bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin and insulin-like growth factor I up-regulate GLUT4 gene expression in fetal brown adipocytes, in a phosphoinositide 3-kinase-dependent manner
Authors: VALVERDE Angela M. , NAVARRO Paloma , TERUEL Teresa , CONEJO Ruben , BENITO Manuel , LORENZO Margarita
Published: August 2015

Fetal brown adipocytes cultured in a serum-free medium, containing 5 mM glucose, expressed both GLUT4 and GLUT1 glucose transporters at the mRNA and protein level. Treatment with either insulin or insulin-like growth factor (IGF)-I at physiological concentrations up-regulates the expression of the GLUT4 gene, producing a time-dependent mRNA accumulation (7-fold increase at 24 h) and a 2.5-fold increase in the amount of protein in the total membrane fraction. However, insulin treatment down-regulates GLUT1 mRNA and protein expression. Moreover, either insulin or IGF-I transactivates a full-promoter GLUT4-chloramphenicol acetyltransferase gene (CAT) construct transiently transfected to the cells, without affecting GLUT1-CAT activity. In consequence, insulin treatment for 24 h increased by 3-fold the basal glucose uptake. Inhibition of phosphoinositide (PI) 3-kinase activity with chemical agents such as wortmannin or LY294002 partially blocked insulin-induced GLUT4 mRNA accumulation, insulin-induced GLUT4 protein content, GLUT4-CAT transactivation and glucose uptake. Furthermore, co-transfection of brown adipocytes with a dominant-negative form of PI 3-kinase precluded the transactivation of the GLUT4 promoter by insulin. However, inhibition of p70S6 kinase (p70s6k) with rapamycin or of mitogen-activated protein kinase (MAPK) with PD098059 does not preclude insulin effects on GLUT4 gene expression or glucose uptake. Our results show for the first time a positive effect of insulin on GLUT4 gene expression in fetal brown adipocytes, suggesting the existence of insulin response element(s) in its promoter. Moreover, PI 3-kinase, but not p70s6k or MAPK, is an essential requirement for insulin regulation of GLUT4 gene expression.
Glutamine synthesis is heterogeneous and differentially regulated along the rabbit renal proximal tubule
Authors: FERRIER Bernard , CONJARD Agnès , MARTIN Mireille , BAVEREL Gabriel
Published: August 2015

Glutamine synthesis, a major process for ammonia detoxification and the control of acid–base balance, occurs from various precursors in suspensions of rabbit proximal tubules. However, no data are currently available on the distribution of glutamine synthesis along the rabbit proximal tubule, and its modulation by changes of substrate concentration. Therefore we have microdissected and incubated the three parts (S1, S2 and S3) of rabbit proximal tubules and measured glutamine synthesis from alanine and aspartate. With a physiological concentration of alanine (0.25 mM) or aspartate (0.05 mM), glutamine synthesis in the S1 segment was about half of that in the S2 and S3 segments, and was greater from alanine than from aspartate along the entire proximal tubule. Elevation of alanine and aspartate concentrations to 5 mM increased glutamine synthesis in both a substrate- and segment-dependent manner. It is concluded that glutamine synthesis occurs from alanine and aspartate along the entire rabbit proximal tubule; however, contrary to what might have been expected on the basis of measurement of glutamine synthetase activity, the basal rate of glutamine synthesis and its adaptation to increased substrate availability are heterogeneous along this nephron segment.
Role of phosphatidylinositol 3,4,5-trisphosphate in regulating the activity and localization of 3-phosphoinositide-dependent protein kinase-1
Authors: CURRIE Richard A. , WALKER Kay S. , GRAY Alex , DEAK Maria , CASAMAYOR Antonio , DOWNES C. Peter , COHEN Philip , ALESSI Dario R. , LUCOCQ John
Published: August 2015

3-Phosphoinositide-dependent protein kinase-1 (PDK1) interacts stereoselectively with the d-enantiomer of PtdIns(3,4,5)P3 (KD 1.6 nM) and PtdIns(3,4)P2 (KD 5.2 nM), but binds with lower affinity to PtdIns3P or PtdIns(4,5)P2. The binding of PtdIns(3,4,5)P3 to PDK1 was greatly decreased by making specific mutations in the pleckstrin homology (PH) domain of PDK1 or by deleting it. The same mutations also greatly decreased the rate at which PDK1 activated protein kinase Bα (PKBα) in vitro in the presence of lipid vesicles containing PtdIns(3,4,5)P3, but did not affect the rate at which PDK1 activated a PKBα mutant lacking the PH domain in the absence of PtdIns(3,4,5)P3. When overexpressed in 293 or PAE cells, PDK1 was located at the plasma membrane and in the cytosol, but was excluded from the nucleus. Mutations that disrupted the interaction of PtdIns(3,4,5)P3 or PtdIns(4,5)P2 with PDK1 abolished the association of PDK1 with the plasma membrane. Growth-factor stimulation promoted the translocation of transfected PKBα to the plasma membrane, but had no effect on the subcellular distribution of PDK1 as judged by immunoelectron microscopy of fixed cells. This conclusion was also supported by confocal microscopy of green fluorescent protein–PDK1 in live cells. These results, together with previous observations, indicate that PtdIns(3,4,5)P3 plays several roles in the PDK1-induced activation of PKBα. First, it binds to the PH domain of PKB, altering its conformation so that it can be activated by PDK1. Secondly, interaction with PtdIns(3,4,5)P3 recruits PKB to the plasma membrane with which PDK1 is localized constitutively by virtue of its much stronger interaction with PtdIns(3,4,5)P3 or PtdIns(4,5)P2. Thirdly, the interaction of PDK1 with PtdIns(3,4,5)P3 facilitates the rate at which it can activate PKB.
Expression and purification of the first nucleotide-binding domain and linker region of human multidrug resistance gene product: comparison of fusions to glutathione S-transferase, thioredoxin and maltose-binding protein
Authors: WANG Changsen , CASTRO Ariel F. , WILKES Denise M. , ALTENBERG Guillermo A.
Published: August 2015

