bluuebunny/crossref_metadata_2025 huggingface dataset:
Botulinum neurotoxins serotypes A and E cleave SNAP‐25 at distinct COOH‐terminal peptide bonds
Authors: Schiavo Giampietro , Santucci Annalisa , Dasgupta Bibhuti R. , Mehta Prashant P. , Jontes Jaime , Benfenati Fabio , Wilson Michael C. , Montecucco Cesare
Published: July 2002

SNAP‐25, a membrane‐associated protein of the nerve terminal, is specifically cleaved by botulinum neurotoxins serotypes A and E, which cause human and animal botulism by blocking neurotransmitter release at the neuromuscular junction. Here we show that these two metallo‐endopeptidase toxins cleave SNAP‐25 at two distinct carboxyl‐terminal sites. Serotype A catalyses the hydrolysis of the Gln197‐Arg198 peptide bond, while serotype E cleaves the Arg180‐Ile181 peptide linkage. These results indicate that the carboxyl‐terminal region of SNAP‐25 plays a crucial role in the multi‐protein complex that mediates vesicle docking and fusion at the nerve terminal.
Effect of okadaic acid on protein phosphorylation patterns of chicken myogenic cells with special reference to creatine kinase
Authors: Hemmer W. , Skarli M. , Perriard J.-C. , Wallimann T.
Published: July 2002

Okadaic acid and other agents affecting cellular phosphorylation and dephosphorylation processes profoundly changed the phosphoprotein pattern of 32Pilabelled chicken embryonic skeletal muscle cells. The phosphorylation states of proteins in the lower molecular weight range were especially increased. Immunoprecipitation of cellular extracts with anti‐creatine kinase antibodies enabled us to identify creatine kinase (CK) phosphoproteins. B‐CK was phosphorylated after treating the cultures with 1‐oleoyl‐2‐acetyl‐sn‐glycerol, dibutyryl‐cAMP, okadaic acid and combinations thereof, but not with 1,2‐dioleoyl‐sn‐glycerol. M‐CK was also shown to be phosphorylated. The results indicated that in vivo, CK isoforms in muscle are subjected to control mediated by phosphorylation and dephosphorylation processes.
Why are the same protein folds used to perform different functions?
Authors: Finkelstein Alexei V. , Gutun Alexander M. , Badretdinov Azat Ya.
Published: July 2002

A small number of folding patterns describe in outline most of the known protein globules, the same folds being found in non‐homologous proteins with different functions. We show that the ‘popular’ folding patterns are those which, due to some thennodynamic advantages of their structure, can be stabilized by a lot of random sequences. In contrast, the folds which are rarely or never observed in natural globular proteins can be stabilized only by a tiny number of random sequences. The advantageous folds are few, they tolerate various primary structures, and therefore they can and ought to perform different functions. A connection between the inherent ‘weak points’ of protein folding patterns and positions of active sites are discussed.
Effects of amino‐terminus truncation in human cytochrome P450IID6 on its insertion into the endoplasmic reticulum membrane of Saccharomyces cerevisiae
Authors: Krynetsky E.Yu. , Drutsa V.L. , Kovaleva I.E. , Luzikov V.N. , Uvarov V.Yu.
Published: July 2002

A truncated form of cytochrome P450IID6 deprived of 22 NH2‐terminal amino acids residues (P450IID6Δ1‐22) was found in both the cytosol and the microsomal fraction of the yeast, Saccharomyces cerevisiae. A reduced CO difference spectrum of this form was characterized by the absence of absorption at 448 nm and weak absorption at 420 nm. Another peculiarity of P450IID6Δ1‐22 expression was its reduced content in the yeast cells compared to that of P450IID6, with the intracellular levels of the corresponding mRNAs being the same. We suggest that the deleted form of P450IID6, i.e. lacking 22 NH2‐terminal amino acid residues, is not inserted properly in the endoplasmic reticulum membrane: it does not take up the proper conformation to enable normal heme binding and is degraded in the yeast cells.
A threshold membrane potential accounts for controversial effects of fatty acids on mitochondrial oxidative phosphorylation
Authors: Köhnke Detlef , Ludwig Bernd , Kadenbach Bernhard
Published: July 2002

The uncoupling effect of free fatty acids on oxidative phosphorylation in mitochondria has been known for more than 35 years. The mechanism of action, however, remains controversial. In this report the physicochemical basis of uncoupling was elucidated by studying the effect of free fatty acids on the proton permeability and membrane potential of proteoliposomes containing reconstituted cytochrome c oxidase (COX). A threshold membrane potential of about 125 mV was identified for fatty acid‐induced proton permeability. Only above this potential do free fatty acids translocate protons across the biological membrane. The data explain the controversial effects of long‐chain fatty acids on oxidative phosphorylation as well as their role on non‐shivering thermogenesis in larger mammals.
Synthesis and farnesylation of a‐factor fusion proteins in Saccharomyces cerevisiae
Authors: Kölling Ralf , Böttner Benjamin , Hollenberg Cornelis P.
Published: July 2002

We have generated in‐frame fusions between the mouse dihydrofolate reductase (DHFR) and parts of the a‐factor MFA1 gene to explore the potential of a‐factor as a secretion signal for larger polypeptides. We demonstrated that the fusion proteins are farnesylated by comparing the mobility of fusion proteins prepared from a wild‐type strain and a farnesyltransferase mutant (ste16/ram1) on SDS‐gels and by an in vitro farnesylation assay. In contrast to unmodified DHFR, the fusion proteins could be sedimented from cell extracts by centrifugation. Solubilization experiments indicated that the highly hydrophobic a‐factor moiety renders the fusion proteins insoluble, explaining why the fusions are not secreted into the culture medium.
Anti‐(p34 protein) antibodies inhibit ribosome binding to and protein translocation across the rough microsomal membrane
Authors: Ichimura Tohru , Shindo Yukiko , Uda Yasuyo , Ohsumi Tomoya , Omata Saburo , Sugano Hiroshi
Published: July 2002

