bluuebunny/crossref_metadata_2025 huggingface dataset:
Separate upstream and convergent downstream pathways of G-protein- and phorbol ester-mediated Ca2+ sensitization of myosin light chain phosphorylation in smooth muscle
Authors: JENSEN Peter E. , GONG Ming C. , SOMLYO Avril V. , SOMLYO Andrew P.
Published: August 2015

The effect of phorbol ester-induced down-regulation of protein kinase C (PKC) on diacylglycerol (sn-1,2-dioctanoylglycerol, diC8)- and G-protein-coupled Ca2+ sensitization and on the relationship between phosphorylation of the regulatory myosin light chains (MLC20) and force during Ca2+ sensitization were investigated in rabbit portal vein (PV), femoral artery (FA) and ileum smooth muscle. The effects of phorbol dibutyrate (PDBu), guanosine 5´-[γ-thio]triphosphate (GTP[S]) and agonists on the membrane versus cytosolic distribution of PKC isoenzymes were also determined. Down-regulation of PKC abolished Ca2+ sensitization of force and the accompanying increases in MLC20 phosphorylation induced by PDBu, as well as Ca2+ sensitization of force by diC8, but not that by GTP[S], aluminum fluoride (AlF4-) or agonists (phenylephrine, endothelin or carbachol). Down-regulation also inhibited the PDBu-, but not the GTP[S]-induced increase in force under Ca2+-free conditions. In ileum, PDBu translocated PKCs α, β1, β2, ϵ and θ to the membrane fraction, and GTP[S] caused a small translocation of PKC-ϵ. Carbachol- and GTP[S]-induced Ca2+ sensitization remained unaffected in down-regulated ileum in which no cytosolic PKC-ϵ was detectable. We conclude that, although both phorbol ester-induced and G-protein-coupled Ca2+ sensitization of force are mediated by increased MLC20 phosphorylation, it is likely that PKCs α, β1, β2, ϵ and θ do not play an essential role in, although they may contribute to, the G-protein-coupled mechanism.
Mediation of interleukin-11-dependent biological responses by a soluble form of the interleukin-11 receptor
Authors: KAROW Julia , HUDSON Keith R. , HALL Mark A. , VERNALLIS Ann B. , TAYLOR Jacky A. , GOSSLER Achim , HEATH John K.
Published: August 2015

Interleukin-11 (IL-11) is a polyfunctional cytokine whose biological actions require a specific IL-11 receptor (IL-11R) and the transmembrane transducer gp130. Here we report the production of a soluble form of the murine IL-11R and demonstrate that it interacts with IL-11 ligand with high affinity. The affinity of IL-11 alone for gp130 is below the level of detection, but a complex of IL-11 and soluble IL-11R interacts with gp130 with high affinity. The addition of soluble IL-11R potentiates the effects of exogenous IL-11 in cells that are normally responsive to IL-11. A biological response to IL-11 can be reconstituted in BAF cells transfected with gp130 by addition of IL-11 and soluble IL-11R. These findings show that the cytoplasmic domain of the IL-11R is not required for the biological effects of IL-11 and that a complex of IL-11 and IL-11R mediates signalling by association with gp130.
Dual mechanism for cAMP-dependent modulation of Ca2+ signalling in articular chondrocytes
Authors: D'ANDREA Paola , PASCHINI Valentina , VITTUR Franco
Published: August 2015

The ability of cAMP to modulate the actions of Ca2+-mobilizing agonists was studied in single Fura-2-loaded pig articular chondrocytes in primary culture. Forskolin and 8-Br-cAMP increased both the frequency and amplitude of Ca2+ oscillations induced by ATP, and, in unstimulated cells, induced single Ca2+ transients or even Ca2+ oscillations. The cAMP-dependent protein kinase inhibitor H89 totally prevented the effect of cAMP-elevating agents on Ca2+ signalling. Forskolin and 8-Br-cAMP promptly increased the rate of Mn2+ quenching, when administered in the presence of ATP, suggesting a potentiation of receptor-mediated Ca2+ influx. In Ca2+-free medium, ATP-induced Ca2+ oscillations decreased and stopped after a few cycles: subsequent ATP additions temporarily resumed the activity, an effect that could be mimicked by forskolin. The same agent induced single Ca2+ transients in 42% of the cell population maintained in Ca2+-free medium. Thapsigargin prevented Ca2+ responses to both ATP and forskolin. The results indicate a dual mechanism for cAMP-induced potentiation of Ca2+ signalling in articular chondrocytes: an increase of receptor-mediated Ca2+ influx and a positive modulation of intracellular Ca2+ release.
Hepatic regeneration induces changes in lipoprotein lipase activity in several tissues and its re-expression in the liver
Authors: SABUGAL Rosa , ROBERT Monique Q , JULVE Josep , AUWERX Johan , LLOBERA Miquel , PEINADO-ONSURBE Julia
Published: August 2015

We examined the expression of lipoprotein lipase (LPL) gene and LPL activity following a two-thirds hepatectomy and during liver regeneration. In most of the tissues studied, LPL activity increased a few hours after partial hepatectomy, but soon returned to normal levels. The greatest increase was found in the adrenal glands, plasma and liver. This increase in LPL activity in the liver could be partially due to an increase in the influx of the enzyme from extrahepatic tissues. There is, however, also a re-expression of LPL mRNA in the liver after partial hepatectomy (during the first hours). It is well known that LPL is expressed in the liver of neonatal animals, but progressively decreases during post-natal development, to reach adult levels around the time of weaning. Our results show by the first time that the remaining liver re-expresses LPL gene during the regeneration process and that the hepatocytes de-differentiate and acquire some of the neonatal characteristics. The increase in LPL mRNA will contribute to the rise in LPL activity after hepatectomy. This presence of LPL could enable the liver to take up fatty acids from the circulating triacylglycerols, which are needed as energetic and plastic substrates during the process of hepatic regeneration.
The proximal element of the human apolipoprotein A-II promoter increases the enhancer activity of the distal region
Authors: LACORTE Jean-Marc , FOURNIAT Esther , PASTIER Danièle , CHAMBAZ Jean , RIBEIRO Agnès , CARDOT Philippe
Published: August 2015

