bluuebunny/crossref_metadata_2025 huggingface dataset:
Cloning and sequencing of rat liver carboxylesterase ES-4 (microsomal palmitoyl-CoA hydrolase)
Authors: ROBBI Mariette , VAN SCHAFTINGEN Emile , BEAUFAY Henri
Published: August 2015

A cDNA which encodes a carboxylesterase of 561 amino acid residues including a cleavable signal peptide is described. The enzyme expressed in COS cells migrates during PAGE (SDS-, and non-denaturing) as a single prominent band in the region of liver ES-4. It ends in the C-terminal cell-retention signal -HNEL, which, in COS cells overexpressing the enzyme, appears to be slightly less efficient than the signals -HTEL and -HVEL of ES-3 and ES-10 respectively. Glycosylation is not essential for intracellular retention, but leads to a higher activity. As do many carboxylesterases, the enzyme expressed in COS cells hydrolyses o-nitrophenyl acetate and α-naphthyl acetate. It also hydrolyses acetanilide, although less efficiently than ES-3, and, distinctively, palmitoyl-CoA. In addition to the four canonical Cys residues of the carboxylesterases, it contains a fifth, unpaired Cys336, which apparently is not essential for the catalytic properties. Indeed, treatment with iodoacetamide or substitution of Cys336 by Phe does not markedly alter the activity of the enzyme on the various substrates. The predicted structure of ES-4 is highly homologous to that of two other recently cloned esterases which also end in -HNEL [Yan, Yang, Brady and Parkinson (1994) J. Biol. Chem. 269, 29688–29696; Yan, Yang and Parkinson (1995) Arch. Biochem. Biophys. 317, 222–234]. Together, these isoenzymes probably account for the closely spaced bands observed in the region of ES-4 in non-denaturing PAGE.
Surfactant-associated protein A is important for maintaining surfactant large-aggregate forms during surface-area cycling
Authors: VELDHUIZEN Ruud A. W. , YAO Li-Juan , HEARN Stephen A. , POSSMAYER Fred , LEWIS James F.
Published: August 2015

Alveolar surfactant can be separated into two major subfractions, the large surfactant aggregates (LAs) and the small surfactant aggregates (SAs). The surface-active LAs are the metabolic precursors of the inactive SAs. This conversion of LAs into SAs can be studied in vitro using a technique called surface-area cycling. We have utilized this technique to examine the effect of trypsin on aggregate conversion. Our results show that trypsin increases the conversion of LAs into SAs in a concentration- and time-dependent manner. Immunoblot analysis revealed that surfactant-associated Protein A (SP-A) was the main target of trypsin. To examine further the role of SP-A in aggregate conversion, we tested the effect of Ca2+ and mannan on this process. The absence of Ca2+ (1 mM EDTA) and the presence of mannan both increased the formation of SAs. Electron microscopy revealed that highly organized multilamellar and tubular myelin structures were present in samples that converted slowly to SAs. We concluded that SP-A is important for maintaining LA forms during surface-area cycling by stabilizing tubular myelin and multilamellar structures.
Effect of channelling on the concentration of bulk-phase intermediates as cytosolic proteins become more concentrated
Authors: KHOLODENKO Boris N. , WESTERHOFF Hans V. , CASCANTE CASCANTE
Published: August 2015

This paper shows that metabolic channelling can provide a mechanism for decreasing the concentration of metabolites in the cytoplasm when cytosolic proteins become more concentrated. A metabolic pathway in which two sequential enzymes can form a dynamic complex catalysing the direct transfer of an intermediate is compared with the analogous pathway lacking a channel (an ‘ideal’ pathway). In an ideal pathway a proportional increase in protein content does not result in a change in the steady-state concentration of the bulk-phase intermediate, whereas in a channelling pathway the bulk-phase intermediate either decreases or increases depending on the elemental rate constants within the enzyme mechanisms. When the concentrations of the enzymes are equal, the pool size decreases with increasing protein concentration if the elemental step depleting the bulk-phase intermediate exerts more control on its concentration than the step supplying the intermediate. Results are illustrated numerically, and a simplified dynamic channel is analysed in which the concentration of the enzyme–enzyme complex is negligible compared with the monomeric enzyme forms. For such a ‘hit-and-run’ channel it is shown that, when the product-releasing step of the enzyme located upstream is close to equilibrium, the pool size decreases as the concentrations of the enzymes increase in proportion, regardless of the rate, equilibrium constants and concentration ratios of the two sequential enzymes.
The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes
Authors: STANDAERT Mary L. , AVIGNON Antoine , YAMADA Kouji , BANDYOPADHYAY Gautam , FARESE Robert V.
Published: August 2015

We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-α and PKC-β in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulated, phospholipase D-dependent PC hydrolysis and subsequent translocation of PKC-α and PKC-β to the plasma membrane. Wortmannin did not inhibit PKC directly in vitro, or the PKC-dependent effects of phorbol esters on glucose transport in intact adipocytes. The PKC inhibitor RO 31-8220 did not inhibit PI 3-kinase directly or its activation in situ by insulin, but inhibited both insulin-stimulated and phorbol ester-stimulated glucose transport. Our findings suggest that insulin acts through PI 3-kinase to activate a PC-specific phospholipase D and causes the translocative activation of PKC-α and PKC-β in plasma membranes of rat adipocytes.
Characterization of Saccharomyces cerevisiae deficient in expression of phospholipase D
Authors: ELLA Krishna M. , DOLAN Joseph W. , QI Chen , MEIER Kathryn E.
Published: August 2015

