bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning of the cDNAs for mast-cell chymases from the jejunum of Mongolian gerbils, Meriones unguiculatus, and their sequence similarities with chymases expressed in the connective-tissue mast cells of mice and rats
Authors: ITOH Hiroshi , MURAKUMO Yuko , TOMITA Masaki , IDE Hisamitsu , KOBAYASHI Takahiko , MARUYAMA Haruhiko , HORII Yoichiro , NAWA Yukifumi
Published: August 2015

By using the combination of reverse-transcription PCR and rapid amplification of cDNA ends methods, two distinct cDNAs encoding mast-cell proteases (chymases; MCPs), designated as gMCP-1 and -2, were successfully cloned and sequenced from the jejunum of Mongolian gerbil, Meriones unguiculatus, infected with Nippostrongylus brasiliensis. On the basis of a comparison of the deduced amino acid sequences with those of known rodent mast-cell chymases, gMCP-1 was found to be highly similar to mouse mast-cell protease (mMCP)-4 and rat mast-cell protease (rMCP)-1, while gMCP-2 was similar to mMCP-5 and rMCP-3. Although mMCP-4 and -5 and rMCP-1 and -3 were restrictedly or mainly expressed in connective-tissue mast cells and serosal mast cells, the gMCP-1 and -2 genes were mainly transcribed in the jejunal mucosa and to a lesser extent in the skin and tongue. Moreover, kinetic study after infection revealed that the amounts of the gMCP-1 and -2 mRNAs in jejunum paralleled well the degree of intestinal mastocytosis. The expression of gMCP-1 and -2 in mucosal mast cells of gerbil jejunum was also confirmed by in situ hybridization. Since a tryptase, another type of MCP, was also expressed in mucosal mast cells of gerbils but not in those of mice and rats, the expression of MCPs in mucosal mast cells of gerbils is different from those of mice and rats. The Mongolian gerbil would be a useful model with which to investigate the physiopathological role of MCPs.
Molecular cloning, expression and potential functions of the human proteinase-activated receptor-2
Authors: BÖHM Stephan K. , KONG Wuyi , BRÖMME Dieter , SMEEKENS Steven P. , ANDERSON David C. , CONNOLLY Andrew , KAHN Mark , NELKEN Nicholas A. , COUGHLIN Shaun R. , PAYAN Donald G. , BUNNETT Nigel W.
Published: August 2015

We used PCR to amplify proteinase activated receptor-2 (PAR-2) from human kidney cDNA. The open reading frame comprised 1191 bp and encoded a protein of 397 residues with 83% identity with mouse PAR-2. In KNRK cells (a line of kirsten murine sarcoma virus-transformed rat kidney epithelial cells) transfected with this cDNA, trypsin and activating peptide (AP) corresponding to the tethered ligand exposed by trypsin cleavage (SLIGKV-NH2) induced a prompt increase in cytosolic calcium ion concentration ([Ca2+]i). Human PAR-2 (hPAR-2) resided both on the plasma membrane and in the Golgi apparatus. hPAR-2 mRNA was highly expressed in human pancreas, kidney, colon, liver and small intestine, and by A549 lung and SW480 colon adenocarcinoma cells. Hybridization in situ revealed high expression in intestinal epithelial cells throughout the gut. Trypsin and AP stimulated an increase in [Ca2+]i in a rat intestinal epithelial cell line (hBRIE 380) and stimulated amylase secretion in isolated pancreatic acini. In A549 cells, which also responded to trypsin and AP with mobilization of cytosolic Ca2+, AP inhibited colony formation. Thus PAR-2 may serve as a trypsin sensor in the gut. Its expression by cells and tissues not normally exposed to pancreatic trypsin suggests that other proteases could serve as physiological activators.
The inter-α-inhibitor family: from structure to regulation
Authors: SALIER Jean-Philippe , ROUET Philippe , RAGUENEZ Gilda , DAVEAU Maryvonne
Published: August 2015

Inter-α-inhibitor (IαI) and related molecules, collectively referred to as the IαI family, are a group of plasma protease inhibitors. They display attractive features such as precursor polypeptides that give rise to mature chains with quite distinct fates and functions, and inter-chain glycosaminoglycan bonds within the various molecules. The discovery of an ever growing number of such molecules has raised pertinent questions about their pathophysiological functions. The knowledge of this family has long been structure-oriented, whereas the structure/function and structure/regulation relationships of the family members and their genes have been largely ignored. These relationships are now being elucidated in events such as gene transcription, precursor processing, changes in plasma protein levels in health and disease and binding capacities that involve hyaluronan as well as other plasma proteins as ligands. This review presents some recent progress made in these fields that paves the way for an understanding of the functions of IαI family members in vivo. Finally, given the wealth of heterogeneous, complicated and sometimes contradictory nomenclatures and acronyms currently in use for this family, a new, uniform, nomenclature is proposed for IαI family genes, precursor polypeptides and assembled proteins.
Synergistic binding of inhibitors to the protease from HIV type 1
Authors: ASANTE-APPIAH Ernest , CHAN William W.-C.
Published: August 2015

Inhibition of the protease in HIV is a potentially useful approach for the treatment of AIDS. In the course of evaluating inhibitors of the HIV-1 protease, we observed a strong synergism between certain inhibitors that might be expected to bind to different sites in this enzyme. The binding affinity of carbobenzyloxyisoleucinylphenylalaninol, for example, is increased 125-fold in the presence of carbobenzyloxyglutaminylisoamylamide. These synergistic effects between inhibitors have specific structural requirements that correlate well with the known substrate preference of the enzyme. The molecular basis for this phenomenon remains to be elucidated but it could involve substrate-induced conformational change as part of the reaction mechanism. Similar effects have been reported previously for several zinc proteases. Thus this work extends the observation to a different class of enzymes and suggests that the phenomenon might be widespread.
Cloning, sequencing and expression of the transferrin-binding protein 1 gene from Actinobacillus pleuropneumoniae
Authors: DABAN Montserrat , MEDRANO Andrés , QUEROL Enrique
Published: August 2015

