bluuebunny/crossref_metadata_2025 huggingface dataset:
Importance of aspartate-70 in organophosphate inhibition, oxime re-activation and aging of human butyrylcholinesterase
Authors: MASSON Patrick , FROMENT Marie-Thérèse , BARTELS Cynthia F. , LOCKRIDGE Oksana
Published: August 2015

Asp-70 is the defining amino acid in the peripheral anionic site of human butyrylcholinesterase (BuChE), whereas acetylcholinesterase has several additional amino acids, the most important one being Trp-277 (Trp-279 in TorpedoAChE). We studied mutants D70G, D70K and A277W to evaluate the role of Asp-70 and Trp-277 in reactions with organophosphates. We found that Asp-70 was important for binding positively charged echothiophate, but not neutral paraoxon and iso-OMPA. Asp-70 was also important for binding of positively charged pralidoxime (2-PAM) and for activation of re-activation by excess 2-PAM. Excess 2-PAM had an effect similar to substrate activation, suggesting the binding of 2 mol of 2-PAM to wild-type but not to the D70G mutant. A surprising result was that Asp-70 was important for irreversible aging, the D70G mutant having a 3-and 8-fold lower rate of aging for paraoxon-inhibited and di-isopropyl fluorophosphate-inhibited BuChE. Mutants of Asp-70 had the same rate constants for phosphorylation and re-activation by 2-PAM as wild-type. The A277W mutant behaved like wild-type in all assays. Our results predict that people with the atypical (D70G) variant of BuChE will be more sensitive to the toxic effects of echothiophate, but will be equally sensitive to paraoxon and di-isopropyl fluorophosphate. People with the D70G mutation will be resistant to re-activation of their inhibited BuChE by 2-PAM, but this will be offset by the lower rate of irreversible aging of inhibited BuChE, allowing some regeneration by spontaneous hydrolysis.
Nitrogen dioxide depletes uric acid and ascorbic acid but not glutathione from lung lining fluid
Authors: KELLY Frank J. , TETLEY Teresa D.
Published: August 2015

The aim of this study was to determine the kinetics of the reactions between the gaseous free-radical pollutant, nitrogen dioxide (NO2), and the water-soluble antioxidants present in respiratory tract lining fluid (RTLF). Samples of RTLF, recovered from 12 subjects (mean age 54.1±16.3 years; eight male, four female) as bronchoalveolar lavage (BAL) fluid were exposed ex vivo to NO2 [50–1000 parts per billion (ppb)] for 4 h. For comparison, similar exposures were carried out with single and composite solutions with relevant RTLF antioxidant concentrations. Ascorbic acid (AA), uric acid (UA), GSH depletion, and GSSG and malondialdehyde (MDA) formation were determined with time. In the three models, UA and AA were consumed in a time- and NO2-concentration-related fashion. In addition, their rate of depletion correlated positively with their initial concentration (UA, r = 0.92, P < 0.05; AA, r = 0.94, P < 0.05). Little difference was found between the rate of loss of AA (2.2±0.2; 1.9±0.5; 1.4±0.3 nmol·l-1·h-1·ppb-1), and that of UA (2.4±0.2; 2.1±0.6; 1.3±0.2 nmol·l-1·h-1·ppb-1) in the three RTLF models examined (single, composite, BAL fluid respectively). GSH loss from BAL fluid (0.2±0.1) was significantly less than that seen in either single (1.4±0.3) or composite (1.2±0.5 nmol·l-1·h-1·ppb-1) antioxidant solutions. In all cases, GSH consumption was significantly less than AA or UA. As model complexity increased, the rate of individual antioxidant loss decreased, such that in BAL fluid, AA, UA and GSH consumption rates were significantly less (P < 0.05) than in the pure or composite antioxidant mixtures. In BAL fluid, little GSSG production was observed at any NO2 concentration. MDA concentration, determined as a measure of lipid peroxidation, did not change following exposure to 50, 150 or 400 ppb NO2, but increased MDA was seen in BAL fluid from 8/12 subjects following exposure to 1000 ppb NO2 for 1 h or more. In conclusion, NO2, at environmentally relevant concentrations, depletes BAL fluid of the antioxidant defences, UA and AA, but not GSH.
Effects of thimerosal on the transient kinetics of inositol 1,4,5-trisphosphate-induced Ca2+ release from cerebellar microsomes
Authors: MEZNA Mokdad , MICHELANGELI Francesco
Published: August 2015

Thimerosal, a thiol-reactive reagent, has been shown to increase the cytosolic Ca2+ concentration in a variety of cells by sensitizing inositol 1,4,5-trisphosphate (InsP3) receptors. Thimerosal can have both sensitizing (at concentrations of < 2 μM) and inhibitory (at concentrations of > 2 μM) effects on InsP3-induced Ca2+ release (IICR) from cerebellar microsomes. Transient kinetic studies were performed by employing a fluorimetric stopped-flow approach using fluo-3. IICR was found to be a bi-exponential process with a fast and a slow component. At a maximal InsP3 concentration (20 μM), the fast phase had a rate constant of 0.9 s-1 and the slow phase had a rate constant of 0.4 s-1. The amplitudes of the two phases were 60% and 40% respectively. When the rate constants for the two phases were plotted as Hill plots, the processes were found to be non-co-operative in both cases (Hill coefficient of 1.0), thus arguing for a simple mechanism linking InsP3 binding to channel opening. At a submaximal InsP3 concentration (0.2 μM), where the sensitizing effects of thimerosal are most pronounced, thimerosal increased the rate constants of both phases in a sigmoidal fashion, with a Hill coefficient of 4.0, suggesting that several cysteine residues (up to four) need to be modified in order for maximum sensitization to occur. The rate constants remained elevated even at thimerosal concentrations that inhibited IICR. The amplitude or extent of Ca2+ release was, however, elevated to a much greater extent in the slow phase, suggesting that the two phases respond differently. At maximal InsP3 concentrations, thimerosal has no effect upon the rate constants but inhibits the amplitude of Ca2+ release.
Interaction of human neutrophil flavocytochrome b with cytosolic proteins: transferred-NOESY NMR studies of a gp91phox C-terminal peptide bound to p47phox
Authors: ADAMS Earle R. , DRATZ Edward A. , GIZACHEW Dawit , DeLEO Frank R. , YU Lixin , VOLPP Bryan D. , VLASES Michael , JESAITIS Algirdas J. , QUINN Mark T.
Published: August 2015