Many membrane proteins that belong to the ATP-binding cassette (ABC) superfamily are clinically important, including the cystic fibrosis transmembrane conductance regulator, the sulphonylurea receptor and P-glycoprotein (multidrug resistance gene product; MDR1). These proteins contain two multispanning transmembrane domains, each followed by one nucleotide-binding domain (NBD) and a linker region distal to the first NBD. ATP hydrolysis by the NBDs is critical for ABC protein function; the linker region seems to have a regulatory role. Previous attempts to express soluble NBDs and/or linker regions without detergent solubilization, or to purify NBDs at high yields as soluble fusion proteins, have been unsuccessful. Here we present a system for the expression in Escherichia coli of the first NBD of MDR1 followed by its linker region (NBD1MLD). A comparison of the expressions of NBD1MLD fused to glutathione S-transferase, thioredoxin and maltose-binding protein (MBP) shows that a high level of expression in the soluble fraction (approx. 8% of total E. coli protein) can be achieved only for MBP–NBD1MLD. The addition of a proteolytic thrombin site just proximal to the N-terminal end of NBD1MLD allows the cleavage of NBD1MLD from MBP, which can be easily purified with retention of its ATPase activity. In summary, success was obtained only when using an MBP fusion protein vector containing a thrombin proteolytic site between MBP and NBD1MLD. The approach described here could be generally applicable to solving the problems of expression and purification of NBDs/linker regions of ABC proteins.
The major phosphorylation site of the NADPH oxidase component p67phox is Thr233
Authors: FORBES Louisa V. , TRUONG Oanh , WIENTJES Frans B. , MOSS Stephen J. , SEGAL Anthony W.
Published: August 2015

Phosphorylation of p67phox was shown to increase two- to three-fold upon stimulation by PMA, N-formylmethionyl-leucylphenylalanine or serum-opsonized zymosan. Phosphopeptide mapping showed one major tryptic peptide for p67phox immunoprecipitated from resting or stimulated cells. In vitro phosphorylation of p67phox by isolated cytosol or mitogen-activated protein kinase also generated the same phosphopeptide. Results of cyanogen bromide digestion and HPLC–MS suggested that Thr233 was the phosphorylated residue. Mutagenesis of Thr233 to alanine resulted in loss of phosphorylation in vitro. In the present work, Thr233 has been identified as the major phosphorylation site of p67phox, which is situated in a proline-rich domain.
Purification of intracellular compartments involved in antigen processing: a new method based on magnetic sorting
Authors: PERRIN-COCON Laure A. , MARCHE Patrice N. , VILLIERS Christian L.
Published: August 2015

In the present study, we describe a method to specifically isolate intracellular compartments containing endocytosed antigen. We have demonstrated that isolated compartments represent a small proportion of the intracellular material, highly enriched in antigen. Antigen-containing vesicles are specifically sorted from other intracellular compartments, such as endoplasmic reticulum or Golgi apparatus, and from the plasma membrane. They remain functional in vitro since they can be acidified, and the antigen inside has been found to be partially proteolysed. In macrophages, kinetic analysis has revealed that the antigen is first found in compartments of endosomal density, carrying Rab 5 and Rab 7, then in late compartments of lysosomal density, which are rich in proteases. The global protein content of the compartments was mapped by two-dimensional electrophoresis. In B lymphocytes, this method has allowed the isolation of endocytic compartments emerging from receptor-mediated endocytosis of the antigen. After 2 h of chase, the antigen reached vesicles containing large amounts of MHC-class II molecules, invariant chain and human leucocyte antigen-DM, where peptide loading can occur.
Characterization of the 70 kDa polypeptide of the Na/Ca exchanger
Authors: SABA Rami I. , BOLLEN Alex , HERCHUELZ André
Published: August 2015

The Na/Ca exchanger is associated with 160, 120 and 70 kDa polypeptides whose nature is poorly understood. We have purified and characterized the Na/Ca exchanger from bovine cardiac sarcolemmal vesicles (SLVs) by using ion-exchange and affinity chromatographies. The Na/Ca exchanger-enriched fraction was reconstituted into asolectin liposomes [lipid to protein ratio 10:1 (w/w)] that showed Na/Ca exchange activity. Under non-reducing conditions, SDS/PAGE showed a single 70 kDa polypeptide, which was further characterized by immunoblots with different antibodies: SWant, raised against the purified exchanger protein; NH2-terminus, residues 1–21; NCX1, residues 393–406; and Exon F, residues 622–644. Immunoblots under reducing conditions with SWant, NH2-terminus and NCX1 showed three bands migrating at 160, 120 and 70 kDa for SLV preparations, whereas Exon F reacted only with the 160 and 120 kDa bands. Under non-reducing conditions, immunoblots with purified reconstituted Na/Ca exchanger showed a single band at 70 kDa reacting with SWant, NH2-terminus and NCX1 but not with Exon F. We conclude that the 70 kDa protein is associated with Na/Ca exchange activity, has the same N-terminal sequence as the cloned bovine cardiac exchanger, and has its length decreased by at least 35% from its C-terminal portion as compared with that of the wild-type exchanger.
Control of sarcoplasmic/endoplasmic-reticulum Ca2+ pump expression in cardiac and smooth muscle
Authors: MISQUITTA Christine M. , SING Angela , GROVER Ashok K.
Published: August 2015

