bluuebunny/crossref_metadata_2025 huggingface dataset:
Torbafylline (HWA 448) inhibits enhanced skeletal muscle ubiquitin–proteasome-dependent proteolysis in cancer and septic rats
Authors: COMBARET Lydie , TILIGNAC Thomas , CLAUSTRE Agnès , VOISIN Laure , TAILLANDIER Daniel , OBLED Christiane , TANAKA Keiji , ATTAIX Didier
Published: August 2015

The development of new pharmacological approaches for preventing muscle wasting in cancer is an important goal because cachectic patients display a reduced response to chemotherapy and radiotherapy. Xanthine derivatives such as pentoxifylline inhibit tumour necrosis factor-α (TNF) production, which has been implicated in the signalling of muscle wasting. However, the effect of pentoxifylline has been inconclusive in clinical trials. We report here the first direct evidence that daily injections of torbafylline (also known as HWA 448), another xanthine derivative, had no effect by itself on muscle proteolysis in control healthy rats. In cancer rats, the drug blocked the lipopolysaccharide-induced hyperproduction of TNF and prevented muscle wasting. In these animals HWA 448 suppressed the enhanced proteasome-dependent proteolysis, which is sensitive to the proteasome inhibitor MG132, and the accumulation of high-molecular-mass ubiquitin (Ub) conjugates in the myofibrillar fraction. The drug also normalized the enhanced muscle expression of Ub, which prevails in the atrophying muscles from cancer rats. In contrast, HWA 448 did not reduce the increased expression of either the 14kDa Ub conjugating enzyme E2 or the ATPase and non-ATPase subunits of the 19S regulatory complex of the 26S proteasome, including the non-ATPase subunit S5a, which recognizes polyUb degradation signals. Finally, the drug also prevented muscle wasting in septic rats (which exhibit increased TNF production), and was much more potent than pentoxifylline or other xanthine derivatives. Taken together, the data indicate that HWA 448 is a powerful inhibitor of muscle wasting that blocks enhanced Ub—proteasome-dependent proteolysis in situations where TNF production rises, including cancer and sepsis.
Endothelial nitric oxide synthase activity is linked to its presence at cell–cell contacts
Authors: GOVERS Roland , BEVERS Lonneke , DE BREE Petra , RABELINK Ton J.
Published: August 2015

The enzyme endothelial nitric oxide synthase (eNOS) is essential for vascular integrity. Many studies have demonstrated a link between the localization and activity of eNOS. Here, we studied the influence of cell—cell contact on this link in the microvascular endothelial bEnd.3 cell line. By immunofluorescence microscopy, eNOS localization at the plasma membrane was found to be dependent on cell—cell contact. In particular, eNOS was highly enriched at the intercellular contact sites. Further analysis showed that the pattern of eNOS localization at the plasma membrane resembled that of PECAM-1 (platelet endothelial cell adhesion molecule 1), but not that of the adherens junction proteins VE (vascular endothelial)-cadherin and plakoglobin. eNOS that was localized at the contact sites was, in part, Triton X-100-insoluble, in contrast with eNOS at the Golgi complex, which may indicate an association of eNOS with the actin cytoskeleton. Interestingly, eNOS activity was up-regulated in confluent monolayers compared with subconfluent cells, while there was no difference in eNOS expression. This correlation between cell confluence and eNOS activity was also found when primary bovine aortic endothelial cells were studied. These data imply that cell—cell contact induces the localization of eNOS at intercellular junctions, which is required for agonist-induced eNOS activation.
Role of glutathione in the multidrug resistance protein 4 (MRP4/ABCC4)-mediated efflux of cAMP and resistance to purine analogues
Authors: LAI Liqi , TAN Theresa M. C.
Published: August 2015

Multidrug resistance protein 4 (MRP4/ABCC4) is a member of the MRP subfamily, which in turn is a member of the superfamily of ATP-binding-cassette (ABC) transporters. Within the MRP subfamily, ABCC4, ABCC5 (MRP5), ABCC11 (MRP8) and ABCC12 (MRP9) have similar predicted membrane topologies. All lack the additional transmembrane domain, TMD0, which is present in the other MRPs. Using cells stably overexpressing ABCC4, this study shows that ABCC4 exports GSH. ABCC4 also facilitates the efflux of cAMP. Depletion of intracellular GSH with dl-buthionine-(S,R)-sulphoximine led to decreased export of cAMP and a corresponding increase in intracellular cAMP was observed. ABCC4 also mediates resistance to purine analogues 9-(2-phosphonylmethoxyethyl)-adenine and 6-thioguanine. This resistance can be reversed by the presence of dl-buthionine-(S,R)-sulphoximine. We conclude that as well as nucleotide and nucleoside analogues, ABCC4 can mediate the export of GSH. In addition, GSH plays an important role in the function of ABCC4. Depletion of intracellular GSH adversely affects the export of cAMP by ABCC4. Resistance to nucleoside analogues is also adversely affected by depletion of cellular GSH.
Phosphorylation of a novel zinc-finger-like protein, ZPR9, by murine protein serine/threonine kinase 38 (MPK38)
Authors: SEONG Hyun-A , GIL Minchan , KIM Kyong-Tai , KIM Sung-Jin , HA Hyunjung
Published: August 2015

