bluuebunny/crossref_metadata_2025 huggingface dataset:
A splice variant acquiring an extra transcript leader region decreases the translation of glutamine synthetase gene
Authors: SHIN Daesung , PARK Sangjin , PARK Chankyu
Published: August 2003

The expression of glutamine synthetase (GS), catalysing the ATP-dependent conversion of glutamate and ammonia into glutamine, is transcriptionally and post-transcriptionally regulated. The genomic structure of dog GS shown in the present study is basically similar to that of other mammals in that it is composed of seven exons and six introns. Using 5′-cRACE (where cRACE stands for circular rapid amplification of cDNA ends) and reverse transcriptase–PCR, we identified an additional exon (120 bp) in the first intron, designated in the present study as exon 1′. By means of alternative splicing, the GS gene produces an altered form of GS transcript with 5′-untranslated region (UTR) containing the exon 1′. This alternative transcript is abundantly expressed in brain, whereas it is found at lower levels in other tissues. In the human and mouse GS genes, extra exons are also found at the corresponding site of the intron 1 but with different sizes. An exon-trapping experiment for the GS gene in COS-7, Madin–Darby canine kidney and SK-N-SH cells revealed that the pattern of alternative splicing is variable in different cell types. The propensity of forming a secondary structure is predicted to be considerably higher in the presence of extra 5′-UTR, suggesting the possibility of a translational effect. To test this, we performed a reporter assay for fusions with different 5′-UTRs, demonstrating that the long form with extra 5′-UTR was translated 20- and 10-fold less than the short one in SK-N-SH and Neuro-2A cells respectively. Similarly, translations of human and mouse transcripts with extra 5′-UTRs were less efficient, showing 6–8-fold reductions in SK-N-SH cells. Furthermore, when we mutated an ATG sequence contained in the exon 1′, the suppression of translation was partially relieved, suggesting that the negative regulation by an extra 5′-UTR is, to some extent, due to an abortive translation from the upstream ATG.
Glycosylation of resveratrol protects it from enzymic oxidation
Authors: REGEV-SHOSHANI Gilly , SHOSEYOV Oded , BILKIS Itzhak , KEREM Zohar
Published: August 2003

Plant polyphenols, including dietary polyphenols such as resveratrol, are important components in the plant antioxidant and defence systems. They are also known to exert beneficial effects on human health through diet. As they are produced, these polyphenols may be subjected to deleterious enzymic oxidation by the plant polyphenol oxidases. They are generally synthesized as glycosides like 5,4′-dihydroxystilbene-3-O-β-d-glucopyranoside, the 3-glucoside of resveratrol. The effects of the glycosylation and methylation of the parent resveratrol on its enzymic oxidation were studied. Methyl and glucosyl derivatives were synthesized using simple one-step methodologies. The kinetics of their enzymic oxidation by tyrosinases were defined. Substitution at the p-hydroxy group, by either glucose or methyl, abolished enzymic oxidation by both mushroom and grape tyrosinases. Substitution at the m-hydroxy group with methyl had a small effect, but substitution with glucose resulted in a much lower affinity of the enzymes for the glycoside. We suggest that glycosylation of polyphenols in nature helps to protect these vital molecules from enzymic oxidation, extending their half-life in the cell and maintaining their beneficial antioxidant capacity and biological properties.
Decreased mitochondrial tRNALys steady-state levels and aminoacylation are associated with the pathogenic G8313A mitochondrial DNA mutation
Authors: BACMAN Sandra R. , ATENCIO David P. , MORAES Carlos T.
Published: August 2003

Mutations in human mitochondrial tRNA genes cause a number of multisystemic disorders. A G-to-A transition at position 8313 (G8313A) transition in the mitochondrial tRNALys gene has been associated with a childhood syndrome characterized by gastrointestinal-system involvement and encephaloneuropathy. We have used transmitochondrial cybrid clones harbouring patient-derived mitochondrial DNA with the G8313A mutation for the study of the molecular pathogenesis. Our results showed that mutant mitochondrial cybrids respired poorly, and had severely defective mitochondrial protein synthesis and respiratory-chain-enzyme activity. Mutant cybrids also showed a marked decrease in tRNALys steady-state levels and aminoacylation, suggesting that these molecular abnormalities may underlie the pathogenesis of the mitochondrial G8313A mutation.
A novel serine protease predominately expressed in macrophages
Authors: CHEN Cailin , DARROW Andrew L. , QI Jian-shen , D'ANDREA Michael R. , ANDRADE-GORDON Patricia
Published: August 2003

We have identified a novel serine protease designated EOS by sequence identity searches. The deduced protein contains 284 amino gcids with an active form containing 248 amino acids starting from an Ile-Val-Gly-Gly motif. The active form comprises a catalytic triad of conserved amino acids: His77, Asp126 and Ser231. It shares 44% identity with β-tryptase and belongs to the S1 trypsin-like serine-protease family. Interestingly, this gene also maps to human chromosome 16p13.3. The purified protease showed amidolytic activity, cleaving its substrates before arginine residues. Tissue distribution by immunohistochemistry analysis demonstrated that EOS is highly expressed in spleen and moderately expressed in intestine, colon, lung and brain. We confirmed this expression pattern at the mRNA level by performing in situ hybridization. The results from both immunohistochemistry and in situ hybridization indicate that EOS is associated with macrophages. We corroborated this observation by double immunofluorescence using the anti-EOS antibody and an anti-CD68 antibody, a macrophage specific marker. Furthermore, we have detected a dramatic increase in immune staining of EOS in cultured U937 cells treated with PMA, which represent activated macrophages. This up-regulation is also reflected by elevated EOS mRNA in the PMA-treated U937 cells detected by Northern blotting. Since macrophages have important roles in various pathological conditions, such as wound healing, atherosclerosis and numerous inflammatory diseases, the localization of this novel serine protease to active macrophages may help to further the elucidation of the roles of this gene product in modulating these disorders.
Characterization of human torsinA and its dystonia-associated mutant form
Authors: LIU Zhonghua , ZOLKIEWSKA Anna , ZOLKIEWSKI Michal
Published: August 2003

