bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mildly oxidized low-density lipoproteins decrease early production of interleukin 2 and nuclear factor κB binding to DNA in activated T-lymphocytes
Authors: CASPAR-BAUGUIL Sylvie , TKACZUK Jean , HAURE Marie-José , DURAND Martine , ALCOUFFE Julie , THOMSEN Mogens , SALVAYRE Robert , BENOIST Hervé 
Published: August 2015

Activated T-lymphocytes are found early in atherosclerosis lesions, but little is known about their role. Oxidized low-density lipoproteins (oxLDLs) are considered to be involved in the pathogenesis of the lesions, and we have previously demonstrated that oxLDLs inhibit not only interleukin (IL)-2-receptor expression on the surface of in vitro-activated T-lymphocytes but also their proliferation. We have now investigated the effect of oxLDLs on blast differentiation, on IL-2 synthesis and on the activation of the nuclear factor κB (NF-κB) system in activated lymphocytes. Mildly oxLDLs (50 and 100 µg/ml) decreased the number of lymphoblasts and the level of IL-2 concentration in the culture supernatants after activation of lymphocytes by phytohaemagglutinin and PMA+ionomycin. The inhibition of IL-2 production was observed in the CD3+ T-lymphocyte cytoplasm as early as 4 h after activation by PMA+ionomycin. The study of NF-κB showed that oxLDLs led to a decrease of activation-induced p65/p50 NF-κB heterodimer binding to DNA, whereas the presence of the constitutive nuclear form of p50 dimer was unchanged. This was correlated with an unchanged level of the active form of the cytosolic inhibitor protein IκB-α. Taken together, these observations suggest that the immunosuppressive effect of oxLDLs might operate via a dysregulation of the T-lymphocyte activation mechanisms.
In vitro analysis of the zinc-finger motif in human replication protein A
Authors: DONG Jiaowang , PARK Jang-Su , LEE Suk-Hee 
Published: August 2015

Human replication protein A (RPA) is composed of 70, 34 and 11 kDa subunits (p70, p34 and p11 respectively) and functions in all three major DNA metabolic processes: replication, repair and recombination. Recent deletion analysis demonstrated that the large subunit of RPA, p70, has multiple functional domains, including a DNA polymerase α-stimulation domain and a single-stranded DNA-binding domain. It also contains a putative metal-binding domain of the 4-cysteine type (Cys-Xaa4-Cys-Xaa13-Cys-Xaa2-Cys) that is highly conserved among eukaryotes. To study the role of this domain in DNA metabolism, we created various p70 mutants that lack the zinc-finger motif (by Cys → Ala substitutions). Mutation at the zinc-finger domain (ZFM) abolished RPA's function in nucleotide excision repair (NER), but had very little impact on DNA replication. The failure of zinc-finger mutant RPA in NER may be explained by the observation that wild-type RPA significantly stimulated DNA polymerase δ activity, whereas only marginal stimulation was observed with zinc-finger mutant RPA. We also observed that ZFM reduced RPA's single-stranded DNA-binding activity by 2–3-fold in the presence of low amounts of RPA. Interestingly, the ZFM abolished phosphorylation of the p34 subunit by DNA-dependent protein kinase, but not that by cyclin-dependent kinase. Taker together, our results strongly suggest a positive role for RPA's zinc finger domain in its function.
Importance of splicing for prosaposin sorting
Authors: MADAR-SHAPIRO Liora , PASMANIK-CHOR Metsada , VACCARO Anna Maria , DINUR Tama , DAGAN Arie , GATT Shimon , HOROWITZ Mia 
Published: August 2015

The prosaposin gene encodes a 70 kDa protein. This protein might either reach the lysosomes and get processed there to four peptides, which are activators of known lysosomal enzymes, or be secreted by cells as a 70 kDa protein, recently anticipated to have several biological activities. The human prosaposin gene has a 9 bp exon (exon 8) that is alternatively spliced, thus encoding three prosaposin forms: one with an extra three amino acid residues, one with an extra two residues and a third form with no extra residues. With the aim of testing whether there is an association between the alternative splicing and the differential sorting of prosaposins, we cloned two human prosaposin cDNA forms in a T7/EMC/vaccinia virus-derived vector and expressed them in human cells. The results indicated that the prosaposin containing the three extra residues accumulated faster and in greater amounts in the medium, whereas the prosaposin with no extra residues was mainly destined for lysosomes. Point mutations created by mutagenesis in vitro in the 9 bp stretch had a diverse effect on prosaposin secretion. When supplied to cells in the medium, both prosaposins were endocytosed and reached the lysosomes, where they were processed to active saposin B and saposin C. The activities of the saposins were monitored qualitatively and quantitatively. Quantitatively, lipids were extracted from the cells, separated on TLC and measured fluorimetrically. Qualitatively, cells were detected by fluorescence microscopy.
Formation of apolipoprotein-specific high-density lipoprotein particles from lipid-free apolipoproteins A-I and A-II
Authors: CLAY Moira A. , CEHIC Daniel A. , PYLE Diana H. , RYE Kerry-Anne , BARTER Philip J. 
Published: August 2015