We have identified previously a new murine protein serine/threonine kinase, MPK38, closely related to the sucrose-non-fermenting protein kinase family [Gil, Yang, Lee, Choi and Ha (1997) Gene 195, 295–301]. Using the C-terminal half of the putative human counterpart of MPK38, HPK38, as a bait in a yeast two-hybrid screen of a human HeLa cDNA library, it was discovered that the zinc-finger-motif-containing protein, termed zinc-finger-like protein 9 (ZPR9), bound both HPK38 and MPK38. In a co-expression assay, ZPR9 associated with MPK38 in vivo, and we showed that the ZPR9 is also phosphorylated by MPK38. In addition, ZPR9 physically interacts with itself in mammalian cells. The ZPR9 cDNA hybridized with a mRNA species of approx. 1.7kb in Northern-blot analysis. The ZPR9 transcript was detected in all tissues examined, including lung, kidney, spleen, liver and brain. Co-expression of ZPR9 with MPK38 caused the accumulation of ZPR9 in the nucleus. These findings suggest a potentially important role for ZPR9 in MPK38-mediated signal transduction, and that ZPR9 is a physiological substrate of MPK38 in vivo.
Overexpression, purification and biochemical characterization of a class A high-molecular-mass penicillin-binding protein (PBP), PBP1∗ and its soluble derivative from Mycobacterium tuberculosis
Authors: BHAKTA Sanjib , BASU Joyoti
Published: August 2015

The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones representing the genome of Mycobacteriumtuberculosis, has been named penicillin-binding protein 1∗ (PBP1∗), by analogy to the previously characterized PBP1∗ of M. leprae. This gene has been overexpressed in Escherichia coli. His6-tagged PBP1∗ localizes to the membranes of induced E. coli cells. Its susceptibility to degradation upon proteinase K digestion of spheroplasts from E. coli expressing the protein supports the view that the majority of the protein translocates to the periplasmic side of the membrane. Recombinant PBP1∗ binds benzylpenicillin and several other β-lactams, notably cefotaxime, with high affinity. Truncation of the N-terminal 64 amino acid residues results in an expressed protein present exclusively in inclusion bodies and unable to associate with the membrane. The C-terminal module encompassing amino acids 272–663 can be extracted from inclusion bodies under denaturing conditions using guanidine/HCl and refolded to give a protein fully competent in penicillin-binding. Deletion of Gly95—Gln143 results in the expression of a protein, which is localized in the cytosol. The soluble derivative of PBP1∗ binds benzylpenicillin with the same efficiency as the full-length protein. This is the first report of a soluble derivative of a class A high-molecular-mass PBP.
Oxidative stress inhibits caveolin-1 palmitoylation and trafficking in endothelial cells
Authors: PARAT Marie-Odile , STACHOWICZ Rafal Z. , FOX Paul L.
Published: August 2015

During normal and pathological conditions, endothelial cells (ECs) are subjected to locally generated reactive oxygen species, produced by themselves or by other vessel wall cells. In excess these molecules cause oxidative injury to the cell but at moderate levels they might modulate intracellular signalling pathways. We have investigated the effect of oxidative stress on the palmitoylation and trafficking of caveolin-1 in bovine aortic ECs. Exogenous H2O2 did not alter the intracellular localization of caveolin-1 in ECs. However, metabolic labelling experiments showed that H2O2 inhibited the trafficking of newly synthesized caveolin-1 to membrane raft domains. Several mechanisms potentially responsible for this inhibition were examined. Impairment of caveolin-1 synthesis by H2O2 was not responsible for diminished trafficking. Similarly, the inhibition was independent of H2O2-induced caveolin-1 phosphorylation as shown by the markedly different concentration dependences. We tested the effect of H2O2 on palmitoylation of caveolin-1 by the incorporation of [3H]palmitic acid. Exposure of ECs to H2O2 markedly inhibited the palmitoylation of caveolin-1. Comparable inhibition was observed after treatment of cells with H2O2 delivered either as a bolus or by continuous delivery with glucose and glucose oxidase. Kinetic studies showed that H2O2 did not alter the rate of caveolin-1 depalmitoylation but instead decreased the ‘on-rate’ of palmitoylation. Together these results show for the first time the modulation of protein palmitoylation by oxidative stress, and suggest a cellular mechanism by which stress might influence caveolin-1-dependent cell activities such as the concentration of signalling proteins and cholesterol trafficking.
Phytanic acid, a novel activator of uncoupling protein-1 gene transcription and brown adipocyte differentiation
Authors: SCHLÜTER Agatha , BARBERÁ Maria José , IGLESIAS Roser , GIRALT Marta , VILLARROYA Francesc
Published: August 2015

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a phytol-derived branched-chain fatty acid present in dietary products. Phytanic acid increased uncoupling protein-1 (UCP1) mRNA expression in brown adipocytes differentiated in culture. Phytanic acid induced the expression of the UCP1 gene promoter, which was enhanced by co-transfection with a retinoid X receptor (RXR) expression vector but not with other expression vectors driving peroxisome proliferator-activated receptor (PPAR) α, PPARγ or a form of RXR devoid of ligand-dependent sensitivity. The effect of phytanic acid on the UCP1 gene required the 5′ enhancer region of the gene and the effects of phytanic acid were mediated in an additive manner by three binding sites for RXR. Moreover, phytanic acid activates brown adipocyte differentiation: long-term exposure of brown preadipocytes to phytanic acid promoted the acquisition of the brown adipocyte morphology and caused a co-ordinate induction of the mRNAs for gene markers of brown adipocyte differentiation, such as UCP1, adipocyte lipid-binding protein aP2, lipoprotein lipase, the glucose transporter GLUT4 or subunit II of cytochrome c oxidase. In conclusion, phytanic acid is a natural product of phytol metabolism that activates brown adipocyte thermogenic function. It constitutes a potential nutritional signal linking dietary status to adaptive thermogenesis.
Allotopic antagonism of the non-peptide atrial natriuretic peptide (ANP) antagonist HS-142-1 on natriuretic peptide receptor NPR-A
Authors: POIRIER Hugo , LABRECQUE Jean , DESCHÊNES Julie , DeLÉAN André
Published: August 2015

