bluuebunny/crossref_metadata_2025 huggingface dataset:
Amplification of the thapsigargin-evoked increase in the cytosolic free Ca2+ concentration by acetylcholine in acutely isolated mouse submandibular acinar cells
Authors: SMITH Peter. M. , REED Helen. E.
Published: August 2015

The intracellular Ca2+ concentration was measured in single, acutely isolated, mouse submandibular acinar cells loaded with fura-2 AM. All experiments were performed in the absence of extracellular Ca2+ in order to eliminate Ca2+ influx. The microsomal ATPase inhibitor, thapsigargin, was used to release Ca2+ from intracellular stores and simultaneously prevent re-uptake into the stores. Sequential application of thapsigargin (2 μM) and the Ca2+ ionophore ionomycin (500 nM) indicated that thapsigargin was able to mobilize practically all intracellular Ca2+. Furthermore, in comparison with results obtained following inhibition of the plasma membrane Ca2+-ATPase by La3+ (2 mM), it may be shown that slowly unloading the intracellular Ca2+ stores using thapsigargin does not normally cause a massive, cytotoxic, increase in the cytosolic Ca2+ concentration, because Ca2+ is rapidly extruded from the cell across the plasma membrane. Application of a submaximal dose of acetylcholine (500 nM) during the rising phase of the response to thapsigargin caused a 3–4-fold increase in the amplitude of the rise in the cytosolic Ca2+ concentration without any significant alteration of the time course of the response. As thapsigargin alone is capable of mobilizing all releasable Ca2+, this increase in amplitude is most likely the result of inhibition of the Ca2+ extrusion process by acetylcholine.
Dual regulation of heat-shock transcription factor (HSF) activation and DNA-binding activity by H2O2: role of thioredoxin
Authors: JACQUIER-SARLIN Muriel R. , POLLA Barbara S
Published: August 2015

The heat-shock (HS) response is a ubiquitous cellular response to stress, involving the transcriptional activation of HS genes. Reactive oxygen species (ROS) have been shown to regulate the activity of a number of transcription factors. We investigated the redox regulation of the stress response and report here that in the human pre-monocytic line U937 cells, H2O2 induced a concentration-dependent transactivation and DNA-binding activity of heat-shock factor-1 (HSF-1). DNA-binding activity was, however, lower with H2O2 than with HS. We thus hypothesized a dual regulation of HSF by oxidants. We found that oxidizing agents, such as H2O2 and diamide, as well as alkylating agents, such as iodoacetic acid, abolished, in vitro, the HSF-DNA-binding activity induced by HS in vivo. The effects of H2O2in vitro were reversed by the sulphydryl reducing agent dithiothreitol and the endogenous reductor thioredoxin (TRX), while the effects of iodoacetic acid were irreversible. In addition, TRX also restored the DNA-binding activity of HSF oxidized in vivo, while it was found to be itself induced in vivo by both HS and H2O2. Thus, H2O2 exerts dual effects on the activation and the DNA-binding activity of HSF: on the one hand, H2O2 favours the nuclear translocation of HSF, while on the other, it alters HSF-DNA-binding activity, most likely by oxidizing critical cysteine residues within the DNA-binding domain. HSF thus belongs to the group of ROS-modulated transcription factors. We propose that the time required for TRX induction, which may restore the DNA-binding activity of oxidized HSF, provides an explanation for the delay in heat-shock protein synthesis upon exposure of cells to ROS.
Identification of a vitamin D3-response element that overlaps a unique inverted TATA box in the rat bone sialoprotein gene
Authors: KIM Richard H. , LI Jack J , OGATA Yorimasa , YAMAUCHI Masato , FREEDMAN Leonard P. , SODEK Jaro
Published: August 2015

Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during bone formation de novo. Our studies, using the osteoblastic cell line ROS 17/2.8, have revealed that rat BSP gene expression is suppressed by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], which is a powerful regulator of bone formation and resorption. To determine the molecular basis of the transcriptional suppression of BSP gene transcription by 1,25(OH)2D3, we have conducted transient transfection analyses with chimaeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene. 1,25(OH)2D3 suppressed expression in all constructs, including a short construct (pLUC 3; nt -116 to +60) that contained a putative vitamin D3-response element (VDRE; AGGGTTTATAGGTCA; nt -28 to -14) that overlaps a unique inverted TATA (TTTATA) box. Mobility shift assays demonstrated strong binding of recombinant human vitamin D3 receptor protein (hVDR) to the VDRE. Point mutations introduced into each half-site and analysed for 1,25(OH)2D3-mediated suppression of transcription and for hVDR binding either decreased or increased both transcriptional suppression and binding. In comparison with activating VDREs, the rat BSP VDRE bound VDR–VDR homodimers more avidly than VDR–RXRα heterodimers (where RXR is retinoid X receptor). These studies have therefore identified a novel 1,25(OH)2D3 suppressor element that overlaps the inverted TATA box in the rat BSP gene and indicate that transcriptional suppression of the rat BSP gene by 1,25(OH)2D3 might involve competition between the VDR and the TATA binding protein (TBP).
Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation
Authors: FERREIRA Ricardo M. B. , FRANCO Emanuel , TEIXEIRA Artur R. N.
Published: August 2015

The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a 65 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of 35S-labelled ribulose bisphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose bisphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose bisphosphate carboxylase. For short periods of time (< 1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1–6 h) resulted in the formation of high-molecular-mass aggregates of ribulose bisphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photosynthetic tissues.
A rat skeletal muscle cell line (L6) expresses specific adrenomedullin binding sites but activates adenylate cyclase via calcitonin gene-related peptide receptors
Authors: COPPOCK Hedley A , OWJI Ali A , BLOOM Stephen R , SMITH David M
Published: August 2015

