bluuebunny/crossref_metadata_2025 huggingface dataset:
Focal adhesion kinase (pp125FAK) cleavage and regulation by calpain
Authors: COORAY Prasad , YUAN Yuping , SCHOENWAELDER Simone M , MITCHELL Christina A , SALEM Hatem H , JACKSON Shaun P.
Published: August 2015

Focal adhesion kinase (125 kDa form; pp125FAK) is a widely expressed non-receptor tyrosine kinase that is implicated in integrin-mediated signal transduction. We have identified a novel means of pp125FAK regulation in human platelets, in which this kinase undergoes sequential proteolytic modification from the native 125 kDa form to 90, 45 and 40 kDa fragments in thrombin-, collagen- and ionophore A23187-stimulated platelets. The proteolysis of pp125FAK was prevented by pretreating platelets with the calpain inhibitors calpeptin or calpain inhibitor-1, and was reproduced in vitro by incubating immunoprecipitated pp125FAK with purified calpain. Proteolysis of pp125FAK resulted in a dramatic reduction in its autokinase activity and led to its dissociation from the cytoskeletal fraction of platelets. These studies define a novel signal-terminating role for calpain, wherein proteolytic modification of pp125FAK attenuates its autokinase activity and induces its subcellular relocation within the cell.
Interleukin 2 is a lectin that associates its receptor with the T-cell receptor complex
Authors: ZANETTA Jean-Pierre , ALONSO Catherine , MICHALSKI Jean-Claude
Published: August 2015

To determine the nature of the mechanism by which the binding of interleukin-2 (IL-2) to its receptor (IL-2Rβ) induces IL-2Rβ phosphorylation by the tyrosine kinase p56lck associated with the T-cell receptor (TCR) complex, we investigated the possibility that this mechanism was due to the putative lectin activity of IL-2 ([Sherblom, Sathyamoorthy, Decker and Muchmore (1989) J. Immunol. 143, 939–944]. Here we demonstrate that IL-2 is a calcium-independent lectin specific for oligomannosidic N-glycans with five and six mannose residues. This lectin activity is preserved after binding of IL-2 to IL-2Rβ. IL-2 behaves as a bifunctional molecule that associates IL-2Rβ with specific glycoprotein ligands of the TCR complex including a glycosylated form of CD3.
Compartment-specific regulation of phosphoinositide 3-kinase by platelet-derived growth factor and insulin in 3T3-L1 adipocytes
Authors: NAVÉ Barbara T. , J RichardHAIGH , HAYWARD Amanda C , SIDDLE Kenneth , SHEPHERD Peter R.
Published: August 2015

To understand how the stimulation of phosphoinositide 3-kinase (PI 3-kinase) by different growth factors can activate different subsets of downstream responses, growth-factor regulation of PI 3-kinase activity at different intracellular locations was investigated in 3T3-L1 adipocytes. Insulin caused a large stimulation of glucose transport and stimulated recruitment of transferrin receptors to the plasma membrane (PM) in these cells, whereas platelet-derived growth factor (PDGF)-bb was virtually without effect on these responses. Subcellular fractionation studies after stimulation with PDGF-bb or insulin revealed a differential effect of these growth factors on subcellular localization of PI 3-kinase activity. PDGF was more effective than insulin in stimulating PI 3-kinase activity and recruiting the p85α PI 3-kinase adaptor subunit in the fraction containing the PM. However, in the microsomal fraction insulin significantly increased PI 3-kinase activity and p85α levels, whereas PDGF was almost without effect. In the microsomal membrane fraction the insulin-stimulated recruitment of p85α closely matched the increase PI 3-kinase activity, indicating that insulin stimulation of PI 3-kinase in this fraction is largely due to recruitment of PI 3-kinase enzyme rather than alterations in specific activity. Insulin-stimulated recruitment of p85α to the microsomal membranes was not inhibited by wortmannin, indicating that PI 3-kinase activity was not required for this process. A further level of compartment-specific regulation of PI 3-kinase in response to PDGF was revealed by the finding that tyrosine phosphorylation of the p85α adaptor was restricted to the PM-containing fraction. Insulin had no effect on p85 tyrosine phosphorylation in either fraction. In summary, these results suggest a basis by which insulin and PDGF could both use PI 3-kinase signalling cascades but achieve different signalling outcomes.
The inositol 1,4,5-trisphosphate-gated Ca2+ channel: effect of the protein thiol reagent thimerosal on channel activity
Authors: THROWER Edwin C. , DUCLOHIER Hervé , LEA Edward J. A. , MOLLE Gérard , DAWSON Alan P
Published: August 2015

The solubilized partially purified Ins(1,4,5)P3-sensitive Ca2+ channel from rat cerebellum has been reconstituted into planar lipid bilayer membranes by the ‘tip-dip’ method [Ehrlich (1992) Methods Enzymol. 207, 463–471] allowing low noise current records. Single-channel events have been recorded. In the presence of 10 µM Ins(1,4,5)P3, 50 µM ATP, and 0.2 µM Ca2+ the Ins(1,4,5)P3 receptor channel opens to a conductance level of 53 pS. In the presence of 100 µM thimerosal (TMS), a sulphydryl-oxidizing agent, three subconductance levels (60 pS, 80 pS and 120 pS) were observed. More than one population of mean open times was found, both in the absence and presence of TMS, although TMS affected the length of the open time by decreasing the short opening significantly from 4.05 ms to 2.78 ms and increasing the longer open time from 27.8 ms to 94.8 ms. The results indicate that TMS enhances Ins(1,4,5)P3-induced Ca2+ release by both altering the open times of the channel significantly and causing a shift to higher subconductance levels.
NMR solution conformation of heparin-derived tetrasaccharide
Authors: MIKHAILOV Dmitri , MAYO Kevin H. , VLAHOV Ioncho R. , TOIDA Toshihiko , PERVIN Azra , LINHARDT Robert J.
Published: August 2015