Deletion of a single glutamate in torsinA correlates with early-onset dystonia, the most severe form of a neurological disorder characterized by uncontrollable muscle contractions. TorsinA is targeted to the ER (endoplasmic reticulum) in eukaryotic cells. We investigated the processing and membrane association of torsinA and the dystonia-associated Glu-deletion mutant (torsinAΔE). We found that the signal sequence of torsinA (residues 1–20 from the 40 amino-acid long N-terminal hydrophobic region) is cleaved in Drosophila S2 cells, as shown by the N-terminal sequencing after partial protein purification. TorsinA is not secreted from S2 cells. Consistently, sodium carbonate extraction and Triton X-114 treatment showed that torsinA is associated with the ER membrane in CHO (Chinese-hamster ovary) cells. In contrast, a variant of torsinA that contains the native signal sequence without the hydrophobic region Ile24–Pro40 does not associate with the membranes in CHO cells, and a truncated torsinA without the 40 N-terminal amino acids is secreted in the S2 culture. Thus the 20-amino-acid-long hydrophobic segment in torsinA, which remains at the N-terminus after signal-peptide cleavage, is responsible for the membrane anchoring of torsinA. TorsinAΔE showed similar cleavage of the 20 N-terminal amino acids and membrane association properties similar to wild-type torsinA but, unlike the wild-type, torsinAΔE was not secreted in the S2 culture even after deletion of the membrane-anchoring segment. This indicates that the dystonia-associated mutation produces a structurally distinct, possibly misfolded, form of torsinA, which cannot be properly processed in the secretory pathway of eukaryotic cells.
Replacement of a cytosolic copper/zinc superoxide dismutase by a novel cytosolic manganese superoxide dismutase in crustaceans that use copper (haemocyanin) for oxygen transport
Authors: BROUWER Marius , HOEXUM BROUWER Thea , GRATER Walter , BROWN-PETERSON Nancy
Published: August 2003

The blue crab, Callinectes sapidus, which uses the copper-dependent protein haemocyanin for oxygen transport, lacks the ubiquitous cytosolic copper-dependent enzyme copper/zinc superoxide dismutase (Cu,ZnSOD) as evidenced by undetectable levels of Cu,ZnSOD activity, protein and mRNA in the hepatopancreas (the site of haemocyanin synthesis) and gills. Instead, the crab has an unusual cytosolic manganese SOD (cytMnSOD), which is retained in the cytosol, because it lacks a mitochondrial transit peptide. A second familiar MnSOD is present in the mitochondria (mtMnSOD). This unique phenomenon occurs in all Crustacea that use haemocyanin for oxygen transport. Molecular phylogeny analysis suggests the MnSOD gene duplication is as old as the origin of the arthropod phylum. cytMnSOD activity in the hepatopancreas changes during the moulting cycle of the crab. Activity is high in intermoult crabs and non-detectable in postmoult papershell crabs. mtMnSOD is present in all stages of the moulting cycle. Despite the lack of cytCu,ZnSOD, crabs have an extracellular Cu,ZnSOD (ecCu,ZnSOD) that is produced by haemocytes, and is part of a large, approx. 160 kDa, covalently-linked protein complex. ecCu,ZnSOD is absent from the hepatopancreas of intermoult crabs, but appears in this tissue at premoult. However, no ecCu,ZnSOD mRNA can be detected, suggesting that the protein is recruited from the haemolymph. Screening of different taxa of the arthropod phylum for Cu,ZnSOD activity shows that those crustaceans that use haemoglobin for oxygen transport have retained cytCu,ZnSOD. It appears, therefore, that the replacement of cytCu,ZnSOD with cytMnSOD is part of an adaptive response to the dynamic, haemocyanin-linked, fluctuations in copper metabolism that occur during the moulting cycle of the crab.
Characterization of the carbohydrate moieties of the functional unit RvH1-a of Rapana venosa haemocyanin using HPLC/electrospray ionization MS and glycosidase digestion
Authors: DOLASHKA-ANGELOVA Pavlina , BECK Alexander , DOLASHKI Alexandar , BELTRAMINI Mariano , STEVANOVIC Stefan , SALVATO Benedetto , VOELTER Wolfgang
Published: August 2003

The primary structures of two biantennary N-glycans of the glycoprotein Rapana venosa (marine snail) haemocyanin were determined. Two different structural subunits have been found in R. venosa haemocyanin: RvH1 and RvH2. The carbohydrate content of the N-terminal functional unit RvH1-a of RvH1 was studied and compared with the N-terminal functional unit RvH2-a of RvH2. Oligosaccharide fragments were released from the glycoprotein by Smith degradation of a haemocyanin pronase digest and separated on a Superdex 300 column. The glycopeptide fragments, giving a positive reaction for the orcinol/H2SO4 method, were separated by HPLC. In order to determine the linked sugar chains to the hinge glycopeptides isolated from the functional unit RvH1-a, several techniques were applied, including capillary electrophoresis, matrix-assisted laser desorption ionization-MS and electrospray ionization-MS in combination with glycosidase digestion. On the basis of these results and amino acid sequence analysis, we concluded that the functional unit RvH1-a contains 7% oligosaccharides N-glycosidically attached to Asn262 and Asn401, and the following structures were suggested:
Characterization of a cysteine-less human reduced folate carrier: localization of a substrate-binding domain by cysteine-scanning mutagenesis and cysteine accessibility methods
Authors: CAO Wei , MATHERLY Larry H.
Published: August 2003