We have shown previously that apolipoprotein A (apoA)-I-containing high-density lipoprotein (HDL) particles are formed by the conjugation of lipid-free apoA-I with lipids derived from other lipoprotein fractions in a process dependent on non-esterified fatty acids, generated by the lipolysis of very-low-density lipoprotein (VLDL) or provided exogenously. In the present study, we show that this process is also able to generate HDL particles containing apoA-II (A-II HDL) and both apoA-I and apoA-II (A-I/A-II HDL). When lipid-free apoA-II was incubated with either VLDLs and lipoprotein lipase or LDLs and sodium oleate, a significant proportion of the apoA-II was recovered in the HDL density fraction. This was associated with the formation of several populations of HDL-sized particles with pre-β2 electrophoretic mobility, which contained phospholipids and unesterified cholesterol as their main lipid constituents. When both lipid-free apoA-I and lipid-free apoA-II were incubated with LDL and sodium oleate, both apolipoproteins were recovered in HDLs that contained phospholipids and unesterified cholesterol as their main lipids. Two populations of particles had diameters of 7.4 and 10.8 nm and pre-β2-migration; there was also a population of pre-β1-migrating particles of diameter 4.7 nm. ApoA-I and apoA-II were both present in the larger HDLs, whereas only apoA-I was present in the smaller particles. Immunoaffinity chromatography on an anti-(apoA-I)–Sepharose column revealed that 10–20% of the apoA-II resided in particles that also contained apoA-I. The majority of the A-I/A-II HDL were present in a population of pre-β2 particles of 10.8 nm diameter. These results in vitro illustrate a potential mechanism for the formation of HDLs containing both apoA-I and apoA-II.
L-ATP is recognized by some cellular and viral enzymes: does chance drive enzymic enantioselectivity?
Authors: VERRI Annalisa , MONTECUCCO Alessandra , GOSSELIN Gilles , BOUDOU Valérie , IMBACH Jean-Louis , SPADARI Silvio , FOCHER Federico 
Published: August 2015

We demonstrate that l-ATP is recognized by some enzymes that are involved in the synthesis of nucleotides and nucleic acids. l-ATP, as well as its natural d-enantiomer, acts as a phosphate donor in the reaction catalysed by human deoxycytidine kinase, whereas it is not recognized by either enantioselective human thymidine kinase or non-enantioselective herpes virus thymidine kinase. l-ATP strongly inhibits (Ki 80 µM) the synthesis of RNA primers catalysed by DNA primase associated with human DNA polymerase α, whereas RNA synthesis catalysed by Escherichia coli RNA polymerase is completely unaffected. Moreover, l-ATP competitively inhibits ATP-dependent T4 DNA ligase (Ki 25 µM), suggesting that it interacts with the ATP-binding site of the enzyme. Kinetic studies demonstrated that l-ATP cannot be used as a cofactor in the ligase-catalysed joining reaction. On the other hand, l-AMP is used by T4 DNA ligase to catalyse the reverse reaction, even though a high level of intermediate circular nicked DNA molecules accumulates. Our results suggest that a lack of enantioselectivity of enzymes is more common than was believed a few years ago, and, given the absence of selective constraints against l-nucleosides in Nature, this may depend on chance more than on evolutionary strategy.
Incorporation of iron by the unusual dodecameric ferritin from Listeria innocua
Authors: STEFANINI Simonetta , CAVALLO Stefano , MONTAGNINI Benedetta , CHIANCONE Emilia 
Published: August 2015

