bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of sarcoplasmic/endoplasmic-reticulum Ca2+-ATPases in mediating Ca2+ waves and local Ca2+-release microdomains in cultured glia
Authors: SIMPSON Peter B. , RUSSELL James T.
Published: August 2015

We have characterized the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) pumps in cultured rat cortical type-1 astrocytes, type-2 astrocytes and oligodendrocytes. Perfusion with 10 μM cyclopiazonic acid (CPA) or 1 μM thapsigargin evoked a large and persistent elevation in cytosolic [Ca2+] in normal Ca2+-containing medium and a small and transient increase in nominally Ca2+-free medium. Subtraction of the response in Ca2+-free medium from that in the control revealed a slow-onset Ca2+-entry response to SERCA inhibition, which began after most of the store depletion had occurred. Thapsigargin- and CPA-induced responses propagated as Ca2+ waves, which began in several distinct cellular sites and travelled throughout the cell and through nearby cells, in confluent cultures. Propagation was supported by specialized Ca2+-release sites where the amplitude of the response was significantly higher and the rate of rise steeper. Such higher Ca2+-release kinetics were observed at these sites during Ins(1,4,5)P3-mediated Ca2+ waves in the same cells. Fluorescently tagged thapsigargin labelled SERCA pumps throughout glial cell bodies and processes. In oligodendrocyte processes, multiple domains with elevated SERCA staining were always associated with mitochondria. Our results are consistent with a model in which only a single Ca2+ store, expressing Ins(1,4,5)P3 receptors and SERCAs sensitive to both thapsigargin and CPA, is present in rat cortical glia, and indicate that inhibition of SERCA activates both Ca2+ release as a wavefront and Ca2+ entry via store-operated channels. The spatial relationship between SERCAs and mitochondria is likely to be important for regulating microdomains of elevated Ca2+-release kinetics.
Preservation of the native structure and function of Ca2+-ATPase from sarcoplasmic reticulum: Solubilization and reconstitution by new short-chain phospholipid detergent 1,2-diheptanoyl-sn-phosphatidylcholine
Authors: SHIVANNA Bannikuppe D. , ROWE Elizabeth S.
Published: August 2015

The properties of Ca2+-ATPase purified and reconstituted from rabbit skeletal sarcoplasmic reticulum (SR) has been studied in comparison with the preparations obtained by the commonly used detergent poly(oxyethylene)8-lauryl ether (C12E8) and the bile salt detergents cholate and deoxycholate. 1,2-Diheptanoyl-sn-phosphatidylcholine (DHPC) has been shown to be excellent for solubilizing a wide variety of membrane proteins [Kessi, Poiree, Wehrli, Bachofen, Semenza and Hauser (1994) Biochemistry 33, 10825–10836]. The DHPC method consistently gave higher yields of purified Ca2+-ATPase with a greater specific activity than the methods with C12E8, cholate, or deoxycholate. DHPC and C12E8 were superior to cholate and deoxycholate in active enzyme yields and specific activity. DHPC-solubilized Ca2+-ATPase purified on a density gradient retained the E1Ca–E1*Ca conformational transition, whereas the enzyme from the C12E8 purification did not retain this transition. The coupling of Ca2+ transported to ATP hydrolysed in the DHPC-purified enzyme was maximal and matched the values obtained with native SR, whereas the coupling was much lower for the C12E8-purified enzyme. The specific activity of Ca2+-ATPase reconstituted into dioleoylphosphatidylcholine vesicles with DHPC was up to 2-fold greater than that achieved with C12E8, and is comparable to that measured in the native SR. Finally, the dissociation of Ca2+-ATPase into monomers by DHPC preserved the ATPase activity, whereas similar dissociation by C12E8 gave only one-sixth the activity of that obtained with DHPC. These studies show that the Ca2+-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C12E8 in significant ways, making it a preparation suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of membrane proteins.
Kinetoplastid glucose transporters
Authors: TETAUD Emmanuel , BARRETT Michael P. , BRINGAUD Frédéric , BALTZ Théo
Published: August 2015

Protozoa of the order kinetoplastida have colonized many habitats, and several species are important parasites of humans. Adaptation to different environments requires an associated adaptation at a cell's interface with its environment, i.e. the plasma membrane. Sugar transport by the kinetoplastida as a phylogenetically related group of organisms offers an exceptional model in which to study the ways by which the carrier proteins involved in this process may evolve to meet differing environmental challenges. Seven genes encoding proteins involved in glucose transport have been cloned from several kinetoplastid species. The transporters all belong to the glucose transporter superfamily exemplified by the mammalian erythrocyte transporter GLUT1. Some species, such as the African trypanosome Trypanosoma brucei, which undergo a life cycle where the parasites are exposed to very different glucose concentrations in the mammalian bloodstream and tsetse-fly midgut, have evolved two different transporters to deal with this fluctuation. Other species, such as the South American trypanosome Trypanosoma cruzi, multiply predominantly in conditions of relative glucose deprivation (intracellularly in the mammalian host, or within the reduviid bug midgut) and have a single, relatively high-affinity type, transporter. All of the kinetoplastid transporters can also transport d-fructose, and are relatively insensitive to the classical inhibitors of GLUT1 transport cytochalasin B and phloretin.
Effect of adenine nucleotides on myo-inositol-1,4,5-trisphosphate-induced calcium release
Authors: MISSIAEN Ludwig , PARYS Jan B. , DE SMEDT Humbert , SIENAERT Ilse , SIPMA Henk , VANLINGEN Sara , MAES Karlien , CASTEELS Rik
Published: August 2015

