bluuebunny/crossref_metadata_2025 huggingface dataset:
Channelling of intermediates in the biosynthesis of phosphatidylcholine and phosphatidylethanolamine in mammalian cells
Authors: BLADERGROEN Bellinda A. , GEELEN Math J. H. , REDDY A. Ch. Pulla , DECLERCQ Peter E. , VAN GOLDE Lambert M. G.
Published: August 2015

Previous studies with electropermeabilized cells have suggested the occurrence of metabolic compartmentation and Ca2+-dependent channeling of intermediates of phosphatidylcholine (PC) biosynthesis in C6 rat glioma cells. With a more accessible permeabilization technique, we investigated whether this is a more general phenomenon also occurring in other cell types and whether channeling is involved in phosphatidylethanolamine (PE) synthesis as well. C6 rat glioma cells, C3H10T½ fibroblasts and rat hepatocytes were permeabilized with Staphylococcus aureus α-toxin, and the incorporation of the radiolabelled precursors choline, phosphocholine (P-choline), ethanolamine and phosphoethanolamine (P-EA) into PC and PE were measured both at high and low Ca2+ concentrations. In glioma cells, permeabilization at high Ca2+ concentration did not affect [14C]choline or [14C]P-choline incorporation into PC. However, reduction of free Ca2+ in the medium from 1.8 mM to < 1 nM resulted in a dramatic increase in [14C]P-choline incorporation into permeabilized cells, whereas [14C]choline incorporation remained unaffected. Also, in fibroblasts, reduction of extracellular Ca2+ increased [14C]P-choline and [14C]P-EA incorporation into PC and PE respectively. In hepatocytes, a combination of α-toxin and low Ca2+ concentration severely impaired [14C]choline incorporation into PC. Therefore, α-toxin-permeabilized hepatocytes are not a good model in which to study channeling of intermediates in PC biosynthesis. In conclusion, our results indicate that channeling is involved in PC synthesis in glioma cells and fibroblasts. PE synthesis in fibroblasts is also at least partly dependent on channeling.
Specific alterations in levels of mannose 6-phosphorylated glycoproteins in different neuronal ceroid lipofuscinoses
Authors: SLEAT David E. , SOHAR Istvan , PULLARKAT Premila S. , LOBEL Peter , PULLARKAT Raju K.
Published: August 2015

Mannose 6-phosphate (Man-6-P) is a carbohydrate modification that is generated on newly synthesized lysosomal proteins. This modification is specifically recognized by two Man-6-P receptors that direct the vesicular transport of the lysosomal enzymes from the Golgi to a prelysosomal compartment. The Man-6-P is rapidly removed in the lysosome of most cell types; however, in neurons the Man-6-P modification persists. In this study we have examined the spectrum of Man-6-P-containing glycoproteins in brain specimens from patients with different neuronal ceroid lipofuscinoses (NCLs), which are progressive neurodegenerative disorders with established links to defects in lysosomal catabolism. We find characteristic alterations in the Man-6-P glycoproteins in specimens from late-infantile (LINCL), juvenile (JNCL) and adult (ANCL) patients. Man-6-P glycoproteins in LINCL patients were similar to controls, with the exception that the band corresponding to CLN2, a recently identified lysosomal enzyme whose deficiency results in this disease, was absent. In an ANCL patient, two Man-6-P glycoproteins were elevated in comparison with normal controls, suggesting that this disease also results from a perturbation in lysosomal hydrolysis. In JNCL, total levels of Man-6-P glycoproteins were 7-fold those of controls. In general this was reflected by increased lysosomal enzyme activities in JNCL but three Man-6-P glycoproteins were elevated to an even greater degree. These are CLN2 and the unidentified proteins that are also highly elevated in the ANCL.
The ‘aromatic patch’ of three proximal residues in the human acetylcholinesterase active centre allows for versatile interaction modes with inhibitors
Authors: ARIEL Naomi , ORDENTLICH Arie , BARAK Dov , BINO Tamar , VELAN Baruch , SHAFFERMAN Avigdor
Published: August 2015

The role of the functional architecture of the human acetylcholinesterase (HuAChE) active centre in accommodating the non-covalent inhibitors tacrine and huperzine A, or the carbamates pyridostigmine and physostigmine, was analysed using 16 mutants of residues lining the active-centre gorge. Despite the structural diversity of the ligands, certain common properties of the complexes could be observed: (a) replacement of aromatic residues Tyr133, Tyr337 and especially Trp86, resulted in pronounced changes in stability of all the complexes examined; (b) effects due to replacements of the five other aromatic residues along the active-centre gorge, such as the acyl pocket (Phe295, Phe297) or at the peripheral anionic site (Tyr124, Trp286, Tyr341) were relatively small; (c) effects due to substitution of the carboxylic residues in the gorge (Glu202, Glu450) were moderate. These results and molecular modelling indicate that the aromatic side chains of residues Trp86, Tyr133 and Tyr337 form together a continuous ‘aromatic patch ’ lining the wall of the active-centre gorge, allowing for the accommodation of the different ligands via multiple modes of interaction. Studies with HuAChE mutants carrying replacements at positions 86, 133 and 337 indicate that the orientations of huperzine A and tacrine in the HuAChE complexes in solution are significantly different from those observed in X-ray structures of the corresponding complexes with Torpedo californica AChE (TcAChE). These discrepancies may be explained in terms of structural differences between the complexes of HuAChE and TcAChE or, more likely, by the enhanced flexibility of the AChE active-centre gorge in solution as compared with the crystalline state.
Modulation of RGD sequence motifs regulates disintegrin recognition of αIIbβ3 and α5β1 integrin complexes
Authors: RAHMAN Salman , AITKEN Alex , FLYNN Geraldine , FORMSTONE Caroline , SAVIDGE Geoffrey F.
Published: August 2015

