bluuebunny/crossref_metadata_2025 huggingface dataset:
Association between the first two immunoglobulin‐like domains of the neural cell adhesion molecule N‐CAM
Authors: Atkins Annette R. , Osborne Michael J. , Lashuel Hilal A. , Edelman Gerald M. , Wright Peter E. , Cunningham Bruce A. , Dyson H.Jane
Published: July 2002

The extracellular domain of N‐CAM contains five immunoglobulin‐like (Ig) and two fibronectin type III‐like domains and facilitates cell‐cell binding through multiple, weak interdomain interactions. NMR spectroscopy indicated that the two N‐terminal Ig‐like domains from chicken N‐CAM (Ig I and Ig II) interact with millimolar affinity. Physico‐chemical studies show that this interaction is significantly amplified when the domains are covalently linked, consistent with an antiparallel domain arrangement. The binding of the two individual domains and the dimerization of the concatenated protein were essentially independent of salt, up to a concentration of 200 mM. The residues in Ig I involved in the interaction map to the BED strands of the β sandwich, and delineate a largely hydrophobic patch.
Sequence of subunit a of the Na+‐translocating F1F0‐ATPase of Acetobacterium woodii: proposal for residues involved in Na+ binding
Authors: Rahlfs Stefan , Müller Volker
Published: July 2002

Na+ transport through the F0 domain of Na+‐F1F0‐ATPases involves the combined action of subunits c and a but the residues involved in Na+ liganding in subunit a are unknown. As a first step towards the identification of these residues, we have cloned and sequenced the gene encoding subunit a of the Na+‐F1F0‐ATPase of Acetobacterium woodii. This is the second sequence available now for this subunit from Na+‐F1F0‐ATPases. A comparison of subunit a from Na+‐F1F0‐ATPases with those from H+‐translocating enzymes unraveled structural similarity in a C‐terminal segment including the ultimate and penultimate transmembrane helix. Seven residues are conserved in this region and, therefore, likely to be involved in Na+ liganding.
Targeting of tail‐anchored proteins to yeast mitochondria in vivo
Authors: Egan Billie , Beilharz Traude , George Rebecca , Isenmann Sandra , Gratzer Sabine , Wattenberg Binks , Lithgow Trevor
Published: July 2002

Tail‐anchored proteins are inserted into intracellular membranes via a C‐terminal transmembrane domain. The topology of the protein is such that insertion must occur post‐translationally, since the insertion sequence is not available for membrane insertion until after translation of the tail‐anchored polypeptide is completed. Here, we show that the targeting information in one such tail‐anchored protein, translocase in the outer mitochondrial membrane 22, is contained in a short region flanking the transmembrane domain. An equivalent region is sufficient to specify the localisation of Bcl2 and SNARE proteins to the secretory membranes. We discuss the targeting process for directing members of this protein family to the secretory and mitochondrial membranes in vivo.
Surfactant protein A and D expression in the porcine Eustachian tube1
Authors: Paananen Reija , Glumoff Virpi , Hallman Mikko
Published: July 2002

Surfactant proteins A and D are collectins which are considered to play an important role in the innate immunity of lungs. Our aim was to investigate whether surfactant protein A or D is expressed in the porcine Eustachian tube originating from the upper airways. Both surfactant proteins A and D were present in the epithelial cells of the Eustachian tube, as shown by strong immunostaining. Using RT‐PCR and Northern hybridization, these collectins were detected in the Eustachian tube. The present study is the first report demonstrating surfactant protein gene expression in the Eustachian tube. Surfactant proteins A and D may be important in the antibody‐independent protection of the middle ear.
The thermodynamics and kinetics of electron transfer in the cytochrome P450cam enzyme system
Authors: Honeychurch Michael J , Hill H.Allen O , Wong Luet-Lok
Published: July 2002

In anaerobic environments the first electron transfer in substrate‐free P450cam is known to be thermodynamically unfavourable, but in the presence of dioxygen the reduction potential for the reaction shifts positively to make electron transfer thermodynamically favourable. Nevertheless a slower rate of electron transfer is observed in the substrate‐free P450cam compared to substrate‐bound P450cam. The ferric haem centre in substrate‐free P450cam changes from six co‐ordinate to five co‐ordinate when reduced whereas in substrate‐bound P450cam the iron centre remains five co‐ordinate in both oxidation states. The slower rate of electron transfer in the substrate‐free P450cam is therefore attributed to a larger reorganisation energy as predicted by Marcus theory.
Caspase‐3 is necessary and sufficient for cleavage of protein synthesis eukaryotic initiation factor 4G during apoptosis
Authors: Bushell Martin , McKendrick Linda , Jänicke Reiner U. , Clemens Michael J. , Morley Simon J.
Published: July 2002

Induction of apoptosis BJAB cells is accompanied by the rapid cleavage of protein synthesis eukaryotic initiation factor 4G and the appearance of a fragment of approximately 76 kDa. Inhibition of apoptotic proteases (caspases) has previously been shown to prevent the cleavage of eukaryotic initiation factor 4G. In MCF‐7 breast carcinoma cells, which are deficient in caspase‐3, eukaryotic initiation factor 4G is not cleaved but in vivo expression of caspase‐3 restores eukaryotic initiation factor 4G cleavage following induction of apoptosis. Recombinant caspase‐3 can also cleave eukaryotic initiation factor 4G to yield the 76 kDa fragment both in cell extracts and when the eukaryotic initiation factor 4G is presented in a purified eukaryotic initiation factor 4F complex. These results indicate that caspase‐3 activity is necessary and sufficient for eukaryotic initiation factor 4G degradation.
3‐Hydroxy‐3‐methylglutaryl CoA reductase inhibitors reduce serum triglyceride levels through modulation of apolipoprotein C‐III and lipoprotein lipase
Authors: Schoonjans Kristina , Peinado-Onsurbe Julia , Fruchart Jean-Charles , Tailleux Anne , Fiévet Catherine , Auwerx Johan
Published: July 2002