The effects of a whole series of adenine nucleotides on Ins(1,4,5)P3-induced Ca2+ release were characterized in permeabilized A7r5 smooth-muscle cells. Several adenine nucleotides activated the Ins(1,4,5)P3 receptor. It was observed that 3′-phosphoadenosine 5′-phosphosulphate, CoA, di(adenosine-5′)tetraphosphate (Ap4A) and di(adenosine-5′)pentaphosphate (Ap5A) were more effective than ATP. Ap4A and Ap5A also interacted with a lower EC50 than ATP. In order to find out how these adenine nucleotides affected Ins(1,4,5)P3-induced Ca2+ release, we have measured their effect on the response of permeabilized A7r5 cells to a progressively increasing Ins(1,4,5)P3 concentration. Stimulatory ATP and Ap5A concentrations had no effect on the threshold Ins(1,4,5)P3 concentration for initiating Ca2+ release, but they stimulated Ca2+ release in the presence of supra-threshold Ins(1,4,5)P3 concentrations by increasing the co-operativity of the release process. Inhibition of the Ins(1,4,5)P3-induced Ca2+ release at higher ATP concentrations was associated with a further increase in co-operativity and also with a shift in threshold towards higher Ins(1,4,5)P3 concentrations. ATP had no effect on the non-specific Ca2+ leak in the absence of Ins(1,4,5)P3. We conclude that the adenine-nucleotide-binding site can be activated by many different adenine nucleotides. Binding of these compounds to the transducing domain of the Ins(1,4,5)P3 receptor increases the efficiency of transmitting Ins(1,4,5)P3 binding to channel opening. The inhibition by high ATP concentrations is exerted at a different site, related to Ins(1,4,5)P3 binding.
Distribution of mRNA for human epiregulin, a differentially expressed member of the epidermal growth factor family
Authors: TOYODA Hitoshi , KOMURASAKI Toshi , UCHIDA Daisuke , MORIMOTO Sigeo
Published: August 2015

We have recently identified epiregulin as a new growth regulator and a member of the epidermal growth factor (EGF) family. Epiregulin has certain characteristics that are different from those of the classical members of the EGF family, EGF and transforming growth factor α, including mitogenic responses on several normal cells and binding to EGF receptors on epidermoid carcinoma A431 cells. In the present study we cloned and identified the expression of human epiregulin transcript. The human epiregulin gene encoded a 163-residue putative transmembrane precursor containing an EGF-like domain in the internal segment, and the structural organization was similar to that of other members of the EGF family that bind to EGF receptors. Northern blot analysis showed the expression of human epiregulin to be mainly on peripheral blood macrophages and the placenta in normal tissues, and was highest on epithelial tumour cell lines in various types of tumour cell lines. The expression profile was quite different from that of other members of the EGF family in normal and tumour cells. Recombinant expression in mammalian cells also showed that human epiregulin was secreted as a soluble form of approx. 5 kDa that is biologically active on the basis of the stimulation of DNA synthesis. Our findings suggest that epiregulin is involved in certain physiological processes such as maintenance or development of normal cell growth, and the progression of carcinomas.
Characterization of the hydrolytic activity of a polyclonal catalytic antibody preparation by pH-dependence and chemical modification studies: evidence for the involvement of Tyr and Arg side chains as hydrogen-bond donors
Authors: RESMINI Marina , VIGNA Roberta , SIMMS Caroline , BARBER Nicola J. , HAGI-PAVLI Eleni P. , WATTS Aaron B. , VERMA Chandra , GALLACHER Gerard , BROCKLEHURST Keith
Published: August 2015

The hydrolyses of 4-nitrophenyl 4′-(3-aza-2-oxoheptyl)phenyl carbonate and of a new, more soluble, substrate, 4-nitrophenyl 4′-(3-aza-7-hydroxy-2-oxoheptyl)phenyl carbonate, each catalysed by a polyclonal antibody preparation elicited in a sheep by use of an analogous phosphate immunogen, were shown to adhere closely to the Michaelis–Menten equation, in accordance with the growing awareness that polyclonal catalytic antibodies may be much less heterogeneous than had been supposed. The particular value of studies on polyclonal catalytic antibodies is discussed briefly. Both the kcat and kcat/Km values were shown to increase with increase in pH across a pKa of approx. 9. Group-selective chemical modification studies established that the side chains of tyrosine and arginine residues are essential for catalytic activity, and provided no evidence for the involvement of side chains of lysine, histidine or cysteine residues. The combination of evidence from the kinetic and chemical modification studies and from studies on the pH-dependence of binding suggests that catalysis involves assistance to the reaction of the substrate with hydroxide ions by hydrogen-bond donation at the reaction centre by tyrosine and arginine side chains. This combination of hydrogen-bond donors appears to be a feature common to a number of other hydrolytic catalytic antibodies. High-pKa acidic side chains may be essential for the effectiveness of catalytic antibodies that utilize hydroxide ions.
Identification of human complement factor H as a chemotactic protein for monocytes
Authors: NABIL Karim , RIHN Bertrand , JAURAND Marie-Claude , VIGNAUD Jean-Michel , RIPOCHE Jean , MARTINET Yves , MARTINET Nadine
Published: August 2015