A gene encoding phospholipase D (PLD) in Saccharomyces cerevisiae was identified. The 195 kDa product of PLD1 has 24% overall sequence identity with a plant PLD. Expression of yeast PLD activity was eliminated by one-step gene disruption. Yeast haploids lacking PLD activity were deficient in growth on non-fermentable carbon sources. Diploids lacking expression of PLD1 were unable to sporulate.
Effects of nucleotide substitutions within the T-loop of precursor tRNAs on interaction with ATP/CTP:tRNA nucleotidyltransferases from Escherichia coli and yeast
Authors: LI Zengji , GILLIS Kimberly A. , HEGG Lisa A. , ZHANG Jianchao , THURLOW David L.
Published: August 2015

Recognition of tRNA and tRNA-like substrates by the enzyme ATP/CTP:tRNA nucleotidyltransferase requires chemically intact nucleotides within the T-loop, especially at positions 57 and 58, which are invariant purines among naturally occurring tRNAs. To test the effects of base substitutions at these positions, which are distant from the site of catalysis, we synthesized mutant tRNAGlu molecules. These in vitro-synthesized RNAs also contained an extra 33 bases at the 5´ end and lacked post-transcriptionally modified bases. The precursor tRNAs were used as substrates for nucleotidyltransferases from Escherichia coli and yeast. Substitution of cytidines at either position 57 or 58 had dramatic inhibitory effects on recognition by both enzymes, including raising the apparent Km and lowering the apparent Vmax.; substitution of an adenosine at position 57 or a uridine at position 58 inhibited the reaction only slightly by comparison. Our results demonstrate that the identities of nucleotides at positions 57 and 58 are relevant to recognition by nucleotidyltransferase, and that a purine is required at position 57. The extra bases at the 5´ end and the lack of post-transcriptionally modified bases did not substantially inhibit interaction with the enzyme, as judged by the wild-type precursor tRNAGlu acting as an effective substrate for both enzymes in the presence of equal concentrations of appropriate tRNA substrates isolated from E. coli.
Overproduction, purification and characterization of M.EcoHK31I, a bacterial methyltransferase with two polypeptides
Authors: LEE Kai-Fai , LIAW Yen-Chywan , SHAW Pang-Chui
Published: August 2015

The two overlapping genes coding for EcoHK31I methyltransferase have previously been cloned, sequenced and expressed [Lee, Kam and Shaw (1995) Nucleic Acids Res. 23, 103–108]. Here we describe protocols developed to purify polypeptides α and β together or separately, to apparent homogeneity by various chromatographic media. M.EcoHK31I is a heterodimer with a native molecular mass of 61 kDa. Its specific activity towards non-methylated lambda DNA was 3.0×105 units per mg of protein. The respective denatured molecular masses of polypeptides α and β were 38 and 23 kDa, and their pI values were 8.7 and 6.8. Initial rate kinetic parameters of the native enzyme were 2.0 nM, 0.58 μM and 3 min-1 for KmDNA, KmAdoMet and kcat. respectively, where AdoMet stands for S-adenosyl-L-methionine. Fully active enzyme was reconstituted by co-purifying the two separately synthesized polypeptides, and activity assays confirmed our previous finding that two polypeptides were needed to methylate substrate DNA.
Purification and characterization of oil-bodies (oleosomes) and oil-body boundary proteins (oleosins) from the developing cotyledons of sunflower (Helianthus annuus L.)
Authors: MILLICHIP Mark , TATHAM Arthur S. , JACKSON Frances , GRIFFITHS Gareth , SHEWRY Peter R. , STOBART A. Keith
Published: August 2015

Oil-bodies, from the immature cotyledons of sunflower (Helianthus annuus L.), were difficult to purify to homogeneity using conventional techniques. The major protein contaminants were albumin and globulin storage proteins. A protocol has been developed, therefore, based upon the stringent washing of the oil-body fraction in 9 M urea, which effectively removed almost all the contaminating protein as judged by SDS/PAGE. The urea-washed oil-bodies were enriched in two major proteins of Mr 19000 and 20000. These proteins were oleosins as demonstrated by their amino acid compositions and the sequence analysis of peptides produced by CNBr cleavage. Far-UV CD spectra of the oleosins in trifluoroethanol, trifluoroethanol/water mixtures and as mixed micelles in SDS, were typical of α-helical proteins with α-helical contents of some 55%. The phospholipid content of the urea-washed preparations was less than 0.1% of that required to form a half-unit membrane surrounding the oil-body. The oil-body surface therefore appears to be an unusual and novel structure, covered largely by an oleosin protein coat or pellicle rather than a conventional fluid membrane, half-unit or otherwise.
Extracellular calcium concentration controls the frequency of intracellular calcium spiking independently of inositol 1,4,5-trisphosphate production in HeLa cells
Authors: BOOTMAN M. D. , YOUNG K. W. , YOUNG J. M. , MORETON R. B. , BERRIDGE M. J.
Published: August 2015

Stimulation of single HeLa cells with histamine evoked repetitive increases of the intracellular calcium ion concentration (Ca2+ spikes). The frequency of Ca2+ spiking increased as the extracellular hormone concentration was elevated. In addition, the frequency of Ca2+ spiking could be accelerated by increasing the extracellular Ca2+ concentration ([Ca2+]o) in the presence of a constant hormone concentration. The range of [Ca2+]o over which the spiking frequency could be titrated was nominally-zero to 10 mM, being half-maximally effective at approx. 1 and 2.5 mM for 37 and 22 °C respectively. The effect of [Ca2+]o on inositol phosphates production was also examined. Changes of [Ca2+]o over a range which had been found to affect the frequency of Ca2+ spiking did not have any effect on the rate of myo-inositol 1,4,5-trisphosphate (InsP3) production, although an increase in inositol phosphates production was observed as [Ca2+]o was increased from zero to values giving less than half-maximal Ca2+ spike frequency. These data suggest that at low Ca2+ spike frequency, Ca2+-stimulated activation of phospholipase C may contribute to Ca2+ spiking in HeLa cells, but under some conditions the availability of Ca2+ to the intracellular stores, rather than changes in the rate of InsP3 production, determines the Ca2+ spike frequency.
Activation of a protein tyrosine phosphatase and inactivation of Raf-1 by somatostatin
Authors: REARDON Dean B. , WOOD Steven L. , BRAUTIGAN David L. , BELL Graeme I. , DENT Paul , STURGILL Thomas W.
Published: August 2015