Two outer-membrane proteins are involved in the uptake of iron from transferrin by certain Gram-negative bacteria, transferrin-binding proteins 1 and 2. The gene encoding transferrin-binding protein 1 from a serotype 1 isolate of the Gram-negative pathogen Actinobacillus pleuropneumoniae was cloned, and a fragment encoding 700 amino acids of Tbp1 was expressed in Escherichia coli. We also report here sequencing of the tbp1 gene and a comparison of the deduced amino acid sequence with Tbp1s from related species. The predicted polypeptide product of tbp1 is a 106 kDa protein with a 22-residue signal peptide.
Substrate specificity of endoglucanase A from Cellulomonas fimi: fundamental differences between endoglucanases and exoglucanases from family 6
Authors: DAMUDE Howard G. , FERRO Vito , WITHERS Stephen G. , WARREN R.
Published: August 2015

Values of kcat. and Km for the hydrolysis of cellotetraose, cellotriose, β-cellobiosyl fluoride and various β-aryl cellobiosides by endoglucanase A (CenA) from Cellulomonas fimi indicate that specific binding interactions between the reducing-end glucose residues of cellotetraose and cellotriose and the enzyme at the transition state provide enormous stabilization, endowing glucose with the ‘effective leaving group ability’ of 2,4-dinitrophenol. As has been seen with several other inverting glycosidases, CenA hydrolyses the ‘wrong’ anomer of its glycosyl fluoride substrate, α-cellobiosyl fluoride, according to non-Michaelian kinetics. This indicates that CenA carries out this hydrolysis by a mechanism involving binding of two substrate molecules in the active site [Hehre, Brewer and Genghof (1979) J. Biol. Chem. 254, 5942–5950] in contrast with that reported for cellobiohydrolase II, another family-6 enzyme [Konstantinidis, Marsden and Sinnott (1993) Biochem. J. 291, 833–838]. The pH profiles for wild-type CenA indicate that kcat. for CenA depends on the presence of both a protonated group and a deprotonated group for full activity, consistent with the presence of an acid and a base catalyst at the active site. By contrast, the profile for the Asp252Ala mutant of CenA shows a dependence only on a base-catalytic group, thereby confirming the role of Asp-252 as an acid catalyst. These results show that hydrolysis by CenA occurs by a typical inverting mechanism involving both acid and base catalysis, as first proposed by Koshland. It also suggests that endoglucanases from family 6 may function by fundamentally different mechanisms from exoglucanases in this family.
The effects of tyrosine nitration on the structure and function of hen egg-white lysozyme
Authors: RICHARDS Paul G. , WALTON David J. , HEPTINSTALL John
Published: August 2015

We have investigated the effects of tyrosine nitration (to form the weak acid, 3-nitrotyrosine) at positions 23 or 20 plus 23, on the structure and function of hen egg-white lysozyme. Enzyme activity against Micrococcus luteus cell-wall fragments or soluble substrates exhibits two phenomena. (a) A decrease in Km and kcat for the hydrolysis of soluble oligo- and poly-saccharides, resulting in only minor changes in the catalytic efficiency (kcat/Km) upon nitration. (b) The hydrolysis of M. luteus cell-wall fragments appeared to be dominated by electrostatic interactions with the protein, giving a decrease in enzyme activity as the 3-nitrotyrosyl group became ionized. Removal of the cell-wall anionic polymer, teichuronic acid, from M. luteus abolished this effect. The 3-nitrotyrosine group was also found to act as a fluorescence quencher of exposed tryptophan residues in lysozyme.
Activation of phosphatidylinositol 3-kinase by concanavalin A through dual signaling pathways, G-protein-coupled and phosphotyrosine-related, and an essential role of the G-protein-coupled signals for the lectin-induced respiratory burst in human monocytic THP-1 cells
Authors: MATSUO Tsuyoshi , HAZEKI Kaoru , HAZEKI Osamu , KATADA Toshiaki , UI Michio
Published: August 2015

Stimulation of monocytic THP-1 cells by a lectin, concanavalin A (Con A), resulted in protein-tyrosine phosphorylation and association of some of the thus phosphorylated proteins with the 85 kDa regulatory subunit of PtdIns 3-kinase. Both actions of Con A were not inhibited by wortmannin, a PtdIns 3-kinase inhibitor, or by prior exposure of cells to pertussis toxin which uncouples certain G-proteins from receptors. The binding of PtdIns 3-kinase to the tyrosine-phosphorylated proteins increased upon Con A stimulation; there was a marked increase in the enzymic activity in the anti-phosphotyrosine immunoprecipitates from Con A-treated cells. The increase was abolished by wortmannin but not affected by pertussis toxin. The incorporation of 32P into PtdInsP3 also increased during incubation of [32P]Pi-prelabelled cells with Con A, reflecting activation of whole-cell PtdIns 3-kinase which could not be accounted for solely by the increase in the phosphotyrosine-bound enzyme activity from the following aspects: (1) different concentration dependencies for Con A; and (2) almost total susceptibility of the incorporation to pertussis toxin. This notion appears to be supported by different time courses between increases in PtdInsP3 production and the phosphotyrosine-bound activity. The susceptibility to the toxin may reflect involvement of the toxin-sensitive G-proteins. In contrast, insulin-induced increases in PtdInsP3 production, as well as increases in phosphotyrosine-bound PtdIns 3-kinase activity, were blocked by wortmannin, but never affected by prior exposure of cells to pertussis toxin, excluding a possible involvement of G-proteins in the insulin-induced activation. Con-A-induced O2- production was almost inhibited by either pertussis toxin or wortmannin. These results suggest that oligomerization of cell-surface glycoproteins with Con A gives rise to activation of G-protein(s) and certain tyrosine kinase(s), both of which were responsible for PtdIns 3-kinase activation; the G-protein-mediated activation led to the respiratory burst.
Detection of gp91-phox precursor protein in B-cell lines from patients with X-linked chronic granulomatous disease as an indicator for mutations impairing cytochrome b558 biosynthesis
Authors: PORTER Colin D. , KURIBAYASHI KURIBAYASHI , PARKAR Mohamed H. , ROOS Dirk , KINNON Christine
Published: August 2015