Statins are hypolipidemic drugs which not only improve cholesterol but also triglyceride levels. Whereas their cholesterol‐reducing effect involves inhibition of de novo biosynthesis of cellular cholesterol through competitive inhibition of its rate‐limiting enzyme 3‐hydroxy‐3‐methylglutaryl CoA reductase, the mechanism by which they lower triglycerides remains unknown and forms the subject of the current study. Treatment of normal rats for 4 days with simvastatin decreased serum triglycerides significantly, whereas it increased high density lipoprotein cholesterol moderately. The decrease in triglyceride concentrations after simvastatin was caused by a reduction in the amount of very low density lipoprotein particles which were of an unchanged lipid composition. Simvastatin administration increased the lipoprotein lipase mRNA and activity in adipose tissue and heart. This effect on lipoprotein lipase was accompanied by decreased mRNA as well as plasma levels of the lipoprotein lipase inhibitor apolipoprotein C‐III. These results suggest that the triglyceride‐lowering effect of statins involves a stimulation of lipoprotein lipase‐mediated clearance of triglyceride‐rich lipoproteins.
Action of thyroid hormones at the cellular level: the mitochondrial target
Authors: Goglia Fernando , Moreno Maria , Lanni Antonia
Published: July 2002

Thyroid hormones exert profound effects on the energy metabolism. An inspection of the early and more recent literature shows that several targets at the cellular level have been identified. Since their effects on the nuclear signalling pathway have already been well‐defined and extensively reviewed, this article focuses on the regulation of mitochondrial activity by thyroid hormones. Mitochondria, by virtue of their biochemical functions, are a natural candidate as a direct target for the calorigenic effects of thyroid hormones. To judge from results coming from various laboratories, it is quite conceivable that mitochondrial activities are regulated both directly and indirectly. Not only triiodo‐L‐thyronine, but also diiodothyronines are active in regulating the energy metabolism. They influence the resting metabolism in rats with 3,5‐diiodo‐L‐thyronine seeming to show a clearer effect.
Genomic organization of three neurotoxins active on small conductance Ca2+‐activated potassium channels from the scorpion Buthus martensi Karsch1
Authors: Wu Jing-Jiang , Dai Li , Lan Zheng-Dao , Chi Cheng-Wu
Published: July 2002

According to the known primary sequences of three neurotoxins active on small conductance Ca2+‐activated potassium channels from the scorpion Buthus martensi Karsch, their corresponding cDNAs were cloned and sequenced using 3′‐ and 5′‐RACE. All of them encoded a signal peptide composed of 28 residues and a mature toxin of 29, 28 and 33 residues, respectively. Their cDNA deduced sequences were totally consistent with those determined, and the C‐terminal amidation of one neurotoxin was confirmed. The genomic DNAs of these three toxins were also amplified by PCR, cloned and sequenced. They all consisted of two exons disrupted by a small single intron. All of these introns were inserted within the signal peptide at the same −10 position upstream from the mature toxin, consisting of 94, 78 and 87 bp, respectively.
Mutational analysis of a conserved tetraloop in the 5′ untranslated region of hepatitis C virus identifies a novel RNA element essential for the internal ribosome entry site function
Authors: Psaridi L , Georgopoulou U , Varaklioti A , Mavromara P
Published: July 2002

The 5′ untranslated region of hepatitis C virus RNA forms an extensive secondary structure including several hairpin motifs and mediates translation initiation by an internal ribosome entry site‐dependent pathway. We report, here, an extensive mutagenesis analysis of a highly conserved tetraloop in the 5′ untranslated region of hepatitis C virus, namely hairpin IIIe (295′‐GAUA‐298′). Our results demonstrate that hairpin IIIe is essential for the internal ribosome entry site function. Moreover, they indicate the importance of the primary structure of this motif because mutations in all four nucleotides of the loop caused a severe loss of internal ribosome entry site activity. These data represent the first experimental evidence for the functional significance of tetraloops in internal ribosome entry site‐driven translation of hepatitis C virus.
Effect of mutations found in carbohydrate‐deficient glycoprotein syndrome type IA on the activity of phosphomannomutase 2
Authors: Pirard Michel , Matthijs Gert , Heykants Leen , Schollen Els , Grünewald Stephanie , Jaeken Jaak , van Schaftingen Emile
Published: July 2002

Seven mutant forms of human phosphomannomutase 2 were produced in Escherichia coli and purified. These mutants had a V
max of 0.2–50% of the wild enzyme and were unstable. The least active protein (R141H) bears a very frequent mutation, which has never been found in the homozygous state whereas the second least active protein (D188G) corresponds to a mutation associated with a particularly severe phenotype. We conclude that total lack of phosphomannomutase 2 is incompatible with life. Another conclusion is that the elevated residual phosphomannomutase activity found in fibroblasts of some patients is contributed by their mutated phosphomannomutase 2.
The small GTPases Rab5a, Rab5b and Rab5c are differentially phosphorylated in vitro
Authors: Chiariello Mario , Bruni Carmelo B , Bucci Cecilia
Published: July 2002