During activation of the neutrophil NADPH oxidase, cytosolic p47phox is translocated to the membrane where it associates with flavocytochrome b via multiple binding regions, including a site in the C-terminus of gp91phox. To investigate this binding site further, we studied the three-dimensional structure of a gp91phox C-terminal peptide (551SNSESGPRGVHFIFNKEN568) bound to p47phox using transferred nuclear Overhauser effect spectroscopy (Tr-NOESY) NMR. Using MARDIGRAS analysis and simulated annealing, five similar sets of structures of the p47phox-bound peptide were obtained, all containing an extended open bend from Ser5 to Phe14 (corresponding to gp91phox residues 555–564). The ends of the peptide were poorly defined, however, suggesting they were more flexible. Therefore further refinement was performed on the Ser5–Phe14 region of the peptide after omitting the ends of the peptide from consideration. In this case, two similar structures were obtained. Both structures again exhibited extended open-bend conformations. In addition, the amino acid side chains that showed evidence of immobilization on binding to p47phox correlated directly with those that were found previously to be essential for biological activity. Thus during NADPH oxidase assembly, the C-terminus of gp91phox binds to 47phox in an extended conformation between gp91phox residues 555 and 564, with immobilization of all of the amino acid side chains in the 558RGVHFIF564 region except for His561.
Localization of human heparan glucosaminyl N-deacetylase/N-sulphotransferase to the trans-Golgi network
Authors: HUMPHRIES Donald E. , SULLIVAN Brandon M. , ALEIXO M. Deize , STOW Jennifer L.
Published: August 2015

In order to determine the intracellular location of heparan N-deacetylase/N-sulphotransferase, cDNAs encoding human heparan glucosaminyl N-deacetylase/N-sulphotransferase were cloned from human umbilical vein endothelial cells. The deduced amino acid sequence was identical to that of the human heparan N-sulphotransferase cloned previously [Dixon, Loftus, Gladwin, Scambler, Wasmuth and Dixon (1995) Genomics 26, 239–244]. RNA blot analysis indicated that two heparan N-sulphotransferase transcripts of approx. 8.5 and 4 kb were produced in all tissues. Expression was most abundant in heart, liver and pancreas. A cDNA encoding a Flag-tagged human heparan N-sulphotransferase (where Flag is an epitope with the sequence DYKDDDDK) was transfected into mouse LTA cells. Immunofluorescence detection using anti-Flag monoclonal antibodies demonstrated that the enzyme was localized to the trans-Golgi network. A truncated Flag-tagged heparan N-sulphotransferase was also retained in the Golgi, indicating that, as for many other Golgi enzymes, the N-terminal region of heparan N-sulphotransferase is sufficient for retention in the Golgi apparatus.
Co-amplification explains linkage disequilibrium of two mosquito esterase genes in insecticide-resistant Culex quinquefasciatus
Authors: VAUGHAN Ashley , HAWKES Nicola , HEMINGWAY Janet
Published: August 2015

The mosquito Culexquinquefasciatus (Say) is a vector of human disease and a world-wide biting nuisance. Organophosphorus insecticides (OPs) have been widely used to control C. quinquefasciatus populations and this has led to the emergence of OP-resistance. Predominantly, resistance is caused by increased production of two non-specific carboxylesterases, Estα21 and Estβ21. Increased abundance of these esterases is associated with the amplification of their respective genes. The estα21 and estβ21 genes were cloned and sequenced from OP-resistant Sri Lankan C. quinquefasciatus; the two adjacent genes are in a head to head configuration, within a single amplification unit (amplicon). The homology between the two genes suggests that they arose from an ancient duplication event. The two genes have different numbers of exons (estα21 has seven and estβ21 has four); however, the intron/exon boundaries in estβ21 are all conserved in estα21. The two genes are co-amplified in three other mosquito strains with the elevated Estα21/Estβ21 phenotype. Their complete linkage disequilibrium is explained by the location of the two genes involved in resistance within a single amplicon. In insecticide-susceptible C. quinquefasciatus, the non-amplified estα and estβ gene loci are also found in a similar head to head configuration, but the size of the intergenic non-coding region is approx. 1 kb less than in the amplicon. The smaller intergenic spacer is also found in a strain with amplified estβ11, which suggests that extensive laboratory selection for this amplified esterase has not eliminated the non-amplified genes. The intergenic spacer regions have been subcloned and sequenced. They contain numerous possible TATA boxes, promoters and a number of possible regulatory elements with high homology to the consensus sequence of the Barbie box. These latter putative regulatory elements are more numerous in the larger intergenic spacer, which differs from the non-amplified spacer by two large (> 420 bp) and one small (5 bp) insertions.
Multiple C-terminal serine phosphorylation accompanies both protein kinase C-dependent and -independent activation of cytosolic 85 kDa phospholipase A2 in macrophages
Authors: WIJKANDER Jonny , GEWERT Karin , SVENSSON Ulf , HOLST Elisabeth , SUNDLER Roger
Published: August 2015