We have previously demonstrated specific binding sites for adrenomedullin, a novel member of the calcitonin family of peptides, in rat muscles. It is unclear whether these receptors are vascular or muscular. Receptors for the structurally similar calcitonin gene-related peptide (CGRP) are present on myocytes and might be involved in the regulation of myocyte glucose metabolism and control by motor neurons. We investigated whether adrenomedullin binding sites were present on L6 myocytes. Specific [125I]adrenomedullin binding sites were demonstrated where adrenomedullin competed with an IC50 of 0.22±0.04 nM (mean±S.E.M.) and a concentration of binding sites (Bmax) of 0.95±0.19 pmol/mg of protein (mean±S.E.M.). CGRP and the specific CGRP receptor antagonist CGRP(8–37) competed weakly at this site (IC50 > 10 and 601±298 nM respectively). Binding studies with [125I]CGRP revealed a binding site for CGRP (IC50 = 0.13±0.01 nM; Bmax = 0.83±0.10 pmol/mg of protein) where both CGRP(8–37) and adrenomedullin competed with [125I]CGRP with IC50 values of 1.15±0.12 and 8.68±0.98 nM respectively. Chemical cross-linking showed the CGRP and adrenomedullin binding site–ligand complexes to have approximate molecular masses of 82 and 76 kDa respectively. Both CGRP and adrenomedullin increased adenylate cyclase activity with similar potencies. In both cases adenylate cyclase activation was blocked by CGRP(8–37). Stimulation with 10 nM adrenomedullin or CGRP caused an increase in the percentage of total activated cellular cAMP-dependent protein kinase from 38% in resting cells to 100% and 98% respectively. Therefore in L6 cells adrenomedullin can bind to CGRP receptors, activating adenylate cyclase and cAMP-dependent protein kinase.
Sequence of bovine carbonic anhydrase VI: potential recognition sites for N-acetylgalactosaminyltransferase
Authors: JIANG Weiping , WOITACH Joseph T , GUPTA Dwijendra
Published: August 2015

Carbonic anhydrases (CAs I–VII) are products of a gene family that encodes seven isoenzymes and several CA-related proteins. We report the cloning and sequencing of the cDNA clones encoding one of these isoenzymes, CA VI, from bovine submaxillary gland. The translated polypeptide consists of 319 amino acids, including a signal peptide (14 amino acids) typical of secreted proteins. The predicted mature protein contains 305 amino acids including a 13-amino-acid C-terminal sequence that is also present in the sheep but absent in human CA VI. The deduced mature bovine protein is 87% and 68% identical to that of sheep and human CA VI, respectively. Active-site residues of the enzyme, as well as the three zinc-binding histidines and the two cysteines involved in an intra-chain disulphide bond, are all conserved in the three species. Two potential Asn-glycosylation sites are also conserved, both of which appear to be glycosylated in sheep and bovine CA VI. Two potential peptide recognition sequences are present in bovine CA VI for the glycoprotein hormone: N-acetylgalactosaminyltransferase (GalNAc-transferase), which is one of the two transferases required to form GalNAc-4-SO4 in bovine CA VI-linked oligosaccharides. Specifically, these two sequences are Asp-Leu-Lys-Met-Lys-Lys and Ile-Thr-Lys-Arg-Lys-Lys. Comparison of these sequences with sheep and human CA VI sequences indicates that distinct glycoforms of CA VI could exist in submaxillary gland from different species.
Regulation of glucose transporters (GLUT-1 and GLUT-3) in human retinal endothelial cells
Authors: KNOTT Rachel M , ROBERTSON Marie , MUCKERSIE Elizabeth , FORRESTER John V
Published: August 2015

The regulation of glucose transporters (GLUT-1 and GLUT-3), in terms of both mRNA and protein, in human retinal endothelial cells was investigated. The cells responded within 1 h of exposure to 5 mM glucose with an increase in the level of GLUT-3 mRNA that was due to an increase in the transcription of the 4.1 kb mRNA of the gene for GLUT-3. In the absence of glucose, the gene for GLUT-1 was not transcribed but the level of GLUT-3 mRNA was increased in these conditions and this was the result of an increase in the transcription of the 4.1 kb mRNA. The level of GLUT-1 and GLUT-3 mRNA was maximal when the cells were exposed to 15 mM glucose. These results are discussed in the light of the glucose regulatory potential of the retinal microvasculature and the implications that this may have for the mechanisms of diabetic retinopathy.
Insulin and secretagogues differentially regulate fluid-phase pinocytosis in insulin-secreting β-cells
Authors: XU Gang , HOWLAND Jennie , ROTHENBERG Paul L
Published: August 2015