Human somatostatin receptor 3 (‘hsstr3’) was transiently expressed in NIH 3T3 cells stably transformed with Ha-Ras (G12V). Somatostatin activated a protein tyrosine phosphatase, and inactivated the constitutively active, membrane-associated form of the Raf-1 serine kinase present in these cells in vivo and in vitro.
Characterization of sheep lacrimal-gland peroxidase and its major physiological electron donor
Authors: MAZUMDAR Abhijit , CHATTERJEE Ratna , ADAK Subrata , GHOSH Anil , MONDAL Chhabinath , BANERJEE Ranajit K.
Published: August 2015

A soluble sheep lacrimal-gland peroxidase was purified to apparent homogeneity. It had a native molecular mass of 75 kDa with a subunit molecular mass of 82 kDa and an isoelectric point of 6.5. Western blotting showed that it shares some of the enzyme antigenic determinants in common with other soluble peroxidases. The enzyme exhibits a Soret peak at 410 nm which is shifted to 431 nm by 5 equiv. of H2O2 due to the formation of compound II. The latter is, however, unstable and gradually returns to the native state. The enzyme forms complexes with CN- and N3- and is reduced by dithionite showing a characteristic reduced peroxidase spectrum. Although the enzyme oxidizes I-, SCN- and Br- optimally at pH 5.5, 5.25 and 5.0 respectively, at physiological pH, it oxidizes I- and SCN- only. Since extracellular SCN- concentration is much higher than I-, SCN- may act as the major electron donor to the enzyme. The second-order rate constants for the reaction of the enzyme with H2O2 (k+1) and of compound I with SCN- (k+2) were 4×107 M-1·s-1 and 8.1×105 M-1·s-1 respectively. A plot of log Vmax. against pH yields a sigmoidal curve consistent with a single ionizable group on the enzyme with a pKa value of 5.75, controlling thiocyanate oxidation. In a coupled system with the peroxidase, H2O2, SCN-, GSH, NADPH and glutathione reductase, peroxidase-catalysed SCN- oxidation by H2O2 could be coupled to NADPH consumption. The system is proposed to operate in vivo for the efficient elimination of endogenous H2O2.
Noradrenaline increases glucose transport into brown adipocytes in culture by a mechanism different from that of insulin
Authors: SHIMIZU Yasutake , KIELAR Danuta , MINOKOSHI Yasuhiko , SHIMAZU Takashi
Published: August 2015

Glucose uptake into brown adipose tissue has been shown to be enhanced directly by noradrenaline (norepinephrine) released from sympathetic nerves. In this study we characterized the glucose transport system in cultured brown adipocytes, which responds to noradrenaline as well as insulin, and analysed the mechanism underlying the noradrenaline-induced increase in glucose transport. Insulin increased 2-deoxyglucose (dGlc) uptake progressively at concentrations from 10-11 to 10-6 M, with maximal stimulation at 10-7 M. Noradrenaline concentrations ranging from 10-8 to 10-6 M also enhanced dGlc uptake, even in the absence of insulin. The effects of noradrenaline and insulin on dGlc uptake were additive. The stimulatory effect of noradrenaline was mimicked by the β3-adrenergic agonist, BRL37344, at concentrations two orders lower than noradrenaline. Dibutyryl cyclic AMP also mimicked the stimulatory effect of noradrenaline, and the antagonist of cyclic AMP, cyclic AMP-S Rp-isomer, blocked the enhancement of glucose uptake due to noradrenaline. Furthermore Western blot analysis with an anti-phosphotyrosine antibody revealed that, in contrast with insulin, noradrenaline apparently does not stimulate intracellular phosphorylation of tyrosine, suggesting that the noradrenaline-induced increase in dGlc uptake depends on elevation of the intracellular cyclic AMP level and not on the signal chain common to insulin. When cells were incubated with insulin, the content of the muscle/adipocyte type of glucose transporter (GLUT4) in the plasma membrane increased, with a corresponding decrease in the amount in the microsomal membrane. In contrast, noradrenaline did not affect the subcellular distribution of GLUT4 or that of the HepG2/erythrocyte type of glucose transporter. Although insulin increased Vmax. and decreased the Km value for glucose uptake, the effect of noradrenaline was restricted to a pronounced decrease in Km. These results suggest that the mechanism by which noradrenaline stimulates glucose transport into brown adipocytes is not due to translocation of GLUT but is probably due to an increase in the intrinsic activity of GLUT, which is mediated by a cyclic AMP-dependent pathway.
P2-purigenic receptors regulate phospholipase C and adenylate cyclase activities in immortalized Schwann cells
Authors: BERTI-MATTERA Liliana N. , WILKINS Pamela L. , MADHUN Zuhayr , SUCHOVSKY Deborah
Published: August 2015