We have previously shown that human apolipoprotein A-II (apoA-II) transcription is controlled by a complex set of regulatory elements. In this study, we demonstrate that the distal region of the apoA-II promoter (-911/-614) acts as an enhancer and results in a 6-fold increase in activity when the proximal AB element is inserted between this enhancer and a TATA box. The AB element alone does not display any transcriptional activity. The combination of the proximal AB element and the enhancer is sufficient to activate transcription to the same level as that achieved with the full-length promoter. DNA binding and competition assays, and binding interference experiments allowed us to identify two adjacent binding sites within the AB element. These bind activities designated CIIIB1 and AIIAB3/4, respectively. Mutation on each of these sites showed that the binding site of CIIIB1 within the AB element played a major role in the interaction with the enhancer. Whereas transcriptional activation of other apolipoprotein genes involves the binding of the liver-enriched hepatocyte nuclear factor 4 (HNF4) on their proximal promoter, the present study demonstrates that neither HNF4 nor ApoA-I regulatory protein 1, its antagonistic orphan receptor, was able to bind the AB element. Instead, apoA-II transcription was driven by the interaction of apoA-II enhancer with proximal AB element, which involves an unidentified activity, CIIIB1.
Purinergic stimulation of rat cardiomyocytes induces tyrosine phosphorylation and membrane association of phospholipase Cγ: a major mechanism for InsP3 generation
Authors: PUCEAT Michel , VASSORT Guy
Published: August 2015

Phospholipase Cγ (PLCγ) expression and activation by a purinergic agonist were investigated in adult rat cardiomyocytes. PLCγ is expressed in isolated cardiomyocytes. Stimulation of cells with extracellular ATP induces a rapid increase in membrane-associated PLCγ immunoreactivity most probably due to redistribution of the lipase from the cytosol to the membrane. The purine triggers a significant phosphorylation on tyrosine residues of a cytosolic pool of PLCγ with a time course that correlates with that of translocation. Extracellular ATP also increases intracellular Ins(1,4,5)P3 content. All these events (translocation and phosphorylation of PLCγ, InsP3 formation) are blocked by genistein, a tyrosine kinase inhibitor. The purinergic effect on both PLCγ translocation and phosphorylation are Ca-sensitive. We thus propose that the purinergic stimulation activates a non-receptor tyrosine kinase that phosphorylates PLCγ in the presence of an increased Ca level and induces PLCγ redistribution to the membrane. There, PLCγ becomes activated leading to the hydrolysis of phosphatidylinositol diphosphate and in turn Ins(1,4,5)P3 formation. This cascade of events may play a significant role in the induction of arrhythmogenesis by purinergic agonists.
Organization, transcription and regulation of the Leishmania infantum histone H3 genes
Authors: SOTO Manuel , REQUENA Jose M , QUIJADA Luis , ALONSO Carlos
Published: August 2015

The genomic organization and transcription of the genes encoding the histone H3 of the protozoan parasite Leishmania infantum have been studied. It was found that there are multiple copies of the histone H3 genes distributed in chromosomal bands XIX and XIV. The nucleotide sequence of two of the L. infantum H3 genes, each one located in a different chromosome, is reported. Although the nucleotide sequence of the coding region of both genes is identical, the sequence of the 3´ untranslated region is highly divergent. It was found also that there exist two different size classes of histone H3 transcripts, each one derived from a different gene, and that they are polyadenylated. The steady-state level of the transcripts dramatically decreases when the parasites enter the stationary phase of growth, suggesting a mode of regulation which is linked to the proliferation status of the cell. Unlike the replication-dependent histones, the L. infantum H3 mRNA levels do not decrease after treatment with DNA synthesis inhibitors. A comparative analysis of the sensitivity of the histone mRNA levels to DNA inhibition in the parasites L. infantum and Trypanosoma cruzi revealed the existence of different control mechanisms in histone expression in these two phylogenetically related protozoan parasites.
Selective uptake of cholesteryl esters of low-density lipoproteins is mediated by the lipoprotein-binding site in HepG2 cells and is followed by the hydrolysis of cholesteryl esters
Authors: BRISSETTE Louise , CHAREST Marie-Claude , FALSTRAULT Louise
Published: August 2015

The study described in this paper shows that 125I-labelled low-density lipoproteins (LDL) interact with high- and low-affinity binding sites on human hepatoma (HepG2) cells. The former site is the LDL receptor and the latter is the lipoprotein-binding site (LBS). The association of 125I-labelled LDL and [3H]cholesteryl ethers–LDL with HepG2 cells revealed a 4-fold selective uptake of cholesteryl esters (CE) in a 4 h incubation period, which correlated with the depletion of CE mass in LDL. This selective uptake was not observed when the cells were incubated in the presence of a 100-fold excess of high-density lipoprotein 3, conditions where only the LDL receptor is being monitored. Also, no reduction in uptake was observed in the presence of IgG-C7, an anti-(LDL receptor) monoclonal antibody. Both findings indicate that the selective uptake occurs through the LBS and that the LBS contributes more to the entry of CE from LDL into the cell than does the LDL receptor. The fates of CE entering the cell via the LDL receptor and the LBS were also followed. To achieve this, LDL were labelled with [3H]cholesteryl oleate and the hydrolysis of [3H]cholesteryl oleate was monitored. The results indicated that 45% of the CE were hydrolysed after a 4 h incubation period, irrespective of the site of entry. Chloroquine (100 µM) was shown to inhibit hydrolysis, indicating that lysosomal enzymes were responsible for the hydrolysis of LDL–CE, whichever pathway was used. Thus our results reveal, for the first time, that the mass of CE entering the cell via the LBS is substantial and that hydrolysis of CE is by lysosomal enzyme activity. Overall, this suggests that the LBS has significant physiological importance.
A regulatory role for cAMP in phosphatidylinositol 3-kinase/p70 ribosomal S6 kinase-mediated DNA synthesis in platelet-derived-growth-factor-stimulated bovine airway smooth-muscle cells
Authors: SCOTT Pamela H , BELHAM Christopher M , AL-HAFIDH Jenan , CHILVERS Edwin R. , PEACOCK Andrew J. , GOULD Gwyn W. , PLEVIN Robin
Published: August 2015