The human reduced folate carrier (hRFC) mediates the transport of reduced folates and classical anti-folates into mammalian cells. Whereas the functionally important domains in hRFC are poorly characterized, previous studies with anti-folate-resistant cells suggest critical roles for transmembrane domain (TMD) 1 and residues (Gly44, Glu45, Ser46 and Ile48) in or flanking this region. An hRFC mutant devoid of cysteine residues was prepared by deleting the C-terminal 56 amino acids, including four cysteine residues, and mutagenizing the remaining cysteine residues to serine residues. A fully functional cysteine-less hRFC protein was expressed in transport-impaired MtxRIIOuaR2-4 Chinese-hamster ovary cells. To explore the role of residues in or flanking TMD1 in transport, all 24 amino acids from Trp25 to Ile48 of hRFC were mutated individually to cysteine residues, and the mutant hRFCs were transfected into MtxRIIOuaR2-4 cells. All of the 24 cysteine mutants were expressed and, with the exception of R42C (Arg42→Cys), were capable of mediating methotrexate uptake above the low level in MtxRIIOuaR2-4 cells. We found that by treating the transfected cells with the small, water-soluble, thiol-reactive anionic reagent, sodium (2-sulphonatoethyl) methanethiosulphonate, methotrexate transport by several of the cysteine-substituted hRFC mutants was significantly inhibited, including Q40C, G44C, E45C and I48C. Sodium (2-sulphonatoethyl) methanethiosulphonate transport inhibition of the Q40C, G44C and I48C mutants was protected by leucovorin [(6R,S)-5-formyltetrahydrofolate], indicating that these residues lie at or near a substrate-binding site. Using surface-labelling reagents [N-biotinylaminoethyl methanethiosulphonate and 3-(N-maleimidylpropionyl)biocytin, combined with 4-acetamido-4′-maleimidylstilbene-2,2′-disulphonic acid] with cysteine mutants from positions 37–48, the extracellular TMD1 boundary was found to lie between residues 39 and 40, and amino acids 44–46 and 48 were localized to the TMD1 exofacial loop. Collectively, our results imply that amino acids 40, 44, 48 and, possibly, 42 serve important roles in hRFC transport, albeit not as structural components of the putative transmembrane channel for folate substrates.
Pigment-epithelium-derived factor (PEDF) occurs at a physiologically relevant concentration in human blood: purification and characterization
Authors: PETERSEN Steen V. , VALNICKOVA Zuzana , ENGHILD Jan J.
Published: August 2003

Pigment epithelium-derived factor (PEDF) inhibits the formation of blood vessels in the eye by inducing apotosis in actively dividing endothelial cells. The activity of PEDF equals or supersedes that of other anti-angiogenic factors, including angiostatin, endostatin and thrombospondin-1. In addition, PEDF has the potential to promote the survival of neurons and affect their differentiation. Here we show that PEDF is present in plasma at a concentration of approx. 100 nM (5 μg/ml) or twice the level required to inhibit aberrant blood-vessel growth in the eye. Thus the systemic delivery of PEDF has the potential to affect angiogenesis or neurotrophic processes throughout the body, significantly expanding the putative physiological role of the protein. A complete map of all post-translational modifications revealed that authentic plasma PEDF carries an N-terminal pyroglutamate blocking group and an N-linked glycan at position Asn266. The pyroglutamate residue may regulate the activity of PEDF analogously to the manner in which it regulates thyrotropin-releasing hormone.
Hepatocyte growth factor activates endothelial nitric oxide synthase by Ca2+- and phosphoinositide 3-kinase/Akt-dependent phosphorylation in aortic endothelial cells
Authors: MAKONDO Kennedy , KIMURA Kazuhiro , KITAMURA Naoki , KITAMURA Takanori , YAMAJI Daisuke , JUNG Bae Dong , SAITO Masayuki
Published: August 2003

Hepatocyte growth factor (HGF) causes endothelium-dependent vasodilation, but its relation to endothelial nitric oxide synthase (eNOS) activity remains to be elucidated. Treatment of bovine aortic endothelial cells with HGF increased eNOS activity within minutes, accompanied by an increase of activity-related site-specific phosphorylation of eNOS. The phosphorylation was completely abolished by pretreatment of the cells with a phosphoinositide 3-kinase (PI3K) inhibitor (wortmannin) and by transfection of dominant-negative Akt, and the enzyme activity was inhibited by wortmannin. In addition, eNOS activity and phosphorylation were abolished by pretreatment of the cells with an intracellular Ca2+-chelator, bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester) (BAPTA/AM), with a suppression of Akt phosphorylation. These results suggest that HGF stimulates eNOS activity by a PI3K/Akt-dependent phosphorylation in a Ca2+-sensitive manner in vascular endothelial cells.
Correction of an enzyme trafficking defect in hereditary kidney stone disease in vitro
Authors: LUMB Michael J. , BIRDSEY Graeme M. , DANPURE Christopher J.
Published: August 2003