Several recent studies have demonstrated that the amino acid residues flanking the RGD sequence of high-affinity ligands modulate their specificity of interaction with integrin complexes. The present study has addressed the role of the residues flanking the RGD sequence in regulating the recognition by disintegrin of the αIIbβ3 and α5β1 complexes by construction of a panel of recombinant molecules of Elegantin (the platelet aggregation inhibitor from the venom of Trimerasurus elegans) expressing specific RGD sequence motifs. Wild-type Elegantin (ARGDNP) and several variants including Eleg. AM (ARGDMP), Eleg. PM (PRGDMP) and Eleg. PN (PRGDNP) were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The inhibitory efficacies of the panel of Elegantin variants were analysed in platelet adhesion assays with substrates immobilized with fibrinogen and fibronectin. Elegantin molecules containing an Ala residue N-terminal to the RGD sequence (wild-type Elegantin and Eleg. AM) showed strong inhibitory activity towards αIIbβ3-dependent platelet adhesion on fibronectin, whereas a Pro residue in this position (Eleg. PM and Kistrin, the inhibitor from the venom of Calloselasma rhodostoma) engendered lower activity. The decreased activity could not be attributed to a decrease in the affinity of the disintegrin for the αIIbβ3 complex because both Eleg. AM and Eleg. PM had similar Kd (app) values. In contrast, Elegantin molecules into which a Met residue was introduced in place of the Asn residue C-terminal to the RGD sequence showed 10–13-fold elevated inhibitory activity towards platelet adhesion on fibrinogen and this was maintained with either a Pro or Ala residue N-terminal to the RGD sequence. In experiments with the α5β1 complex on K562 cells, the inhibitory efficacies of the panel of Elegantin molecules were analysed under two different cation conditions. First, in the presence of Ca2+/Mg2+, K562 cell adhesion on fibronectin was inhibited equally well by Elegantin and Eleg. AM but inhibited poorly by Eleg. PM and Kistrin. In contrast with platelets, the decreased inhibitory efficacy of the PRGDMP disintegrins was due to poor recognition of the α5β1 complex. In the presence of Mn2+ cation, K562 cell adhesion on fibrinogen was observed in an α5β1-dependent manner. Under these conditions both PRGD and ARGD containing disintegrins were strong inhibitors of K562 cell adhesion on fibrinogen and this was due to a markedly improved recognition of the α5β1 complex by the PRGD molecules. These observations demonstrate the pivotal role of the amino acids flanking the RGD sequence for disintegrin recognition of integrin complexes and highlight the subtle nature by which integrin-ligand binding specificity can be modulated by both cation and adhesive motif.
Evidence for the presence of multiple forms of Sph kinase in human platelets
Authors: BANNO Yoshiko , KATO Makiko , HARA Akira , NOZAWA Yoshinori
Published: August 2015

The intracellular distribution of sphingosine (Sph) kinase activity was examined in human platelets. A large proportion (72%) of the total activity was found to be associated with the membrane fraction, and the membrane-associated fraction had higher specific activity compared with the cytosolic enzyme. Most of the membrane-associated activity could be extracted with 1 M NaCl. The cytosolic activity was unstable upon heat treatment, with 80% of the activity being lost during incubation at 45 °C for 1 h, whereas the NaCl-extractable fraction was stable under the same conditions. When subjected to Mono Q column chromatography, the cytosolic fraction produced two activity peaks and the NaCl-extractable fraction gave a single peak. These three Sph kinase activities showed different responses to stimulation by β-octylglucoside and inhibition by N,N-dimethylsphingosine and l-threo-dihydrosphingosine, suggesting the presence of multiple enzyme forms in human platelets.
Energy requirements for two aspects of phospholipid metabolism in mammalian brain
Authors: PURDON A. David , RAPOPORT Stanley I.
Published: August 2015

Previous estimates have placed the energy requirements of total phospholipid metabolism in mammalian brain at 2% or less of total ATP consumption. This low estimate was consistent with the very long half-lives (up to days) reported for fatty acids esterified within phospholipids. However, using an approach featuring analysis of brain acyl-CoA, which takes into account dilution of the precursor acyl-CoA pool by recycling of fatty acids, we reported that half-lives of fatty acids in phospholipids are some 100 times shorter (min–h) than previously thought. Based on these new estimates of short half-lives, palmitic acid and arachidonic acid were used as prototype fatty acids to calculate energy consumption by fatty acid recycling at the sn-1 and sn-2 positions of brain phospholipids. We calculated that the energy requirements for reacylation of fatty acids into lysophospholipids are 5% of net brain ATP consumption. We also calculated ATP requirements for maintaining asymmetry of the aminophospholipids, phosphatidylserine and phosphatidylethanolamine across brain membrane bilayers. This asymmetry is maintained by a translocase at a stoichiometry of 1 mol of ATP per mol of phospholipid transferred in either direction across the membrane. The energy cost of maintaining membrane bilayer asymmetry of aminophospholipids at steady-state was calculated to be 8% of total ATP consumed. Taken together, deacylation–reacylation and maintenance of membrane asymmetry of phosphatidylserine and phosphatidylethanolamine require about 13% of ATP consumed by brain as a whole. This is a lower limit for energy consumption by processes involving phospholipids, as other processes, including phosphorylation of polyphosphoinositides and de novo phospholipid biosynthesis, were not considered.
Sulphated glycosaminoglycans prevent the neurotoxicity of a human prion protein fragment
Authors: PÉREZ Mar , WANDOSELL Francisco , COLAÇO Camilo , AVILA Jesús
Published: August 2015

