bluuebunny/crossref_metadata_2025 huggingface dataset:
cADP-ribose potentiates cytosolic Ca2+ elevation and Ca2+ entry via L-type voltage-activated Ca2+ channels in NG108-15 neuronal cells
Authors: HASHII Minako , MINABE Yoshio , HIGASHIDA Haruhiro
Published: August 2015

The effects of cADP-ribose (cADPR), a metabolite of β-NAD+, on the elevation of cytoplasmic free Ca2+ concentration ([Ca2+]i) and Ca2+ influx through voltage-activated Ca2+ channels (VACCs) were studied in NG108-15 neuroblastoma×glioma hybrid cells. NG108-15 cells were pre-loaded with fura-2 and whole-cell patch-clamped. Application of cADPR through patch pipettes did not by itself trigger any [Ca2+]i rise at the resting membrane potential. A rise in [Ca2+]i was evoked upon sustained membrane depolarization, and was significantly larger in cADPR-infused cells than in non-infused cells. This potentiation in the [Ca2+]i elevation was reproduced by infusion of β-NAD+, and was blocked by 8-bromo-cADPR and antagonized by external application of ryanodine or by pretreatment of cells with FK506. Nicotinamide inhibited β-NAD+-induced, but not cADPR-elicited, potentiation. [Ca2+]i increases or Ca2+ influx, measured by Mn2+ quenching, elicited by the same protocol of depolarization was blocked completely by nifedipine but not by Ω-conotoxin. Ca2+ influx in cADPR- or β-NAD+-infused cells was steeper and greater than that in control cells, and was inhibited partly by ryanodine. In contrast, ryanodine accelerated Ca2+ influx in non-infused cells. These results show that cADPR amplifies both depolarization-induced [Ca2+]i increase and Ca2+ influx through L-type VACCs. These results suggest that cADPR functions on ryanodine receptors as a direct agonist and also interacts with L-type VACCs as an indirect agonist, i.e. via a retrograde signal.
Proteins isolated from lucerne roots by affinity chromatography with sugars analogous to Nod factor moieties
Authors: MINIC Zoran , LEPROUST-LECOESTER Lydie , LAPORTE Jean , DE KOUCHKOVSKY Yaroslav , BROWN Spencer C.
Published: August 2015

Nod factors are important elicitors in legume-bacterium symbiosis. Any candidate plant receptor(s) for these lipo-oligosaccharides can be expected to show some lectin-like properties. A novel protein (P60), a native tetramer with 60 kDa monomers, has been isolated from a membrane fraction of Medicago sativa (lucerne, alfalfa) roots by using affinity chromatography with either GlcNAc or N,Nʹ,Nʹʹ-triacetyl-(1 → 4)-β-D-chitotriose [(GlcNAc)3] grafted to agarose beads as the matrix and, in a second step, Sephadex G-200 gel filtration. With (GlcNAc)3-agarose an additional protein of 78 kDa was isolated. P60 showed haemagglutination activity with specificity for GalNAc, GalN, GlcNAc and GlcN. Binding experiments with radioactive GlcNAc gave a Kd of 95 nM and one binding site per monomer of P60; Nod factor competed strongly for this binding. In native PAGE, protein incubated with O-sulphated Nod factors had a higher electrophoretic mobility as a consequence of binding. However, the largest modification was observed with a natural mixture of Nod factors, containing the O-acetylated and O-sulphated tetrasaccharidic NodRm-IV(Ac,S) (in which Ac stands for an O-acetylated group at the non-reducing end and S for O-sulphation at the reducing end) in addition to the non-O-acetylated NodRm-IV(S) (which alone had little effect) and NodRm-V(S). The native PAGE study was also performed with known lectins from other sources, but only the 34 kDa lectin of Phytolacca americana (pokeweed) showed any such interaction, although without discrimination between Nod factors. Finally, one peptide of each isolated protein was sequenced; the peptide from P60 showed some similarity with dihydrolipoamide dehydrogenase and ferric leghaemoglobin reductase, whereas the peptide from P78 was identical with an analogous region of 70 kDa heat shock protein.
Endolyn is a mucin-like type I membrane protein targeted to lysosomes by its cytoplasmic tail
Authors: IHRKE Gudrun , GRAY Sally R. , LUZIO J. Paul
Published: August 2015

Endolyn (endolyn-78) is a membrane protein found in lysosomal and endosomal compartments of mammalian cells. Unlike ‘classical’ lysosomal membrane proteins, such as lysosome-associated membrane protein (lamp)-1, it is also present in a subapical compartment in polarized WIF-B hepatocytes. The structural features that determine sorting of endolyn are unknown. We have identified a rat endolyn cDNA by expression screening. The cDNA encodes a ubiquitously expressed type I membrane protein with a short cytoplasmic tail of 13 amino acids and many putative sites for N- and O-linked glycosylation in the predicted luminal domain. Endolyn is closely related to two human mucin-like proteins, multi-glycosylated core protein (MGC)-24 and CD164 (MGC-24v), expressed in gastric carcinoma cells and bone marrow stromal and haematopoietic precursor cells respectively. The predicted transmembrane and cytoplasmic tail domains of endolyn, as well as parts of its luminal domain, also show some similarities with lamp-1 and lamp-2. Like these and other known lysosomal membrane proteins, endolyn contains a YXXØ motif at the C-terminus of its cytoplasmic tail (where Ø is a bulky hydrophobic amino acid), but with no preceding glycine. Nonetheless, the last ten amino acids of this tail, when transplanted on to human CD8, caused efficient targeting of the chimaeric protein to endosomes and lysosomes in transfected normal rat kidney cells.
Hydrolyses of α- and β-cellobiosyl fluorides by Cel6A (cellobiohydrolase II) of Trichoderma reesei and Humicola insolens
Authors: BECKER Dieter , JOHNSON Karin S. H. , KOIVULA Anu , SCHÜLEIN Martin , SINNOTT Michael L.
Published: August 2015