The microbial polysaccharide HS-142-1 has been documented as an antagonist of natriuretic peptides. It inhibits activation and peptide binding to both guanylate receptors natriuretic peptide receptor (NPR)-A and NPR-B, but has no effect on the non-cyclase receptor NPR-C. At first sight the effect of HS-142-1 on peptide binding appears to be surmountable, suggesting that it might be competitive despite its chemically divergent nature. We explored its mode of action on wild-type NPR-A (WT), on a disulphide-bridged constitutively active mutant (C423S) and on truncated mutants lacking either their cytoplasmic domain (ΔKC) or both the cytoplasmic and the transmembrane domains (ECD). On the WT, HS-142-1 inhibited atrial natriuretic peptide (ANP) binding with a pK value of 6.51±0.07 (Kd = 0.31μM). It displayed a similar effect on the C423S mutant (pK = 6.31±0.11), indicating that its action might not be due to interference with receptor dimerization. HS-142-1 also inhibited ANP binding to ΔKC with a pK of 7.05±0.05 (Kd = 0.089μM), but it was inactive on ANP binding to ECD at a concentration of 10−4M, suggesting that the antagonism was not competitive at the peptide-binding site located on the ECD and that the transmembrane domain might be required. HS-142-1 also enhanced dissociation of NPR-A-bound 125I-ANP in the presence of excess unlabelled ANP, implying an allotopic (allosteric) mode of action for the antagonist.
Differentiation regulates interleukin-1β-induced cyclo-oxygenase-2 in human articular chondrocytes: role of p38 mitogen-activated protein kinase
Authors: THOMAS Béatrice , THIRION Sylvie , HUMBERT Lydie , TAN Lujian , GOLDRING Mary B. , BÉRÉZIAT Gilbert , BERENBAUM Francis
Published: August 2015

Chondrocyte dedifferentiation has been noted in osteoarthritic cartilage, but the contribution of this phenomenon is poorly understood. Interleukin (IL)-1β, the major pro-inflammatory cytokine found in osteoarthritic synovial fluid, induces the dedifferentiation of cultured articular chondrocytes, whereas E-series prostaglandins (PGE) are capable of inducing cell differentiation. Since PGE2 synthesis is up-regulated by IL-1β, we addressed the question of whether the state of chondrocyte differentiation may influence the production of IL-1-induced PGE2 by modulating cyclooxygenase (COX)-2 expression. Immortalized human articular chondrocytes, (tsT/AC62) cultured in monolayer after passage through alginate matrix (alg+) produced 5-fold greater amounts of PGE2 than continuous monolayer cultures (alg-) after stimulation with IL-1β. Moreover, IL-1β induced COX-2 expression at 0.01ng/ml in (alg+) cells, whereas a 100-fold higher dose of cytokine was necessary for stimulation in (alg-) cells. SB203580, a selective p38 mitogen-activated protein kinase (MAPK) inhibitor, completely abolished the IL-1β-induced COX-2 mRNA. Overexpression of p38 MAPK induces a COX-2 reporter, whereas overexpression of dominant negative p38 MAPK represses IL-1β-induced promoter expression. Interestingly, IL-1β-induced p38 MAPK activity was greatly enhanced in (alg+) compared with (alg-) cells. Our results suggest that differentiated articular chondrocytes are highly responsive to IL-1β and that p38 MAPK mediates this response by inducing COX-2 gene expression.
Measurement of ferrochelatase activity using a novel assay suggests that plastids are the major site of haem biosynthesis in both photosynthetic and non-photosynthetic cells of pea (Pisum sativum L.)
Authors: CORNAH Johanna E. , ROPER Jennifer M. , SINGH Davinder Pal , SMITH Alison G.
Published: August 2015

Ferrochelatase is the terminal enzyme of haem biosynthesis, catalysing the insertion of ferrous iron into the macrocycle of protoporphyrin IX, the last common intermediate of haem and chlorophyll synthesis. Its activity has been reported in both plastids and mitochondria of higher plants, but the relative amounts of the enzyme in the two organelles are unknown. Ferrochelatase is difficult to assay since ferrous iron requires strict anaerobic conditions to prevent oxidation, and in photosynthetic tissues chlorophyll interferes with the quantification of the product. Accordingly, we developed a sensitive fluorimetric assay for ferrochelatase that employs Co2+ and deuteroporphyrin in place of the natural substrates, and measures the decrease in deuteroporphyrin fluorescence. A hexane-extraction step to remove chlorophyll is included for green tissue. The assay is linear over a range of chloroplast protein concentrations, with an average specific activity of 0.68nmol·min−1·mg of protein−1, the highest yet reported. The corresponding value for mitochondria is 0.19nmol·min−1·mg of protein−1. The enzyme is inhibited by N-methylprotoporphyrin, with an estimated IC50 value of ≈ 1nM. Using this assay we have quantified ferrochelatase activity in plastids and mitochondria from green pea leaves, etiolated pea leaves and pea roots to determine the relative amounts in the two organelles. We found that, in all three tissues, greater than 90% of the activity was associated with plastids, but ferrochelatase was reproducibly detected in mitochondria, at levels greater than the contaminating plastid marker enzyme, and was latent. Our results indicate that plastids are the major site of haem biosynthesis in higher plant cells, but that mitochondria also have the capacity for haem production.
Hyaluronate degradation as an alternative mechanism for proteoglycan release from cartilage during interleukin-1β-stimulated catabolism
Authors: SZTROLOVICS Robert , RECKLIES Anneliese D. , ROUGHLEY Peter J. , MORT John S.
Published: August 2015

