bluuebunny/crossref_metadata_2025 huggingface dataset:
The intracellular triacylglycerol/fatty acid cycle: a comparison of its activity in hepatocytes which secrete exclusively apolipoprotein (apo) B100 very-low-density lipoprotein (VLDL) and in those which secrete predominantly apoB48 VLDL
Authors: SALTER Andrew M. , WIGGINS David , SESSIONS Victoria A. , GIBBONS Geoffrey F.
Published: August 2015

Hamster hepatocytes, like human hepatocytes, secrete triacylglycerol (TAG) as very-low-density lipoprotein (VLDL) in association with apolipoprotein (apo) B100, whereas in the rat, TAG is secreted predominantly in association with apoB48. Nevertheless, in hepatocytes from both species, a minimum of between 60% and 70% [69.1±1.4% (hamster), 60.6±2.5% (rat)] of the VLDL TAG was secreted following lipolysis and re-esterification of intracellular TAG. The fractional rates of hepatocellular TAG turnover (lipolysis and re-esterification) were similar in both species [1.83±0.28 pools/24 h (hamster), 1.39±0.23 pools/24 h (rat)]. Comparison of the relative changes in the 3H and 14C specific radioactivities of the VLDL and cellular TAG, pre-labelled with [3H]glycerol and [4C]oleate, suggested that fatty acids released by lipolysis either were recruited directly into a VLDL assembly pool or were recycled to the cellular pool following re-esterification. Recycling in the hamster was somewhat greater than in the rat (66.1±5.7% versus 53.7±4.8% of TAG lipolysed respectively). Similarly, a larger proportion of newly synthesized TAG was retained within the cell, rather than secreted as VLDL, in the hamster compared with the rat (37.9±2.8% versus 20±3.8%, P< 0.01). These factors may have contributed to the somewhat lower rate of VLDL TAG secretion in the hamster hepatocytes compared with those from the rat (43.3±4.2 versus 96.4±3.4 µg/24 h per mg of cell protein). Rat hepatocytes were more sensitive to inhibition of VLDL secretion by insulin than were those from hamster. In neither case did insulin affect total or fractional TAG turnover. The results suggest that assembly of both apoB100 VLDL and apoB48 VLDL is associated with efficient intracellular TAG lipolysis.
Functional analysis of the human annexin I and VI gene promoters
Authors: DONNELLY Shaun R. , MOSS Stephen E.
Published: August 2015

To gain insight into the molecular basis of annexin gene expression we have analysed the annexin I and VI gene promoters. A previously described 881 bp sequence immediately upstream of the annexin I transcription start site and a similar size fragment proximal to the annexin VI transcription start site both drove expression of the luciferase reporter gene in fibroblasts and epithelial cells. Neither promoter displayed any sensitivity to dexamethasone, suggesting that the putative glucocorticoid response element in the annexin I promoter is non-functional. Consistent with this, endogenous annexin I gene expression was unaffected by dexamethasone at the mRNA and protein levels in A431 cells. A series of 5´ deletions of the two promoters were examined to define the minimal active sequences. For annexin I this corresponded to a sequence approx. 150 bp upstream of the transcription start site that included CAAT and TATA boxes. Unexpectedly, the annexin VI promoter, which also contains CAAT and TATA boxes, was fully active in the absence of these elements, a 53 bp sequence between these boxes and the transcription start site having maximal activity. Electrophoretic mobility-shift assays with nuclear extracts from A431 and HeLa cells with probes corresponding to this region revealed an SP1-binding site. These results show that the annexin I and VI genes have individual modes of transcriptional regulation and that if either annexin I or annexin VI has an anti-inflammatory role, then this is in the absence of steroid-induced gene expression.
Molecular basis of non-responsiveness to peroxisome proliferators: the guinea-pig PPARα is functional and mediates peroxisome proliferator-induced hypolipidaemia
Authors: BELL Alex R. , SAVORY Richard , HORLEY Neill J. , CHOUDHURY Agharul I. , DICKINS Maurice , GRAY Tim J. B. , SALTER Andrew M. , BELL David R.
Published: August 2015