The p34 protein is a non‐glycosylated, integral membrane protein characteristic of rough microsomes and is believed to play a role in the ribosome‐membrane association. Here, antibodies directed against p34 were examined as to their inhibitory effect on ribosome binding to and protein translocation across the microsomal membrane. Preincubation of the stripped (ribosome‐depleted) membrane with anti‐p34 immunoglobulins (IgGs) or their Fab fragments led to more than 80% inhibition of the binding of ribosomes and their large (60S) subunit to the membrane. The inhibition was dependent on the amount of antibodies used, but comparable amounts of IgGs and Fab fragments from nonimmune serum had less effect. The p34 antibodies were also inhibitory for cotranslational translocation of secretory proteins, i.e. placental lactogen and serum albumin, across the membrane. These results suggest that p34 is involved in the binding of ribosomes to the microsomal membrane and that it is in close proximity to the protein translocation site in the microsomal membrane.
Calcium currents recorded from a neuronal α1C L‐type calcium channel in Xenopus oocytes
Authors: Charnet Pierre , Bourinet Emmanuel , Dubel Stefan J. , Snutch Terry P. , Nargeot Joel
Published: July 2002

Xenopus oocytes expressing neuronal α1C, α2 and β1b calcium channel subunit cDNAs were used in this study. During two‐electric voltage clamp recording the oocyte was injected with 10–20 nl of a 100 mM BAPTA solution. Under these conditions, the endogenous Ca‐activated Cl current was completely suppressed resulting in an α1C Ba current free from Cl current contamination. BAPTA injection also allowed α1C currents with different permeating ions, including Ca, to be examined. Compared to Ba and Sr, α1C whole cell Ca currents were smaller in magnitude and showed kinetic and voltage‐dependent properties more similar to those for L‐type Ca currents recorded in native cells. That Ca‐dependent inactivation occurs in BAPTA‐buffered cells suggests that the Ca‐binding site involved in this type of inactivation is very close to the pore of the channel.
Purification and properties of the α‐acetolactate decarboxylase from Lactococcus lactis subsp. lactis NCDO 2118
Authors: Phalip Vincent , Monnet Christophe , Schmitt Philippe , Renault Pierre , Godon Jean-Jacques , Diviès Charles
Published: July 2002

α‐Acetolactate decarboxylase from Lactococcus lactis subsp. lactis NCDO 2118 was expressed at low levels in cell extracts and was also unstable. The purification was carried out from E. coli in which the enzyme was expressed 36‐fold higher. The specific activity was 24‐fold enhanced after purification. The main characteristics of α‐acetolactate decarboxylase were: (i) activation by the three branched chain amino acids leucine, valine and isoleucine; (ii) allosteric properties displayed in absence and Michaelis kinetics in the presence of leucine. The enzyme is composed of six identical subunits of 26,500 Da.
Complete Golgi passage of glycotripeptides generated in the endoplasmic reticulum of mammalian cells
Authors: van Leyen Klaus , Wieland Felix
Published: July 2002

The tripeptide, N‐octanoyl‐Asn‐[125I]Tyr‐Thr‐NH2, which contains the acceptor sequence for N‐glycosylation, is readily taken up by cell culture cells, glycosylated in the endoplasmic reticulum (ER), and secreted into the medium. Therefore such glycosylated tripeptides have been used as markers for the vesicular flow from the endoplasmic reticulum to the plasma membrane [(1987) Cell 50, 289–300; (1990) J. Biol. Chem. 265, 20027‐20032]. We have now studied the pathway taken by the glycotripeptides in mammals in more detail. In the perfused rat liver, the glycotripeptides secreted to the medium were analyzed by digestion with exoglycosidases, and a significant fraction was found to contain the terminating sequence ‐Gal‐Sial, which is generated by processing enzymes that reside in the late Golgi apparatus. Thus we conclude that these glycotripeptides have passed through the complete Golgi complex on their way from the ER to the cell surface.
Determination of the vitronectin binding site on plasminogen activator inhibitor 1 (PAI‐1)
Authors: van Meijer Marja , Gebbink Raymond Klein , Preissner Klaus T. , Pannekoek Hans
Published: July 2002

Vitronectin is the carrier protein of plasminogen activator inhibitor 1 (PAI‐1). We used a well‐characterized panel of anti‐human PAI‐1 monoclonal antibodies (MoAbs) to localize the vitronectin‐binding site on PAI‐1. By employing a direct vitronectin/PAI‐1 binding assay and two vitronectin‐dependent inhibition assays, we demonstrate that the anti‐PAI‐1 MoAbs CLB‐5, CLB‐10, CLB‐2C8 and I1, directed against different epitopes in the region between amino acids 110 and 145, prevent the interaction of PAI‐1 with vitronectin. We conclude that the region between amino acids 110 and 145 of PAI‐1 harbours an important determinant for the interaction with vitronectin.
Involvement of plasma membrane‐located calmodulin in the response decay of cyclic nucleotide‐gated cation channel of cultured carrot cells
Authors: Kurosaki Fumiya , Kaburaki Hiroshi , Nishi Arasuke
Published: July 2002