In bovine airway smooth-muscle cells platelet-derived growth factor (PDGF) and endothelin (Et-1) stimulate sustained and comparable activation of mitogen-activated protein kinase (MAP kinase) but display very different mitogenic efficacies, with PDGF inducing 50 times more DNA synthesis than Et-1. To examine additional signalling pathways which may be involved in this response, we investigated the role of phosphatidylinositol 3-kinase (PtdIns 3-kinase)/p70 ribosomal protein S6 kinase (p70s6k) in mediating PDGF- and Et-1-induced mitogenesis, and whether inhibition of this pathway may underly the ability of cAMP to inhibit cell proliferation. PDGF stimulated an increase in PtdIns 3-kinase activity and a sustained 15-fold increase in p70s6k activity that was abolished by both wortmannin and rapamycin. Et-1, however, stimulated only a 2-fold increase in p70s6k activity that was rapamycin-sensitive but wortmannin-insensitive. DNA synthesis stimulated by PDGF (50-fold) and Et-1 (2-fold) followed a similar pattern of inhibition. Pretreatment with phorbol ester did not affect p70s6k activation in response to PDGF. Raising intracellular cAMP levels using forskolin, however, resulted in a marked time-dependent inhibition of p70s6k activity, a decrease in the tyrosine phosphorylation of the PtdIns 3-kinase p85 subunit and reduced PtdIns 3-kinase activity. Forskolin also inhibited PDGF-stimulated DNA synthesis. These results suggest that PtdIns 3-kinase-dependent activation of p70s6k may determine mitogenic efficacy of agonists that generate comparable MAP kinase signals. Negative regulation of PtdIns 3-kinase by cAMP may play an important role in the inhibition of airway smooth-muscle cell proliferation.
Ca2+-independent fusion of synaptic vesicles with phospholipase A2-treated presynaptic membranes in vitro
Authors: NISHIO Hideaki , TAKEUCHI Tadayoshi , HATA Fumiaki , YAGASAKI Osamu
Published: August 2015

To clarify the mechanism of exocytosis in neurotransmitter release, the fusion of synaptic vesicles with presynaptic membranes prepared from rat brain synaptosomes and concomitant acetylcholine (ACh) release induced by fusion of them were studied in vitro. Fusion of the synaptic vesicles with presynaptic membranes was measured by a fluorescence-dequenching assay with octadecyl rhodamine B. Synaptic vesicles fused with presynaptic membranes which had been pretreated with porcine phospholipase A2 (PLA2) in the presence of 20 µM Ca2+ and released ACh, whereas synaptic vesicles did not interact with non-pretreated membranes. The fusion followed by ACh release depended (i) on the activity of PLA2 during the membrane pretreatment, (ii) on the amount of pretreated membrane and (iii) on the duration of the pretreatment. The presence of Ca2+ ions during the pretreatment was essential for inducing a fusogenic activity of the membranes, but Ca2+ ions were not required for the fusion itself because the fusion experiment was carried out in the presence of 5 mM EGTA without added Ca2+. The presence of quinacrine, an antagonist of PLA2, during the membrane pretreatment inhibited their fusogenic activity, suggesting the importance of activation of PLA2. Presence of albumin during the pretreatment, which is an adsorbent of free fatty acids, also inhibited the fusogenic activity. Arachidonic acid, when added during the pretreatment, potentiated the fusogenic activity of the membrane. These findings suggest that the conformational change in the presynaptic membrane phospholipids induced by PLA2 and the presence of arachidonic acid produced by PLA2 are important in the process of fusion of synaptic vesicles with the presynaptic membranes of rat brain, and that the fusion process itself is independent of Ca2+.
Attenuation of oxidation and nitration reactions of peroxynitrite by selenomethionine, selenocystine and ebselen
Authors: BRIVIBA Karlis , ROUSSYN Ivan , SHAROV Victor S , SIES Helmut
Published: August 2015

The effect of the selenium-containing compounds selenomethionine, selenocystine and ebselen [2-phenyl-1,2-benzisoselenazol-3(2H)-one] on the oxidation of dihydrorhodamine 123 caused by peroxynitrite and on the nitration of 4-hydroxyphenylacetate by peroxynitrite was studied in comparison with their sulphur analogues methionine, cystine and ebsulfur [2-phenyl-1,2-benzisothiazol-3(2H)-one]. The selenocompounds protected dihydrorhodamine 123 from oxidation and 4-hydroxyphenylacetate from nitration more effectively than their sulphur analogues. Sodium selenite exhibited no effect. These observations are corollaries to the recent finding [Roussyn, Briviba, Masumoto and Sies (1996) Arch. Biochem. Biophys. 330, 216–218] that selenium-containing compounds are efficient in protecting against peroxynitrite-induced DNA single-strand breaks.
Inhibition of hsc70-catalysed clathrin uncoating by HSJ1 proteins
Authors: CHEETHAM Michael E , ANDERTON Brian H. , JACKSON Antony P.
Published: August 2015