Although a number of features distinguish the disease isoform of the prion protein (PrPSc) from its normal cellular counterpart (PrPC) in the transmissible spongiform encephalopathies (TSEs), the neuropathogenesis of these diseases remains an enigma. The amyloid fibrils formed by fragments of human PrP have, however, been shown to be directly neurotoxic in vitro. We show here that sulphated polysaccharides (heparin, keratan and chondroitin) inhibit the neurotoxicity of these amyloid fibrils and this appears to be mediated via inhibition of the polymerization of the PrP peptide into fibrils. This provides a rationale for the therapeutic effects of sulphated polysaccharides and suggests a rapid in vitro functional screen for TSE therapeutics.
Altered glutathione transferase levels in rat skin inflamed due to contact hypersensitivity: induction of the Alpha-class subunit 1
Authors: KIMURA Junya , HAYAKARI Makoto , KUMANO Takayuki , NAKANO Hajime , SATOH Kimihiko , TSUCHIDA Shigeki
Published: August 2015

Since glutathione transferases (GSTs) are suggested to be involved in the prevention of tissue damage by oxidative stress, quantitative and qualitative alterations of GST forms were examined in rat skin after induction of inflammation by 0.6 and 1% 1-chloro-2,4-dinitrobenzene (CDNB) treatment. With 0.6% CDNB, the GST activity in supernatant preparations was 1.8-fold higher than that for control skin, with most GSTs in both cases being bound to S-hexyl-GSH–Sepharose. Major GST subunits of control skin were identified as subunits 7, 4 and 2 by HPLC and chromatofocusing at pH 11–7. These subunits were increased in inflamed skin by 0.6% CDNB and, in addition, the subunit 1 of the Alpha class and subunit 6, both hardly detectable in control skin, were expressed. The specific activity value for GST 7-7 from the inflamed skin by 0.6% CDNB was 2.4-fold lower than that from control skin. However, in the case of inflamed skin after application of 1% CDNB, GST activity was decreased to 69% of the control value and most activity was recovered in fractions binding to a GSH–Sepharose but not a S-hexyl-GSH–Sepharose column. GSTs eluted from the former column demonstrated a restored capacity to bind to the latter, suggesting the GSTs in inflamed skin to be partly inactivated and that they regained activity on exposure to GSH. The Km and Vmax values for GSH of GST 4-4 from inflamed skin after 1% CDNB treatment were 6-fold and 2-fold higher, respectively, than those for the enzyme from control skin, suggesting partial enzyme modification. These results suggest that not only quantitative but also qualitative alterations of GST subunits occur with CDNB-induced inflammation in vivo.
Increased bioactivation of dihaloalkanes in rat liver due to induction of class Theta glutathione S-transferase T1-1
Authors: SHERRATT Philip J. , MANSON Margaret M. , THOMSON Anne M. , HISSINK Erna A. M. , NEAL Gordon E. , BLADEREN Peter J. van , GREEN Trevor , HAYES John D.
Published: August 2015

A characteristic feature of the class Theta glutathione S-transferase (GST) T1-1 is its ability to activate dichloromethane and dibromoethane by catalysing the formation of mutagenic conjugates. The level of the GSTT1 subunit within tissues is an important determinant of susceptibility to the carcinogenic effects of these dihaloalkanes. In the present study it is demonstrated that hepatic GST activity towards these compounds can be elevated significantly in female and male Fischer-344 rats by feeding these animals on diets supplemented with cancer chemopreventive agents. Immunoblotting experiments showed that increased activity towards the dihaloalkanes is associated with elevated levels of the GSTT1 subunit in rat liver. Sex-specific effects were observed in the induction of GSTT1 protein. Amongst the chemopreventive agents tested, indole-3-carbinol proved to be the most potent inducer of hepatic GSTT1 in male rats (6.2-fold), whereas coumarin was the most potent inducer of this subunit in the livers of female rats (3.5-fold). Phenobarbital showed significant induction of GSTT1 only in male rat liver and had little effect in female rat liver. Western blotting showed that class Alpha, Mu and Pi GST subunits are not co-ordinately induced with GSTT1, indicating that the expression of GSTT1 is determined, at least in part, by mechanisms distinct from those that regulate levels of other transferases. The increase in amount of hepatic GSTT1 protein was also reflected by an increase in the steady-state level of mRNA in response to treatment with chemopreventive agents and model inducers. Immunohistochemical detection of GSTT1 in rat liver supported the Western blotting data, but showed, in addition to cytoplasmic staining, significant nuclear localization of the enzyme in hepatocytes from some treated animals, including those fed on an oltipraz-containing diet. Significantly, the hepatic level of cytochrome P-450 2E1, an enzyme which offers a detoxification pathway for dihaloalkanes, was unchanged by the various inducing agents studied. It is concluded that the induction of GSTT1 by dietary components and its localization within cells are important factors that should be considered when assessing the risk dihaloalkanes pose to human health.
Insulin-stimulated kinase from rat fat cells that phosphorylates initiation factor 4E-binding protein 1 on the rapamycin-insensitive site (serine-111)
Authors: HEESOM Kate J. , AVISON Matthew B. , DIGGLE Tricia A. , DENTON Richard M.
Published: August 2015