Exposure of mouse macrophages to either phorbol ester or certain bacteria was previously shown to cause increased phosphorylation of the cytosolic 85 kDa phospholipase A2 as well as a stable increase in its catalytic activity. We have now attempted to map the major phosphorylation sites on the enzyme in such cells. Phosphorylation occurred on serine residues without a detectable increase in either phosphothreonine or phosphotyrosine. After CNBr cleavage five fragments showed increased 32P labelling. Among those the most heavily labelled fragment was identified as the most C-terminal (residues 698–749), containing six serine residues. This was true whether phorbol ester or bacteria, causing protein kinase C-independent phospholipase A2 activation, was used as stimulus. The heavy phosphorylation of the most C-terminal fragment and an analysis of tryptic peptides derived from it suggested that more than one of the six serine residues became phosphorylated. Smaller increases also occurred in other CNBr-cleaved fragments from the C-terminal part of the protein, including that carrying Ser-505, a known target of the mitogen-activated protein kinase ERK-2 (extracellular-signal regulated kinase). Dexamethasone treatment (1–100 nM for 20 h), which was earlier shown to dose-dependently down-regulate the 85 kDa phospholipase A2 and its activation by phorbol ester and zymosan, was here shown also to counteract the protein kinase C-independent activation and arachidonate release elicited by bacteria. It remains to be determined whether all phosphorylation sites are equally affected under those conditions.
Perfluorodecanoic acid enhances the formation of oleic acid in rat liver
Authors: YAMAMOTO Aya , KAWASHIMA Yoichi
Published: August 2015

The feeding of perfluorodecanoic acid (PFDA) to male rats at a dietary concentration of 0.005% (w/w) for 7 days resulted in a marked increase in the activity of microsomal stearoyl-CoA desaturation in the liver. This increase in the overall desaturation activity was due to the induction of terminal desaturase among the components comprising the desaturation system. In contrast, PFDA inhibited desaturation invitro, seemingly due to interference with electron transport through the desaturation system. Accordingly, PFDA can be an inducer and also an inhibitor of Δ9-desaturation. PFDA feeding enhanced the conversion of radioactive stearic acid into oleic acid in the liver invivo, indicating that the induction of δ9-desaturase by PFDA functions invivo. PFDA feeding increased the mass of octadecenoic acid (C18:1) in the liver and the proportion of C18:1 in microsomal lipid. A highly significant linear correlation existed between the microsomal desaturase activity and the proportion of C18:1 in microsomal lipid when compared using rats in five different physiological states: control, PFDA-fed, p-chlorophenoxyisobutyric acid (clofibric acid)-fed, starved and starved/refed. These results suggest that the increase in the hepatic level of C18:1 caused by feeding of PFDA to rats can be explained by the common concept of regulation, i.e. the hepatic level of C18:1 is under the control of δ9-desaturase. The dietary administration of PFDA also increased the content of cytochrome P-450 and the activity of 7-ethoxycoumarin O-de-ethylase in the liver.
Rabbit DNase I: purification from urine, immunological and proteochemical characterization, nucleotide sequence, expression in tissues, relationships with other mammalian DNases I and phylogenetic analysis
Authors: YASUDA Toshihiro , TAKESHITA Haruo , NAKAJIMA Tamiko , HOSOMI Osamu , NAKASHIMA Yoshimitsu , KISHI Koichiro
Published: August 2015

DNase I from rabbit urine was purified approx. 3600-fold to apparent homogeneity with a 41% yield by affinity chromatography utilizing DNA–cellulose; the purity of the final preparation was assessed by SDS/PAGE, lack of contamination by other nucleases and production of a monospecific antibody against the enzyme. Although the proteochemical and enzymological properties of the purified enzyme resembled those of other mammalian DNases I, the enzymic activity of rabbit DNase I was less efficiently inhibited by monomeric actin than was that of human DNase I, probably due to substitution of an amino acid residue involved in actin binding (Tyr-65 to Phe). The effects of specific antibodies to human, rabbit and rat DNases I on the activities of the corresponding purified enzymes revealed that human DNase I lies between the rat and rabbit enzymes with regard to its immunological properties. An 1158 bp full-length cDNA encoding rabbit DNase I was constructed from the total RNA of rabbit pancreas using a combination of reverse transcriptase-PCR and rapid amplification of cDNA ends, followed by sequencing. This identified a 17- or 21-amino-acid signal sequence, with the mature enzyme containing 260 amino acids and a single N-glycosylation site at Asn-18. The amino acid sequence deduced from the cDNA sequence exactly matched that determined proteochemically from the purified enzyme up to residue 20. A systematic survey of DNase I distribution as measured by both enzymic activity and DNase I gene transcripts in 12 rabbit tissues showed the pancreas and parotid gland to produce equivalent levels, higher than those in other tissues. Enzymic activity and DNase I gene expression levels in each tissue correlated well. The results of phylogenetic and sequence identity analysis, immunological properties and tissue-distribution patterns of DNase I indicated a closer relationship between the rabbit and human enzymes than for other mammalian DNases I. Furthermore, differences between the enzymic activities expressed in mammalian parotid gland and pancreas suggest that the distribution of DNase I in mammalian tissue is species-specific.
ADP-ribosylation-factor-regulated phospholipase D activity localizes to secretory vesicles and mobilizes to the plasma membrane following N-formylmethionyl-leucyl-phenylalanine stimulation of human neutrophils
Authors: MORGAN C. P. , SENGELOV H. , WHATMORE J. , BORREGAARD N. , COCKCROFT S.
Published: August 2015

