bluuebunny/crossref_metadata_2025 huggingface dataset:
Vasoactive intestinal peptide receptors in rat liver after partial hepatectomy
Authors: Guijarro L G , Couvineau A , Rodriguez-Pena M S , Juarranz M G , Rodriguez-Henche N , Arilla E , Laburthe M , Prieto J C
Published: August 2015

We describe the status of vasoactive intestinal peptide (VIP) receptors in regenerating liver. VIP-stimulated adenylate cyclase activity was markedly decreased in proliferating liver 3 days after partial (70%) hepatectomy. This was associated with a reduced efficacy of VIP (53% compared with controls), with no change in the potency of the peptide (ED50 0.8 nM). In contrast, forskolin- and guanosine 5′-[beta gamma-imido]triphosphate (Gpp[NH]p)-stimulated enzyme activities were not decreased after hepatectomy. The expression of Gs protein subunits (alpha and beta) was studied by cholera toxin-catalysed ADP ribosylation of alpha s and by immunoblotting of alpha s and beta subunits. Both subunits were increased in regenerating liver, further suggesting that the decreased response to VIP was not related to a decreased expression of Gs proteins. In fact, the reduced adenylate cyclase response to VIP in regenerating liver was associated with quantitative and structural changes in VIP receptors. Equilibrium binding data obtained with 125I-VIP indicated the presence of two classes of binding sites, the Kds of which were not altered after hepatectomy. In contrast, changes in binding capacity (Bmax.) were as follows: 0.11 +/- 0.01 and 0.05 +/- 0.01 pmol/mg of protein for high-affinity sites in control and hepatectomized rats respectively; and 2.3 +/- 0.2 and 0.65 +/- 0.03 pmol/mg of protein for low-affinity sites in control and hepatectomized rats respectively. Moreover, affinity labelling experiments showed that the M(r) value of 125I-VIP-receptor complexes was higher in regenerating liver than in quiescent hepatocytes, e.g. 58,000 and 53,000 respectively. It is concluded that VIP receptors are altered in regenerating liver, resulting in a decreased response of adenylate cyclase to the neuropeptide.
Cloning and sequence analysis of a cDNA encoding the α-subunit of mouse β-N-acetylhexosaminidase and comparison with the human enzyme
Authors: Beccari T , Hoade J , Orlacchio A , Stirling J L
Published: August 2015

cDNAs encoding the mouse beta-N-acetylhexosaminidase alpha-subunit were isolated from a mouse testis library. The longest of these (1.7 kb) was sequenced and showed 83% similarity with the human alpha-subunit cDNA sequence. The 5′ end of the coding sequence was obtained from a genomic DNA clone. Alignment of the human and mouse sequences showed that all three putative N-glycosylation sites are conserved, but that the mouse alpha-subunit has an additional site towards the C-terminus. All eight cysteines in the human sequence are conserved in the mouse. There are an additional two cysteines in the mouse alpha-subunit signal peptide. All amino acids affected in Tay-Sachs-disease mutations are conserved in the mouse.
Inhibition of protein N-glycosylation by 2-deoxy-2-fluoro-d-galactose
Authors: Gross V , Hull W E , Berger U , Andus T , Kreisel W , Gerok W , Keppler D
Published: August 2015