The polypeptide chain that assembles into the unusual dodecameric shell of Listeria innocua apoferritin lacks the ferroxidase centre characteristic of H-type mammalian chains, but is able to catalyse both Fe(II) oxidation and nucleation of the iron core. A cluster of five carboxylate residues, which correspond in part to the site of iron core nucleation typical of L-type mammalian ferritins, has been proposed to be involved in both functions. The features of the iron uptake kinetics and of Fe(II) autoxidation in the presence of citrate followed spectrophotometrically confirm this assignment. In Listeria the kinetics of iron uptake is hyperbolic at low Fe(II)-to-dodecamer ratios and becomes sigmoidal when iron exceeds 150 Fe(II) atoms per dodecamer, namely when a fast crystal growth phase follows a slow initial nucleation step. Iron autoxidation in the presence of citrate displays a similar behaviour. Thus the time course is sigmoidal at low citrate-to-Fe ratios at which Fe(III) polymerization is predominant, but is hyperbolic at ligand concentrations high enough to prevent polymerization. The marked inhibitory effect of Tb(III) on the kinetics of iron incorporation confirms that carboxylates provide the iron ligands in L. innocua apoferritin. Iron uptake followed in steady-state fluorescence experiments allows one to distinguish Fe(II) binding and oxidation from the subsequent movement of Fe(III) into the apoferritin cavity as in mammalian ferritins despite the different localization of the tryptophan residues.
Presence of enolase in the M-band of skeletal muscle and possible indirect interaction with the cytosolic muscle isoform of creatine kinase
Authors: FOUCAULT Georges , VACHER Monique , MERKULOVA Tatyana , KELLER Angelica , ARRIO-DUPONT Martine 
Published: August 2015

Glycerol-skinned skeletal muscle fibres retain the defined sarcomeric structure of the myofibrils. We show here that a small fraction of two enzymes important for energy metabolism, the cytosolic muscle isoform of creatine kinase (EC 2.7.3.2), MM-creatine kinase (MM-CK), and enolase (EC 4.2.1.11), remains bound to skinned fibres. CK is slowly exchangeable, whereas enolase is firmly bound. Two-dimensional gel electrophoresis followed by Western blot analyses demonstrates that both α (ubiquitous) and β (muscle-specific) subunits of enolase are present in these preparations. Enolase and CK were co-localized at the M-band of the sarcomeres, as observed by indirect immunofluorescence and confocal microscopy. Cross-linking experiments were performed on skinned fibres with three bifunctional succinimidyl esters of different lengths and yielded a protein complex of 150 kDa that reacted with antibodies directed against either M-CK or β-enolase. The cross-linking efficiency was greatest for the longest reagent and zero for the shortest one. The length of the cross-linker giving a covalent complex between the two enzymes does not support the notion of a direct interaction between M-CK and enolase. This is the first demonstration of the presence of an enzyme of energy metabolism other than CK at the M-band of myofibres.
Predominance of the acylation route in the metabolic processing of exogenous sphingosine in neural and extraneural cells in culture
Authors: RIBONI Laura , BASSI Rosaria , PRINETTI Alessandro , VIANI Paola , TETTAMANTI Guido 
Published: August 2015

The metabolic fate of exogenous [3H]sphingosine was investigated in five types of cultured cells: primary cultures of neurons and astrocytes, murine and human neuroblastoma cells and human skin fibroblasts. After administration of 40 nM [3-3H]sphingosine into a cell-conditioned medium containing fetal calf serum, all cell types rapidly and efficiently incorporated the long-chain base in a time-dependent fashion. In all cases, after a 120 min pulse, the amount of radioactivity taken up was in the range of the endogenous sphingosine content. However, unchanged [3H]sphingosine represented only a very minor portion of the label incorporated into cells throughout the pulse period (10–120 min), indicating rapid and efficient sphingosine metabolism in these cells. Most of the [3H]sphingosine taken up was metabolically processed, either by degradation (assessed as 3H2O release into the culture medium) or by N-acylation (mainly to radioactive ceramide, sphingomyelin, neutral glycolipids and gangliosides). [3H]Sphingosine 1-phosphate accounted for less than 2% of the total radioactivity incorporated in all cases. Throughout the pulse period and in all cell types, 3H-labelled organic metabolites largely prevailed over 3H2O, indicating that N-acylation is the major metabolic fate of sphingosine in these cells under apparently physiological conditions. These results are consistent with the notion that sphingosine has a rapid turnover in the cells studied, and indicate that regulation of the basal level of this bioactive molecule occurs mainly through N-acylation.
Proteoglycan involvement in polyamine uptake
Authors: BELTING Mattias , PERSSON Susanne , FRANSSON Lars-Åke 
Published: August 2015