Phospholipase D (PLD) is responsible for the hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. Human neutrophils contain PLD activity which is regulated by the small GTPases, ADP-ribosylation factor (ARF) and Rho proteins. In this study we have examined the subcellular localization of the ARF-regulated PLD activity in non-activated neutrophils and cells ‘primed‘ with N-formylmethionyl-leucyl-phenylalanine (fMetLeuPhe). We report that PLD activity is localized at the secretory vesicles in control cells and is mobilized to the plasma membrane upon stimulation with fMetLeuPhe. We conclude that the ARF-regulated PLD activity is translocated to the plasma membrane by secretory vesicles upon stimulation of neutrophils with fMetLeuPhe in inflammatory/priming doses. We propose that this relocalization of PLD is important for the subsequent events occurring during neutrophil activation.
Interleukin-6 is necessary, but not sufficient, for induction of the humanC-reactive protein gene in vivo
Authors: WEINHOLD Birgit , BADER Augustinus , POLI Valeria , RÜTHER Ulrich
Published: August 2015

We have investigated the involvement of interleukin-6 (IL-6) in the induction of the gene encoding the acute-phase protein human C-reactive protein (hCRP). In transgenic mice the hCRP gene can be induced by lipopolysaccharide (LPS), but not by IL-6. In contrast, hCRP was inducible by IL-6 in primary human hepatocytes and in primary hepatocytes isolated from transgenic mice. To further evaluate the role of IL-6, we introduced the hCRP transgene into animals lacking endogenous IL-6 (IL-6-negative mice). Here, hCRP was not inducible by LPS, but was induced by a combination of LPS and IL-6. These results clearly demonstrate that IL-6 is necessary, but not sufficient, for the induction of hCRP expression. These animal models will allow further dissection of the cytokine network responsible for the regulation of the major human acute-phase reactant CRP.
Receptor recognition sites reside in both lobes of human serum transferrin
Authors: MASON Anne B. , TAM Beatrice M. , WOODWORTH Robert C. , OLIVER Ronald W. A. , GREEN Brian N. , LIN Lung-Nan , BRANDTS John F. , SAVAGE Kerry J. , LINEBACK Janet A. , MACGILLIVRAY Ross T. A.
Published: August 2015

The binding of iron by transferrin leads to a significant conformational change in each lobe of the protein. Numerous studies have shown that the transferrin receptor discriminates between iron-saturated and iron-free transferrin and that it modulates the release of iron. Given these observations, it seems likely that there is contact between each lobe of transferrin and the receptor. This is the case with chicken transferrin, in which it has been demonstrated unambiguously that both lobes are required for binding and iron donation to occur [Brown-Mason and Woodworth (1984) J. Biol. Chem. 259, 1866–1873]. Further support to this contention is added by the ability of both N- and C-domain-specific monoclonal antibodies to block the binding of a solution containing both lobes [Mason, Brown and Church (1987) J. Biol. Chem. 262, 9011–9015]. In the present study a similar conclusion is reached for the binding of human serum transferrin to the transferrin receptor. With the use of recombinant N- and C-lobes of human transferrin produced in a mammalian expression system, we show that both lobes are required to achieve full binding. (Production of recombinant C-lobe in the baby hamster kidney cell system is reported here for the first time.) Each lobe is able to donate iron to transferrin receptors on HeLa S3 cells in the presence of the contralateral lobe. The results are not identical with the chicken system, because the C-lobe alone shows a limited ability to bind to receptors and to donate iron. Further complications arise from the relatively weak re-association between the two lobes of human transferrin compared with the re-association of the ovotransferrin lobes. However, domain-specific monoclonal antibodies to either lobe block the binding of N- and C-lobe mixtures in the human system, thus substantiating the need for both.
Inhibition of cytokine-induced inducible nitric oxide synthase expression by glucagon and cAMP in cultured hepatocytes
Authors: SMITH Fiona S. , CEPPI Enrico D. , TITHERADGE Michael A.
Published: August 2015

Addition of lipopolysaccharide plus interferon γ, tumour necrosis factor α and interleukin 1β to cultured hepatocytes resulted in the induction of inducible nitric oxide synthase (iNOS) activity as measured by NO3-+NO2- formation, the conversion of L-[U-14C]arginine into citrulline and Western blotting of the iNOS protein. The inclusion of 1 μM glucagon during the induction period significantly decreased the effect of the cytokines on iNOS activity, the major effect being at the level of the total amount of protein, rather than alterations in substrate supply or covalent modification of the existing protein. In contrast, 1 μM insulin was without effect. The effect of glucagon was mediated via cAMP and could be mimicked by the presence of either dibutyryl cAMP or forskolin to activate adenylate cyclase directly. It was rapid in onset and long-lived, a 30 min pre-treatment period protecting the cells from the induction of NO synthesis over the next 21 h in the presence of cytokines. Addition of glucagon at any time point up to 9 h after treatment of the cells with lipopolysaccharide plus the cytokines resulted in a significant inhibition of iNOS activity, glucagon being most potent when added during the first 3 h.
Co-operation of the 5′ and 3′ untranslated regions of ornithine decarboxylase mRNA and inhibitory role of its 3′ untranslated region in regulating the translational efficiency of hybrid RNA species via cellular factor(s)
Authors: LORENZINI Erna C. , SCHEFFLER Immo E.
Published: August 2015