Cardiac muscle expresses sarcoplasmic/endoplasmic-reticulum Ca2+ pump isoform SERCA2a; stomach smooth muscle expresses SERCA2b. In 2-day-old rabbits, cardiac muscle contained levels of SERCA2 protein that were 100–200-fold those in the stomach smooth muscle. In nuclear run-on assays, the rate of SERCA2 gene transcription in heart nuclei was not significantly higher than in the stomach smooth-muscle nuclei. However, the SERCA2 mRNA levels (mean±S.E.M.) were (29±4)-fold higher in the heart. In both tissues the SERCA2 mRNA was associated with polyribosomes. In a sucrose-density-gradient sedimentation velocity experiment on polyribosomes, there was no difference in the sedimentation pattern of SERCA2 mRNA between the two tissues, suggesting that the translation efficiency of SERCA2 RNA in the two tissues is quite similar. Thus the main difference in the control of SERCA2 expression in the two tissues is post-transcriptional and pretranslational.
Purification, characterization and molecular cloning of trichoanguin, a novel type I ribosome-inactivating protein from the seeds of Trichosanthes anguina
Authors: CHOW Lu-Ping , CHOU Ming-Huei , HO Cheng-Ying , CHUANG Chyh-Chong , PAN Fu-Ming , WU Shih-Hsiung , LIN Jung-Yaw
Published: August 2015

The seeds of the plant Trichosanthes anguina contain a type I ribosome-inactivating protein (RIP), designated trichoanguin, which was purified to apparent homogeneity by the combined use of ion-exchange chromatographies, i.e. first with DE-52 cellulose and then with CM-52 cellulose. The protein was found to be a glycoprotein with a molecular mass of 35 kDa and a pI of 9.1. It strongly inhibits the protein synthesis of rabbit reticulocyte lysate, with an IC50 of 0.08 nM, but only weakly that of HeLa cells, with an IC50 of 6 µM. Trichoanguin cleaves at the A4324 site of rat 28 S rRNA by its N-glycosidase activity. The cDNA of trichoanguin consists of 1039 nt and encodes an open reading frame coding for a polypeptide of 294 amino acid residues. The first 19 residues of this polypeptide encode a signal peptide sequence and the last 30 residues comprise an extension at its C-terminus. There are four potential glycosylation sites, located at Asn-51, Asn-65, Asn-201 and Asn-226. A comparison of the amino acid sequence of trichoanguin with those of RIPs such as trichosanthin, α-momorcharin, ricin A-chain and abrin A-chain reveals 55%, 48%, 36% and 34% identity respectively. Molecular homology modelling of trichoanguin indicates that its tertiary structure closely resembles those of trichosanthin and α-momorcharin. The large structural similarities might account for their common biological effects such as an abortifacient, an anti-tumour agent and anti-HIV-1 activities. Trichoanguin contains two cysteine residues, Cys-32 and Cys-155, with the former being likely to be located on the protein surface, which is directly amenable for conjugation with antibodies to form immunoconjugates. It is therefore conceivable that trichoanguin might be a better type I RIP than any other so far examined for the preparation of immunotoxins, with a great potential for application as an effective chemotherapeutic agent for the treatment of cancer.
Spontaneous activation of NADPH oxidase in a cell-free system: unexpected multiple effects of magnesium ion concentrations
Authors: CROSS Andrew R. , ERICKSON Richard W. , ELLIS Beverly A. , CURNUTTE John T.
Published: August 2015

The role of magnesium ions in the activation of NADPH oxidase has been investigated using flavocytochrome b-245 and either neutrophil cytosol or mixtures of recombinant p40phox, p47phox, p67phox and Rac2. Purified flavocytochrome b-245 is highly active (turnover number 120–150 mol of O2-/s per mol of cytochrome haem) in the absence of Mg2+, in marked contrast to neutrophil membranes or detergent-solubilized membranes, which have an absolute requirement for Mg2+ for NADPH oxidase activity. It was also found that Mg2+ affected the anionic amphiphile requirement for oxidase activation, and this was dependent on whether neutrophil cytosol or mixtures of recombinant cytosolic proteins were used in the assay. Unexpectedly we found that, using purified flavocytochrome b-245 and recombinant cytosolic proteins, NADPH oxidase undergoes spontaneous activation in the absence of anionic amphiphiles under Mg2+-free conditions. The results suggest that Mg2+ ions play an important role in NADPH oxidase function, perhaps stabilizing the 260 kDa complex of cytosolic phox proteins or the regulation of a guanine nucleotide-binding protein. We provide evidence that if the latter explanation is correct, the identity of the guanine nucleotide-binding protein is unlikely to be Rap1a.
The kinetics of acylation and deacylation of penicillin acylase from Escherichia coli ATCC 11105: evidence for lowered pKa values of groups near the catalytic centre
Authors: MORILLAS Manuel , GOBLE Martin L. , VIRDEN Richard
Published: August 2015

Penicillin G acylase catalysed the hydrolysis of 4-nitrophenyl acetate with a kcat of 0.8 s-1 and a Km of 10 µM at pH 7.5 and 20 °C. Results from stopped-flow experiments fitted a dissociation constant of 0.16 mM for the Michaelis complex, formation of an acetyl enzyme with a rate constant of 32 s-1 and a subsequent deacylation step with a rate constant of 0.81 s-1. Non-linear Van't Hoff and Arrhenius plots for these parameters, measured at pH 7.5, may be partly explained by a conformational transition affecting catalytic groups, but a linear Arrhenius plot for the ratio of the rate constant for acylation relative to KS was consistent with energy-compensation between the binding of the substrate and catalysis of the formation of the transition state. At 20 °C, the pH-dependence of kcat was similar to that of kcat/Km, indicating that formation of the acyl-enzyme did not affect the pKa values (6.5 and 9.0) of an acidic and basic group in the active enzyme. The heats of ionization deduced from values of pKa for kcat, which measures the rate of deacylation, are consistent with α-amino and guanidinium groups whose pKa values are decreased in a non-polar environment. It is proposed that, for catalytic activity, the α-amino group of the catalytic SerB1 and the guanidinium group of ArgB263 are required in neutral and protonated states respectively.
Phosphorylation by protein kinase C decreases catalytic activity of avian phospholipase C-β
Authors: FILTZ Theresa M. , CUNNINGHAM Michelle L. , STANIG Kara J. , PATERSON Andrew , HARDEN T. Kendall
Published: August 2015

