bluuebunny/crossref_metadata_2025_split huggingface dataset:
Recombinant human endothelin-converting enzyme ECE-1b is located in an intracellular compartment when expressed in polarized Madin–Darby canine kidney cells
Authors: AZARANI Arezou , BOILEAU Guy , CRINE Philippe 
Published: August 2015

Endothelin-converting enzyme (ECE) is a phosphoramidon-sensitive membrane-bound metalloprotease responsible for the conversion of big-endothelins into endothelins [Yanagisawa, Kurihara, Kimura, Tomobe, Kobayashi, Mitsui, Yazaki, Goto and Masaki (1988) Nature (London) 332, 411–415]. Several distinct isoforms of ECE have been cloned and identified. ECE-1a, b and c have the same ectodomain and differ only by their cytosolic tails [Schweizer, Valdenaire, Nelböck, Deuschle, Edwards, Stumpf and Löffler (1997) Biochem. J. 328, 871–877]. The ectodomain common to ECE-1 a, b and c shares extensive sequence similarities with neprilysin, a major kidney brush border metallopeptidase. To study the sorting of ECE in polarized cells, ECE-1b cDNA was expressed by transfection in polarized Madin–Darby canine kidney (MDCK) cells. Cell-surface biotinylation and immunofluorescence studies showed that ECE-1b is not expressed on the cell-surface but was rather located in intracellular compartments that could also be labelled with anti-Rab-5 and Rab-7 antibodies and was thus tentatively identified as early and late endosomes. Similar results were also obtained when ECE-1b was expressed in non-polarized Chinese hamster ovary cells for comparison purposes. When MDCK or Chinese hamster ovary transfected cells were pre-treated with the ECE inhibitor phosphoramidon, a 3-fold increase in the level of ECE-1b was observed both by Western blotting and by enzymic activity. However, no change in the level of neprilysin or the β-chain of meprin, two apical membrane metallopeptidases, was observed in MDCK cells transfected under similar conditions. Northern blotting showed that the increase in the level of ECE-1b was not owing to changes in the ECE mRNA transcription rate or stability. Rather, pulse-chase experiments followed by immunoprecipitation showed a decrease in the rate of degradation of ECE-1b in phosphoramidon-treated cells. Half-lives were determined to be 2.8 and 7.5 h for non-treated and phosphoramidon-treated cells, respectively. Confocal microscopy showed accumulation of ECE-1b immunoreactive material in the lysosomes of phosphoramidon-treated cells. Taken together, these results suggest that ECE-1b turns over very rapidly between endosomal and lysosomal compartments and that lysosomal degradation of the enzyme is slowed down by phosphoramidon.
Tryprostatin A, a specific and novel inhibitor of microtubule assembly
Authors: USUI Takeo , KONDOH Masuo , CUI Cheng-Bin , MAYUMI Tadanori , OSADA Hiroyuki 
Published: August 2015

We have investigated the cell cycle inhibition mechanism and primary target of tryprostatin A (TPS-A) purified from Aspergillus fumigatus. TPS-A inhibited cell cycle progression of asynchronously cultured 3Y1 cells in the M phase in a dose- and time-dependent manner. In contrast, TPS-B (the demethoxy analogue of TPS-A) showed cell-cycle non-specific inhibition on cell growth even though it inhibited cell growth at lower concentrations than TPS-A. TPS-A treatment induced the reversible disruption of the cytoplasmic microtubules of 3Y1 cells as observed by indirect immunofluorescence microscopy in the range of concentrations that specifically inhibited M-phase progression. TPS-A inhibited the assembly in vitro of microtubules purified from bovine brains (40% inhibition at 250 µM); however, there was little or no effect on the self-assembly of purified tubulin when polymerization was induced by glutamate even at 250 µM TPS-A. TPS-A did not inhibit assembly promoted by taxol or by digestion of the C-terminal domain of tubulin. However, TPS-A blocked the tubulin assembly induced by inducers interacting with the C-terminal domain, microtubule-associated protein 2 (MAP2), tau and poly-(l-lysine). These results indicate that TPS-A is a novel inhibitor of MAP-dependent microtubule assembly and, through the disruption of the microtubule spindle, specifically inhibits cell cycle progression at the M phase.
Mutational analysis of the two ATP-binding sites in ClpB, a heat shock protein with protein-activated ATPase activity in Escherichia coli
Authors: KIM Keun Il , WOO Kee Min , SEONG Ihn Sik , LEE Zee-Won , BAEK Sung Hee , CHUNG Chin Ha 
Published: August 2015