Schwann cells play an important role in both the development and regeneration of peripheral nerves. Proliferation and differentiation of Schwann cells are critically dependent on changes in the levels of cAMP. ATP is a fast excitatory transmitter in the peripheral nervous system, inducing depolarization of the vagus nerve through occupancy of P2-purinergic receptors. In the present study we demonstrate that extracellular ATP stimulates phospholipase C and inhibits adenylate cyclase activities in cultured Schwann cells. Addition of ATP inhibited, in a concentration-dependent manner, forskolin- or isoprenaline-stimulated adenylate cyclase activity. The rank order of potency corresponding to different purinergic receptor agonists was 2 - methylthio-ATP > ATP = ADP ⩾ adenosin eFS685´-[γ-thio]triphosphate (ATP[S]) > UTP, consistent with the involvement of a P2y subtype. Adenosine and adenosine 5´-[α,β-methylene]triphosphate (pp[CH2pA) were ineffective. Preincubation with pertussis toxin completely blocked this inhibitory effect. When Schwann cells were pre-labelled with myo-[3H]inositol and incubated in Hanks' balanced salt solution containing Ca2+ and Mg2+, addition of ATP[S] resulted in a concentration-dependent increase in the release of InsP with a concomitant increase in intracellular free [Ca2+] ([Ca2+]i). Under these conditions, the effects of both ATP and UTP were of lower magnitude. Removal of Ca2+ and Mg2+ from the assay medium resulted in a significant increase in the effects of ATP[S], ATP and UTP. The decreased response observed in the presence of both bivalent cations (1.2 mM Ca2+ and 1 mMMg2+) could not be explained either by increased degradation of ATP by Ca2+/Mg2+-dependent nucleotidases or by cation influx. The rank order of potency for the effects of agonists on phospholipase C activity was ATP[S] = adenosine 5´-[γ-imido]triphosphate > ATP = UTP > ADP, indicating the involvement of a P2u receptor subtype in this response. Adenosine, AMP and pp[CH2]pA were ineffective. These results demonstrate that immortalized Schwann cells express P2u and P2y purinoceptors, which are coupled to stimulation of phospholipase C and inhibition of adenylate cyclase, respectively. Our observations unveil signal-transduction pathways that may be used by ATP to regulate proliferation and differentiation of Schwann cells, and ultimately to influence nerve homoeostasis.
Thapsigargin activates univalent- and bivalent-cation entry in human neutrophils by a SK&F I3 96365- and Gd3+-sensitive pathway and is a partial secretagogue: involvement of pertussis-toxin-sensitive G-proteins and protein phosphatases 1/2A and 2B in the signal-transduction pathway
Authors: WENZEL-SEIFERT Katharina , KRAUTWURST Dietmar , MUSGRAVE Ian , SEIFERT Roland
Published: August 2015

The Ca2+-ATPase inhibitor thapsigargin (TG) activates bivalent-cation entry in human neutrophils via depletion of intracellular Ca2+ stores, but little is known about the underlying mechanism and the functional role of TG-induced cation entry. We studied the effects of TG on univalent- and bivalent-cation entry, lysozyme release and superoxide-anion (O2-) formation in human neutrophils. TG, like the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), stimulated entry of Ca2+, Mn2+, Ba2+, Sr2+ and Na+ in a 1-{β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SK&F 96365)- and Gd3+-sensitive manner. The inhibitors of protein phosphatases 1/2A, calyculin A and okadaic acid, diminished TG-induced cation influxes, whereas the inhibitors of protein phosphatase 2B, cyclosporin A and FK-506, were potentiators. Pertussis toxin (PTX) partially inhibited the effects of TG on Ca2+ and Mn2+ entry. TG and fMLP activated inward currents with a linear current–voltage relationship and a reversal potential at about 0 mV. TG activated lysozyme release and potentiated fMLP-induced O2- formation. TG-induced lysozyme release was inhibited by SK&F 96365, PTX and the removal of extracellular Ca2+ or Na+. Our data show that TG activates a non-selective and SK&F 96365- and Gd3+-sensitive cation entry pathway and is a partial secretagogue. TG-stimulated cation entry involves PTX-sensitive G-proteins and protein phosphatases, with protein phosphatases 1/2A and 2B playing opposite roles.
Formation of secretory vesicles in permeabilized cells: a salt extract from yeast membranes promotes budding of nascent secretory vesicles from the trans-Golgi network of endocrine cells
Authors: LING Wai Lam W. , SHIELDS Dennis
Published: August 2015

The mechanism of secretory-vesicle formation from the trans-Golgi network (TGN) of endocrine cells is poorly understood. To identify cytosolic activities that facilitate the formation and fission of nascent secretory vesicles, we treated permeabilized pituitary GH3 cells with high salt to remove endogenous budding factors. Using this cell preparation, secretory-vesicle budding from the TGN required addition of exogenous cytosol and energy. Mammalian cytosols (GH3 cells and bovine brain) promoted post-TGN vesicle formation. Most significantly, a salt extract of membranes from the yeast Saccharomyces cerevisiae, a cell lacking a regulated secretory pathway, stimulated secretory vesicle budding in the absence of mammalian cytosolic factors. These results demonstrate that the factors which promote secretory-vesicle release from the TGN are conserved between yeast and mammalian cells.
Rapid reduction and removal of HDL- but not LDL-associated cholesteryl ester hydroperoxides by rat liver perfused in situ
Authors: CHRISTISON Julie , KARJALAINEN Ari , BRAUMAN Julie , BYGRAVE Fyfe , STOCKER Roland
Published: August 2015