Increase in cytoplasmic cyclic AMP concentration stimulates Ca2+ influx through the cyclic AMP‐gated cation channel in the plasma membrane of cultured carrot cells. However, the Ca2+ current terminated after a few minutes even in the presence of high concentrations of cyclic AMP indicating that hydrolysis of the nucleotide is not responsible for stop of the Ca2+ influx. Cyclic AMP evoked discharge of Ca2+ from inside‐out sealed vesicles of carrot plasma membrane, and it was strongly inhibited when the suspension of the vesicles was supplemented with 1 μM of free Ca2+, while Ca2+ lower than 0.1 μM did not affect the Ca2+‐release. The Ca2+ flux across plasma membrane was restored from this Ca2+‐induced inhibition by the addition of calmodulin inhibitors or anti‐calmodulin. These results suggest that Ca2+ influx initiated by the increase in intracellular cAMP in cultured carrot cells is terminated when the cytosolic Ca2+ concentration reaches the excitatory level in the cells, and calmodulin located in the plasma membrane plays an important role in the response decay of the cyclic nucleotide‐gated Ca2+ channel.
Solubility of artificial proteins with random sequences
Authors: Prijambada Irfan D , Yomo Tetsuya , Tanaka Fumihiro , Kawama Toshihiro , Yamamoto Keizo , Hasegawa Akihisa , Shima Yasufumi , Negoro Seiji , Urabe Itaru
Published: July 2002

A library of artificial random proteins of 141 amino acid residues of which 95 are random and which includes the 20 kinds of amino acids was prepared. Out of the 25 identified random proteins, 5 were soluble in the cell lysate, indicating that about 20% of the random proteins expressed in Escherichia coli are expected to be soluble. The soluble random proteins RP3–42 and RP3–45 and insoluble RP3–70 were purified. The solubility of the purified form is the same as that in the cell lysate.
Phase specific association of heterotrimeric GTP‐binding proteins with the actin‐based cytoskeleton during thrombin receptor‐mediated platelet activation
Authors: Ozawa Kentaro , Takahashi Masahiko , Sobue Kenji
Published: July 2002

Subcellular distribution of heterotrimeric GTP‐binding proteins during thrombin receptor‐mediated platelet activation was examined, revealing two phases of translocation to the cytoskeleton. A part of Gi2α and Gsα shows first phase translocation to the low‐speed pellet (15000 × g pellet) within 1 min after activation, suggesting involvement in platelet shape change or granule secretion. In the second phase, Gi2α, Gsα, Gqα, and Gβ translocate to the low‐speed pellet, depending on platelet aggregation. These translocation correlated with the reorganization of the actin‐cytoskeleton and were inhibited by cytochalasin D. Reconstitution experiments also revealed that G proteins are associated with the actin‐cytoskeleton during platelet activation.
Substrate specificity of monomeric and dimeric α‐sarcin
Authors: Cheung Jin-I. , Wang Yuh-Ru , Lin Alan
Published: July 2002

The substrate specificity of monomeric and dimeric forms of α‐sarcin was investigated by membrane blotting procedures. Dimeric α‐sarcin fails to inactivate ribosomes as well as to hydrolyze mini‐stem‐loop RNA, whereas monomeric α‐sarcin catalyzes both substrates. Both monomeric and dimeric α‐sarcin are effective ribonucleases that are displayed by in situ RNA‐impregnated gel electrophoresis. The same purine base specificity was detected for both dimeric and monomeric forms. α‐Sarcin is also an effective deoxyribonuclease to supercoiled DNA. The action of α‐sarcin as deoxyribonuclease and ribonuclease is inhibited by the presence of SDS (3.5 × 10−6 M); the inhibition on ribonuclease, but not on deoxyribonuclease, is reversible if the proteins are renatured.
A randomized, double-blind study of alpidem vs placebo in the prevention and treatment of benzodiazepine withdrawal syndrome
Authors: Cassano GB , Petracca A , Borghi C , Chiroli S , Didoni G , Garreau M
Published: July 2002

The aim of the trial was to assess alpidem efficacy in preventing and treating the benzodiazepine (BZ) withdrawal syndrome (WS). A multicentre, double-blind, randomized versus placebo, parallel group study of six-week duration was carried out in outpatients suffering from generalized anxiety or adjustment disorder with an anxious mood and taking non-hypnotic BZ as continuous course of therapy of at least one-year duration. At the entry, the patients abruptly discontinued BZs and were treated with 50 mg/bid/tid of alpidem or placebo. Withdrawal syndrome diagnosis was (regarding treatment allocation) formulated by an independent psychiatrist, according to DSM-III-R and an appropriate scale, the SESSB. One hundred seventy-three patients were randomized and 148 completed the study. Withdrawal syndrome occurred in 27 patients of the alpidem group (31.0%) and in 38 patients of the placebo group (44.2%). A severe WS was diagnosed in 11.1% of the patients in the alpidem group and in 31.6% of the placebo group. If not having been withdrawn from the market, alpidem could have been useful for the prevention of BZ withdrawal syndrome.
The role of histidine‐114 of Sulfolobus acidocaldarius geranylgeranyl diphosphate synthase in chain‐length determination
Authors: Hirooka Kazutake , Kato Tatsuya , Matsu-ura Jun-ichiro , Hemmi Hisashi , Nishino Tokuzo
Published: July 2002

Sulfolobus acidocaldarius geranylgeranyl diphosphate synthase yields (all‐E)‐C20 prenyl diphosphate as a final product. The three‐dimensional model of the enzyme suggested that removing two bulky residues at 77 and 114 would allow additional prenyl‐chain elongation. To test this, we examined several mutants with substitutions at 77 and/or 114. As a result, the mutants, F77G, F77G and H114A, F77G and H114G, H114A, and H114G gave C30, C45, C50, C30 and C40 as the main long product, respectively. These observations indicate that histidine‐114 plays a crucial role in chain‐length determination along with phenylalanine‐77.
A reversible protein phosphorylation system is present at the surface of infective larvae of the parasitic nematode Trichinella spiralis
Authors: Smith Vincent P. , Selkirk Murray E. , Gounaris Kleoniki
Published: July 2002