The uncoating of clathrin-coated vesicles can be mediated in vitro by the ‘uncoating ATPase’ that has been identified as the constitutive 70 kDa heat shock protein (hsp70), hsc70. It is now established that the activity of hsp70 proteins can be regulated by another family of molecular chaperones, the DnaJ family. In this study, we have investigated the effects of DnaJ-like proteins (the human neuron-specific proteins HSJ1a and HSJ1b) on clathrin uncoating. In order to measure the kinetics of clathrin release from coated vesicles, we have developed a quantitative, two-site ELISA for clathrin triskelions and demonstrated that stoichiometric amounts of HSJ1 proteins inhibit the initial burst of hsc70-mediated clathrin uncoating by over 40%. This inhibition is not a consequence of ADP binding by hsc70 or the aggregation of hsc70, but correlates with an increase in the hsc70 associated with the coated vesicle fraction, suggesting that the inhibition is a consequence of a non-productive stabilization of hsc70 with a component of the coated vesicle fraction. These results strongly suggest that HSJ1 proteins interfere with an endogenous DnaJ-like protein that is involved in uncoating. Recent evidence suggests that the brain-specific vesicle-associated protein auxilin could play such a role. Athough we find no evidence for auxilin in our coated vesicle preparation, our results predict that an auxilin-like protein will be a general factor in clathrin uncoating.
Molecular cloning and expression in COS-1 cells of pig kidney aminopeptidase P
Authors: HYDE Ralph J , HOOPER Nigel M , TURNER Anthony J
Published: August 2015

Aminopeptidase P (AP-P; X-Pro aminopeptidase; EC 3.4.11.9), a key enzyme in the metabolism of the vasodilator bradykinin, has been cloned from a pig kidney cortex cDNA library following the use of the PCR to identify sub-libraries enriched in AP-P clones. The complete primary sequence of the enzyme has been deduced from a full-length cDNA clone. This predicts a protein of 673 amino acids with a cleavable N-terminal signal sequence and six potential N-linked glycosylation sites. A stretch of mainly hydrophobic amino acids at the C-terminus is predicted to co-ordinate the attachment of a glycosyl-phosphatidylinositol (GPI) membrane anchor. Although AP-P is a zinc metallopeptidase, the predicted primary sequence does not contain any recognizable zinc-binding motif. Transient expression of AP-P cDNA in COS-1 cells resulted in enzymic activity characteristic of AP-P, namely apstatin- and EDTA-sensitive hydrolysis of bradykinin and Gly-Pro-Hyp. The expressed protein was recognized as a polypeptide of Mr 91000 under reducing conditions, following immunoblotting of COS-1 membranes with a polyclonal antibody raised against purified pig kidney AP-P. The presence of a GPI anchor on expressed AP-P was established by demonstrating release of the enzyme from a membrane fraction following treatment with bacterial phosphatidylinositol-specific phospholipase C and its corresponding conversion from an amphipathic to a hydrophilic form, as assessed by phase separation in Triton X-114. Sequence comparisons confirm that AP-P is a member of the proline peptidase family of hydrolytic enzymes and is unrelated in sequence to other brush-border membrane peptidases.
Alterations in nutritional status regulate acetyl-CoA carboxylase expression in avian liver by a transcriptional mechanism
Authors: HILLGARTNER F. Bradley , CHARRON Tina , CHESNUT Kye A
Published: August 2015

Feeding previously starved chicks with a high-carbohydrate, low-fat diet stimulates a 9-fold increase in both the rate of synthesis of acetyl-CoA carboxylase (ACC) and the abundance of its mRNA in liver. To define the steps involved in mediating diet-induced changes in the abundance of ACC mRNA, transcriptional activity was measured with the nuclear run-on assay and multiple DNA probes specific to the ACC gene. ACC transcription was low in livers of starved chicks; feeding them with a high-carbohydrate, low-fat diet induced ACC transcription, increasing it 11-fold. An increase in transcription was detectable at 1 h, was maximal at 5 h and remained high for 26 h. Feeding previously starved chicks with a low-carbohydrate, high-fat diet stimulated a smaller increase (4-fold) in the abundance of ACC mRNA and the transcription of ACC than feeding with a high-carbohydrate, low-fat diet. The half-life of ACC mRNA in liver, as estimated from the kinetics of accumulation and decay of ACC mRNA during high-carbohydrate feeding and starvation, was not changed significantly by dietary manipulation. ACC mRNA was expressed at low levels in heart, pectoral muscle, kidney and brain. The abundance of ACC mRNA in these tissues was not affected by nutritional manipulation. These results demonstrate that nutritional control of the abundance of ACC mRNA in the chicken is liver-specific and is mediated primarily by changes in the rate of transcription of the ACC gene.
Evidence from kinetic isotope studies for an enolate intermediate in the mechanism of type II dehydroquinases
Authors: HARRIS Joanna M , GONZALEZ-BELLO Concepcion , KLEANTHOUS Colin , HAWKINS Alastair R. , COGGINS John R. , ABELL Chris
Published: August 2015

Substrate isotope studies, solvent isotope studies, proton in-ventories and studies of Vmax and Km as a function of pH suggest an E1CB (V. E. Anderson (1991) in Enzyme Mechanisms for Isotope Effects (Cook, P. F., ed.), pp. 389–417, CRC Press, Boca Raton, FL) mechanism via an enolate intermediate for type II dehydroquinases involved in biosynthetic or catabolic pathways.
Purification and characterization of two components of epoxypropane isomerase/carboxylase from Xanthobacter Py2
Authors: CHION CHAN KWO Chan K. N. , LEAK David J
Published: August 2015