In normal human hepatocytes, the intermediary-metabolic enzyme alanine:glyoxylate aminotransferase (AGT) is located within the peroxisomes. However, in approx. one-third of patients suffering from the hereditary kidney stone disease primary hyperoxaluria type 1, AGT is mistargeted to the mitochondria. AGT mistargeting results from the synergistic interaction between a common P11L (Pro11→Leu) polymorphism and a disease-specific G170R mutation. The polymorphism generates a functionally weak mitochondrial targeting sequence, the efficiency of which is increased by the mutation. The two substitutions together, but not in isolation, inhibit AGT dimerization, highlighting the different structural requirements of the peroxisomal and mitochondrial protein-import machineries. In the present study, we show that treatments known to increase the stability of proteins non-specifically (i.e. lowering the temperature from 37 to 30 °C or by the addition of glycerol) completely normalize the intracellular targeting of mutant AGT expressed in transfected COS cells. On the other hand, treatments known to decrease protein stability (e.g. increasing the temperature from 37 to 42 °C) exacerbate the targeting defect. Neither of the treatments affects the relative efficiencies of the peroxisomal and mitochondrial protein-import pathways intrinsically. Results are discussed in the light of the known structural requirements of the two protein trafficking pathways and the formulation of possible treatment strategies for primary hyperoxaluria type 1.
Evaluation of the symmetric model for myosin-linked regulation: effect of site-directed mutations in the regulatory light chain on scallop myosin
Authors: COLEGRAVE Melanie , PATEL Hitesh , OFFER Gerald , CHANTLER Peter D.
Published: August 2003

Regulatory myosins are controlled through mechanisms intrinsic to their structures and can alternate between activated and inhibited states. However, the structural difference between these two states is unclear. Scallop (Pecten maximus) striated adductor myosin is activated directly by calcium. It has been proposed that the two heads of scallop myosin are symmetrically arranged and interact through their regulatory light chains [Offer and Knight (1996) J. Mol. Biol. 256, 407–416], the interface being strengthened in the inhibited state. By contrast, vertebrate smooth-muscle myosin is activated by phosphorylation. Its structure in the inhibited state has been determined from two-dimensional crystalline arrays [Wendt, Taylor, Trybus and Taylor (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 4361–4366] and is asymmetric, requiring no interaction between regulatory light chains. Using site-directed mutagenesis of the scallop regulatory light chain, we have tested the symmetric model for scallop adductor muscle myosin. Specifically, we have made myosin hybrid molecules from scallop (P. maximus) myosin, in which the normal regulatory light chains have been replaced by expressed light chains containing mutations in three residues proposed to participate in the interaction between regulatory light chains. The mutations were R126A (Arg126→Ala), K130A and E131A; made singly, in pairs or all three together, these mutations were designed to eliminate hydrogen bonding or salt linkages between heads, which are key features of this model. Functional assays to address the competence of these hybrid myosins to bind calcium specifically, to exhibit a calcium-regulated myofibrillar Mg-ATPase and to display calcium-dependent actin sliding were performed. We conclude that the symmetrical model does not describe the inhibited state of scallop regulatory myosin and that an asymmetric structure is a plausible alternative.
Principles of interleukin (IL)-6-type cytokine signalling and its regulation
Authors: HEINRICH Peter C. , BEHRMANN Iris , HAAN Serge , HERMANNS Heike M. , MÜLLER-NEWEN Gerhard , SCHAPER Fred
Published: August 2003

The IL (interleukin)-6-type cytokines IL-6, IL-11, LIF (leukaemia inhibitory factor), OSM (oncostatin M), ciliary neurotrophic factor, cardiotrophin-1 and cardiotrophin-like cytokine are an important family of mediators involved in the regulation of the acute-phase response to injury and infection. Besides their functions in inflammation and the immune response, these cytokines play also a crucial role in haematopoiesis, liver and neuronal regeneration, embryonal development and fertility. Dysregulation of IL-6-type cytokine signalling contributes to the onset and maintenance of several diseases, such as rheumatoid arthritis, inflammatory bowel disease, osteoporosis, multiple sclerosis and various types of cancer (e.g. multiple myeloma and prostate cancer). IL-6-type cytokines exert their action via the signal transducers gp (glycoprotein) 130, LIF receptor and OSM receptor leading to the activation of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) and MAPK (mitogen-activated protein kinase) cascades. This review focuses on recent progress in the understanding of the molecular mechanisms of IL-6-type cytokine signal transduction. Emphasis is put on the termination and modulation of the JAK/STAT signalling pathway mediated by tyrosine phosphatases, the SOCS (suppressor of cytokine signalling) feedback inhibitors and PIAS (protein inhibitor of activated STAT) proteins. Also the cross-talk between the JAK/STAT pathway with other signalling cascades is discussed.
Investigation of the pore-forming mechanism of a cytolytic δ-endotoxin from Bacillus thuringiensis
Authors: PROMDONKOY Boonhiang , ELLAR David J.
Published: August 2003

Cyt2Aa1 is a cytolytic protein produced by Bacillus thuringiensis subsp. kyushuensis. Penetration of the toxin into membranes has been studied to learn more about membrane-insertion mechanisms and transmembrane-pore formation. The haemolysis assay of Cyt2Aa1 showed a steep and sigmoidal dose–response curve, indicating that toxin aggregation or oligomerization is required for pore formation. Studies of the effect of temperature on pore formation and fluorimetric studies of acrylodan-labelled toxin suggest that toxin inserts into the membrane before oligomerizing to form a pore. Low temperature neither inhibited membrane binding nor closed pores that have been formed, but markedly inhibited oligomerization of the toxin molecules. When toxin-treated red blood cells at 4 °C were transferred to a toxin-free solution at 37 °C, no significant increase in haemolysis was observed. This result suggests that membrane-bound toxin could not diffuse laterally and interact with other molecules to form a pore. From these results, we propose that Cyt2Aa1 binds and inserts into the membrane as a monomer. Oligomerization occurs when toxin molecules have bound in close proximity to each other and pores are formed from large oligomers.
Cloning of an emopamil-binding protein (EBP)-like protein that lacks sterol Δ8-Δ7 isomerase activity
Authors: MOEBIUS Fabian F. , FITZKY Barbara U. , WIETZORREK Georg , HAIDEKKER Alexander , EDER Andrea , GLOSSMANN Hartmut
Published: August 2003