The potential role of protein kinase C (PKC)-promoted phosphorylation has been examined in the G-protein-regulated inositol lipid signalling pathway. Incubation of [32P]Pi-labelled turkey erythrocytes with either the P2Y1 receptor agonist 2-methylthioadenosine triphosphate (2MeSATP) or with PMA resulted in a marked increase in incorporation of 32P into the G-protein-activated phospholipase C PLC-βT. Purified PLC-βT also was phosphorylated by PKC in vitro to a stoichiometry (mean±S.E.M.) of 1.06±0.2 mol of phosphate/mol of PLC-βT. Phosphorylation by PKC was isoenzyme-specific because, under identical conditions, mammalian PLC-β2 also was phosphorylated to a stoichiometry near unity, whereas mammalian PLC-β1 was not phosphorylated by PKC. The effects of PKC-promoted phosphorylation on enzyme activity were assessed by reconstituting purified PLC-βT with turkey erythrocyte membranes devoid of endogenous PLC activity. Phosphorylation resulted in a decrease in basal activity, AlF4--stimulated activity, and activity stimulated by 2MeSATP plus guanosine 5´-[γ-thio]triphosphate in the reconstituted membranes. The decreases in enzyme activities were proportional to the extent of PKC-promoted phosphorylation. Catalytic activity assessed by using mixed detergent/phospholipid micelles also was decreased by up to 60% by phosphorylation. The effect of phosphorylation on Gqα-stimulated PLC-βT in reconstitution experiments with purified proteins was not greater than that observed on basal activity alone. Taken together, these results illustrate that PKC phosphorylates PLC-βT in vivo and to a physiologically relevant stoichiometry in vitro. Phosphorylation is accompanied by a concomitant loss of enzyme activity, reflected as a decrease in overall catalytic activity rather than as a specific modification of G-protein-regulated activity.
Isolation and complete covalent structure of liver microsomal paraoxonase
Authors: OZOLS Juris
Published: August 2015

Paraoxonase (PON1) is a serum esterase exclusively associated with high-density lipoproteins; it might confer protection against coronary artery disease by destroying pro-inflammatory oxidized lipids in oxidized low-density lipoproteins. Here I show that rabbit liver microsomes contain a PON analogue (MsPON) and report the isolation and complete covalent structure of MsPON. In detergent-solubilized microsomes, MsPON co-purifies with the microsomal triacylglycerol transfer protein (MTP) complex. MsPON was separated from the complex and purified to homogeneity under non-denaturing conditions. Automated sequence analysis of intact MsPON and peptides obtained from enzymic and chemical cleavages led to the elucidation of the complete covalent structure of MsPON. The protein is a single polypeptide consisting of 350 residues. The sequence of rabbit liver microsomal MsPON is 60% identical with that of rabbit serum PON1, and 84% identical with the sequence predicted by a human cDNA of unknown function, designated PON3. MsPON has a hydrophobic segment at the N-terminus that might serve to anchor the protein to the microsomal membrane or to the MTP complex. Unlike in the serum enzyme, two potential N-glycan acceptor sites in MsPON are not glycosylated. An absence of N-glycans was also indicated in the rabbit liver MTP. MsPON has a single free cysteine residue at position 38 and a disulphide bond between Cys-279 and Cys-348. The microsomal enzyme lacks three residues at the N-terminus that are present in the serum protein. MsPON lacks four residues at the C-terminus that are present in the rabbit serum protein but absent from human serum PON1. On the basis of the observation that MsPON displays a high degree of similarity with serum PON1, it is proposed that MsPON might have a function related to that of PON1 in serum high-density lipoprotein complexes.
Calorimetric studies on the stability of the ribosome-inactivating protein abrin II: effects of pH and ligand binding
Authors: KRUPAKAR Jayarapu , SWAMINATHAN Chittoor P. , DAS Puspendu K. , SUROLIA Avadhesha , PODDER Sunil K.
Published: August 2015

The effects of pH and ligand binding on the stability of abrin II, a heterodimeric ribosome-inactivating protein, and its subunits have been studied using high-sensitivity differential scanning calorimetry. At pH 7.2, the calorimetric scan consists of two transitions, which correspond to the B-subunit [transition temperature (Tm) 319.2 K] and the A-subunit (Tm 324.6 K) of abrin II, as also confirmed by studies on the isolated A-subunit. The calorimetric enthalpy of the isolated A-subunit of abrin II is similar to that of the higher-temperature transition. However, its Tm is 2.4 K lower than that of the higher-temperature peak of intact abrin II. This indicates that there is some interaction between the two subunits. Abrin II displays increased stability as the pH is decreased to 4.5. Lactose increases the Tm values as well as the enthalpies of both transitions. This effect is more pronounced at pH 7.2 than at pH 4.5. This suggests that ligand binding stabilizes the native conformation of abrin II. Analysis of the B-subunit transition temperature as a function of lactose concentration suggests that two lactose molecules bind to one molecule of abrin II at pH 7.2. The presence of two binding sites for lactose on the abrin II molecule is also indicated by isothermal titration calorimetry. Plotting ΔHm (the molar transition enthalpy at Tm) against Tm yielded values for ΔCp (change in excess heat capacity) of 27±2 kJ·mol-1·K-1 for the B-subunit and 20±1 kJ·mol-1·K-1 for the A-subunit. These values have been used to calculate the thermal stability of abrin II and to surmise the mechanism of its transmembrane translocation.
Efficient binding of regulated secretory protein aggregates to membrane phospholipids at acidic pH
Authors: LAINÉ Jean , LeBEL Denis
Published: August 2015