Epoxypropane isomerase from Xanthobacter Py2 has been resolved into at least two components (A and B) by ion-exchange chromatography. Both components were required for the degradation of epoxypropane and were purified further. Component A was apparently homohexameric with a subunit Mr of about 44000, and possessed NAD+-dependent dihydrolipoamide dehydrogenase activity and lipoamide reductase activity. It was sensitive to inhibition by o-phenanthroline and the thiol-specific reagents N-ethylmaleimide (NEM) and p-chloromercuribenzoate. Component B was homodimeric with a subunit Mr of 62170 and contained 2 mol·mol-1 FAD. It had an NADPH-dependent lipoamide reductase activity which was sensitive to NEM and p-chloromercuribenzoate. The N-terminal amino acid sequences and monomer sizes of components A and B correspond to those of ORF1 and ORF3 respectively (ORF = open reading frame) of a recently published sequence of a clone which complements mutants unable to degrade epoxypropane. NADPH was found to replace the need for a low-Mr fraction in epoxypropane degradation assays containing components A and B and NAD+. The predicted amino acid sequence of component A (ORF1) has been analysed and shown to contain a potential ADP binding site near the N-terminus and a putative cofactor binding domain near the C-terminus, with sequence similarity to the biotinyl and lipoyl binding domains of biotin-dependent carboxylases and 2-oxoacid dehydrogenases respectively. A reaction mechanism for epoxypropane degradation, incorporating recent evidence for combined isomerization and carboxylation to acetoacetate, is discussed.
Inhibition by calponin of isometric force in demembranated vascular smooth muscle strips: the critical role of serine-175
Authors: UYAMA Yoshiaki , IMAIZUMI Yuji , WATANABE Minoru , WALSH Michael P.
Published: August 2015

α-Calponin is a thin-filament-associated protein which has been implicated in the regulation of smooth muscle contraction. Quantification of the tissue content of rat tail arterial smooth muscle revealed approximately half the amount of α-calponin relative to actin compared with chicken gizzard and other smooth muscles, suggesting that this tissue would be particularly suitable for investigation of the effects of exogenous α-calponin on the contractile properties of permeabilized muscle strips. Rat tail arterial strips demembranated with Triton X-100 retained ≈ 90% of their complement of α-calponin, and exogenous chicken gizzard α-calponin (which conveniently has a slightly lower molecular mass than the rat arterial protein) bound to the permeabilized muscle, presumably through its high affinity for actin. Exogenous α-calponin inhibited force in demembranated muscle strips in a concentration-dependent manner when added at the peak of a submaximal Ca2+-induced contraction, with a half-maximal effect at ≈ 3 µM α-calponin. Pretreatment of demembranated muscle strips with α-calponin inhibited subsequent force development at all concentrations of Ca2+ examined over the activation range. The inhibitory effect of α-calponin was shown to be Ca2+-independent, since exogenous α-calponin also inhibited force in the absence of Ca2+ in demembranated muscle strips containing thiophosphorylated myosin. Phosphorylation of α-calponin on Ser-175 by protein kinase C has been suggested to alleviate the inhibitory effect of α-calponin on smooth muscle contraction. To test this hypothesis, the effects on Ca2+-induced and Ca2+-independent contractions of demembranated muscle strips of phosphorylated α-calponin and three site-specific mutants of α-calponin (in which Ser-175 was replaced by Ala, Asp or Thr) were compared with the effects of unphosphorylated tissue-purified and recombinant wild-type α-calponins. The recombinant wild-type protein behaved identically to the unphosphorylated tissue-purified protein, as did the S175T mutant, which is known to bind actin with high affinity and to inhibit the actin-activated myosin MgATPase in vitro. On the other hand, phosphorylated α-calponin and the S175A and S175D mutants, which bind weakly to actin and have little effect on the actin-activated myosin MgATPase in vitro, failed to cause significant inhibition of force induced by Ca2+ or myosin thiophosphorylation. These results support a role for α-calponin in the regulation of smooth muscle contraction and indicate the functional importance of Ser-175 of α-calponin as a regulatory site of phosphorylation.
Steroid hormone receptors and their regulation by phosphorylation
Authors: WEIGEL Nancy L
Published: August 2015

The steroid/thyroid hormone receptor superfamily of ligand-activated transcription factors encompasses not only the receptors for steroids, thyroid hormone, retinoids and vitamin D, but also a large number of proteins whose functions and/or ligands are unknown and which are thus termed orphan receptors. Recent studies have highlighted the importance of phosphorylation in receptor function. Although most of the phosphorylation sites are serine and threonine residues, a few of the family members are also phosphorylated on tyrosine. Those steroid receptor family members that are bound to heat-shock proteins in the absence of ligand typically are basally phosphorylated and exhibit increases in phosphorylation upon ligand binding. Most of these sites contain Ser-Pro motifs, and there is evidence that cyclin-dependent kinases and MAP kinases (mitogen-activated protein kinases) phosphorylate subsets of these sites. In contrast, phosphorylation sites identified thus far in members of the family that bind to DNA in the absence of hormone typically do not contain Ser-Pro motifs and are frequently casein kinase II or protein kinase A sites. Phosphorylation has been implicated in DNA binding, transcriptional activation and stability of the receptors. The finding that some of the steroid receptor family members can be activated in the absence of ligand by growth factors or neurotransmitters that modulate kinase and/or phosphatase pathways underscores the role of phosphorylation in receptor function. Hence this family of transcription factors integrates signals from ligands as well as from signal transduction pathways, resulting in alterations in mRNA and protein expression that are unique to the complex signals received.
Expression of recombinant rat myo-inositol 1,4,5-trisphosphate 3-kinase B suggests a regulatory role for its N-terminus
Authors: THOMAS Stephen , SORIANO Salvador , d'SANTOS Clive , BANTING George
Published: August 2015