We used chromatographic separation to purify to homogeneity a monomeric monocyte chemotactic protein of 150 kDa contained in mesothelioma pleural effusions. It was identified by N-terminal amino acid sequencing and immunoblotting as complement factor H, an inhibitor of the alternative complement pathway. Specific antibodies against factor H inhibited the monocyte chemotactic activity of the purified protein, which was most active at 10 nM. Factor H is a restrictive factor of alternative complement pathway activation. The new chemotactic function assigned to factor H in recruiting monocytes to the mesothelioma site might contribute to malignant cell phagocytosis via the iC3b/complement receptor type 3 pathway. These functions link the humoral and cellular immune systems.
Exposure of endothelial cells to cyclic strain induces elevations of cytosolic Ca2+ concentration through mobilization of intracellular and extracellular pools
Authors: ROSALES Oscar R. , ISALES Carlos M. , BARRETT Paula Q. , BROPHY Colleen , SUMPIO Bauer E.
Published: August 2015

We have previously reported that exposure of endothelial cells to cyclic strain elicited a rapid but transient generation of inositol 1,4,5-trisphosphate (IP3), which reached a peak 10 s after the initiation of cyclic deformation. To address the effect of cyclic strain on intracellular Ca2+ concentration ([Ca2+]i) and its temporal relationship to IP3 generation, confluent bovine aortic endothelial cells were grown on flexible membranes, loaded with aequorin and the membranes placed in a custom-designed flow-through chamber. The chamber was housed inside a photomultiplier tube, and vacuum was utilized to deform the membranes. Our results indicate that the initiation of 10% average strain induced a rapid increase in [Ca2+]i which contained two distinct components: a large initial peak 12 s after the initiation of stretch which closely followed the IP3 peak, and a subsequent lower but sustained phase. Pretreatment with 5 μMGdCl3 for 10 min or nominally Ca2+-free medium (CFM) for 3 min reduced the magnitude of the initial rise and abolished the sustained phase. Repetitive 10% average strain at a frequency of 60 cycles/min also elicited a single IP3 peak at 10 s. However, there was also a large initial [Ca2+]i peak followed by multiple smaller transient [Ca2+]i elevations. Preincubation with 5 μMGdCl3 or CFM diminished the initial [Ca2+]i transient and markedly inhibited the late-phase component. Preincubation with 25 μM 2,5-di-(t-butyl)-1,4-benzohydroquinone (BHQ) attenuated the initial [Ca2+]i transient. Cyclic-strain-mediated IP3 formation in confluent endothelial cells at 10 s, however, was not modified by pretreatment with 25 μM BHQ, 500 μM NiCl2, 10 nM charybdotoxin, 5 μM GdCl3 or CFM. We conclude that in endothelial cells exposed to cyclic strain, Ca2+ enters the cytosol from intracellular and extracellular pools but IP3 formation is not dependent on Ca2+ entry via the plasma membrane.
Identification of three internalization sequences in the cytoplasmic tail of the 46 kDa mannose 6-phosphate receptor
Authors: DENZER Kristin , WEBER Birgit , HILLE-REHFELD Annette , VON FIGURA Kurt , POHLMANN Regina
Published: August 2015

The cytoplasmic tail of the human 46 kDa mannose 6-phosphate receptor (MPR 46) is necessary for rapid internalization of the receptor and sufficient to mediate internalization of a resident plasma membrane protein. To localize the internalization sequences within the 67 amino acids of the cytoplasmic tail, the tail was progressively shortened from its C-terminus, internal deletions of between four and eight amino acids were introduced into the tail, and individual residues were substituted by alanine, glycine or serine. Three sequences were identified that contribute to the internalization of MPR 46. The first is located within the 23 juxtamembrane cytoplasmic residues of the tail. It contains four essential residues within a heptapeptide and does not resemble known internalization signals. The second sequence contains as a critical residue Tyr-45. The third region is located within the C-terminal seven residues and contains a di-leucine pair as essential residues. The first and third sequences were shown to function as autonomous internalization sequences. Substitution of critically important residues within a single internalization sequence was tolerated, with no or only a moderate decrease in the internalization rate. When essential residues from two or all three internalization sequences were substituted, however, the internalization rate was decreased by more than 60% and 90% respectively. This indicates that the autonomous internalization signals in the cytoplasmic tail of MPR 46 function in an additive manner, but are partly redundant.
Regulation of rat AMP deaminase 3 (isoform C) by development and skeletal muscle fibre type
Authors: MAHNKE-ZIZELMAN Donna K. , D'CUNHA Jonathan , WOJNAR Jean M. , BROGLEY Michele A. , SABINA Richard L.
Published: August 2015

AMP deaminase (AMPD) is characterized by a multigene family in rodents and man. Highly conserved rat and human AMPD1 and AMPD2 genes produce protein products that exhibit cross-species immunoreactivities (AMPD1, rat isoform A and human isoform M; AMPD2, rat isoform B and human isoform L). A third gene, AMPD3, has been described in humans, but antisera raised against its purified protein product (isoform E) reportedly does not cross-react with a third activity purified from rat tissues (isoform C). This study was designed to address this latter issue by cloning, sequencing and expressing rat AMPD3 cDNA species. Similarly to the human AMPD3 gene, the rat AMPD3 gene produces multiple transcripts that differ at or near their 5′ ends. The boundary at which these alternative sequences diverge is precisely conserved in both species. Across the region that is common to all rat and human AMPD3 cDNA species, nucleotide and predicted amino acid sequences are 89% and 93% identical respectively, although the rat open reading frame is lacking two separate in-frame codons in the 5′ end. Extreme 5′ regions between the two species are entirely divergent, and one alternative rat sequence is predicted to confer at least 36 additional N-terminal residues to its encoded AMPD3 polypeptide. A comparison of 3′ untranslated regions indicates that the rat sequence is 250 bp longer and contains multiple consensus polyadenylation signals. Examination of relative rat AMPD3 gene expression shows (1) variable patterns of alternative mRNA abundance across adult tissues, (2) developmental regulation in skeletal muscle and liver, and (3) greater mRNA abundance in adult red (soleus) than in mixed (plantaris) and white (outer gastrocnemius) skeletal muscle. Finally, baculoviral expression of rat and human AMPD3 proteins produces enzymes that are chromatographically and kinetically similar. Moreover, both recombinant activities immunoreact with anti-C and anti-E serum. These combined results demonstrate that rat isoform C and human isoform E are homologous cross-species AMPD3 proteins.
Regulatory role of prostaglandin E2 in induction of cyclo-oxygenase-2 by a thromboxane A2 analogue (U46619) and basic fibroblast growth factor in porcine aortic smooth-muscle cells
Authors: KARIM Souad , BERROU Eliane , LÉVY-TOLEDANO Sylviane , BRYCKAERT Marijke , MACLOUF Jacques
Published: August 2015