Some regulated secretory proteins are thought to be targeted to secretory granules through an acidic-dependent aggregation in the trans-Golgi network. In this report we use pancreatic zymogens, a paradigm of regulated proteins, to test this hypothesis, because they qualitatively aggregate upon acidification in vitro. Pig zymogens were found to start to aggregate significantly at pH ∼ 6.0, a pH slightly lower than that at which rat zymogens aggregate, but still compatible with the pH of the cell-sorting compartments. When pig zymogen granule membranes were mixed with the zymogens in the aggregation assay, membranes that normally floated on 1 M sucrose were observed to be pelleted by the aggregating zymogens. Rat membranes were pelleted by pig zymogens and vice versa. Igs, typical constitutively secreted proteins, which needed chemical cross-linking to serve as an aggregated protein control, pelleted membranes almost independently of pH. Corresponding cross-linked zymogen-binding ability and pH dependence was unaffected by the chemical modification. Membranes treated with sodium carbonate, pH 11, or with protease K, were still pelleted by zymogens, suggesting that the aggregated zymogens bound to membrane lipids. This hypothesis was confirmed by the efficient pelleting of unilamellar vesicles composed of granule membrane lipids. Vesicles composed of single classes of phospholipids were also pelleted, but with various efficacies. We conclude that pancreatic zymogen aggregates, formed under the acidic conditions of the secretory pathway sorting compartments, have the capacity to bind firmly to membranes through their phospholipid constituents.
Overexpression of spermidine/spermine N1-acetyltransferase under the control of mouse metallothionein I promoter in transgenic mice: evidence for a striking post-transcriptional regulation of transgene expression by a polyamine analogue
Authors: SUPPOLA Suvikki , PIETILÄ Marko , PARKKINEN Jyrki J. , KORHONEN Veli-Pekka , ALHONEN Leena , HALMEKYTÖ Maria , PORTER Carl W. , JÄNNE Juhani
Published: August 2015

We recently generated a transgenic mouse line overexpressing spermidine/spermine N1-acetyltransferase (SSAT) gene under its own promoter. The tissue polyamine pools of these animals were profoundly affected and the mice were hairless from early age. We have now generated another transgenic-mouse line overexpressing the SSAT gene under the control of a heavy-metal-inducible mouse metallothionein I (MT) promoter. Even in the absence of heavy metals, changes in the tissue polyamine pools indicated that a marked activation of polyamine catabolism had occurred in the transgenic animals. As with the SSAT transgenic mice generated previously, the mice of the new line (MT-SSAT) suffered permanent hair loss, but this occurred considerably later than in the previous SSAT transgenic animals. Liver was the most affected tissue in the MT–SSAT transgenic animals, revealed by putrescine overaccumulation, significant decrease in spermidine concentration and > 90% reduction in the spermine pool. Even though hepatic SSAT mRNA accumulated to massive levels in non-induced transgenic animals, SSAT activity was only moderately elevated. Administration of ZnSO4 further elevated the level of hepatic SSAT message and induced enzyme activity, but not more than 2- to 3-fold. Treatment of the transgenic animals with the polyamine analogue N1,N11-diethylnorspermine (DENSPM) resulted in an immense induction, more than 40000-fold, of enzyme activity in the liver of transgenic animals, and minor changes in the SSAT mRNA level. Liver spermidine and spermine pools were virtually depleted within 1–2 days in response to the treatment with the analogue. The treatment also resulted in a marked mortality (up to 60%) among the transgenic animals which showed ultrastructural changes in the liver, most notably mitochondrial swelling, one of the earliest signs of cell injury. These results indicated that, even without its own promoter, SSAT is powerfully induced by the polyamine analogue through a mechanism that appears to involve a direct translational and/or heterogenous nuclear RNA processing control. It is likewise significant that overexpression of SSAT renders the animals extremely sensitive to polyamine analogues.
Differential regulation of the human nidogen gene promoter region by a novel cell-type-specific silencer element
Authors: ZEDLACHER Marlies , SCHMOLL Marion , ZIMMERMANN Katrin , HORSTKORTE Oliver , NISCHT Roswitha
Published: August 2015

Transfection analyses of the human nidogen promoter region in nidogen-producing fibroblasts from adult skin revealed multiple positive and negative cis-acting elements controlling nidogen gene expression. Characterization of the positive regulatory domains by gel mobility-shift assays and co-transfection studies in Drosophila SL2 cells unequivocally demonstrated that Sp1-like transcription factors are essential for a high expression of the human nidogen gene. Analysis of the negative regulatory domains identified a novel silencer element between nt -1333 and -1322, which is bound by a distinct nuclear factor, by using extracts from adult but not from embryonal fibroblasts. In embryonal fibroblasts, which express significantly higher amounts of nidogen mRNA as compared with adult fibroblasts, this inhibitory nidogen promoter region did not affect nidogen and SV40 promoter activities. The silencer element seems to be active only in nidogen-producing cells. Therefore this regulatory element might function in vivo to limit nidogen gene expression in response to external stimuli. However, none of the identified regulatory elements, including the silencer, contribute significantly to cell-specific expression of the human nidogen gene. Instead we provide evidence that gene expression in epidermal keratinocytes that are not producing nidogen is repressed by methylation-specific and chromatin-dependent mechanisms.
Synergistic activation of JNK/SAPK by interleukin-1 and platelet-derived growth factor is independent of Rac and Cdc42
Authors: DAVIS W. , STEPHENS Leonard R. , HAWKINS Phillip T. , SAKLATVALA Jeremy
Published: August 2015