We have expressed rat myo-inositol 1,4,5-trisphosphate (IP3) 3-kinase B as both a full-length, recombinant, non-fusion protein and a full-length, recombinant, fusion protein with maltose-binding protein (MBP) in Escherichia coli. The fusion protein with MBP is soluble, binds calmodulin and is enzymically active whereas the non-fusion protein is insoluble and does not bind calmodulin unless co-expressed with bacterial chaperone proteins (either GroES and GroEL, or DnaK, DnaJ and GrpE). However, soluble, calmodulin-binding non-fusion IP3 3-kinase B is enzymically inactive. The catalytic domain of the enzyme has previously been shown to reside near the C-terminus; the results we present suggest an auto-regulatory role for the N-terminus.
Folate binding protein: molecular characterization and transcript distribution in pig liver, kidney and jejunum
Authors: HOOZEN Chi Mai VAN , LING Erh-hsin , HALSTED Charles H
Published: August 2015

Folate-binding protein (FBP) was identified and characterized in a pig liver cDNA library by screening with a 0.6 kb fragment from the cDNA of FBP from a human KB cell cancer line. The cDNA of pig liver FBP included 1230 bp containing 759 bp in the open reading frame with 80% similarity to the human placenta FBP. The deduced 253 amino acid sequence showed 67–73% similarity to previous sequences and contained 16 conserved cysteine residues, 11 tryptophan potential folate-binding sites, three sites for N-linked glycosylation and 14 hydrophobic C-terminal residues. Northern analysis and reverse transcriptase PCR identified transcripts in pig liver and kidney, but not in jejunal mucosa. Although defining the molecular structure of pig liver FBP, these studies suggest that this protein participates in the regulation of folate uptake by liver and kidney membranes but is not involved in folate absorption.
Reconstitution of the quinoprotein methanol dehydrogenase from inactive Ca2+-free enzyme with Ca2+, Sr2+ or Ba2+
Authors: GOODWIN Matthew G , AVEZOUX Alain , DALES Simon L , ANTHONY Christopher
Published: August 2015

The reconstitution of active holoenzyme containing calcium from inactive calcium-free methanol dehydrogenase, isolated from a moxA mutant of Methylobacterium extorquens, has a pH optimum of about pH 10, with a well defined pK for the process at pH 9.3. Two Ca2+ ions were irreversibly incorporated per α2β2 tetramer. Calcium could be replaced in the incorporation process by strontium or barium, the affinities for these ions being similar to that for Ca2+. Arrhenius plots for measurement of the activation energy of reconstitution were biphasic; the lower activation energy was typical of most biological processes, while the higher activation energy was at least three times greater, implying the involvement of a large conformational change during incorporation of the cations. The activation energy for incorporation of Ba2+ was considerably higher than that for incorporation of Ca2+. The novel disulphide bridge that is at the active site of the enzyme was not involved in the incorporation process. Studies of the time courses for incorporation of 45Ca2+, production of active enzyme and changes in absorption spectra failed to show any intermediates in the incorporation process.
Didecanoyl phosphatidylcholine is a superior substrate for assaying mammalian phospholipase D
Authors: VINGGAARD Anne M , JENSEN Torben , MORGAN Clive P. , COCKCROFT Shamshad , HANSEN Harald S.
Published: August 2015

Phospholipase D (PLD) activity in crude or solubilized membranes from mammalian tissues is difficult to detect with the current assay techniques, unless a high radioactive concentration of substrate and/or long incubation times are employed. Generally, the enzyme has to be extracted and partially purified on one column before easy detection of activity. Furthermore, PLD activity in cultured cells can only be detected by the available assay techniques in the presence of guanosine 5´-[γ-thio]triphosphate (GTP[S]) and a cytosolic factor [usually ADP-ribosylation factor (Arf)]. In this paper we report that the use of didecanoyl phosphatidylcholine (C10-PC) in mammalian PLD assays considerably increases the detection limit. C10-PC was compared with the commonly used dipalmitoyl phosphatidylcholine (C16-PC) as a substrate for PLD activity from membranes of human neutrophils, human placenta and pig brain, and from placental cytosol. C10-PC was superior to C16-PC by a factor of 2–28 depending on assay conditions and tissue, and it allowed the detection of GTP[S]-and Arf-stimulated PLD activity without addition of phosphatidylinositol 4,5-bisphosphate.
Species-specific alternative splicing of the epidermal growth factor-like domain 1 of cartilage aggrecan
Authors: FÜLÖP Csaba , CS-SZABÓ Gabriella , GLANT Tibor T.
Published: August 2015

Aggrecan transcripts contain two alternatively spliced exons that code for two epidermal growth factor-like domains (EGF1 and EGF2). Whereas the EGF2 sequence is expressed at a uniform level among different species, the EGF1 sequence has been detected only in human aggrecan transcripts. In this study we have used the nested primer reverse transcription-PCR (RT-PCR) method to compare the expression of the EGF1 exon in human, bovine and dog aggrecan transcripts. Our results indicate that this exon is expressed in a species-specific manner. In addition to its significant expression level in human transcripts, the EGF1 sequence can be detected in a small portion of bovine aggrecan transcripts as shown with nested primer RT-PCR. In contrast, the same module is not detectable in dog aggrecan transcripts, although an EGF1 sequence is present in the dog aggrecan gene. The expression level of the EGF1 exon in the aggrecan transcripts correlates with the strength of the polypyrimidine tract upstream of the exon. The EGF1 sequence also shows much less conservation between the species than the EGF2 sequence. The species-specific expression and high sequence variation of the EGF1 exon imply that this sequence is likely to code for an aggrecan domain having no cartilage-specific function.
Chloroplastic aspartate aminotransferase from Arabidopsis thaliana: an examination of the relationship between the structure of the gene and the spatial structure of the protein
Authors: WILKIE Susan E , LAMBERT Richard , WARREN Martin J.
Published: August 2015