The effects of 2-deoxy-2-fluoro-D-galactose (dGalF) on N- and O-glycosylation of proteins was studied in rat hepatocyte primary cultures and in human monocytes. In hepatocytes, dGalF at concentrations of 1 mM or higher completely inhibited N-glycosylation of alpha 1-antitrypsin and alpha 1-acid glycoprotein, whereas 4 mM-2-deoxy-D-galactose (dGal) only slightly impaired N-glycosylation. In monocytes, 1 mM- or 4 mM-dGalF blocked N-glycosylation of alpha 1-antitrypsin and of interleukin-6, while O-glycosylation of interleukin-6 remained unaffected. In monocytes, dGal had no effect on protein N-glycosylation. Addition of uridine effectively prevented the UTP deficiency induced by dGalF, but had no effect on the inhibition of protein N-glycosylation by dGalF. Using 19F-n.m.r. spectroscopy, 2-deoxy-2-fluoro-D-galactose 1-phosphate (dGalF-1-P), UDP-dGalF and UDP-dGlcF could be identified as the major metabolites of dGalF in hepatocytes as well as in monocytes. In conclusion, compared with dGal, dGalF is a more efficient inhibitor of protein N-glycosylation. The effect is not caused by the depletion of UTP induced by dGalF, but rather by metabolites of dGalF. dGalF is metabolized not only in hepatocytes but also in peripheral blood monocytes, which can be used for ex vivo studies of disturbances in D-galactose metabolism.
Rat surfactant protein D enhances the production of oxygen radicals by rat alveolar macrophages
Authors: Van Iwaarden J F , Shimizu H , Van Golde P H M , Voelker D R , Van Golde L M G
Published: August 2015

Rat surfactant protein D (SP-D) was shown to enhance the production of oxygen radicals by rat alveolar macrophages. This enhancement, which was determined by a lucigenin-dependent chemiluminescence assay, was maximal after 18 min at an SP-D concentration of 0.2 micrograms/ml. Surfactant lipids did not influence the stimulation of alveolar macrophages by SP-D, whereas the oxygen-radical production of these cells induced by surfactant protein A was inhibited by the lipids in a concentration-dependent manner.
Reconstitution of the human placental 5-hydroxytryptamine transporter in a catalytically active form after detergent solubilization
Authors: Ramamoorthy S , Cool D R , Leibach F H , Mahesh V B , Ganapathy V
Published: August 2015

The 5-hydroxytryptamine (5-HT; serotonin) transporter was solubilized from purified human placental brush border membranes by cholate in the presence of urea, and the solubilized transporter was reconstituted into proteoliposomes in a functionally active form. Solubilization of the membranes with cholate in the absence of urea inactivated the transporter. The reconstitution procedure involved precipitation of the solubilized proteins and simultaneous removal of cholate and urea by poly(ethylene glycol), and incorporation of the precipitated proteins into proteoliposomes in the presence of asolectin by a freeze-thaw/sonication technique. Optimal conditions included the use of 6% poly(ethylene glycol) during the precipitation step and an asolectin/protein ratio of 10:1 during the reconstitution step. K+ was present in the reconstitution medium. The reconstituted proteoliposomes showed the ability to transport 5-HT against a concentration gradient when an inwardly directed NaCl gradient was imposed. The 5-HT transport system in the proteoliposomes had an absolute requirement for Na+ and Cl-. The system was specific for 5-HT and was inhibited by imipramine, paroxetine and fluoxetine. The Na+/Cl-/5-HT stoichiometry was found to be 1:1:1. The transport process was electrically silent, indicating that one K+ ion was countertransported for each 5-HT molecule. The reconstituted 5-HT transporter showed high affinity for 5-HT, with an apparent Michaelis-Menten constant of 0.34 +/- 0.01 microM. It is concluded that the human placental 5-HT transporter can be solubilized and reconstituted into proteoliposomes in a transport-competent form and that the characteristics of the reconstituted transporter are similar to those of the native transporter.
l-tryptophan uptake by segment-specific membrane vesicles from the proximal tubule of rabbit kidney
Authors: Jessen H , Sheikh M I
Published: August 2015