The solution conformation of the homogeneous, heparin-derived tetrasaccharide ΔUA2S(1 → 4)-α-d-GlcNpS6S(1 → 4)-α-l-IdoAp2S(1 → 4)-α-d-GlcNpS6S (residues A, B, C and D respectively, where IdoA is iduronic acid) has been investigated by using 1H- and 13C-NMR. Ring conformations have been defined by J-coupling constants and inter-proton nuclear Overhauser effects (NOEs), and the orientation of one ring with respect to the other has been defined by inter-ring NOEs. NOE-based conformational modelling has been done by using the iterative relaxation matrix approach (IRMA), restrained molecular dynamics simulations and energy minimization to refine structures and to distinguish between minor structural differences and equilibria between various ring forms. Both glucosamine residues B and D are in the 4C1 chair conformation. The 6-O-sulphate group is oriented in the gauche–trans configuration in the D ring, whereas in the B ring the gauche–gauche rotomer predominates. Uronate (A) and iduronate (C) residues are mostly represented by 1H2 and 2S0 twisted boat forms, respectively, with small deviations in expected coupling constants and NOEs suggesting minor contributions from other A and C ring conformations.
Molecular cloning and overexpression of a glutathione transferase gene from Proteus mirabilis
Authors: PERITO Brunella , ALLOCATI Nerino , CASALONE Enrico , MASULLI Michele , DRAGANI Beatrice , POLSINELLI Mario , ACETO Antonio , ILIO Carmine DI
Published: August 2015

The structural gene of the Proteus mirabilis glutathione transferase GSTB1-1 (gstB) has been isolated from genomic DNA. A nucleotide sequence determination of gstB predicted a translational product of 203 amino acid residues, perfectly matching the sequence of the previously purified protein [Mignogna, Allocati, Aceto, Piccolomini, Di Ilio, Barra and Martini (1993) Eur. J. Biochem. 211, 421–425]. The P. mirabilis GST sequence revealed 56% identity with the Escherichia coli GST at DNA level and 54% amino acid identity. Similarity has been revealed also with the translation products of the recently cloned gene bphH from Haemophilus influenzae (28% identity) and ORF3 of Burkholderia cepacia (27% identity). Putative promoter sequences with high similarity to the E. coli σ70 consensus promoter and to promoters of P. mirabiliscat and glnA genes preceded the ATG of the gstB open reading frame (ORF). gstB was brought under control of the tac promoter and overexpressed in E. coli by induction with isopropyl-β-d-thiogalactopyranoside and growth at 37 °C. The physicochemical and catalytic properties of overexpressed protein were indistinguishable from those of the enzyme purified from P. mirabilis extract. Unlike the GST belonging to Mu and Theta classes, GSTB1-1 was unable to metabolize dichloromethane. The study of the interaction of cloned GSTB1-1 with a number of antibiotics indicates that this enzyme actively participates in the binding of tetracyclines and rifamycin.
Chromatin condensation during apoptosis requires ATP
Authors: KASS George E. N. , ERIKSSON John E. , WEIS Marianne , ORRENIUS Sten , CHOW Sek C.
Published: August 2015

The processes leading to morphological changes of the chromatin in cells that undergo apoptosis are presently unclear. We have recently shown that chromatin fragmentation and the nuclear morphological changes typically seen in apoptosis were reproduced in an in vitro system comprised of isolated rat thymocyte nuclei incubated in the presence of a lysate from Fas/APO-1-stimulated JURKAT cells [Chow, Weis, Kass, Holmström, Eriksson and Orrenius (1995) FEBS Lett. 364, 134–138]. Using this in vitro system, we now report that the presence of ATP is necessary for chromatin condensation, its movement to the nuclear periphery and apoptotic body formation. In clear contrast, chromatin cleavage into high-molecular-mass and oligonucleosomal-length DNA fragments induced by lysates derived from Fas/APO-1-activated JURKAT cells did not require the presence of ATP. The induction of these morphological changes by ATP could not be substituted by the analogues, adenosine 5´-[β,γ-methylene]triphosphate and adenosine 5´-[α,β-methylene]-triphosphate, AMP, cAMP and UTP. However, adenosine 5´-[γ-thio]triphosphate, and to a lesser degree GTP and ADP, could partially replace ATP in inducing nuclear apoptotic morphological changes. It is concluded that ATP is essential for the morphological changes occurring in nuclei during apoptosis, but not for DNA fragmentation.
Alternative splicing of the human IgA Fc receptor CD89 in neutrophils and eosinophils
Authors: PLEASS Richard J , ANDREWS Paul D. , KERR Michael A , WOOF Jenny M.
Published: August 2015

Receptors for the Fc portion of IgA (FcαR) trigger important immunological elimination processes against IgA-coated targets. Investigation of human FcαR (CD89) transcripts in neutrophils, eosinophils and a monocyte-like cell line, THP-1, with the use of reverse transcriptase PCR, Northern blotting and RNase protection analysis, has provided evidence in these cell types for at least two distinct transcripts generated by alternative splicing. The cDNAs derived from the two major transcripts of both neutrophils and eosinophils have been cloned and sequenced. For both cell types, the larger clone represents the previously described full-length receptor, whereas the second, shorter, splice variant lacks the entire second, membrane-proximal, Ig-like domain. Stable CHO-K1 transfectants have been obtained for both full-length and truncated variant neutrophil receptors. Whereas the full-length receptor is recognized by a panel of five anti-FcαR monoclonal antibodies (mAbs), the shorter variant is bound weakly by only two of the antibodies, suggesting that the epitopes recognized by the majority of the mAbs lie at least in part in the second Ig-like domain of FcαR. Both full-length and splice variant forms of the receptor bind secretory IgA, but the weak binding to serum IgA seen with the full-length receptor is not evident with the shorter variant. Alternative splicing might therefore serve as a means of diversifying FcαR structure and function.
High rates of extracellular superoxide generation by cultured human fibroblasts: involvement of a lipid-metabolizing enzyme
Authors: O'DONNELL Valerie B. , AZZI Angelo
Published: August 2015

