomerulonephritis.

Conclusion:  For Australians born in the Pacific Island nations, Asia, the Middle East or Southern Europe, excess prevalence of, and/or susceptibility to, diseases that cause ESRD has more than offset any ‘healthy migrant’ effect.
Pentoxifylline: A potential therapy for chronic kidney disease
Authors: LIN SHUEI‐LIONG , CHEN YUNG‐MING , CHIANG WEN‐CHIH , TSAI TUN‐JUN , CHEN WAN‐YU
Published: September 2004

SUMMARY:  Almost all forms of chronic kidney disease progressing to end‐stage kidney failure are characterized by diffuse fibrosis, a final common pathway converging from multiple pathogenetic networks regardless of the initial injury. Four principal interventions including glycaemic and blood pressure control, dietary protein restriction, and angiotensin II blockade have been proven to slow progression of diabetic and/or non‐diabetic chronic kidney disease. However, the ultimate solution to halt disease progression in the long term is still pending. Because of the pathogenetic complexity of kidney disease, multidrug intervention with the least side‐effects should, without doubt, be the next step to stop kidney disease progression. Animal and cellular studies have demonstrated the rationale for pentoxifylline (i.e. its effects against cell proliferation, inflammation, and extracellular matrix accumulation) in the treatment of chronic kidney disease induced by immune‐ or non‐immune‐mediated mechanisms. Limited human studies have proven its efficacy in reducing proteinuria in patients with diabetes receiving angiotensin‐converting enzyme inhibitors, and in patients with nephrotic syndrome refractory to conventional immunosuppressive therapy. Moreover, monotherapy with pentoxifylline markedly reduces proteinuria in patients with membranous nephropathy. Further studies are required to examine whether pentoxifylline can improve the renal outcome in patients receiving interventions with proven efficacy.
MULTICENTRE COLLABORATIVE PROSPECTIVE STUDY OF ENDOSCOPIC TREATMENT OF EARLY GASTRIC CANCER
Authors: Ida Kazunori , Nakazawa Saburo , Yoshino Junji , Hiki Yoshiki , Akamatsu Taiji , Asaki Shigeru , Kurihara Minoru , Shimao Hitoshi , Tada Masahiro , Misumi Atsunobu , Kato Takahiro , Niwa Hirohumi
Published: September 2004

Aims:  The present study was conducted with the aims of elucidating the present state of endoscopic treatment, in particular endoscopic mucosal resection (EMR) of early gastric cancer, as well as any associated problems, and the prospects for further broadening of the indications for EMR.

Subjects and methods:  This study was begun in 1994 and completed in 2002. Lesions were classiﬁed into Group I, with macroscopic appearance of 0–I, IIa, IIb or IIc·U1(–), well‐ to moderately differentiated intramucosal carcinoma ≤ 20 mm in diameter, and Group II, with the same macroscopic appearance as Group I, intramucosal carcinoma either the same histological type with diameter 21–40 mm, or undifferentiated carcinoma ≤ 40 mm in diameter. Of the 412 subjects registered (Group I, 305 subjects; Group II, 107 subjects, including seven undifferentiated carcinoma cases), 258 were evaluated (Group I, 199 subjects; Group II, 59 subjects including seven undifferentiated carcinoma).

Results:  The overall complete excision rate for Group I was 71.9%, and was 82.4% for ≤ 10 mm, 67.2% for 11–15 mm, and 45.5% for 16–20 mm, thus declining with increasing lesion size. The long‐term results, including cases of additional treatment for residual cancer following the original EMR, showed that recurrence occurred in eight out of 199 cases (4.0%), in ﬁve of whom local cure was achieved with endoscopic treatment, giving a ﬁnal local cure in 196 cases (98.5%). The overall complete excision rate for Group II differentiated carcinoma was 46.2%. With the inclusion of additional treatment, local cure was achieved with endoscopic treatment in 57 out of 59 cases (96.6%). The remaining two cases of recurrence underwent open surgery.