The 5′ untranslated region (UTR) has an inhibitory role in the translatability of ornithine decarboxylase (ODC) mRNA and of hybrid mRNA species, whereas the ODC 3′ UTR causes a partial release of this inhibition. We designed experiments to explore whether the co-operation between ODC 5′ UTR and 3′ UTR in the translational regulation is due to a direct interaction of those sequences or whether it is mediated by their interaction with cellular factor(s). We stably transfected Chinese hamster ovary (CHO)-K1 cells and transiently transfected COS-1 cells with expression vectors carrying different chimaeric DNAs having the luciferase (LUC) coding sequence as reporter gene, the ODC 5′ UTR or the ODC 3′ UTR, or both, in the appropriate positions. We compared the results obtained by assaying the LUC activities of both transfected cell lines with each chimaeric DNA with those observed by translating the hybrid RNAs in a translation system in vitro. When the ODC 3′ UTR was present, we observed a partial release of the translation inhibition owing to the ODC 5′ UTR only in vivo. The releasing effect was restored in vitro by the addition of cytoplasmic extracts from wild-type CHO-K1 or COS-1 cells, prepared 2 and 8 h after their release from serum starvation. We also observed a partial inhibition of the translatability of the hybrid RNA owing to the presence of the ODC 3′ UTR itself; the translational efficiency could be rescued by cell extract from 8 h serum-stimulated cells. The co-operation between the ODC-UTRs might be mediated by factors expressed by cells during particular phases of the cell cycle. Excess copies of the ODC-UTRs, expressed in trans, could compete in binding limited amounts of such regulatory factors and remove them from interaction with the endogenous ODC mRNA. This phenomenon should be reflected by modifications of the kinetics of ODC and/or LUC activities during serum stimulation. The overexpression of the ODC 3′ UTR determined an increase in both endogenous ODC activity and LUC activity. Moreover, in the transfectants expressing the hybrid RNA species bearing the ODC 3′ UTR the basal ODC activity is higher than that observed in control cells. We suggest that excess copies of the ODC 3′ UTR mis-regulate the endogenous ODC translatability, probably by tying up regulatory molecules expressed by cells in limited amounts and sequestering them from the ODC mRNA species they should interact with.
An Aspergillus awamori acetylesterase: purification of the enzyme, and cloning and sequencing of the gene
Authors: KOSEKI Takuya , FURUSE Shinji , IWANO Kimio , SAKAI Hiroshi , MATSUZAWA Hiroshi
Published: August 2015

An inducible acetylesterase was purified from the culture medium of Aspergillus awamori strain IFO4033 growing on wheat-bran culture by ion-exchange, gel-filtration and hydrophobic-interaction chromatographies. The purified enzyme had an Mr of 31000 and contained Asn-linked oligosaccharides. The enzyme liberated acetic acid from wheat bran, hydrolysed only α-naphthyl acetate and propionate when aromatic esters were used for the substrate, and was tentatively classified as a carboxylic esterase (EC 3.1.1.1). The gene encoding acetylesterase was cloned and sequenced. The deduced amino acid sequence showed that acetylesterase was produced as a 304-amino-acid-residue precursor, which was converted post-translationally into a 275-amino-acid-residue mature protein. Part of the sequence of acetylesterase was similar to the region near the active-site serine of lipases of Geotrichum candidum and Candida cylindracea. A unique site of putative Asn-linked oligosaccharides was presented.
Direct modulation of G-proteins by polyunsaturated fatty acids: a novel eicosanoid-independent regulatory mechanism in the developing lung
Authors: MUKHOPADHYAY Somnath , RAMMINGER Sarah J. , McLAUGHLIN Mark , GAMBLING Lorraine , OLVER Richard E. , KEMP Paul J.
Published: August 2015

Basal and fatty-acid-modulated G-protein function was studied in 1–3-day-pre-term, fetal guinea-pig, type II (fATII) pneumocyte apical membrane. Unstimulated (tonic) high-affinity GTPase activity (measured as [γ-32P]GTP hydrolysis rate) was high and 77% pertussis toxin-insensitive. Alteration of this activity was used as a marker of G-protein regulation. Arachidonic acid (AA) showed a dose-dependent (IC50 = 48±8 μM) inhibition of activity at concentrations significantly below critical micellar concentrations; this effect was mimicked by other polyunsaturated fatty acids (IC50 for linoleic acid = 47±2 μM; IC50 for oleic acid= 106±11 μM). Saturated fatty acids showed no effect. The effect of AA on ouabain-insensitive ATPases in the same preparation was significantly lower, suggesting a specificity of the GTPase modulation effect. AA modulation of GTPase activity was not attenuated by blocking eicosanoid metabolism with inhibitors of 5′-lipoxygenase, cyclo-oxygenase and P-450. In order to explore further the mechanism of AA–G-protein interaction, the effect of AA on the time course and equilibrium binding of [35S]GTP[S] to apical membrane was studied. Consistent with our GTPase assay data, AA inhibited binding with an IC50 value of 71±1 μM; stearic acid did not mimic this effect. This is the first report of unsaturated-fatty-acid-specific modulation of lung G-protein function: since AA also up-regulates perinatal lung alveolar Na+ transport, we suggest this lipid/G-protein switch helps maintain pulmonary fluid homoeostasis around birth.
Stat1 associates with c-kit and is activated in response to stem cell factor
Authors: DeBERRY Candy , MOU Sherry , LINNEKIN Diana
Published: August 2015