NADPH oxidase cytochrome b558 consists of two subunits, gp91-phox and p22-phox, defects of which result in chronic granulomatous disease (CGD). The nature of the interaction between these subunits has yet to be determined. Absence of p22-phox in autosomal CGD patient-derived B-cell lines results in detectable levels of an incompletely glycosylated gp91-phox precursor. We have detected this same precursor species in four cell lines from patients with the X-linked form of the disease due to mutations in gp91-phox. Such mutations should delineate regions of gp91-phox important for its biosynthesis, including stable association with p22-phox. One mutation mapped to the putative FAD-binding domain, one mapped to a potential haem-binding domain, and two involved the region encoded by exon 3.
Pituitary adenylate cyclase-activating polypeptide induces expression of corticosteroid-binding globulin in cultured fetal hepatocytes: synergy with tri-iodothyronine
Authors: FAHIME Elmostafa EL , LUTZ-BUCHER Bernadette , FELIX Jean-Marc , KOCH Bernard
Published: August 2015

The purpose of the present study was to determine whether functional receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) are expressed in cultured rat fetal hepatocytes and eventually play a role in regulating gene expression of corticosteroid-binding globulin (CBG). We found PACAP38 and PACAP27 to elevate cAMP levels in hepatocytes in a dose-dependent manner, with a plateau being achieved at 10 nM and EC50 values of about 0.5–1 nM. PACAP failed to alter the turnover of inositol phosphates, whereas PACAP and VIP stimulated cAMP accumulation in an equipotent manner, suggesting the presence in these cells of type II receptor isoforms. As revealed by measurements of both CBG mRNA levels and concentrations of binding sites, long-term treatment of fetal cells with 10 nM PACAP, although resulting in partial desensitization of peptide-induced cAMP accumulation, caused a significant 3-fold elevation in CBG synthesis. This stimulatory influence of PACAP was mimicked by the cell permeant N6,2´-O-dibutyryladenosine 3´,5´-phosphate (dbcAMP). Treatment of hepatocytes with tri-iodothyronine (T3) enhanced CBG expression and, most interestingly, appeared to synergize with PACAP to elicit a 2–3-fold amplification of CBG synthesis. This study thus provides first evidence for the up-regulation by PACAP and cAMP of CBG expression in fetal hepatocytes and for T3's playing a synergistic role in enhancing PACAP-induced synthesis of the binder.
Substrate modulation of aldolase B binding in hepatocytes
Authors: AGIUS Loranne
Published: August 2015

The binding properties of hepatic aldolase (B) were determined in digitonin-permeabilized rat hepatocytes after the cells had been preincubated with either glycolytic or gluconeogenic substrates. In hepatocytes that had been preincubated in medium containing 5 mM glucose as sole carbohydrate substrate, binding of aldolase to the hepatocyte matrix was maximal at low KCl concentrations (20 mM) or bivalent cation concentrations (1 mM Mg2+) and half-maximal dissociation occurred at 50 mM KCl. Preincubation of hepatocytes (for 10–30 min) with glucose or mannose (10–40 mM), fructose, sorbitol, dihydroxyacetone or glycerol (1–10 mM), caused a leftward shift of the salt dissociation curve (maximum binding at 10 mM KCl; half-maximum dissociation at 35 mM KCl) but did not affect the proportion of bound enzyme at low or high KCl concentrations. Galactose and 2-deoxyglucose had no effect on aldolase binding. Inhibitors of glucokinase (mannoheptulose and glucosamine) suppressed the effects of glucose but not the effects of sorbitol, glycerol or dihydroxyacetone. Glucagon suppressed the effects of glucose, fructose and dihydroxyacetone but not glycerol. Poly(ethylene glycol) (PEG) (2–10%), added to the permeabilization medium, increased aldolase binding and caused a rightward shift in the salt dissociation curve. In the presence of PEG (6–8%), the effects of substrates on aldolase dissociation were shifted to higher salt concentrations (50–100 mM versus 35 mM KCl). The effects of substrates (added to the intact cell) on aldolase binding to the permeabilized cell could be mimicked by addition of the phosphorylated derivatives of these substrates to the permeabilized cell. Of the intermediates tested dihydroxyacetone phosphate and fructose 1,6-bisphosphate were the most effective at dissociating aldolase (A50 values of 20 μM and 40 μM respectively). Other effective intermediates in order of decreasing potency were fructose 1-phosphate, glycerol 3-phosphate, glucose 1,6-bisphosphate/fructose 2,6-bisphosphate. These results show that aldolase B binds to the hepatocyte matrix by a salt-dependent mechanism that is influenced by macromolecular crowding and metabolic intermediates. Maximum binding occurs when hepatocytes are incubated in the absence of glycolytic and gluconeogenic substrates and minimum binding occurs in the presence of substrates that are precursors of either fructose 1,6-bisphosphate or triose phosphates. Since the bound form of aldolase represents a kinetically less active state it is proposed that aldolase binding and dissociation may be a mechanism for buffering the concentrations of metabolic intermediates.
Characterization of mitochondria from pig muscle: higher activity of exo-NADH oxidase in animals suffering from malignant hyperthermia
Authors: RASMUSSEN Ulla F. , RASMUSSEN Hans N. , ANDERSEN Anette J. , JØRGENSEN Preben FOGD , QUISTORFF Bj⊘rn
Published: August 2015