Conclusion:  Regardless of Group I or II classiﬁcation, if the diagnosis of intramucosal cancer from the specimen resected at the initial EMR is histologically correct, then local cure can be achieved with endoscopic treatment, including cases of recurrence, with appropriate follow up and use of concomitant techniques such as piecemeal resection and coagulation therapy. The indications for endoscopic treatment can now reasonably be widened to include Group II lesions.
SUCCESSFUL TREATMENT USING A SELF‐EXPANDABLE METALLIC STENT IN THE PALLIATION FOR UNRESECTABLE MALIGNANT OBSTRUCTION OF THE COLON AND RECTUM
Authors: Maeda Kiyoshi , Inoue Toru , Yashiro Masakazu , Nishihara Tamahiro , Nishiguchi Yukio , Hirakawa Kosei
Published: September 2004

Background:  We attempted the placement of a self‐expandable metallic stent (SEMS) for unresectable malignant obstruction of the proximal colon as well as obstruction of the distal colon and rectum, using a technical device.

Patients and methods:  Thirty patients were selected to place SEMS. The procedure was performed under endoscopic and fluoroscopic guidance. As a technical device, we used an angiographic introducer to straighten the rectosigmoid region. Moreover, we used a transparent hood to easily obtain a front view of the stricture.

Results:  Of 30 cases of attempted SEMS placement, SEMS was successfully placed in 26 (87%). The technical success rate was 82% (9/11) in strictures of the proximal colon, while it was 89% (17/19) in those of the distal colon or rectum. According to complications, stent migration occurred in one patient (3%) and restenosis was also observed in one patient (3%).

Conclusion:  Placement of SEMS is a feasible and effective adjunct and alternative to stoma in malignant large bowel obstruction.
COMPARING BALLOON DIAMETER ON PERFORMING ENDOSCOPIC PAPILLARY BALLOON DILATATION WITH ISOSORBIDE DINITRATE DRIP INFUSION FOR REMOVAL OF BILE DUCT STONES
Authors: Nakagawa Hiroshi
Published: September 2004

Background:  Endoscopic papillary balloon dilatation (EPBD) is one of the methods to remove bile duct stones. EPBD might preserve the function of the sphincter of Oddi despite the potential risk of acute pancreatitis. There are only a few reports of EPBD reducing the risk of acute pancreatitis and, at same time, preserving the function of the sphincter of Oddi.

Methods:  We performed EPBD for bile duct stone removal in 60 patients using two balloons with different diameters. Patients were randomized to EPBD with a 6 mm balloon (n = 30) or an 8 mm balloon (n = 30). In both groups, isosorbide dinitrate (ISDN) was infused in a rate of 5 mg/h while low pressure EPBD were being performed. The pressure of the sphincter of Oddi was observed before and after the EPBD procedures. Also, serum amylase level after EPBD was observed for both groups.

Results:  Serum amylase level of the 6 mm group was signiﬁcantly higher than that of the 8 mm group (P < 0.05). Acute pancreatitis occurred in two patients ( 6.7%) in the 6 mm group whereas no case was observed for the 8 mm group. The rates of duct clearance were 93% in the 6 mm group and 100% in the 8 mm group. Stone removals were difﬁcult in seven cases with 6 mm balloon dilatations due to the narrow oriﬁces of the papilla. In the 6 mm group, there was no signiﬁcant difference between the basal sphincter of Oddi pressure (BSOP) and the phasic sphincter of Oddi pressure (PSOP) before and after EPBD. However in the 8 mm group, the BSOP observed after the EPBD procedure was signiﬁcantly higher than BSOP before the treatments. Within this group, BSOP values after EPBD were preserved by approximately 80% of the BSOP values before the treatments. In contrast, there was no signiﬁcant difference in PSOP before and after the treatments. Regarding the stone numbers, no signiﬁcant difference was observed in BSOP before and after the treatments for the 6 mm group with less than two stones. Also, as for stone size, no signiﬁcant difference was observed in BSOP before and after the treatments for the 6 mm group with stones of less than 6 mm in diameter.