Rab GTPases play a fundamental role in the regulation of membrane traffic. Three different Rab5 isoforms have been reported but no differences in their function in endocytosis have been discovered. As the Rab5 isoforms show a conserved consensus site for Ser/Thr phosphorylation, we investigated whether this site was phosphorylated. Here, we report that the three Rab5 proteins are differentially recognized by different kinases. Rab5a is efficiently phosphorylated by extracellular‐regulated kinase 1 but not by extracellular‐regulated kinase 2, while cdc2 kinase preferentially phosphorylates Ser‐123 of Rab5b. These findings strongly suggest that phosphorylation could be important to differentially regulate the function of the Rab5 isoforms.
The 3′ non‐coding region of the Drosophila melanogaster HeT‐A telomeric retrotransposon contains sequences with propensity to form G‐quadruplex DNA
Authors: Abad José P. , Villasante Alfredo
Published: July 2002

HeT‐A elements are non‐long terminal repeat retrotransposons added onto the Drosophila chromosome ends. We have investigated the formation in vitro of higher order structures by oligonucleotides derived from the 3′ non‐coding region of HeT‐A elements and found that they are capable of forming G‐quadruplex DNA. These results suggest that the 3′ repeat region of HeT‐A may structurally behave as the telomeric repeats common to a majority of eukaryotes. The presence of structural motifs shared by telomeres and centromeres and the implications of these findings for chromosome evolution are discussed.
Hyperphosphorylated tau in SY5Y cells: similarities and dissimilarities to abnormally hyperphosphorylated tau from Alzheimer disease brain
Authors: Zhong J , Iqbal K , Grundke-Iqbal I
Published: July 2002

Unlike normal tau, abnormally hyperphosphorylated tau (AD P‐tau) from Alzheimer disease (AD) does not promote but instead inhibits microtubule assembly and disrupts already formed microtubules. Tau in the human neuroblastoma cell line SH‐SY5Y is hyperphosphorylated at several of the same sites as AD P‐tau, and accumulates in the cell body without any association to the cellular microtubule network. The aim of the present study was to elucidate why the SY5Y tau does not affect the viability of the cells. We found that, like AD P‐tau, SY5Y tau because of hyperphosphorylation does not bind to microtubules and inhibits the tau‐promoted assembly of microtubules. However, the tau/HMW MAP ratio is about 10 times less in SY5Y cells than in AD brain. These findings suggest that the hyperphosphorylated tau from SY5Y cells has similar biological characteristics as AD P‐tau from AD brain, but is not lethal to the SY5Y cells because of its low tau/HMW MAP ratio.
Mechanism of cytochrome P450 reductase from the house fly: evidence for an FMN semiquinone as electron donor
Authors: Murataliev Marat B , Feyereisen René
Published: July 2002

The interaction of recombinant house fly (Musca domestica) P450 reductase with NADPH and the role of the FMN semiquinone in reducing cytochrome c have been investigated. House fly P450 reductase can rapidly oxidize only one molecule of NADPH, whereas the rate of oxidation of a second molecule of NADPH is too slow to account for the observed rates of catalysis. This demonstrates that house fly P450 reductase does not require a priming reaction with NADPH for catalysis. Kinetics of cytochrome c reduction and EPR spectroscopy revealed that the enzyme forms two types of neutral FMN semiquinone. One serves as the catalytic intermediate of cytochrome c reduction, and another one is an ‘air‐stable’ semiquinone, which reduces cytochrome c 3000 times more slowly. The results show that the reduction state of the house fly P450 reductase during catalysis cycles in a 0‐2‐1‐0 sequence.
A novel substrate for analyzing Alzheimer's disease γ‐secretase
Authors: Lichtenthaler Stefan F. , Multhaup Gerd , Masters Colin L. , Beyreuther Konrad
Published: July 2002

Proteolytic processing of Alzheimer's disease amyloid precursor protein (APP) by β‐secretase leads to A4CT (C99), which is further cleaved by the as yet unknown protease called γ‐secretase. To study the enzymatic properties of γ‐secretase independently of β‐secretase, A4CT together with an N‐terminal signal peptide (SPA4CT) may be expressed in eukaryotic cells. However, in all existing SPA4CT proteins the signal peptide is not correctly cleaved upon membrane insertion. Here, we report the generation of a mutated SPA4CT protein that is correctly cleaved by signal peptidase and, thus, identical to the APP‐derived A4CT. This novel SPA4CT protein is processed by γ‐secretase in the same manner as APP‐derived A4CT and might be valuable for the generation of transgenic animals showing amyloid pathology.
High molecular weight hyaluronic acid inhibits advanced glycation endproduct‐induced NF‐κB activation and cytokine expression
Authors: Neumann Arne , Schinzel Reinhard , Palm Dieter , Riederer Peter , Münch Gerald
Published: July 2002

Advanced glycation endproducts (AGEs), which accumulate on long‐lived proteins and protein deposits (amyloids), induce the expression of proinflammatory cytokines through NF‐κB‐dependent pathways. Hyaluronic acid with a molecular weight above 1.2 MDa (HMW‐HA) inhibits the AGE‐induced activation of the transcription factor NF‐κB and the NF‐κB‐regulated cytokines interleukin‐1α, interleukin‐6 and tumor necrosis factor‐α. Since the molecular weight of hyaluronic acid in humans decreases with age and under conditions of oxidative stress, it is likely that the protective effect of HMW‐HA against AGE‐induced cellular activation is lost at sites of chronic inflammation and in older age.
Multiple TGF‐β receptor related genes in sponge and ancient gene duplications before the parazoan–eumetazoan split1
Authors: Suga Hiroshi , Ono Kanako , Miyata Takashi
Published: July 2002