The guinea pig does not undergo peroxisome proliferation in response to peroxisome proliferators, in contrast with other rodents. To understand the molecular basis of this phenotype, the peroxisome proliferator activated receptor α (PPARα) from guinea-pig liver was cloned; it encodes a protein of 467 amino acid residues that is similar to rodent and human PPARα. The guinea-pig PPARα showed a high substitution rate: maximum likelihood analysis was consistent with rodent monophyly, but could not exclude rodent polyphyly (P≈ 0.06). The guinea-pig PPARα cDNA was expressed in 293 cells and mediated the induction of the luciferase reporter gene by the peroxisome proliferator, Wy-14,643, dependent on the presence of a peroxisome proliferator response element. Moreover the PPARα RNA and protein were expressed in guinea-pig liver, although at lower levels than in a species which is responsive to peroxisome proliferators, the mouse. To determine whether the guinea-pig PPARα mediated any physiological effects, guinea pigs were exposed to two selective PPARα agonists, Wy-14,643 and methylclofenapate; both compounds induced hypolipidaemia. Thus the guinea pig is a useful model for human responses to peroxisome proliferators.
Functional expression of a vacuolar-type H+-ATPase in the plasma membrane and intracellular vacuoles of Trypanosoma cruzi
Authors: BENCHIMOL Marlene , SOUZA Wanderley de , VANDERHEYDEN Nicole , ZHONG Li , LU Hong-Gang , MORENO Silvia N.J. , DOCAMPO Roberto
Published: August 2015

Acid-loaded Trypanosoma cruzi amastigotes and trypomastigotes regained normal cytoplasmic pH (pHi), as measured in cells loaded with 2´,7´-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), by a process that was sensitive to bafilomycin A1 at concentrations comparable to those that inhibited vacuolar (V) H+-ATPases from different sources. Steady-state pHi was also decreased by similar concentrations of bafilomycin A1 in a concentration-dependent manner. The efflux of H+ equivalents from amastigotes and trypomastigotes was measured by following changes in the fluorescence of extracellular BCECF. Basal H+ extrusion in the presence of glucose was 15.4±2.8 (S.D.) nmol of H+/min per 108 amastigotes and 6.37±0.8 nmol of H+/min per 108 trypomastigotes. Bafilomycin A1 treatment significantly decreased the efflux of H+ equivalents by amastigotes (8.9±2.2 nmol of H+/min per 108 cells), but not by trypomastigotes (5.1±1.7 nmol of H+/min per 108 cells). The localization of the V-H+-ATPase of T. cruziwas investigated by immunocytochemistry. Confocal and electron microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the V-H+-ATPase of different stages of T. cruzi is also located in the plasma membrane. However, no labelling was detected in the plasma membrane lining the flagellar pocket of the different developmental stages. Surface localization of the V-H+-ATPase was confirmed by experiments involving the biotinylation of cell surface proteins and immunoprecipitation with antibodies against the V-H+-ATPase. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of amastigotes and with an important role for intracellular acidic compartments in the maintenance of pHi in different stages of T. cruzi.
Adenoviral expression of murine serum amyloid A proteins to study amyloid fibrillogenesis
Authors: KINDY Mark S. , KING Amy R. , YU Jin , GERARDOT Connie , WHITLEY Joel , BEER Frederick C. De
Published: August 2015