Expression of NADPH oxidase and low superoxide generation (approx. 0.06 nmol/min per 106 cells) by cytokine- or ionophore-stimulated human fibroblasts is known. However, we here show that these cells also contain an ectoplasmic enzyme, distinct from NADPH oxidase, which can generate superoxide (2.19±0.14 nmol/min per 106 cells) at levels similar to phorbol ester-stimulated monocytes on exogenous NADH addition. Superoxide generation was temperature-dependent, insensitive to chelation (desferal), and had a Km(app)(NADH) of 11.5 µM. Inhibitor studies showed that there was no involvement of NADPH oxidase (diphenylene iodonium, diphenyl iodonium), prostaglandin H synthase (indomethacin), xanthine oxidase (allopurinol), cytochrome P-450 (metyrapone) or mitochondrial respiration (rotenone, antimycin A). NAD+ was a competitive inhibitor, whereas NADPH supported 40% of the rate seen with NADH. No luminescence was observed after the addition of lactate, malate, pyruvate, GSH or l-cysteine. NADH-stimulated superoxide generation was enhanced by the addition of (3–30 µM) arachidonic acid, linoleic acid or (5S)-hydroxyeicosatetraenoic acid [(5S)-HETE] but not palmitic acid, (15S)-hydroperoxyeicosatetraenoic acid [(15S)-HPETE], (15S)-HETE or (12S)-HETE. Several features suggest involvement of an enzyme related to 15-lipoxygenase, and, in support of this, we show superoxide generation and NADH oxidation by recombinant rabbit reticulocyte 15-lipoxygenase. The large amounts of superoxide measured suggest that the fibroblast extracellular enzyme could be a major source of reactive oxygen species after tissue damage.
Regulation of NAD+ glycohydrolase activity by NAD+-dependent auto-ADP-ribosylation
Authors: HAN Myung-Kwan , LEE Ji-Young , CHO Yee-Sook , SONG Young M , AN Nyeon-Hyoung , KIM Hyung-Rho , KIM Uh-Hyun
Published: August 2015

NAD+ glycohydrolase (NADase; EC 3.2.2.5) is an enzyme that catalyses hydrolysis of NAD+ to produce ADP-ribose and nicotinamide. Its physiological role and the regulation of its enzymic activity have not been fully elucidated. In the present study, the mechanism of self-inactivation of NADase by its substrate, NAD+, was investigated by using intact rabbit erythrocytes and purified NADase. Our results suggest that inactivation of NADase was due an auto-ADP-ribosylation reaction. ADP-ribosylated NADase of rabbit erythrocytes was deADP-ribosylated when incubated without NAD+, and thus enzyme activity was simultaneously restored. These findings suggest that reversible auto-ADP-ribosylation of NADase might regulate the enzyme's activity in vivo.
An investigation of the mechanism of inhibition of the Ca2+-ATPase by phospholamban
Authors: HUGHES Glen , STARLING Anthony P , SHARMA Ram P , EAST Malcolm , LEE Anthony G. J.
Published: August 2015

The Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum has been reconstituted with peptides corresponding to the hydrophobic domain of phospholamban (PLB) with or without the three Cys residues replaced by Ala, and with PLB with the three Cys residues replaced by Ala [PLBCys-(1–52)]. Reconstitution with the hydrophobic domain of PLB [PLB(25–52)] was found to decrease the apparent affinity of the ATPase for Ca2+ with no effect on the maximal rate of ATP hydrolysis observed at saturating concentrations of Ca2+. Reconstitution with PLBCys-(1–52) decreased both the apparent affinity for Ca2+ and the maximal activity; the effect on maximal activity followed from a decrease in the rate of the Ca2+ transport step (E1PCa2 → E2P) as observed with the hydrophilic domain PLB(1–25). The concentration dependences of the effects of the hydrophobic domain and of the whole PLB molecule were very similar, suggesting that the hydrophilic domain made little contribution to the affinity of the ATPase for PLB. The effect of PLB on the ATPase was dependent on the molar ratio of phospholipid to ATPase, suggesting partition of the PLB between its binding site on the ATPase and the bulk lipid phase in the membrane. Neither PLB nor its hydrophobic domain affected the rates of phosphorylation or dephosphorylation of the ATPase. Despite their effects on the apparent affinity of the ATPase for Ca2+, neither PLB nor its hydrophobic domain had any effect on the true affinity of the ATPase for Ca2+, as measured from changes in the tryptophan fluorescence of the ATPase. The effects of PLB on the activity of the ATPase are the sum of the effects of its hydrophilic and hydrophobic domains.
Expression of recombinant human serum amyloid A in mammalian cells and demonstration of the region necessary for high-density lipoprotein binding and amyloid fibril formation by site-directed mutagenesis
Authors: PATEL Himakshi , BRAMALL Jo , WATERS Helen , BEER Maria C. DE , WOO Patricia
Published: August 2015