The physiological role of the β-cell insulin receptor is unknown. To evaluate a candidate function, the insulin regulation of fluid-phase pinocytosis was investigated in a clonal insulinoma cell line (βTC6-F7) and, for comparison, also in Chinese hamster ovary cells transfected with the human insulin receptor (CHO-T cells). In CHO-T cells, the net rate of fluid-phase pinocytosis was rapidly increased 3–4-fold over the basal rate by 100 nM insulin, with half-maximal stimulation at 2 nM insulin, as assayed by cellular uptake of horseradish peroxidase from the medium. Wortmannin, an inhibitor of phosphatidylinositol (PI)-3-kinase, blocked insulin-stimulated pinocytosis with an IC50 of 7.5 nM without affecting the basal rate of pinocytosis. In insulin-secreting βTC6-F7 cells, the secretagogues glucose and carbachol (at maximally effective concentrations of 15 mM and 0.5 mM respectively) augmented fluid-phase pinocytosis 1.65-fold over the basal rate. Wortmannin also inhibited secretagogue-stimulated pinocytosis in these β-cells with an IC50 of 7 nM but did not affect the basal rate of pinocytosis measured in the absence of secretagogues. Wortmannin did not influence either basal or secretagogue-induced insulin secretion. Although these βTC6-F7 cells have cell-surface insulin receptors, adding exogenous insulin or insulin-like growth factor 1 did not affect their rate of fluid-phase pinocytosis, either in the absence or presence of secretagogues. From these observations, we conclude that: (1) in both insulin-secreting β-cells and in conventional, insulin-responsive CHO-T cells, a common, wortmannin-sensitive reaction, which probably involves PI-3-kinase, regulates fluid-phase pinocytosis; (2) the insulin-receptor signal transduction pathway is dissociated from the regulation of fluid-phase pinocytosis in the insulin-secreting β-cell line we studied; and (3) the enhancement of fluid-phase pinocytosis associated with secretagogue-induced insulin release in βTC6-F7 cells is not attributable to autocrine activation of β-cell surface insulin receptors.
Efficacy of agonist-stimulated MEK activation determines the susceptibility of mitogen-activated protein (MAP) kinase to inhibition in rat aortic smooth muscle cells
Authors: PLEVIN Robin , SCOTT Pamela H , ROBINSON Casper J. M. , GOULD Gwyn W.
Published: August 2015

In rat aortic smooth muscle cells, platelet-derived growth factor (PDGF) stimulated a sustained activation of mitogen-activated protein kinase (MAP kinase) while the response to angiotensin II (AII) was transient. This was due to a relatively greater initial activation of MAP kinase kinase (MEK) and a correspondingly greater residual MEK activity at later time points. Pretreatment of cells with the novel MEK inhibitor PD 098059 reduced MEK activation at 5 min in response to each agonist by a similar proportion (70%); however, at this time point MAP kinase activation in response to PDGF was only marginally affected while the response to AII was substantially reduced. PD 098059 did, however, reduce PDGF-stimulated MEK activity after 30 min and this correlated with a loss in MAP kinase activity and DNA synthesis. Pretreatment with forskolin also caused a similar pattern of inhibition of agonist-stimulated MEK and MAP kinase activity. Only following protein kinase C down-regulation were both AII- and PDGF-stimulated MAP kinase activation substantially reduced and this correlated with the virtual loss of both MEK and c-Raf-1 activity in response to both agents. The differential inhibition of MAP kinase activation by forskolin was not due to specific activation of A-Raf by PDGF; both PDGF and AII stimulated A-Raf kinase and this activity was strongly inhibited by forskolin. These results suggest that the efficacy of MEK activation determines the duration of MAP kinase activation and the susceptibility of MAP kinase activation to inhibition by different agents. The results also argue against the selective activation of A-Raf by PDGF as a mechanism to explain the differences in the kinetics of MAP kinase activity stimulated by AII and PDGF.
Interferon γ differentially affects the synthesis of chondroitin/dermatan sulphate and heparan sulphate by human skin fibroblasts
Authors: PRAILLET Christel , LORTAT-JACOB Hugues , GRIMAUD Jean-Alexis
Published: August 2015

Interferon γ (IFNγ) is often considered to be an antifibrotic cytokine because it inhibits collagen synthesis in fibroblasts. Here we report the effects of recombinant human IFNγ on sulphated glycosaminoglycan chains produced by normal skin fibroblasts from adult donors. IFNγ (250 i.u./ml) induced an increase in incorporation of d-[1-3H]glucosamine into glycosaminoglycans, either secreted into the culture medium or associated with the cell layer. The structures of these molecules were analysed by using various cleavage agents (heparinases I and II, heparitinase/chondroitinases ABC and AC/periodate oxidation) followed by size-exclusion and anion-exchange HPLC. No modification was detected in the structure of the heparan sulphate chains. In contrast, the cytokine induced changes in the microcomposition of chondroitin/dermatan sulphate chains. More precisely, we found a decrease in the iduronic acid content, associated with down-regulation of the 4-O-sulphation on the GalNAc residues. In contrast, the 6-O-sulphation on these GalNAc residues was potentiated by the cytokine. These results indicate that IFNγ is able to modulate not only collagen but also the structure of galactosaminoglycans synthesized by human skin fibroblasts.
Isolation and characterization of the gene encoding an aminopeptidase involved in the selective toxicity of ascamycin toward Xanthomonas campestris pv. citri
Authors: SUDO Tatsuhiko , SHINOHARA Kiwako , DOHMAE Naoshi , TAKIO Koji , USAMI Ron , HORIKOSHI Koki , OSADA Hiroyuki
Published: August 2015

An aminopeptidase gene named XAP has been isolated from Xanthomonas campestris pv. citri, a plant pathogenic bacterium. The bacterium is one of the rare micro-organisms susceptible to ascamycin, an aminoacyl nucleoside antibiotic that inhibits protein synthesis. Sequence analysis reveals that the gene encodes a 311 amino acid protein with a calculated molecular mass of 35134 Da and approx. 50% identity for amino acids to the proline iminopeptidase from Neisseria gonorrhoeae. The XAP gene product, Xap, expressed in Escherichia coli has proline iminopeptidase activity as well as ascamycin dealanylating activity in vitro.
Molecular cloning and characterization of PTPπ, a novel receptor-like protein-tyrosine phosphatase
Authors: CROSSLAND Susan , SMITH Paul D , CROMPTON Mark R
Published: August 2015