Members of the transforming growth factor β (TGF‐β) family mediate key events in cell growth and development. Various receptors for diverse members of the TGF‐β family have recently been isolated and sequenced. These receptors form a family (TβR family) with a Ser/Thr kinase domain in common. To understand the divergence pattern of the TβR family during animal evolution, we have conducted cloning of cDNAs encoding the TβR family members from Ephydatia fluviatilis, a freshwater sponge. We obtained seven cDNAs (sALK‐1–sALK‐7) which are closely related in structure to known family members. Including these sponge sequences, a phylogenetic tree of the family members was inferred by a maximum likelihood method. The phylogenetic tree suggests that the sponge receptors sALK‐1–sALK‐3, which are closely related to each other, are sponge homologs of vertebrate activin type I receptor (ActR‐I). sALK‐5 is likely to be a homolog of TGF‐β type II receptor. sALK‐4 and sALK‐6 might be ancestral precursors of type I and type II receptors, respectively, and sALK‐7 is possibly an ancestral precursor of both types. The tree revealed that most, if not all, of the gene duplications that gave rise to known subtypes with distinct ligand specificities antedate the divergence of parazoans and eumetazoans, the earliest divergence of extant animal phyla.
Efficient expression, purification and crystallisation of two hyperthermostable enzymes of histidine biosynthesis
Authors: Thoma Ralf , Obmolova Galina , Lang Dietmar A. , Schwander Martin , Jenö Paul , Sterner Reinhard , Wilmanns Matthias
Published: July 2002

Enzymes from hyperthermophiles can be efficiently purified after expression in mesophilic hosts and are well‐suited for crystallisation attempts. Two enzymes of histidine biosynthesis fromThermotoga maritima,N′‐((5′‐phosphoribosyl)‐formimino)‐5‐aminoimidazol‐4‐carboxamid ribonucleotide isomerase and the cyclase moiety of imidazoleglycerol phosphate synthase, were overexpressed inEscherichia coli, both in their native and seleno‐methionine‐labelled forms, purified by heat precipitation of host proteins and crystallised.N′‐((5′‐phosphoribosyl)‐formimino)‐5‐aminoimidazol‐4‐carboxamid ribonucleotide isomerase crystallised in four different forms, all suitable for X‐ray structure solution, and the cyclase moiety of imidazoleglycerol phosphate synthase yielded one crystal form that diffracted to atomic resolution. The obtained crystals will enable the determination of the first three‐dimensional structures of enzymes from the histidine biosynthetic pathway.
Evolutionary aspects of inorganic pyrophosphatase
Authors: Sivula Toni , Salminen Anu , Parfenyev Alexey N. , Pohjanjoki Pekka , Goldman Adrian , Cooperman Barry S. , Baykov Alexander A. , Lahti Reijo
Published: July 2002

Based on the primary structure, soluble inorganic pyrophosphatases can be divided into two families which exhibit no sequence similarity to each other. Family I, comprising most of the known pyrophosphatase sequences, can be further divided into prokaryotic, plant and animal/fungal pyrophosphatases. Interestingly, plant pyrophosphatases bear a closer similarity to prokaryotic than to animal/fungal pyrophosphatases. Only 17 residues are conserved in all 37 pyrophosphatases of family I and remarkably, 15 of these residues are located at the active site. Subunit interface residues are conserved in animal/fungal but not in prokaryotic pyrophosphatases.
Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme
Authors: Emanuelli Monica , Carnevali Francesco , Lorenzi Maria , Raffaelli Nadia , Amici Adolfo , Ruggieri Silverio , Magni Giulio
Published: July 2002

The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD. A new purification procedure for NMN adenylyltransferase from Saccharomyces cerevisiae provided sufficient amounts of enzyme for tryptic fragmentation. Through data‐base search a full matching was found between the sequence of tryptic fragments and the sequence of a hypothetical protein encoded by the S. cerevisiae YLR328W open reading frame (GenBank accession number U20618). The YLR328W gene was isolated, cloned into a T7‐based vector and successfully expressed in Escherichia coli BL21 cells, yielding a high level of NMN adenylyltransferase activity. The purification of recombinant protein, by a two‐step chromatographic procedure, resulted in a single polypeptide of 48 kDa under SDS‐PAGE, in agreement with the molecular mass of the hypothetical protein encoded by YLR328W ORF. The N‐terminal sequence of the purified recombinant NMN adenylyltransferase exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant NMN adenylyltransferase are reported and compared with those already known for the enzyme obtained from different sources.
Orientational behavior of phospholipid membranes with mastoparan studied by 31P solid state NMR
Authors: Hori Yumiko , Demura Makoto , Niidome Takuro , Aoyagi Haruhiko , Asakura Tetsuo
Published: July 2002

Solid state 31P NMR spectroscopy was used to study the perturbing effect of the wasp venom peptide mastoparan (MP) on lipid bilayers composed of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG). The 31P chemical shift anisotropy of multilamellar vesicles decreased with increasing peptide concentration, indicating that MP interacts strongly and selectively with the charged DMPG head group. Macroscopically oriented MP‐lipid samples between glass plates were studied by 31P NMR as a function of tilt angle. These spectra showed the coexistence of orientation‐dependent lamellar signals as well as an isotropic peak, suggesting that MP can induce non‐lamellar phases in DMPC/DMPG membranes.
Ubiquitous and cell‐specific members of the avian small heat shock protein family
Authors: Kawazoe Yoshinori , Tanabe Masako , Nakai Akira
Published: July 2002