The c-Jun N-terminal kinases (JNKs) are activated strongly by inflammatory cytokines and environmental stresses, but only weakly by growth factors. Here we show that platelet-derived growth factor (PDGF) strongly potentiates activation of JNK by interleukin 1 (IL-1) in human fibroblasts and a pig aortic endothelial (PAE) cell line. This synergistic activation of JNK by IL-1 and PDGF was unaffected by bacterial toxins that inactivate Rho proteins and Ras. Since Rho proteins have been implicated in JNK activation, their possible involvement was investigated further using stably expressed, inducible N17 or V12 mutants in PAE cell lines. N17 Rac non-selectively reduced JNK activity by 30% in resting or stimulated cells (IL-1 alone, or with PDGF). N17 Cdc42 had no effect. V12 Rac weakly activated JNK and synergized with IL-1, but not with PDGF. V12 Cdc42 weakly activated JNK, but synergized with PDGF and not IL-1. Our results imply that Rho GTPases are not directly involved in mediating IL-1-induced JNK activation, or in the potentiation of this activation by PDGF.
Heterogeneous nuclear ribonucleoprotein D0B is a sequence-specificDNA-binding protein
Authors: TOLNAY Mate , VERESHCHAGINA Lyudmila A. , TSOKOS George C.
Published: August 2015

Complement receptor 2 (CR2) is important in the regulation of the B lymphocyte response; the regulation of its expression is therefore of central importance. We recently reported that a 42 kDa heterogeneous nuclear ribonucleoprotein (hnRNP) is involved in the transcriptional regulation of the human CR2 gene [Tolnay, Lambris and Tsokos (1997) J. Immunol. 159, 5492–5501]. We cloned the cDNA encoding this protein and found it to be identical with hnRNP D0B, a sequence-specific RNA-binding protein. By using a set of mutated oligonucleotides, we demonstrated that the recombinant hnRNP D0B displays sequence specificity for double-stranded oligonucleotide defined by the CR2 promoter. We conducted electrophoretic mobility-shift assays to estimate the apparent Kd of hnRNP D0B for the double-stranded DNA motif and found it to be 59 nM. Interestingly, hnRNP D0B displayed affinities of 28 and 18 nM for the sense and anti-sense strands of the CR2 promoter-defined oligonucleotide respectively. The significantly greater binding affinity of hnRNP D0B for single-stranded DNA than for double-stranded DNA suggests that the protein might melt the double helix. The intranuclear concentration of sequence-specific protein was estimated to be 250–400 nM, indicating that the protein binds to the CR2 promoter in vivo. Co-precipitation of a complex formed in vivo between hnRNP D0B and the TATA-binding protein demonstrates that hnRNP D0B interacts with the basal transcription apparatus. Our results suggest a new physiological role for hnRNP D0B that involves binding to double- and single-stranded DNA sequences in a specific manner and functioning as a transcription factor.
Cloning and functional characterization of the 5′-flanking region of the human bone morphogenetic protein-2 gene
Authors: SUGIURA Takeyuki
Published: August 2015

Bone morphogenetic protein-2 (BMP-2) is involved in bone formation, organogenesis or pattern formation during development. The expression of BMP-2 is regulated accurately and coordinately with that of other transforming growth factor-β (TGF-β) superfamily members. To elucidate the mechanism underlying the regulation of BMP-2 expression, a 6.7 kb SpeI–SalI fragment, from the P1 phage library, encompassing the 5´-flanking region of the human BMP-2 gene, was isolated and sequenced. Transcription start sites were mapped by the 5´-rapid amplification of cDNA ends (RACE) method. It has been found that the human BMP-2 gene contains, largely, two promoter regions surrounded by GC-rich sequences with several Sp1 consensus motifs. The proximal promoter possesses a single start site, whereas several start sites are clustered in the distal promoter region. Neither TATA nor CAAT consensus sequences are found in the proximity of the start sites for either promoter. Interestingly, in no case is the transcription-initiation site common between the human and mouse BMP-2 genes, although the sequence of the BMP-2 gene is well conserved in the promoter region between two species. Transient transfection experiments with the reporter fused with various lengths of the BMP-2 promoter sequence demonstrated that there exist enhancer elements in an 1.1 kb GC-rich fragment covering both promoter regions. It is noteworthy that the enhancer elements are 5´-flanked by a 790 bp strong repressor element that is characterized by numerous AT stretches. This intriguing organization may be amenable to the tight control of the expression of BMP-2 that is essential for development or bone morphogenesis.
Contributions to gene activation by multiple functions of Bicoid
Authors: MA Xiuguang , YUAN Dong , SCARBOROUGH Tom , MA Jun
Published: August 2015

Bicoid is a Drosophila morphogenetic protein required for the development of anterior structures in the embryo. To gain a better understanding of how Bicoid works as a transcriptional activator, we systematically analysed various functions of Bicoid required for gene activation. We provide evidence suggesting that Bicoid is an intrinsically weak activator. First, our biochemical experiments demonstrate that the Bicoid–DNA complexes are very unstable, suggesting a weak DNA-binding function of Bicoid. This idea is further supported by our experiments demonstrating that the same number of LexA–Bicoid fusion molecules can activate transcription more effectively from LexA sites than from Bicoid sites. Secondly, we demonstrate that transcriptional activation by the weak activator Bicoid is readily influenced by the local enhancer environment. These influences are decreased when the Bicoid function is enforced by attaching to it either a known dimerization domain or the strong activation domain VP16. VP16 can also compensate for the loss of some Bicoid sites in an enhancer element. Our experiments demonstrate that the outcome of transcriptional activation by Bicoid is determined by multiple weak functions that are interconnected, a finding that can further help us to understand how this morphogenetic protein achieves its molecular functions.
Hyperoxia induces the neuronal differentiated phenotype of PC12 cells via a sustained activity of mitogen-activated protein kinase induced by Bcl-2
Authors: KATOH Shinsuke , MITSUI Youji , KITANI Kenichi , SUZUKI Takahiko
Published: August 2015