Conclusion:  We are now conducting EPBD with ISDN infusion using a 6 mm balloon for a patient who has less than two stones with size not exceeding 6 mm in diameter. An 8 mm balloon is used for a patient with more than two stones or a stone greater than 6 mm in size.
NEW ENDOSCOPIC TECHNIQUE TO CLOSE LARGE MUCOSAL DEFECTS AFTER ENDOSCOPIC MUCOSAL RESECTION IN PATIENTS WITH GASTRIC MUCOSAL TUMORS
Authors: Endo Masaki , Inomata Masaaki , Terui Torahiko , Oana Syuhei , Kudara Norihiko , Obara Hirohiko , Hashimoto Yasuto , Chiba Toshimi , Orii Seishi , Suzuki Kazuyuki
Published: September 2004

Endoscopic mucosal resection has been recognized as a standard method for treating mucosal tumors of the stomach in Japan. In our department, we have treated mucosal defects after this procedure by using metallic clips to prevent and manage complications related to endoscopic mucosal resection. In the present study, we explain the new technique, the ‘loop‐and‐clips’ method, which uses clips and a detachable snare to close large mucosal defects after endoscopic mucosal resection.
THE HEALING PROCESS OF GASTRIC ARTIFICIAL ULCERS AFTER ENDOSCOPIC SUBMUCOSAL DISSECTION
Authors: Kakushima Naomi , Yahagi Naohisa , Fujishiro Mitsuhiro , Iguchi Mikitaka , Oka Masashi , Kobayashi Katsuya , Hashimoto Takuhei , Omata Masao
Published: September 2004

Background:  Due to the remarkable progress of endoscopic resection techniques, endoscopic submucosal dissection (ESD) has been widely performed for larger mucosal tumors that would result in large artiﬁcial ulcers. The healing process of peptic ulcers has been previously studied in detail; however, no precise investigation for artiﬁcial ulcers after ESD has been reported. To conﬁrm the validity of the treatment from the aspect of wound healing, we aimed to clarify the healing process of large gastric artiﬁcial ulcers after ESD.

Methods:  Seventy patients with gastric mucosal tumors treated by ESD were enrolled. The size, location and time of scar formation of the ulcers were reviewed using endoscopic pictures taken from the same view and angle. Follow‐up endoscopy was performed at 1, 4, 8 and 12 weeks after ESD. For postoperative medication, all patients received normal doses of proton pump inhibitors and sucralfate for 8 weeks.

Results:  The average size of the resected specimen was 34.7 mm (20–90 mm). Irrespective of ulcer size and location, all of the cases healed up to scarring stages within 8 weeks.

Conclusions:  Gastric artiﬁcial ulcers after ESD healed within 8 weeks regardless of size and location using normal doses of medication as peptic ulcers. The fact that even giant ulcers after ESD heal within 8 weeks could be helpful information for candidates for ESD and for postoperative management of patients after ESD.
EOSINOPHILIC GASTROENTERITIS WITH PREDOMINANT MUSCULAR LAYER DISEASE: FEATURE OF ENDOSCOPIC ULTRASONOGRAPHY
Authors: Fukuda Akihisa , Kajiyama Toru , Kishimoto Hiroyuki , Someda Hitoshi , Sakai Masahiko , Seno Hiroshi , Chiba Tsutomu
Published: September 2004

A 34‐year‐old man was admitted to Kansai‐Denryoku Hospital because of severe epigastric pain and vomiting. Laboratory studies revealed peripheral blood eosinophilia and elevated immunoglobulin E. Endoscopy showed narrowing of the antrum and pylorus with edematous mucosa. Endoscopic ultrasonography (EUS) demonstrated a transmural circumferential thickening of the antrum predominantly in the muscularis propria. The overall thickness of the antrum was 1.2 cm. Mucosal biopsies of the antrum showed a small number of eosinophilic infiltrations. A diagnosis of eosinophilic gastroenteritis with predominant muscular layer disease was made, and predonisone (40 mg/day) was given. Thereafter, symptoms and peripheral eosinophilia resolved rapidly. Repeated endoscopy showed improvement of the narrowing and EUS revealed that the thickness of the antrum improved to 0.7 cm and 0.5 cm at 1 and 3 months later, respectively. Thus, EUS is valuable in evaluating and diagnosing muscular involvement in eosinophilic gastroenteritis with predominant muscular layer disease.
The Origins of Wittgenstein's Imaginary Scenarios: Something Old, Something New
Authors: Peach Andrew J.
Published: September 2004