U46619, a thromboxane A2 analogue, and basic fibroblast growth factor (FGF-2) both induced the expression of the inducible cyclo-oxygenase (Cox)-2 in porcine aortic smooth-muscle cells. This induction was dose-dependent (submaximal at 300 nM for U46619 and 1 ng/ml for FGF-2) and time-dependent, with similar intensity and maximal expression at 2 h. Under these conditions, both inducers stimulated rapid activation of extracellular signal-regulated kinase (ERK2) at 5–10 min, a transient and lower intensity being induced by U46619 whereas that induced by FGF-2 was sustained (> 1 h). PD98059, an inhibitor of the ERK pathway, inhibited the expression of Cox-2. In contrast, activation of Jun-N-terminal kinase (JNK1) was sustained with U46619 but poorly induced by FGF-2. Cox-2 expression induced by U46619 or FGF-2 was similarly reduced by prostaglandin (PGE2), forskolin or dibutyryl-cAMP, suggesting a regulatory effect of adenylate cyclase on Cox-2 expression. However, activation of ERK2 by FGF-2 was not affected by PGE2 whereas that of JNK1 by U46619 was inhibited, suggesting that inhibition of COX-2 expression by cAMP may be downstream of ERK2. The effects of PGE2 and forskolin on Cox-2 and phosphorylation of JNK1 were reversed with the protein kinase A inhibitor H89. In addition, endogenous PGE2 down-regulated the expression of Cox-2 by the two inducers, as stimulation of the cells in the presence of different Cox inhibitors increased the expression of the protein. Overall, these results suggest that exogenous and endogenous PGE2 exert negative inhibitory effects on Cox-2 expression mediated by stimulation of protein kinase A.
Absorption of kininogen from human plasma by Streptococcus pyogenes is followed by the release of bradykinin
Authors: BEN NASR Abdelhakim , HERWALD Heiko , SJÖBRING Ulf , RENNÉ Thomas , MÜLLER-ESTERL Werner , BJÖRCK Lars
Published: August 2015

H-kininogen (high-molecular-mass kininogen, HK) is the precursor of the vasoactive peptide hormone bradykinin (BK). Previous work has demonstrated that HK binds to Streptococcus pyogenesthrough M-proteins, fibrous surface proteins and important virulence factors of these bacteria. Here we find that M-protein-expressing bacteria absorb HK from human plasma. The HK bound to the bacteria was found to be cleaved, and analysis of the degradation pattern suggested that the cleavage of HK at the bacterial surface is associated with the release of BK. Moreover, addition of activated plasma prekallikrein to bacteria preincubated with human plasma, resulted in BK release. This mechanism, by which a potent vasoactive and proinflammatory peptide is generated at the site of infection, should influence the host–parasite relationship during S. pyogenes infections.
Nuclear factor κB is required for the transcriptional control of type II NO synthase in regenerating liver
Authors: DÍAZ-GUERRA Mara J. M. , VELASCO Marta , MARTÍN-SANZ Paloma , BOSCÁ Lisardo
Published: August 2015

A concerted activation of transcription factors involved in the transactivation of type II NO synthase (iNOS) gene occurred after partial hepatectomy (PH), resulting in the transient expression of iNOS. The corresponding mRNA and protein levels of iNOS reached a maximum at 4 h and 8 h post-PH respectively. This induction was preceded by an early and transient activation of nuclear factor κB (NF-κB). Analysis of the κB inhibitory (I) proteins showed an important role for IκBα in the process of NF-κB activation, whereas the contribution of IκBβ was less evident. Interferon regulatory factor 1, which has been described as an important activator of iNOS expression, was up-regulated after PH but failed to bind to the corresponding DNA binding sequences of the iNOS promoter. The transcriptional control of iNOS after PH, was compared with the events associated with the hepatic expression of this enzyme in animals challenged with lipopolysaccharide, showing a differential pattern of transcription-factor activation and IκB degradation between both models. Transfection of hepatoma cell lines with iNOS promoter constructs, followed by stimulation with post-PH sera, revealed the requirement of NF-κB activation for iNOS expression. These data suggest that there is an important role for the restricted NF-κB activation in the temporal pattern of iNOS expression in regenerating liver.
Two forms of Wilson disease protein produced by alternative splicing are localized in distinct cellular compartments
Authors: YANG Xiao-Li , MIURA Naoyuki , KAWARADA Yoshihiko , TERADA Kunihiko , PETRUKHIN Konstantin , GILLIAM T. Conrad , SUGIYAMA Toshihiro
Published: August 2015