Serum amyloid A (SAA) proteins are one of the most inducible acute-phase reactants and are precursors of secondary amyloidosis. In the mouse, SAA1 and SAA2 are induced in approximately equal quantities in response to amyloid induction models. These two isotypes differ in only 9 of 103 amino acid residues; however, only SAA2 is selectively deposited into amyloid fibrils. SAA expression in the CE/J mouse species is an exception in that gene duplication did not occur and the CE/J variant is a hybrid molecule sharing features of SAA1 and SAA2. However, even though it is more closely related to SAA2 it is not deposited as amyloid fibrils. We have developed an adenoviral vector system to overexpress SAA proteins in cell culture to determine the ability of these proteins to form amyloid fibrils, and to study the structural features in relation to amyloid formation. Both the SAA2 and CE/J SAA proteins were synthesized in large quantities and purified to homogeneity. Electron microscopic analysis of the SAA proteins revealed that the SAA2 protein was capable of forming amyloid fibrils, whereas the CE/J SAA was incapable. Radiolabelled SAAs were associated with normal or acute-phase high-density lipoproteins (HDLs); we examined them for their clearance from the circulation. In normal mice, SAA2 had a half-life of 70 min and CE/J SAA had a half-life of 120 min; however, in amyloid mice 50% of the SAA2 cleared in 55 min, compared with 135 min for the CE/J protein. When the SAA proteins were associated with acute-phase HDLs, SAA2 clearance was decreased to 60 min in normal mice compared with 30 min in amyloidogenic mice. Both normal and acute-phase HDLs were capable of depositing SAA2 into preformed amyloid fibrils, whereas the CE/J protein did not become associated with amyloid fibrils. This established approach opens the doors for large-scale SAA production and for the examination of specific amino acids involved in the fibrillogenic capability of the SAA2 molecule in vitro and in vivo.
Characterization of authentic recombinant pea-seed lipoxygenases with distinct properties and reaction mechanisms
Authors: HUGHES Richard K. , WU Zecai , ROBINSON David S. , HARDY David , WEST Stuart I. , FAIRHURST Shirley A. , CASEY Rod
Published: August 2015

The two major isoforms of lipoxygenase (LOX-2 and LOX-3) from pea (Pisum sativum L. cv. Birte) seeds have been cloned and expressed from full-length cDNAs as soluble, active, non-fusion proteins in Escherichia coli. A comparison of both isoforms purified to apparent homogeneity from E. coli and pea seeds has confirmed the authenticity of the recombinant products and established the properties of the native enzymes. Despite 86% similarity at the amino acid sequence level, the enzymes have distinct properties. They have been characterized in terms of specific activity, Fe content, optimum pH, substrate and product specificity, apparent Km and Vmax for the preferred substrate, linoleic acid, and interfacial behaviour with linoleic acid. We have used this evidence, in addition to EPR spectroscopy of the hydroperoxide-activated enzymes and estimates of kcat/Km, to propose different reaction mechanisms for linoleic acid oxidation for the two isoforms. The differences relate primarily to carbonyl production from linoleic acid for which we propose a mechanism. This implicates the release of a peroxyl radical in an aerobic hydroperoxidase reaction, as the source of the carbonyl compounds formed by dismutation of the liberated peroxyl radical.
The tumour necrosis factor-sensitive pool of sphingomyelin is resynthesized in a distinct compartment of the plasma membrane
Authors: ANDRIEU-ABADIE Nathalie , CARPENTIER Stéphane , SALVAYRE Robert , LEVADE Thierry
Published: August 2015

Sphingomyelin (SM) biosynthesis is believed to occur in the early Golgi apparatus, plasma membrane and recycling endosomes. In the present study, the localization of the SM synthesis that follows its hydrolysis upon activation of the SM signal-transduction pathway was investigated in human skin fibroblasts treated with tumour necrosis factor (TNF) α. After TNFα-induced degradation, the intracellular SM levels returned to baseline levels within 30–60 min in cells treated at 37 °C. Pretreatment or co-incubation of cells with bacterial sphingomyelinase or phospholipase C, decreasing the SM and phosphatidylcholine content in the external leaflet of the plasma membrane respectively, did not inhibit SM resynthesis. However, SM resynthesis was not observed when TNFα-treated cells were continuously exposed to exogenous sphingomyelinase, suggesting that under these particular conditions the resynthesized SM becomes accessible to the enzyme. Furthermore, whereas inhibition of vesicular traffic/endocytosis at 4 °C blocked exoplasmic SM resynthesis, it did not alter SM resynthesis in TNFα-treated fibroblasts, negating the role of endosomes and the Golgi apparatus. This was further evidenced by the finding that after SM resynthesis, TNFα was again able to promote SM turnover, even at 4 °C. In addition, when the exoplasmic leaflet SM was hydrolysed by treating fibroblasts with bacterial sphingomyelinase, resynthesis of SM occurred at 37 °C much more slowly than after TNFα treatment. These findings support strongly the conclusion that the SM, which is resynthesized after TNFα-induced hydrolysis, resides in the cytosolic leaflet of the plasma membrane, and that the process involved in this resynthesis displays characteristics different from those of the previously described SM synthases.
A 211-bp enhancer of the rat uncoupling protein-1 (UCP-1) gene controls specific and regulated expression in brown adipose tissue
Authors: CASSARD-DOULCIER Anne-Marie , GELLY Chantal , BOUILLAUD Frédéric , RICQUIER Daniel
Published: August 2015