Novel cDNAs encoding a receptor-like protein-tyrosine phosphatase (rPTP) have been isolated from human breast tumour cells and foetal brain. The predicted protein of ∼160 kDa, called PTPπ, comprises an extracellular portion with a MAM (meprin-A5 antigen-PTPµ) domain, an IgG-like domain and four fibronectin III-like repeats, a hydrophobic transmembrane domain and an intracellular portion consisting of two PTP catalytic units. The predicted amino acid sequence shows high identity with those of the two homophilic binding rPTPs, PTPµ and PTPκ. A variant of PTPπ potentially encoding a protein lacking three amino acids within the N-terminal tyrosine phosphatase domain has been identified. Reverse transcription-PCR has been used to confirm the expression of the variant in human foetal brain tissue. Expression analysis has shown that PTPπ is expressed in a variety of tissue types. Both forms of the N-terminal catalytic domain, the C-terminal catalytic domain and both catalytic domains in tandem were expressed in bacteria as fusion proteins. Intrinsic phosphatase activity was detected for all protein products with an artificial substrate. The fusion protein comprising both domains in tandem was also shown to dephosphorylate purified autophosphorylated epidermal growth factor receptor in vitro.
Improvements on the purification of mannan-binding lectin and demonstration of its Ca2+-independent association with a C1s-like serine protease
Authors: TAN Suet Mien , CHUNG Maxey C. M. , KON Oi Lian , THIEL Steffen , LEE Szu Hee , LU Jinhua
Published: August 2015

Mannan-binding lectin (MBL), previously called ‘mannan-binding protein’ or MBP, is a plasma C-type lectin which, upon binding to carbohydrate structures on micro-organisms, activates the classical pathway of complement. Purification of MBL relies on its Ca2+-dependent affinity for carbohydrate, but existing methods are susceptible to contamination by anti-carbohydrate antibodies. In the present study a sequential-sugar-elution method has been developed which can achieve a preparation of virtually pure MBL and its associated serine protease (MBL-associated serine protease, MASP) by two steps of affinity chromatography. In further separation of MASP from MBL, it was found that activated MASP was associated with MBL independent of Ca2+. Since MBL was found to bind to underivatized Sepharose 4B, the MBL-MASP complex was purified using Sepharose 4B and protease inhibitors were included to purify the complex with MASP in its proenzyme form. Analysis of thus-purified MBL-MASP complex by gel filtration on a Sephacryl S-300 column at pH 7.8 showed that the proenzyme MASP was also associated with MBL independently of Ca2+, but that the complex could be disrupted at a low pH (5.0). Therefore the mechanism of MBL-MASP-mediated complement activation appears to be significantly different from the C1-mediated classical pathway.
The effect of respiratory chain impairment of β-oxidation in rat heart mitochondria
Authors: EATON Simon , POURFARZAM Morteza , BARTLETT Kim
Published: August 2015

Cardiac ischaemia leads to an inhibition of β-oxidation flux and an accumulation of acyl-CoA and acyl-carnitine esters in the myocardium. However, there remains some uncertainty as to which esters accumulate during cardiac ischaemia and therefore the site of inhibition of β-oxidation [Moore, Radloff, Hull and Sweely (1980) Am. J. Physiol. 239, H257-H265; Latipää (1989) J. Mol. Cell. Cardiol. 21, 765–771]. When β-oxidation of hexadecanoyl-CoA in state III rat heart mitochondria was inhibited by titration of complex III activity, flux measured as 14CO2 release, acid-soluble radioactivity or as acetyl-carnitine was progressively decreased. Low concentrations of myxothiazol caused reduction of the ubiquinone pool whereas the NAD+/NADH redox state was less responsive. Measurement of the CoA and carnitine esters generated under these conditions showed that there was a progressive decrease in the amounts of chain-shortened saturated acyl esters with increasing amounts of myxothiazol. The concentrations of 3-hydroxyacyl and 2-enoyl esters, however, were increased between 0 and 0.2 µM myxothiazol but were lowered at higher myxothiazol concentrations. More hexadecanoyl-CoA and hexadecanoyl-carnitine were present with increasing concentrations of myxothiazol. We conclude that 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA dehydrogenase activities are inhibited by reduction of the ubiquinone pool, and that this explains the confusion over which esters of CoA and carnitine accumulate during cardiac ischaemia. Furthermore these studies demonstrate that the site of the control exerted by the respiratory chain over β-oxidation is shifted depending on the extent of the inhibition of the respiratory chain.
Non-erythroid α-spectrin breakdown by calpain and interleukin 1 β-converting-enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis
Authors: NATH Rathna , RASER Kadee J , STAFFORD Daniel , HAJIMOHAMMADREZA Iradj , POSNER Avigail , ALLEN Hamish , TALANIAN Robert V. , YUEN Po-wai , GILBERTSEN Richard B. , WANG Kevin K. W.
Published: August 2015

The cytoskeletal protein non-erythroid α-spectrin is well documented as an endogenous calpain substrate, especially under pathophysiological conditions. In cell necrosis (e.g. maitotoxin-treated neuroblastoma SH-SY5Y cells), α-spectrin breakdown products (SBDPs) of 150 kDa and 145 kDa were produced by cellular calpains. In contrast, in neuronal cells undergoing apoptosis (cerebellar granule neurons subjected to low potassium and SH-SY5Y cells treated with staurosporine), an additional SBDP of 120 kDa was also observed. The formation of the 120 kDa SBDP was insensitive to calpain inhibitors but was completely blocked by an interleukin 1β-converting-enzyme (ICE)-like protease inhibitor, Z-Asp-CH2OC(O)-2,6-dichlorobenzene. Autolytic activation of both calpain and the ICE homologue CPP32 was also observed in apoptotic cells. α-Spectrin can also be cleaved in vitro by purified calpains to produce the SBDP doublet of 150/145 kDa and by ICE and ICE homologues [ICH-1, ICH-2 and CPP32(β)] to produce a 150 kDa SBDP. In addition, CPP32 and ICE also produced a 120 kDa SBDP. Furthermore inhibition of either ICE-like protease(s) or calpain protects both granule neurons and SH-SY5Y cells against apoptosis. Our results suggest that both protease families participate in the expression of neuronal apoptosis.
The transcription of human α1(I) procollagen gene (COL1A1) is suppressed by tumour necrosis factor-α through proximal short promoter elements: evidence for suppression mechanisms mediated by two nuclear-factorbinding sites
Authors: MORI Kenichi , HATAMOCHI Atsushi , UEKI Hiroaki , OLSEN Anne , JIMENEZ Sergio A.
Published: August 2015