We have measured the hydrolyses of α- and β-cellobiosyl fluorides by the Cel6A [cellobiohydrolase II (CBHII)] enzymes of Humicola insolens and Trichoderma reesei, which have essentially identical crystal structures [Varrot, Hastrup, Schülein and Davies (1999) Biochem. J. 337, 297-304]. The β-fluoride is hydrolysed according to Michaelis-Menten kinetics by both enzymes. When the ~ 2.0% of β-fluoride which is an inevitable contaminant in all preparations of the α-fluoride is hydrolysed by Cel7A (CBHI) of T. reesei before initial-rate measurements are made, both Cel6A enzymes show a sigmoidal dependence of rate on substrate concentration, as well as activation by cellobiose. These kinetics are consistent with the classic Hehre resynthesis-hydrolysis mechanism for glycosidase-catalysed hydrolysis of the ‘wrong’ glycosyl fluoride for both enzymes. The Michaelis-Menten kinetics of α-cellobiosyl fluoride hydrolysis by the T. reesei enzyme, and its inhibition by cellobiose, previously reported [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 883-888] are withdrawn. 1H NMR monitoring of the hydrolysis of α-cellobiosyl fluoride by both enzymes reveals that in neither case is α-cellobiosyl fluoride released into solution in detectable quantities, but instead it appears to be hydrolysed in the enzyme active site as soon as it is formed.
Development and characterization of novel potent and stable inhibitors of endopeptidase EC 3.4.24.15
Authors: SHRIMPTON Corie N. , ABBENANTE Giovanni , LEW Rebecca A. , SMITH A. Ian
Published: August 2015

Solid-phase synthesis was used to prepare a series of modifications to the selective and potent inhibitor of endopeptidase EC 3.4.24.15 (EP24.15), N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP), which is degraded at the Ala-Tyr bond, thus severely limiting its utility in vivo. Reducing the amide bond between the Ala and Tyr decreased the potency of the inhibitor to 1/1000. However, the replacement of the second alanine residue immediately adjacent to the tyrosine with α-aminoisobutyric acid gave a compound (JA-2) that was equipotent with cFP, with a Ki of 23 nM. Like cFP, JA-2 inhibited the closely related endopeptidase EC 3.4.24.16 1/20 to 1/30 as potently as it did EP24.15, and did not inhibit the other thermolysin-like endopeptidases angiotensin-converting enzyme, endothelin-converting enzyme and neutral endopeptidase. The biological stability of JA-2 was investigated by incubation with a number of membrane and soluble sheep tissue extracts. In contrast with cFP, JA-2 remained intact after 48 h of incubation with all tissues examined. Further modifications to the JA-2 compound failed to improve the potency of this inhibitor. Hence JA-2 is a potent, EP24.15-preferential and biologically stable inhibitor, therefore providing a valuable tool for further assessing the biological functions of EP24.15.
Mitochondrial aspartate aminotransferase: direction of a single protein with two distinct functions to two subcellular sites does not require alternative splicing of the mRNA
Authors: BRADBURY Michael W. , BERK Paul D.
Published: August 2015

During differentiation of mouse 3T3-L1 fibroblasts to an adipocyte phenotype, the mitochondrial isoform of aspartate aminotransferase accumulates on the plasma membrane. The determination of whether this reflects translation of an alternatively spliced message lacking the mitochondrial leader sequence required cloning of the enzyme's uncommon a allele, for which these cells are homozygous. The 1.4-kb cDNA sequence of the a allele was obtained from oligo-dT-primed reverse-transcriptase PCR products amplified from FVB mouse RNA. It differed from the b allele at only 2 bp and one amino acid. By contrast, gene-specific primers generated an additional 1.4-kb fragment that differed from the b allele by ≈ 1% of nucleotides, encoding four amino acid substitutions. This sequence proved to represent a recently diverged processed pseudogene. The presence of such pseudogenes can complicate interpretation of expressed-sequence-tag data and single-nucleotide-polymorphism genotyping studies. Using probes derived from the a allele, RNase protection analyses indicated that only a single message for the enzyme was present in 3T3-L1 fibroblasts and adipocytes, despite differences in subcellular protein distribution.
L-Pipecolic acid oxidase, a human enzyme essential for the degradation of L-pipecolic acid, is most similar to the monomeric sarcosine oxidases
Authors: DODT Gabriele , KIM Do G. , REIMANN Sylvia A. , REUBER Bernadette E. , MCCABE Katherine , GOULD Stephen J. , MIHALIK Stephanie J.
Published: August 2015

L-Pipecolic acid oxidase activity is deficient in patients with peroxisome biogenesis disorders (PBDs). Because its role, if any, in these disorders is unknown, we cloned the associated human gene and expressed its protein product. The cDNA was cloned with the use of a reverse genetics approach based on the amino acid sequence obtained from purified L-pipecolic acid oxidase from monkey. The complete cDNA, obtained by conventional library screening and 5ʹ rapid amplification of cDNA ends, encompassed an open reading frame of 1170 bases, translating to a 390-residue protein. The translated protein terminated with the sequence AHL, a peroxisomal targeting signal 1. Indirect immunofluorescence studies showed that the protein product was expressed in human fibroblasts in a punctate pattern that co-localized with the peroxisomal enzyme catalase. A BLAST search with the amino acid sequence showed 31% identity and 53% similarity with Bacillus sp. NS-129 monomeric sarcosine oxidase, as well as similarity to all sarcosine oxidases and dehydrogenases. No similarity was found to the peroxisomal D-amino acid oxidases. The recombinant enzyme oxidized both L-pipecolic acid and sarcosine. However, PBD patients who lack the enzyme activity accumulate only L-pipecolic acid, suggesting that in humans in vivo, this enzyme is involved mainly in the degradation of L-pipecolic acid.
The gene for human TATA-binding-protein-associated factor (TAFII) 170: structure, promoter and chromosomal localization
Authors: VAN DER KNAAP Jan A. , VAN DEN BOOM Vincent , KUIPERS Jeroen , VAN EIJK Michiel J. T. , VAN DER VLIET Peter C. , TIMMERS H. Th. Marc
Published: August 2015