The uncoupling protein-1 gene is uniquely expressed in brown adipose tissue (BAT) and is positively regulated by cold exposure of animals and the sympathetic nervous system. To analyse the importance of a previously identified 211-bp enhancer [Cassard-Doulcier, Gelly, Fox, Schrementi, Raimbault, Klaus, Forest, Bouillaud and Ricquier (1993) Mol. Endocrinol. 7, 497–506] in the tissue-specific expression of this gene, transgenic mice were generated using the chloramphenicol acetyltransferase (CAT) gene as a reporter gene. One out of fourteen lines of the control transgenic mice bearing the Herpes simplex thymidine kinase (TK) promoter expressed weakly the CAT reporter gene in several tissues, whereas the other lines did not express CAT. Eight founders bearing the 211-bp enhancer-TK transgene were obtained. In six lines, no expression of CAT was detected. In one line, the expression of CAT was restricted to BAT. In another line, the expression of CAT was found in BAT and, to a lesser extent, in testis. Moreover, in these lines a marked and specific increase in the expression of the reporter gene in BAT was observed either after exposure of mice to the cold or by treating them with a β-adrenoceptor agonist drug. These results demonstrate that the 211-bp enhancer alone is sufficient to both direct and restrict expression to BAT. This enhancer also mediates the transcriptional response of the gene to β-adrenergic stimulation, although it does not contain conserved cAMP response element.
Interplay between cytoplasmic Ca2+ and the ATP/ADP ratio: a feedback control mechanism in mouse pancreatic islets
Authors: DETIMARY Philippe , GILON Patrick , HENQUIN Jean-Claude
Published: August 2015

In pancreatic β cells, the increase in the ATP/ADP ratio that follows a stimulation by glucose is thought to play an important role in the Ca2+-dependent increase in insulin secretion. Here we have investigated the possible interactions between Ca2+ and adenine nucleotides in mouse islets. Measurements of both parameters in the same single islet showed that the rise in the ATP/ADP ratio precedes any rise in the cytoplasmic free-Ca2+ concentration ([Ca2+]i) and is already present during the initial transient lowering of [Ca2+]i produced by the sugar. Blockade of Ca2+ influx with nimodipine did not prevent the concentration-dependent increase in the ATP/ADP ratio produced by glucose and even augmented the ratio at all glucose concentrations which normally stimulate Ca2+ influx. In contrast, stimulation of Ca2+ influx by 30 mM K+ or 100 µM tolbutamide lowered the ATP/ADP ratio. This lowering was of rapid onset and reversibility, sustained and prevented by nimodipine or omission of extracellular Ca2+. It was, however, not attenuated after blockade of secretion by activation of α2-adrenoceptors. The difference in islet ATP/ADP ratio during blockade and stimulation of Ca2+ influx was similar to that observed between threshold and submaximal glucose concentrations. The results suggest that the following feedback loop could control the oscillations of membrane potential and [Ca2+]i in β cells. Glucose metabolism increases the ATP/ADP ratio in a Ca2+-independent manner, which leads to closure of ATP-sensitive K+ channels, depolarization and stimulation of Ca2+ influx. The resulting increase in [Ca2+]i causes a larger consumption than production of ATP, which induces reopening of ATP-sensitive K+ channels and arrest of Ca2+ influx. Upon lowering of [Ca2+]i the ATP/ADP ratio increases again and a new cycle may start.
Insight into naphthoquinone metabolism: β-glucosidase-catalysed hydrolysis of hydrojuglone β-d-glucopyranoside
Authors: DUROUX Laurent , DELMOTTE Francis M. , LANCELIN Jean-Marc , KÉRAVIS Gérard , JAY-ALLEMAND Christian
Published: August 2015