Copper is an essential trace element in prokaryotes and eukaryotes and is strictly regulated by biological mechanisms. Menkes and Wilson diseases are human disorders that arise from disruption of the normal process of copper export from the cytosol to the extracellular environment. Recently a gene for Wilson disease (WD) (also named the ATP7B gene) was cloned. This gene encodes a copper transporter of the P-type ATPase. We prepared monoclonal and polyclonal anti-(WD protein) antibodies and characterized the full-length WD protein as well as a shorter form that is produced by alternative splicing in the human brain. We found that the WD protein is localized mainly in the Golgi apparatus, whereas the shorter form is present in the cytosol. These results suggest that the alternative WD proteins act as key regulators of copper metabolism, perhaps by performing distinct roles in the intracellular transport and export of copper.
Sequence of human carboxypeptidase D reveals it to be a member of the regulatory carboxypeptidase family with three tandem active site domains
Authors: TAN Fulong , REHLI Michael , KRAUSE Stefan W. , SKIDGEL Randal A.
Published: August 2015

We have cloned the cDNA for human carboxypeptidase D (CPD), a new B-type metallocarboxypeptidase that is membrane bound and has an acidic pH optimum. The 5.8 kb of cDNA sequenced contains an open reading frame of 4131 bp encoding 1377 amino acid residues. The sequence is similar (75% identity) to duck gp180, a protein that was isolated, cloned and sequenced as a hepatitis B virus-binding protein but not characterized as a carboxypeptidase. Hydropathic analysis revealed a hydrophobic region at the N-terminus, representing the signal peptide, and one near the C-terminus that probably represents the transmembrane anchor. The most striking feature is the presence of three tandem carboxypeptidase homology domains that have sequence similarity to the regulatory B-type carboxypeptidase family, typified by carboxypeptidases M, E and N. Because of the three repeats, CPD is about three times larger (175–180 kDa) than other members of this family (approx. 50–62 kDa). Domain 2 is most closely related to carboxypeptidases M, E and N (45–48% identity), followed by domain 1 (37–38%) and domain 3 (20–27%). There is much higher sequence identity in regions containing putative active site residues, and all catalytically important residues are strictly conserved in domains 1 and 2. In domain 3, however, only 1 of 8 active site residues is conserved, indicating that this portion might not be catalytically active. Northern blotting of mRNA from human tissues and cells showed high levels of CPD mRNA in placenta, pancreas and Hep G2 hepatoma cells, and smaller amounts in skeletal muscle, heart and HT-29 colon carcinoma and melanoma cell lines.
Expression, purification and characterization of recombinant caprine N-acetylglucosamine-6-sulphatase
Authors: LITJENS Tom , BIELICKI Julie , ANSON Donald S. , FRIDERICI Karen , JONES Margaret Z. , HOPWOOD John J.
Published: August 2015

Mucopolysaccharidosis type IIID or Sanfilippo D syndrome is a lysosomal storage disorder caused by the deficiency of N-acetylglucosamine-6-sulphatase (Glc6S). In addition to human patients, a Nubian goat with this disorder has been described and the caprine Glc6S (cGlc6S) cDNA cloned. In this study, the full-length cGlc6S cDNA was inserted into the expression vector, pEFNeo, which placed the cGlc6S cDNA under the transcriptional control of the human polypeptide chain elongation factor promoter. The pEFNeo expression vector also contains the human growth hormone polyadenylation signal and the genes encoding resistance to ampicillin and G418. The cGlc6S expression construct was electroporated into Chinese hamster ovary (CHO-K1) cells, and stably transfected clones were isolated. One clone, CHOrcGlc6S.17, which secreted the highest Glc6S activity into the culture medium, was selected and cultured in cell factories. The secreted recombinant cGlc6S (rcGlc6S) precursor was purified to homogeneity from conditioned medium by a two-column procedure which consisted of a Cu2+-chelating Sepharose column followed by TSK G3000SW gel filtration. The native molecular mass of rcGlc6S was estimated to be 102 kDa and the subunit size was 94 kDa. The kinetic properties of cGlc6S were similar to those of human Glc6S isolated from liver. rcGlc6S was endocytosed by fibroblasts from patients with mucopolysaccharidosis type IIID via the mannose 6-phosphate receptor-mediated pathway resulting in correction of the storage phenotype of these cells.
Role of Mg2+ in the structure and activity of maize (Zea mays L.) isocitrate lyase: indications for hysteretic behaviour
Authors: BEECKMANS Sonia , KHAN A. Salam , VAN DRIESSCHE Edilbert
Published: August 2015

The role of Mg2+ in the structure and activity of maize isocitrate lyase has been studied by CD, limited proteolysis, protection by ligands against inactivation, and activity measurements at various metal concentrations. From CD and trypsinolysis experiments, the existence of high-affinity binding sites for Mg2+ was demonstrated, and a KdME of 200 μM was determined. Both free enzyme (E) and enzyme molecules with high-affinity sites occupied (ME) are catalytically competent, the former showing 40% of the activity of the latter. Mg2+ thus acts as a non-essential activator. A second Mg2+-binding site with a KdMEM of 6 mM was revealed from protection experiments by increasing Mg2+ concentrations against inactivation. From activity measurements at different Mg2+ concentrations, the affinity of the enzyme for the Mg2+–isocitrate complex (MI) was determined to be KdE(MI) = 9 μM. Maize isocitrate lyase was shown to display hysteretic behaviour. Filling the low-affinity binding sites with Mg2+ induces a conformational change in the high-affinity binding sites resulting in an even higher affinity for Mg2+ (KdME* = 40 μM). On lowering the Mg2+ concentration again, the enzyme only responds slowly: the time needed for all enzyme molecules to return to the conformation at which KdME is 200 μM was found to be 60 min. Finally it was shown that the high-affinity binding site for Mg2+ is not formed at low (4 °C) temperature.
Cucurbitacins are insect steroid hormone antagonists acting at the ecdysteroid receptor
Authors: DINAN Laurence , WHITING Pensri , GIRAULT Jean-Pierre , LAFONT René , DHADIALLA S. Tarlochan , CRESS E. Dean , MUGAT Bruno , ANTONIEWSKI Christophe , LEPESANT Jean-Antoine
Published: August 2015