The effects of insulin and rapamycin on the phosphorylation of the translation regulator, initiation factor 4E-binding protein 1 (4E-BP1) have been studied in rat fat cells by following changes in the incorporation of 32P from [32P]Pi under steady-state conditions. Both unbound 4E-BP1 and 4E-BP1 bound to eukaryotic initiation factor 4E (eIF4E) were isolated from the cells and then digested with trypsin and other proteases; the radiolabelled phosphopeptides were then separated by two-dimensional thin- layer analysis and HPLC. The results provide confirmation of the conclusion of Fadden, Haystead and Lawrence [J. Biol. Chem. (1997) 272, 10240–10247] that insulin increases the phosphorylation of four sites that fit a Ser/Thr-Pro motif (Thr-36, Thr-45, Ser-64 and Thr-69) and that taken together these phosphorylations result in the dissociation of 4E-BP1 from eIF4E. The effects of insulin on the phosphorylation of these sites, and hence dissociation from eIF4E, are blocked by rapamycin. However, the present study also provides evidence that insulin increases the phosphorylation of 4E-BP1 bound to eIF4E on a further site (Ser-111) and that this is by a rapamycin-insensitive mechanism. Extraction of rat epididymal fat cells followed by chromatography on Mono-S and Superose 12 columns resulted in the separation of both an insulin-stimulated eIF4E kinase and an apparently novel kinase that is highly specific for Ser-111 of 4E-BP1. The 4E-BP1 kinase was activated more than 10-fold by incubation of the cells with insulin and was markedly more active towards 4E-BP1 bound to eIF4E than towards unbound 4E-BP1. The effects of insulin were blocked by wortmannin, but not by rapamycin. A 14-mer peptide based on the sequence surrounding Ser-111 of 4E-BP1 was also a substrate for the kinase, but peptide substrates for other known protein kinases were not. The kinase is quite distinct from casein kinase 2, which also phosphorylates Ser-111 of 4E-BP1. The possible importance of these kinases in the phosphorylation of 4E-BP1 in fat cells is discussed. It is suggested that the phosphorylation of Ser-111 might be a priming event that facilitates the subsequent phosphorylation of Thr-36, Thr-45, Ser-64 and Thr69 by a rapamycin-sensitive process that initiates the dissociation of 4E-BP1 from eIF4E and hence the formation of the eIF4F complex.
The activation of protein kinase B by H2O2 or heat shock is mediated by phosphoinositide 3-kinase and not by mitogen-activated protein kinase-activated protein kinase-2
Authors: SHAW Morag , COHEN Philip , ALESSI Dario R.
Published: August 2015

Protein kinase B (PKB) isoforms became activated [and glycogen synthase kinase-3 (GSK3) became inhibited] when mouse Swiss 3T3 fibroblasts were exposed to oxidative stress (H2O2) or heat shock, but not when they were exposed to osmotic shock (0.5 M sorbitol or 0.7 M NaCl), chemical stress (sodium arsenite), the protein-synthesis inhibitor anisomycin, or UV radiation. In contrast, all seven stimuli activated mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP-K2). The activation of MAPKAP-K2 was suppressed by the drug SB 203580, but not by inhibitors of phosphoinositide (phosphatidylinositide, PI) 3-kinase. In contrast, the activation of PKB isoforms and the inhibition of GSK3 by oxidative stress or heat shock were prevented by inhibitors of PI 3-kinase, but not by SB 203580. Thus the activation of PKB by oxidative stress or heat shock is mediated by PI 3-kinase and not by MAPKAP-K2. PKBα and PKBγ were also activated by heat shock and oxidative stress in human embryonic kidney 293 cells and PKBγ was activated by heat shock in NIH 3T3 cells; in each case activation was suppressed by inhibitors of PI 3-kinase. The activation of PKB isoforms by H2O2 may underlie some of the insulin-mimetic effects of this compound.
Molecular aspects of the endocytic pathway
Authors: CLAGUE Michael J.
Published: August 2015

Observation of the flow of material along the endocytic pathway has lead to the description of the basic architecture of the pathway and provided insight into the relationship between compartments. Significant advances have been made in the study of endocytic transport steps at the molecular level, of which studies of cargo selection, vesicle budding and membrane fusion events comprise the major part. Progress in this area has been driven by two approaches, yeast genetics and in vitro or cell-free assays, which reconstitute particular transport steps and allow biochemical manipulation. The complex protein machineries that control vesicle budding and fusion are significantly conserved between the secretory and endocytic pathways such that proteins that regulate particular steps are often part of a larger family of proteins which exercise a conserved function at other locations within the cell. Well characterized examples include vesicle coat proteins, rabs (small GTPases) and soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs). Intracompartmental pH, lipid composition and cytoskeletal organization have also been identified as important determinants of the orderly flow of material within the endocytic pathway.
Molecular study of the Na+/Ca2+ exchanger in bovine adrenal chromaffin cells
Authors: PAN Chien-Yuan , CHU Yeh-Shiu , KAO Lung-Sen
Published: August 2015

To identify the Na+/Ca2+ exchanger expressed in bovine chromaffin cells, the ncx gene was cloned from a bovine chromaffin cell cDNA library. Five partial clones were obtained and their nucleotide sequences showed that there were at least three isoforms containing different intracellular loops. The 3´-untranslated region was the same in all the clones. To examine the Na+/Ca2+ exchange activity of the clones, full-length ncx1 genes were constructed by replacing the corresponding region of bovine cardiac ncx1 clone p17 with the different regions from two bovine chromaffin cell clones; these were designated p17c and p17h. p17h, but not p17c, showed Na+/Ca2+ exchange activity when expressed in Chinese hamster ovary cells and Xenopus oocytes. The expressed exchange activity of p17 was inhibited by 8-bromoadenosine 3´:5´-cyclic monophosphate (8-Br-cAMP) but was not affected by PMA. However, the activity of p17h was inhibited by PMA but enhanced by 8-Br-cAMP. The agents that changed the activity of protein kinase C and cAMP-dependent protein kinase modulated the endogenous Na+/Ca2+ exchange current of chromaffin cells in a manner similar to that of p17h. Our results suggest that the p17h clone is the major isoform of the exchanger in chromaffin cells and is similar to the major ncx1 isoform in kidney. The exchange activity could be regulated by phosphorylation, and the variable region in the intracellular loop is important for the different effects of phosphorylation on the different isoforms.
Protein kinase C-dependent activation of KATP channel enhances adenosine-induced cardioprotection
Authors: LIANG Bruce T.
Published: August 2015