To test whether high-density lipoproteins (HDL) could aid in the removal in vivo of potentially atherogenic oxidized lipids, we perfused rat liver in situ with buffer supplemented with isolated human HDL containing small amounts of cholesteryl linoleate hydro(pero)xides [Ch18:2-O(O)H]. Perfusion resulted in the rapid removal of Ch18:2-O(O)H from HDL with a half-life (t½) of 11.4 min, faster than that of unoxidized cholesteryl linoleate, and dependent on the presence of the liver. In addition, the liver enhanced the reduction of Ch18:2-OOH associated with HDL remaining in the perfusate buffer. Perfusion resulted in the release of a hepatic activity that enhanced the reduction of HDL-associated Ch18:2-OOH and was resistant to heat treatment. In contrast with the situation with HDL, low-density lipoprotein (LDL)-associated Ch18:2-O(O)H were neither removed nor reduced by perfused rat liver within the time course studied, in support of a possible role for HDL in the detoxification of circulating lipid hydroperoxides in vivo.
Fibrates induce mdr2 gene expression and biliary phospholipid secretion in the mouse
Authors: CHIANALE José , VOLLRATH Valeska , WIELANDT Ana M. , AMIGO Ludwig , RIGOTTI Attilio , NERVI Flavio , GONZALEZ Sergio , ANDRADE Leonardo , PIZARRO Margarita , ACCATINO Luigi
Published: August 2015

Disruption of the murine mdr2 gene leads to the complete absence of biliary phospholipids. We tested the hypothesis that the increase in biliary phospholipid output induced by fibrates is mediated via induction of the hepatic mdr2 gene and its encoded product, the P-glycoprotein canalicular flippase. Increased levels of mdr2 mRNA were observed in the liver of mice treated with different fibrates: ciprofibrate, 660±155% (as compared with control group); clofibrate, 611±77%; bezafibrate, 410±47%; fenofibrate, 310±52%; gemfibrozil, 190±25% (P < 0.05 compared with control group). Induction of expression of the mdr gene family was specific to the mdr2 gene. Two- to three-fold increases in P-glycoprotein immunodetection were evident on the canalicular plasma-membrane domain of clofibrate- and ciprofibrate-treated mice. Biliary phospholipid output increased from 4.2±1.2 nmol/min per g of liver in the control group to 8.5±0.6, 7.1±2.9 and 5.8±2.5 in ciprofibrate-, clofibrate- and bezafibrate-treated mice respectively (P < 0.05 compared with control group). Moreover, a significant correlation between biliary phospholipid output and the relative levels of mdr2 mRNA was found (r = 0.86; P < 0.05). In treated animals, bile flow as well as cholesterol and bile acid outputs remained unchanged. Our findings constitute the first evidence that pharmacological modulation of biliary lipid secretion mediated by fibrates can be related to the overexpression of a specific liver gene product, the mdr2 P-glycoprotein, and are consistent with the hypothesis that the mdr2 P-glycoprotein isoform plays a crucial role in the secretion of biliary phospholipid.
Regulation of phosphatidylcholine synthesis in maturing type II cells: increased mRNA stability of CTP:phosphocholine cytidylyltransferase
Authors: HOGAN Martina , KULISZEWSKI Maciej , LEE Wensu , POST Martin
Published: August 2015

Pulmonary surfactant phosphatidylcholine synthesis increases in fetal lung type II cells with advancing gestation. This increase is accompanied by an increase in gene and protein expression of CTP:phosphocholine cytidylyltransferase (CT; EC 2.7.7.15), which catalyses a regulatory step in de novo phosphatidylcholine synthesis by fetal type II cells. In the present study we investigated the role of transcriptional and post-transcriptional mechanisms in the developmental induction of CT mRNA in maturing type II cells. We found that CT mRNA increased 2-fold from days 18 to 21 of fetal rat gestation (term 22 d). This increase in CT mRNA was not accompanied by a developmental increase in CT gene transcription. However, CT mRNA was more stable on day 21 (t½ 48 h) compared with that on day 18 (t½ 17 h). Glucocorticoids have been shown to enhance surfactant phosphatidylcholine synthesis in fetal type II cells. Therefore we also examined the effect of maternal glucocorticoid administration to pregnant rats at 19 d of gestation on CT mRNA expression in fetal type II cells isolated 24 h later. Glucocorticoid treatment did not increase type II cell CT mRNA. As reported previously, however, glucocorticoids increased CT activity in the microsomal membrane fraction of fetal type II cells, whereas no differences in cytosolic CT activity were observed. We conclude that the developmental increase in CT mRNA in fetal type II cells is due to a decreased breakdown of the CT transcript and that glucocorticoids regulate fetal type II cell CT activity at a post-translational level.
Studies on lipids and the activity of Na,K-ATPase in lens fibre cells
Authors: DEAN William L. , DELAMERE Nicholas A. , BORCHMAN Douglas , MOSELEY Amy E. , AHUJA Rajesh P.
Published: August 2015

Na,K-ATPase was studied in the two cell types that make up the lens of the eye. Membrane material was isolated from lens fibre cells, which make up the bulk of the lens cell mass, and also from lens epithelial cells, which are present only as a monolayer on the anterior lens surface. Judged by immunoblotting, greater amounts of Na,K-ATPase α1 and β1 polypeptides were found in fibre cell membrane material than in epithelial cell membrane material. However, the Na,K-ATPase activity in epithelial cell membrane material was 20 times that measured in fibre cell membrane material. In 86Rb uptake experiments with intact lenses, ouabain-inhibitable 86Rb uptake was observed for lens epithelium but not for lens fibres. These findings are consistent with a low Na,K-ATPase activity in lens fibre cells even though these cells express a considerable amount of Na,K-ATPase α1 and β1 polypeptides. The lipid composition of lens fibre cell membranes causes them to be more ordered than epithelial cell membranes; this was confirmed by measurements of the infrared CH2 symmetric stretching band frequency. Because lipid composition can influence Na,K-ATPase activity, experiments were conducted to determine whether the activity of Na,K-ATPase α1β1 is inhibited by lens fibre lipid. However, no significant difference in Na,K-ATPase activity was detected when Na,K-ATPase α1β1 was purified from rabbit kidney and then reconstituted with lipid that had been isolated from either lens epithelium or lens fibre cells. These studies indicate that lens fibre cells contain both Na,K-ATPase α1 and β1 polypeptides but have low Na,K-ATPase activity. However, the results do not support the notion that this is due to the lipid composition of lens fibre cell membranes.
Cloning and expression of a cDNA encoding a mouse brain orphanin FQ/nociceptin precursor
Authors: PAN Ying-Xian , XU Jin , PASTERNAK Gavril W.
Published: August 2015