Two triterpenoids, cucurbitacins B and D, have been isolated from seeds of Iberis umbellata (Cruciferae) and shown to be responsible for the antagonistic activity of a methanolic extract of this species in preventing the 20-hydroxyecdysone (20E)-induced morphological changes in the Drosophila melanogaster BII permanent cell line. With a 20E concentration of 50 nM, cucurbitacins B and D give 50% responses at 1.5 and 10 μM respectively. Both cucurbitacins are able to displace specifically bound radiolabelled 25-deoxy-20-hydroxyecdysone (ponasterone A) from a cell-free preparation of the BII cells containing ecdysteroid receptors. The Kd values for cucurbitacins B and D (5 and 50 μM respectively) are similar to the concentrations required to antagonize 20E activity with whole cells. Cucurbitacin B (cucB) prevents stimulation by 20E of an ecdysteroid-responsive reporter gene in a transfection assay. CucB also prevents the formation of the Drosophila ecdysteroid receptor/Ultraspiracle/20E complex with the hsp27 ecdysteroid response element as demonstrated by gel-shift assay. This is therefore the first definitive evidence for the existence of antagonists acting at the ecdysteroid receptor. Preliminary structure/activity studies indicate the importance of the Δ23-22-oxo functional grouping in the side chain for antagonistic activity. Hexanorcucurbitacin D, which lacks carbon atoms C-22 to C-27, is found to be a weak agonist rather than an antagonist. Moreover, the side chain analogue 5-methylhex-3-en-2-one possesses weak antagonistic activity.
Expression levels of functional folate receptors α and β are related to the number of N-glycosylated sites
Authors: SHEN Feng , WANG Huiquan , ZHENG Xuan , RATNAM Manohar
Published: August 2015

In a previous study with inhibitors of N-glycosylation, it was proposed that core glycosylation of the folate receptor (FR) is required for the proper folding of the protein [Luhrs (1991) Blood 77, 1171-1180]. The human FR isoforms type α and type β have three and two candidate sites for N-glycosylation respectively, only one of which is conserved. The significance of N-glycosylation at each of these loci in the expression and function of FR was examined by eliminating the sites both individually and in combination by introducing Asn → Gln substitutions. Translation experiments in vitro showed that the mutations did not alter the synthetic rates of the polypeptides. The recombinant proteins were expressed in human 293 fibroblasts. Treatment with N-glycanase and analysis by Western blotting of the wild-type and mutant proteins revealed that all of the candidate sites in both FR-α and FR-β are glycosylated. When all of the N-glycosylation sites were abolished, 2% and 8% of FR-α and FR-β respectively were expressed on the cell surface compared with the corresponding wild-type proteins; the residual FR polypeptides in the cell lysates were unable to bind [3H]folic acid. In both the proteins, the inclusion of each additional N-glycosylation site partly contributed to restoration of cell surface [3H]folic acid binding and receptor-mediated folate transport. Further, in FR-β the introduction of an additional unnatural site of N-glycosylation resulted in the enhancement of the expression of the cell surface receptor compared with the wild-type protein. The results indicate that the total mass of N-glycosylation, not a specific locus of the modification, is critical for the efficient folding and optimal expression of functional FR-α and FR-β.
Characterization of maize (Zea mays) pollen profilin function in vitro and in live cells
Authors: GIBBON C. Bryan , REN Haiyun , STAIGER J. Christopher
Published: August 2015

Profilin is a small, 12-15 kDa, actin-binding protein that interacts with at least three different ligands. The 1:1 interaction of profilin with globular actin (G-actin) was originally thought to provide a mechanism for sequestering actin monomers in the cytoplasm. It has recently become clear that the role of profilin in the cell is more complex, perhaps due to interactions with polyphosphoinositides and proline-rich proteins, or due to the ability to lower the critical concentration for actin assembly at the fast-growing barbed end of actin filaments. Because actin-binding proteins have been shown to behave differently with heterologous sources of actin, we characterized the interaction between maize pollen profilins and plant G-actin. The equilibrium dissociation constants measured by tryptophan fluorescence quenching were similar to those of other CaATP-G-actin-profilin complexes (Kd = 1.0-1.5 μM). The ability of maize profilin isoforms to bind poly-L-proline was analysed, and the Kd values for recombinant pollen and human profilins were similar when determined by two independent methods. However, the affinity of native maize pollen profilin for poly-l-proline was substantially lower than that of any of the recombinant proteins by one of these assays. The possibility of post-translational modification of profilin in the mature pollen grain is discussed. Finally, we quantified the effects of microinjection of each profilin isoform on the cytoarchitecture of Tradescantia stamen hair cells and show that the resultant disruption can be used to compare actin-binding proteins in living cells. The results are discussed in relation to a recent model of the interphase actin array in these plant cells.
Adrenergic activation of vascular endothelial growth factor mRNA expression in rat brown adipose tissue: implication in cold-induced angiogenesis
Authors: ASANO Atsushi , MORIMATSU Masami , NIKAMI Hideki , YOSHIDA Toshihide , SAITO Masayuki
Published: August 2015