We previously reported that rat pheochromocytoma PC12 cells express the neuronal differentiated phenotype under hyperoxia through the production of reactive oxygen species (ROS). In the present study, we found that in this phenotype, Bcl-2, an apoptosis inhibitor, affects mitogen-activated protein (MAP)-kinase activity, which is known as a key enzyme of the signal-transduction cascade for differentiation. When PC12 cells were cultured under hyperoxia, a rapid increase in MAP-kinase activity, including that of both p42 and p44, was observed. Although the activity level then decreased quickly, activity higher than the control level was observed for 48 h. PD98059, an inhibitor of MAP kinase, suppressed the hyperoxia-induced neurite extensions, suggesting the involvement of MAP-kinase activity in the mechanism of differentiation induced by ROS. An elevation of Bcl-2 expression was observed after culturing PC12 cells for 24 h under hyperoxia. This Bcl-2 elevation was not affected by treatment with PD98059, suggesting that it did not directly induce neurite extension under hyperoxia. However, the blockade of the Bcl-2 elevation by an antisense oligonucleotide inhibited the sustained MAP-kinase activity and neurite extensions under hyperoxia. Further, in PC12 cells highly expressing Bcl-2, the sustained MAP-kinase activity and neurite extensions under hyperoxia were enhanced. These results suggested that MAP kinase is activated through the production of ROS, and the subsequent elevation of Bcl-2 expression sustains the MAP-kinase activity, resulting in the induction of the neuronal-differentiation phenotype of PC12 cells under hyperoxia.
Dephosphorylation of the catenins p120 and p100 in endothelial cells in response to inflammatory stimuli
Authors: RATCLIFFE Marianne J. , SMALES Caroline , STADDON James M.
Published: August 2015

Inflammatory mediators such as histamine and thrombin increase the tight-junction permeability of endothelial cells. Tight-junction permeability may be independently controlled, but is dependent on the adherens junction, where adhesion is achieved through homotypic interaction of cadherins, which in turn are associated with cytoplasmic proteins, the catenins. p120, also termed p120cas/p120ctn, and its splice variant, p100, are catenins. p120, originally discovered as a substrate of the tyrosine kinase Src, is also a target for a protein kinase C-stimulated pathway in epithelial cells, causing its serine/threonine dephosphorylation. The present study shows that pharmacological activation of protein kinase C stimulated a similar pathway in endothelial cells. Activation of receptors for agents such as histamine (H1), thrombin and lysophosphatidic acid in the endothelial cells also caused serine/threonine dephosphorylation of p120 and p100, suggesting physiological relevance. However, protein kinase C inhibitors, although blocking the effect of pharmacological activation of protein kinase C, did not block the effects due to receptor activation. Calcium mobilization and the myosin-light-chain-kinase pathway do not participate in p120/p100 signalling. In conclusion, endothelial cells possess protein kinase C-dependent and -independent pathways regulating p120/p100 serine/threonine phosphorylation. These data describe a new connection between inflammatory agents, receptor-stimulated signalling and pathways potentially influencing intercellular adhesion in endothelial cells.
Identification and characterization of a recombinant metallothionein protein from a marine alga, Fucus vesiculosus
Authors: MORRIS Ceri A. , NICOLAUS Beate , SAMPSON Viola , HARWOOD John L. , KILLE Peter
Published: August 2015

A cDNA library was constructed from macroalgae adapted to prolonged elevated environmental copper levels. To investigate the possible existence of a metallothionein (MT) gene, the library was screened with degenerate probes designed using plant MT cysteine-rich motifs. A gene was identified (1229 bp) with a putative open reading frame (204 bp) encoding a 67-amino-acid protein exhibiting several characteristic features of MT proteins, including 16 cysteine residues (24%) and only one aromatic residue. Although the protein sequence showed high identity with plant and invertebrate MTs, it contained a unique ‘linker ’ region (14 amino acid residues) between the two putative metal-binding domains which contained no cysteine residues. This extended linker is larger than the tripeptide found in archetypal vertebrate MTs, but does not conform either with the 40-amino-acid linkers commonly found in plant MT sequences. An S-peptide Fucus MT fusion protein expressed in Escherichia coli exhibited a relative molecular mass of ∼ 14 kDa. The recombinant fusion bound seven Cd ions, of which 50% were dissociated at pH 4.1. Under anaerobic conditions, the Cd ions were displaced by Cu(I), which associated with the protein at a ratio of 13:1. Laboratory exposure of F. vesiculosus to elevated copper resulted in induction of the MT gene. Thus this paper describes, for the first time, an MT gene identified from macroalgae which is induced by copper exposure and whose encoded protein product binds cadmium and copper.
NMR structure and dynamics of monomeric neutrophil-activating peptide 2
Authors: YOUNG Helen , ROONGTA Vikram , DALY Thomas J. , MAYO Kevin H.
Published: August 2015