Mitochondria were isolated from biopsies of the biceps femoris muscle of Danish landrace pigs. Three groups of animals were compared: (1) normal pigs; (2) pigs that were homozygous with respect to the gene Haln/Haln coding for the porcine malignant hyperthermia syndrome; and (3) heterozygote animals. A newly developed micro-method for preparation and assaying of small quantities of intact mitochondria was employed. With this technique mitochondria from biopsies weighing less than 100 mg were examined with respect to cytochrome content as well as phosphorylating and respiratory activities, including the non-phosphorylating exo-NADH oxidase activity. The mitochondria, prepared in a yield of 48%, showed high respiratory activities with tricarboxylic acid-cycle intermediates and pyruvate, and somewhat lower activity with palmitoyl-carnitine as substrate. The ATP synthase activity was about 1000 μmol ATP/min per g of protein and the maximal respiratory activity approx. 700 μmol of O2/min per g of protein. No differences among the three groups of animals were detected, except for the exo-NADH oxidase activities, which were 43, 78 and 107 μmol of O2/ min per g of protein in the groups of normal, heterozygous and homozygous animals respectively. It is concluded that the exo-NADH oxidase activity may be a genetic manifestation of malignant hyperthermia and may play a significant role in the heat production characteristic of the syndrome.
Structures of diphospho-myo-inositol pentakisphosphate and bisdiphospho-myo-inositol tetrakisphosphate from Dictyostelium resolved by NMR analysis
Authors: LAUSSMANN Tim , EUJEN Reint , Michael WEISSHUHN C. , THIEL Ulrich , VOGEL Günter
Published: August 2015

Diphospho-myo-inositol phosphates (PP-InsP5 and bis-PP-InsP4) were isolated from Dictyostelium in order to clarify the precise positional isomerism by two-dimensional 1H/31P-NMR analysis. The diphosphorylated inositol phosphates are 4-PP-Ins(1,2,3,5,6)P5 and 4,5-bis-PP-Ins(1,2,3,6)P4 or their corresponding enantiomers. The vicinal arrangement of the diphospho groups with its steric and electrostatic constraints possibly qualifies bis-PP-InsP4 as a metabolite with high phosphate-group-transfer potential in phosphotransferase reactions.
Growth inhibition signalled through the interleukin-4/interleukin-13 receptor complex is associated with tyrosine phosphorylation of insulin receptor substrate-1
Authors: SCHNYDER Bruno , LAHM Harald , WOERLY Gaëtane , ODARTCHENKO Nicolas , RYFFEL Bernhard , CAR Bruce D.
Published: August 2015

Induction of growth inhibition in human colorectal carcinoma cell lines by interleukin (IL)-4 and IL-13 was associated with the neophosphorylation of a 170 kDa cellular protein, identified as insulin receptor substrate-1 (IRS-1) by immunoprecipitation. Tyrosine phosphorylation of IRS-1 was also induced by insulin and insulin-like growth factor I. Sublines of colorectal carcinoma cells unresponsive to growth modulation by IL-4, IL-13 or insulin-like growth factor I-induced growth did not phosphorylate IRS-1. A functional, multimeric IL-4 receptor complex was present on all carcinoma cell lines with a subunit composition of 65 kDa, 75 kDa and the previously characterized 130 kDa band as demonstrated by affinity cross-linking with 125I-labelled IL-4. The 65 kDa subunit is novel whereas the 75 kDa band represents the common IL-2 receptor γ-chain. The novel 65 kDa receptor was present as a double band and bound primarily 125I-labelled IL-13. The present study demonstrates the involvement of a novel chain other than the γ-chain in the receptor complexes of IL-4 and IL-13 and post-receptor tyrosine phosphorylation of IRS-1. The association of IRS-1 with growth inhibitory signals in carcinoma cells suggests a novel mechanism of tumour growth control.
Purification and characterization of cytosolic and microsomal cyclophilins from maize (Zea mays)
Authors: SHELDON Philip S. , VENIS Michael A.
Published: August 2015

Methods for the purification and separation of peptidyl prolyl cis–trans isomerase (PPI) from cytosolic and microsomal fractions of etiolated maize are described. On SDS/PAGE, the purified preparations appear as single polypeptides with molecular masses of 17.5 kDa and 17.7 kDa respectively. Instead of using immobilized cyclosporin A derivatives as affinity adsorbents, our methods employ conventional techniques enabling purification of the proteins on a much larger scale than previously described. An antiserum raised against the cytosolic PPI recognizes polypeptides of similar molecular mass from a wide range of plant species on an immunoblot. There is virtually no recognition of the microsomal PPI. The cytosolic and microsomal PPIs are inhibited by cyclosporin A (Ki = 6 nM in both cases), indicating that they are cyclophilins. The cytosolic enzyme is inactivated by 5 mM N-ethylmaleimide and 2 mM phenylglyoxal. N-terminal sequencing of the microsomal PPI indicates a high level of sequence similarity with the N-terminal sequence of mature animal s-cyclophilin (cyclophilin B).
A novel structural class of K+-channel blocking toxin from the scorpion Pandinus imperator
Authors: OLAMENDI-PORTUGAL Timoteo , GÓMEZ-LAGUNAS Froylan , GURROLA Georgina B. , POSSANI Lourival D.
Published: August 2015