In plants, the naphthoquinone juglone is known to be involved in pathogenic defence mechanisms, but it may also take part in plant developmental processes. This naphthoquinone can accumulate in a glycosylated form, namely hydrojuglone β-d-glucopyranoside. The structural configuration of this compound was shown to be 1,5-dihydroxy-4-naphthalenyl-β-d-glucopyranoside by means of MS, NMR and nuclear Overhauser effect spectroscopy analyses. A hydrojuglone β-d-glucopyranoside β-glucosidase (EC 3.2.1.21) was purified to homogeneity from Juglans regia L. The enzyme catalysed the release of juglone from hydrojuglone β-d-glucopyranoside with high specificity and showed Michaelis–Menten kinetics with Km = 0.62 mM and Vmax = 14.5 µkat/mg of protein. This enzyme also showed a higher activity towards β-d-fucosyl than β-d-glucosyl bonds. The purified enzyme had an apparent Mr of 64000 by SDS/PAGE and a pI 8.9 by isoelectrofocusing PAGE. The purified enzyme was inhibited by several bivalent cations, such as Cu2+, Fe2+, Hg2+, and by d-glucono-1,5-lactone, showing non-competitive inhibition of the mixed type.
Cloning of subunits of convulxin, a collagen-like platelet-aggregating protein from Crotalus durissus terrificus venom
Authors: LEDUC Mireille , BON Cassian
Published: August 2015

Convulxin (CVX) is a potent platelet-aggregating glycoprotein from the venom of the snake Crotalus durissus terrificus. It consists of two subunits, α and β, joined by disulphide bridges in a hexameric structure. A cDNA library from venom gland was constructed in the vector pT3T7. The cloned cDNAs encoding the two chains of CVX were sequenced. Both are preceded by an identical 23-amino acid peptide signal sequence and encode sequences of 135 amino acids for the α chain and 125 amino acids for the β chain. These polypeptides include a carbohydrate-recognition domain (CRD) in which some of the specific amino acids required for binding Ca2+ and galactose or mannose are absent. The presence of such a domain means that CVX can be included in the family of C-type lectins along with other snake venom proteins, although it is not a true lectin. Assuming that the localization of intracatenary disulphide bridges of each CVX chain is similar to that of the CRD and that an intercatenary bridge between the α and β chains is similar to that of the C-type lectin botrocetin, we postulate the existence of an additional intercatenary bridge, which explains the tridimeric structure (αβ)3 of CVX.
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.
Discrimination of two amino acid transport activities in 4F2 heavy chain- expressing Xenopus laevis oocytes
Authors: BRÖER Angelika , HAMPRECHT Bernd , BRÖER Stefan
Published: August 2015

Expression of the type II membrane proteins of the rbAT/4F2hc family in Xenopus laevisoocytes results in the induction of amino acid transport activity. To elucidate the mechanism of action, amino acid transport was investigated in oocytes expressing the surface antigen 4F2hc. Leucine transport was mediated by a Na+-independent and a Na+-dependent transport mechanism. Both systems could be further discriminated by their stereochemical constraints. Isoleucine, with a branch at the β-position, shared only the Na+-independent transport system with leucine. Both transport systems were sensitive to inhibition by arginine, but only the Na+-independent system was sensitive to inhibition by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. When compared with known transport systems the two transport activities could be described as similar to, but not identical with, mammalian systems b0,+ and y+L. The Na+-independent b0,+-like transport system was found both in rbAT and 4F2hc expressing oocytes, indicating that both proteins act in a similar way.
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.
Identification of upstream stimulatory factor as transcriptional activator of the liver promoter of the glucokinase gene
Authors: IYNEDJIAN Patrick B.
Published: August 2015