Cold exposure produces adaptive hyperplasia and growth of brown adipose tissue (BAT), the major site of non-shivering thermogenesis in rodents, associated with increased angiogenesis in this tissue. Vascular endothelial growth factor (VEGF), one of the most potent angiogenic factors, was found to be expressed abundantly in BAT of the rat. When rats were exposed to cold at 4 °C, the VEGF mRNA level in BAT was increased by 2-3-fold in 1-4 h, but returned to the basal level within 24 h. VEGF expression in other tissues such as heart, kidney and lung did not change after cold exposure. The cold-induced increase in VEGF mRNA was abolished by surgical sympathetic denervation, but mimicked by administration of noradrenaline or a β3-adrenoceptor agonist CL316,243, indicating the critical role of the β-adrenergic pathway in VEGF expression in BAT. Among three isoforms of VEGF, the mRNA of a short form (VEGF120) lacking heparin-binding activity was preferentially increased after cold exposure and treatment with the adrenergic agonists. These results suggest that cold exposure activates the sympathetic nerves and leads to a rapid increase in synthesis of VEGF in BAT, which in turn stimulates the proliferation of surrounding vascular endothelial cells.
Primary structure of a copper-binding metallothionein from mantle tissue of the terrestrial gastropod Helix pomatia L
Authors: BERGER Burkhard , DALLINGER Reinhard , GEHRIG Peter , HUNZIKER E. Peter
Published: August 2015

A novel copper-binding metallothionein (MT) has been purified from mantle tissue of the terrestrial snail Helix pomatia using gel-permeation chromatography, ion-exchange chromatography and reverse-phase HPLC. Copper was removed from the thionein by addition of ammonium tetrathiomolybdate. The resulting apothionein (molecular mass 6247 Da) was S-methylated and digested with trypsin, endoproteinase Arg-C and endoproteinase Lys-C. Amino acid sequences of the resulting peptides were determined by collision-induced dissociation tandem MS. The protein is acetylated at its N-terminus, and consists of 64 amino acids, 18 of which are cysteine residues. A comparison with the cadmium-binding MT isolated from the midgut gland of the same species shows an identical arrangement of the cysteines, but an unexpectedly high variability in the other amino acids. The two MT isoforms differ in total length and at 26 positions of their peptide chains. We suggest that the copper-binding MT isoform from the mantle of H. pomatia is responsible for regulatory functions in favour of copper, probably in connection with the metabolism of the copper-bearing protein, haemocyanin.
Characterization of α-conotoxin interactions with the nicotinic acetylcholine receptor and monoclonal antibodies
Authors: ASHCOM D. James , STILES G. Bradley
Published: August 2015

The venoms of predatory marine cone snails, Conus species, contain numerous peptides and proteins with remarkably diverse pharmacological properties. One group of peptides are the α-conotoxins, which consist of 13-19 amino acids constrained by two disulphide bonds. A biologically active fluorescein derivative of Conus geographus α-conotoxin GI (FGI) was used in novel solution-phase-binding assays with purified Torpedo californica nicotinic acetylcholine receptor (nAchR) and monoclonal antibodies developed against the toxin. The binding of FGI to nAchR or antibody had apparent dissociation constants of 10-100 nM. Structure-function studies with α-conotoxin GI analogues composed of a single disulphide loop revealed that different conformational restraints are necessary for effective toxin interactions with nAchR or antibodies.
Thrombin produces phosphorylation of cytosolic phospholipase A2 by a mitogen-activated protein kinase kinase-independent mechanism in the human astrocytoma cell line 1321N1
Authors: HERNÁNDEZ Marita , BAYÓN Yolanda , SÁNCHEZ CRESPO Mariano , NIETO María Luisa
Published: August 2015

The release of [3H]arachidonic acid was studied in the 1321N1 astrocytoma cell line upon stimulation with thrombin. The effect of thrombin was antagonized by hirudin only when both compounds were added simultaneously, which suggests activation of thrombin receptor. Evidence that the cytosolic phospholipase A2 (cPLA2) takes part in thrombin-induced arachidonate release was provided by the finding that thrombin induced retardation of the mobility of cPLA2 in SDS/polyacrylamide gels, which is a feature of the activation of cPLA2 by mitogen-activated protein (MAP) kinases. Thrombin induced activation of two members of the MAP kinase family whose consensus primary sequence appears in cPLA2, namely p42-MAP kinase and c-Jun kinase. However, the activation of c-Jun kinase preceded the phosphorylation of cPLA2 more clearly than the activation of p42-MAK kinase did. Both cPLA2 and c-Jun kinase activation were not affected by PD-98059, a specific inhibitor of MAP kinase kinases, which indeed completely blocked p42-MAP kinase shift. Heat shock, a well-known activator of c-Jun kinase, also phosphorylated cPLA2 but not p42-MAP kinase. These data indicate the existence in astrocytoma cells of a signalling pathway triggered by thrombin receptor stimulation that activates a kinase cascade acting on the Pro-Leu-Ser-Pro consensus primary sequence, activates cPLA2, and associates the release of arachidonate with nuclear signalling pathways.
Chromatin structure of the Saccharomyces cerevisiae DNA topoisomerase I promoter in different growth phases
Authors: RUBBI Liudmilla , CAMILLONI Giorgio , CASERTA Micaela , MAURO Ernesto DI , VENDITTI Sabrina
Published: August 2015