Small heat shock proteins (sHsps) have been suggested to act as molecular chaperones for many kinds of substrates and have protective roles in cells exposed to external stresses. Unlike other major Hsps such as Hsp70 and Hsp90, expression of many vertebrate sHsps is restricted to the muscle tissues and/or eye lens. Among the sHsps, the heat‐inducible human Hsp27 (hHsp27) homologue is believed to be expressed ubiquitously in various cell types. Here, we distinguished the chicken homologue of hHsp27 (cHsp24) from the chicken major heat‐inducible protein of molecular size 25 kDa (cHsp25). cHsp25 is not expressed in the absence of stress, but is highly expressed after hyperthermia in all tissues of developing embryos. In contrast, expression of cHsp24 is restricted to some specific tissues even in the presence of stress. Thus, cHsp25 is the first member of the sHsps in vertebrates the expression of which is ubiquitous in tissues exposed to external stresses similar to Hsp70.
A missense mutation in canine ClC‐1 causes recessive myotonia congenita in the dog1
Authors: Rhodes Thomas H. , Vite Charles H. , Giger Urs , Patterson Donald F. , Fahlke Christoph , George Alfred L.
Published: July 2002

Myotonia congenita is an inherited disorder of sarcolemmal excitation leading to delayed relaxation of skeletal muscle following contractions. Mutations in a skeletal muscle voltage‐dependent chloride channel, ClC‐1, have been identified as the molecular genetic basis for the syndrome in humans, and in two well characterized animal models of the disease: the myotonic goat, and the arrested development of righting (adr) mouse. We now report the molecular genetic and electrophysiological characterization of a canine ClC‐1 mutation that causes autosomal recessive myotonia congenita in miniature Schnauzers. The mutation results in replacement of a threonine residue in the D5 transmembrane segment with methionine. Functional characterization of the mutation introduced into a recombinant ClC‐1 and heterologously expressed in a cultured mammalian cell line demonstrates a profound effect on the voltage‐dependence of activation such that mutant channels have a greatly reduced open probability at voltages near the resting membrane potential of skeletal muscle. The degree of this dysfunction is greatly diminished when heterodimeric channels containing a wild‐type and mutant subunit are expressed together as a covalent concatemer strongly supporting the observed recessive inheritance in affected dog pedigrees. Genetic and electrophysiological characterization of the myotonic dog provides a new and potentially valuable animal model of an inherited skeletal muscle disease that has advantages over existing models of myotonia congenita.
MAP kinase activation by mu opioid receptor involves phosphatidylinositol 3‐kinase but not the cAMP/PKA pathway
Authors: Ai Wandong , Gong Jianhua , Yu Lei
Published: July 2002

The involvement of protein kinases was studied in mu opioid receptor activation of mitogen‐activated protein (MAP) kinase using cells transfected with the receptor clone. The cAMP/protein kinase A (PKA) pathway is known to be the major biochemical pathway for mu opioid receptor signaling. However, our data showed that stimulating adenylyl cyclase or activating PKA had no effect on mu receptor enhancement of MAP kinase activity, suggesting that the cAMP/PKA pathway is not involved in mediating the mu receptor activation of MAP kinase. Inhibition of phosphatidylinositol (PI) 3‐kinase reduced mu receptor enhancement of MAP kinase activity, suggesting PI 3‐kinase involvement. Together, these results show that cross‐talk between the mu opioid receptor and the MAP kinase cascade is not mediated by the cAMP/PKA pathway, but involves PI 3‐kinase.
Low iron concentration and aconitase deficiency in a yeast frataxin homologue deficient strain
Authors: Foury Françoise
Published: July 2002

Deletion of the yeast frataxin homologue, YFH1, elicits accumulation of iron in mitochondria and mitochondrial defects. We report here that in the presence of an iron chelator in the culture medium, the concentration of iron in mitochondria is the same in wild‐type and YFH1 deletant strains. Under these conditions, the activity of the respiratory complexes is restored. However, the activity of the mitochondrial aconitase, a 4Fe‐4S cluster‐containing protein, remains low. The frataxin family bears homology to a bacterial protein family which confers resistance to tellurium, a metal closely related to sulfur. Yfh1p might control the synthesis of iron‐sulfur clusters in mitochondria.
Phosphorylation of phenylalanine ammonia‐lyase: evidence for a novel protein kinase and identification of the phosphorylated residue
Authors: Allwood Ellen G. , Davies Dewi R. , Gerrish Chris , Ellis Brian E. , Bolwell G.Paul
Published: July 2002

The site of phosphorylation of phenylalanine ammonia‐lyase (PAL) has been identified as a threonine residue. A Ca2+‐stimulated protein kinase of approximately 55 kDa has been partially purified from elicited cells. The kinase can phosphorylate a synthetic peptide derived from PAL and a recombinant poplar PAL. PAL phosphorylation was associated with a decrease inVmaxin agreement with the suggestion that protein phosphorylation is involved in marking PAL subunits for turnover. The phosphorylation site in French bean PAL is most likely Thr545in the sequence VAKRTLT (539–546). Conservation of the phosphorylation site in PAL from diverse species suggests that phosphorylation of PAL may be a ubiquitous regulatory mechanism in higher plants.
Circularly permuted variants of the green fluorescent protein
Authors: Topell Simon , Hennecke Jens , Glockshuber Rudi
Published: July 2002