A novel peptide was purified and characterized from the venom of the scorpion Pandinus imperator. Analysis of its primary structure reveals that it belongs to a new structural class of K+-channel blocking peptide, composed of only 35 amino acids, but cross-linked by four disulphide bridges. It is 40, 43 and 46% identical to noxiustoxin, margatoxin and toxin 1 of Centruroides limpidus respectively. However, it is less similar (26 to 37% identity) to toxins from scorpions of the geni Leiurus, Androctonus and Buthus. The disulphide pairing was determined by sequencing heterodimers produced by mild enzymic hydrolysis. They are formed between Cys-4–Cys-25, Cys-10–Cys-30, Cys-14–Cys-32 and Cys-20–Cys-35. Three-dimensional modelling, using the parameters determined for charybdotoxin, showed that is it possible to accommodate the four disulphide bridges in the same general structure of the other K+-channel blocking peptides. The new peptide (Pi1) blocks Shaker B K+ channels reversibly. It also displaces the binding of a known K+-channel blocker, [125I]noxiustoxin, from rat brain synaptosomal membranes with an IC50 of about 10 nM.
prICE: a downstream target for ceramide-induced apoptosis and for the inhibitory action of Bcl-2
Authors: SMYTH Miriam J. , PERRY David K. , ZHANG Jiandi , POIRIER Guy G. , HANNUN Yusuf A. , OBEID Lina M.
Published: August 2015

The novel lipid second messenger, ceramide, specifically induced poly(ADP-ribose) polymerase cleavage through activation of the protease prICE. Over-expression of Bcl-2 inhibited ceramide-induced poly(ADP-ribose) polymerase proteolysis and protected cells from ceramide-induced death. These data provide the first insight into the mechanism by which ceramide mediates apoptosis and suggest a mechanism by which Bcl-2 protects from cell death.
Translocation of annexin I to plasma membranes and phagosomes in human neutrophils upon stimulation with opsonized zymosan: possible role in phagosome function
Authors: KAUFMAN Michaela , LETO Thomas , LEVY Rachel
Published: August 2015

Annexin I in the cytosol of resting neutrophils was translocated to the plasma membranes upon addition of opsonized zymosan (OZ). Maximum translocation could be detected 1 min after stimulation with OZ, and decreased thereafter. Subcellular fractionation studies demonstrated that annexin I could not be detected in the granule fractions in either resting or activated cells, but was found in association with the phagosome fraction. The marked translocation of annexin I was unique to OZ, since formyl-Met-Leu-Phe induced only slight translocation of annexin I to the plasma membranes, and phorbol 12-myristate 13-acetate had no effect at all. The mechanism regulating the translocation of annexin I is not clear. Annexin I is not phosphorylated in resting or stimulated cells. The correlation between the elevation in the intracellular calcium ion concentration ([Ca2+]i) and the degree of translocation of annexin I to the plasma membranes induced by the different stimuli, together with the inhibition of these processes by the addition of EGTA, indicate that the translocation of annexin I can probably be attributed to the rise in [Ca2+]i. However, this cannot be the sole mechanism since ionomycin, which caused an increase in [Ca2+]i similar to that induced by OZ, was less efficient than OZ in inducing translocation of annexin I. The induction of annexin I translocation to the plasma membrane by OZ, which was the only agent that induced phagosome formation, and the detection of annexin I in the phagosome fraction, suggest that annexin I participates in phagosome function.
Isolation of the murine S100 protein MRP14 (14 kDa migration-inhibitory-factor-related protein) from activated spleen cells: characterization of post-translational modifications and zinc binding
Authors: RAFTERY Mark J. , HARRISON Craig A. , ALEWOOD Paul , JONES Alun , GECZY Carolyn L.
Published: August 2015

MRP14 (macrophage migration-inhibitory factor-related protein of molecular mass 14 kDa) is an S100 calcium binding protein constitutively expressed in human neutrophils which may be associated with cellular activation/inflammation. Murine MRP14 expression was up-regulated following concanavalin A activation of spleen cells, and the protein was isolated from conditioned medium in high yield (approx. 500 ng/ml). MRP14 had a mass of 12972±2 Da by electrospray ionization MS, whereas the theoretical mass derived from the cDNA sequence, after removal of the initiator Met, was 12918 Da, suggesting that the protein was post-translationally modified. We identified four post-translational modifications of MRP14: removal of the N-terminal Met, N-terminal acetylation, disulphide bond formation between Cys79 and Cys90, and 1-methylation of His106; the calculated mass was then 12971.8 Da. Methylation of His106 was further characterized after incubation of spleen cells with L-[methyl-3H]Met during concanavalin A stimulation. Sequential analysis of a peptide (obtained by digestion with Lys C) containing methylated His indicated that > 80% of the label in the cycle corresponded to His106, suggesting that the methyl residue was transferred from S-adenosyl-L-methionine. Comparison of the C18 reverse-phase HPLC retention times of phenylthiocarbamoyl derivatives of a hydrolysed digest peptide of MRP14 with those of standards confirmed methyl substitution on the 1-position of the imidazole ring. MRP14 bound more 65Zn2+ than the same amounts of the 10 kDa chemotactic protein (CP10) or S100β. Ca2+ decreased Zn2+ binding in S100β but it did not influence binding to MRP14, suggesting that the Zn2+ binding site was distinct from and independent of the two Ca2+ binding domains.
Function and membrane topology of wild-type and mutated cytochrome P-450c21
Authors: HU Meng-Chun , HSU Li-Chung , HSU Nai-Chi , CHUNG Bon-chu
Published: August 2015