Site-directed mutagenesis of the acute-phase human serum amyloid A (SAA1α) protein was used to evaluate the importance of the N-terminal amino acid residues, namely RSFFSFLGEAF. The full-length cDNA clone of SAA1α (pA1.mod.) was used to create two mutations, namely Gly-8 to Asp-8 and an 11 amino acid truncation between Arg-1 and Phe-11 respectively. Wild-type and mutant cDNAs were expressed in Chinese hamster ovary (CHO) cells under the control of the human cytomegalovirus promoter, which resulted in the secretion of the processed proteins into the culture media. Wild-type recombinant human SAA (rSAA) protein was shown to have pI values of 6.0 and 6.4, similar to the human SAA isoform SAA1α and SAA1α desArg found in acute-phase plasma. N-terminal sequencing of 56 residues confirmed its identity with human SAA1α. The total yield of wild-type rSAA measured by ELISA was between 3.5 and 30 mg/l. The two mutations resulted in reduced expression levels of the mutant SAA proteins (3–10 mg/l). Further measurements of rSAA concentration in lipid fractions of culture medium collected at a density of 1.21 g/ml (high-density lipoprotein; HDL) and 1.063–1.18 g/ml (very-low-density lipoprotein/low-density lipoprotein; VLDL/LDL) showed that 76% of the wild-type protein was found in the HDL fraction and the remaining 24% in the infranatant non-lipid fraction. In contrast the relative concentration of mutant rSAA in HDL and infranatant fractions was reversed. This is consistent with the previously proposed involvement of the 11 amino acid peptide in anchoring SAA protein on to HDL3 [Turnell, Sarra, Glover, Baum, Caspi, Baltz and Pepys (1986) Mol. Biol. Med.3, 387–407]. Wild-type rSAA protein was shown to form amyloid fibrils in vitro under acidic conditions as shown by electron microscopy, and stained positive with Congo Red and exhibited apple-green birefringence when viewed under polarized light. Under the same conditions mutSAA(G8D) and mutSAAΔ1–11 did not form amyloid fibrils. In conclusion, replacement of Gly-8 by Asp-8 or deletion of the first 11 amino acid residues at the N-terminus of rSAA diminishes its capacity to bind to HDL and decreases amyloid fibril formation.
Tumour necrosis factor-α-induced phosphorylation and activation of cytosolic phospholipase A2 are abrogated by an inhibitor of the p38 mitogen-activated protein kinase cascade in human neutrophils
Authors: WATERMAN Waltraut H , MOLSKI Thaddeus F. P. , HUANG Chi-Kuang , ADAMS Jerry L. , SHA'AFI Ramadan I.
Published: August 2015

The role of the newly identified p38 mitogen-activated protein kinase (MAP kinase) in terminally differentiated cells, such as human neutrophils, is totally unknown. In order to examine the possible role of this MAP kinase in the phosphorylation and activation of cytoplasmic phospholipase A2 (cPLA2), we tested the effect of the recently synthesized inhibitor of p38 MAP kinase, SB 203580, on the phosphorylation and activation of both p38 MAP kinase and cPLA2. We found that while tumour necrosis factor-α (TNF-α)-stimulated tyrosine phosphorylation of p38 MAP kinase is affected only slightly by SB 203580, its stimulated kinase activity is greatly reduced in human neutrophils in suspension treated with this inhibitor. Furthermore, the TNF-α-stimulated phosphorylation and activation of cPLA2 are completely abolished in cells treated with SB 203580. Based on these data, it is reasonable to conclude that an SB 203580-sensitive kinase, or kinases and/or phosphatases, are involved in the phosphorylation and activation of cPLA2 in intact human neutrophils in suspension stimulated by TNF-α. The possible role of the p38 MAP kinase cascade in the phosphorylation and activation of cPLA2 is discussed.
Theoretical studies on the control of oxidative phosphorylation in muscle mitochondria: application to mitochondrial deficiencies
Authors: KORZENIEWSKI Bernard , MAZAT Jean-Pierre
Published: August 2015

1. The dynamic model of oxidative phosphorylation developed previously for rat liver mitochondria incubated with succinate was adapted for muscle mitochondria respiring on pyruvate. We introduced the following changes considering: (1) a higher external ATP/ADP ratio and an ATP/ADP carrier less displaced from equilibrium; (2) a substrate dehydrogenation more sensitive to the NADH/NAD+ ratio; and (3) the respiratory chain, ATP synthase and phosphate carrier being more displaced from equilibrium. The experimental flux control coefficients already determined in state 3 for respiratory rate and ATP synthesis were used to adjust some parameters. This new oxidative phosphorylation model enabled us to simulate the whole titration curves obtained experimentally in state 3. These curves, which mimic the effect of mitochondrial complex deficiencies on oxidative phosphorylation, show a threshold effect, which is reproduced by the model. 2. The model was also used to simulate other physiological conditions such as: (i) state 3.5, conditions inbetween state 4 and state 3; and (ii) hypoxic conditions. In both cases a profound change in the pattern of the control coefficients was shown. 3. This model was thus found useful in investigating a variety of new conditions, the most interesting of which can then be experimentally studied.
Lipid mediators of insulin resistance: ceramide signalling down-regulates GLUT4 gene transcription in 3T3-L1 adipocytes
Authors: LONG Sheree D , PEKALA Phillip H
Published: August 2015

We have previously demonstrated that chronic exposure of 3T3-L1 adipocytes to tumour necrosis factor-α (TNF) resulted in a marked decrease (∼ 90%) in cellular GLUT4 (insulin-responsive glucose transporter) mRNA content as a result of a decreased transcription rate of the GLUT4 gene (∼ 75%) and a reduced half-life of its mRNA (9 to 4.5 h). Investigation of the signalling mechanism responsible for this regulation demonstrated that in the 3T3-L1 adipocytes, sphingomyelin levels decreased to 50% of control levels within 40 min of exposure to TNF, consistent with activation of a sphingomyelinase. In the same manner as with TNF, treatment of the adipocytes with 1-3 µM C6-ceramide, a membrane-permeable analogue of ceramide, decreased GLUT4 mRNA content by ∼ 60%. Subsequent investigations revealed that transcription of the GLUT4 gene was reduced by ∼ 65% in response to C6-ceramide, demonstrating that the decrease in mRNA content is mediated by a reduction in the transcription of the gene. No effect on GLUT4 mRNA stability was observed after exposure of the adipocytes to C6-ceramide. These observations are interesting in light of our previous data demonstrating that TNF affects both GLUT4 transcription and mRNA stability in the 3T3-L1 adipocytes. In conclusion, the effect of ceramide on GLUT4 gene expression is at the level of transcription, suggesting that another pathway controls mRNA stability. These data establish that ceramide-initiated signal transduction pathways exist within the adipocyte, and provide a potential mechanism for control of GLUT4 gene expression.
Y-box proteins interact with the S1 nuclease-sensitive site in the chicken α 2(I) collagen gene promoter
Authors: BAYARSAIHAN Dashzeveg , ENKHMANDAKH Badam , LUKENS Lewis N
Published: August 2015