EBP (emopamil-binding protein) is a high-affinity binding protein for [3H]emopamil and belongs to the family of so-called sigma receptors. Mutations that disrupt EBP's 3β-hydroxysteroid sterol Δ8-Δ7 isomerase activity (EC 5.3.3.5) impair cholesterol biosynthesis and cause X-chromosomal dominant chondrodysplasia punctata. We identified a human cDNA for a novel EBPL (EBP-like protein) with a calculated mass of 23.2 kDa. Amino acid sequence alignments and phylogenetic analysis revealed that EBPL is distantly related to EBP (31% identity and 52% similarity) and found in animals but not in plants. EBPL is encoded by four exons on human chromosome 13q14.2 covering 30.7 kb, and a partially processed EBPL pseudogene was found on 16q21. The EBPL mRNA was expressed ubiquitously and most abundant in liver, lung and kidney. Upon heterologous expression in yeast EBPL had no detectable 3β-hydroxysteroid sterol Δ8-Δ7 isomerase and sigma-ligand-binding activity. Nine out of ten amino acid residues essential for catalytic activity of EBP were conserved in EBPL. Replacement of the only differing residue (EBP-Y111W) reduced catalytic activity of EBP. Transfer of the divergent residue from EBP to EBPL (EBPL-W91Y) and chimaerization of EBP and EBPL at various positions failed to restore catalytic activity of EBPL. Chemical cross-linking induced homodimerization of EBPL and EBP. Whereas mevinolin increased the mRNA for EBP and DHCR7 (Δ7-sterol reductase) in HepG2 cells, it had no effect on mRNAs for EBPL and sigma1 receptor, indicating that EBP and EBPL expression are not co-ordinated. We propose that EBPL has a yet-to-be-discovered function other than cholesterol biosynthesis.
Protein–protein, protein–RNA and protein–lipid interactions of signal-recognition particle components in the hyperthermoacidophilic archaeon Acidianus ambivalens
Authors: MOLL Ralf G.
Published: August 2003

The signal-recognition particle (SRP) of one of the most acidophilic and hyperthermophilic archaeal cells, Acidianus ambivalens, and its putative receptor component, FtsY (prokaryotic SRP receptor), were investigated in detail. A. ambivalens Ffh (fifty-four-homologous protein) was shown to be a soluble protein with strong affinity to membranes. In its membrane-residing form, Ffh was extracted from plasma membranes with chaotropic agents like urea, but not with agents diminishing electrostatic interactions. Using unilamellar tetraether phospholipid vesicles, both Ffh and FtsY associate independently from each other in the absence of other factors, suggesting an equilibrium of soluble and membrane-bound protein forms under in vivo conditions. The Ffh protein precipitated from cytosolic cell supernatants with anti-Ffh antibodies, together with an 7 S-alike SRP–RNA, suggesting a stable core ribonucleoprotein composed of both components under native conditions. The SRP RNA of A. ambivalens depicted a size of about 309 nucleotides like the SRP RNA of the related organism Sulfolobus acidocaldarius. A stable heterodimeric complex composed of Ffh and FtsY was absent in cytosolic super-natants, indicating a transiently formed complex during archaeal SRP targeting. The FtsY protein precipitated in cytosolic super-natants with anti-FtsY antisera as a homomeric protein lacking accessory protein components. However, under in vitro conditions, recombinantly generated Ffh and FtsY associate in a nucleotide-independent manner, supporting a structural receptor model with two interacting apoproteins.
Comparative membrane interaction study of viscotoxins A3, A2 and B from mistletoe (Viscum album) and connections with their structures
Authors: COULON Alexandre , MOSBAH Amor , LOPEZ André , SAUTEREAU Anne-Marie , SCHALLER Gerhard , URECH Konrad , ROUGÉ Pierre , DARBON Hervé
Published: August 2003

Viscotoxins A2 (VA2) and B (VB) are, together with viscotoxin A3 (VA3), among the most abundant viscotoxin isoforms that occur in mistletoe-derived medicines used in anti-cancer therapy. Although these isoforms have a high degree of amino-acid-sequence similarity, they are strikingly different from each other in their in vitro cytotoxic potency towards tumour cells. First, as VA3 is the only viscotoxin whose three-dimensional (3D) structure has been solved to date, we report the NMR determination of the 3D structures of VA2 and VB. Secondly, to account for the in vitro cytotoxicity discrepancy, we carried out a comparative study of the interaction of the three viscotoxins with model membranes. Although the overall 3D structure is highly conserved among the three isoforms, some discrete structural features and associated surface properties readily account for the different affinity and perturbation of model membranes. VA3 and VA2 interact in a similar way, but the weaker hydrophobic character of VA2 is thought to be mainly responsible for the apparent different affinity towards membranes. VB is much less active than the other two viscotoxins and does not insert into model membranes. This could be related to the occurrence of a single residue (Arg25) protruding outside the hydrophobic plane formed by the two amphipathic α-helices, through which viscotoxins are supposed to interact with plasma membranes.
XB51 isoforms mediate Alzheimer's β-amyloid peptide production by X11L (X11-like protein)-dependent and -independent mechanisms
Authors: SUMIOKA Akio , IMOTO Seiyu , MARTINS Ralph N. , KIRINO Yutaka , SUZUKI Toshiharu
Published: August 2003