A clone encoding a plastid isoenzyme of aspartate aminotransferase (AAT5) was isolated from an Arabidopsis genomic library and its complete sequence determined. The gene for AAT5 (asp5) contains an open reading frame of 2447 bp comprising 11 exons separated by introns ranging in length from 74 to 207 bp. The upstream regulatory region contains a putative TATA box and multiple copies of two sequence motifs, CTCTT and AAAGAT, previously associated with nodule-specific gene activity in legumes. The deduced primary amino acid sequence of the protein product of asp5 was used to generate a three-dimensional structure of the AAT5 protein by using the computer program Sybyl: Biopolymer Composer and known AAT structures on the protein databases. Both the mature protein and its precursor protein containing a putative N-terminal transit peptide were modelled. The resulting structure of the precursor protein indicated that the transit peptide might also inhibit dimerization of the protein until after its translocation across the chloroplast membrane. The derived structure of the mature protein was then analysed in terms of its component elements of secondary structure, and the positions on the polypeptide backbone corresponding to intron insertion sites were determined. It is observed that the introns tend to map to regions between structural subdomains of the protein and also map to sites on the surface of the molecule. The asp5 gene in Arabidopsis is thus consistent with Gilbert's exon-shuffling theory of gene evolution [Gilbert (1985) Science 228, 823–824]. A high degree of conservation of intron insertion sites between AAT genes from different plants and animals is observed, particularly within the part of the gene encoding a large β-sheet structure that forms the structural and functional core of the protein. This β-sheet structure is thus believed to comprise an ancient and very highly conserved moiety of the molecule.
Purification and characterization of a thermostable ADP-glucose pyrophosphorylase from Thermus caldophilus GK-24
Authors: KO Jeong Heon , KIM Cheorl Ho , LEE Dae-Sil , KIM Yu Sam
Published: August 2015

An extremely thermostable ADP-glucose pyrophosphorylase (AGPase) has been purified from Thermus caldophilus GK-24 to homogeneity by chromatographic methods, including gel filtration and ion-exchange and affinity chromatography. The specific activity of the enzyme was enriched 134.8-fold with a recovery of 10.5%. The purified enzyme was a single band by SDS/PAGE with a molecular mass of 52 kDa. The homotetrameric structure of the native enzyme was determined by gel filtration analysis, which showed a molecular mass of 230 kDa on a Superose-12 column, indicating that the structure of the enzyme is different from the heterotetrameric structures of higher-plant AGPases. The enzyme was most active at pH 6.0. The activity was maximal at 73–78 °C and its half-life was 30 min at 95 °C. Kinetic and regulatory properties were characterized. It was found that AGPase activity could be stimulated by a number of glycolytic intermediates. Fructose 6-phosphate, fructose 1,6-bisphosphate, phenylglyoxal and glucose 6-phosphate were effective activators, of which fructose 1,6-bisphosphate was the most effective. The enzyme was inhibited by phosphate, AMP or ADP. ATP and glucose 1-phosphate gave hyperbolic-shaped rate-concentration curves in the presence or absence of activator. A remarkable aspect of the amino acid composition was the existence of the hydrophobic and Ala+Gly residues. The N-terminal and internal peptide sequences were determined and compared with known sequences of various sources. It was apparently similar to those of AGPases from other bacterial and plant sources, suggesting that the enzymes are structurally related.
LP2, a differentiation-associated lipid-binding protein expressed in bovine lens
Authors: JAWORSKI Cynthia , WISTOW Graeme
Published: August 2015

A 13 kDa protein from bovine lens was identified and characterized by protein microsequencing and by rapid amplification of cDNA ends (RACE) PCR. Its complete sequence shows that this protein belongs to a family of fatty acid-binding proteins (FABPs), including myelin and adipocyte P2, that are associated with cellular differentiation. The bovine lens protein, designated LP2, shows very close similarity to human epidermal FABP (eFABP) and human eFABP was detected in human lens, suggesting that the two proteins might be orthologous. Reverse transcriptase–PCR (RT–PCR) was used to compare expression patterns of LP2 with those for actin and for the differentiation markers γB-crystallin and γs-crystallin in lens. Actin was most abundant in the relatively undifferentiated epithelial cells and decreased with lens cell differentiation. In contrast γB-crystallin and γs-crystallin were detected only in fibres (nuclear and cortical respectively). LP2 transcripts were detected most abundantly in fibre cells and apparently increased with cellular differentiation. Molecular modelling confirms that the sequence of LP2 fits the tertiary template of adipocyte P2 but reveals the presence of two close pairs of cysteine residues that might be susceptible to intramolecular disulphide bond formation under appropriate oxidizing conditions. LP2 is thus another potential target for oxidative stress during cataract formation in lens.
Desensitization of P2U receptor in neuronal cell line. Different control by the agonists ATP and UTP, as demonstrated by single-cell Ca2+ responses
Authors: CZUBAYKO Uwe , REISER Georg
Published: August 2015