The sequence of the chicken α2(I) collagen promoter from -712 to -85, relative to exon 1, has been shown to be important for transcriptional activity. Within this region a pyrimidine/purine asymmetrical element at -200 bp forms an in vitro S1 nuclease-sensitive site. The pyrimidine-rich strand of this element interacts specifically with single-stranded DNA-binding proteins present in fibroblast nuclear extracts [Bayarsaihan and Lukens (1996) Biochem. J. 314, 293–296]. To identify these proteins we performed expression screening of a chick embryo fibroblast cDNA library using a single-stranded polypyrimidine sequence derived from this element. One of the isolated clones was found to encode a member of the cold-shock gene family, either chicken YB-1 or a highly homologous protein. This protein and a known chicken Y-box protein were both found to bind sequence-specifically to the pyrimidine-rich strand of the pyrimidine/purine asymmetrical element in the chicken α2(I) collagen promoter. The binding mechanism of these proteins could be based on the formation of a non-canonical triplex DNA structure (H-DNA). Although members of this widespread and conserved protein family have been reported to modulate the expression of a number of genes, the findings reported here provide the first evidence for a possible role of cold-shock proteins in the regulation of type I collagen genes.
Time-course studies by synchrotron X-ray solution scattering of the structure of human low-density lipoprotein during Cu2+-induced oxidation in relation to changes in lipid composition
Authors: MEYER David F , NEALIS Adam S , MacPHEE Colin H. , GROOT Pieter H. E. , SUCKLING Keith E. , BRUCKDORFER K. Richard , PERKINS Stephen J.
Published: August 2015

Low-density lipoproteins (LDLs) in plasma are constructed from a single molecule of apolipoprotein B-100 (apoB) (Mr 512000) in association with lipid [approximate Mr (2–3)×106]. LDL oxidation is an important process in the development of atherosclerosis, and can be imitated by the addition of Cu2+ ions. Synchrotron X-ray scattering of LDL yields curves without radiation damage effects at concentrations close to physiological. The radius of gyration RG for preparations of LDL from different donors ranged between 12.1 and 16.0 nm, with a mean of 13.9 nm. At 4 °C, the distance distribution curve P(r) indicated a maximum dimension of 25–27 nm for LDL, a peak at 19.5 nm which corresponds to a surface shell of protein and phospholipid head groups in LDL, and submaxima between 1.7 and 13.5 nm, which correspond to an ordered lipid core in LDL. LDL from different donors exhibited distinct P(r) curves. For oxidation studies of LDL by X-rays, data are best obtained at 4 °C at a concentration of ⩾ 2 mg of LDL protein/ml together with controls based on non-oxidized LDL. LDL oxidation (2 mg of apoB/ml) was studied at 37 °C in the presence of 6.4, 25.6 and 51.2 µmol of Cu2+/g of apoB. Large changes in P(r) were reproducibly observed in the inter-particle distance range between 13 and 16 nm shortly after initiation of oxidation. This corresponds to the phospholipid hydrocarbon in LDL, which has either increased in electron density during oxidation or become increasingly disordered. After 25 h, the structural changes subsequently spread to regions of the P(r) curves assigned to surface apoB and the central core of cholesteryl esters and triacylglycerols. Lipid analyses were carried out under the same solution conditions. The α-tocopherol and β-carotene antioxidant contents of LDL were consumed within 1–2 h. Analyses of the formation of thiobarbituric acid-reactive substances and lipid hydroperoxides indicated that arachidonic acid was preferentially oxidized before the maximal formation of lipid hydroperoxides at 8–12 h after initiation of oxidation. High-performance TLC showed that phosphatidylcholine was continuously converted into lysophosphatidylcholine during oxidation, which is consistent with the early changes in the X-ray P(r) curves. The neutral core lipids became modified only after 12–15 h of oxidation. The combination of X-ray scattering structural analyses with biochemical analyses shows that the oxidation of LDL first affects the outer shell of surface phospholipid, then it spreads towards damage of apoB and the internal neutral lipid core of LDL.
Regulation of Na+,K+-ATPase and the Na+/K+/Cl+ co-transporter in the renal epithelial cell line NBL-1 under osmotic stress
Authors: FERRER-MARTINEZ Andreu , Javier CASADO F. , FELIPE Antonio , PASTOR-ANGLADA Marçal
Published: August 2015