Trichinella spiralis infective larvae have externally oriented enzymes catalysing reversible protein phosphorylation on their surface. Incubation of larvae with exogenous ATP resulted in phosphorylation of surface bound and released proteins. Exposure of the parasites to bile, a treatment which renders them infective for intestinal epithelia, resulted in increased release of protein and an altered profile of phosphorylation. Both serine/threonine and tyrosine phosphorylation and dephosphorylation reactions took place at the parasite surface. Examination of the structural characteristics of the larvae following exposure to bile showed that the non‐bilayer surface coat was not shed but was structurally reorganised.
Folding nuclei in proteins
Authors: Galzitskaya Oxana V , Ivankov Dmitry N , Finkelstein Alexei V
Published: July 2002

When a protein folds or unfolds, it passes through many half‐folded microstates. Only a few of them can accumulate and be seen experimentally, and this happens only when the folding (or unfolding) occurs far from the point of thermodynamic equilibrium between the native and denatured states. The universal features of folding, though, are observed just close to the equilibrium point. Here the ‘two‐state’ transition proceeds without any accumulation of metastable intermediates, and only the transition state (‘folding nucleus’) is outlined by its key influence on the folding–unfolding kinetics. Our aim is to review recent experimental and theoretical studies of the folding nuclei.
The dimerization motif of cytosolic sulfotransferases
Authors: Petrotchenko Evgeniy V. , Pedersen Lars C. , Borchers Christoph H. , Tomer Kenneth B. , Negishi Masahiko
Published: July 2002

Cytosolic sulfotransferases sulfate steroids such as estrogens and hydroxysteroids. The enzymes, including human estrogen sulfotransferase (hEST) and hydroxysteroid sulfotransferase (hHST), are generally homodimers in solution with mouse estrogen sulfotransferase (mEST) being one of few exceptions. To identify the amino acid residues responsible for the dimerization, eight residues on the surface of hEST were mutated to their counterparts in mEST and mutated hESTs were then analyzed by gel filtration chromatography. A single mutation of Val269 to Glu was sufficient to convert hEST to a monomer and the corresponding mutation of Val260 also altered hHST to a monomer. The hHST crystal structure revealed a short stretch of peptide with the side‐chains from two hHST monomers forming a hydrophobic zipper‐like structure enforced by ion pairs at both ends. This peptide consisted of 10 residues near the C‐terminus that, including the critical Val residue, is conserved as KXXXTVXXXE in nearly all cytosolic sulfotransferases. When mEST underwent the double mutations Pro269Thr/Glu270Val dimerization resulted. Thus, the KXXXTVXXXE sequence appears to be the common protein–protein interaction motif that mediates the homo‐ as well as heterodimerization of cytosolic sulfotransferases.
Different efficiencies of the Tag and AlkA DNA glycosylases from Escherichia coli in the removal of 3‐methyladenine from single‐stranded DNA
Authors: Bjelland Svein , Seeberg Erling
Published: July 2002

Escherichia coli possesses two different DNA repair glycoselases, Tag and AlkA, which have similar ability to remove the alkylation product 3‐methyladenine from double‐stranded DNA. In this study we show that these enzymes have quite different activities for the excision of 3‐methyladenine from single‐stranded DNA, AlkA being 10–20 times more efficient than Tag. We propose that AlkA and perhaps other glycosylases as well may have an important role in the excision of base damage from single‐stranded regions transiently formed in DNA during transcription and replication.
Effects of carbon dioxide/bicarbonate on induction of DNA single‐strand breaks and formation of 8‐nitroguanine, 8‐oxoguanine and base‐propenal mediated by peroxynitrite
Authors: Yermilov Vladimir , Yoshie Yumiko , Rubio Julieta , Ohshima Hiroshi
Published: July 2002

Carbon dioxide has been reported to react with peroxynitrite (ONOO−), a strong oxidant and nitrating agent, to form an ONO2CO2
− adduct, altering the reactivity characteristic of peroxynitrite. We found that bicarbonate (0–10 mM) caused a dose‐dependent increase of up to 6‐fold in the formation of 8‐nitroguanine in calf‐thymus DNA incubated with 0.1 mM peroxynitrite, whereas it produced no apparent effect on 8‐oxoguanine formation. In contrast, bicarbonate inhibited peroxynitrite‐induced strand breakage in plasmid pBR322 DNA and thymine‐propenal formation from thymidine. We conclude that reacts with peroxynitrite to form a potent nitrating agent, but also to inactivate hydroxyl‐radical‐like activity of peroxynitrous acid.
Tissue‐specific expression of the two isoforms of the mitochondrial phosphate carrier in bovine tissues
Authors: Dolce V. , Fiermonte G. , Palmieri F.
Published: July 2002

Comparison of the sequence of the human mitochondrial phosphate carrier (PiC) gene with cDNA clones characterised from a human heart cDNA library suggested the existence of two isoforms of the PiC, which were generated by alternative splicing of exon IIIA or exon IIIB and which differed in 13 amino acids [Dolce et al. (1994) J. Biol. Chem. 269,10451]. In this work the expression of isoforms A and B of the PiC was investigated in different bovine tissues by Northern blot analysis using two probes that are specific for bovine exon IIIA and exon IIIB, respectively. Isoform A is highly expressed in heart and skeletal muscle. Isoform B is ubiquitously expressed in all tissues that were examined, although at different levels. The tissue‐specific expression pattern of the two PiC isoforms is similar to that reported for the isoforms of several mitochondrial proteins required for energy production.
Conversion of glutaconate CoA‐transferase from Acidaminococcus fermentans into an acyl‐CoA hydrolase by site‐directed mutagenesis
Authors: Mack Matthias , Buckel Wolfgang
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Kinetics of calcium and calmodulin‐dependent protein kinase III from embryonic chicken leg muscle cells
Authors: Riis Bent , Nygård Odd
Published: July 2002