A functionally important cis-acting element termed P2 was identified in the liver promoter of the glucokinase gene. Element P2 was delineated by footprinting in vitro with nuclear proteins from rat liver and spleen. Its core sequence in the rat gene is a canonical CACGTG E-box. In the electrophoretic mobility-shift assay with nuclear proteins from rat liver, hepatocytes and hepatoma cells, an oligonucleotide with P2 in the context of the glucokinase promoter sequence gave rise to a DNA–protein complex shown to contain the upstream stimulatory factor (USF) by specific competition experiments and by reactivity with anti-USF antibodies. Transient transfection of hepatoma HepG2 cells, combined with site-directed mutagenesis, demonstrated that the P2 element was important for liver glucokinase promoter activity. Co-transfection of an expression plasmid coding for USF1 activated reporter gene expression in a manner dependent on an intact P2 element, whereas an expression plasmid for c-Myc was ineffective. Expression of a truncated form of USF1 lacking the transcription activation domain and the basic region decreased reporter activity by a dominant-negative effect. The functional significance of the P2 element was also demonstrated in transient transfection of primary hepatocytes.
Glycosylation pattern of human inter-α-inhibitor heavy chains
Authors: FLAHAUT Christophe , CAPON Calliope , BALDUYCK Malika , RICART Guy , SAUTIERE Pierre , MIZON Jacques
Published: August 2015

Human inter-α-inhibitor (IαI) is a plasma serine-proteinase inhibitor. It consists of three polypeptide chains covalently linked by a glycosaminoglycan chain: a light chain named bikunin carrying the anti-proteinase activity and two heavy chains, H1 and H2, which exhibit specific properties, e.g. they interact with hyaluronan thus stabilizing the extracellular matrix. In this study, using matrix-assisted laser desorption ionization–time-of-flight MS and amino acid sequencing of tryptic peptides, we provide a detailed analysis of the glycosylation pattern of both heavy chains. H1 carries two complex-type N-glycans of predominantly biantennary structure linked to asparagine residues at positions 256 and 559 respectively. In contrast, the oligosaccharides attached to H2 are a complex-type N-glycan in the N-terminal region of the protein (Asn64) and three to four type-1 core-structure O-glycans mono- or di-sialylated, clustered in the C-terminal region. We propose that these O-glycans might function as a recognition signal for the H2 heavy chain. The biological implications of this hypothesis, notably for the biosynthetic pathway of IαI, are discussed.
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.
Vitronectin binds to the gonococcal adhesin OpaA through a glycosaminoglycan molecular bridge
Authors: DUENSING Thomas D. , PUTTEN Jos P. M. van
Published: August 2015

Several bacterial pathogens including Neisseria gonorrhoeae bind the human serum glycoprotein vitronectin. We aimed at defining the gonococcal receptor for vitronectin. Ligand blots demonstrated that vitronectin bound specifically to the heparin-binding outer-membrane protein OpaA, but that coating OpaA with the sulphated polysaccharide heparin was required for the interaction to occur. Bound vitronectin could be dissociated from OpaA–heparin–vitronectin complexes by the addition of excess heparin, indicating that sulphated polysaccharides provided the main linkage between the two proteins. Binding assays with intact micro-organisms substantiated the requirement of sulphated polysaccharides such as heparin and dextran sulphate for the efficient binding of vitronectin to OpaA+ gonococci. This was underscored by the increased binding of vitronectin to gonococci that had been preincubated with saturating concentrations of dextran sulphate, as opposed to the inhibition of vitronectin binding observed when bacteria were incubated simultaneously with vitronectin and saturating concentrations of dextran sulphate. Binding assays with dextran sulphates of various sizes indicated that vitronectin binding correlated with the size of the polysaccharide rather than with the amount of OpaA produced by the bacteria. The inability of zero-length cross-linking agents to couple vitronectin to OpaA provided further evidence that sulphated polysaccharides formed the linkage between vitronectin and OpaA. Infection experiments demonstrated that proteoglycan-deficient Chinese hamster ovary cells efficiently internalized dextran sulphate/vitronectin-coated gonococci, suggesting that soluble sulphated polysaccharides could substitute for cell surface glycosaminoglycans in the internalization process. On the basis of our results, we propose a novel mechanism of vitronectin binding in which sulphated polysaccharides act as molecular bridges, linking the glycosaminoglycan-binding sites of vitronectin and gonococcal OpaA.
AMP-activated protein kinase: greater AMP dependence, and preferential nuclear localization, of complexes containing the α2 isoform
Authors: SALT Ian , CELLER Jakub W. , HAWLEY Simon A. , PRESCOTT Alan , WOODS Angela , CARLING David , HARDIE D. Grahame
Published: August 2015