We have studied membrane topology of cytochrome P-450c21 (P450c21) using the approaches of mutagenesis and protease digestion. P450c21 is located at the cytoplasm with an N-terminal hydrophobic domain integrated into microsomal membranes. When this hydrophobic domain was replaced by a secretory signal peptide, P450c21 was translocated into the lumen and lost enzymic activity. No other topogenic sequence was detected in the bulk of the P450c21 peptide. A mutant protein with Pro-30 replaced by Leu (L30) corresponding to the mutation found in the diseased state was created. L30 protein lost 90% of enzymic activity, while a double mutant (L30R32) with an additional Leu-32 to Arg mutation had slightly higher residual enzymic activity. Apart from lower activity, L30 was also present in the cell at a lower level than wild-type P450c21. This lower level is probably due to increased degradation, as L30 is synthesized at a normal rate. Both L30 and L30R32 proteins, however, were integrated into membranes normally. Therefore the Pro-30 → Leu mutation did not affect membrane integration, but affected the abundance and enzymic activity of P450c21.
Non-oxidative modification of native low-density lipoprotein by oxidized low-density lipoprotein
Authors: YANG Min , LEAKE David S. , RICE-EVANS Catherine A.
Published: August 2015

The oxidative modification of low-density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis, although little is known as yet about the precise mechanism of oxidation in vivo. The studies presented here demonstrate that, in the absence of cells or transition metals, oxidized LDL can modify native LDL through co-incubation in vitro such as to increase its net negative charge, in a concentration-dependent manner. The interaction is not inhibited by peroxyl radical scavengers or metal chelators, precluding the possibility that the modification of native LDL by oxidized LDL is through an oxidative process. Studies with radioiodinated oxidized LDL showed no transfer of radioactivity to the native LDL, demonstrating that fragmentation of protein and the transfer of some of the fragments does not account for the modified charge on the native LDL particle. The adjacency of native to oxidized LDL in the arterial wall may be a potential mechanism by which the altered recognition properties of the apolipoprotein B-100 may arise rapidly without oxidation or extensive modification of the native LDL lipid itself.
2-Carboxyarabinitol 1-phosphate (CA1P) formation through a phosphate exchange reaction catalysed by the CA1P phosphatase from French bean (Phaseolus vulgaris L.)
Authors: ANDRALOJC P. John , RYAN Jane E. , PARRY Martin A. J. , KEYS Alfred J.
Published: August 2015

Transfer of the phosphate group of 2-carboxy-D-arabinitol 1-phosphate (CA1P) to 14C-labelled 2-carboxy-D-arabinitol (CA) was catalysed by extracts from leaves of Phaseolus vulgaris. This phosphotransferase activity co-purified with CA1P phosphatase, described previously. This activity was increased, up to 16-fold, by addition of bicarbonate ions at pH 9–10, suggesting rate enhancement by enzyme carbamylation. A Vmax of 1.5 μmol/min per mg of protein and a Km (for CA) of 1.8 mM were estimated for the exchange reaction, with the purified phosphatase. 2-Carboxy-D-arabinitol 1,5-bisphosphate and 2-carboxy-D-ribitol 1,5-bisphosphate, but not D-ribulose 1,5-bisphosphate, could replace CA1P as phosphate donor to [14C]CA.
Both rapamycin-sensitive and -insensitive pathways are involved in the phosphorylation of the initiation factor-4E-binding protein (4E-BP1) in response to insulin in rat epididymal fat-cells
Authors: DIGGLE Tricia A. , MOULE S. Kelly , AVISON Matthew B. , FLYNN Andrea , FOULSTONE Emily J. , PROUD Christopher G. , DENTON Richard M.
Published: August 2015

There is mounting evidence that in fat and other insulin-sensitive cells activation of protein synthesis may involve the dissociation of a protein (4E-BP1) from eukaryotic initiation factor (eIF)-4E thus allowing formation of the eIF-4F complex. This study compares the effects of insulin and epidermal growth factor (EGF) on the phosphorylation of 4E-BP1 in fat-cells (followed by gel-shift assays and incorporation of 32P) and on its association with eIF-4E. Several lines of evidence suggest that mitogen-activated protein kinase (MAP kinase) is not involved in these effects of insulin. Insulin causes much more extensive phosphorylation and dissociation of 4E-BP1 from eIF-4E than EGF, although EGF activates MAP kinase to a much greater extent than insulin. Moreover, MAP kinase does not phosphorylate 4E-BP1 when it is complexed with eIF-4E. In contrast, insulin activates the 40S ribosomal protein S6 kinase (p70S6K) 18-fold compared with a 2-fold activation by EGF, and the time course of this activation is similar to the phosphorylation and dissociation of 4E-BP1. Rapamycin, a specific inhibitor of the activation of this latter kinase, inhibits dissociation of 4E-BP1 from eIF-4E in cells incubated with insulin but reveals a phosphorylated form of 4E-BP1 which remains bound to eIF-4E. It is concluded that in rat epididymal fat-cells, the effects of insulin on 4E-BP1 involves multiple phosphorylation events. One phosphorylation event is rapamycin-insensitive, occurs only on bound 4E-BP1 and does not initiate dissociation. The second event does result in dissociation and is blocked by rapamycin, suggesting that the p70S6K signalling pathway is involved: p70S6K itself is probably not involved directly as this kinase does not phosphorylate 4E-BP1 in vitro.
Degradation of aggrecan precursors within a specialized subcompartment of the chicken chondrocyte endoplasmic reticulum
Authors: ALONSO Manuel , HIDALGO Josefina , HENDRICKS Linda , VELASCO Angel
Published: August 2015

