bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning and expression of a cDNA encoding a new neurocalcin isoform (neurocalcin α) from bovine brain
Authors: KATO Masumi , WATANABE Yasuo , IINO Satoshi , TAKAOKA Yuri , KOBAYASHI Shigeru , HAGA Tatsuya , HIDAKA Hiroyoshi 
Published: August 2015

Neurocalcin (NC), a neuron-specific EF-hand Ca2+-binding protein, purified from bovine brain [Terasawa, Nakano, Kobayashi and Hidaka (1992) J. Biol. Chem. 267, 19596–19599] contains multiple isoforms. We previously cloned NCδ from bovine brain and showed high expression in neuronal tissues [Okazaki, Watanabe, Ando, Hagiwara, Terasawa and Hidaka (1992) Biochem. Biophys. Res. Commun. 185, 147–153]. We report here the molecular cloning and expression of a cDNA encoding bovine brain NCα. The translated bovine protein is 191 amino acids long and shares 69.1% of its amino acid sequence with NCδ. Recombinant NCα migrates as a single 23 kDa band and exhibits a Ca2+-dependent mobility shift on SDS/PAGE. Analysis of fluorescence emission spectra showed the Ca2+-induced peak at 337 nm. Interestingly, the mobility shift and the fluorescence intensity at 337 nm were larger for NCα than for NCδ. In Ca2+-overlay experiments, however, the apparent affinity of NCα for 45Ca2+ was similar to that of NCδ. Immunohistochemical analysis revealed NCα expression in the granular layer of the rat cerebellar cortex whereas NCδ was found in the Purkinje cell layer. In the rat olfactory bulb, NCα was located in external tufted cells, and NCδ was found in the periglomerular cells. These data demonstrate that NC isoforms differ in their tissue distribution and conformational changes induced by Ca2+ binding. Thus differential regulation of the two NC isoforms may be involved in control of neuron function.
Molecular cloning and expression of a cDNA encoding an olfactory-specific mouse phenol sulphotransferase
Authors: TAMURA Hiro-omi , HARADA Yuki , MIYAWAKI Atsushi , MIKOSHIBA Katsuhiko , MATSUI Michio 
Published: August 2015

Previously we demonstrated the presence of phenol sulphotransferase (P-ST) in mouse nasal cytosols and identified its zonal location in mouse nasal cavity by staining with an antiserum raised against a rat liver P-ST isoenzyme, PSTg. In the present study a cDNA was isolated from a mouse olfactory cDNA library by immunological screening with the antiserum. The isolated cDNA consisted of 1347 bp with a 912 bp open reading frame encoding a 304-residue polypeptide. Both the nucleotide and deduced amino acid sequences of the cDNA were 94% identical with those of a rat liver P-ST isoenzyme, ST1C1. The expressed enzyme in Escherichia coli displayed high P-ST activity towards phenolic odorants such as eugenol and guaiacol, and it showed a high N-hydroxy-2-acetylaminofluorene sulphation activity in comparison with the rat ST1C1 enzyme. These results indicate that the olfactory P-ST encoded by the cDNA is a mouse orthologue of rat ST1C1; however, expression of the olfactory P-ST mRNA is specific for nasal tissues as revealed by reverse transcriptase-mediated PCR (RT–PCR).
Activation of gelatinase–tissue-inhibitors-of-metalloproteinase complexes by matrilysin
Authors: BREDOW Dorothea C. von , CRESS Anne E. , HOWARD Eric W. , BOWDEN Timothy G. , NAGLE Raymond B. 
Published: August 2015

Matrilysin, gelatinase A and gelatinase B are matrix metalloproteinases (MMPs) implicated in normal and pathological processes that require remodelling of the extracellular matrix. In human prostate tissue, matrilysin is synthesized in ducts surrounded by inflammatory cells, and focally in prostate carcinoma, but not in normal glands. Gelatinase B expression is restricted to inflammatory cells. Gelatinase A can be found in both benign and malignant prostate tissue. MMP activities are regulated by their transition from latent to activated forms, as well as by the presence of tissue inhibitors of metalloproteinases (TIMPs). We investigated whether matrilysin can activate progelatinases A and B in the presence of their bound inhibitors TIMP2 and TIMP1 respectively. Incubation of progelatinase B–TIMP1 complex with active matrilysin resulted in 78 and 68 kDa active forms, as measured by SDS–PAGE and enzyme activity assays. TIMP-free gelatinase B was also activated by matrilysin. In addition, activation of progelatinase B by matrilysin was demonstrated in the conditioned medium of phorbol ester-treated HT1080 cells, confirming the results obtained in the in vitro experiments. In contrast, matrilysin did not proteolytically cleave gelatinase A–TIMP2 complex, but led to a transient increase in gelatinolytic activity of the proenzyme. Matrilysin did not enhance the autocatalytic conversion of its own proform. The data presented here suggest that matrilysin participates in a proteolytic cascade and can activate gelatinases in the presence of TIMPs.
The iron ligand sphere geometry of mammalian 15-lipoxygenases
Authors: KUBAN Ralf Jürgen , WIESNER Rainer , RATHMAN Jörg , VELDINK Gerrit , NOLTING Hans , SOLÉ V. A. , KÜHN Hartmut 
Published: August 2015