1. The mechanism of the renal transport of L-tryptophan by basolateral and luminal membrane vesicles prepared from either the pars convoluta or the pars recta of the rabbit proximal tubule was studied. The uptake of L-tryptophan by basolateral membrane vesicles from the pars convoluta was found to be an Na(+)-dependent transport event. The Na(+)-conditional influx of the amino acid was stimulated in the presence of an inwardly directed H+ gradient. Lowering the pH without an H+ gradient had no effect, indicating that L-tryptophan is co-transported with H+. 3. On the other hand, no transient accumulation of L-tryptophan was observed in the presence or absence of Na+ in basolateral membrane vesicles from the pars recta. 4. In luminal membrane vesicles from the pars recta, the transient Na(+)-dependent accumulation of L-tryptophan occurred via a dual transport system. In addition, an inwardly directed H+ gradient could drive the uphill transport of L-tryptophan into these vesicles in both the presence and the absence of an Na+ gradient. 5. By contrast, the uptake of L-tryptophan by luminal membrane vesicles from the pars convoluta was a strictly Na(+)-dependent and electrogenic transport process, mediated by a single transport component. 6. Investigation of the coupling ratio in luminal membrane vesicles suggested that 1 Na+:1 L-tryptophan are co-transported in the pars convoluta. In the pars recta, examination of the stoichiometry indicated that approx. 1 H+ and 2 Na+ (high affinity) or 1 Na+ (low affinity) are involved in the uptake of L-tryptophan.
Intrinsic factor receptor during fetal development of the human intestine
Authors: Schohn H , Guéant J L , Leheup B , Saunier M , Grignon G , Nicolas J P
Published: August 2015

Intrinsic factor receptor activity was observed in mucosal homogenates from whole small intestine and colon of 10-19-week fetuses, whereas it was only detected in the distal part of the small intestine of a 25-week fetus. The receptor-intrinsic factor-cobalamin complex was eluted into the void-volume position when ileum mucosal extract was assayed for receptor activity by gel filtration after incubation with either fetal gastric extract or human gastric juice. The intrinsic-factor-binding capacity of intestinal mucosal extracts ranged from 2.6 to 30.5 fmol/mg and was correlated with the gestational age of six fetuses. The dissociation constant of the receptor for the intrinsic factor-cobalamin complex was estimated at 0.24-0.36 nM at pH 7.4. In conclusion, intrinsic-factor-receptor activity was detected in the whole intestine in 10-19-week fetuses, whereas it was only present in the distal ileum at the end of fetal development.
Degradation of articular cartilage keratan sulphates using hydrazinolysis and nitrous acid. Environment of fucose residues
Authors: Brown G M , Huckerby T N , Morris H G , Nieduszynski I A
Published: August 2015

Alkaline borohydride-reduced keratan sulphate (KS) chains from bovine articular cartilage (6-8-year-old animals) were fragmented by an anhydrous hydrazine/nitrous acid procedure, previously used on KS by Hopwood & Elliott to isolate the major disaccharides from the poly-N-acetyl-lactosamine repeat sequence [Hopwood & Elliott (1983) Carbohydr. Res. 117, 263-274]. The resulting oligosaccharides were reduced with NaB3H4 or NaBH4 and subjected to ion-exchange chromatography on a Nucleosil 5SB column. In addition to the major disaccharides, two fucose-containing oligosaccharides were examined by high-field 1H n.m.r. spectroscopy, and shown to have the following structures (where AnManOH is 2,5-anhydro-D-mannitol): [formula: see text] It is evident that the presence of fucose protects the N-acetylglucosamine residue from de-N-acetylation, and therefore fragments are produced which preserve the immediate environment of the fucose residue. It may be of biosynthetic significance that these two oligosaccharides contain an unsulphated galactose on the non-reducing side of the fucose residue. The hydrazine/nitrous acid/NaB3H4 method followed by h.p.l.c. provides a sensitive fingerprinting technique for the assay of KS composition and sub-populations.
Expression in Escherichia coli and characterization of a recombinant 7Fe ferredoxin of Rhodobacter capsulatus
Authors: Jouanneau Y , Duport C , Meyer C , Gaillard J
Published: August 2015

The 7Fe ferredoxin of Rhodobacter capsulatus (FdII) could be expressed in Escherichia coli by cloning the fdxA gene coding for FdII downstream from the lac promoter. The expressed recombinant ferredoxin appeared as a brown protein which was specifically recognized in E. coli cell-free extracts by anti-FdII serum. The purified recombinant ferredoxin was indistinguishable from R. capsulatus FdII on the basis of its molecular, redox and spectroscopic properties. These results indicate that the [3Fe-4S] and [4Fe-4S] clusters were correctly inserted into the recombinant ferredoxin.
Structure and expression of the Drosophila ubiquitin-52-amino-acid fusion-protein gene
Authors: Cabrera H L , Barrio R , Arribas C
Published: August 2015