Mammalian AMP-activated protein kinase (AMPK) is the downstream component of a cascade that is activated by cellular stresses associated with ATP depletion. AMPK exists as heterotrimeric αβγ complexes, where the catalytic subunit has two isoforms (α1 and α2) with different tissue distributions. The budding yeast homologue is the SNF1 kinase complex, which is essential for derepression of glucose-repressed genes, and seems to act by the direct phosphorylation of transcription factors in the nucleus. AMPK complexes containing the α2 rather than the α1 isoform have a greater dependence on AMP (approx. 5-fold stimulation compared with approx. 2-fold) both in direct allosteric activation and in reactivation by the upstream kinase. We have also examined their subcellular localization by using Western blotting of nuclear preparations, and by using two detection methods in the confocal microscope, i.e. indirect immunofluorescence of endogenous proteins and transfection of DNA species encoding green fluorescent protein–α-subunit fusions. By all three methods a significant proportion of α2, but not α1, is localized in the nucleus. Like SNF1, AMPK-α2 complexes could therefore be involved in the direct regulation of gene expression. The observed differences in the regulation of α1 and α2 complexes by AMP might result in differential responses to ATP depletion in distinct cellular and subcellular locations.
Expression of the genes encoding CCAAT-enhancer binding protein isoforms in the mouse mammary gland during lactation and involution
Authors: SABATAKOS Georgios , DAVIES Gareth E. , GROSSE Maria , CRYER Anthony , RAMJI Dipak P.
Published: August 2015

Transcription factors belonging to the CCAAT-enhancer binding protein (C/EBP) family have been implicated in the activation of gene expression in the mammary gland during lactation. We have therefore investigated the detailed expression profile of the C/EBP family during lactation and involution of the mouse mammary gland. The expression of C/EBPβ and C/EBPδ mRNA was low during lactation, increased dramatically at the beginning of involution and remained constant thereafter. In contrast, C/EBPα mRNA expression was relatively high during the early stages of lactation, declined to low levels during the late stages of lactation and at the start of involution, and increased again during involution. Electrophoretic mobility-shift assays showed a close correlation between the expression of the C/EBP genes and the functional C/EBP DNA-binding activity and, additionally, demonstrated the participation of heterodimers, formed from among the three proteins, in DNA–protein interactions. The DNA-binding activity of the activator protein 1 (AP1) family of transcription factors was also induced during involution. These results therefore point to potentially important regulatory roles for both the C/EBP and the AP1 family during lactation and involution of the mammary gland.
Determination of the non-ionic detergent insolubility and phosphoprotein associations of glycosylphosphatidylinositol-anchored proteins expressed on T cells
Authors: SOLOMON Keith R. , MALLORY Mark A. , FINBERG Robert W.
Published: August 2015

Glycosylphosphatidylinositol (GPI)-anchored proteins are poorly solublized in non-ionic detergents such as Triton X-100 and Nonidet P40, but are easily solublized by detergents with high critical micelle concentrations such as octylglucoside. This solubility profile has been suggested to be due to the localization of GPI-anchored proteins to lipid microdomains rich in cholesterol and sphingolipids. Additionally, GPI-anchored proteins expressed on haemopoietic cells have been shown to associate with src-family tyrosine kinases and heterotrimeric G proteins. Despite these observations, the non-ionic detergent insolubility of GPI-anchored proteins on haemopoietic cells has not been quantified nor has a relationship between the non-ionic detergent insolubility of these proteins and their association with signal-transduction molecules been identified. Here we show that GPI-anchored proteins found on T-cell tumours and activated T cells, although significantly more insoluble then transmembrane proteins, are not uniform in their detergent insolubility. Whereas CD59 was between 4% and 13% soluble, CD48 was between 13% and 25% soluble, CD55 was between 20% and 30% soluble, and CD109 was between 34% and 75% soluble. The ability of these GPI-anchored proteins to associate with phosphoproteins was correlated with their detergent insolubility: the more detergent-insoluble that a GPI-anchored protein was, the greater the level of phosphoprotein associations. These experiments reveal a relationship between non-ionic detergent insolubility and association with signal-transduction molecules and suggest a cause-and-effect relationship between these two properties. In total, these experiments support the hypothesis that the association of GPI-anchored proteins with signalling molecules is due to their sorting to lipid microdomains.
Sequence of the cDNA of a human dihydrodiol dehydrogenase isoform (AKR1C2) and tissue distribution of its mRNA
Authors: SHIRAISHI Hiroaki , ISHIKURA Syuhei , MATSUURA Kazuya , DEYASHIKI Yoshihiro , NINOMIYA Mitsuo , SAKAI Syunsuke , HARA Akira
Published: August 2015