Prior activation of protein kinase C (PKC) can precondition the cardiac cell against injury during subsequent ischaemia. By using cultured chick ventricular cell model for simulated ischaemia and preconditioning, the present study investigated the biochemical mechanism underlying the PKC-mediated preconditioning. A 5 min exposure to PMA enhanced the ability of pinacidil to mediate cardioprotection during a subsequent 90 min period of ischaemia, which is consistent with a sustained activation of the KATP channel initiated by PKC. The brief prior exposure to PMA was also associated with an enhanced ability of the adenosine A1 or A3 receptor agonist 2-chloro-N6-cyclopentyladenosine or N6-(3-iodobenzyl)adenosine-5´-N-methyluronamide to elicit a cardioprotective response during the subsequent ischaemia. In myocytes pretreated with PMA, the cardioprotection mediated by receptor agonist was blocked by the concomitant presence of KATP-channel antagonists glibenclamide or 5-hydroxydecanoic acid during the ischaemia. Thus the KATP channel acts downstream of the adenosine A1 and A3 receptors in mediating the protective effect due to prior PMA exposure. KATP channel activation is responsible for the adenosine receptor-mediated effect. PMA treatment had no effect on other A1 or A3 receptor-mediated effects such as the inhibition of adenylate cyclase, ruling out a direct stimulation of the receptor or G-protein by PMA. The present results indicate that prior stimulation of PKC causes a sustained KATP channel activation, which in turn renders the myocyte more responsive to the protective action of adenosine A1 and A3 receptor agonists during the subsequent ischaemia.
Biochemical and functional characterization of the GLUT5 fructose transporter in rat skeletal muscle
Authors: DARAKHSHAN Froogh , HAJDUCH Eric , KRISTIANSEN Søren , RICHTER Erik A. , HUNDAL Harinder S.
Published: August 2015

Previous work has demonstrated that human skeletal muscle and adipose tissue both express the GLUT5 fructose transporter, but to date the issue of whether this protein is also expressed in skeletal muscle and adipose tissue of rodents has remained unresolved. In the present study we have used a combination of biochemical and molecular approaches to ascertain whether rat skeletal muscle expresses GLUT5 protein and, if so, whether it possesses the capacity to transport fructose. An isoform-specific antibody against rat GLUT5 reacted positively with crude membranes prepared from rat skeletal muscle. A single immunoreactive band of approx. 50 kDa was visualized on immunoblots which was lost when using anti-(rat GLUT5) serum that had been pre-adsorbed with the antigenic peptide. Subcellular fractionation of skeletal muscle localized this immunoreactivity to a single membrane fraction that was enriched with sarcolemma. Plasma membranes, but not low-density microsomes, from rat adipose tissue also displayed a single protein band of equivalent molecular mass to that seen in muscle. Reverse transcription–PCR analyses, using rat-specific GLUT5 primers, of muscle and jejunal RNA revealed a single PCR fragment of the expected size in jejunum and in four different skeletal muscle types. Sarcolemmal vesicles from rat muscle displayed fructose and glucose uptake. Vesicular uptake of glucose was inhibited by nearly 90% in the presence of cytochalasin B, whereas that of fructose was unaffected. To determine whether fructose could regulate GLUT5 expression in skeletal muscle, rats were maintained on a fructose-enriched diet for 4 days. This procedure increased jejunal and renal GLUT5 protein expression by approx. 4- and 2-fold respectively, but had no detectable effects on the abundance of GLUT5 protein in skeletal muscle or on fructose uptake in rat adipocytes. The present results show that GLUT5 is expressed in the sarcolemma of rat skeletal muscle and that it is likely to mediate fructose uptake in this tissue. Furthermore, unlike the situation in absorptive and re-absorptive epithelia, GLUT5 expression in insulin-sensitive tissues is not regulated by increased substrate supply.
C-terminal tripeptide Ser-Asn-Leu (SNL) of human D-aspartate oxidase is a functional peroxisome-targeting signal
Authors: AMERY Leen , BREES Chantal , BAES Myriam , SETOYAMA Chiaki , MIURA Retsu , MANNAERTS Guy P. , VELDHOVEN Paul P. Van
Published: August 2015

The functionality of the C-terminus (Ser-Asn-Leu; SNL) of human d-aspartate oxidase, an enzyme proposed to have a role in the inactivation of synaptically released d-aspartate, as a peroxisome-targeting signal (PTS1) was investigated in vivoand in vitro. Bacterially expressed human d-aspartate oxidase was shown to interact with the human PTS1-binding protein, peroxin protein 5 (PEX5p). Binding was gradually abolished by carboxypeptidase treatment of the oxidase and competitively inhibited by a Ser-Lys-Leu (SKL)-containing peptide. After transfection of mouse fibroblasts with a plasmid encoding green fluorescent protein (GFP) extended by PKSNL (the C-terminal pentapeptide of the oxidase), a punctate fluorescent pattern was evident. The modified GFP co-localized with peroxisomal thiolase as shown by indirect immunofluorescence. On transfection in fibroblasts lacking PEX5p receptor, GFP–PKSNL staining was cytosolic. Peroxisomal import of GFP extended by PGSNL (replacement of the positively charged fourth-last amino acid by glycine) seemed to be slower than that of GFP–PKSNL, whereas extension by PKSNG abolished the import of the modified GFP. Taken together, these results indicate that SNL, a tripeptide not fitting the PTS1 consensus currently defined in mammalian systems, acts as a functional PTS1 in mammalian systems, and that the consensus sequence, based on this work and that of other groups, has to be broadened to (S/A/C/K/N)-(K/R/H/Q/N/S)-L.
Cell proliferation, apoptosis and accumulation of lipid droplets in U937-1 cells incubated with eicosapentaenoic acid
Authors: FINSTAD Hanne S. , DREVON Christian A. , KULSETH Mari Ann , SYNSTAD Anne V. , KNUDSEN Eirunn , KOLSET Svein Olav
Published: August 2015