By using a reverse transcription–PCR approach we have cloned a peptide precursor from mouse brain which contains the sequence of orphanin FQ/nociceptin. The mouse sequence of orphanin FQ/nociceptin is identical at the amino acid level with that isolated from rat and porcine brain. Northern analysis of the mRNA encoding the precursor reveals a single band of approx. 1 kb, with the highest levels in the brain and much lower levels in kidney and spleen. Southern analysis is consistent with a single gene. The precursor peptide from mouse contains two other putative peptides. Upstream from the orphanin FQ/nociceptin is a 41-amino-acid peptide which is almost identical, except for a six-amino-acid insertion, with the corresponding 35-amino-acid peptide predicted from the rat sequence. Interestingly, the mouse contains a triple AEPGAD repeat within this peptide that is not seen in the rat sequence. Downstream from the orphanin FQ/nociceptin sequence is another 17-amino-acid peptide which is identical with that found in the rat.
Ligand binding kinetics and dissociation of the human embryonic haemoglobins
Authors: HOFMANN Oliver , BRITTAIN Thomas
Published: August 2015

The three human embryonic haemoglobins have been studied using a range of stopped-flow and flash photolysis experiments. The association and dissociation kinetics and equilibrium constants for the tetramer–dimer reactions of the deoxy and oxygenated forms have been investigated and found to be characterized by constants similar to those of the human adult protein. The rates of oxygen dissociation from the embryonic haemoglobins have been measured and appear to be responsible for the high oxygen-binding affinity associated with the embryonic proteins compared with the adult protein. The pH dependence of the oxygen dissociation rate constants also accounts for the rather unusual, previously described, Bohr effects characteristic of the embryonic haemoglobins. A general scheme has been developed coupling both the dimer–tetramer equilibria and ligand-binding steps observed following photolysis of the liganded forms of the human embryonic haemoglobins.
Tissue Factor residue Asp44 regulates catalytic function of the bound proteinase Factor VIIa
Authors: KELLY Curtis R. , SCHULLEK John R. , RUF Wolfram , EDGINGTON Thomas S.
Published: August 2015

The coagulation pathways are initiated by the cell-surface receptor Tissue Factor (TF), which binds the serine proteinase coagulation Factor VIIa (VIIa), resulting in enhanced catalytic function, both amidolytic, towards small pseudo-substrates, and proteolytic, towards macromolecular substrates. Here we implicate Asp44 in TF as a ligand-interactive residue that, in contrast with previously characterized binding residues, is involved in the enhancement of VIIa catalytic function. Whereas charge neutralization by replacement of Asp44 with Asn did not reduce function of human TF, the exchange by Ala resulted in mutants with 8-fold reduced affinity for binding of VIIa. Enhancement of VIIa amidolytic function by TF Ala44 was reduced by 20–25% relative to wild-type and support of proteolytic function was diminished 6-fold indicating that this cofactor residue is significantly enhancing proteolysis of the macromolecular substrate by VIIa. Replacement of Asp44 by Glu, Thr and Arg exhibited a less severe phenotype with an approx. 4-fold reduced affinity for VIIa and a 2–3-fold diminished activation of Factor X. The improved activity of these mutants as compared with the Ala replacement is consistent with functional importance of an extended side chain at this position. The specific influence of the Asp44 exchange on catalytic function of the TF·VIIa complex indicates fine specificity of the TF ligand interface in mediating receptor and cofactor function.
High-glucose incubation of human umbilical-vein endothelial cells does not alter expression and function either of G-protein α-subunits or of endothelial NO synthase
Authors: MANCUSI Gudrun , HUTTER Caroline , BAUMGARTNER-PARZER Sabina , SCHMIDT Kurt , SCHÜTZ Wolfgang , SEXL Veronika
Published: August 2015

Alterations in G-protein-controlled signalling pathways (primarily pathways controlled by Gs and Gi) have been reported to occur in animal models of diabetes mellitus. We have therefore studied the effect of a long-term exposure of human umbilical vein endothelial cells to elevated concentrations of glucose on expression and function of G-protein subunits and endothelial NO synthase. Long-term incubation in high glucose (30 mM for 15 days) did not affect the levels of Giα-2, Gqα, the splice variants (long and short form) of Gsα, and the G-protein β-subunits or adenylate cyclase activity: basal, as well as isoprenaline-, forskolin- and guanosine 5´-[γ-thio]triphosphate-stimulated enzyme activities were comparable in high- and low-glucose-treated cells, thus ruling out any functional changes in the stimulatory pathway. Pretreatment of endothelial cells with pertussis toxin blocked a substantial fraction (50%) of the mitogenic response to serum factor(s) which depend(s) on functional Gi2. The sensitivity of cells cultured in high glucose was comparable with that of the paired controls maintained in normal glucose (EC50 = 3.1±0.5 and 3.3±0.4 ng/ml respectively). Similarly, we failed to detect any differences in endothelial NO synthase expression, or intracellular distribution and basal activity of the enzyme in endothelial cells cultured in high glucose. Stimulation of NO synthase in intact cells revealed a comparable response to the calcium ionophore (A23187). In contrast, stimulation with histamine (which acts via H1-receptors predominantly coupled to Gq) resulted in a significantly increased response in the cells maintained in high glucose. These data are suggestive of an altered H1-histamine receptor–Gq–phospholipase C pathway in endothelial cells cultured in high glucose concentrations, but rule out any glucose-induced functional changes in Gs- and Gi-controlled signalling pathways.
Rapid reduction of nitric oxide by mitochondria, and reversible inhibition of mitochondrial respiration by nitric oxide
Authors: BORUTAITÉ Vilmanté , BROWN Guy C.
Published: August 2015