Data presented previously suggest that release of components of the cartilage matrix, in response to catabolic agents, cannot be accounted for by proteolytic mechanisms alone. In the present study, the release of glycosaminoglycan-containing components from bovine nasal cartilage cultured in the presence of interleukin-1β, and from bovine nasal, fetal bovine epiphyseal and adult human articular cartilage cultured in the presence of retinoic acid, was accompanied by the loss of link protein and hyaluronate into the culture medium. Chromatographic analysis of the released hyaluronate showed it to be markedly reduced in size relative to that extracted from the corresponding tissue. It is proposed that, under stimulation by catabolic agents, two independent, but concurrent, mechanisms act to promote the release of aggrecan from the cartilage matrix. First, proteolytic cleavage of the aggrecan core protein results in the production of glycosaminoglycan-containing fragments that are free to diffuse from the tissue. Secondly, cleavage of hyaluronate renders portions of the proteoglycan aggregate small enough so that complexes of aggrecan (or fragments containing its G1 domain) and link protein are released from the tissue. It is likely that both mechanisms contribute to cartilage metabolism in normal physiology and pathology.
A region in the first exon/intron of rat carnitine palmitoyltransferase Iβ is involved in enhancement of basal transcription
Authors: WANG Guo-Li , MOORE Meredith L. , McMILLIN Jeanie B.
Published: August 2015

Carnitine palmitoyltransferase-Iβ (CPT-Iβ) catalyses the transfer of long-chain fatty acids to the enzymes of β-oxidation of muscle and heart. Transcriptional control of this regulatory protein is relevant to disorders of fatty acid oxidation and the switch to glucose metabolism that occurs in cardiac pathology. The presence of a transcriptional enhancer sequence in the first untranslated exon and first intron of the CPT-Iβ gene was identified using deletional and mutational analysis, and by ligation of an oleate responsive element (fatty acid response element) to a minimal promoter. The enhancer sequences are contained in the first 40 bases downstream of the transcription start site and increase CPT-Iβ reporter gene expression independent of any 5′ cis-acting elements. Deletion of the first 40 bases of the 3′-untranslated region does not affect the up-regulation of transcription by 10μM phenylephrine. However, mutation and/or deletion of bases between +11 and +30 dramatically decreases reporter gene expression. Electrophoretic mobility-shift assays reveal two DNA (+11 to +36)—protein complexes that appear cardiac specific. The exon/intron element enhances activation of the heterologous thymidine kinase promoter in a position- and orientation-dependent manner. Therefore we have identified a novel region in the first exon/intron of the CPT-Iβ gene that acts as a non-classical transcriptional enhancer downstream of regulatory elements characterized previously in the 5′-flanking region of the minimal promoter.
Comparison between Ca2+-induced scrambling of various fluorescently labelled lipid analogues in red blood cells
Authors: DEKKERS David W. C. , COMFURIUS Paul , BEVERS Edouard M. , ZWAAL Robert F. A.
Published: August 2015

Treatment of red blood cells with calcium and ionomycin causes activation of the lipid scramblase, a putative membrane protein catalysing flip-flop of (phospho)lipids. Various fluorescent 1-oleoyl-2-[6(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl (C6-NBD) analogues were tested for transbilayer movement across the plasma membrane of red blood cells. Among these phospholipid analogues were phosphatidylgalactose, phosphatidylmaltose and phosphatidylmaltotriose, which were obtained from C6-NBD-phosphatidylcholine by phospholipase D-catalysed transphosphatidylation. The inward movement after the onset of scrambling was monitored by extraction of the non-internalized probe with BSA. We demonstrate that both the amino group and the size of the headgroup determine the kinetics of lipid scrambling, and that lipids with a ceramide backbone migrate much more slowly than glycerophospholipids with the same headgroup.
Platelet-derived growth factor-BB-mediated glycosaminoglycan synthesis is transduced through Akt
Authors: CARTEL Nicholas J. , WANG Jinxia , POST Martin
Published: August 2015

Previously we have demonstrated that the phosphoinositide 3-kinase (PI-3K) signal-transduction pathway mediates platelet-derived growth factor (PDGF)-BB-induced glycosaminoglycan (GAG) synthesis in fetal lung fibroblasts. In the present study we further investigated the signal-transduction pathway(s) that results in PDGF-BB-induced GAG synthesis. Over-expression of a soluble PDGF β-receptor as well as a mutated form of the β-receptor, unable to bind PI-3K, diminished GAG synthesis in fetal lung fibroblasts subsequent to PDGF-BB stimulation. The PI-3K inhibitor wortmannin blocked PDGF-BB-induced Akt activity as well as significantly diminishing PDGF-BB-mediated GAG synthesis. Expression of dominant-negative PI-3K also abrogated Akt activity and GAG synthesis. Furthermore, expression of dominant-negative Akt abrogated endogenous Akt activity, Rab3D phosphorylation and GAG synthesis, whereas expression of constitutively activated Akt stimulated Rab3D phosphorylation and GAG synthesis in the absence of PDGF-BB. Over-expression of wild-type PTEN (phosphatase and tensin homologue deleted in chromosome 10) inhibited Akt activity and concomitantly attenuated GAG synthesis in fibroblasts stimulated with PDGF-BB. These data suggest that Akt is an integral protein involved in PDGF-BB-mediated GAG regulation in fetal lung fibroblasts.
Involvement of protein kinase D in Fcγ-receptor activation of the NADPH oxidase in neutrophils
Authors: DAVIDSON-MONCADA Jan K. , LOPEZ-LLUCH Guillermo , SEGAL Anthony W. , DEKKER Lodewijk V.
Published: August 2015