Neutrophil-activating peptide 2 (NAP-2), which demonstrates a range of proinflammatory activities, is a 72-residue protein belonging to the α-chemokine family. Although NAP-2, like other α-chemokines, is known to self-associate into dimers and tetramers, it has been shown that the monomeric form is physiologically active. Here we investigate the solution structure of monomeric NAP-2 by multi-dimensional 1H-NMR and 15N-NMR spectroscopy and computational modelling. The NAP-2 monomer consists of an amphipathic, triple-stranded, anti-parallel β-sheet on which is folded a C-terminal α-helix and an aperiodic N-terminal segment. The backbone fold is essentially the same as that found in other α-chemokines. 15N T1, T2 and nuclear Overhauser effects (NOEs) have been measured for backbone NH groups and used in a model free approach to calculate order parameters and conformational exchange terms that map out motions of the backbone. N-terminal residues 1 to 17 and the C-terminus are relatively highly flexible, whereas the β-sheet domain forms the most motionally restricted part of the fold. Conformational exchange occurring on the millisecond time scale is noted at the top of the C-terminal helix and at proximal residues from β-strands 1 and 2 and the connecting loop. Dissociation to the monomeric state is apparently responsible for increased internal mobility in NAP-2 compared with dimeric and tetrameric states in other α-chemokines.
Identification of an enhancer element of class Pi glutathione S-transferase gene required for expression by a co-planar polychlorinated biphenyl
Authors: MATSUMOTO Michi , IMAGAWA Masayoshi , AOKI Yasunobu
Published: August 2015

3,3´,4,4´,5-Pentachlorobiphenyl (PenCB), one of the most toxic co-planar polychlorinated biphenyl congeners, specifically induces class Pi glutathione S-transferase (GSTP1) as well as cytochrome P-450 1A1 in primary cultured rat liver parenchymal cells [Aoki, Matsumoto and Suzuki (1993) FEBS Lett. 333, 114–118]. However, the 5´-flanking sequence of the GSTP1 gene does not contain a xenobiotic responsive element, to which arylhydrocarbon receptor binds. Using a chloramphenicol acetyltransferase assay we demonstrate here that the enhancer termed GSTP1 enhancer I (GPEI) is necessary for the stimulation by PenCB of GSTP1 gene expression in primary cultured rat liver parenchymal cells. GPEI is already known to contain a dyad of PMA responsive element-like elements oriented palindromically. It is suggested that a novel signal transduction pathway activated by PenCB contributes to the stimulation of GSTP1 expression.
UV-A-induced decrease in nuclear factor-κB activity in human keratinocytes
Authors: DJAVAHERI-MERGNY Mojgan , GRAS Marie-Pierre , MERGNY Jean-Louis , DUBERTRET Louis
Published: August 2015

Previous reports have demonstrated an increase in nuclear factor-κB (NF-κB) activity in response to UV radiation. These studies have essentially focused on the DNA-damaging fraction of solar UV radiation (UV-B and UV-C). In contrast, the effects of UV-A radiation (320–400 nm) on NF-κB are not well known. In this study, we present evidence that UV-A radiation induces a marked decrease in NF-κB DNA-binding activity in NCTC 2544 human keratinocytes. In addition, NCTC 2544 keratinocytes pretreated with UV-A fail to respond to NF-κB inducers. Moreover, UV-A radiation induces a decrease in NF-κB-driven luciferase reporter gene expression in NCTC 2544 keratinocytes. The expression of the gene encoding IκBα (IκB is the NF-κB inhibitor), which is closely associated with NF-κB activity, is also reduced (3-fold) upon UV-A treatment. Our results indicate that the UV-A-induced decrease in NF-κB DNA-binding activity is associated with a decrease in the levels of the p50 and p65 protein subunits. This is the first evidence that an oxidative stress, such as UV-A radiation, may induce a specific decrease in NF-κB activity in mammalian cells, probably through degradation of NF-κB protein subunits. These findings suggest that UV-A could modulate the NF-κB-dependent gene expression.
Activation of integrin-β3-associated syk in platelets
Authors: SARKAR Sibaji , ROONEY Michael M. , LORD Susan T.
Published: August 2015

Published data suggest that the tyrosine kinase syk participates in platelet signalling through the integrin αIIbβ3. Our data show an association of syk and integrin β3 in immunoprecipitates from unstimulated and stimulated platelets. We detected syk in anti-β3 precipitates and, conversely, β3 in anti-syk precipitates. In vitrokinase assays with anti-β3 precipitates demonstrated that syk activity was enhanced in ADP-stimulated platelets.
The role of Saccharomyces cerevisiae Met1p and Met8p in sirohaem and cobalamin biosynthesis
Authors: RAUX Evelyne , McVEIGH Treasa , PETERS Sarah E. , LEUSTEK Thomas , WARREN Martin J.
Published: August 2015

MET1 and MET8 mutants of Saccharomyces cerevisiae can be complemented by Salmonella typhimurium cysG, indicating that the genes are involved in the transformation of uroporphyrinogen III into sirohaem. In the present study, we have demonstrated complementation of defined cysG mutants of Sal. typhimurium and Escherichia coli, with either MET1 or MET8 cloned in tandem with Pseudomonas denitrificans cobA. The conclusion drawn from these experiments is that MET1 encodes the S-adenosyl-l-methionine uroporphyrinogen III transmethylase activity, and MET8 encodes the dehydrogenase and chelatase activities (all three functions are encoded by Sal. typhimurium and E. coli cysG). MET8 was further cloned into pET14b to allow expression of the protein with an N-terminal His-tag. After purification, the functions of the His-tagged Met8p were studied in vitro by assay with precorrin-2 in the presence of NAD+ and Co2+. The results demonstrated that Met8p acts as a dehydrogenase and chelatase in the biosynthesis of sirohaem. Moreover, despite the fact that S. cerevisiae does not make cobalamins de novo, we have shown also that MET8 is able to complement cobalamin cobaltochelatase mutants and have revealed a subtle difference in the early stages of the anaerobic cobalamin biosynthetic pathways between Sal. typhimurium and Bacillus megaterium.