The monocytic cell line U937-1 was cultured in the presence of eicosapentaenoic acid (20:5, n-3) (EPA) or oleic acid (18:1, n-9) (OA). EPA caused a dose-dependent inhibition of cell proliferation, whereas OA had no effect. At the highest EPA concentrations, 120 and 240 µM, inhibition of cell proliferation was accompanied by initiation of apoptosis. A concentration of 60 µM EPA caused a 35% reduction in cell proliferation without inducing apoptosis, and was therefore used for further studies. Addition of antioxidants or inhibitors of eicosanoid synthesis had no influence on the reduced cell proliferation after EPA treatment. The inhibition required continuous presence of EPA in the incubation medium as the cells resumed a normal proliferation rate when they were placed in EPA-free medium. The inhibition of proliferation was not accompanied by differentiation into macrophage-like cells, as expression of serglycin and the ability to perform respiratory burst was unaffected by EPA. Expression of CD23 mRNA increased when the cells were incubated with EPA, but to a smaller extent than after retinoic acid (RA) or PMA treatment. Furthermore, expression of the monocytic differentiation markers CD36 and CD68 was lower in cells treated with EPA or OA when compared with untreated cells. The cell cycle distribution of U937-1 cells was similar in cells incubated with EPA or PMA, whereas RA-treated cells accumulated in the G1 phase. Side scatter increased in cells incubated with EPA and OA, which was ascribed to an accumulation of lipid droplets after examination of the cells by electron microscopy. The number of droplets per cell was higher in cells exposed to EPA than OA. The cellular triacylglycerol (TAG) increased 5.5- and 15.5-fold after incubation with OA and EPA respectively. No difference in the cellular content of cholesterol compared with untreated cells was observed. The TAG fraction in EPA-treated cells contained high amounts of EPA and docosapentaenoic acid and minor amounts of docosahexaenoic acid, whereas OA-treated cells had high levels of OA in the TAG. In cells incubated with a sulphur-substituted EPA, only minor effects on cell proliferation and no accumulation of cellular TAG were observed. These findings may indicate the existence of other mechanisms for regulation of cell behaviour by very-long-chain polyunsaturated n-3 fatty acids than the well established lipid peroxide and eicosanoid pathways.
Purification and characterization of a lysophosphatidic acid-specific phosphatase
Authors: HIROYAMA Masami , TAKENAWA Tadaomi
Published: August 2015

Lysophosphatidic acid (LPA)-specific phosphatase was purified 3300-fold from bovine brain cytosol. The purification was achieved by (NH4)2SO4 fractionation and several chromatography steps, such as Q-Sepharose, DEAE-5PW, Superdex 200 and heparin–Sepharose. The final enzyme preparation showed a single band of molecular mass 44 kDa on SDS/PAGE under reducing conditions. The enzyme activity was completely dependent on the presence of detergents such as Triton X-100, CHAPS, cholate and octyl-β-glucoside. The activity was independent of Mg2+; other cations were inhibitory. The enzyme hydrolysed LPA specifically but not cardiolipin, tetraoleoyl-bisphosphatidic acid, ceramide 1-phosphate or sphingosine 1-phosphate, although phosphatidic acid was hydrolysed slightly. The purified enzyme hydrolysed 1-oleoyl LPA at a rate of 1.1 µmol/min per mg of protein when assayed with LPA as Triton X-100 mixed micelles. The Km value for LPA was 38 µM. NaF and N-ethylmaleimide markedly inhibited the activity, but propranolol had a less potent inhibitory effect. The LPA-specific phosphatase might have an important role in LPA elimination.
P2 receptor subtypes in the cardiovascular system
Authors: KUNAPULI Satya P. , DANIEL James L.
Published: August 2015

Extracellular nucleotides have been implicated in a number of physiological functions. Nucleotides act on cell-surface receptors known as P2 receptors, of which several subtypes have been cloned. Both ATP and ADP are stored in platelets and are released upon platelet activation. Furthermore, nucleotides are also released from damaged or broken cells. Thus during vascular injury nucleotides play an important role in haemostasis through activation of platelets, modulation of vascular tone, recruitment of neutrophils and monocytes to the site of injury, and facilitation of adhesion of leucocytes to the endothelium. Nucleotides also moderate these functions by generating nitric oxide and prostaglandin I2 through activation of endothelial cells, and by activating different receptor subtypes on vascular smooth muscle cells. In the heart, P2 receptors regulate contractility through modulation of L-type Ca2+ channels, although the molecular mechanisms involved are still under investigation. Classical pharmacological studies have identified several P2 receptor subtypes in the cardiovascular system. Molecular pharmacological studies have clarified the nature of some of these receptors, but have complicated the picture with others. In platelets, the classical P2T receptor has now been resolved into three P2 receptor subtypes: the P2Y1, P2X1 and P2TAC receptors (the last of these, which is coupled to the inhibition of adenylate cyclase, is yet to be cloned). In peripheral blood leucocytes, endothelial cells, vascular smooth muscle cells and cardiomyocytes, the effects of classical P2X, P2Y and P2U receptors have been found to be mediated by more than one P2 receptor subtype. However, the exact functions of these multiple receptor subtypes remain to be understood, as P2-receptor-selective agonists and antagonists are still under development.
Anti-cancer-prostaglandin-induced cell-cycle arrest and its modulation by an inhibitor of the ATP-dependent glutathione S-conjugate export pump (GS-X pump)
Authors: ISHIKAWA Toshihisa , AKIMARU Kunihiro , NAKANISHI Makoto , TOMOKIYO Keiichiro , FURUTA Kyoji , SUZUKI Masaaki , NOYORI Ryoji
Published: August 2015