Ubiquitin belongs to a multigene family. In Drosophila two members of this family have been previously described. We report here the organization and expression of a third member, the DUb52 gene, isolated by screening a Drosophila melanogaster genomic library. This gene encodes an ubiquitin monomer fused to a 52-amino acid extension protein. There are no introns interrupting the coding sequence. Recently, it has been described that this extension encodes a ribosomal protein in Saccharomyces, Dictyostelium, and Arabidopsis. The present results show that the 5′ regulatory region of DUb52 shares common features with the ribosomal protein genes of Drosophila, Xenopus and mouse, including GC- and pyrimidine-rich regions. Moreover, sequences similar to the consensus Ribo-box in Neurospora crassa have been identified. Furthermore, a sequence has been found that is similar to the binding site for the TFIIIA distal element factor from Xenopus laevis. The DUb52 gene is transcribed to a 0.9 kb mRNA that is expressed constitutively throughout development and is particularly abundant in ovaries. In addition, the DUb52 gene has been found to be preferentially transcribed in exponentially growing Drosophila cells.
Inhibition by heparin of Fe(II)-catalysed free-radical peroxidation of linolenic acid
Authors: Ross M A , Long W F , Williamson F B
Published: August 2015

Heparin, in a concentration-dependent manner, inhibited the generation of conjugated dienes and thiobarbituric acid-positive substances when incubated with Fe2+ and gamma-linolenic acid. In the conjugated diene assay, other glycosaminoglycans, on a molar basis calculated with respect to their respective hydrated disaccharide repeat units, were less effective than heparin. Heparin which had been re-N-sulphonated after removal of both N-sulphonates and O-sulphates, and heparin in which iduronate residues had been reduced to idose residues, were largely unaffected in their activity. Removal of both N-sulphonates and O-sulphates greatly reduced the effectiveness of the heparin. Analysis of the effects of heparin fragments generated by heparinase I treatment of heparin showed that depolymerization decreased the effectiveness of the heparin. It is possible that heparins and related strongly acidic polysaccharides may function as endogenous antioxidants, and that sequestration by them, or harmless oxidation by them, of ions such as Fe2+, contributes to their effectiveness.
Quantification of the accumulation and degradation of β-very-low-density lipoproteins in vivo using a 19F-containing residualizing label and n.m.r. spectroscopy
Authors: Meeh L A , Ackerman J J H , Thorpe S R , Daugherty A
Published: August 2015

beta-Very-low-density lipoproteins (beta-VLDL)O were conjugated to the 19F-containing residualizing label, NN-dilactitol-3,5-bis(trifluoromethyl)benzylamine (DLBA), to determine whether the metabolism of this lipoprotein fraction could be characterized in vivo with n.m.r. spectroscopy. Solution state 19F high-resolution n.m.r. spectroscopy of DLBA-beta-VLDL, containing either intact apoproteins or selectively enzymically digested products, demonstrated that the extent of degradation could be distinguished by differences in spin-spin relaxation times (T2 times). DLBA-beta-VLDL was injected intravenously into rabbits, and accumulation of 19F in hepatic tissue was quantified non-invasively by n.m.r. spectroscopy 5 and 30 h after injection. In addition to quantifying the accumulation of DLBA-beta-VLDL in hepatic tissue, a marked decrease (approx. 100 Hz) in the linewidth of 19F resonance from labelled lipoproteins was observed at 30 h compared with the 5 h interval in continuously monitored animals. The change in linewidth was consistent with a decrease in molecular size that occurred during protein degradation, resulting in increased T2 times. To demonstrate that T2 times can be used as an index to quantify apoprotein degradation in vivo, relaxation measurements were performed on livers excised 20 h after injection of DLBA-beta-VLDL into rabbits. Two molecular motional fractions were revealed by relaxation profiles representing either an intact or an extensively degraded form of apoprotein. The amplitudes of each component were compared with results from trichloroacetic acid precipitation of liver homogenates acquired from rabbits 20 h after injection of beta-VLDL labelled with the radioiodinated analogue of DLBA, dilactitol-125I-tyramine. The amount of degraded apoprotein determined by n.m.r. spectroscopy and acid precipitation was 68.6 +/- 7.0% and 58.7 +/- 7.5% (n = 4) respectively. The results of this study demonstrate that 19F n.m.r. spectroscopy can be used to define the temporal characteristics of the hepatic metabolism of lipoproteins in vivo by quantifying both the tissue-specific accumulation and extent of apoprotein degradation. The methodology developed offers promise for the non-invasive, sequential and longitudinal evaluation of lipoprotein metabolism in vivo.
Localization of methyltransferase activities throughout the endomembrane system of flax (Linum usitatissimum L) hypocotyls
Authors: Vannier M P , Thoiron B , Morvan C , Demarty M
Published: August 2015