Recent studies have demonstrated that tumour necrosis factor-α (TNF-α) decreases α1(I) procollagen gene (COL1A1) expression in cultured human dermal fibroblasts. The purpose of this study was to analyse the transcriptional control of COL1A1 by TNF-α. Cultured human dermal fibroblasts were transiently transfected with plasmids containing 5´ flanking sequences of COL1A1 fused to the chloramphenicol acetyltransferase (CAT) gene, and were incubated for 48 h in medium with or without TNF-α. TNF-α inhibited the CAT activity of fibroblasts transfected with plasmids containing 2.3 kb of 5´ flanking sequences of COL1A1, whereas the activity of control plasmids containing the herpes simplex thymidine kinase promoter gene (pBLCAT) was unaltered. A series of deletion constructs or various small substitution mutations of the COL1A1 5´ flanking region fused to the CAT gene were also transfected, and CAT activity was measured after incubation with TNF-α. TNF-α suppressed COL1A1 promoter activity through proximal short promoter elements containing only 107 bp. Short substitution mutations between -101 and -97 bp or between -46 and -38 bp abolished TNF-α suppression of COL1A1 promoter activity. DNA–protein complex formation was observed involving both sites in gel retardation assays. These results suggest that TNF-α suppressed COL1A1 promoter activity through elements located between -101 and -97 bp and between -46 and -38 bp of the COL1A1 promoter, and that the suppression involved DNA-protein interactions.
Characterization of a novel pterin intermediate formed in the catalytic cycle of tyrosine hydroxylase
Authors: ALMÅS Bjørg , HAAVIK Jan , FLATMARK Torgeir
Published: August 2015

A novel pterin intermediate, in addition to the expected 4a-hydroxytetrahydrobiopterin (4a-OH-BH4) and quinonoid dihydrobiopterin, was generated during catalytic turnover of tyrosine hydroxylase (TH) with tetrahydrobiopterin as the cofactor. Based on chromatographic, spectroscopic and stability properties its structure is proposed to be similar to the product formed by the non-enzymic conversion of synthetic 4a-OH-BH4 [Bailey, Rebrin, Boerth and Ayling (1995) J. Am. Chem. Soc. 117, 10203–10211]. This compound was tentatively described as a 4a-adduct of a side-chain hydroxy group, i.e. the O2´,4a-cyclic-tetrahydrobiopterin (4a-Cyc-BH4). The intermediate generated in the TH reaction has a UV spectrum which is similar to that of 4a-OH-BH4, but elutes with a longer retention time (tR = 1.69 min compared with 1.06 min) on reversed-phase chromatography. Its conversion into quinonoid dihydrobiopterin is catalysed by pterin-4a-carbinolamine dehydratase (EC 4.2.1.96), although 4a-OH-BH4 is the preferred substrate for that enzyme. A precursor-product relationship was demonstrated between 4a-OH-BH4 and the putative 4a-Cyc-BH4 intermediate. The apparent stability of this compound is dependent on pH as well as on the nature of the buffer ions. At pH 8.0 a large amount was generated in Hepes and Tris, but little in phosphate buffer. At pH 7.0 in Hepes (standard assay conditions) and Tris buffer the putative 4a-Cyc-BH4, but no 4a-OH-BH4, was observed. None of the intermediates was observed at pH 6.0. The accumulation of these intermediates in the absence of dehydratase has important implications for the assay of TH and phenylalanine hydroxylase activities, and is also compatible with a possible physiological role of the dehydratase in the synthesis of catecholamines in vivo.
Stoichiometry of the subunits of flavocytochrome b558 of the NADPH oxidase of phagocytes
Authors: WALLACH Timothy M , SEGAL Anthony W
Published: August 2015

Flavocytochrome b558, the membrane-spanning component of the NADPH oxidase system of phagocytic cells, is composed of two subunits, p22phox and gp91phox (where phox stands for phagocyte oxidase). The stoichiometry of the subunits has been determined for purified flavocytochrome b558 by: (1) densitometry of Coomassie Blue-stained proteins separated by SDS/PAGE, (2) aromatic absorbance at 280 nm by the subunits after separation by gel filtration under denaturing conditions, (3) cross-linking studies with bis[sulphosuccinimidyl]suberate, where the molecular mass of the cross-linked complex was determined by Western blotting, and (4) radiolabelling of pure flavocytochrome b558 on lysine residues with 125I-labelled Bolton–Hunter reagent (N-succinimidyl-3-(4-hydroxy-5-[125I]iodophenyl)propionate) followed by SDS/PAGE and determination of the radioactivity on each subunit. The ratio of p22phox to gp91phox in the purified flavocytochrome b558 was related back to that in the neutrophil membrane by quantitative Western and dot-blotting to ensure that the stoichiometry was maintained during purification. These measurements showed that the two subunits were present in neutrophil membranes in a molar ratio of 1:1.
Calcium-dependent activation of Erk-1 and Erk-2 after hypo-osmotic astrocyte swelling
Authors: SCHLIESS Freimut , SINNING Ralf , FISCHER Richard , SCHMALENBACH Corinne , HÄUSSINGER Dieter
Published: August 2015