Embryonic chicken muscle cells (CELM) contain the calmodulin‐dependent protein kinase that specifically phosphorylates eukaryotic elongation factor 2. The kinase requires Ca2+ and maximum activity in CELM was observed at 10 μM Ca2+. The ATP concentration required for half the maximum activity of CaM PKIII in CELM was calculated to be 0.15 mM. In CELM, dephosphorylation of eEF‐2 was catalyzed by phosphoprotein phosphatase PP2A alone. The activity of PP2A was relatively low and the half‐life of added phosphorylated eEF‐2 was more than 15 min. Due to the low phosphoprotein phosphatase activity, inhibition of the PP2A activity by addition of okadaic acid had little effect on the eEF‐2 phosphorylation kinetics.
A hairpin‐loop conformation in tandem repeat sequence of the ice nucleation protein revealed by NMR spectroscopy
Authors: Tsuda Sakae , Ito Ai , Matsushima Norio
Published: July 2002

The 1H‐NMR spectrum of a synthetic 24‐residue peptide (A1‐G‐V‐D‐S‐S‐L‐I‐A‐G‐Y‐G‐S‐T‐Q‐T‐S‐G‐S‐D‐S‐A‐L‐T24; INP24), comprising three repeats of the 8‐residue consensus sequence of Pseudomonas syringae ice nucleation protein, was fully assigned using 2‐dimensional (2D) NMR spectroscopy at 4°C and 30°C. Close proximity of the aliphatic protons between Leu7, Ile8, Ala9, and the ring‐protons of Tyr11 was indicated from the observation of the inter‐molecular nuclear Overhauser enhancement (NOE) effect. Hydrogen‐bonding was strongly suggested for the NH group of Leu7 from its extremely low‐temperature coefficient estimated from the temperature dependence of the chemical shift. These results indicate the formation of a hairpin‐loop conformation constructed by a hexapeptide segment of INP24, ‐Leu7‐Ile8‐Ala9‐Gly10‐Tyr11‐Gly12.
Folding of the Fab fragment within the intact antibody
Authors: Lilie Hauke
Published: July 2002

At present it is not clear to which extent the Fab fragment and the Fc part of an antibody interact in the intact immunoglobulin structure. To determine such potential interactions the unfolding and refolding of an isolated Fab fragment and the respective antibody MAK 33 (κ/IgG1) are compared. It could be shown that the proline independent renaturation kinetics of both an unfolding intermediate and the fully denatured form of both proteins are identical. Upon denaturation, the loss of antigen binding activity occurs with the same rate for both the Fab fragment and the intact antibody. However, the complete structural unfolding of the Fab part of the antibody is significantly slower than that of the isolated Fab fragment. These kinetic data suggest that the structure of the Fab fragment within the intact antibody is stabilized by interactions, presumably with the Fc part, missing in the isolated Fab.
Induction of UCP2 mRNA by thyroid hormones in rat heart
Authors: Lanni Antonia , De Felice Mario , Lombardi Assunta , Moreno Maria , Fleury Christophe , Ricquier Daniel , Goglia Fernando
Published: July 2002

The possible regulation of the expression of uncoupling protein‐2 (UCP2) mRNA by thyroid hormones in different tissues was examined in rats. Triiodothyronine (T3) was found to produce an organ‐specific enhancement of UCP2 expression in rat tissues. The effect of T3 was markedly observed in heart, whereas a moderate effect was seen in skeletal muscle and no effect in kidney or liver. These results suggest that UCP2 is a protein that may be involved in the nuclear‐mediated effect of T3 on resting metabolic rate in the rat.
Primary structure and expression of a naturally truncated human P2X ATP receptor subunit from brain and immune system
Authors: Lê Khanh-Tuoc , Paquet Michel , Nouel Dominique , Babinski Kazimierz , Séguéla Philippe
Published: July 2002

A novel member of the ionotropic ATP receptor gene family has been identified in human brain. This 422 amino acid long P2X receptor subunit has 62% sequence identity with rat P2X5. Several characteristic motifs of ATP‐gated channels are present in its primary structure, but this P2X5‐related subunit displays a single transmembrane domain. Heterologous expression of chimeric subunits containing the C‐terminal domain of rat P2X5 leads to the formation of desensitizing functional ATP‐gated channels in Xenopus oocytes. The developmentally regulated mRNA, found in two splicing variant forms, is expressed at high levels in brain and immune system.
c‐Rel and p65 subunits bind to an upstream NF‐κB site in human granulocyte macrophage‐colony stimulating factor promoter involved in phorbol ester response in 5637 cells
Authors: Ghiorzo Paola , Musso Marco , Mantelli Michela , Garré Cecilia , Ravazzolo Roberto , Bianchi-Scarrà Giovanna
Published: July 2002

To further clarify the complex transcriptional regulation of the human GM‐CSF gene, which was extensively investigated in activated T cells, we have studied the role of an upstream NF‐κB like site in the 5637 non‐lymphoid cell line, which derives from a bladder carcinoma and constitutively produces GM‐CSF. This sequence, named the A element, has an active role on GM‐CSF transcription and is responsive to the tumor promoter PMA in transient transfection experiments. We describe here a heterodimeric binding complex of NF‐κB subunits (c‐Rel and p65) which is identical to the one obtained using the HIV‐LTR‐κB site as recognition sequence and different from the one (c‐Rel and p50) observed with nuclear extracts from Mo T‐lymphoid HTLV‐II infected cells.
Exon 2 of human cathepsin B derives from an Alu element
Authors: Berquin Isabelle M , Ahram Mamoun , Sloane Bonnie F
Published: July 2002