The TATA-binding protein (TBP) plays a central role in eukaryotic transcription and forms protein complexes with TBP-associated factors (TAFs). The genes encoding TAFII proteins frequently map to chromosomal regions altered in human neoplasias. TAFII170 of B-TFIID is a member of the SF2 superfamily of putative helicases. Members of this superfamily have also been implicated in several human genetic disorders. In this study we have isolated human genomic clones encoding TAFII170 and we show that the gene contains 37 introns. Ribonuclease-protection experiments revealed that TAFII170 has multiple transcription start sites, consistent with the observation that the promoter lacks a canonical TATA box and initiator element. Deletion analysis of the promoter region showed that a fragment of 264 bp is sufficient to direct transcription. In addition, we determined the chromosomal localization by two independent methods which mapped the gene to human chromosome 10q22-q23 between the markers D10S185 and WI-1183. The region surrounding these markers has been implicated in several human disorders.
Induction of Myc-intron-binding polypeptides MIBP1 and RFX1 during retinoic acid-mediated differentiation of haemopoietic cells
Authors: ZAJAC-KAYE Maria , BEN-BARUCH Noa , KASTANOS Evdokia , KAYE Frederic J. , ALLEGRA Carmen
Published: August 2015

Retinoic acid-mediated differentiation of HL60 cells is associated with an alteration of chromatin structure that maps to protein-binding sequences within intron I of the c-myc gene and with down-regulation of c-myc expression. By using HeLa cell extracts, we previously identified two polypeptides, designated MIBP1 (for Myc-intron-binding peptide) and RFX1, that interact in vivo and bind to the intron I element; we showed that tandem repeats of an MIBP1/RFX1-binding site can exhibit silencer activity on a heterologous promoter. Here we demonstrate that p160 MIBP1 and p130 RFX1 are absent from undifferentiated HL60 cells. In addition, we show that treatment with retinoic acid induces both MIBP1 and RFX1 protein, as well as their DNA-binding activity, upon granulocytic differentiation of HL60 cells, with a gel mobility pattern identical to that of HeLa cells. In the absence of p160 MIBP1 and p130 RFX1, we observed that the altered gel mobility-shift pattern detected in undifferentiated HL60 cells reflects the binding of two novel polypeptides, p30 and p97, that can be cross-linked to the same recognition intron sequence. We also show that the time course of MIBP1 and RFX1 induction is inversely correlated with the down-regulation of c-myc levels during the treatment of HL60 cells with retinoic acid.
Gi-mediated translocation of GLUT4 is independent of p85/p110α and p110γ phosphoinositide 3-kinases but might involve the activation of Akt kinase
Authors: WANG Lihong , HAYASHI Hideki , KISHI Kazuhiro , HUANG Luping , HAGI Akifumi , TAMAOKA Keisuke , HAWKINS Phillip T. , EBINA Yousuke
Published: August 2015

Activation of phosphoinositide 3-kinase (PI-3K) is essential for insulin-stimulated translocation of GLUT4 and glucose transport in insulin target tissues. A novel p110γ PI-3K was reported to be activated by Gi-coupled receptors via Gβγ subunits. We asked whether the stimulation of Gi-coupled receptors would trigger GLUT4 translocation and glucose uptake by the activation of Gβγ-dependent p110γ PI-3K. We find that this translocation and glucose uptake can be induced by the ligand stimulation of Gi-coupled α2A adrenergic receptor and fMet-Leu-Phe receptor in cells stably expressing these receptors. The noradrenaline (‘noradrenaline’)- and fMet-Leu-Phe-stimulated GLUT4 translocations were abolished by pretreatment with pertussis toxin. Pretreatment with wortmannin or genistein also inhibited the Gi-mediated GLUT4 translocation. On ligand stimulation of these two kinds of Gi-coupled receptor, although there was a slight increase in PtdIns(3,4,5)P3 production, activation of either the p85/p110α PI-3K or Gβγ-dependent p110γ PI-3K was not observed even in Chinese hamster ovary cells stably overexpressing exogenous p101/p110γ. The Gi-mediated GLUT4 translocation was accompanied by activation of the serine-threonine kinase Akt; the inhibitory effects of pertussis toxin, wortmannin and genistein on Gi-mediated GLUT4 translocation paralleled their inhibitory effects on Akt activation. In contrast, the activation of some other Gi-coupled receptors, such as prostaglandin EP3α receptor and platelet-activating factor receptor, did not cause either pertussis-toxin-sensitive translocation of GLUT4myc or activation of Akt kinase. These results indicate that the ligand stimulation of some Gi-coupled receptors triggers GLUT4 translocation that occurs independently of p85/p110α-type and p110γ-type PI-3Ks but might involve the activation of Akt kinase.
pgaA and pgaB encode two constitutively expressed endopolygalacturonases of Aspergillus niger
Authors: PAŘENICOVÁ Lucie , BENEN Jacques A. E. , KESTER Harry C. M. , VISSER Jaap
Published: August 2015