The 93 kDa ClpB (ClpB93) is a heat shock protein and has a protein-activated ATPase activity. To define the role of the two ATP-binding sites in ClpB93, site-directed mutagenesis was performed to replace Lys212 or Lys611 with Thr or Glu. All of the mutant proteins hydrolysed ATP at a higher rate than that seen with ClpB93 at ATP concentrations up to 2 mM. However, ClpB93 carrying mutations in both of the ATP-binding sites could not cleave ATP. Thus any of the two ATP-binding sites seems to be capable of supporting the ATPase activity of ClpB93. The ATPase activities of both ClpB93/K212T and ClpB93/K212E were gradually decreased when ATP concentrations were increased above 2 mM, unlike those of ClpB93, ClpB93/K611T and ClpB93/K611E, which showed a typical saturation curve. Furthermore ADP inhibited ATP hydrolysis by ClpB93/K212T and ClpB93/K212E more effectively than that by the latter proteins, suggesting that the mutations in the first ATP-binding site result in an increase in the affinity of ADP for the second site in ClpB93. In addition, all of the purified ClpB93 and its mutant forms behaved as an oligomer of 400–450 kDa on a Sephacryl S-300 gel-filtration column, whether or not ATP was present. Thus the binding of ATP to either of the two sites seems not to be essential for oligomerization of ClpB93. Although a low-copy plasmid carrying clpB93 could rescue the sensitivity of a clpB-null mutant cell at 52 °C, none of the plasmids carrying the mutations in the ATP-binding sites could. Furthermore, incubation at 52 °C resulted in a gradual loss of the ATPase activity of ClpB93 carrying the mutations in either of the two ATP-binding sites, but not of the parental ClpB93, indicating that the mutant proteins have a greater tendency to denature at this temperature than the parental ClpB93. These results suggest that both of the ATP-binding sites in ClpB have an important role in maintaining the thermotolerance of the protein and hence in the survival of Escherichia coli at high temperatures.
Bacillus thuringiensis Cry1Ac toxin interaction with Manduca sexta aminopeptidase N in a model membrane environment
Authors: COOPER Matthew A. , CARROLL Joe , TRAVIS Emma R. , WILLIAMS Dudley H. , ELLAR David J. 
Published: August 2015

The Bacillus thuringiensis Cry1Ac δ-endotoxin was shown to bind in a biphasic manner to Manduca sexta aminopeptidase N (APN) present in a novel model membrane. Surface plasmon resonance analysis allowed the quantification of toxin binding to M. sexta APN in a supported lipid monolayer. The initial binding was rapid and reversible, with an affinity constant of 110 nM. The second phase was slower and resulted in an overall affinity constant of 3.0 nM. Reagents used to disrupt protein–protein interactions did not dissociate the toxin after high-affinity binding was attained. The initial association between Cry1Ac and APN was inhibited by the sugar GalNAc, but the higher-affinity state was resistant to GalNAc-induced dissociation. The results suggest that after binding to M. sexta APN, the Cry1Ac toxin undergoes a rate-limiting step leading to a high-affinity state. A site-directed Cry1Ac mutant, N135Q, exhibited a similar initial binding affinity for APN but did not show the second slower phase. This inability to form an irreversible association with the APN-lipid monolayer helps explain the lack of toxicity of this protein towards M. sexta larvae and its deficient membrane-permeabilizing activity on M. sexta midgut brush border membrane vesicles.
Sheep mast-cell proteinases-1 and -3: cDNA cloning, primary structure and molecular modelling of the enzymes and further studies on substrate specificity
Authors: McALEESE Sybil M. , PEMBERTON Alan D. , McGRATH Mary E. , HUNTLEY John F. , MILLER Hugh R. P. 
Published: August 2015