Transcripts for the cysteine protease cathepsin B are alternatively spliced in the untranslated regions (UTRs). We show that a cathepsin B probe containing 5′‐UTR sequences hybridized to an RNA of ∼300 nt in addition to the typical 2.2 and 4.0 kbp mRNAs. Within this 5′‐UTR, exon 2 was found to be homologous to Alu repetitive elements. Specifically, exon 2 was part of an Alu element interspersed with the cathepsin B gene. The ∼300 nt band that hybridized to our cathepsin B probe likely corresponds to Alu transcripts, which are known to accumulate in human cells. Indeed, a similarly migrating band was detected with an authentic Alu probe. Thus, we suggest that primary transcripts for cathepsin B contain Alu sequences which are preserved as exon 2 in some fully spliced mRNAs.
A high diffusion coefficient for coenzyme Q10 might be related to a folded structure
Authors: Di Bernardo Salvatore , Fato Romana , Casadio Rita , Fariselli Piero , Lenaz Giorgio
Published: July 2002

We measured the lateral diffusion of different coenzyme Q homologues and analogues in model lipid vesicles using the fluorescence collisional quenching technique with pyrene derivatives and found diffusion coefficients in the range of 10−6 cm2/s. Theoretical diffusion coefficients for these highly hydrophobic components were calculated according to the free volume theory. An important parameter in the free volume theory is the relative dimension between diffusant and solvent: a molecular dynamics computer simulation of the coenzymes yielded their most probable geometries and volumes and revealed surprisingly similar sizes of the short and long homologues, due to a folded structure of the isoprenoid chain in the latter, with a length for coenzyme Q10 of 21 Å. Using this information we were able to calculate diffusion coefficients in the range of 10−6 cm2/s, in good agreement with those found experimentally.
The chloroplast Ndh complex mediates the dark reduction of the plastoquinone pool in response to heat stress in tobacco leaves
Authors: Sazanov Leonid A , Burrows Paul A , Nixon Peter J
Published: July 2002

We have examined the effects of heat stress on electron transfer in the thylakoid membrane of an engineered plastid ndh deletion mutant, Δ1, incapable of performing the Ndh‐mediated reduction of the plastoquinone pool in the chloroplast. Upon heat stress in the dark, the rate of PSII‐independent reduction of PSI after subsequent illumination by far‐red light is dramatically enhanced in both Δ1 and a wild‐type control plant (WT). In contrast, in the dark, only the WT shows an increase in the reduction state of the plastoquinone pool. We conclude that the heat stress‐induced reduction of the intersystem electron transport chain can be mediated by Ndh‐independent pathways in the light but that in the dark the dominant pathway for reduction of the plastoquinone pool is catalysed by the Ndh complex. Our results therefore demonstrate a functional role for the Ndh complex in the dark.
Characterization of genes for two‐component phosphorelay mediators with a single HPt domain in Arabidopsis thaliana
Authors: Miyata Shin-ichi , Urao Takeshi , Yamaguchi-Shinozaki Kazuko , Shinozaki Kazuo
Published: July 2002

Three cDNAs that encode two‐component phosphorelay‐mediator‐like proteins were cloned from Arabidopsis thaliana. Putative proteins (ATHP1–3) contain an HPt (istidine‐containing hospho ransfer)‐like domain with a conserved histidine and some invariant residues that are involved in phosphorelay. Growth retardation of YPD1‐disrupted yeast cells was reversed with ATHPs, which indicates that ATHPs function as phosphorelay mediators in yeast cells. The ATHP genes are expressed more in roots than in other tissues, similar to the expression of genes for a sensor histidine kinase, ATHK1, and response regulators ATRR1–4. These results suggest that ATHPs function as two‐component phosphorelay mediators between sensor histidine kinase and response regulators in Arabidopsis.
Human monocyte derived dendritic cells express functional P2X and P2Y receptors as well as ecto‐nucleotidases
Authors: Berchtold Susanne , Ogilvie Alexandra L.J. , Bogdan Cornelia , Mühl-Zürbes Petra , Ogilvie Adaling , Schuler Gerold , Steinkasserer Alexander
Published: July 2002

We investigated the expression and function of P2 receptors and ecto‐nucleotidases on human monocyte derived dendritic cells (DC). In addition we analyzed the effect of extracellular ATP on the maturation of DC. By RT‐PCR, DC were found to express mRNA for several P2X (P2X1, P2X4, P2X5, P2X7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y5, P2Y6, P2Y10, P2Y11) receptors. As shown by FURA‐2 measurement, triggering of P2 receptors resulted in an increase in free intracellular Ca2+. In combination with Tumor necrosis factor‐α, ATP increased the expression of the DC surface markers CD80, CD83 and CD86 indicating a maturation promoting effect. DC expressed the ecto‐apyrase CD39 and the ecto‐5′‐nucleotidase CD73 as demonstrated by RT‐PCR. Extracellular ATP was rapidly hydrolyzed by these ecto‐enzymes as shown by separation of 3H‐labeled ATP metabolites using a thin layer technique. These data suggest that ATP acts as a costimulatory factor on DC maturation.
A novel function for a ubiquitous plant enzyme pectin methylesterase: the host‐cell receptor for the tobacco mosaic virus movement protein
Authors: Dorokhov Yu.L. , Mäkinen K. , Frolova O.Yu. , Merits A. , Saarinen J. , Kalkkinen N. , Atabekov J.G. , Saarma M.
Published: July 2002