Human liver contains three isoforms (DD1, DD2 and DD4) of dihydrodiol dehydrogenase with 20α- or 3α-hydroxysteroid dehydrogenase activity; the dehydrogenases belong to the aldo–oxo reductase (AKR) superfamily. cDNA species encoding DD1 and DD4 have been identified. However, four cDNA species with more than 99% sequence identity have been cloned and are compatible with a partial amino acid sequence of DD2. In this study we have isolated a cDNA clone encoding DD2, which was confirmed by comparison of the properties of the recombinant and hepatic enzymes. This cDNA showed differences of one, two, four and five nucleotides from the previously reported four cDNA species for a dehydrogenase of human colon carcinoma HT29 cells, human prostatic 3α-hydroxysteroid dehydrogenase, a human liver 3α-hydroxysteroid dehydrogenase-like protein and chlordecone reductase-like protein respectively. Expression of mRNA species for the five similar cDNA species in 20 liver samples and 10 other different tissue samples was examined by reverse transcriptase-mediated PCR with specific primers followed by diagnostic restriction with endonucleases. All the tissues expressed only one mRNA species corresponding to the newly identified cDNA for DD2: mRNA transcripts corresponding to the other cDNA species were not detected. We suggest that the new cDNA is derived from the principal gene for DD2, which has been named AKR1C2 by a new nomenclature for the AKR superfamily. It is possible that some of the other cDNA species previously reported are rare allelic variants of this gene.
A novel role for calmodulin: Ca2+-independent inhibition of type-1 inositol trisphosphate receptors
Authors: CARDY Thomas J. A. , TAYLOR Colin W.
Published: August 2015

Calmodulin inhibits both inositol 1,4,5-trisphosphate (IP3) binding to, and IP3-evoked Ca2+ release by, cerebellar IP3 receptors [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 11627–11632]. In the present study, full-length rat type-1 and -3 IP3 receptors were expressed at high levels in insect Spodoptera frugiperda 9 cells and the effects of calmodulin were examined. In the absence of Ca2+, calmodulin caused a concentration-dependent and reversible inhibition of [3H]IP3 binding to type-1 IP3 receptors by decreasing their apparent affinity for IP3. The effect was not reproduced by high concentrations of troponin C, parvalbumin or S-100. Increasing the medium free [Ca2+] ([Ca2+]m) inhibited [3H]IP3 binding to type-1 receptors, but the further inhibition caused by a submaximal concentration of calmodulin was similar at each [Ca2+]m. In the absence of Ca2+, 125I-calmodulin bound to a single site on each type-1 receptor subunit and to an additional site in the presence of Ca2+. There was no detectable binding of 125I-calmodulin to type-3 receptors and binding of [3H]IP3 was insensitive to calmodulin at all [Ca2+]m. Both peptide and conventional Ca2+–calmodulin antagonists affected neither [3H]IP3 binding directly nor the inhibitory effect of calmodulin in the absence of Ca2+, but each caused a [Ca2+]m-dependent reversal of the inhibition of [3H]IP3 binding caused by calmodulin. Camstatin, a peptide that binds to calmodulin equally well in the presence or absence of Ca2+, reversed the inhibitory effects of calmodulin on [3H]IP3 binding at all [Ca2+]m. We conclude that calmodulin specifically inhibits [3H]IP3 binding to type-1 IP3 receptors: the first example of a protein regulated by calmodulin in an entirely Ca2+-independent manner. Inhibition of type-1 IP3 receptors by calmodulin may dynamically regulate their sensitivity to IP3 in response to the changes in cytosolic free calmodulin concentration thought to accompany stimulation of neurones.