XB51 (derived from X11-like binding protein of clone number 51) was isolated by yeast two-hybrid cDNA screening using the N-terminal domain of X11L (X11-like protein) as a bait. X11L is a neuron-specific adaptor protein that is known to down-regulate APP (β-amyloid precursor protein) metabolism by associating with the cytoplasmic domain of APP, but the detailed mechanisms are still unknown. Thus the X11L-associated protein XB51 is believed to regulate APP metabolism by modifying X11L function through its interaction with X11L. Here we report that the hXB51 (human XB51) gene can yield two transcripts, one with exon 9 spliced out (resulting in the hXB51β isoform) and the other containing exon 9 (yielding the hXB51α isoform). hXB51α binds to X11L to form a tripartite complex composed of hXB51α, X11L and APP. Complex-formation results in blocking X11L's suppression of Aβ (β-amyloid) generation from APP. hXB51β associates with X11L and inhibits its interaction with APP. However, hXB51β suppresses Aβ generation and secretion in an X11L-independent manner. Thus the hXB51 isoforms regulate Aβ generation differently, either enhancing it by modifying the association of X11L with APP or suppressing it in an X11L-independent manner. These observations advance our understanding of the molecular mechanisms regulating intracellular Aβ production and the pathogenesis of Alzheimer's disease.
Training-induced adaptation of oxidative phosphorylation in skeletal muscles
Authors: KORZENIEWSKI Bernard , ZOLADZ Jerzy A.
Published: August 2003

Muscle training/conditioning improves the adaptation of oxidative phosphorylation in skeletal muscles to physical exercise. However, the mechanisms underlying this adaptation are still not understood fully. By quantitative analysis of the existing experimental results, we show that training-induced acceleration of oxygen-uptake kinetics at the onset of exercise and improvement of ATP/ADP stability due to physical training are mainly caused by an increase in the amount of mitochondrial proteins and by an intensification of the parallel activation of ATP usage and ATP supply (increase in direct stimulation of oxidative phosphorylation complexes accompanying stimulation of ATP consumption) during exercise.
P2X7 receptor activates extracellular signal-regulated kinases ERK1 and ERK2 independently of Ca2+ influx
Authors: AMSTRUP Jan , NOVAK Ivana
Published: August 2003

P2X7 nucleotide receptors modulate a spectrum of cellular events in various cells including epithelia, such as exocrine pancreas. Although the pharmacology and channel properties of the P2X7 receptors have been studied intensively, signal transduction pathways are relatively unknown. In this study we applied a heterologous expression system of rat P2X7 receptors in HEK-293 cells. We followed the receptor expression and function using the enhanced green fluorescent protein (EGFP) tag, activation of intracellular proteins and increases in cellular Ca2+. EGFP-P2X7 receptors localized to the plasma membrane, clusters within the membrane and intracellularly. Stimulation of P2X7 receptors in HEK-293 cells led to an activation of extracellular signal-regulated kinases ERK1 and ERK2 and this activation was seen after just 1 min of stimulation with ATP. Using C- and N-terminal P2X7-receptor mutants we show that the N-terminus is important in activation of ERKs, whereas deletion of the last 230 amino acids in the C-terminus did not effect ERK activation. On the other hand, Ca2+ entry was impaired in C-terminal but not in N-terminal mutants. In cell suspensions prepared from rat pancreas we show that P2X7 receptors also activate ERK1 and ERK2, indicating that these signalling pathways are also turned on in native epithelium.
A role for Sec1/Munc18 proteins in platelet exocytosis
Authors: SCHRAW Todd D. , LEMONS Paula P. , DEAN William L. , WHITEHEART Sidney W.
Published: August 2003

A critical aspect of haemostasis is the release of clot-forming components from the three intra-platelet stores: dense-core granules, α granules and lysosomes. Exocytosis from these granules is mediated by soluble proteins [N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (SNAPs)] and integral membrane proteins [vesicle and target SNAP receptors (v- and t-SNAREs)]. Three Sec1/Munc18 proteins (SM proteins) are present in platelets (Munc18a, Munc18b and Munc18c) and they bind to and potentially regulate specific syntaxin t-SNAREs. In resting platelets, these SM proteins associate with granules and open canalicular system membranes predominantly but not with the plasma membrane. Munc18a binds to syntaxin 2 alone and does not associate with other members of the core SNARE complex. Munc18b associates with a larger complex that contains synaptosome-associated protein of 23 kDa (SNAP-23) and cellubrevin/vesicle-associated membrane protein 3. Munc18c associates with both syntaxins 2 and 4, with synaptosome-associated protein of 23 kDa (SNAP-23) and with a v-SNARE. On stimulation, most of the platelet SM proteins are still found in membrane fractions. Phosphorylation of each Munc18 increases in thrombin-treated cells and phosphorylated Munc18c remains associated with syntaxins 2 and 4, but its affinity for the SNAREs appears to be reduced. To determine the functional role of the platelet SM proteins, we examined the effects of Munc18-based peptides (Munc18a peptide 3 and Munc18c peptide 3). Addition of the peptides to permeabilized platelets inhibits secretion from all three platelet granules. These peptides also inhibit agonist-induced aggregation in saponin-permeabilized platelets. These studies demonstrate a clear role for SM proteins in platelet exocytosis and aggregation and suggest a dominant role for Munc18c in all three granule-release events.
Definition of structural elements in Plasmodium vivax and P. knowlesi Duffy-binding domains necessary for erythrocyte invasion
Authors: SINGH Saurabh K. , SINGH Agam P. , PANDEY Sunita , YAZDANI Syed S. , CHITNIS Chetan E. , SHARMA Amit
Published: August 2003