The influence of hypo-osmotic cell swelling on the activity of the mitogen-activated protein (MAP) kinases Erk-1 and Erk-2 (where Erk stands for extracellular signal-regulated protein kinase) was studied in cultured rat astrocytes. Hypo-osmotic treatment led within 10 min to an increased activity of Erk-1 and Erk-2, which became maximal at 20 min and returned to the basal level within 60 min. Moreover, exposure to hypo-osmotic conditions induced a biphasic increase in cytosolic Ca2+ concentration ([Ca2+]i): a rapid peak-like increase was followed by a sustained plateau. The absence of extracellular Ca2+ completely abolished Erk activation as well as the plateau of the [Ca2+]i response after hypo-osmotic stimulation. Application of wortmannin and agents to elevate intracellular cAMP levels also completely blocked Erk activation but were without effect on the biphasic [Ca2+]i response to hypo-osmotic treatment of the cells, suggesting a role of PtdIns 3-kinase and the Ras/Raf pathway downstream of the calcium signal. Protein kinase C (PKC) and Ca2+/calmodulin (CaM)-dependent kinases are unlikely to play a role in the hypo-osmolarity-induced signalling towards MAP kinases, as revealed by the blockage of PKC and CaM kinases. Inhibition of tyrosine kinases, pertussis-toxin- or cholera-toxin-sensitive G-proteins and phospholipase C had no effect on the [Ca2+]i response; the Erk response to hypo-osmolarity was also largely unaltered. This is different from the swelling-induced MAP kinase activation in hepatocytes, which was shown to occur via a calcium-independent but G-protein- and tyrosine kinase-dependent mechanism. Thus osmo-signalling towards MAP kinases might exhibit cell-type-specific features.
Activation effects of a prion protein fragment [PrP-(106-126)] on human leucocytes
Authors: DIOMEDE Luisa , SOZZANI Silvano , LUINI Walter , ALGERI Marina , DE GIOIA Luca , CHIESA Roberto , LIEVENS Patricia M. J. , BUGIANI Orso , FORLONI Gianluigi , TAGLIAVINI Fabrizio , SALMONA Mario
Published: August 2015

Prion-related encephalopathies are characterized by the intracerebral accumulation of an abnormal isoform of the cellular prion protein (PrPC) named scrapie prion protein (PrPSc). The pathological forms of this protein and its cellular precursor are not only expressed in the brain but also, at lower concentrations, in peripheral tissues. We recently showed that a synthetic peptide corresponding to residues 106–126 [PrP-(106–126)] of the human PrP is toxic to neurons and trophic to astrocytes in vitro. Our experiments were aimed at verifying whether PrP-(106–126) and other peptides corresponding to fragments of the amyloid protein purified from brains of patients with Gerstmann–Sträussler–Scheinker disease – namely PrP-(89–106), PrP-(106–114), PrP-(127–147) – were capable of stimulating circulating leucocytes. Native PrP expression in human lymphocytes, monocytes and neutrophils was first confirmed using PCR amplification of total RNA, after reverse transcription, and immunoblot analysis of cell extracts with anti-PrP antibodies. PrP-(106–126), but not the other peptides, increased membrane microviscosity, intracellular Ca2+ concentration and cell migration in circulating leucocytes, and O2-•production in monocytes and neutrophils. Membrane microviscosity was determined by the fluorescence polarization technique, using diphenylhexatriene as a probe, 300 s after the addition of PrP-(106–126) to the cell suspension in the concentration range 5–50 µM. The increase in intracellular Ca2+ elicited by PrP-(106–126) was dose-dependent in the range 5–500 µM. PrP-(106–126) stimulated O2-•production in monocytes and neutrophils in a dose- (10–300 µM) and time-(5–30 min) dependent manner in the presence of 10 µM dihydrocytochalasin B. Both the increase in Ca2+ concentration and the O2-•production were partially sensitive to pertussis toxin. PrP-(106–126) stimulated leucocyte migration in a dose-dependent (30–300 µM) manner and, at the highest concentration used, this migration was comparable with that elicited by 2.5 nM interleukin 8 or 10 nM fMet-Leu-Phe peptide.
Phorbol ester-sensitive phospholipase D is mainly localized in the endoplasmic reticulum of BHK cells
Authors: DECKER Christina , MIRO OBRADORS Maria Jesus , SILLENCE Daniel J. , ALLAN David
Published: August 2015

The localization of phorbol ester-sensitive phospholipase D (PLD) in baby hamster kidney cells has been investigated by determining the subcellular distribution of the phosphatidylbutanol produced when the cells are incubated with phorbol 12-myristate 13-acetate and n-butanol. Results derived by isolation of plasma membrane vesicles from intact cells or by subcellular fractionation on a sucrose density gradient suggest the PLD is specific for phosphatidylcholine and its primary site of action is not the plasma membrane but the endoplasmic reticulum.
Overexpression of phosphatidylethanolamine N-methyltransferase 2 in CHO-K1 cells does not attenuate the activity of the CDP-choline pathway for phosphatidylcholine biosynthesis
Authors: LEE Mark W. , BAKOVIC Marica , VANCE Dennis E.
Published: August 2015