We have determined the chromatin organization of the Saccharomyces cerevisiae DNA topoisomerase I promoter. Three nucleosomal core particles have been mapped at nucleotide level over the promoter region, encompassing the presumptive TATA sequence and the two RNA initiation sites; the most upstream nucleosome particle forms on to a 29 bp-long poly(dA-dT) element. This simple organization remains constant throughout both the logarithmic and the linear phase of growth, with the exception of an increased accessibility to micrococcal nuclease of the nucleosome covering the TATA box and the RNA initiation sites during the diauxic shift (the switching from the fermentative to the respiratory metabolism) in parallel with an increase of the DNA topoisomerase I mRNA. In addition, a strong disorganization of the bulk chromatin structure in the late stationary phase is also reported.
Relocation of annexin V to platelet membranes is a phosphorylation-dependent process
Authors: TROTTER J. Patrick , ORCHARD A. Margaret , WALKER H. John
Published: August 2015

Annexins are a family of calcium-binding proteins that have been implicated in a wide range of intracellular processes. We have previously reported that stimulation of platelets with agents that increase intracellular [Ca2+] induces the relocation of annexin V to membranes, and that this annexin V may be binding to a 50 kDa protein located within platelet membranes. We report here, using an in vitro reconstitution system, that the relocation of annexin V to membranes is enhanced by ATP. We also demonstrate that when adenosine 5ʹ-[γ-thio]-triphosphate, which can replace ATP in phosphorylation reactions, is substituted for ATP, the amount of annexin V that binds to membranes is further increased. In separate experiments using intact cells, we show that the protein phosphatase inhibitor okadaic acid mimics the action of the physiological agonist thrombin, in that it induces annexin V to bind to membranes and that the addition of the protein kinase inhibitor staurosporine inhibits A23187-induced relocation of annexin V. In addition, alkaline phosphatase, when added to isolated membranes, was found to remove endogenous annexin V from the membranes. Furthermore, immunoprecipitation of 33P-labelled proteins indicated that annexin V may form a multi-protein complex including phosphoproteins of 25, 50 and 83 kDa. Taken together these observations suggest that, following physiological activation, the phosphorylation of one or more proteins is responsible for the tight association of annexin V with platelet membranes and the subsequent regulation of membrane localized processes.
Evidence that a low-molecular-mass GTP-binding protein is required for store-activated Ca2+ inflow in hepatocytes
Authors: FERNANDO C. Kekulu , GREGORY B. Roland , KATSIS Frosa , KEMP E. Bruce , BARRITT J. Greg
Published: August 2015

The roles of a monomeric GTP-binding regulatory protein in the activation of store-activated plasma membrane Ca2+ channels and in the release of Ca2+ from the smooth endoplasmic reticulum (SER) in rat liver parenchymal cells were investigated with the use of freshly isolated rat hepatocytes and rat liver microsomes. A low concentration (approx. 130 μM intracellular) of guanosine 5ʹ-[γ-thio]triphosphate (GTP[S]) activated Ca2+ inflow in intact hepatocytes in the absence of an agonist, whereas a high concentration (approx. 530 μM intracellular) of GTP[S] or guanosine 5ʹ-[βγ-imido]triphosphate (p[NH]ppG) inhibited the Ca2+ inflow induced by inhibitors of the activity of the endoplasmic-reticulum Ca2+-ATPase (SERCA) and by vasopressin. GTP (530 μM) prevented the inhibition of Ca2+ inflow by GTP[S] and p[NH]ppG. Brefeldin A and the peptide human Arf-1-(2-17), which inhibit many functions of ADP ribosylation factor (Arf) proteins, inhibited the Ca2+ inflow induced by SERCA inhibitors and vasopressin, and altered the profile of Ca2+ release from the SER. These effects were observed at concentrations of Brefeldin A and Arf-1-(2-17) comparable with those that inhibit the functions of Arf proteins in other systems. Succinylated Arf-1-(2-17) had a negligible effect on Ca2+ inflow. GTP[S] and Arf-1-(2-17) completely inhibited the synergistic action of GTP and Ins(1,4,5)P3 in releasing 45Ca2+ from rat liver microsomes loaded with 45Ca2+. AlF4- (under conditions expected to activate trimeric G-proteins) and succinylated Arf-1-(2-17) had no effect on GTP/Ins(1,4,5)P3-induced 45Ca2+ release, and a mastoparan analogue caused partial inhibition. Arf-1-(2-17) did not inhibit 45Ca2+ release induced by either thapsigargin or ionomycin. It is concluded that a low-molecular-mass G-protein, most probably a member of the Arf protein family, is required for store-activated Ca2+ inflow in rat hepatocytes. The idea that the role of this G-protein is to maintain a region of the SER in the correct intracellular location is discussed briefly.
Effect of aging on the chaperone-like function of human α-crystallin assessed by three methods
Authors: DERHAM K. Barry , HARDING J. John
Published: August 2015

α-Crystallin can function as a molecular chaperone by preventing unwanted interactions. This paper presents the effects of aging and cataract on the chaperone-like properties of α-crystallin from soluble fractions from the cortex and nucleus of human lenses by using three assays: enzyme inactivation and two turbidity experiments. The three methods complemented each other. There was no decrease with age of chaperone-like function of cortical α-low and α-high crystallin. Nuclear α-low crystallin showed a decrease, whereas α-high crystallin showed no age-related change but its protective effect was diminished. Results from the nucleus of 40-year-old cataractous lenses seemed similar to those for clear lenses of equivalent age, whereas 80-year-old cataractous lenses showed decreased chaperone-like behaviour.