Nitric oxide (NO) inhibited the respiration rate of mitochondria isolated from rat heart at sub-micromolar concentrations of NO. The inhibition was rapidly and completely reversible, indicating that NO does not damage mitochondria. The sensitivity of respiration to NO depended on the oxygen concentration, substrate type and respiratory state of the mitochondria, consistent with NO competing with oxygen at cytochrome oxidase. Mitochondria catalysed a rapid rate of NO breakdown, which was greater in the absence of oxygen and was partly inhibited by cyanide and azide, suggesting that at least part of the NO breakdown was due to reduction of NO by cytochrome oxidase. The rapid rate of this breakdown suggests that mitochondrial breakdown of NO may be significant physiologically.
Peptide models for membrane channels
Authors: MARSH Derek
Published: August 2015

Peptides may be synthesized with sequences corresponding to putative transmembrane domains and/or pore-lining regions that are deduced from the primary structures of ion channel proteins. These can then be incorporated into lipid bilayer membranes for structural and functional studies. In addition to the ability to invoke ion channel activity, critical issues are the secondary structures adopted and the mode of assembly of these short transmembrane peptides in the reconstituted systems. The present review concentrates on results obtained with peptides from ligand-gated and voltage-gated ion channels, as well as proton-conducting channels. These are considered within the context of current molecular models and the limited data available on the structure of native ion channels and natural channel-forming peptides.
Streptozotocin-induced diabetes elicits the phosphorylation of hepatocyte Gi2α at the protein kinase C site but not at the protein kinase A-controlled site
Authors: MORRIS Nicholas J. , BUSHFIELD Mark , HOUSLAY Miles D.
Published: August 2015

Streptozotocin-induced diabetes caused a profound increase in the steady-state level of phosphorylation of the α-subunit of the adenylate cyclase inhibitory protein Gi2 in hepatocytes. Unlike hepatocytes from control animals, those from streptozotocin-diabetic animals showed no increase in the phosphorylation of Gi2α in response to a challenge with the protein kinase C activator phorbol myristate acetate. However, a stimulatory effect of 8-bromo-cAMP on Gi2α phosphorylation was evident in hepatocytes from diabetic animals but this was severely reduced compared with that observed in hepatocytes from normal animals. Two-dimensional tryptic phosphopeptide mapping showed that Gi2α in resting hepatocytes from diabetic animals was phosphorylated exclusively at the protein kinase C site (C-site) but no labelling was evident at the protein kinase A-regulated site (AN-site). Treatment of hepatocytes from diabetic animals with phorbol myristate acetate did not change this pattern of labelling. In contrast, challenge of hepatocytes from diabetic animals with 8-bromo-cAMP led to the appearance of a new labelled phosphopeptide that was consistent with labelling at the AN-site. Analysis of the C-site and AN-site phosphopeptides from hepatocytes of diabetic animals treated with 8-bromo-cAMP showed that the increase in labelling of Gi2α caused by this ligand could be attributed almost entirely to labelling at the AN-site. Thus streptozotocin diabetes appears to cause enhanced labelling of hepatocyte Gi2α by exclusively increasing phosphorylation at the C-site. It is suggested that the increased labelling at the C-site reflects an augmentation of the protein kinase C signalling system in hepatocytes from streptozotocin-induced diabetic animals. This may have widespread functional consequences for these cells and may result either from an increased protein kinase C activity and/or a reduction in protein phosphatase 1 and/or 2A activity.
Glycogen synthesis in amphibian oocytes: evidence for an indirect pathway
Authors: KESSI Eduardo , GUIXÉ Victoria , PRELLER Ana , URETA URETA
Published: August 2015

Glycogen is the main end product of glucose metabolism in amphibian oocytes. However, in the first few minutes after [U-14C]glucose microinjection most of the label is found in lactate. The burst of lactate production and the shape of the time curves for the labelling of glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate and glycogen suggest a precursor–product relationship of lactate with respect to glycogen and its intermediates. Expansion (by microinjection) of the pool of glycolytic intermediates, such as dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 3-phosphoglycerate or phosphoenolpyruvate, results in a marked decrease in [U-14C]glucose incorporation into glycogen. After co-injection of doubly labelled glucoses, extensive detritiation (93%) of the glucosyl units of glycogen was observed with [2-3H,U-14C]glucose and partial detritiation with [3-3H,U-14C]glucose (34%) or [5-3H,U-14C]glucose (46%). After injection of [6-3H,U-14C]glucose, a small but significant and reproducible detritiation (13%) in glycogen was observed. Co-injection of [U-14C]glucose and 3-mercaptopicolinate resulted in marked inhibition of glycogen labelling. Half-maximal inhibition was observed at 0.58 mM 3-mercaptopicolinate, which agrees with the IC50 value (0.47 mM) for the inhibition in vitro of phosphoenolpyruvate carboxykinase activity. We conclude that in frog oocytes most of the glucosyl units are incorporated into glycogen by an indirect pathway involving breakdown of glucose to lactate, which is then converted into glycogen via gluconeogenesis. Both processes, glycolytic degradation of glucose to lactate and subsequent reconversion of the latter into hexose phosphates and glycogen, occur in the same cell.
Prediction from sequence comparisons of residues of factor H involved in the interaction with complement component C3b
Authors: SOAMES Candida J. , DAY Antony J. , SIM Robert B.
Published: August 2015