The A and J series of prostaglandins (PGs) accumulate in the nuclei to suppress the proliferation of cancer cells. Here we report that Δ7-PGA1 methyl ester, a synthetic anti-cancer PG, increased the level of mRNA for the cyclin-dependent kinase inhibitor p21 in human leukaemia HL-60 cells. The induction of p21 was associated with the accumulation of hypophosphorylated retinoblastoma protein (pRB) and the suppression of c-myc gene expression. Since the p53 gene is deleted in HL-60 cells, the anti-cancer PG is suggested to inhibit cancer cell growth by inducing p21 via a p53-independent pathway. Unlike HL-60 cells, cisplatin-resistant HL-60/R-CP cells were insensitive to Δ7-PGA1 methyl ester. While c-myc expression was transiently suppressed, neither G1 arrest nor hypophosphorylation of pRB was observed with the anti-cancer PG. Plasma membrane vesicles from HL-60/R-CP cells showed an enhanced level of GS-X pump (ATP-dependent glutathione S-conjugate export pump) activity towards the glutathione S-conjugate of Δ7-PGA1 methyl ester (Km 110 nM). GIF-0019 {N-carbomethoxy-S-[5-(4-benzoylphenyl)pentyl]glutathione dimethyl ester}, a specific inhibitor of the GS-X pump, dose-dependently enhanced the cellular sensitivity of HL-60/R-CP cells to Δ7-PGA1 methyl ester and induced G1 arrest. The GS-X pump is suggested to play a pivotal role in modulating the biological action of the anti-cancer PG.
Activation of phospholipase A2 in human neutrophils by polyunsaturated fatty acids and its role in stimulation of superoxide production
Authors: ROBINSON Brenton S. , HII Charles S. T. , FERRANTE Antonio
Published: August 2015

Although polyunsaturated fatty acids (PUFA) have been shown to stimulate neutrophil responses such as the oxygen-dependent respiratory burst (superoxide production), the mechanisms involved still remain undefined. Here we investigate the effect of PUFA on the phospholipase A2 (PLA2)-signal transduction process in human neutrophils. Exogenous eicosatetraenoic acid [arachidonic acid; C20:4(n-6)] or docosahexaenoic acid [C22:6(n-3)] promoted the release of [3H]C20:4(n-6) from prelabelled neutrophils in a time- and dose-dependent manner, which is indicative of PLA2 activation. The release of [3H]C20:4(n-6) from the cells by C20:4(n-6) and C22:6(n-3) was suppressed by PLA2 inhibitors. Other PUFA {eicosapentaenoic [C20:5(n-3)], octadecatrienoic [γ-linolenic; C18:3(n-6)] and octadecadienoic [linoleic; C18:2(n-6)] acids} also had the ability to release [3H]C20:4(n-6); however, certain C20:4(n-6) derivatives [15-hydroperoxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid and C20:4(n-6) methyl ester] and saturated fatty acids [octadecanoic (stearic; C18:0) and eicosanoic (arachidic; C20:0) acids] had no significant effect. Treatment of the neutrophils with exogenous C22:6(n-3) caused the mass of endogenous unesterified C20:4(n-6) to increase. Incubation of the leucocytes with C20:4(n-6) or C22:6(n-3) evoked activation of the 85 kDa cytosolic PLA2 (cPLA2) and the 14 kDa secretory PLA2 (sPLA2), but not the cytosolic Ca2+-independent PLA2. In contrast, C20:0 did not activate any of the PLA2 isoforms. Activation of cPLA2 by PUFA was found to precede that of sPLA2. C22:6(n-3), C20:4(n-6) and other PUFA induced punctate localization of cPLA2 in the cells, which was not observed with saturated fatty acids. Pretreatment of the leucocytes with PLA2 inhibitors markedly decreased superoxide production induced by C20:4(n-6). These results show that PUFA activate PLA2 in neutrophils, which might have a mandatory role in biological responses.
Structure–function relationships in novel peptide dodecamers with broad-spectrum bactericidal and endotoxin-neutralizing activities
Authors: MAYO Kevin H. , HASEMAN Judith , YOUNG Helen C. , MAYO Josef W.
Published: August 2015

A series of designed peptide 33-mers (βpep peptides) are known to be bactericidal [Mayo, Haseman, Ilyina and Gray (1998) Biochim. Biophys. Acta 1425, 81–92]. Here dodecapeptides (SC-1–SC-8), which ‘walk through’the sequence of βpep-25, were investigated for their ability to kill Gram-negative and -positive bacteria and to neutralize endotoxin. SC-4 (KLFKRHLKWKI I-NH2; the -NH2 at the right of each sequence indicates amidation of the C-terminal carboxylate group) is the most effective, more so than βpep-25, at killing Gram-negative bacteria with nanomolar LD50 values. Against Gram-positive bacteria, SC-4 also shows good activity with submicromolar LD50 values. Leakage studies indicate rapid bacterial membrane permeability, with t½ values of 10–15min. SC-4 in the micromolar range also effectively neutralizes endotoxin and is not haemolytic below 10-4 M. For all SC peptides, CD and NMR data indicate the presence of both 310- and α-helix. For SC-4, nuclear-Overhauser-effect-based computational modelling yields an amphipathic helix with K1, K4, R5, and K8 arrayed on the same face (K is lysine, R is arginine). Activity differences among SC peptides and single-site variants of SC-4 allow some structure–function relationships to be deduced. Relative to other known bactericidal peptides in the linear peptide, helix-forming category, SC-4 is the most potent broad-spectrum antibacterial identified to date. The present study contributes to the development of agents involved in combating the ever-recurring problem of drug-resistant micro-organisms.
Fusion of the tissue factor extracellular domain to a tumour stroma specific single-chain fragment variable antibody results in an antigen-specific coagulation-promoting molecule
Authors: RIPPMANN Jörg F. , PFIZENMAIER Klaus , MATTES Ralf , RETTIG Wolfgang J. , MOOSMAYER Dieter
Published: August 2015