A microsomal fraction from flax hypocotyls (Linum usitatissimum L) showed a methylation ability from S-adenosyl-methionine on to the cell wall polysaccharides. Two kinds of methylation were found: (i) a methyl esterification of uronic acids in the oxalate extracts and (ii) an O-methylation of the hydroxyl groups in the NaOH extracts. The methyltransferase study showed a rapid decrease of the methyl esterification abilities, whereas the O-methylation on to the hydroxyl groups was maintained throughout the culture duration. The localization of such activities in the flax endomembrane system was performed using isopycnic centrifugation. Enzymic marker tests allowed us to identify the different membrane types. Methyltransferase activities in the different enriched fractions appeared to be associated with the Golgi apparatus for the O-methylation, and with the plasma membrane, Golgi apparatus and endoplasmic reticulum compartments for the carboxymethyl esterification.
Turnover of glycogen phosphorylase in the pectoralis muscle of broiler and layer chickens
Authors: Flannery A V , Easterby J S , Beynon R J
Published: August 2015

Glycogen phosphorylase is a major sarcoplasmic protein in chicken pectoralis muscle, constituting approx. 4% of the total protein complement. In slow-growing layer chicks phosphorylase accumulated in parallel with muscle accretion, but in fast-growing broiler chicks the concentration of phosphorylase in the muscle increased (from 5 to 8 mg/g wet wt.) with time. In a 5-week period, the total amount of phosphorylase in the pectoralis muscles increased 18-fold in broiler chicks (from approx. 75 to 1400 mg total), but only 3-fold (from approx. 100 to 270 mg total) in layers. Pyridoxal phosphate, the cofactor of the enzyme glycogen phosphorylase, was used as a specific label to measure the rate of degradation of the enzyme in the pectoralis muscle of growing broiler and layer chickens in vivo. In young animals, the fractional rate of phosphorylase synthesis was similar in broiler and layer chickens (approx. 15%/day), but the rate of degradation in layers (5%/day) was 5-fold higher than in broilers (1%/day). As the animals aged, the rate of synthesis decreased, but more so in layers than in broilers. The rate of degradation of phosphorylase also decreased in layers, but in broilers it remained at the low level seen in young animals. The dramatically higher rate of phosphorylase accretion in the pectoralis muscles of the broilers is therefore achieved by an initial lower rate of degradation combined with a sustained difference between rates of synthesis and degradation.
Kinetic and inhibition studies on catechol-O-methyltransferase affinity labelling by N-(3,4-dihydroxyphenyl)maleimide
Authors: Piedrafita F J , Fernandez-Alvarez E , Nieto O , Tipton K F
Published: August 2015