pgaA and pgaB, two genes encoding endopolygalacturonases (PGs, EC 3.2.1.15) A and B, were isolated from a phage genomic library of Aspergillusniger N400. The 1167 bp protein coding region of the pgaA gene is interrupted by one intron, whereas the 1234 bp coding region of the pgaB gene contains two introns. The corresponding proteins, PGA and PGB, consist of 370 and 362 amino acid residues respectively. Northern-blot analysis revealed that pgaA- and pgaB-specific mRNA accumulate in mycelia grown on sucrose. mRNAs are also present upon transfer to media containing D-galacturonic acid and pectin. Recombinant PGA and PGB were characterized with respect to pH optimum, activity on polygalacturonic acid, and mode of action and kinetics on oligogalacturonates of different chain length (n = 3-7). At their pH optimum the specific activities in a standard assay for PGA (pH 4.2) and PGB (pH 5.0) were 16.5 μkat·mg-1 and 8.3 μkat·mg-1 respectively. Product progression analysis, using polygalacturonate as a substrate, revealed a random cleavage pattern for both enzymes and indicated processive behaviour for PGA. This result was confirmed by analysis of the mode of action using oligogalacturonates. Processivity was observed when the degree of polymerization of the substrate exceeded 6. Using pectins of various degrees of methyl esterification, it was shown that PGA and PGB both preferred partially methylated substrates.
Heat shock cognate protein 70 chaperone-binding site in the co-chaperone murine stress-inducible protein 1 maps to within three consecutive tetratricopeptide repeat motifs
Authors: VAN DER SPUY Jacqueline , KANA Bavesh D. , DIRR Heini W. , BLATCH Gregory L.
Published: August 2015

Murine stress-inducible protein 1 (mSTI1) is a co-chaperone homologous with the human heat shock cognate protein 70 (hsc70)/heat shock protein 90 (hsp90)-organizing protein (Hop). The concomitant interaction of mSTI1 with hsp70 and hsp90 at its N- and C-termini respectively is mediated by the tetratricopeptide repeat (TPR) motifs in these regions. With the use of co-precipitation assays, we show here that the N-terminal TPR domain of mSTI1 without extensive flanking regions is both necessary and sufficient to mediate a specific interaction with hsc70. In contrast, other TPR-containing co-chaperones require TPR flanking regions for target substrate recognition, suggesting different mechanisms of TPR-mediated chaperone-co-chaperone interactions. Furthermore, the interaction between mSTI1 and hsc70 was analysed to ascertain the effect of replacing or deleting conserved amino acid residues and sequences within the three TPR motifs constituting the N-terminal TPR domain of full-length mSTI1. Replacement of a bulky hydrophobic residue in TPR1 disrupted the interaction of mSTI1 with hsc70. A highly conserved sequence in TPR2 was altered by deletion or single amino acid replacement. These derivatives retained a specific interaction with hsc70. These results are consistent with a model in which conserved residues within the N-terminal TPR region of mSTI1 contribute differentially to the interaction with hsc70, and in which TPR1 has a significant role in targeting mSTI1 to hsc70. The contribution of the TPR domain mutations and deletions are discussed with respect to their effect on target substrate interactions.
Androctonin, a hydrophilic disulphide-bridged non-haemolytic anti-microbial peptide: a plausible mode of action
Authors: HETRU Charles , LETELLIER Lucienne , OREN Ziv , HOFFMANN Jules A. , SHAI Yechiel
Published: August 2015

Androctonin is a 25-residue non-haemolytic anti-microbial peptide isolated from the scorpion Androctonus australis and contains two disulphide bridges. Androctonin is different from known native anti-microbial peptides, being a relatively hydrophilic and non-amphipathic molecule. This raises the possibility that the target of androctonin might not be the bacterial membrane, shown to be a target for most amphipathic lytic peptides. To shed light on its mode of action on bacteria and its non-haemolytic activity, we synthesized androctonin, its fluorescent derivatives and its all-D-amino acid enantiomer. The enantiomer preserved high activity, suggesting a lipid-peptide interaction between androctonin and bacterial membranes. In Gram-positive and (at higher concentrations) Gram-negative bacteria, androctonin induced an immediate perturbation of the permeability properties of the cytoplasmic membrane of the bacterial energetic state, concomitant with perturbation of the morphology of the cell envelope as revealed by electron microscopy. Androctonin binds only to negatively charged lipid vesicles and induces the leakage of markers at high concentrations and with a slow kinetics, in contrast with amphipathic α-helical anti-microbial peptides that bind and permeate negatively charged vesicles, and to a smaller extent also zwitterionic ones. This might explain the selective lytic activity of androctonin towards bacteria but not red blood cells. Polarized attenuated total reflection-Fourier transform infrared spectroscopy revealed that androctonin adopts a β-sheet structure in membranes and did not affect the lipid acyl chain order, which supports a detergent-like effect. The small size of androctonin, its hydrophilic character and its physicochemical properties are favourable features for its potential application as a replacement for commercially available antibiotics to which bacteria have developed resistance.
Differential recognition of snake venom proteins expressing specific Arg-Gly-Asp (RGD) sequence motifs by wild-type and variant integrin αIIbβ3: further evidence for distinct sites of RGD ligand recognition exhibiting negative allostery
Authors: RAHMAN Salman , FLYNN Geraldine , AITKEN Alex , PATEL Yatin , HUSSAIN Farhana , LU Xinjie , LOFTUS Joseph C. , FRENCH Deborah , WIJELATH Errol , STRAND Kurt , SAVIDGE Geoffrey F.
Published: August 2015