Protein kinases involved in the activation of the NADPH oxidase by Fcγ receptors in neutrophils were studied. Of three different protein kinase C (PKC) inhibitors, Gö 6976 inhibited the NADPH oxidase completely, whereas bisindolylmaleimide I and Ro 31-8220 caused a 70–80% inhibition. Thus a Gö 6976-sensitive, bisindolylmaleimide I/Ro 31-8220-insensitive component contributes to NADPH oxidase activation induced by Fcγ receptors. Down-regulation of PKC isotypes resulted in inhibition of Fcγ-receptor-activated NADPH oxidase, but a down-regulation-insensitive component was still present. This component was sensitive to Gö 6976, but insensitive to Ro 31-8220. It has been shown previously that protein kinase D/PKC-μ (PKD) shows this same pharmacology in vitro. We show that PKD is present in neutrophils and that, in contrast with PKC isotypes, PKD is not down-regulated. Therefore PKD may participate in NADPH oxidase activation. To obtain direct evidence for this we adopted an antisense approach. Antisense PKD inhibited NADPH oxidase induced by Fcγ-receptor stimulation by 50% and the Ro 31-8220-insensitive component in the activation was inhibited by antisense PKD. In vitro kinase assays showed that PKD is activated by presenting IgG-opsonized particles to neutrophils. Furthermore, PKD localizes to the area of particle intake in the cell and phosphorylates two of the three cytosolic components of the NADPH oxidase, p40phox and p47phox. Taken together, these data indicate that Fcγ receptors engage PKD in the regulation of the NADPH oxidase.
Glycosylation and epitope mapping of the 5T4 glycoprotein oncofoetal antigen
Authors: SHAW David M. , WOODS Andrew M. , MYERS Kevin A. , WESTWATER Caroline , RAHI-SAUND Veena , DAVIES Michael J. , RENOUF David V. , HOUNSELL Elizabeth F. , STERN Peter L.
Published: August 2015

The human 5T4 oncofoetal antigen is a focus for development of several antibody-directed therapies on the basis of the murine monoclonal antibody against 5T4 (mAb5T4), which recognizes a conformational epitope. 5T4 molecules are highly N-glycosylated transmembrane glycoproteins whose extracellular domain contains two regions of leucine-rich repeats (LRRs) and associated flanking regions, separated by an intervening hydrophilic sequence. Using a series of deletion and mutated cDNA constructs as well as chimaeras with the murine homologue, we have mapped the mAb5T4 epitope to the more membrane-proximal LRR2 or its flanking region. Analysis of the glycosylation of the seven consensus Asp-Xaa-Ser/Thr sites was consistent with all of the sites being glycosylated. A combination of two high-mannose chains (predominantly octasaccharide) and five mostly sialylated bi-, tri- and tetra-antennary complex chains with minor quantities of core fucose were detected. The two glycosylation sites, which are the most likely to have predominantly high-mannose chains, are in the only two regions that show significant differences between the human and the 81% identical mouse sequence. A site-directed mutation, which abolished glycosylation at one of these sites (position 192), did not alter antigenicity. The other, which is nearest to the N-terminus in the human, has an Asn-Leu-Thr to Asn-Leu-Leu conversion in the mouse, so cannot be glycosylated in the latter species. The large complex glycosylation at the other sites is likely to influence the antigenicity and tertiary structure generating the 5T4 epitope.
Regulation and recruitment of phosphatidylinositol 4-kinase on immature secretory granules is independent of ADP-ribosylation factor 1
Authors: PANARETOU Christina , TOOZE Sharon A.
Published: August 2015

Heterotrimeric G-proteins, as well as small GTPases of the Rho and ADP-ribosylation factor (ARF) family, are implicated in the regulation of lipid kinases, including PtdIns 4-kinases and PtdIns(4)P 5-kinases. Here, we describe a PtdIns 4-kinase activity on immature secretory granules (ISGs), regulated secretory organelles formed from the trans-Golgi network (TGN), and investigate the regulation of PtdIns4P levels on these membranes. Over 50% of the PtdIns 4-kinase activity on ISGs is inhibited by both a low concentration of adenosine and the monoclonal antibody 4C5G, a specific inhibitor of the type II PtdIns 4-kinase. Treatment of ISGs with mastoparan 7 (M7) stimulates the type II PtdIns 4-kinase via pertussis-toxin-sensitive Gi/G0 proteins, which, in contrast with previous results obtained with chromaffin granules [Gasman, Chasserot-Golaz, Hubert, Aunis and Bader (1998) J. Biol. Chem. 273, 16913–16920], does not require Rho A, B or C. M7 treatment also leads to an inhibition in the recruitment of ARF to ISG membranes: this inhibition is not dependent on Gi/G0 activation, and is not linked to the stimulation of PtdIns 4-kinase observed with M7. PtdIns 4-kinase activity on ISGs is not regulated by myristoylated ARF1—GTP, in contrast with results obtained with Golgi membranes [Godi, Pertile, Meyers, Marra, Di Tullio, Iurisci, Luini, Corda and De Matteis (1999) Nat. Cell Biol. 1, 280–287; Jones, Morris, Morgan, Kondo, Irvine and Cockcroft (2000) J. Biol. Chem. 275, 13962–13170], whereas ARF1—GTP does regulate the production of PtdIns(4,5)P2. Our results suggest that the regulation of PtdIns 4-kinase on the ISGs differs in comparison with that on the TGN, and might be related to a specific requirement of ISG maturation.
The reassembling process of the nonameric Mycobacterium tuberculosis small heat-shock protein Hsp16.3 occurs via a stepwise mechanism
Authors: FENG Xiuguang , HUANG Sufang , FU Xinmiao , ABULIMITI Abuduaini , CHANG Zengyi
Published: August 2015

Conditions are reported under which the reassembled intermediates of the heat-shock protein Hsp16.3 after being denatured in 8M urea were detected by mainly using urea-gradient PAGE (with modifications) and urea-denaturing pore-gradient PAGE. Hsp16.3 is the small heat-shock protein from Mycobacterium tuberculosis, which exists as a specific nonamer and was proposed to form a trimer-of-trimers structure. The refolding and reassembling of this protein was achieved rapidly by dilution or dialysis, suggesting an effectively spontaneous recovery of quaternary structure. Data presented in this report demonstrate that the in vitro reassembling process of Hsp16.3 protein occurs through a spontaneous and effective stepwise mechanism. Modified urea-gradient PAGE may provide a general method for studying the reassembling processes of other oligomeric proteins.
Phenylalanine-24 in the N-terminal region of ammodytoxins is important for both enzymic activity and presynaptic toxicity
Authors: PETAN Toni , KRIŽAJ Igor , GUBENŠEK Franc , PUNGERČAR Jože
Published: August 2015