Chinese hamster ovary (CHO) cells express only a trace amount of phosphatidylethanolamine N-methyltransferase (PEMT) activity. CHO cells make their phosphatidylcholine (PC) via the CDP-choline pathway. We investigated whether or not overexpression of PEMT2, an isoform of PEMT, in these cells would down-regulate the activity of the CDP-choline pathway. Transfection of CHO cells with PEMT2 cDNA behind the cytomegalovirus promoter resulted in a series of cell lines that overexpressed PEMT2. Phospholipid metabolism was characterized in cell lines that expressed a medium (281 pmol/min per mg of protein) and a high (1300 pmol/min per mg of protein) level of PEMT activity. The expression of the regulated enzyme (CTP:phosphocholine cytidylyltransferase) in the CDP-choline pathway was increased, not decreased, in these cell lines as judged by immunoblot analysis and enzymic activity. Conversion of phosphatidylethanolamine to PC was enhanced in CHO cells that expressed PEMT2 activity. PC mass was not increased in the transfected compared with the control cells. The rate of PC catabolism made by either the CDP-choline or methylation pathways was unaffected by PEMT2 expression. We conclude that expression of PEMT2 in CHO cells does not down-regulate, but rather enhances, the expression of CTP:phosphocholine cytidylyltransferase.
Interactions between organic anions, micelles and vesicles in model bile systems
Authors: VERKADE Henkjan J. , de BRUIJN Marjan A. C. , BRINK Menno A. , TALSMA Herre , VONK Roel J. , KUIPERS Folkert , GROEN Albert K.
Published: August 2015

Biliary lipid secretion probably involves both ‘micellization’ and ‘vesiculization’ of bile-canalicular membrane lipids. Several hydrophilic organic anions inhibit the secretion of lipids into the bile without altering bile salt secretion [Verkade, Vonk and Kuipers (1995) Hepatology 21, 1174–1189]. Hydrophobic organic anions do not interfere with biliary lipid secretion. We investigated whether the organic-anion-induced inhibition of biliary lipid secretion in vivo could be attributed to inhibition of micellization, by the application of in vitro models of micellization. Carboxyfluorescein was entrapped in a self-quenching concentration in small unilamellar vesicles (SUV) composed of cholesterol/egg phosphatidylcholine (molar ratios 0, 0.2 and 0.5). Certain organic anions clearly affected the bile-salt-induced release of fluorescence from these SUV, reflecting interference with micellization. However, the effects of hydrophilic and hydrophobic organic anions did not correspond with their effects on biliary lipid secretion in vivo, irrespective of the bile salt species used (taurocholate, taurodeoxycholate or tauroursodeoxycholate) and of the lipid composition of the SUV. Ultracentrifugation and dynamic light-scattering studies indicated that organic anions do interact with bile salt/phosphatidylcholine/cholesterol mixed micelles, but that they do not inhibit micellization, for example by competing with phosphatidylcholine and/or cholesterol for incorporation into mixed micelles. In conclusion, the present in vitro data indicate that the in vivo mechanism of organic-anion-induced inhibition of biliary lipid secretion is not mediated by inhibition of micellization.
Trypsin stimulates proteinase-activated receptor-2-dependent and -independent activation of mitogen-activated protein kinases
Authors: BELHAM Christopher M. , TATE Rothwelle J. , SCOTT Pamela H. , PEMBERTON Alan D. , MILLER Hugh R. P. , WADSWORTH Roger M. , GOULD Gwyn W. , PLEVIN Robin
Published: August 2015

We have examined protease-mediated activation of the mitogen-activated protein (MAP) kinase cascade in rat aortic smooth-muscle cells and bovine pulmonary arterial fibroblasts. Exposure of smooth-muscle cells to trypsin evoked rapid and transient activation of c-Raf-1, MAP kinase kinase 1 and 2 and MAP kinase that was sensitive to inhibition by soybean trypsin inhibitor. The actions of trypsin were closely mimicked by the proteinase-activated receptor 2 (PAR-2)-activating peptide sequence SLIGRL but not LSIGRL. Peak MAP kinase activation in response to both trypsin and SLIGRL was also dependent on concentration, with EC50 values of 12.1±3.4 nM and 62.5±4.5 µM respectively. Under conditions where MAP kinase activation by SLIGRL was completely desensitized by prior exposure of smooth-muscle cells to the peptide, trypsin-stimulated MAP kinase activity was markedly attenuated (78.9±15.1% desensitization), whereas the response to thrombin was only marginally affected (16.6±12.1% desensitization). Trypsin and SLIGRL also weakly stimulated the activation of the MAP kinase homologue p38 in smooth-muscle cells without any detectable activation of c-Jun N-terminal kinase. Strong activation of the MAP kinase cascade and modest activation of p38 by trypsin were also observed in fibroblasts, although in this cell type these effects were not mimicked by SLIGRL nor by the thrombin receptor-activating peptide SFLLRNPNDKYEPF. Reverse transcriptase–PCR analysis confirmed the presence of PAR-2 mRNA in smooth-muscle cells but not fibroblasts. Our results suggest that in vascular smooth-muscle cells, trypsin stimulates the activation of the MAP kinase cascade relatively selectively, in a manner consistent with an interaction with the recently described PAR-2. Activation of MAP kinase by trypsin in vascular fibroblasts, however, seems to be independent of PAR-2 and occurs by an undefined mechanism possibly involving novel receptor species.
The human EBNA-2 coactivator p100: multidomain organization and relationship to the staphylococcal nuclease fold and to the tudor protein involved in Drosophila melanogaster development
Authors: CALLEBAUT Isabelle , MORNON Jean Paul
Published: August 2015