Several studies have demonstrated that the amino acid residues flanking the Arg-Gly-Asp (RGD) sequence of high-affinity ligands modulate their specificity of interaction with integrin complexes. Because of the absence of structural data for integrin complexes with bound ligand, the molecular basis for this specificity modulation remains obscure. In a previous paper [Rahman, Lu, Kakkar and Authi (1995) Biochem. J. 312, 223-232] we demonstrated that two genetically distinct venom-derived RGD proteins, kistrin and dendroaspin (both containing the sequence PRGDMP), were simple competitors, indicating the recognition of an identical binding site on the αIIbβ3 complex. Furthermore, both kistrin and dendroaspin inhibited the binding of the disintegrin elegantin (containing the sequence ARGDNP) via a non-competitive mechanism, suggesting that the binding of elegantin to the αIIbβ3 complex was at a remote site and down-regulated via an allosteric mechanism. Here we present further evidence for distinct RGD ligand recognition sites on the αIIbβ3 complex that exhibit a negative allosteric relationship. A panel of well-characterized recombinant dendroaspin and elegantin derivatives were employed for this study. These recombinant molecules were constructed as glutathione S-transferase fusion proteins with either an Ala or Pro residue N-terminal to the RGD sequence in combination with either a Met or an Asn residue immediately C-terminal. Equilibrium competition experiments showed that elegantin binding to ADP-treated platelets was inhibited by derivatives Eleg. AM (ARGDMP) and Eleg. PM (PRGDMP) via an allosteric competitive mechanism, providing direct evidence that modulation of the RGD motif can alter competitive behaviour. In addition, recombinant kistrin and dendroaspin both inhibited elegantin binding via a non-competitive mechanism, confirming our previous observations. Further evidence for distinct binding sites employing an independent approach was obtained by analysing the binding of the panel of venom proteins to the functionally defective heterodimer αIIbβ3 Ser123 → Ala expressed on Chinese hamster ovary cells. These studies demonstrated that simple competitors kistrin and dendroaspin bound with high affinity to the variant integrin complex. In contrast, the binding of elegantin and most significantly, recombinant Dendro. PN (PRGDNP) and Dendro. AN (ARGDNP) were abolished. These observations, taken together, are consistent with a model depicting the presence of distinct sites of RGD ligand recognition on the αIIbβ3 complex that show the preferential recognition of specific RGD motifs. Competition experiments demonstrate a negative allosteric relationship between these RGD recognition sites.
Thioredoxin reductase
Authors: MUSTACICH Debbie , POWIS Garth
Published: August 2015

The mammalian thioredoxin reductases (TrxRs) are a family of selenium-containing pyridine nucleotide-disulphide oxidoreductases with mechanistic and sequence identity, including a conserved -Cys-Val-Asn-Val-Gly-Cys- redox catalytic site, to glutathione reductases. TrxRs catalyse the NADPH-dependent reduction of the redox protein thioredoxin (Trx), as well as of other endogenous and exogenous compounds. The broad substrate specificity of mammalian TrxRs is due to a second redox-active site, a C-terminal -Cys-SeCys- (where SeCys is selenocysteine), that is not found in glutathione reductase or Escherichia coli TrxR. There are currently two confirmed forms of mammalian TrxRs, TrxR1 and TrxR2, and it is possible that other forms will be identified. The availability of Se is a key factor determining TrxR activity both in cell culture and in vivo, and the mechanism(s) for the incorporation of Se into TrxRs, as well as the regulation of TrxR activity, have only recently begun to be investigated. The importance of Trx to many aspects of cell function make it likely that TrxRs also play a role in protection against oxidant injury, cell growth and transformation, and the recycling of ascorbate from its oxidized form. Since TrxRs are able to reduce a number of substrates other than Trx, it is likely that additional biological effects will be discovered for TrxR. Furthermore, inhibiting TrxR with drugs may lead to new treatments for human diseases such as cancer, AIDS and autoimmune diseases.
Quantitative analysis of gene amplification in insecticide-resistant Culex mosquitoes
Authors: PATON Michael G. , KARUNARATNE S. H. P. Parakrama , GIAKOUMAKI Elsa , ROBERTS Neil , HEMINGWAY Janet
Published: August 2015

The amplification of carboxylesterase structural genes followed by their overexpression is the most common mechanism of resistance to organophosphorus insecticides in Culex mosquitoes. Most resistant Culex quinquefasciatus mosquitoes have co-amplified estα21 and estβ21 genes. Recently, Southern, DNA dot-blot analysis and phosphorimaging technology were used to quantify the est gene copy number in aphids and mosquitoes. Although more accurate than autoradiography, this method relies on probe hybridization, which can be variable. We have directly measured gene and mRNA copy number by using real-time quantitative PCRs in mosquitoes. The acquisition of fluorescence from incorporation of the double-strand-specific dye SYBR GreenI into a PCR product once per cycle is used to provide an absolute quantification of the initial template copy number. Thus it has been possible to show that estα21 and estβ21 are co-amplified approx. 80-fold in the genome of the resistant PelRR strain of C. quinquefasciatus. The two genes, although co-amplified in a 1:1 ratio, are differentially transcribed: the estβ21 gene from this amplicon has greater transcription than estα21 in all individual mosquito larvae tested, with an average ratio of 10:1. Purified esterases from mosquito homogenates were found in a ratio of 3:1, which, combined with the quantitative mRNA data, suggests the operation of both transcriptional and translational control mechanisms to regulate the expression of the amplified genes in C. quinquefasciatus insecticide-resistant mosquitoes.
Sp1 and chromatin environment are important contributors to the formation of repressive chromatin structures on the transfected human adenine nucleotide translocase-2 promoter
Authors: HODNY Zdenek , LI Ronggui , BARATH Peter , NELSON B. Dean
Published: August 2015