The neuronal cell line NG108-15 (180CC15) responds to extracellular stimuli of ATP or UTP with a transient increase in the level of cytosolic Ca2+. Desensitization was investigated by recording single-cell Ca2+ responses induced by consecutive, regularly spaced (100 s intervals), brief pulses of the nucleotides. The two natural ligands of the P2U receptor, ATP and UTP, were applied at a concentration that evoked responses of a comparable size. With two pulses of UTP (10 µM), a substantial decrease (of 43%) was observed in the size of the second response. The magnitude of response was determined by measuring either the maximal amplitude or the total response, represented by the area of the Ca2+ transient. The analogous studies with ATP pulses showed a much smaller decrease (of 12%). Comparable experiments performed to investigate the mutual interaction between ATP and UTP revealed that after stimulation with ATP the response to UTP was slightly (12%) diminished, whereas the response to ATP after UTP was greatly (52%) decreased. The different degree of desensitization by either UTP or ATP of P2U receptors could be due to (1) a difference in the mode of activation of the receptor by the two ligands or (2) recruitment of another effector mechanism besides elevated Ca2+. Our results indicate the existence of a novel mechanism of receptor control, involving different modes of the receptor, that are induced by two different, activating ligands. We also investigated the cross-talk between the bradykinin B2 receptor and the nucleotide receptor. ATP and UTP, even when eliciting responses of comparable size in the neuronal cell line, affect the desensitization of the bradykinin receptor differently. This suggests regulatory binding sites for the nucleotides on either the nucleotide receptor or the peptide receptor.
Proteinases are isoform-specific regulators of the binding of transforming growth factor β to α2-macroglobulin
Authors: WEBB Donna J. , WEAVER Alissa M. , ATKINS-BRADY Tara L. , GONIAS Steven L.
Published: August 2015

α2-Macroglobulin (α2M) regulates growth and gene expression in many cell types by binding and neutralizing transforming growth factor β (TGF-β). In this study we characterized the effects of the serine proteinase, plasmin, on the interaction of α2M with TGF-β1 and TGF-β2. Binding of both TGF-β isoforms to purified α2M–plasmin complex was primarily non-covalent and reversible. The binding affinity of α2M for TGF-β1 was increased by plasmin; the Kd values were 320 and 84 nM for native α2M and α2M–plasmin respectively. In contrast the affinity of α2M for TGF-β2 was decreased by plasmin; the Kd values were 14 and 80 nM for native α2M and α2M–plasmin respectively. Thrombin decreased the affinity of α2M for TGF-β2 in a similar manner to plasmin. In assays of DNA synthesis in fetal bovine heart endothelial cells, native α2M neutralized the activity of exogenously added TGF-β2, whereas α2M–plasmin, at equivalent concentrations, had almost no effect. Native α2M and methylamine-modified α2M increased platelet-derived growth factor α-receptor expression in vascular smooth-muscle cells, an activity attributed to the neutralization of autocrine TGF-β activity, whereas α2M–plasmin was less effective at the same concentration. These studies demonstrate that the effects of proteinases on the cytokine-binding and cytokine-neutralizing activities of α2M are cytokine-dependent. By reacting with α2M, proteinases might regulate not only the availability of cytokines in the extracellular spaces but also the composition of the cytokine milieu.
Fatty acids as modulators of cytochrome c oxidase in proteoliposomes
Authors: SHARPE Martyn , PERIN Ivano , WRIGGLESWORTH John , NICHOLLS Peter
Published: August 2015

The control of cytochrome c oxidase turnover in proteoliposomes by membrane potential (ΔΨ) and by pH gradient (ΔpH) is probably kinetic in nature, and inhibition by valinomycin and stimulation by nigericin indicate that ΔpH exerts a greater influence than does an equivalent ΔΨ. Oleic acid at 100 µM removes all ΔΨ and ΔpH control, whereas a similar concentration of palmitic acid increases turnover but does not completely abolish control. Valinomycin acts synergistically with both fatty acids, indicating that the latter can act as H+/K+ exchangers, but neither fatty acid alone markedly affects ΔpH, showing that they cannot fully mimic nigericin. Oleate, but not palmitate, diminishes ΔΨ, and can move electrophoretically as oleate anion. Submicromolar palmitic acid concentrations partly stimulate turnover in ΔΨ- and ΔpH-controlled proteoliposomes, as reported by Labonia, Muller and Azzi [(1988) Biochem. J. 254, 130–145], which might represent a direct effect on cytochrome c oxidase. The ubiquity of fatty acids in biological membranes suggests that these substances might be responsible for limiting respiratory control and enzyme activity in vivo.
Human microglia convert l-tryptophan into the neurotoxin quinolinic acid
Authors: HEYES Melvyn P. , ACHIM Cristian L. , WILEY Clayton A. , MAJOR Eugene O. , SAITO Kuniaki , MARKEY Sanford P.
Published: August 2015

Immune activation leads to accumulations of the neurotoxin and kynurenine pathway metabolite quinolinic acid within the central nervous system of human patients. Whereas macrophages can convert l-tryptophan to quinolinic acid, it is not known whether human brain microglia can synthesize quinolinic acid. Human microglia, peripheral blood macrophages and cultures of human fetal brain cells (astrocytes and neurons) were incubated with [13C6]l-tryptophan in the absence or presence of interferon γ. [13C6]Quinolinic acid was identified and quantified by gas chromatography and electron-capture negative-chemical ionization mass spectrometry. Both l-kynurenine and [13C6]quinolinic acid were produced by unstimulated cultures of microglia and macrophages. Interferon γ, an inducer of indoleamine 2,3-dioxygenase, increased the accumulation of l-kynurenine by all three cell types (to more than 40 µM). Whereas large quantities of [13C6]quinolinic acid were produced by microglia and macrophages (to 438 and 1410 nM respectively), minute quantities of [13C6]quinolinic acid were produced in human fetal brain cultures (not more than 2 nM). Activated microglia and macrophage infiltrates into the brain might be an important source of accelerated conversion of l-tryptophan into quinolinic acid within the central nervous system in inflammatory diseases.