Interaction of stem cell factor (SCF), a haematopoietic growth factor, with the receptor tyrosine kinase c-kit leads to autophosphorylation of c-kit as well as tyrosine phosphorylation of various substrates. Little is known about the role of the JAK/STAT pathway in signal transduction via receptor tyrosine kinases, although this pathway has been well characterized in cytokine receptor signal transduction. We recently found that the Janus kinase Jak2 associates with c-kit and that SCF induces rapid and transient phosphorylation of Jak2. Here we present evidence that SCF activates the transcription factor Stat1. Phosphorylated c-kit co-immunoprecipitates with Stat1 within 1 min of SCF stimulation of the human cell line MO7e. Co-precipitation experiments using glutathione S-transferase fusion proteins indicate that association with c-kit is mediated by the Stat1 SH2 domain. Stat1 is rapidly tyrosine-phosphorylated in response to SCF in MO7e cells, the murine cell line FDCP-1 and normal progenitor cells. SCF-induced phosphorylation of Jak2 and Stat1 was also observed in murine 3T3 fibroblasts stably transfected with full-length human c-kit receptor. Furthermore c-kit directly phosphorylates Stat1 fusion proteins in in vitro kinase assays. Electrophoretic mobility-shift assays with nuclear extracts from SCF-stimulated cell lines and normal progenitor cells indicate that activated Stat1 binds the m67 oligonucleotide, a high-affinity SIE promoter sequence. These results demonstrate that Stat1 is activated in response to SCF, and suggest that Stat1 is a component of the SCF signal-transduction pathway.
Homodimerization and hetero-oligomerization of the single-domain trefoil protein pNR-2/pS2 through cysteine 58
Authors: CHADWICK Mark P. , WESTLEY Bruce R. , MAY Felicity E. B.
Published: August 2015

The single-domain human trefoil proteins [pNR-2/pS2 and human intestinal trefoil factor (hITF)] have seven cysteine residues, of which six are involved in maintaining the structure of the trefoil domain. The seventh does not form part of the trefoil domain and is located three residues from the C-terminus. The ability of the pNR-2/pS2 single trefoil domain protein to dimerize was examined by using recombinant protein with either a cysteine or a serine residue at this position by equilibrium ultracentrifugation, laser-assisted desorption MS, gel filtration and PAGE. pNR-2/pS2 Cys58 formed dimers, whereas pNR-2/pS2 Ser58 did not. Experiments in which the dimer was treated with thiol agents demonstrated that the dimer was linked via a disulphide bond and that the intermolecular disulphide bond was more susceptible to reduction than the intramolecular disulphide bonds. To examine whether dimeric pNR-2/pS2 was secreted by oestrogen-responsive breast cancer cells, which are known to express pNR-2/pS2 mRNA, conditioned medium was separated on non-denaturing polyacrylamide gels, transferred to PVDF membrane and reacted with antiserum against pNR-2/pS2. Monomeric and dimeric pNR-2/pS2 were detected but the majority of the protein reactivity was associated with a larger protein. Treatment of this protein with thiol agents suggested that it is an oligomer containing pNR-2/pS2 linked to another protein by a disulphide bond. These studies suggest that the biological action of pNR-2/pS2 single-domain trefoil protein might involve the formation of homodimers or oligomers with other proteins.
Co-oxidation of NADH and NADPH by a mammalian 15-lipoxygenase: inhibition of lipoxygenase activity at near-physiological NADH concentrations
Authors: O'DONNELL Valerie B. , KÜHN Hartmut
Published: August 2015

The purified 15-lipoxygenase from rabbit reticulocytes is capable of oxidizing NADH in the presence of linoleic acid and oxygen. This co-oxidation proceeds at a rate that amounts to approx. 7% of linoleic acid oxygenation rates. Although NADH inhibits the lipoxygenase reaction with linoleic acid as substrate (46% inhibition at 0.2 mM NADH), the reaction specificity of the enzyme was not altered since (13S)-hydroperoxy-(9Z,11E)-octadecadienoic acid was identified as the major reaction product. NADH oxidation was inhibited by NAD+ (uncompetitive with respect to linoleate and mixed/competitive with respect to NADH), and NADPH or NMNH could substitute for NADH with slightly different apparent Km values. NADH oxidation was enhanced at lower oxygen tension, but was completely prevented under anaerobic conditions. Computer-assisted modelling of 15-lipoxygenase/NADH interaction and sequence alignments of mammalian lipoxygenases with NADH-dependent enzymes suggested that there is no specific binding of the coenzyme at the putative fatty acid-binding site of lipoxygenases. These results suggest that NAD(P)H might be oxidized by a radical intermediate formed during the dioxygenase cycle of the lipoxygenase reaction but that NADH oxidation might not proceed at the active site of the enzyme. The mechanism and possible biological consequences of 15-lipoxygenase-catalysed NAD(P)H oxidation are discussed.
Mechanism of cAMP-induced Ca2+ influx in Dictyostelium: role of phospholipase A2
Authors: SCHALOSKE Ralph , MALCHOW Dieter
Published: August 2015

cAMP-induced Ca2+ influx in Dictyostelium follows two pathways: a G-protein-dependent pathway where influx is reduced by 50–70% in Gα2 and Gβ-negative strains and a heterotrimeric G-protein-independent pathway. Using a pharmacological approach, we found that phospholipase A2 (PLA2) is the target of both pathways. The products of PLA2 activity, arachidonic acid (AA) and palmitic acid, induced Ca2+ influx to a similar extent as cAMP. Half-maximal activation occurred at 3 μM AA and saturation at 10 μM AA. The response to AA was quantitatively similar throughout early differentiation and thusindependent of cAMP-receptor concentration. Synergy experiments revealed that cAMP and AA acted through identical pathways. The PLA2-activating peptide, a peptide with sequence similarity to the G-protein β-subunit, activated Ca2+ influx. The G-protein-independent pathway was sensitive to genistein but not to blockers of protein kinase C and other kinases, suggesting that tyrosine kinase may directly or indirectly activate PLA2 in this case.
Stromal concentrations of coenzyme A and its esters are insufficient to account for rates of chloroplast fatty acid synthesis: evidence for substrate channelling within the chloroplast fatty acid synthase
Authors: ROUGHAN P. Grattan
Published: August 2015