Plasmodium vivax and P. knowlesi use the Duffy antigen as a receptor to invade human erythrocytes. Duffy-binding ligands belong to a family of erythrocyte-binding proteins that bind erythrocyte receptors to mediate invasion. Receptor-binding domains in erythrocyte-binding proteins lie in conserved cysteine-rich regions called Duffy-binding-like domains. In the present study, we report an analysis of the overall three-dimensional architecture of P. vivax and P. knowlesi Duffy-binding domains based on mild proteolysis and supportive-functional assays. Our proteolysis experiments indicate that these domains are built of two distinct subdomains. The N-terminal region from Cys-1–4 (C1–C4) forms a stable non-functional subdomain. The region spanning C5–C12 forms another subdomain, which is capable of binding Duffy antigen. These subdomains are joined by a protease-sensitive linker. Results from deletion constructs, designed for expression of truncated proteins on COS cell surface, show that regions containing C5–C8 of the Duffy-binding domains are sufficient for the binding receptor. Therefore the central region of Duffy-binding domains, which is flanked by two non-functional regions, is responsible for receptor recognition. Moreover, the minimal Duffy-binding region identified here is capable of folding into a functionally competent module. These studies pave the way for understanding the architecture of Duffy-binding domains and their interactions with host receptors.
Human sodium-coupled citrate transporter, the orthologue of Drosophila Indy, as a novel target for lithium action
Authors: INOUE Katsuhisa , ZHUANG Lina , MADDOX Dennis M. , SMITH Sylvia B. , GANAPATHY Vadivel
Published: August 2003

NaCT (sodium-coupled citrate transporter) is an Na+-coupled citrate transporter identified recently in mammals that mediates the cellular uptake of citrate. It is expressed predominantly in the liver. NaCT is structurally and functionally related to the product of the Indy (I'm not dead yet) gene in Drosophila, the dysfunction of which leads to lifespan extension. Here, we show that NaCT mediates the utilization of extracellular citrate for fat synthesis in human liver cells, and that the process is stimulated by lithium. The transport function of NaCT is enhanced by lithium at concentrations found in humans treated with lithium for bipolar disorders. Valproate and carbamazepine, two other drugs that are used for the treatment of bipolar disorder, do not affect the function of NaCT. The stimulatory effect of Li+ is specific for human NaCT, since NaCTs from other animal species are either inhibited or unaffected by Li+. The data also suggest that two of the four Na+-binding sites in human NaCT may become occupied by Li+ to produce the stimulatory effect. The stimulation of NaCT in humans by lithium at therapeutically relevant concentrations has potential clinical implications. We also show here that a single base mutation in codon-500 (TTT→CTT) in the human NaCT gene, leading to the replacement of phenylalanine with leucine, stimulates the transport function and abolishes the stimulatory effect of lithium. This raises the possibility that genetic mutations in humans may lead to alterations in the constitutive activity of the transporter, with associated clinical consequences.
The effect of rebamipide on the expression of proinflammatory mediators and apoptosis in human neutrophils by Helicobacter pylori water‐soluble surface proteins
Authors: Kim J. S. , Kim J. M. , Jung H. C. , Song I. S.
Published: August 2003

Background : Helicobacter pylori infection elicits persistent neutrophil infiltration in gastric mucosa. The expression of cyclooxygenase (COX)‐2 and inhibition of apoptosis in the neutrophils could contribute to the pathogenesis of H. pylori infection. Rebamipide, a mucosal protective and ulcer‐healing drug, has been known to inhibit neutrophil activation.

Aim : To evaluate the effect of rebamipide on the neutrophils activated by H. pylori water‐soluble proteins.

Methods : After neutrophils were stimulated with H. pylori water extract (HPWE) or pre‐treated with rebamipide, the expression of COX‐2 mRNA and protein was assessed by quantitative RT‐PCR and Western blotting, respectively. Prostaglandin (PG) E2 synthesis was determined by radioimmunoassay. Neutrophil apoptosis was evaluated by cytosolic oligonucleosome‐bound DNA ELISA and caspase‐3 activity was measured by the detection of p‐nitroanilide after cleavage from labelled substrate.

Results : Stimulation with HPWE up‐regulated COX‐2 expression and PGE2 secretion, and inhibited neutrophil apoptosis. Rebamipide suppressed PGE2 secretion from neutrophils dose‐dependently. Rebamipide, however, did not affect neutrophil apoptosis and caspase‐3 activity.

Conclusions : Rebamipide effectively suppressed PGE2 secretion from neutrophils activated by H. pylori water‐soluble proteins. This is another possible mechanism of gastric mucosal protection by rebamipide.
The specific expression of Hypoxia Inducible Factor‐1α in human gastric mucosa induced by nonsteroidal anti‐inflammatory drugs
Authors: Ito M. , Tanaka S. , Kim S. , Kuwai T. , Matsutani N. , Kamada T. , Kitadai Y. , Sumii M. , Yoshihara M. , Haruma K. , Chayama K.
Published: August 2003

Background : Hypoxia is a cause of gastric mucosal damage induced by nonsteroidal anti‐inflammatory drugs (NSAIDs). The expression of hypoxia inducible factor‐1α (HIF‐1α) reflects the status of tissue ischaemia.

Aim : To investigate the effect of NSAID administration on the expression of HIF‐1α in human gastric mucosa.

Methods : We employed 71 patients including 14 with NSAID administration. The HIF‐1α expression was estimated by immunohistochemistry using monoclonal antibody (H1α67) and raised antiserum (HI‐3). Vascular endothelial growth factor expression was also examined by immunohistochemistry. HI‐3 recognized hypoxia‐induced protein in HeLa cells.