Folding of the green fluorescent protein (GFP) from Aequorea victoria is characterized by autocatalytic formation of its p‐hydroxybenzylideneimidazolidone chromophore, which is located in the center of an 11‐stranded β‐barrel. We have analyzed the in vivo folding of 20 circularly permuted variants of GFP and find a relatively low tolerance towards disruption of the polypeptide chain by introduction of new termini. All permuted variants with termini in strands of the β‐barrel and about half of the variants with termini in loops lost the ability to form the chromophore. The thermal stability of the permuted GFPs with intact chromophore is very similar to that of the wild‐type, indicating that chromophore‐side chain interactions strongly contribute to the extraordinary stability of GFP.
Lysophosphatidylcholine phosphorylates CREB and activates the jun2TRE site of c‐jun promoter in vascular endothelial cells
Authors: Ueno Yasushi , Kume Noriaki , Miyamoto Susumu , Morimoto Masahumi , Kataoka Hiroharu , Ochi Hiroshi , Nishi Eiichiro , Moriwaki Hideaki , Minami Manabu , Hashimoto Nobuo , Kita Toru
Published: July 2002

Lysophosphatidylcholine (lyso‐PC), a polar phospholipid increased in atherogenic lipoproteins and atherosclerotic lesions, has been shown to induce transcription of a variety of endothelial genes relevant to atherogenesis. Lyso‐PC has been shown to activate c‐jun N‐terminal kinase (JNK) and activator protein 1 (AP‐1) and thereby stimulate transcription of the c‐jun gene. Here we provide evidence that lyso‐PC can phosphorylate cyclic AMP responsive element binding protein (CREB) and thereby activate the jun2 12‐O‐tetradecanoylphorbol 13‐acetate response element (jun2TRE) site of the c‐jun promoter, which appears to be the major molecular mechanism involved in lyso‐PC‐induced c‐jun gene expression in cultured bovine aortic endothelial cells (BAEC). Transient transfection of BAEC with a 1.6‐kbp c‐jun promoter and luciferase reporter fusion gene resulted in a 12.9‐fold increase in luciferase activity by lyso‐PC treatment. Serial deletion mutation in c‐jun promoter and luciferase reporter gene assay revealed that the 5′ promoter region between nucleotide numbers −268 and −127, which contains a jun2TRE binding sequence, was most crucial for lyso‐PC‐induced transcription. The 5′ promoter region between −76 and −27, which contains an AP‐1 site, also affected lyso‐PC‐induced transcription of the c‐jun gene. Point mutation in the jun2TRE site reduced lyso‐PC‐induced transcription of the c‐jun promoter‐luciferase fusion gene by a 70.3% decrease in c‐jun promoter activity. Electrophoretic mobility shift assays showed increased binding of 32P‐labeled oligonucleotides with jun2TRE in nuclear extracts isolated from lyso‐PC‐treated BAEC, which was abolished or supershifted by anti‐CREB antibody. Immunoblotting with anti‐phosphorylated CREB antibody showed rapid phosphorylation of this protein after lyso‐PC treatment. These results indicate that lyso‐PC phosphorylates CREB, which was then bound to the jun2TRE site of the c‐jun promoter and activated transcription. Activation of jun2TRE may play a key role in the transcriptional activation of c‐jun as well as other endothelial genes depending upon these transcription factors.
Human membrane type‐4 matrix metalloproteinase (MT4‐MMP) is encoded by a novel major transcript: isolation of complementary DNA clones for human and mouse mt4‐mmp transcripts
Authors: Kajita Masahiro , Kinoh Hiroaki , Ito Noriko , Takamura Akiko , Itoh Yoshifumi , Okada Akiko , Sato Hiroshi , Seiki Motoharu
Published: July 2002

Five distinct membrane‐type matrix metalloproteinases (MT‐MMP) have been reported by cDNA cloning. However, the mt4‐mmp gene product (MMP‐17) has not been identified yet in spite of the cDNA isolation [Puente et al. (1996), Cancer Res. 56, 944–949]. In this study, we re‐examined the transcripts for human mt4‐mmp by 5′ RACE and identified two types of transcripts. The minor one corresponded to the cDNA reported by Puente et al. and failed to express protein, and the other is the major transcript that has an extended open reading frame and expressed 67 and 71 kDa translation products. Thus, functional mt4‐mmp has been identified for the first time.
Microcystin affinity purification of plant protein phosphatases: PP1C, PP5 and a regulatory A‐subunit of PP2A
Authors: Meek Sarah , Morrice Nick , MacKintosh Carol
Published: July 2002

Proteins of ∼35, 55 and 65 kDa were purified from cauliflower extracts by microcystin‐Sepharose chromatography and identified by amino acid sequencing as plant forms of protein (serine/threonine) phosphatase 1 (PP1) catalytic subunit, PP5 and a regulatory A‐subunit of PP2A, respectively. Peptides that corresponded both to the tetratricopeptide (TPR) repeat and catalytic domains of PP5 were identified. Similar to mammalian PP5, the casein phosphatase activity of plant PP5 was activated >10‐fold by arachidonic acid, with half‐maximal stimulation occurring at ∼100 μM lipid.
Topological analysis of an RND family transporter, MexD of Pseudomonas aeruginosa
Authors: Gotoh Naomasa , Kusumi Toshiyuki , Tsujimoto Hideto , Wada Takaomi , Nishino Takeshi
Published: July 2002