Chicken chondrocytes in culture synthesize aggrecan proteoglycan as a 370 kDa precursor that is glycosylated and secreted into the medium with a half-life of 30 min. In metabolic studies the 370 kDa precursor was shown to render a degradation intermediate of 190 kDa, which appeared with no measurable lag phase; it was dependent on temperature (> 20 °C) and inhibited by certain serine and serine/cysteine protease inhibitors such as leupeptin and PMSF. By contrast, degradation was unaffected by treatment of the cells with brefeldin A or with lysosomotropic agents. Aggrecan precursors were detected by immunofluorescence analysis within a subcompartment of the endoplasmic reticulum (ER), previously characterized as a smooth-membrane-bound subregion [Vertel, Velasco, LaFrance, Walters and Kaczman-Daniel (1989) J. Cell Biol. 109, 1827–1836]. Analysis of the subcellular fractions derived from chondrocytes indicated that the degradation intermediate was concentrated in the ER subcompartment. Degradation was dependent on the Ca2+ concentration and the redox state in the ER. Treatment of the cells with agents or conditions that alter the degradation rate of aggrecan precursors, such as protease inhibitors, decreased temperature or dithiothreitol, also modified the retention of these molecules in the ER subcompartment, as seen by immunofluorescence. These results indicate that a fraction of the 370 kDa aggrecan precursor is targeted to a smooth ER subcompartment where it undergoes degradation.
Characterization of the inhibitory effects of bile acids on very-low-density lipoprotein secretion by rat hepatocytes in primary culture
Authors: LIN Yuguang , HAVINGA Rick , SCHIPPERS Ingrid J. , VERKADE Henkjan J. , VONK Roel J. , KUIPERS Folkert
Published: August 2015

In this study the effects of bile acids and other organic anions on the secretion of very-low-density lipoproteins (VLDLs) were evaluated in rat hepatocytes in primary culture. Incubation of cells with portal blood concentrations (10–200 μM) of bile acids resulted in dose-dependent suppression of secretion of VLDL-associated [3H]triglyceride (TG) formed from [3H]glycerol, and also of TG mass. The degree of the inhibition was highly correlated with intracellular bile acid concentration. Prolonged incubation with 100 μM extracellular taurocholic acid (TC) decreased the secretion of [3H]TG and TG mass to 35% and 50% of the controls respectively. Cellular content of mass and of [3H]TG during prolonged incubation with TC were about 20% and 60% higher than the controls respectively. The inhibitory effect remained for at least 24 h in the presence of TC without altering VLDL-lipid and VLDL-apolipoprotein compositions or the size distribution of the particles. Secretion of apoB-100 and of apoB-48 was inhibited to a similar extent. Cells largely lost their capacity to accumulate bile acids intracellularly after 48 h in culture. In these cells TC was unable to exert its suppressive effects. Taurine and glycine conjugates of all common bile acids were capable of suppressing [3H]TG secretion. Trihydroxylated (cholic acid) and various dihydroxylated (deoxycholic, chenodeoxycholic and ursodeoxycholic acids) bile acids had similar capacities in this respect, suggesting that their common sterol-3α-OH structure is required for the suppressive effect. Neither non-bile acid organic anions, e.g. bilirubin ditaurate and dibromosulphthalein, nor dianionic bile acid metabolites, e.g. sulphated taurolithocholic acid and lithocholate-3-O-glucuronide, showed any effect on [3H]TG secretion. These results indicate that bile acids might play a physiological role in regulating VLDL production by the liver, especially in the postprandial state when their enterohepatic circulation is stimulated.
Autoprocessing: an essential step for the activation of HIV-1 protease
Authors: WAN Min , TAKAGI Masahiro , LOH Boon-Nee , XU Xing-Zhi , IMANAKA Tadayuki
Published: August 2015

Human immunodeficiency virus type 1 (HIV-1) expresses its structural and functional proteins within Gag-Pol precursor polyproteins. Specific proteolytic processing of the precursors by the viral protease is critical for the maturation and infectivity of viral particles. To observe the influence of autoprocessing on the activation of recombinant HIV-1 protease, we constructed different HIV-1 protease forms, with or without the Phe-Pro bond directly upstream of the protease domain, and expressed them in Escherichia coli systems. We found that the presence of a short upstream sequence of the protease domain, which could generate the original N-terminus of the protease by autoproteolysis of the Phe-Pro bond, resulted in processing of active protease, whereas for a wild-type protease extended only with the initiator methionine, the proteolytic activity was not recovered. Our results suggested that autoprocessing of the direct upstream sequence of the protease domain is an essential step for the activation of recombinant HIV-1 protease in the E. coli expression system. Expression of HIV-1 protease as fusion proteins revealed that the existence of a fusion portion increased the accumulation of expressed protease by affecting its homotypic dimer formation.
Decreased sensitivity of very-low-density lipoprotein secretion to the inhibitory effect of insulin in cultured hepatocytes from lactating rats
Authors: BOURGEOIS Catherine S. , GIBBONS Geoffrey F.
Published: August 2015

Hepatocytes were prepared from 10–11-day lactating rat dams and from lactating dams which had been weaned for periods of either 1–2 days or 7 days. Hepatocytes from each group were cultured for periods of up to 48 h in a chemically defined medium. Compared with those from the 7-day weaned animals, hepatocytes from the lactating rats were resistant to the inhibitory effects of insulin on the secretion of very-low-density lipoprotein (VLDL) triacylglycerol (TAG). These differences persisted for up to 48 h in culture. Hepatocytes from the 1–2-day weaned animals remained relatively insulin-resistant in this respect. Similar differences in the response to insulin were not observed for the secretion of VLDL apolipoprotein B. TAG production increased and ketogenesis decreased in the hepatocytes from the lactating compared with those from the 7-day weaned rats. Insensitivity of the liver to the normal effects of insulin on the secretion of VLDL TAG may arise from a need to maintain an adequate flux of hepatic lipids to the lactating mammary gland in order to meet the large demand for milk-fat production.
Bile acid stimulation of early growth response gene and mitogen-activated protein kinase is protein kinase C-dependent
Authors: BRADY Lynda M. , BENO David W. A. , DAVIS Bernard H.
Published: August 2015