The influence of chromatin on the human adenine nucleotide translocase isoform 2 (ANT2) promoter was investigated in transfected cells treated with the deacetylase inhibitors butyrate and trichostatin A (TSA). Both inhibitors activated the expression of reporter plasmids transfected into HeLa cells, indicating that the promoter was suppressed by hypoacetylated chromatin and activated by hyperacetylation. Inhibitor-dependent activation was traced to the two Sp1-activation elements within the proximal promoter region, indicating that the Sp1 elements are repressed by chromatin structure. Repressive chromatin structures were also formed on the promoter integrated into a stable chromatin environment, as shown by the effects of TSA and butyrate on 14 single-cell-derived NIH3T3 clones bearing the stable integrated ANT2 promoter. Both the basal expression of the luciferase reporter gene and the response to TSA and butyrate varied widely between clones. The range of basal expression (4000-fold) was due partially to variation in the formation of repressive chromatin, since clones with low basal expression were induced by TSA, but those with high basal expression were less effected. These data indicate that chromatin environment surrounding the integrated DNA exerts a strong influence on chromatin-dependent repression of the ANT2 promoter, and that the ability of Sp1 to activate ANT2 expression is compromised in the repressed state.
Changes in gene expression in response to polyamine depletion indicates selective stabilization of mRNAs
Authors: VERESS Ildiko , HAGHIGHI Saeid , PULKKA Anitta , PAJUNEN Antti
Published: August 2015

We used differential display analysis to identify mRNAs responsive to changes in polyamine synthesis. As an overproducing model we used the kidneys of transgenic hybrid mice overexpressing ornithine decarboxylase and S-adenosylmethionine decarboxylase, two key enzymes in polyamine biosynthesis. To identify mRNAs that respond to polyamine starvation, we treated Rat-2 cells with α-difluoromethylornithine, a specific inhibitor of polyamine biosynthesis. We isolated 41 partial cDNA clones, representing 37 differentially expressed mRNAs. Of these, 15 have similarity with known genes, five appear to be similar to reported expressed sequence tags and seventeen clones were novel sequences. Of the 35 mRNAs expressed differentially after α-difluoromethylornithine treatment, 26 were up-regulated. The expression of only three mRNAs was altered in the transgenic animals and all three were down-regulated. Determination of mRNA half-life of three of the mRNAs up-regulated in response to polyamine depletion revealed that the accumulation results from stabilization of the messages. Because most of the transcripts identified from Rat-2 cells suffering polyamine starvation were accumulated, we conclude that polyamine depletion, while blocking cell growth, is stabilizing mRNAs. This may be due to the lack of spermidine for post-translational modification of the eukaryotic initiation factor 5A, which plays a major role in mRNA turnover. The coupling of mRNA stabilization with cell-growth arrest in response to polyamine starvation provides cells with an economical way to resume growth after recovery from polyamine deficiency.
Mechanisms of cGMP-dependent mesangial-cell relaxation: a role for myosin light-chain phosphatase activation
Authors: TORRECILLAS Guadalupe , DÍEZ-MARQUÉS María L. , GARCÍA-ESCRIBANO Carmen , BOSCH Ricardo J. , RODRÍGUEZ-PUYOL Diego , RODRÍGUEZ-PUYOL Manuel
Published: August 2015

Although the cGMP-dependent relaxation of contractile cells seems to depend on the ability of the cyclic nucleotide to interfere with intracellular calcium, this does not appear to be the only mechanism involved. The present experiments were designed to analyse alternative mechanisms, trying to test the hypothesis that cGMP could relax rat mesangial cells by activating myosin light-chain phosphatase (MLC-PP), with the subsequent dephosphorylation of myosin light chain (MLC). The effect of a cGMP analogue, dibutyryl cGMP (dbcGMP), on angiotensin II-(AII) and PMA-induced MLC phosphorylation (MLCP) was tested, in the presence of calyculin A (CA), an inhibitor of MLC-PP. MLCP was measured, after cell labelling with 32P, by immunoprecipitation. dbcGMP prevented the increased MLCP induced by AII or PMA, and this inhibition was blocked by CA. dbcGMP also increased the MLC dephosphorylation observed in cells incubated with AII and in which MLC kinase and protein kinase C activities were blocked. The AII-elicited increased intracellular calcium concentration was only partially inhibited by dbcGMP. These results suggest that the cGMP-induced mesangial-cell relaxation could be due, at least partially, to the stimulation of MLC-PP.
Ethanol, Zn2+ and insulin interact as progression factors to enhance DNA synthesis synergistically in the presence of Ca2+ and other cell cycle initiators in fibroblasts
Authors: HUANG Jin-Sheng , SHE Qing-Bai , CRILLY Karan S. , KISS Zoltan
Published: August 2015

In serum-starved NIH 3T3 fibroblasts, ethanol (30-80 mM) promoted the effects of insulin and insulin-like growth factor I (IGF-I) on DNA synthesis in a Zn2+-dependent manner. Ethanol and Zn2+ were most effective when added shortly before or after insulin, indicating that all these agents facilitated cell cycle progression. The synergistic effects of ethanol, Zn2+ and insulin (or IGF-I) on DNA synthesis required 1.1-2.3 mM Ca2+, which seemed to act as the cell cycle initiator. When serum-starved cells were pretreated for 2 h with other cell cycle initiators such as 10% (v/v) serum, 50 ng/ml platelet-derived growth factor or 2 ng/ml fibroblast growth factor, subsequent co-treatments with 60 mM ethanol, Zn2+ and insulin for an 18 h period again synergistically increased DNA synthesis. Of the various signal transducing events examined, ethanol stimulated cellular uptake of 45Ca and it enhanced the stimulatory effects of insulin on p70 S6 kinase activity in a Zn2+-dependent manner. In contrast, ethanol inhibited insulin-induced activating phosphorylation of p42/p44 mitogen-activated protein kinases; these inhibitory ethanol effects were prevented by Zn2+. The results show that, in NIH 3T3 fibroblasts, ethanol can promote cell cycle progression in the presence of a cell cycle initiator as well as Zn2+ and insulin (or IGF-I).
Actin filaments play a critical role in insulin-induced exocytotic recruitment but not in endocytosis of GLUT4 in isolated rat adipocytes
Authors: OMATA Waka , SHIBATA Hiroshi , LI Lu , TAKATA Kuniaki , KOJIMA Itaru
Published: August 2015