The membrane topology of a resistance‐nodulation‐division (RND) family transporter, MexD of Pseudomonas aeruginosa, was determined. Although it had been predicted previously that most RND proteins contain 12 transmembrane helices, three independent computer programs used in the present study predicted that MexD possessed 11, 14 or 17 transmembrane segments. To investigate the topology of MexD more thoroughly, 25 MexD‐PhoA (alkaline phosphatase) and 18 MexD‐Bla (β‐lactamase) fusion plasmids were constructed and analyzed. The resulting topological model had just 12 transmembrane helices and two periplasmic loops of about 300 residues between helices 1 and 2 and helices 7 and 8. It is therefore proposed that the N‐ and C‐termini are located in the cytoplasm and the predicted orientation is consistent with the ‘positive‐inside rule’. This topological model can be applied to other RND proteins.
Tissue‐specific alternative splicing of rat brain fructose 6‐phosphate 2‐kinase/fructose 2,6‐bisphosphatase
Authors: Watanabe Fusao , Furuya Eisuke
Published: July 2002

We have reported the occurrence of eight splice variants of rat brain fructose 6‐phosphate 2‐kinase/fructose 2,6‐bisphosphatase (RB2K). In the present study, we quantified these splice variants in various tissues using a RNAse protection assay and found a tissue‐specific pattern of alternative splicing of the RB2K gene. Splice variants containing exon F were specifically expressed in brain. Moreover, exons D and E were spliced in brain, skeletal muscle and heart. Consequently, eight, six, four and two splice variants were expressed in brain, skeletal muscle, heart and liver plus testis, respectively. These results suggest that distinct RB2K isoforms could be involved in regulation of glycolysis in a tissue‐specific manner.
Induction and peroxisomal appearance of gulonolactone oxidase upon clofibrate treatment in mouse liver
Authors: Braun László , Mile Valéria , Schaff Zsuzsa , Csala Miklós , Kardon Tamás , Mandl József , Bánhegyi Gábor
Published: July 2002

Various antihyperlipemic peroxisome proliferators are known to be carcinogenic in rodents but not in human, other primates and guinea pig, which species lost their ability to synthesize ascorbate due to mutations in the gulonolactone oxidase gene. Ascorbate synthesis is accompanied by H2O2 production, consequently its induction can be potentially harmful; therefore, the in vivo effect of the peroxisome proliferator clofibrate was investigated on gulonolactone oxidase expression in mouse liver. Liver weights and peroxisomal protein contents were increased upon clofibrate treatment. Elevated plasma ascorbate concentrations were found in clofibrate‐treated mice due to the higher microsomal gulonolactone oxidase activities. Remarkable gulonolactone oxidase activity appeared in the peroxisomal fraction upon the treatment. Increased activity of the enzyme was associated with an elevation of its mRNA level. According to the present results the evolutionary loss of gulonolactone oxidase may contribute to the explanation of the missing carcinogenic effect of peroxisome proliferators in humans.
Co‐operativity between modules within a C3b‐binding site of complement receptor type 1
Authors: Kirkitadze M.D. , Dryden D.T.F. , Kelly Sharon M. , Price Nicholas C. , Wang X. , Krych M. , Atkinson J.P. , Barlow P.N.
Published: July 2002

Complement receptor type 1 (CR1) has 30 modules in its extracellular portion. An understanding of structure‐function relationships within CR1 is being assembled gradually from studies of overlapping protein fragments. A CR1 fragment corresponding to modules 16 and 17 was expressed recombinantly as a non‐glycosylated protein and its stability and unfolding characteristics studied using biophysical techniques. The results were compared with data collected previously on a CR1 fragment encompassing modules 15, 16 and 17 which together constitute a C3b‐binding site (Kirkitadze, M.D., Krych, M., Uhrin, D., Dryden, D.T.F., Smith, B.O., Wang, X., Hauhart, R., Atkinson, J.P. and Barlow, P.N. (1999) Biochemistry 38, 7019–7031). Modules within CR1 were found to co‐operate during unfolding. The folding, stability and flexibility of this protein is therefore likely to be a complex function, and not just the sum, of contributions from individual modules.
A kinetic study of the oxidation effects of amphotericin B on human low‐density lipoproteins
Authors: Barwicz Joanna , Gruda Ilona , Tancrède Pierre
Published: July 2002

The UV‐visible results of this kinetic study show that amphothericin B as Fungizone is a much stronger oxidant than CuSO4, itself a powerful oxidant of low‐density lipoprotein (LDL). Amphotericin B as AmBisome alone has no oxidizing effect on LDL while a mixture of both AmBisome and CuSO4 induces an important potentialization of the LDL oxidation. These results allow us to believe that the high toxicity of amphotericin B is related to its capacity to modify and to weaken the structure of LDL. In addition, differential scanning calorimetry experiments show that the oxidative modifications of LDL by CuSO4 or by amphotericin B proceed through different mechanisms.
Safe motherhood in Sri Lanka: a 100‐year march
Authors: Seneviratne H.R. , Rajapaksa L.C.
Published: July 2002

The present safe motherhood status in Sri Lanka has been achieved by a series of activities for health and social development during the past century. While ancient and traditional religious, as well as cultural concepts provided a sound foundation, it was the policies that focused on human development that resulted in reducing the maternal mortality rate to 2.4 per 10 000 live births, and the infant mortality rate to 16.5 per (1000) live births. Maternal and Child Health (MCH) services were initially closely linked to improvements in the healthcare infrastructure and social development, while family planning introduced in 1953 enhanced its progress further. With MCH integrated into reproductive health, the march towards a safer motherhood in Sri Lanka will require the implementation of new policies and strategies.
Vaginal candidosis: epidemiological and etiological factors
Authors: Ferrer J
Published: July 2002