Hepatic stellate cells are exposed to elevated bile acid levels during hepatic injury and fibrogenesis. Upon activation, the stellate cell becomes a major effector cell during the development of hepatic fibrosis and cirrhosis. Bile acids may function as co-stimulatory signalling molecules. This hypothesis was tested in vitro using rat-derived hepatic stellate cells. Bile acids were studied at concentrations that occur during cirrhosis in vivo. Conjugated and unconjugated bile acids rapidly induced egr and fos gene expression as well as cytoplasmic mitogen-activated protein kinase (MAPK) activation. Protein kinase C was required for both egr induction and MAPK activation. These studies imply that bile acids could contribute to the perpetuation of hepatic fibrosis by helping to keep the stellate cell in an activated state.
Mechanism of bile salt-induced mucin secretion by cultured dog gallbladder epithelial cells
Authors: KLINKSPOOR J. Henriëtte , TYTGAT Guido N. J. , LEE Sum P. , GROEN Albert K.
Published: August 2015

1. Hypersecretion of gallbladder mucin has been proposed to be a pathogenic factor in cholesterol gallstone formation. Using cultured gallbladder epithelial cells, we demonstrated that bile salts regulate mucin secretion by the gallbladder epithelium. In the present study we have investigated whether established second messenger pathways are involved in bile salt-induced mucin secretion. 2. The effect of activators and inhibitors on mucin secretion was studied by measuring the secretion of [3H]N-acetyl-D-glucosamine-labelled glycoproteins. Intracellular cAMP content of the cells was measured using a radioimmunoassay. 3. Incubation of the cells with 10 mM taurocholate did not increase the intracellular cAMP content (25.7 versus control 22.8 pmol of cAMP/mg of protein). No stimulation of mucin secretion was observed after incubation with 1–100 μM concentrations of the calcium ionophores ionomycin and A23187. The stimulatory effect of 10 mM tauroursodeoxycholate (TUDC) on mucin secretion could not be inhibited by the addition of EDTA. Activation of protein kinase C (PKC) by 1 μg/ml phorbol 12-myristate 13-acetate (PMA) caused an increase in mucin secretion (342% versus control 100%), comparable with the effect of 40 mM TUDC. The effect of 10 ng/ml PMA could partially be inhibited by a concentration of 2 μM of the PKC inhibitor staurosporin. Staurosporin had no inhibitory effect on mucin secretion induced by TUDC. 4. In gallbladder epithelial cells bile salts do not stimulate mucin secretion via one of the classical signal transduction pathways. We hypothesize that bile salts act on mucin secretion via a direct interaction with the apical membrane.
Characterization of cell-specific modulatory element in the murine ornithine decarboxylase promoter
Authors: PALVIMO Jorma J. , PARTANEN Maija , JÄNNE Olli A.
Published: August 2015

The promoter of the murine ornithine decarboxylase (ODC) gene contains, adjacent to the TATA box, a cAMP response element (CRE)-like motif that interacts with specific nuclear proteins. Here we examine the role of this CRE-like element (CREL) in ODC promoter activation in proliferating cells. Mutations that abolished binding of nuclear proteins to CREL influenced only marginally the cAMP induction of the reporter constructs driven by 1.6 kb of the ODC promoter. Instead, these mutations altered the basal promoter function in a cell-specific manner, in that they reduced the promoter activity in CV-1 cells, but increased it in NIH/3T3, CHO and HeLa cells. Thus, depending on the cell type, the CREL motif is able to confer either repression or activation on ODC gene transcription. In contrast with 1.6 kb promoter constructs, the same mutations in the context of a shorter sequence (proximal 133 nt) reduced the promoter strength in all cell types studied. The ability of the CREL element to attenuate transcription seems to be connected with the function of some upstream regulatory elements. Differences in nuclear proteins binding to CREL, as studied by electrophoretic mobility shift assays (EMSAs), did not explain the findings on cell-type specificity in transcriptional activation, as mutations in CREL abrogated formation of specific CREL–protein complexes in all cell lines examined. The protein complexes interacting with CREL were not recognized by antibodies specific for CRE-binding proteins CREB-1 and CREB-2, or activating transcription factors ATF-1, ATF-2 and ATF-3. EMSA experiments also demonstrated co-operative interactions between the CREL motif-binding proteins and other nuclear proteins, such as Sp1, interacting with CG-rich sequences of the promoter. In conclusion, the proximal ODC promoter contains a well-conserved regulatory element, which is clearly different from the CRE/ATF element. This motif acts in concert with other distal and proximal elements in a complex cell-specific manner.
The plasma membrane Ca2+ pump mutant lysine591 → arginine retains some activity, but is still inactivated by fluorescein isothiocyanate
Authors: ADAMO Hugo P. , FILOTEO Adelaida G. , PENNISTON John T.
Published: August 2015

Inactivation of the wild-type human plasma membrane Ca2+ pump (isoform 4b) by fluorescein isothiocyanate is accompanied by covalent modification of Lys591. The mutation of Lys591 to arginine reduced the Ca2+ transport activity to 35% of the wild-type, and diminished the amount of acylphosphate formed from ATP by a corresponding amount. When this mutant was treated with fluorescein isothiocyanate, the enzyme was still irreversibly inactivated, even though no reactive residue was available at position 591. The results show that, although Ca2+ pump function is sensitive to the residue at position 591, Lys591 is not essential for enzyme activity. They also demonstrate that irreversible inhibition of the plasma membrane Ca2+ pump by fluorescein isothiocyanate does not require the covalent modification of Lys591. This indicates that fluorescein isothiocyanate reacts with lysine residues at other positions in addition to Lys591.