We have evaluated the possible role of proteoglycans in the uptake of spermine by human lung fibroblasts. Exogenous glycosaminoglycans behaved as competitive inhibitors of spermine uptake, the most efficient being heparan sulphate (Ki = 0.16±0.04 µM). Treatment of fibroblasts with either heparan sulphate lyase, p-nitrophenyl-O-β-d-xylopyranoside or chlorate reduced spermine uptake considerably, whereas chondroitin sulphate lyase had a limited effect. Inhibition of polyamine biosynthesis with α-difluoromethylornithine resulted in an increase of cell-associated heparan sulphate proteoglycans exhibiting higher affinity for spermine. The data indicate a specific role for heparan sulphate proteoglycans in the uptake of spermine by fibroblasts. Spermine uptake by pgsD-677, a mutant Chinese hamster ovary cell defective in heparan sulphate biosynthesis, was only moderately reduced (20%) compared with wild-type cells. Treatment of mutant cells with the above-mentioned xyloside resulted in a greater reduction of endogenous proteoglycan production as well as a higher inhibition of spermine uptake than in wild-type cells. Moreover, treatment with chondroitin sulphate lyase resulted in a selective inhibition of uptake in mutant cells, indicating a role for chondroitin/dermatan sulphate proteoglycans in the uptake of spermine by these cells. Fibroblasts, made growth-dependent on exogenous spermine by α-difluoromethylornithine treatment, were growth-inhibited by heparan sulphate or β-d-xyloside, which might have future therapeutical implications.
Role of retinoid receptors in the regulation of mucin gene expression by retinoic acid in human tracheobronchial epithelial cells
Authors: KOO Ja Seok , JETTEN Anton M. , BELLONI Paula , YOON Joo-Heon , KIM Yong-Dae , NETTESHEIM Paul 
Published: August 2015

To investigate which retinoid receptors are critical in the regulation by all-trans-retinoic acid (RA) of the mucin genes MUC2, MUC5AC and MUC5B in cultured normal human tracheobronchial epithelial (NHTBE) cells, we used pan-RAR-, pan-RXR- and RAR- isotype (α, β and γ)-selective agonists and RARα- and RARγ-selective antagonists (RAR is RA receptor and RXR is retinoid X receptor). RAR-, RARα- and RARγ-selective agonists strongly induced mucin mRNAs in a dose-dependent manner, while the RARβ-selective retinoid only weakly induced mucin gene expression at very high concentrations (1 µM). The pan-RXR-selective agonist by itself did not induce mucin gene expression, but acted synergistically with suboptimal concentrations of the pan-RAR agonist. A retinoid with selective anti-activator-protein-1 activity only marginally induced mucin gene expression. The RARα antagonist strongly inhibited mucin gene induction and mucous cell differentiation caused by RA and by the RARα- and RARγ-selective retinoids. In contrast, the RARγ antagonist only weakly inhibited RARα-selective-retinoid-induced mucin gene expression, but completely blocked mucin gene expression induced by the RARγ-selective retinoid. Our studies indicate that RARα is the major retinoid receptor subtype mediating RA-dependent mucin gene expression and mucous cell differentiation, but that the RARγ isotype can also induce mucin genes. Furthermore these studies suggest that RARβ is probably not (directly) involved in RA-induced mucin gene expression.
Canalicular multispecific organic anion transporter/multidrug resistance protein 2 mediates low-affinity transport of reduced glutathione
Authors: PAULUSMA Coen C. , GEER Michael A. van , EVERS Raymond , HEIJN Marc , OTTENHOFF Roelof , BORST Piet , OUDE ELFERINK Ronald P. J. 
Published: August 2015