A review is presented of the emerging problem of candidal colonization regarding epidemiological and etiological factors. In recent years a change in epidemiological trends has been observed. Vaginal candidosis seems to show a higher frequency to recur and a significant increase in infections caused by non albicans species of candidas has been stated. The three stage mechanisms of adhesion, blastopore germination and epithelium invasion are emphasized. There is a balance between candidal organisms and vaginal defense factors (lactobacilli, cellular and humoral immunity) controlling and limiting fungal growth. Vaginitis appears because of an increased number or an enhanced virulence of candidas. In some other patients, a decreased vaginal defense mechanism is a determinant factor. There are still a number of factors involved in clinical candidal vulvovaginitis that need to be clarified.
The American College of Obstetricians and Gynecologists: a decade of responding to violence against women
Authors: Jones R.F. , Horan D.L.
Published: July 2002

Violence against women is a common phenomenon worldwide. Effects can be severe and life‐long. As physicians who treat women exclusively, the obstetrician‐gynecologist has a medical and ethical obligation to recognize and intervene on behalf of their abused patients. At the same time, because of the special nature of the patient‐physician relationship, the obstetrician‐gynecologist is in a unique position to provide such assistance. This article presents an overview of violence against women and its consequences, highlights responses to the problem, and details the activities of the American College of Obstetricians and Gynecologists to educate its members on domestic violence. ACOG activities may serve as a model of response to domestic violence for similar organizations.
Commercial availability of misoprostol and induced abortion in Brazil
Authors: Costa S.H.
Published: July 2002

In Brazil, abortion is only permitted to save the woman's life or in cases of rape. The principal effect of legal restrictions is not to make induced abortion practice less prevalent but to force poor women to resort to abortions performed under unhygience conditions or attempt self‐induced abortion. Within this context, misoprostol, a synthetic analogue of prostaglandin E1, was introduced in the country in 1986. Purchased over the counter in pharmacies, misoprostol has became a popular abortifacient method among Brazilian women. By 1990, about 70% of women hospitalized with abortion‐related diagnoses reported use of the drug. In 1991, the Ministry of Health restricted the sale of misoprostol, and in some states its use was totally banned. While the proportion of abortions induced with misoprostol has decreased, the drug continues to be sold on the black market at an inflated value. Research indicates that women have acquired more experience with the drug over time, resulting in lower doses and more effective administration. Several studies show that the rate and severity of complications are significantly less among women who used misoprostol compared with women who used invasive methods. Research also suggests that about half of the women have complete abortion with misoprostol, but seek medical care as soon as they have vaginal bleeding. The experience of Brazilian women with misoprostol is an example of how women when faced with unwanted pregnancy will resort to illegal abortion whatever the costs are to their health.
Tetanus toxoid and congenital abnormalities
Authors: Czeizel A.E , Rockenbauer M
Published: July 2002

Objective: To study the human teratogenic potential of tetanus vaccination during pregnancy. Methods: Pair analysis of cases with congenital abnormalities and matched healthy controls was performed in the large population‐based dataset of the Hungarian Case–Control Surveillance of Congenital Abnormalities, 1980–1994. Results: Of 35 727 pregnant women who had babies without any defects in the study period (control group), 33 (0.09%) were vaccinated with tetanus. Of 21 563 pregnant women who had offspring with congenital abnormalities, 25 (0.12%) had tetanus vaccination. This difference was not significant (P=0.39). The case–control pair analysis confirmed the safety of tetanus vaccination during pregnancy, particularly in the second and third months of gestation, i.e. during the critical period for congenital abnormalities. Conclusion: Tetanus vaccination during pregnancy appears not be teratogenic to the fetus. Thus, there is no contraindication, if the use of tetanus toxoid is necessary during pregnancy.
On the symmetry of blowup solutions to a mean field equation
Authors: Chen Chuin Chuan , Lin Chang-Shou
Published: July 2002

In this article, we consider the mean field equation

\Delta u + \rho \left(\frac{e^{u}}{\int e^{u}}−\frac{1}{A}\right) = 0 \text{ in }Σ,

where

Σ*

is a flat torus and

A

is the area of

Σ

. This paper is concerned with the symmetry induced by the phenomenon of concentration. By using the method of moving planes, we prove that blowup solutions often possess certain symmetry. In this paper, we consider cases when solutions blowup at one or two points. We also consider related problems for annulu domains of

\mathbf R^2

.

Nous considérons l’équation de champ moyen

\Delta u + \rho \left(\frac{e^{u}}{\int e^{u}}−\frac{1}{A}\right) = 0 \text{ dans }Σ,

où

Σ

est un tore plat et

A

est l’aire de

Σ

. Cet article se rapporte à la symétrie induite par le phénomène de concentration. En utilisant la méthode de déplacement de plans, nous démontrons que les solutions avec singularités possèdent dans la plupart des cas des propriétés de symétrie. Dans cet article, nous considérons des cas où les solutions explosent en un ou deux points. Nous traitons également de problèmes reliés pour des domaines annulaires de

\mathbf R^2

avec conditions de Dirichlet an berd.
Asymptotic behaviour of holomorphic strips
Authors: Robbin Joel W , Salamon Dietmar A
Published: July 2002

The asymptotic behaviour of a finite energy pseudoholomorphic strip with Lagrangian boundary conditions in a symplectic manifold is determined by an eigenfunction of the linearized operator at the (transverse) intersection.

Le comportement asymptotique d’une bande pseudoholomorphe d’énergie finie, à frontière dans une sous-variété Lagrangienne d’une variété symplectique, est déterminé par une fonction propre du problème linéarisé le long de l’intersection (transverse).