Ammodytoxins (Atxs) are group II phospholipases A2 (PLA2s) with presynaptic toxicity from venom of the snake Vipera ammodytes ammodytes. The molecular basis of their neurotoxicity, and that of similar PLA2 toxins, is still to be explained. To address this problem, a surface-exposed aromatic residue, Phe24, in the N-terminal region of the most potent Atx, AtxA, was replaced by other aromatic (tyrosine, tryptophan), hydrophobic (alanine) and polar uncharged (serine, asparagine) residues. The mutants were produced in the bacterial expression system, refolded in vitro and purified to homogeneity. All but the Trp24 mutant, whose activity was similar to that of the wild type, showed a considerable decrease (40–80%) in enzymic activity on a micellar phosphatidylcholine substrate. This result indicates an important role for the aromatic side chains of phenylalanine or tryptophan, but not tyrosine, in PLA2 activity, very likely at a stage of interfacial adsorption of the enzyme to zwitterionic aggregated substrates. The substitutions of Phe24 also significantly decreased toxicity in mice, with the most prominent decrease, of 130-fold, observed in the case of the Asn24 mutant. The results with the mutants show that there is no correlation between enzymic activity, lethality and binding affinity for three AtxA neuronal receptors (R180, R25 and calmodulin). Our results suggest a critical involvement of Phe24 in the neurotoxicity of AtxA, apparently at a stage which does not involve the interaction with the known Atx-binding neuronal proteins and catalytic activity.
Intracellular trafficking of glycosylphosphatidylinositol (GPI)-anchored proteins and free GPIs in Leishmania mexicana
Authors: RALTON Julie E. , MULLIN Kylie A. , McCONVILLE Malcolm J.
Published: August 2015

Free glycosylphosphatidylinositols (GPIs) are an important class of membrane lipids in many pathogenic protozoa. In this study, we have investigated the subcellular distribution and intracellular trafficking of an abundant class of free GPIs [termed glycosylinositolphospholipids (GIPLs)] in Leishmania mexicana promastigotes. The intracellular transport of the GIPLs and the major GPI-anchored glycoprotein gp63 was measured by following the incorporation of these molecules into sphingolipid-rich, detergent-resistant membranes (DRMs) in the plasma membrane. In metabolic-labelling experiments, mature GIPLs and gp63 were transported to DRMs in the plasma membrane with a t1/2 of 70 and 40min, respectively. Probably, GIPL transport to the DRMs involves a vesicular mechanism, as transport of both the GIPLs and gp63 was inhibited similarly at 10°C. All GIPL intermediates were quantitatively recovered in Triton X-100-soluble membranes and were largely orientated on the cytoplasmic face of the endoplasmic reticulum, as shown by their sensitivity to exogenous phosphatidylinositol-specific phospho-lipase C. On the contrary, a significant proportion of the mature GIPLs (≈50% of iM4) were accessible to membrane-impermeable probes on the surface of live promastigotes. These results suggest that the GIPLs are flipped across intracellular or plasma membranes during surface transport and that a significant fraction may populate the cytoplasmic leaflet of the plasma membrane. Finally, treatment of L. mexicana promastigotes with myriocin, an inhibitor of sphingolipid biosynthesis, demonstrated that ongoing sphingolipid biosynthesis is not required for the plasma-membrane transport of either gp63 or the GIPLs and that DRMs persist even when cellular levels of the major sphingolipid are depleted by 70%.
The Aspergillus niger faeB gene encodes a second feruloyl esterase involved in pectin and xylan degradation and is specifically induced in the presence of aromatic compounds
Authors: de VRIES Ronald P. , vanKUYK Patricia A. , KESTER Harry C.M. , VISSER Jaap
Published: August 2015

The faeB gene encoding a second feruloyl esterase from Aspergillus niger has been cloned and characterized. It consists of an open reading frame of 1644bp containing one intron. The gene encodes a protein of 521 amino acids that has sequence similarity to that of an Aspergillus oryzae tannase. However, the encoded enzyme, feruloyl esterase B (FAEB), does not have tannase activity. Comparison of the physical characteristics and substrate specificity of FAEB with those of a cinnamoyl esterase from A. niger [Kroon, Faulds and Williamson (1996) Biotechnol. Appl. Biochem. 23, 255–262] suggests that they are in fact the same enzyme. The expression of faeB is specifically induced in the presence of certain aromatic compounds, but not in the presence of other constituents present in plant-cell-wall polysaccharides such as arabinoxylan or pectin. The expression profile of faeB in the presence of aromatic compounds was compared with the expression of A. nigerfaeA, encoding feruloyl esterase A (FAEA), and A. nigerbphA, the gene encoding a benzoate-p-hydroxylase. All three genes have different subsets of aromatic compounds that induce their expression, indicating the presence of different transcription activating systems in A. niger that respond to aromatic compounds. Comparison of the activity of FAEA and FAEB on sugar-beet pectin and wheat arabinoxylan demonstrated that they are both involved in the degradation of both polysaccharides, but have opposite preferences for these substrates. FAEA is more active than FAEB towards wheat arabinoxylan, whereas FAEB is more active than FAEA towards sugar-beet pectin.
Fusion of two distinct peptide exosite inhibitors of Factor VIIa
Authors: ROBERGE Martin , PEEK Mark , KIRCHHOFER Daniel , DENNIS Mark S. , LAZARUS Robert A.
Published: August 2015