Actin-based cytoskeletons have been implicated in insulin-stimulated glucose transport and translocation of the insulin-regulated glucose transporter, GLUT4, from the intracellular pool to the plasma membrane. However, most previous studies were done using adherent cell systems such as L6 myotubes and 3T3-L1 adipocytes, and very little information is available on the significance of the actin filaments to the insulin action in isolated adipocytes, a widely used experimental system. In the present study, we investigated the physiological role of actin filaments in the subcellular trafficking of GLUT4 in isolated rat adipocytes. We first compared the effects of two actin-disrupting reagents, latrunculin A and cytochalasin D, on the organization of the actin filaments as well as on the insulin action on glucose transport by laser confocal microscopy combined with biochemical analysis of the insulin action. Treatment of the cells with latrunculin A induced dose- and time-dependent disappearance of the filamentous actin, which correlated very well with inhibition of the insulin effect on glucose transport. Although cytochalasin D at 50 μM significantly inhibited insulin-stimulated glucose transport, it was not effective in disassembly of the actin filaments; rather, many intense punctate signals were observed in cytochalasin D-treated cells. In the actin-disrupted adipocytes treated with latrunculin A, insulin-induced GLUT4 translocation was inhibited completely. In addition, latrunculin A remarkably inhibited both insulin-induced glucose transport and GLUT4 translocation in the presense of Dk-(62-85), a potent inhibitor of GLUT4 endocytosis, suggesting that intactness of the actin filaments was necessary for insulin-induced exocytosis of the GLUT4-containing vesicles. On the other hand, latrunculin A showed little inhibitory effect on either endocytosis of the trypsin-cleaved 35-kDa fragment of GLUT4 or decay of the glucose transport activity after addition of wortmannin in insulin-stimulated cells. The results of our experiment show clearly that, in rat adipocytes, (i) latrunculin A may be a more suitable tool than cytochalasin D for disruption of actin filaments, and (ii) actin filaments play a crucial role in exocytotic recruitment of GLUT4 to the plasma membrane from the intracellular pool, but not in its endocytosis.
Down-regulation of cyclic-nucleotide phosphodiesterase 3B in 3T3-L1 adipocytes induced by tumour necrosis factor α and cAMP
Authors: LANDSTRÖM Tova RAHN , MEI Jie , KARLSSON Marie , MANGANIELLO Vincent , DEGERMAN Eva
Published: August 2015

We have used murine 3T3-L1 cells, which differentiate in culture and acquire morphological and biochemical features of mature adipocytes, as a model for studying the expression of cyclic-nucleotide phosphodiesterase (PDE) 3B activity, protein and mRNA during differentiation and during long-term treatment of the cells with tumour necrosis factor α (TNF-α), a cytokine associated with insulin resistance, and a cAMP analogue, N6,2ʹ-O-dibutyryl cAMP (dbcAMP). PDE3B activity, protein and mRNA could be detected 4 days after the initiation of differentiation of 3T3-L1 preadipocytes. Treatment of 3T3-L1 adipocytes with 10 ng/ml TNF-α for 24 h produced a maximal (50%) decrease in PDE3B activity, protein and mRNA, which was well correlated with both activation of protein kinase A (PKA) and stimulation of lipolysis, presumably reflecting an increase in intracellular cAMP concentration. To investigate the effect of cAMP on PDE3B we treated 3T3-L1 adipocytes with dbcAMP. After 4 h with 0.5 mM dbcAMP, PDE3B activity was decreased by 80%, which was also correlated with a decrease in PDE3B protein and mRNA. This effect was abolished in the presence of N-[2-(bromocinnamylamino)ethyl]-5-isoquinolinesulphonamide] (H-89), a specific PKA inhibitor. We conclude that the lipolytic effect of TNF-α involves the down-regulation of PDE3B, which is associated with increased activation of PKA, presumably owing to increased levels of cAMP. In addition, the PKA activation induced by dbcAMP resulted in the down-regulation of PDE3B. These results, which suggest that PDE3B is a novel target for long-term regulation by TNF-α and cAMP, could contribute to the understanding of the mechanisms of insulin resistance.
Involvement of Giα2 in sodium butyrate-induced erythroblastic differentiation of K562 cells
Authors: DAVIS Mark G. , KAWAI Yumiko , ARINZE Ifeanyi J.
Published: August 2015

The chronic myelogenous leukaemia cell line K562 can be triggered in culture to differentiate along the erythrocytic pathway in response to a variety of stimulatory agents. In the presence of sodium butyrate, these cells differentiate to erythroblasts and acquire the capability to synthesize haemoglobin. We used this cell system to study alterations in the levels of several G-protein subunits during the cell differentiation programme and to assess the involvement of Giα2 in this process. Western immunoblot analysis revealed the presence of Gsα1, Gsα2, Giα2, Gqα, Gα12, Gβ1 and Gβ2 in K562 cells. Goα, Gzα, Gα13 and Gα16 were not detected. Although the levels of several G-protein subunits were altered after treatment with sodium butyrate, the most striking change was the robust increase in the levels of Giα2, which was accompanied by an increase in the mRNA for Giα2. Inactivation of Giα2 by adding Bordetella pertussis toxin to the cultures inhibited erythroblastic differentiation by as much as 62%, as measured by haemoglobin accumulation. Furthermore, the addition of an oligonucleotide anti-sense to Giα2 inhibited the sodium butyrate-induced robust increase in Giα2 levels, decreasing it to the basal levels seen in control cells; this treatment decreased the erythroblastic differentiation of the cells (as measured by haemoglobin expression) by 50%. Taken together, these findings imply that increased levels of Giα2 contribute to the sodium butyrate-induced erythroblastic differentiation of K562 cells.
GTPase mechanism and function: new insights from systematic mutational analysis of the phosphate-binding loop residue Ala30 of Rab5
Authors: LIANG Zhimin , MATHER Timothy , LI Guangpu
Published: August 2015