The long-term adaptation of the Na+,K+-ATPase to hypertonicity was studied using the bovine renal epithelial cell line NBL-1. Na+,K+-ATPase activity measured in intact cells as the ouabain-sensitive fraction of Rb+ uptake was stimulated (40% above controls) after incubating the cells in hypertonic medium. This stimulation was not correlated with significant changes in the amount of Na+,K+-ATPase α1 subunit protein. Nevertheless, the amount of α1 but not β1 subunit mRNA progressively increased after hypertonic shock (3–4-fold above basal values). These results suggest that the α1 subunit gene is modulated by medium osmolarity, although this does not necessarily involve enhanced translation of the mRNA into active α1 protein. Indeed, the increase in the biological activity of the Na+,K+-ATPase is abolished when the electrochemical Na+ transmembrane gradient is depleted by monensin, which is consistent with a post-translational effect on the activity of the sodium pump. A furosemide-sensitive component of Rb+ uptake, attributable to Na+/K+/Cl- co-transporter activity, was very low when cells were cultured in a regular medium, but was greatly induced after hypertonic shock. This induction could not be blocked by cycloheximide. Colcemide addition slightly reduced the absolute increase in Na+/K+/Cl- co-transporter activity, while cytochalasin B significantly potentiated the effect triggered by hypertonic shock. It is concluded: (i) that in NBL-1 cells the α1 but not the β1 subunit of the Na+,K+-ATPase is encoded by an osmotically sensitive gene, and (ii) that the Na+/K+/Cl- co-transporter, although an osmotically sensitive carrier, is induced by a mechanism that is independent of protein synthesis but may rely, in an undetermined manner, on the structure of the cytoskeletal network.
Electron transfer in zinc-reconstituted nitrite reductase from Pseudomonas aeruginosa
Authors: BELLELLI Andrea , BRZEZINSKI Peter , ARESE Marzia , CUTRUZZOLÀ Francesca , SILVESTRINI Maria Chiara , BRUNORI Maurizio
Published: August 2015

1. The catalytic cycle of the haem-containing nitrite reductase (NIR) from Pseudomonas aeruginosa involves electron transfer between the two prosthetic groups of the enzyme, the c-haem and the d1-haem; this reaction was shown to be slow by stopped-flow analysis. The recombinant enzyme, expressed in Pseudomonas putida, contains the c-haem but no d1-haem; we have reconstituted this protein with Zn-protoporphyrin IX in the place of the d1-haem. 2. Photoexcitation of Zn-NIR is followed by electron transfer from the triplet excited state of the Zn-porphyrin to the oxidized c-haem, with a rate constant of 7×105 s-1; since the intermediate with reduced c-haem is not significantly populated, we conclude that the back reaction is probably as fast. 3. Even taking into account that in the native NIR the driving force is close to zero, the rate constant for the c → d1 electron transfer, estimated from our experiments, is still much higher than that observed by stopped flow (k = 0.3 s-1) using reduced azurin as the electron donor. This finding may be a direct kinetic indication that reduction of the d1-haem is associated with a substantial reorganization of the co-ordination of the metal, as shown by spectroscopy of the oxidized and reduced NIR.
Calmodulin sensitivity of the sarcoplasmic reticulum ryanodine receptor from normal and malignant-hyperthermia-susceptible muscle
Authors: O'DRISCOLL Sean , McCARTHY Tommie V. , EICHINGER Hans M. , ERHARDT Wolf , LEHMANN-HORN Frank , HERRMANN-FRANK Annegret
Published: August 2015

Ca2+ release from sarcoplasmic reticulum (SR) of malignant-hyperthermia-susceptible (MHS) muscle is hypersensitive to Ca2+ and caffeine. To determine if an abnormal calmodulin (CaM) regulation of the SR Ca2+-release-channel-ryanodine-receptor complex (RYR1) contributes to this hypersensitivity, we investigated the effect of CaM on high-affinity [3H]ryanodine binding to isolated SR vesicles from normal and MHS pig skeletal muscle. CaM modulated [3H]ryanodine binding in a Ca2+-dependent manner. In the presence of maximally activating Ca2+ concentrations, CaM inhibited [3H]ryanodine binding with no differences between normal and MHS vesicles. In the absence of Ca2+, however, CaM activated [3H]ryanodine binding with a 2-fold-higher potency in MHS vesicles. Significant differences between normal and MHS tissue were observed for CaM concentrations between 50 nM and 10 µM. A polyclonal antibody raised against the central region of RYR1 specifically inhibited this activating effect of CaM without affecting the inhibition by CaM. This indicates that the central region of RYR1 is a potential binding domain for CaM in the absence of Ca2+. It is suggested that in vivo an enhanced CaM sensitivity of RYR1 might contribute to the abnormal high release of Ca2+ from the SR of MHS muscle.
Structural and functional analysis of a promoter of the human granulin/epithelin gene
Authors: BHANDARI Vijay , DANIEL Rachael , LIM Pheng Siew , BATEMAN Andrew
Published: August 2015

Granulins (grns) or epithelins (epis) are peptides with molecular masses of approx. 6 kDa that modulate the growth of cells. The precursor for the grns/epis, which might itself be biologically active, is a secreted glycoprotein containing multiple repeats of the grn/epi motif. Grn/epi mRNA occurs widely in vivo, particularly in tissues rich in epithelial and haematopoietic cells. To understand better the role of the gene products for grn/epi it is important to determine the patterns of grn/epi gene expression and how this is regulated. To assist in this we have obtained the 5´ sequence of the human grn/epi gene, and using chimaeras of the grn/epi -5´ sequence and the chloramphenicol acetyltransferase gene we have shown a strong promoter activity associated with the 5´ sequence of the human grn/epi gene. We have further delineated regions of the 5´ sequence that confer high-level expression on the chimaeric gene.
Insulin-like growth factor I stimulates degradation of an mRNA transcript encoding the 14 kDa ubiquitin-conjugating enzyme
Authors: WING Simon S , BEDARD Nathalie
Published: August 2015