We investigated the geometry of the iron ligand sphere of the native rabbit 15-lipoxygenase (15-LOX) by X-ray absorption spectroscopy using synchrotron radiation. The soybean LOX-1 was used as a reference compound because its iron ligand sphere is well characterized. For structural information the X-ray absorption spectra were evaluated using the Excurve Program (CCLRC Daresbury Laboratory, Warrington, U.K.). From the positions of the absorption edges and from the intensities of the 1s–3d pre-edge transition peaks a six-coordinate ferrous iron was concluded for the rabbit 15-LOX. Evaluation of the extended region of the absorption spectra suggested six nitrogen and/or oxygen atoms as direct iron ligands, and the following binding distances were determined (means±S.D.; estimated accuracy is ±0.001 nm for bond distances, on the basis of more than 22 X-ray absorption spectra): 0.213±0.001 nm, 0.213±0.001 nm, 0.236±0.001 nm, 0.293±0.001 nm, 0.189±0.001 nm and 0.242±0.001. Lyophilization of the LOX altered the binding distances but did not destroy the octahedral iron ligand sphere. For construction of a structural model of the iron ligand sphere the binding distances extracted from the X-ray spectra were assigned to specific amino acids (His-360, -365, -540, -544 and the C-terminal Ile-662) by molecular modelling using the crystal coordinates of the soybean LOX-1 and of a rabbit 15-LOX–inhibitor complex.
Oxidative scission of plant cell wall polysaccharides by ascorbate-induced hydroxyl radicals
Authors: FRY Stephen C. 
Published: August 2015

Scission of plant cell wall polysaccharides in vivo has generally been assumed to be enzymic. However, in the presence of l-ascorbate, such polysaccharides are shown to undergo non-enzymic scission under physiologically relevant conditions. Scission of xyloglucan by 1 mM ascorbate had a pH optimum of 4.5, and the maximum scission rate was reached after a 10–25-min delay. Catalase prevented the scission, whereas added H2O2 (0.1–10 mM) increased the scission rate and shortened the delay. Ascorbate caused detectable xyloglucan scission above approx. 5 µM. Dehydroascorbate was much less effective. Added Cu2+ (&gt; 0.3 µM) also increased the rate of ascorbate-induced scission; EDTA was inhibitory. The rate of scission in the absence of added metals appeared to be attributable to the traces of Cu (2.8 mg·kg-1) present in the xyloglucan. Ascorbate-induced scission of xyloglucan was inhibited by radical scavengers; their effectiveness was proportional to their rate constants for reaction with hydroxyl radicals (•OH). It is proposed that ascorbate non-enzymically reduces O2 to H2O2, and Cu2+ to Cu+, and that H2O2 and Cu+ react to form •OH, which causes oxidative scission of polysaccharide chains. Evidence is reviewed to suggest that, in the wall of a living plant cell, Cu+ and H2O2 are formed by reactions involving ascorbate and its products, dehydroascorbate and oxalate. Systems may thus be in place to produce apoplastic •OH radicals in vivo. Although •OH radicals are often regarded as detrimental, they are so short-lived that they could act as site-specific oxidants targeted to play a useful role in loosening the cell wall, e.g. during cell expansion, fruit ripening and organ abscission.
Binding of human α-thrombin to platelet GpIb: energetics and functional effects
Authors: CRISTOFARO Raimondo de , CANDIA Erica de , CROCE Giovanni , MOROSETTI Roberta , LANDOLFI Raffaele 
Published: August 2015

Thrombin interaction with platelet glycocalicin (GC), the 140 kDa extracytoplasmic fragment of the membrane glycoprotein Ib, was investigated by using a solid-phase assay. Thrombin bound to GC-coated polystyrene wells was detected by measuring the hydrolysis of a chromogenic substrate. The monoclonal antibody LJ-Ib10, which specifically binds to the thrombin-binding site of GC, could displace thrombin from immobilized GC, whereas the monoclonal antibody LJ-Ib1, which interacts with the von Willebrand factor-binding domain of GC, did not affect thrombin binding to GC. Competitive inhibition of thrombin binding to immobilized GC was also observed using GC in solution or ligands that bind to the thrombin heparin-binding site, such as heparin and prothrombin fragment 2. Furthermore functional experiments demonstrated that GC binding to thrombin competes with heparin for thrombin inactivation by the antithrombin III–heparin complex as well. Thrombin–GC interaction was also studied as a function of temperature over the range 4–37 °C. A large negative heat capacity change (ΔCp), of -4.14±0.8 kJ·mol-1·K-1, was demonstrated to dominate the thermodynamics of thrombin–GC complex-formation. Finally it was demonstrated that GC binding to thrombin can allosterically decrease the enzyme affinity for hirudin via a simultaneous decrease in association rate and increase in the dissociation velocity of the enzyme–inhibitor adduct. Together these observations indicate the GC binding to the heparin-binding domain of thrombin is largely driven by a hydrophobic effect and that such interaction can protect the enzyme from inhibition by the heparin–anti-thrombin III complex.
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&lt; 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.
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.
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.
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.