The human p100 protein was recently identified as a coactivator of the Epstein-Barr virus nuclear antigen 2. On the basis of sequence analysis with the hydrophobic cluster analysis method, we predict that this protein consists of a repeat of four similar domains. Their fold can be related to the staphylococcal nuclease structure whose first subdomain belongs to the large oligonucleotide/oligosaccharide-binding (OB)-fold superfamily. The catalytic amino acids present in nucleases are missing, however, suggesting that these repeated motifs could only serve to bind DNA without catalytic activity, as in many other OB-folds. A highly modified fifth domain follows the four nuclease-like domains, conserving the second subdomain of the nuclease structure but not the first one (the OB-fold), which is replaced by an original domain found in multiple copies in the tudor protein, a Drosophila melanogaster protein required during oogenesis for establishment of a functional posterior organizing centre. We named this heretofore undescribed domain the ‘tudor domain’ and highlight within it five invariant residues which could be involved in one of the essential roles played by these proteins.
Involvement of Ca2+/calmodulin-dependent protein kinase II in the activation of carnitine palmitoyltransferase I by okadaic acid in rat hepatocytes
Authors: VELASCO Guillermo , GUZMÁN Manuel , ZAMMIT Victor A. , GEELEN Math J. H.
Published: August 2015

The present work was undertaken to study the mechanism by which okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, stimulates carnitine palmitoyltransferase I (CPT-I) in isolated rat hepatocytes [Guzmán, Kolodziej, Caldwell, Costorphine and Zammit (1994) Biochem. J. 300, 693–699]. The OA-induced stimulation of CPT-I was abolished by the general protein kinase inhibitor K-252a as well as by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (Ca2+/CM-PKII). However, neither the protein kinase C-specific inhibitor bisindolylmaleimide nor the protein kinase A/protein kinase C inhibitor H-7 was able to prevent the OA-induced stimulation of CPT-I. Hepatocyte-shrinkage-induced stimulation of CPT-I as well as OA-induced hepatocyte shrinkage was prevented by KN-62. KN-62 also antagonized the OA-enhanced release of lactate dehydrogenase from digitonin-permeabilized hepatocytes. Exposure of 32P-labelled hepatocytes to OA increased the degree of phosphorylation of Ca2+/CM-PKII, as immunoprecipitated by a monoclonal antibody raised against the α-subunit of rat brain kinase. This effect of OA was also antagonized by KN-62. The results thus indicate that the OA-dependent stimulation of CPT-I may be mediated (at least in part) by increased phosphorylation and subsequent activation of Ca2+/CM-PKII.
Membrane protein secretases
Authors: HOOPER Nigel M. , KARRAN Eric H. , TURNER Anthony J.
Published: August 2015

A diverse range of membrane proteins of Type I or Type II topology also occur as a circulating, soluble form. These soluble forms are often derived from the membrane form by proteolysis by a group of enzymes referred to collectively as ‘secretases‘ or ‘sheddases’. The cleavage generally occurs close to the extracellular face of the membrane, releasing physiologically active protein. This secretion process also provides a mechanism for down-regulating the protein at the cell surface. Examples of such post-translational proteolysis are seen in the Alzheimer's amyloid precursor protein, the vasoregulatory enzyme angiotensin converting enzyme, transforming growth factor-α, the tumour necrosis factor ligand and receptor superfamilies, certain cytokine receptors, and others. Since the proteins concerned are involved in pathophysiological processes such as neurodegeneration, apoptosis, oncogenesis and inflammation, the secretases could provide novel therapeutic targets. Recent characterization of these individual secretases has revealed common features, particularly sensitivity to certain metalloprotease inhibitors and up-regulation of activity by phorbol esters. It is therefore likely that a closely related family of metallosecretases controls the surface expression of multiple integral membrane proteins. Current knowledge of the various secretases are compared in this Review, and strategies for cell-free assays of such proteases are outlined as a prelude to their ultimate purification and cloning.
Purification of a protein from Agrobacterium tumefaciens strain A348 that binds phenolic compounds
Authors: DYÉ Florence , DELMOTTE Francis M.
Published: August 2015

In order to induce tumours on dicotyledonous plants, the bacterium Agrobacterium tumefaciens needs to be able to sense signal molecules, i.e. phenolic compounds. In order to identify putative chemoreceptors or environmental sensors involved in vir gene induction, we undertook the purification of a phenol-binding protein by affinity chromatography on a syringamideŐUltrogel A4 column equilibrated at pH 5.6. A mild extraction of bacterial proteins with a Tris/HCl buffer at pH 9.0 led to the purification of a 39 kDa protein (Pbp39) with a pI of 4.3 after specific elution of the affinity matrix with sodium syringate. When the affinity chromatography was performed at neutral pH, barely any protein was isolated, indicating the importance of an acidic pH for optimal affinity. A microplate binding experiment revealed that both syringylŐbiotinylated-BSA and sinapylŐbiotinylated-BSA bound at pH 5.6 to the plate coated with Pbp39.
Purification and characterization of paraoxon hydrolase from rat liver
Authors: RODRIGO Lourdes , GIL Fernando , HERNANDEZ Antonio F. , MARINA Anabel , VAZQUEZ Jesus , PLA Antonio
Published: August 2015

Paraoxonase (paraoxon hydrolase), an enzyme that hydrolyses paraoxon (O,O-diethyl O-p-nitrophenyl phosphate), is located in mammals primarily in the serum and liver. Although considerable information is available regarding serum paraoxonase, little is known about the hepatic form of this enzyme. The present work represents the first study on the purification of rat liver paraoxonase. This enzyme has been purified 415-fold to apparent homogeneity with a final specific activity of 1370 units/mg using a protocol consisting of five steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA and anion exchange on Mono Q HR 5/5. The presence of Ca2+ and Triton X-100 in the buffers throughout the purification procedure was essential for maintaining enzyme activity. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent Mr of 45000. N-terminal and internal amino acid sequences were determined and compared with those of paraoxonases from human and rabbit serum and mouse liver, showing a high similarity. The pH profile showed optimum activity at pH 8.5. The pH stability and heat inactivation of the enzyme were also studied. The Km for liver paraoxonase was 1.69 mM.