Highly potent bifunctional inhibitors of Factor VIIa (FVIIa) were generated by linking two distinct peptides, recently shown to bind to two discrete exosites on the FVIIa protease domain [Dennis, Eigenbrot, Skelton, Ultsch, Santell, Dwyer, O'Connell and Lazarus (2000) Nature (London) 404, 465–470; Dennis, Roberge, Quan and Lazarus (2001) Biochemistry 40, 9513–9521; Roberge, Santell, Dennis, Eigenbrot, Dwyer and Lazarus (2001) Biochemistry 40, 9522–9531]. Fusion peptides consisting of an N-terminal A-series peptide followed by flexible linkers, an E-series peptide, and the Z-domain of protein A were expressed in Escherichia coli and purified using IgG—Sepharose affinity chromatography. The fusion peptides were potent anticoagulants and had steep concentration dependence curves in tissue factor-dependent prothrombin time (PT) assays in comparison to the individual peptides or their noncovalent combination. This phenomenon was dependent on the length of the linker joining the A- and E-peptides. The fusion of the peptides increased the extent of inhibition of Factor X (FX) activation to 100% at saturating peptide concentrations, but did not improve the binding affinity for Factor VIIa (FVIIa) at the A- and E- binding sites or the IC50 for the inhibition of FX activation. Differences between the peptides in the PT fold prolongation in normal and FVII-deficient plasma, in conjunction with the inhibition of 125I-FVII activation, suggest that the enhanced effects of the fusion peptides involve the inhibition of FVII autoactivation.
A frequent human coagulation Factor VII mutation (A294V, c152) in loop 140s affects the interaction with activators, tissue factor and substrates
Authors: TOSO Raffaella , PINOTTI Mirko , HIGH Katherine A. , POLLAK Eleanor S. , BERNARDI Francesco
Published: August 2015

Activated Factor VII (FVIIa) is a vitamin-K-dependent serine protease that initiates blood clotting after interacting with its cofactor tissue factor (TF). The complex FVIIa—TF is responsible for the activation of Factor IX (FIX) and Factor X (FX), leading ultimately to the formation of a stable fibrin clot. Activated FX (FXa), a product of FVIIa enzymic activity, is also the most efficient activator of zymogen FVII. Interactions of FVII/FVIIa with its activators, cofactor and substrates have been investigated extensively to define contact regions and residues involved in the formation of the complexes. Site-directed mutagenesis and inhibition assays led to the identification of sites removed from the FVIIa active site that influence binding specificity and affinity of the enzyme. In this study we report the characterization of a frequent naturally occurring human FVII mutant, A294V (residue 152 in the chymotrypsin numbering system), located in loop 140s. This region undergoes major rearrangements after FVII activation and is relevant to the development of substrate specificity. FVII A294V shows delayed activation by FXa as well as reduced activity towards peptidyl and macromolecular substrates without impairing the catalytic efficiency of the triad. Also, the interaction of this FVII variant with TF was altered, suggesting that this residue, and more likely loop 140s, plays a pivotal role not only in the recognition of FX by the FVIIa—TF complex, but also in the interaction of FVII with both its activators and cofactor TF.
Constitutive expression of hZnT4 zinc transporter in human breast epithelial cells
Authors: MICHALCZYK Agnes A. , ALLEN Justin , BLOMELEY Rachael C. , ACKLAND M. Leigh
Published: August 2015

Zinc is an essential trace element required by all living organisms. An adequate supply of zinc is particularly important in the neonatal period. Zinc is a significant component of breast milk, which is transported across the maternal epithelia during lactation. The mechanisms by which zinc becomes a constituent of breast milk have not been elucidated. The function of the zinc transporter ZnT4 in the transport of zinc into milk during lactation was previously demonstrated by studies of a mouse mutant, the ‘lethal milk’ mouse, where a mutation in the ZnT4 gene decreased the transport of zinc into milk. In the present study, we have investigated the expression of the human orthologue of ZnT4 (hZnT4) in the human breast. We detected hZnT4 mRNA expression in the tissue from the resting and lactating human breast, using reverse-transcriptase PCR. Western-blot analysis using antibodies to peptide sequences of hZnT4 detected a major band of the predicted size of 47kDa and a minor band of 77kDa, in extracts from the resting and lactating breast tissues. There was no difference in the hZnT4 expression levels between lactating and resting breasts. The hZnT4 protein was present in the luminal cells of the ducts and alveoli where it had a granular distribution. A cultured human breast epithelial cell line PMC42 was used to investigate the subcellular distribution of hZnT4 and this showed a granular label throughout the cytoplasm, consistent with a vesicular localization. The presence of zinc-containing intracellular vesicles was demonstrated by using the zinc-specific fluorphore Zinquin (ethyl-[2-methyl-8-p-toluenesulphonamido-6-quinolyloxy]acetate). Double labelling indicated that there was no obvious overlap between Zinquin and the hZnT4 protein, suggesting that hZnT4 was not directly involved in the transport of zinc into vesicles. We detected expression of two other members of the hZnT family, hZnT1 and hZnT3, in human breast epithelial cells. We conclude that hZnT4 is constitutively expressed in the human breast and may be one of the several members of the ZnT family involved in the transport of zinc into milk.
Ca2+ uptake by the endoplasmic reticulum Ca2+-ATPase in rat microvascular endothelial cells
Authors: MOCCIA Francesco , BERRA-ROMANI Roberto , BARUFFI Silvana , SPAGGIARI Santina , SIGNORELLI Silvia , CASTELLI Loretta , MAGISTRETTI Jacopo , TAGLIETTI Vanni , TANZI Franco
Published: August 2015