Plant virus‐encoded movement proteins promote viral spread between plant cells via plasmodesmata. The movement is assumed to require a plasmodesmata targeting signal to interact with still unidentified host factors presumably located on plasmodesmata and cell walls. The present work indicates that a ubiquitous cell wall‐associated plant enzyme pectin methylesterase of Nicotiana tabacum L. specifically binds to the movement protein encoded by tobacco mosaic virus. We also show that pectin methylesterase is an RNA binding protein. These data suggest that pectin methylesterase is a host cell receptor involved in cell‐to‐cell movement of tobacco mosaic virus.
Speculoscopy vs. the acetic acid test for cervical neoplasia
Authors: Cronjé H.S , van Rensburg E , Cooreman B.F , Niemand I , Beyer E
Published: July 2002

Objective: To compare the specificity of speculoscopy and the acetic acid test (AAT). Methods: A randomized controlled trial was performed on 1150 women in a primary healthcare clinic. The main end‐points were the sensitivity and specificity of speculoscopy and the AAT. Results: The mean age of the patients was 37.7 years in the speculoscopy group (n=545) and 37.5 years in the AAT group (n=605). Cervicography (the golden standard) was positive in 11.3% of the participants, speculoscopy in 20.4% and the AAT in 25.1%. The sensitivity, specificity and the kappa value for speculoscopy were 53.5%, 83.6% and 0.23, respectively. For the AAT, the corresponding figures were 51.1%, 77.3% and 0.15. No statistically significant differences were found between these two groups. Of clinical importance, however, was a finding that speculoscopy did not miss a single case of high grade squamous intra‐epithelial lesion or cancer whilst the AAT missed five such lesions. Conclusion: Speculoscopy could not be shown to have an improved specificity when compared with the acetic acid test. The low specificity of both the tests will result in the referral of too many patients for colposcopy.
Women's health problems in the Arab World: a holistic policy perspective
Authors: Zurayk H. , Sholkamy H. , Younis N. , Khattab H.
Published: July 2002

The paper reviews key health problems of women in the Arab World. It relies on data and information from international yearbooks, regional data bases, and small scale field studies. The relevant context in which women live; of lingering illiteracy rates, lack of access to cash income, and increasing poverty is described. Reproductive health is reviewed within this context pointing to trends of delayed marriage and declining fertility in some countries while other countries maintain high levels. Similar variability is observed in contraceptive use rates and the interaction of contraception and health is discussed. Evidence points to high levels of reproductive morbidity. The socio‐cultural context is found particularly relevant to pregnancy and childbirth, seen as natural processes by women, to experiences of menopause, and to violence against women, particularly female circumcision. A holistic policy perspective is suggested to address these problems.
Ultrasound‐guided aspiration in management of cystic lesions of the ovary
Authors: Mittal S , Kumar S , Verma A
Published: July 2002

Objective: To evaluate the role of ultrasound‐guided aspiration (UGA) as an alternative therapeutic modality in patients with cystic lesions of ovary (CLO). Methods: Forty‐six patients (age 14–65 years) of CLO were managed by UGA. Patients suspected of having malignancy either clinically or on ultrasound were excluded. The aspiration was done transvaginally (11 patients) or transabdominally (35 patients). All aspirates were subjected to cytological examination. Patients have been followed up for 10–48 months. Results: The cyst size varied from 4 to 30 cm and the volume aspirated ranged from 30 to 1600 ml. There were no procedure‐related complications. No further intervention were required in 33 (71.7%) patients. Re‐aspiration was required in six (13%) and, to date, seven patients (15.3%) have been operated on for recurrence or persistence of lesion. Recurrence rate was not influenced by cyst size or volume aspirated. Mucinous or hemorrhagic aspirate correlated significantly with a higher risk of recurrence. Conclusion: Ultrasound‐guided aspiration is a simple, safe and effective alternative to surgery in management of selected patients with CLO. Simple unilocular cysts can be safely aspirated, but it should not be carried out in patients with multilocular cysts. If the aspirate is hemorrhagic or mucinous, the patient should be considered for surgical intervention.
Service quality: a tutorial
Authors: Harvey Jean
Published: July 2002

This article is about quality of service. It presents an overview and a summary model of current thinking on the topic. Many examples and illustrations are presented. Aspects of quality include: Quality of results and quality of process; search, experiential and credence quality; reality vs. perception; expectations vs. perceptions; customer satisfaction and technical quality. Various approaches and techniques are presented to improve performance quality—quality functions deployment, moving the line of visibility, blueprinting and failsafing—and conformance quality—guaranteeing, mystery shopping, recovering and measuring.
An investigation of the relationship between global manufacturing practices and outcomes in machine tools and textile industries
Authors: Kadipasaoglu Sukran N , Hurley Simon F , Foote Jeffrey L , Khumawala Basheer M
Published: July 2002

This empirical research study investigates the manufacturing planning and control (MPC) practices associated with successful company performance. To form a database of global manufacturing practices, data collected from machine tools and textile industries in 12 countries around the world via the Global Manufacturing Research Survey was combined. An exploratory factor analysis was first conducted to reduce the dimensionality of the data. Three Discriminant Analyses were then performed on the factor scores of successful and non‐successful companies, between free and planned economies and among the 12 countries represented in the database, with an emphasis on the factors found to be associated with success. The results show that close coordination of several business functions with the MPC system is essential for success. Significant differences were found between state‐managed and free‐market economies and among the countries represented in the database. In general, countries with free‐market economies had high success coefficients, led by Japan, which had the most combined use of essential success factors, while countries with planned economies had low success coefficients.
Fibroblast expression of C—C chemokines in response to orthopaedic biomaterial particle challenge in vitro
Authors: Yaszay Burt , Trindade Michael C. D. , Lind Martin , Goodman Stuart B. , Smith R. Lane
Published: July 2002