Structural and biochemical data indicate the importance of the phosphate-binding loop residues Gly12 and Gly13 of Ras both in the GTP hydrolysis reaction and in biological activity, but these two residues are not conserved in other Ras-related GTPases. To gain a better understanding of this region in GTP hydrolysis and GTPase function, we used the Ras-related Rab5 GTPase as a model for comparison, and substituted the Ala30 residue (the equivalent of Gly13 of Ras) with all the other 19 amino acids. The resulting mutants were analysed for GTP hydrolysis, GTP binding, GTP dissociation and biological activity. Only the substitution of alanine with proline reduced the GTPase activity by an order of magnitude. This effect is in sharp contrast with the observation that a proline substitution at the neighbouring position (Gly12 of Ras) has little effect on the GTPase activity. Whereas most other substitutions showed either a small negative effect or no effect on the GTPase activity, the arginine substitution surprisingly stimulated the GTPase activity by 5-fold. Molecular modelling suggests that this built-in arginine mimics the catalytic arginine residues found in trimeric GTPases and GTPase-activating proteins in providing the positive charge to facilitate the GTP hydrolysis reaction. We investigated further the biological activity of the Rab5 mutants in relation to stimulating endocytosis. When expressed in cultured baby hamster kidney cells, both arginine and proline mutants, like wild-type Rab5, stimulated endocytosis. However, the arginine mutant was a more potent stimulator than the proline mutant (3-fold stimulation as against 1.7-fold). The tryptophan mutant, on the other hand, was completely deficient in activity in terms of the stimulation of endocytosis, demonstrating the importance of the phosphate-binding loop in Rab GTPase function.
Modulation of the glutathione S-transferase in Ochrobactrum anthropi: function of xenobiotic substrates and other forms of stress
Authors: FAVALORO Bartolo , TAMBURRO Antonio , TROFINO Massimo A. , BOLOGNA Luciano , ROTILIO Domenico , HEIPIEPER Hermann J.
Published: August 2015

The gluthathione S-transferase gene of the atrazine-degrading bacterium Ochrobactrum anthropi (OaGST) encodes a single-subunit polypeptide of 201 amino acid residues (Favaloro et al. 1998, Biochem. J. 335, 573-579). RNA blot analysis showed that the gene is transcribed into an mRNA of about 800 nucleotides, indicating a monocistronic transcription of the OaGST gene. The modulation of OaGST in this bacterium, in the presence of different stimulants, was investigated. The level of expression of OaGST was detected both by measuring the mRNA level and by immunoblotting experiments. OaGST is a constitutive enzyme which is also inducible by several stimulants. In fact, atrazine caused an increase in the expression of OaGST even at concentrations which had no effect on growth rates of the bacteria. Moreover, the presence of other aromatic substrates of this bacterium, such as phenol and chlorophenols, leads to a marked enhancement in OaGST expression. In this case, the expression of OaGST was related to growth inhibition and membrane damage caused by these hydrophobic compounds, and to the adaptive responses of the cell membranes. On the other hand, toluene and xylene, two aromatic compounds not degradable by this bacterium, did not induce the OaGST expression. The same was observed for other stress conditions such as low pH, heat shock, hydrogen peroxide, osmotic stress, starvation, the presence of aliphatic alcohols or heavy metals. These results suggest a co-regulation of the OaGST gene by the catabolic pathways of phenols and chlorophenols in this bacterium. Therefore, OaGST could function as a detoxifying agent within the catabolism of these xenobiotics.
The PI3K–PDK1 connection: more than just a road to PKB
Authors: VANHAESEBROECK Bart , ALESSI Dario R.
Published: August 2015

Phosphoinositide 3-kinases (PI3Ks) generate specific inositol lipids that have been implicated in the regulation of cell growth, proliferation, survival, differentiation and cytoskeletal changes. One of the best characterized targets of PI3K lipid products is the protein kinase Akt or protein kinase B (PKB). In quiescent cells, PKB resides in the cytosol in a low-activity conformation. Upon cellular stimulation, PKB is activated through recruitment to cellular membranes by PI3K lipid products and phosphorylation by 3ʹ-phosphoinositide-dependent kinase-1 (PDK1). Here we review the mechanism by which PKB is activated and the downstream actions of this multifunctional kinase. We also discuss the evidence that PDK1 may be involved in the activation of protein kinases other than PKB, the mechanisms by which this activity of PDK1 could be regulated and the possibility that some of the currently postulated PKB substrates targets might in fact be phosphorylated by PDK1-regulated kinases other than PKB.
The thermostabilizing domain, XynA, of Caldibacillus cellulovorans xylanase is a xylan binding domain
Authors: SUNNA Anwar , GIBBS Moreland D. , BERGQUIST Peter L.
Published: August 2015

We show that the N-terminal ‘thermostabilizing domain’ (TSD) of the xylanase, XynA, from the thermophilic bacterium Caldibacillus cellulovorans also acts as a xylan binding domain. Affinity electrophoresis experiments show that this TSD selectively binds soluble xylan and binds weakly to hydroxyethylcellulose. Based on this, and previously reported evidence, we propose that xylanase-associated TSDs are xylan binding domains.