In non-excitable cells, many agonists increase the intracellular Ca2+ concentration ([Ca2+]i) by inducing an inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the intracellular stores. Ca2+ influx from the extracellular medium may then sustain the Ca2+ signal. [Ca2+]i recovers its resting level as a consequence of Ca2+-removing mechanisms, i.e. plasma-membrane Ca2+-ATPase (PMCA) pump, Na+/Ca2+ exchanger (NCX) and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) pump. In a study performed in pancreatic acinar cells, evidence has been provided suggesting that, during the decay phase of the agonist-evoked Ca2+ transients, the Ca2+ concentration within the intracellular stores remains essentially constant [Mogami, Tepikin and Petersen (1998) EMBO J. 17, 435–442]. It was therefore hypothesized that, in such a situation, intracellular Ca2+ is not only picked up by the SERCA pump, but is also newly released through IP3-sensitive Ca2+ channels, with the balance between these two processes being approximately null. The main aim of the present work was to test this hypothesis by a different experimental approach. Using cardiac microvascular endothelial cells, we found that inhibition of the SERCA pump has no effect on the time course of agonist-evoked Ca2+ transients. This result was not due to a low capacity of the SERCA pump since, after agonist removal, this pump proved to be very powerful in clearing the excess of intracellular Ca2+. We showed further that: (i) in order to avoid a rapid removal of Ca2+ by the SERCA pump, continuous IP3 production appears to be required throughout all of the decay phase of the Ca2+ transient; and (ii) Ca2+ picked up by the SERCA pump can be fully and immediately released by agonist application. All these results support the model of Mogami, Tepikin and Petersen [(1998) EMBO J. 17, 435–442]. Since the SERCA pump did not appear to be involved in shaping the decay phase of the agonist-evoked Ca2+ transient, we inhibited the PMCA pump with carboxyeosin, and NCX with benzamil and by removing extracellular Na+. The results indicate that, during the decay phase of the agonist-evoked Ca2+ transient, the intracellular Ca2+ is removed by both the PMCA pump and NCX. Finally, we provide evidence indicating that mitochondria have no role in clearing intracellular Ca2+ during agonist-evoked Ca2+ transients.
Cardiolipin prevents rate-dependent uncoupling and provides osmotic stability in yeast mitochondria
Authors: KOSHKIN Vasilij , GREENBERG Miriam L.
Published: August 2015

The role of cardiolipin in mitochondrial function was studied by comparing the energy-transforming and osmotic properties of mitochondria isolated from the Saccharomyces cerevisiae cardiolipin synthase-null mutant crd1Δ, which has no cardiolipin, and the isogenic wild type. The results indicated that the importance of cardiolipin for energetic coupling strongly depends on the rate of oxidative phosphorylation, which was set by using NADH (maximal rate limited by coupling mechanism) or ethanol (moderate rate limited by electron supply) as a respiratory substrate, or by modulating the steady-state rate of NADH supply. The absence of cardiolipin resulted in only a small effect on oxidative phosphorylation proceeding at a moderate rate, but led to significant uncoupling (decreased ADP/O and increased state 4 respiration) at the maximal rate of respiration. This indicates that cardiolipin prevents rate-dependent uncoupling in the energy-transforming apparatus. This role of cardiolipin may derive from its strong interaction with, and modulation of the function of, respiratory complexes, and from its effects on the physical properties of the membrane. The importance of cardiolipin for mitochondrial osmotic properties was determined by comparing oxidative phosphorylation, release of matrix enzyme, shrinking ability and volume dynamics upon hypotonically induced swelling in crd1Δ and wild-type mitochondria. Opening of the yeast mitochondrial unspecific channel (YMUC) in the wild-type and mutant mitochondria was also tested. It was found that the lack of cardiolipin strongly undermines the osmotic stability of the mitochondrial membrane.
Methionine aminopeptidases and angiogenesis
Authors: Bradshaw Ralph A , Yi Elizabeth
Published: August 2015

The initiator methionine residue of proteins is removed during synthesis by a specific and ubiquitous enzyme, methionine aminopeptidase (MetAP). Prokaryotes have a single gene, while eukaryotes have two isoforms. This family of metalloenzymes generally cleaves substrates in which the penultimate residue is one of the seven smaller amino acids (glycine, alanine, serine, threonine, proline, cysteine and valine). One of the eukaryotic isoforms (MetAP2) has an additional non-proteolytic function and is the principle target of a family of anti-angiogenic drugs that are related to fumagillin. The resulting covalent modification inhibits the protease activity of MetAP2 and blocks cell-cycle function in endothelial and some cancer cells. The role of MetAP2 in the mitogenic activity of these cells is unknown.
Proteases in blood clotting
Authors: Walsh Peter N , Ahmad Syed S
Published: August 2015

The serine proteases, cofactors and cell-receptor molecules that comprise the haemostatic mechanism are highly conserved modular proteins that have evolved to participate in biochemical reactions in blood coagulation, anticoagulation and fibrinolysis. Blood coagulation is initiated by exposure of tissue factor, which forms a complex with factor VIIa and factor X, which results in the generation of small quantities of thrombin and is rapidly shutdown by the tissue factor pathway inhibitor. The generation of these small quantities of thrombin then activates factor XI, resulting in a sequence of events that lead to the activation of factor IX, factor X and prothrombin. Sufficient thrombin is generated to effect normal haemostasis by converting fibrinogen into fibrin. The anticoagulant pathways that regulate blood coagulation include the protein C anticoagulant mechanism, the serine protease inhibitors in plasma, and the Kunitz-like inhibitors, tissue factor pathway inhibitor and protease nexin 2. Finally, the fibrinolytic mechanism that comprises the activation of plasminogen into plasmin prevents excessive fibrin accumulation by promoting local dissolution of thrombi and promoting wound healing by reestablishment of blood flow.