Concentrations of total CoAs in chloroplasts freshly isolated from spinach and peas were 10–20 μM, assuming a stromal volume of 66 μl per mg of chlorophyll. Acetyl-CoA and CoASH constituted at least 90% of the total CoA in freshly isolated chloroplasts. For a given chloroplast preparation, the concentration of endogenous acetyl-CoA was the same when extractions were performed using HClO4, trichloroacetic acid, propan-2-ol or chloroform/methanol, and the extracts analysed by quantitative HPLC after minimal processing. During fatty acid synthesis from acetate, concentrations of CoASH within spinach and pea chloroplasts varied from less than 0.1 to 5.0 μM. Malonyl-CoA concentrations were also very low (< 0.1–3.0 μM) during fatty acid synthesis but could be calculated from radioactivity incorporated from [1-14C]acetate. Concentrations of CoASH in chloroplasts synthesizing fatty acids could be doubled in the presence of Triton X-100, suggesting that the detergent stimulates fatty acid synthesis by increasing the turnover rate of acyl-CoA. However, although taken up, exogenous CoASH (1 μM) did not stimulate fatty acid synthesis by permeabilized spinach chloroplasts. Calculated rates for acetyl-CoA synthetase, acetyl-CoA carboxylase and malonyl-CoA–acyl-carrier-protein transacylase reactions at the concentrations of metabolites measured here are < 0.1–4% of the observed rates of fatty acid synthesis from acetate by isolated chloroplasts. The results suggest that CoA and its esters are probably confined within, and channelled through, the initial stages of a fatty acid synthase multienzyme complex.
Characterization of endoproteases from plant peroxisomes
Authors: DISTEFANO Stefania , PALMA M. José , GÓMEZ Manuel , del RÍO A. Luis
Published: August 2015

In this work, the characterization of endoprotease (EP) isoenzymes in peroxisomes is reported for the first time in cell organelles purified from pea leaves (Pisum sativum L.). A comparative analysis of the endo-proteolytic activity in peroxisomes purified from young (15-day-old) and senescent (50-day-old) leaves was carried out. Peroxisomes purified from senescent leaves showed a much higher endo-proteolytic activity than organelles from young plants. A 16 h incubation with exogenous substrates was the threshold time for the detection of a linear increase in the endo-proteolytic activity of peroxisomes from senescent leaves. Three EP isoenzymes (EP2, EP4 and EP5), having molecular masses of 88, 64 and 50 kDa respectively, were found in young plants by using SDS/polyacrylamide-gradient gels co-polymerized with gelatin. However, four additional isoenzymes (EP1, EP3, EP6 and EP7), with molecular masses of 220, 76, 46 and 34 kDa respectively, were detected in senescent plants. All the isoenzymes detected in peroxisomes from both young and senescent leaves were neutral proteases. By using different class-specific inhibitors, the electrophoretically separated EP isoenzymes were characterized as three serine-proteinases (EP1, EP3 and EP4), two cysteine-proteinases (EP2 and EP6) and a metallo-proteinase (EP7), and EP5 might be a metal-dependent serine-proteinase. Moreover, a peroxisomal polypeptide of 64 kDa was recognized by an antibody against a thiol-protease. The serine-proteinase isoenzymes (EP1, EP3 and EP4), which represent approx. 70% of the total EP activity of peroxisomes, showed a notable thermal stability, not being inhibited by incubation at 50 °C for 1 h.
Modification of the mitochondrial F1-ATPase ∊ subunit, enhancement of the ATPase activity of the IF1–F1 complex and IF1-binding dependence of the conformation of the ∊ subunit
Authors: SOLAINI Giancarlo , BARACCA Alessandra , GABELLIERI Edi , LENAZ Giorgio
Published: August 2015

Treatment of bovine heart submitochondrial particles with a low concentration of 2-hydroxy-5-nitrobenzyl bromide (HNB), a selective reagent for the Trp residue of the ε subunit [Baracca, Barogi, Lenaz and Solaini (1993) Int. J. Biochem. 25, 1269-1275], enhances the ATP hydrolytic activity of the particles exclusively when the natural inhibitor protein IF1 is present. Similarly, isolated F1 [the catalytic sector of the mitochondrial H+-ATPase complex (ATP synthase)] treated with the reagent has the ATPase activity enhanced exclusively if IF1 is bound to it. These experiments suggest that the modification of the ε subunit decreases the inhibitory activity of IF1, eliciting the search for a relationship between the ε subunit and the inhibitory protein. Certainly, a reverse relationship exists because HNB binds covalently to the isolated F1 exclusively when the inhibitory protein is present. This finding is consistent with the existence of the ε subunit in different conformational states depending on whether IF1 is bound to F1 or not. Support for this assertion is obtained by measurements of the intrinsic phosphorescence decay rate of F1, a probe of the Trp ε subunit conformation in situ [Solaini, Baracca, Parenti-Castelli and Strambini (1993) Eur. J. Biochem. 214, 729-734]. A significant difference in phosphorescence decay rate is detected when IF1 is added to preparations of F1 previously devoid of the inhibitory protein. These studies indicate that IF1 and the ε subunit of the mitochondrial F1-ATPase complex are related, suggesting a possible role of the ε subunit in the mechanism of regulation of the mitochondrial ATP synthase.
Association of bile-salt-dependent lipase with membranes of human pancreatic microsomes is under the control of ATP and phosphorylation
Authors: PASQUALINI Eric , CAILLOL Nathalie , MAS Eric , BRUNEAU Nadine , LEXA Doris , LOMBARDO Dominique
Published: August 2015