Sheep mast-cell proteinase-1 (sMCP-1) is a serine proteinase expressed predominantly by mucosal mast cells, with specificity for cleavage C-terminal to basic and hydrophobic amino acid residues. A cDNA encoding sMCP-1 has been cloned using reverse transcriptase (RT)-PCR. It appears to be translated as a pre-proenzyme with a 17-amino-acid signal peptide, a basic 2-amino-acid propeptide and a 226-amino-acid catalytic domain. A second cDNA, encoding a serine proteinase 90% identical with sMCP-1, was also cloned and named sMCP-3. Molecular models were constructed for both enzymes using coordinates for the refined X-ray structures of human cathepsin G, chymase and rat mast-cell proteinase-2. The model for sMCP-1 suggests that the acidic Asp-226 side chain extends into the substrate-binding pocket, hydrogen-bonding with Ser-190 on the opposite side and bisecting the pocket. The location of an acidic moiety in this position would favour interaction with basic substrate residues and binding of aromatic residues is rationalized by interaction of the positively charged equatorial plane with Asp-226. The balance between chymotryptic and tryptic activities of sMCP-1 was found to be sensitive to salt concentration, with increasing univalent cation concentration favouring chymotryptic activity relative to the tryptic. Using a peptide substrate representing residues 36–59 of the human thrombin receptor, increasing salt concentration favoured cleavage at Phe-43 rather than at Arg-41.
Synthesis of calbindin-D28K during mineralization in human bone marrow stromal cells
Authors: FAUCHEUX Corinne , BAREILLE Reine , AMEDEE Joëlle 
Published: August 2015

1α,25-Dihydroxyvitamin D3 [1,25(OH)2D3] is known to modulate Ca2+ metabolism in several cell types. Vitamin-D-dependent calcium binding proteins such as calbindin-D28K (28 kDa calcium binding proteins) have been shown to be regulated by 1,25(OH)2D3 but the mechanisms controlling calbindin synthesis are still poorly understood in human osteoblast cell culture models. The human bone marrow stromal cells (HBMSC) described in this paper developed a calcified matrix, expressed osteocalcin (OC), osteopontin (OP) and responded to 1,25(OH)2D3. The expression of vitamin D receptor mRNA was demonstrated by reverse transcription-PCR. Calbindin-D28K protein was identified only in cells arising from the sixth subculture, which exhibited a calcified matrix and all of the osteoblastic markers, e.g. OC and OP. It was demonstrated by dot-immunodetection using immunological probes, and by in situ hybridization using labelled cDNA probes. Moreover, vitamin D3 enhanced calbindin-D28K synthesis as well as OC synthesis and alkaline phosphatase activity. Uptake of 45Ca induced into the matrix by 1,25(OH)2D3 supports the hypothesis that the calcium-enriched matrix could trap calbindin-D proteins. In conclusion, the studies in vitro described in the present paper indicate, for the first time, a possible role of calbindin-D28K in mineralized matrix formation in HBMSC.
Molecular characterization of a sarco(endo)plasmic reticulum Ca2+-ATPase gene from Paramecium tetraurelia and localization of its gene product to sub-plasmalemmal calcium stores
Authors: HAUSER Karin , PAVLOVIC Nada , KISSMEHL Roland , PLATTNER Helmut 
Published: August 2015

A cDNA encoding the gene for a sarco(endo)plasmic reticulum-type Ca2+-ATPase (SERCA) was isolated from a cDNA library of Paramecium tetraurelia by using degenerated primers according to conserved domains of SERCA-type ATPases. The identified nucleotide sequence (PtSERCA) is 3114 nucleotides in length with an open reading frame of 1037 amino acids. An intron of only 22 nucleotides occurs. Homology searches for the deduced amino acid sequence revealed 38–49% similarity to SERCA-type ATPases from organisms ranging from protozoans to mammals, with no more similarity to some parasitic protozoa of the same phylum. The calculated molecular mass of the encoded protein is 114.7 kDa. It contains the typical 10 transmembrane domains of SERCA-type ATPases and other conserved domains, such as the phosphorylation site and the ATP binding site. However, there are no binding sites for phospholamban and thapsigargin present in the PtSERCA. Antibodies raised against a cytoplasmic loop peptide between the phosphorylation site and the ATP binding site recognize on Western blots a protein of 106 kDa, exclusively in the fraction of sub-plasmalemmal calcium stores (‘alveolar sacs’). In immunofluorescence studies the antibodies show labelling exclusively in the cell cortex of permeabilized cells in a pattern characteristic of the arrangement of alveolar sacs. When alveolar sacs where tested for phosphoenzyme-intermediate formation a phosphoprotein of the same molecular mass (106 kDa) could be identified.
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number Y17496.
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 &lt; 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.
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.
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.
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.