Results : In human gastric mucosa, HIF‐1α was mainly expressed in the nuclei of the surface epithelial cells and in the neck zone both by use of HI‐3 and of H1α67. The expression of vascular endothelial growth factor correlated well with that of HIF‐1α. The level of HIF‐1α in the surface epithelium was significantly higher in patients with administration of NSAIDs than those without NSAID use (P < 0.001) both in the gastric corpus and antrum. Helicobacter pylori infection did not affected the levels of HIF‐1α. Long‐term administration of rebamipide reduced the level of HIF‐1α.

Conclusion : HIF‐1α expression is a new biological marker of ischaemia especially in NSAID‐related gastric lesions.
Interaction of leptin with gastric myofibroblast transdifferentiation in Helicobacter pylori‐infected Mongolian gerbils: the effect of rebamipide
Authors: Nakamura M. , Akiba Y. , Matsui H. , Tsuchimoto K. , Ishii H.
Published: August 2003

Background : Our recent histochemical studies have revealed the marked increase of myofibroblasts in the Helicobacter pylori‐infected Mongolian gerbil fundic mucosa, while the mediators, which facilitate the conversion of fibroblasts to the myofibroblasts have remained unknown.

Aim : The present study was undertaken to clarify the alteration of leptin in the control and H. pylori‐infected Mongolian gerbil stomach. The effector sites of rebamipide were also investigated in relation to leptin.

Methods : The localization of leptin was investigated by the indirect immunofluorescence. Plasma leptin levels were determined by ELISA method. The localization of 3H‐rebamipide binding sites was investigated by autoradiography.

Results : Serum leptin content in H. pylori‐infected Mongolian gerbils was significantly increased. The presence of leptin immunoreactivity was recognized in the endothelial cells of the microcirculatory network and very weakly in the glandular cells in the control group, while in the H. pylori‐infected group leptin was markedly recognized in the mesenchymal cells. Rebamipide bound to the fibroblasts and surface mucous cells and decreased the leptin immunoreactivity in the gastric mucosa.

Conclusions : Leptin was mostly found in the mesenchymal cells. Rebamipide administration brought about the decrease of leptin in the gastric mucosaof the H. pylori‐infected gerbils.
Expression of midkine and receptor‐like protein tyrosine phosphatase (RPTP)‐β genes in the rat stomach and the influence of rebamipide
Authors: Yuki T. , Ishihara S. , Rumi M. , Ortega‐Cava Cesar F. , Kadowaki Y. , Kazumori H. , Yuki M. , Wada T. , Miyaoka Y. , Yoshino N. , Kinoshita Y.
Published: August 2003

Background : Midkine has been reported to bind to receptor‐like protein tyrosine phosphatase (RPTP)‐β and to play important roles in growth and differentiation of various cells. Midkine is expressed in rat stomach during experimental ulcer healing, suggesting that the midkine‐RPTP‐β system has some physiological functions in the stomach. Rebamipide is a mucoprotective drug used for the treatment of gastric ulcers. We have tested the hypothesis that the ulcer healing mechanism stimulated by rebamipide is linked physiologically to the gastric midkine‐RPTP‐β system.

Materials and methods : Seven‐week‐old‐male Wistar rats were used. Midkine and RPTP‐β gene expression in rat stomach was investigated by laser capture microdissection coupled with the reverse transcription‐polymerase chain reaction (RT‐PCR). The effects of rebamipide on midkine and RPTP‐β expression in rat stomach and the gastric epithelial cell line RGM1 were evaluated by RT‐PCR and Northern blot analyses.

Results : Midkine and RPTP‐β expression was detected in the gastric mucosal, submucosal and muscle layers. Rebamipide stimulated both midkine and RPTP‐β expression in rat stomach and RGM1 cells.

Conclusion : Rebamipide may protect the gastric mucosa by regulating midkine and RPTP‐β expression.
Effect of rebamipide on prostaglandin receptors‐mediated increase of inflammatory cytokine production by macrophages
Authors: Bamba H. , Ota S. , Kato A. , Miyatani H. , Kawamoto C. , Yoshida Y. , Fujiwara K.
Published: August 2003

Background : Rebamipide (Reb) is an anti‐ulcer drug, and has unique properties such as anti‐inflammatory action. We previously reported that prostaglandins (PGs) dramatically increased vascular endothelial growth factor (VEGF), a known angiogenic factor and a vascular permeable factor, by activated macrophages through specific PGE receptor and peroxisome proliferator‐activated receptor γ (PPARγ, a nuclear receptor of PG) mediated process. Effects of PGs on the production of other cytokines such as interleukin (IL)‐6 and IL‐8 have been controversial.

Aim : To clarify the anti‐inflammatory roles of Reb, we examined the effect of Reb on PGE1‐ and 15‐deoxy‐Δ12, 14‐PGJ2 (a potent PPARγ ligand, 15d‐PGJ2) ‐induced increase of VEGF production by macrophages. Additionally, effects of these PGs on the production of IL‐6 and IL‐8, and modulation of these actions by Reb were studied.

Methods : Phorbol 12‐myristate 13‐acetate‐differentiated U937 cells were used as a human macrophage model (H‐Mac). VEGF, IL‐6, IL‐8 and cAMP were measured by EIA.

Results : Reb suppressed PGE1‐, but not 15d‐PGJ2‐, induced increase of VEGF production partially through decrease of cAMP formation. Reb suppressed PGE1 ‐, but not 15d‐PGJ2‐, induced increase of IL‐6 and IL‐8 production.

Conclusion : Reb suppresses membrane, but not nuclear PG receptors mediated increase of inflammatory cytokine production, which may be involved in anti‐ulcer action of this drug.