Bile-salt-dependent lipase (BSDL) is secreted by the pancreas into the duodenum, where it catalyses the hydrolysis of dietary lipid esters on activation by bile salts. The secretion pathway of BSDL is comparable with that of other digestive enzymes produced by pancreatic acinar cells. However, in contrast with these other enzymes, BSDL is partly associated with endoplasmic reticulum membranes as part of a folding complex, including a Grp94-related protein to which BSDL is transiently linked. The release of BSDL from membranes occurs once its glycosylation is completed [Bruneau and Lombardo (1995) J. Biol. Chem. 270, 13524-13533]. In the present study, investigations concerning the mechanism of association/dissociation of BSDL with membranes of microsomes were performed. For this purpose the role of ATP and that of the possible phosphorylation of BSDL were examined. For the first time, it is shown that human pancreatic BSDL is phosphorylated, probably at a serine residue, during its transport within the acinar cell. The phosphorylation of BSDL is provoked by calphostin C, an inhibitor of protein kinase C. In the presence of 1-(isoquinolinesulphonyl)2-methylpiperazine, a non-specific inhibitor of serine/threonine protein kinase A, C or G, or of calcium chelator 1,2-bis(O-aminophenoxy)ethane-N,N,Nʹ,Nʹ-tetra-acetic tetra(acetoxymethyl)ester, the phosphorylation of BSDL elicited by calphostin C is abolished. These data suggested that the phosphorylation of BSDL within human pancreatic microsomes is under the control of a cascade of protein kinases. We have also shown that the phosphorylation of BSDL appears to be involved in the release of the enzyme from microsome membranes. Nevertheless ATP, which modifies the conformation of BSDL, triggers this association, and an unhydrolysable ATP analogue was unable to promote it.
Regulation of inositol lipid-specific phospholipase Cδ by changes in Ca2+ ion concentrations
Authors: ALLEN Victoria , SWIGART Philip , CHEUNG Robert , COCKCROFT Shamshad , KATAN Matilda
Published: August 2015

Studies of inositol lipid-specific phospholipase C (PLC) have elucidated the main regulatory pathways for PLCβ and PLCγ but the regulation of PLCΔ isoenzymes still remains obscure. Here we demonstrate that an increase in Ca2+ ion concentration within the physiological range (0.1-10 μM) is sufficient to stimulate PLCΔ1, but not PLCγ1 and PLCβ1, to hydrolyse cellular inositol lipids present in permeabilized cells. The activity of PLCΔ1 is further enhanced in the presence of phosphatidylinositol transfer protein (PI-TP). Both full activation by Ca2+ ions and stimulation in the presence of PI-TP require an intact PH domain involved in the membrane attachment of PLCδ1. The physiological implication of this study is that PLCΔ1 could correspond to a previously uncharacterized PLC responsible for Ca2+ ion-stimulated inositol lipid hydrolysis observed in many cellular systems.
Functional conformations of the nuclear 1α,25-dihydroxyvitamin D3 receptor
Authors: NAYERI Sepideh , CARLBERG Carsten
Published: August 2015

The nuclear hormone 1α,25-dihydroxyvitamin D3 (VD) has important cell regulatory functions. Various synthetic VD analogues are under investigation to identify candidates with an improved therapeutic profile against hyperproliferative diseases. VD directly activates the transcription factor VD receptor (VDR), which in turn stimulates the expression of a cascade of primary and secondary VD-responsive genes. The activation of the VDR through binding of its natural and synthetic ligands is linked to a conformational change presenting the interface with co-activator proteins, referred to as the (trans)activation function 2 (AF-2) domain. Multiple conformations of the VDR might be the key to understanding a selective action of VD analogues. The method of limited protease digestion was used here to characterize up to three different functional VDR conformations stabilized individually by VD and its analogues. The relative potency of VDR ligands can be quantified in the interaction with these VDR conformations by determination of a functional dissociation constant, where a two-concentration-point comparison has already provided important information. In this way seven amino acid residues in the AF-2 domain have been analysed as potential ligand contact points. Interestingly, residues Phe-422 and Val-418 seem to interact with all tested VDR ligands, whereas VD analogues such as the anti-psoriatic drug MC903 displayed additional contact points within the AF-2 domain. Taken together, limited protease digestion is a powerful method for studying functional VDR conformations and seems to be very appropriate for screening VD analogues.
Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins
Authors: NAKAYAMA Kazuhisa
Published: August 2015

Limited endoproteolysis of inactive precursor proteins at sites marked by paired or multiple basic amino acids is a widespread process by which biologically active peptides and proteins are produced within the secretory pathway in eukaryotic cells. The identification of a novel family of endoproteases homologous with bacterial subtilisins and yeast Kex2p has accelerated progress in understanding the complex mechanisms underlying the production of the bioactive materials. Seven distinct proprotein convertases of this family (furin, PC2, PC1/PC3, PC4, PACE4, PC5/PC6, LPC/PC7/PC8/SPC7) have been identified in mammalian species, some having isoforms generated via alternative splicing. The family has been shown to be responsible for conversion of precursors of peptide hormones, neuropeptides, and many other proteins into their biologically active forms. Furin, the first proprotein convertase to be identified, has been most extensively studied. It has been shown to be expressed in all tissues and cell lines examined and to be mainly localized in the trans-Golgi network, although some proportion of the furin molecules cycle between this compartment and the cell surface. This endoprotease is capable of cleaving precursors of a wide variety of proteins, including growth factors, serum proteins, including proteases of the blood-clotting and complement systems, matrix metalloproteinases, receptors, viral-envelope glycoproteins and bacterial exotoxins, typically at sites marked by the consensus Arg-Xaa-(Lys/Arg)-Arg sequence. The present review covers the structure and function of mammalian subtilisin/Kex2p-like proprotein convertases, focusing on furin (EC 3.4.21.85)