C—C chemokines are soluble mediators that occur in a periprosthetic granuloma and influence recruitment, localization and activation of inflammatory cells. This study tested effects of titanium and polymethylmethacrylate (PMMA) particles on expression of selected C—C chemokines in cultured human fibroblasts. The C—C chemokines analyzed included monocyte chemoattractant protein‐1, 2 (MCP‐1, 2), monocyte inflammatory protein‐1 alpha (MIP‐1 alpha), and regulated on activation, normal T‐cell expressed and secreted protein (RANTES). Interleukin‐1 beta (IL‐1 beta) served as a known stimulator of chemokine release while interleukin‐6 (IL‐6) expression served as a marker for fibroblast activation. Protein and mRNA signal levels were determined by ELISA and RT‐PCR, respectively. The results demonstrated that exposure of fibroblasts to titanium and PMMA particles resulted in increased release of MCP‐1 in a dose‐ and time‐dependent manner. After 24 h, titanium particles maximally upregulated MCP‐1 release 7‐fold while PMMA particles increased MCP‐1 levels 2‐fold, when compared to unchallenged fibroblasts. MCP‐2, MIP‐1 alpha and RANTES levels remained unchanged following exposure of fibroblasts to titanium or PMMA particles at any concentration or time point tested. However, IL‐1 beta stimulated release of MCP‐1, MCP‐2, and RANTES, but not MIP‐1 alpha from the fibroblasts. IL‐1 beta, not particles, exhibited the most prominent effect on MCP‐1 mRNA levels. Increased release of MCP‐1 from fibroblasts exposed to titanium and PMMA particles coincided with increased release of IL‐6. This study suggests that release of chemoattractant factors from fibroblasts localized in periprosthetic membranes enhances the chronic inflammatory process leading to bone resorption and implant loosening. © 2001 Orthopaedic Research Society. Punlished by Elsevier Science Ltd. All rights reserved.
Expressed emotion in an 8 year follow-up
Authors: Schulze Mönking H , Hornung WP , Stricker K , Buchkremer G
Published: July 2002

This study addresses the questions if and for which patients expressed emotion (EE) has a predictive validity in an 8 year follow-up study and if this is dependent on the residential form, especially on patients and relatives living together. Eight years after a baseline examination (Camberwell Family Interviews [CFI], global assessment scale [GAS], symptom score) 69 of 99 patients were reexamined. The number and duration of rehospitalizations as well as the symptom and GAS scores were recorded. These was no difference between outcome of high and low EE patients with a duration of illness of less than 4.5 years and for those living with partners. Patients from high EE parents with a longer duration of illness (> 4.5 years) at the outset of the study were significantly more often and longer in hospital. They had significantly higher symptom and GAS scores at the outset of the study and after 8 years. The EE effect was equal in the first and second 4 years of the follow-up and was independent from patients and parents living together. The results emphasize the significance of the EE index as a long-term predictor for the course of schizophrenic illness. The independence of the prediction from living together is an argument against a simple causal interpretation of the connection between EE and the course of schizophrenia.
Methodological issues raised by clinical trials on dysthymia: assessment instruments and response criteria
Authors: Guelfi J.D. , Corruble E
Published: July 2002

Dysthymia clinical trials raise several methodological issues. The validity of the concept remains a matter of concern, as does the need for specific instruments, the difficulties in assessment of change and long-term assessments. Diagnostic criteria for inclusion should be used in a polydiagnostic approach. This paper summarises the main assessment tools and diagnostic criteria used in clinical trials on dysthymia. Severity criterion for inclusion could be a Hamilton Depression Rating Scale score (HAMD-17) between 13 and 17. The main response criteria should be a decrease of at least 50% of the total score on this scale and a final score under a predetermined limit. However, other response criteria may be useful: depression self-ratings, global assessments, general psychopathology assessments, personality and defense mechanisms, quality of life, psychosocial and functional impairment, diagnostic criteria (presence or absence) and side-effect assessment.
Psychotic disorders among inpatients with abuse of cannabis, amphetamine and opiates. Do dopaminergic stimulants facilitate psychiatric illness?
Authors: Dalmau A. , Bergman B. , Brismar B.
Published: July 2002

We have studied the occurrence of dual diagnoses (psychoses as well as abuse of either amphetamine, cannabis or opiates) during a 15-year period, among patients treated at Huddinge Hospital, Stockholm, Sweden. The purpose of the study is to evaluate if the different drugs were coupled to different rates of psychiatric co-morbidity. During the period in question, 461, 425 and 371 different patients respectively had been admitted at least once due to dependency on amphetamine, cannabis and opiates. Approximately 30% of the patients with a pure abuse of amphetamine or cannabis and less than 6% of the opiate abusers had been diagnosed at least once with any of the psychoses studied. Comparing the frequency of psychoses among mixed and pure abusers of illegal drugs, with and without a concomitant abuse of alcohol, we found that the co-morbidity rate for mixed opiate abusers increased significantly from 7.2 to 20.2% when alcohol abuse was also present. For abusers of amphetamine and cannabis (both pure and mixed), no differences in co-morbidity rates were seen when an abuse of alcohol was added to that of the drugs. It is difficult to find an explanation for the significant difference between the co-morbidity of pure abuse of amphetamine or cannabis on the one hand and opiates on the other. In conclusion, our findings show that the distribution of psychotic illness is high among abusers of amphetamine and cannabis, in contrast to the generally lower co-morbidity among abusers of opiates. Although these findings are consistent with earlier studies that have shown a propensity for developing psychoses among abusers of amphetamine and cannabis, one should bear in mind that this study is based on inpatients, and is not necessarily representative for all abusers of the drugs in question.