The imaginary scenarios that appear in nearly every work of the later Wittgenstein – ones involving laughing cattle, disembodied eyes that see, and the like – are decidedly absent from the Tractatus. What necessitated this change in methodology? A comparison of the Tractatus with the Philosophical Remarks, Wittgenstein's first major work after his return to philosophy, reveals that these devices are the product of something old and something new. The rationale for these devices is already present in the notion of a “propositional variable,” but Wittgenstein had little use for them until he rejected the phenomenological language and laconic style of the Tractatus.
Faith, Belief and Perspective: Peter Winch's Philosophy of Religion
Authors: Springsted Eric O.
Published: September 2004

Peter Winch's philosophy of religion is controversial, accused of mere “perspectivism” and fideism, and for avoiding discussion of any existential reference for the object of belief. This essay examines what Winch meant by a “perspective.” It first deals with problems of first person propositions of belief. For Wittgenstein and Winch belief and the fact it believes are inextricably bound together. Thus Winch argues that what is said cannot be divorced from the situation of the sayer; understanding requires making shifts in perspective. Finally I compare Winch's use of religious language to Augustine's doctrine of the “inner word,” arguing that there are important parallels in Winch to pre‐Lockean theological understandings of faith.
Is Knowing‐how Simply a Case of Knowing‐that?
Authors: Rosefeldt Tobias
Published: September 2004

Jason Stanley and Timothy Williamson have argued that there is no fundamental distinction between what Gilbert Ryle famously called ‘knowing how’ and ‘knowing that’, and that the former can be treated as a special kind of the latter. I will endeavour to show that sentences of the form ‘a knows how to F’ are ambiguous between a reading in which we ascribe knowledge‐that to a and another in which we ascribe something to a which is irreducible to any kind of knowledge‐that and can most appropriately be characterized as an ability. The authors’ attempt to reduce also the latter reading to an ascription of knowledge‐that fails because it rests on an unexplained conception of practical modes of presentation.
Metal ion transporters in mammals: structure, function and pathological implications
Authors: Rolfs Andreas , Hediger Matthias A.
Published: September 2004

Despite the importance of metal ions in several catalytic functions, there has been, until recently, little molecular information available on the mechanisms whereby metal ions are actively taken up by mammalian cells. The classical concept for iron uptake into mammalian cells has been the endocytosis of transferrin‐bound Fe3+ by the transferrin receptor. Studies with hypotransferrinaemic mice revealed that in the intestine mucosal transferrin is derived from the plasma and that its presence is not required in the intestinal lumen for dietary iron absorption. This suggests that, at least in the intestine, other non‐receptor‐mediated uptake systems exist. The molecular identification of metal ion transporters is of great importance, in particular since an increasing number of human diseases are thought to be related to disturbances in metal ion homeostasis, including metal ion overload and deficiency disorders (i.e. anaemia, haemochromatosis, Menkes disease, Wilson's disease), and neurodegenerative diseases (i.e. Alzheimer's, Friedreich's ataxia and Parkinson's diseases). Furthermore, susceptibilities to mycobacterial infections are caused by metal ion transporter defects. The pathological implications of disturbed metal ion homeostasis confirm the vital roles these metal ions play in the catalytic function of many enzymes, in gene regulation (zinc‐finger proteins), and in free radical homeostasis. Recent insights have significantly advanced our knowledge of how metal ions are taken up or released by mammalian cells. The purpose of this review is to summarize these advances and to give an overview on the growing number of mammalian metal ion transporters.
G‐protein coupled receptor kinases as modulators of G‐protein signalling
Authors: Bünemann Moritz , Hosey M. Marlene
Published: September 2004

G‐protein coupled receptors (GPCRs) comprise one of the largest classes of signalling molecules. A wide diversity of activating ligands induce the active conformation of GPCRs and lead to signalling via heterotrimeric G‐proteins and downstream effectors. In addition, a complex series of reactions participate in the ‘turn‐off’ of GPCRs in both physiological and pharmacological settings. Some key players in the inactivation or ‘desensitization’ of GPCRs have been identified, whereas others remain the target of ongoing studies. G‐protein coupled receptor kinases (GRKs) specifically phosphorylate activated GPCRs and initiate homologous desensitization. Uncoupling proteins, such as members of the arrestin family, bind to the phosphorylated and activated GPCRs and cause desensitization by precluding further interactions of the GPCRs and G‐proteins. Adaptor proteins, including arrestins, and endocytic machinery participate in the internalization of GPCRs away from their normal signalling milieu. In this review we discuss the roles of these regulatory molecules as modulators of GPCR signalling.