The canalicular multispecific organic anion transporter (cMOAT), a member of the ATP-binding cassette transporter family, mediates the transport of a broad range of non-bile salt organic anions from liver into bile. cMOAT-deficient Wistar rats (TR-) are mutated in the gene encoding cMOAT, leading to defective hepatobiliary transport of a whole range of substrates, including bilirubin glucuronide. These mutants also have impaired hepatobiliary excretion of GSH and, as a result, the bile flow in these animals is reduced. In the present work we demonstrate a role for cMOAT in the excretion of GSH both in vivo and in vitro. Biliary GSH excretion in rats heterozygous for the cMOAT mutation (TR/tr) was decreased to 63% of controls (TR/TR) (114±24 versus 181±20 nmol/min per kg body weight). Madin-Darby canine kidney (MDCK) II cells stably expressing the human cMOAT protein displayed &gt; 10-fold increase in apical GSH excretion compared with wild-type MDCKII cells (141±6.1 pmol/min per mg of protein versus 13.2±1.3 pmol/min per mg of protein in wild-type MDCKII cells). Similarly, MDCKII cells expressing the human multidrug resistance protein 1 showed a 4-fold increase in GSH excretion across the basolateral membrane. In several independent cMOAT-transfectants, the level of GSH excretion correlated with the expression level of the protein. Furthermore, we have shown, in cMOAT-transfected cells, that GSH is a low-affinity substrate for the transporter and that its excretion is reduced upon ATP depletion. In membrane vesicles isolated from cMOAT-expressing MDCKII cells, ATP-dependent S-(2,4-dinitrophenyl)glutathione uptake is competitively inhibited by high concentrations of GSH (Ki≈ 20 mM). We concluded that cMOAT mediates low-affinity transport of GSH. However, since hepatocellular GSH concentrations are high (5–10 mM), cMOAT might serve an important physiological function in maintenance of bile flow in addition to hepatic GSH turnover.
Cloning and characterization of a maize cytochrome-b5 reductase withFe3+-chelate reduction capability
Authors: BAGNARESI Paolo , THOIRON Séverine , MANSION Monique , ROSSIGNOL Michel , PUPILLO Paolo , BRIAT Jean-François 
Published: August 2015

We previously purified an NADH-dependent Fe3+-chelate reductase (NFR) from maize roots with biochemical features of a cytochrome-b5 reductase (b5R) [Sparla, Bagnaresi, Scagliarini and Trost (1997) FEBS Lett. 414, 571–575]. We have now cloned a maize root cDNA that, on the basis of sequence information, calculated parameters and functional assay, codes for NFR. Maize NFR has 66% and 65% similarity to mammal and yeast b5R respectively. It has a deduced molecular mass of 31.17 kDa and a pI of 8.53. An uncharged region is observed at its N-terminus but no myristoylation consensus site is present. Taken together, these results, coupled with previous biochemical evidence, prove that NFR belongs to the b5R class and document b5R from a plant at the molecular level for the first time. We have also identified a putative Arabidopsis thaliana NFR gene. Its organization (nine exons) closely resembles mammalian b5Rs. Several NFR isoforms are expected to exist in maize. They are probably not produced by alternative translational mechanisms as occur in mammals, because of specific constraints observed in the maize NFR cDNA sequence. In contrast with yeast and mammals, tissue-specific and various subcellular localizations of maize b5R isoforms could result from differential expression of the various members of a multigene family. The first molecular characterization of a plant b5R indicates an overall remarkable evolutionary conservation for these versatile reductase systems. In addition, the well-characterized Fe3+-chelate reduction capabilities of NFR, in addition to known Fe3+-haemoglobin reduction roles for mammal b5R isoforms, suggest further and more generalized roles for the b5R class in endocellular iron reduction.
Purification and characterization of an α-galactosyltransferase from Trypanosoma brucei
Authors: PINGEL Sabine , RHEINWEILER Uta , KOLB Volker , DUSZENKO Michael 
Published: August 2015

A membrane-associated galactosyltransferase from Trypanosoma brucei was purified 34000-fold by affinity chromatography on UDP-hexanolamine–Sepharose™. Using SDS/PAGE under reducing conditions, the isolated enzyme ran as a relatively broad band with apparent molecular masses of 53 kDa and 52 kDa, indicative of glycosylation and the existence of two isoforms. N-Glycosylation of the enzyme was subsequently confirmed using Western blotting and either specific binding of concanavalin A or peptide-N4-(N-acetylglucosaminyl)asparagine amidase digestion. The de-N-glycosylated enzyme ran with apparent molecular masses of 51 kDa and 50 kDa, indicative of a single N-glycosylation site. The galactosyltransferase exhibited a pH optimum at 7.2 and had a pronounced requirement for Mn2+ ions (KM = 2.5 mM) for its action. The transferase activity was independent of the concentration of Triton X-100. The enzyme was capable of transferring galactose from UDP-galactose to a variety of galactose-based acceptors in α-glycosidic linkages. The apparent KM values for UDP-galactose and for the preferred acceptor substrate N-acetyl-lactosamine are 46 µM and 4.5 mM respectively. From these results we would like to suggest that the galactosyltransferase functions in the processing of terminal N-acetyl-lactosamine structures of trypanosomal glycoproteins.