Solid tumours growing beyond a size of 1–2mm in diameter induce supporting connective tissue structures, the tumour stroma, comprising activated fibroblasts and newly formed blood vessels, embedded in an extracellular matrix. The selective destruction of this tissue or the inhibition of its function (e.g. tumour neoangiogenesis) may result in the destruction of tumour nodules, thus providing novel opportunities for tumour therapy. Our approach aims at an antibody-mediated induction of coagulation in tumour nodules to cut off their blood supply. As a target structure the fibroblast activation protein (FAP) is used, which is specifically and abundantly expressed on the activated fibroblasts of the tumour stroma. We constructed a fusion protein comprising a single-chain module of a FAP-specific humanized antibody [single-chain fragment variable (scFv) OS4] and the extracellular domain of human tissue factor. The fusion protein, designated TFOS4, was produced in the Proteus mirabilis protoplast expression system with a yield of 15µg/ml. Biochemical characterization of TFOS4 revealed high-affinity binding to cellular FAP. Further, TFOS4 bound to factor VIIa and also exerted allosteric activation of factor VIIa. A complex of TFOS4 and factor VIIa bound to FAP-expressing cells efficiently generated activated factor X. Finally, cell-bound TFOS4 selectively induced plasma coagulation, implying its activity under physiological conditions, notably with relevant concentrations of coagulation factors and their natural inhibitors. These findings suggest that TFOS4 has the potential to increase the procoagulant state in a cell-type-specific fashion. No systemic coagulation or side effects were observed when TFOS4 was injected intravenously into normal mice, indicating the biosafety and specificity of the recombinant protein.
Role of Arg-410 and Tyr-411 in human serum albumin for ligand binding and esterase-like activity
Authors: WATANABE Hiroshi , TANASE Sumio , NAKAJOU Keisuke , MARUYAMA Toru , KRAGH-HANSEN Ulrich , OTAGIRI Masaki
Published: August 2015

Recombinant wild-type human serum albumin (rHSA), the single-residue mutants R410A, Y411A, Y411S and Y411F and the double mutant R410A/Y411A were produced using a yeast expression system. The recombinant proteins were correctly folded, as they had the same stability towards guanidine hydrochloride and the same CD spectrum as HSA isolated from serum (native HSA). Thus the global structures of the recombinant proteins are probably very similar to that of native HSA. We investigated, by ultrafiltration and CD, the high-affinity binding of two representative site II ligands, namely ketoprofen and diazepam. According to the crystal structure of HSA, the residues Arg-410 and Tyr-411 protrude into the centre of site II (in subdomain 3A), and the binding results showed that the guanidino moiety of Arg-410, the phenolic oxygen and the aromatic ring of Tyr-411 are important for ketoprofen binding. The guanidino moiety probably interacts electrostatically with the carboxy group of ketoprofen, the phenolic oxygen could make a hydrogen-bond with the keto group of the ligand, and the aromatic ring may participate in a specific stacking interaction with one of or both of the aromatic rings of ketoprofen. By contrast, Arg-410 is not important for diazepam binding. The two parts of Tyr-411 interact favourably with diazepam, and probably do so in the same way as with ketoprofen. In addition to its unique ligand binding properties, HSA also possesses an esterase-like activity, and studies with p-nitrophenyl acetate as a substrate showed that, although Arg-410 is important, the enzymic activity of HSA is much more dependent on the presence of Tyr-411. A minor activity could be registered when serine, but not alanine or phenylalanine, was present at position 411.
The membrane insertion of trichosanthin is membrane-surface-pH dependent
Authors: XIA Xiao-feng , SUI Sen-fang
Published: August 2015

Trichosanthin (TCS) is the active component extracted from Tianhuafen, a traditional herbal medicine that has been used for abortion in China for centuries. It belongs to the type-I ribosome-inactivating protein (RIP) family and can inactivate the eukaryotic ribosome through its RNA N-glycosidase activity. Recent studies have shown TCS to be multifunctional, its pharmacological properties including immunomodulatory, anti-tumour and anti-HIV activities. The membrane-insertion property of TCS is thought to be essential for its physiological effect, for it must get across the membrane before it can enter the cytoplasm and exert its RIP function. In this paper, the membrane-insertion mechanism of TCS was studied. The monolayer experiment revealed that TCS's membrane-insertion ability was dependent on low pH. Fluorescence spectroscopy using 1-anilinonaphthalene-8-sulphonic acid as a probe showed that low pH may induce the conformational change of TCS that leads to the hydrophobic-site exposure, and the CD result showed that this conformational change did not alter its secondary structure. Such conformational change leads to an intermediate state, called the ‘molten globular state’by previous investigators. The pH-dependent membrane insertion and conformational change were related by the fact that the optimal membrane-surface pH needed was the same for the two events. From these and other results, a membrane-insertion model was proposed.