Upon fasting, the ubiquitin-dependent proteolytic system is activated in skeletal muscle in parallel with the increases in rates of proteolysis. Levels of mRNA encoding the 14 kDa ubiquitin-conjugating enzyme (E214k), which can catalyse the first irreversible reaction in this pathway, rise and fall in parallel with the rates of proteolysis [Wing and Banville (1994) Am. J. Physiol. 267, E39-E48], indicating that the conjugation of ubiquitin to proteins is a regulated step. To characterize the mechanisms of this regulation, we have examined the effects of insulin, insulin-like growth factor I (IGF-I) and des(1–3) insulin-like growth factor I (DES-IGF-I), which does not bind IGF-binding proteins, on E214k mRNA levels in L6 myotubes. Insulin suppressed levels of E214k mRNA with an IC50 of 4×10-9 M, but had no effects on mRNAs encoding polyubiquitin and proteasome subunits C2 and C8, which, like E214k, also increase in skeletal muscle upon fasting. Reduction of E214k mRNA levels was more sensitive to IGF-I with an IC50 of approx. 5×10-10 M. During the incubation of these cells for 12 h there was significant secretion of IGF-I-binding proteins into the medium. DES-IGF-I, which has markedly reduced affinity for these binding proteins, was found to potently reduce E214k mRNA levels with an IC50 of 3×10-11 M. DES-IGF-I did not alter rates of transcription of the E214k gene, but enhanced the rate of degradation of the 1.2 kb mRNA transcript. The half-life of the 1.2 kb transcript was approximately one-third that of the 1.8 kb transcript and can explain the more marked regulation of this transcript observed previously. This indicates that the additional 3´ non-coding sequence in the 1.8 kb transcript confers stability. These observations suggest that IGF-I is an important regulator of E214k expression and demonstrate, for the first time, stimulation of degradation of a specific mRNA transcript by this hormone, while overall RNA accumulates.
Changes induced by fasting and dietetic obesity in thermogenic parameters of rat brown adipose tissue mitochondrial subpopulations
Authors: MATAMALA Juan C , GIANOTTI Magdalena , PERICÁS Jordi , QUEVEDO Santiago , ROCA Pilar , PALOU Andreu , GARCÍA-PALMER Francisco J.
Published: August 2015

The effects of starvation on the thermogenic parameters of three different mitochondrial subpopulations in brown adipose tissue (BAT) of both post-cafeteria obese and lean rats were investigated. Tissue from different BAT depots from fed and 24 h starved rats were collected, pooled and three mitochondrial subpopulations were isolated by differential centrifugation; the M1 fraction (1000 g), the M3 fraction (3000 g) and the M15 fraction (15000 g). Thermogenic parameters were measured in the three mitochondrial subtypes, and uncoupling protein (UCP) mRNA was determined in BAT. The results showed that starvation induced a decrease in mitochondrial turnover in BAT from both lean and obese rats. Moreover, a selective net loss of UCP from the lightest mitochondrial fraction (M15) in lean rats, with a concomitant reduction of UCP mRNA was observed. The reductions did not occur in obese rats and, as a result, a change in UCP distribution between the mitochondrial subpopulations was produced, with an increase in the M1 mitochondrial subtype. The lack of response of UCP to starvation observed in BAT of obese rats compared with the decrease seen in lean animals, is a consequence of a different mitochondrial subpopulation composition and/or a different response of a particular subpopulation to starvation.
Interaction of aspartic acid-104 and proline-287 with the active site of m-calpain
Authors: ARTHUR J. Simon C. , ELCE John S
Published: August 2015

In an ongoing study of the mechanisms of calpain catalysis and Ca2+-induced activation, the effects of Asp-104 → Ser and Pro-287 → Ser large subunit mutations on m-calpain activity, the pH-activity profile, Ca2+-sensitivity, and autolysis were measured. The importance of these positions was suggested by sequence comparisons between the calpain and papain families of cysteine proteinases. Asp-104 is adjacent to the active-site Cys-105, and Pro-287 is adjacent to the active-site Asn-286 and probably to the active-site His-262; both Asp-104 and Pro-287 are absolutely conserved in the known calpains, but are replaced by highly conserved serine residues in the papains. The single mutants had approx. 10–15% of wild-type activity, due mainly to a decrease in kcat, since Km was only slightly increased. The Pro-287 → Ser mutation appeared to cause a local perturbation of the catalytic Cys-105/His-262 catalytic ion pair, reducing its efficiency without major effect on the conformation and stability of the enzyme. The Asp-104 → Ser mutation caused a marked narrowing of the pH-activity curve, a 9-fold increase in Ca2+ requirement, and an acceleration of autolysis, when compared with the wild-type enzyme. The results indicated that Asp-104 alters the nature of its interaction with the catalytic ion pair during Ca2+-induced conformational change in calpain. This interaction may be direct or indirect, but is important in activation of the enzyme.
D-3 phosphoinositide metabolism in cells treated with platelet-derived growth factor
Authors: WHITEFORD Craig C , BEST Christie , KAZLAUSKAS Andrius , ULUG Emin T.
Published: August 2015

Despite extensive analysis of phosphoinositide 3-hydroxykinases (PI 3-kinases) at the molecular level, comparatively little is known about the mechanisms by which products of these enzymes exert their expected second-messenger functions. This study examines the metabolism of D-3 phosphoinositides in mouse Ph-N2 fibroblasts lacking the platelet-derived growth factor (PDGF) α-receptor. Treatment of these cultures with BB PDGF, but not AA PDGF, resulted in transient activation of PI 3-kinase activity measured in vitro. Treatment of myo-[3H]inositol-labelled Ph-N2 cells with BB PDGF resulted in the rapid induction of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 and, to a smaller extent, PtdIns3P. The appearance of PtdIns(3,4,5)P3 preceded that of PtdIns(3,4)P2 and PtdIns3P after the addition of PDGF, suggesting that PtdIns(4,5)P2 is the preferred substrate of the agonist-stimulated PI 3-kinase in intact cells. Treatment of both resting and PDGF-stimulated cells with the fungal metabolite wortmannin resulted in pronounced, selective effects on the levels of all D-3 phosphoinositides. Kinetic studies with this PI 3-kinase inhibitor revealed the presence of at least two independent routes for the biosynthesis of D-3 phosphoinositides in PDGF-treated cells.
Activation of Mac-1 (CD11b/CD18)-bound factor X by released cathepsin G defines an alternative pathway of leucocyte initiation of coagulation
Authors: PLESCIA Janet , ALTIERI Dario C
Published: August 2015