The amino acid sequence of the region of bovine factor H containing the C3b binding site has been derived from sequencing overlapping cDNA clones. A cDNA sequence encoding 669 amino acids was obtained. Like human and mouse factor H the sequence can be arranged into a number of internally homologous units (CPs), each of which is about 60 amino acids long and is based on a framework of four conserved cysteine residues. Bovine factor H is of the same molecular mass as human and mouse factor H, and is therefore likely to be composed of 20 contiguous CPs. Comparisons with human and mouse factor H indicate that the partial bovine sequence encodes CPs 2–12 inclusive of bovine factor H. Bovine factor H binds to human ammonia-treated C3 (causing thiolester cleavage) [C3(NH3)] and promotes the cleavage of human C3(NH3) in the presence of bovine factor I. Other studies indicate that CPs 2–5 of human factor H encompass the C3b binding and factor I cofactor activity site. Multiple sequence alignments of human factor H, mouse factor H (which also interacts with human C3b) and bovine factor H with CP modules whose structures have been determined experimentally, have been used to predict residues in the hypervariable loops of CPs 2–5 and to identify residues of potential importance in human C3 binding and factor I cofactor activity. Leu-17 and Gly-20 of CP 2, Ser-17, Ala-19, Glu-21, Asp-23 and Glu-25 of CP 3 and Lys-18 of CP 4 are all conserved between the three species. It may be that CPs 3 and 4 interact with C3(NH3) directly, whilst CPs 2 and 5 maintain the correct orientation for CPs 3 and 4 to interact.
Differential regulation of transcription and transcript stability of pro-α 1(I) collagen and fibronectin in activated fibroblasts derived from patients with systemic scleroderma
Authors: ECKES Beate , MAUCH Cornelia , HÜPPE Gabriele , KRIEG Thomas
Published: August 2015

Activated fibroblasts were derived from the skin of patients with systemic scleroderma (SSc), used as a model for fibrosis. Such cells are characterized by increased production of collagens and other matrix constituents. Increased collagen and fibronectin production has been correlated with similarly elevated mRNA steady-state levels. In the present study we analysed the contribution of transcriptional activity and post-transcriptional transcript stability to the increases in pro-α1(I) collagen and fibronectin mRNA steady-state levels in activated (scleroderma) fibroblasts. Fibroblasts, when cultured in close contact with a three-dimensional collagenous matrix, down-regulate collagen synthesis. Culture of skin fibroblasts from two patients with SSc in three-dimensional collagen lattices, however, showed 4-fold elevated pro-α1(I) collagen mRNA levels over fibroblasts from healthy donors. Transcription of the COL1A1 gene in SSc fibroblasts was induced 2–3-fold over that in controls in both monolayer and lattice cultures, accounting in part for the elevated steady-state level. A 50% decrease in transcription rate in lattice compared with monolayer culture occurred, as in control cells. In contrast, whereas control cells in lattices responded with decreased (50%) pro-α1(I) collagen mRNA stability, in SSc cells these transcripts were found to be more stable (half-life of 5 h compared with 2 h in control cells). Fibronectin steady-state mRNA levels, in contrast, were not significantly regulated by the three-dimensional environment. In SSc fibroblasts, fibronectin mRNA levels were induced 1.5–4.9-fold over controls. In part, this increase appears to be due to elevated transcription, and an increase in fibronectin transcript stability was also detected. We therefore conclude that activated fibroblasts such as those derived from scleroderma patients utilize transcriptional and post-transcriptional mechanisms to maintain increased collagen and fibronectin production, which contribute to the pathogenesis of the disease.
Transcriptional regulation by glucocorticoids of mitochondrial oxidative enzyme genes in the developing rat kidney
Authors: DJOUADI Fatima , BASTIN Jean , KELLY Daniel P. , MERLET-BENICHOU Claudie
Published: August 2015

Mitochondrial fatty acid β-oxidation plays a major role in providing the ATP required for reabsorptive processes in the adult rat kidney. However, the molecular mechanisms and signals involved in induction of the enzymes of fatty acid oxidation during development in this and other organs are unknown. We therefore studied the changes in the steady-state levels of mRNA encoding medium-chain acyl-CoA dehydrogenase (MCAD), which catalyses the initial step in mitochondrial fatty acid β-oxidation, in the rat kidney cortex and medulla between postnatal days 10 and 30. Furthermore, we investigated whether the expression of MCAD and of mitochondrial malate dehydrogenase (mMDH), a key enzyme in the tricarboxylic acid cycle, might be co-ordinately regulated by circulating glucocorticoids in the immature kidney during development. In the cortex, the levels of MCAD mRNA rose 4-fold between day 10 and day 21, and then decreased from day 21 to day 30. In the medulla a postnatal increase in the concentration of MCAD mRNA (8-fold) was observed during the same period. Adrenalectomy prevented the 16–21-day developmental increases in MCAD and mMDH mRNA levels in the cortex and medulla; these could be restored by dexamethasone treatment. A single injection of dexamethasone into 10-day-old rats led to a rise in MCAD and mMDH mRNA levels in the renal cortex due to stimulation of gene transcription, as shown by nuclear run-on assays. Therefore MCAD and mMDH gene expression is tightly regulated at the transcriptional level by developmental changes in circulating glucocorticoid levels. These hormones might thus represent a good candidate as a co-ordinating factor in the expression of nuclear genes encoding mitochondrial enzymes in the kidney during postnatal development.