Leucocyte initiation of coagulation preserves the haemostatic balance and may aberrantly contribute to vascular injury. In addition to the extrinsic activation mediated by tissue factor: factor VIIa, monocytes express an alternative procoagulant response after binding of the zymogen factor X to the integrin Mac-1 (CD11b/CD18). Here, factor X-activating activity was found in purified monocyte granules, and coincided with size-chromatographed fractions containing cathepsin G. In contrast, elastase-containing granule fractions did not activate factor X. In the presence of Ca2+ ions, purified cathepsin G, but not elastase, cleaved factor X to a ∼ 54 kDa catalytically active derivative, structurally indistinguishable from the procoagulant product generated on monocytes after binding to Mac-1. Factor X activation by purified cathepsin G involved limited proteolysis of a novel Leu177-Leu178 peptide bond in the zymogen's activation peptide. Cathepsin G activation of factor X was completely inhibited by α1 antitrypsin, α1 antichymotrypsin, or soybean trypsin inhibitor, or by a neutralizing antiserum to cathepsin G, while eglin, or an anti-elastase antibody, were ineffective. Affinity chromatography on active-site-dependent inhibitors Glu-Gly-Arg-chloromethyl ketone or benzamidine completely abolished factor Xa activity generated by cathepsin G. Cathepsin G was not constitutively detected on the monocyte surface by flow cytometry. However, inflammatory stimuli, including formyl peptide or phorbol ester, or Mac-1 engagement with its ligands fibrinogen, factor X or serum-opsonized zymosan, triggered monocyte degranulation and cathepsin G activation of factor X. These findings demonstrate that monocytes can alternatively initiate coagulation in a sequential three-step cascade, including (i) binding of factor X to Mac-1, (ii) discharge of azurophil granules, and (iii) limited proteolytic activation of membrane-bound factor X by cathepsin G. By rapidly forming thrombin and factor Xa in a protected membrane microenvironment, this pathway may contribute a ‘priming’ signal for clotting, anticoagulation and vascular cell signal transduction, in vivo.
Recombinant human phenylalanine hydroxylase is a substrate for the ubiquitin-conjugating enzyme system
Authors: DØSKELAND Anne P. , FLATMARK Torgeir
Published: August 2015

Mammalian phenylalanine hydroxylase (PAH) catalyses the conversion of L-phenylalanine to L-tyrosine in the presence of dioxygen and tetrahydrobiopterin; it is a highly regulated enzyme. Little is known about the rates of synthesis and degradation of PAH in vivo. The enzyme has been reported to have a half-life of approx. 2 days in rat liver and 7–8 h in rat hepatoma cells, but the mechanism of its degradation is not known. In the present study it is shown that the tetrameric form of the recombinant wild-type human enzyme is a substrate for the ubiquitin-conjugating enzyme system in the cytosolic fraction of rat testis. Our findings support the conclusion that multi-/poly-ubiquitination of human PAH plays a key role in the turnover of this cytosolic liver enzyme and provides a mechanism for the increased turnover observed for a number of recombinant mutant forms of the enzyme related to the metabolic disorder phenylketonuria, when expressed in eukaryotic cells.
Primary structures of decapod crustacean metallothioneins with special emphasis on freshwater and semi-terrestrial species
Authors: PEDERSEN Søren N. , PEDERSEN Knud L , HØJRUP Peter , DEPLEDGE Michael H. , KNUDSEN Jens
Published: August 2015

Cadmium injections induced only a single form of metallothionein (MT) in the midgut gland of Potamon potamios, whereas the same treatment induced two isoforms in Astacus astacus. The only difference between the two latter isoforms was that one had an extra N-terminal methionine residue. MT from P. potamios showed structural differences from other decapod crustacean MTs. It contained a Gly-Thr motif at positions 8 and 8a, which had previously been found only in certain vertebrate and molluscan MTs. Furthermore P. potamios MT contained two to three times as many glutamic acid residues as normally found in decapod crustacean MT. The primary structure of MT from the freshwater crayfish A. astacus showed a high degree of sequence identity with MT from other decapod crustaceans, especially the marine astacidean Homarus americanus, although two valine residues were unexpectedly found at positions 8 and 21, where lysine residues are normally found.
Proinsulin processing in the rat insulinoma cell line INS after overexpression of the endoproteases PC2 or PC3 by recombinant adenovirus
Authors: IRMINGER Jean-Claude , MEYER Katharina , HALBAN Philippe
Published: August 2015

Proinsulin is converted to insulin by the two endoproteases PC2 and PC3. For complete conversion to insulin, cleavage must occur at both the B-chain/C-peptide and C-peptide/A-chain junctions of proinsulin. Studies in vitro have shown the specificity of PC3 for the B-chain/C-peptide junction and that of PC2 for the C-peptide/A-chain junction. In contrast, studies in vivo have suggested that the proinsulin cleavage substrate specificity of these two endoproteases might be more complex. We have now used recombinant adenovirus to overexpress PC2 or PC3 in the rat insulinoma cell line INS. These cells convert proinsulin more slowly than primary pancreatic β-cells, possibly reflecting their lower levels of PC3. Infection of INS cells with the corresponding recombinant adenovirus led to 5–10-fold and 20–40-fold increases in PC2 and PC3 expression respectively. Recombinant adenovirus is thus an effective tool for expressing proteins at high levels in slow-growing INS cells. Overexpression of either PC2 or PC3 in INS cells led to a striking acceleration of conversion of proinsulin to insulin and to a decreased accumulation of the conversion intermediate des-64.65-split proinsulin (cleaved only at the A-chain/C-peptide junction). There was no detectable accumulation of des-31.32-split proinsulin (cleaved only at the B-chain/C-peptide junction) after overexpression of either enzyme. Taken together, the data indicate that when expressed at high levels, both PC2 and PC3 seem to be able to cleave proinsulin at both its junctions in vivo.