Initial velocity and product inhibition studies have been performed on soluble catechol-O-methyltransferase which has been partially purified from pig liver. The results are consistent with an ordered reaction mechanism, in which S-adenosyl-L-methionine (AdoMet) is the leading substrate. The enzyme is irreversibly inhibited by maleimide derivatives in a biphasic manner, which suggests a differential reaction with two thiol groups. N-(3,4-Dihydroxyphenyl)maleimide, which has a reactive moiety (maleimide ring) and an affinity moiety (catechol ring), acts as an affinity labelling compound on the more reactive SH group; AdoMet and Mg2+ protect against this modification. Total protection of this SH group results in a pseudo-first-order inhibition of the enzyme, with the apparent rate constant being proportional to the inhibitor concentration. All the other maleimide derivatives studied inhibited the enzyme by reacting with one of the two SH groups in a non-specific manner. The reaction of the other, more reactive, SH group was either specific (active-site-directed) or non-specific, depending on the substituent present in the affinity moiety and also on the length of an intermediate chain of methylene groups present between this moiety and the reactive maleimide ring. In the presence of both AdoMet and Mg2+, 3,5-dinitrocatechol, a reversible inhibitor of the enzyme which is competitive with respect to the catechol substrate, protects the enzyme from inactivation by any of the maleimide derivatives. The adducts of these maleimide derivatives formed with dithiothreitol inhibit the enzyme reversibly, showing inhibition patterns that are consistent with the mechanism deduced from the initial velocity and product inhibition studies.
Carbachol stimulation of phospholipase A2 and insulin secretion in pancreatic islets
Authors: Konrad R J , Jolly Y C , Major C , Wolf B A
Published: August 2015

Arachidonic acid has been implicated as a second messenger in insulin secretion by islets of Langerhans. D-Glucose, the major physiological stimulus, increases unesterified arachidonate accumulation in islets. We now show, for the first time, that the muscarinic agonist carbachol, at concentrations which stimulate insulin secretion, causes a rapid and nearly 3-fold increase in arachidonic acid accumulation in islets. The combination of glucose and carbachol has an additive effect on unesterified arachidonate release. There is a large component of secretagogue-induced arachidonate accumulation that is independent of extracellular Ca2+. Carbachol stimulation of arachidonic acid release is mediated by activation of phospholipase A2, as demonstrated by early increases in endogenous lysophosphatidylcholine. In addition to phospholipase A2 activation, carbachol-induced arachidonic acid accumulation also appears to involve diacylglycerol hydrolysis, since the diacylglycerol lipase inhibitor RG80267 partly inhibited arachidonic acid accumulation. In contrast, glucose-induced arachidonic acid accumulation appears to reflect diacylglycerol hydrolysis entirely. Our observations indicate that phospholipase A2 has an important role in muscarinic-induced insulin secretion.
Structure of the bovine ETB endothelin receptor gene
Authors: Mizuno T , Saito Y , Itakura M , Ito F , Ito T , Moriyama E N , Hagiwara H , Hirose S
Published: August 2015

The structure of the gene encoding the bovine type B endothelin receptor (ETB) has been established and compared with those of other heptahelical receptors. The gene is present as a single copy in the bovine genome, as demonstrated by Southern blot analysis, and spans at least 36 kb. The coding region is divided into 7 exons separated by 6 introns, one of which is more than 23 kb in length. The exons correspond well to the structural domains of the receptor: the first exon encodes the first and second transmembrane domains, and each of the following transmembrane domains is encoded by a separate exon. The portion of the ETB protein sequence encoded by exon 3 is quite different from the corresponding ETA sequence, suggesting that this region is responsible for the distinct ligand specificities of the two receptor subtypes. The second intron interrupts the canonical Asp-Arg-Tyr sequence, which is located at the end of the third transmembrane domain of the heptahelical receptors, as with the substance P, substance K, dopamine D2 and dopamine D3 receptor genes. To map the 5′ region of the gene and determine the start of transcription, primer-extended cDNAs were cloned and sequenced: multiple start sites were deduced with no apparent TATA box in the expected upstream region. Similar results were obtained by ribonuclease protection analysis.
Phosphatidylcholine-specific phospholipase D-derived 1,2-diacylglycerol does not initiate protein kinase C activation in the RBL 2H3 mast-cell line
Authors: Lin P , Fung W J , Gilfillan A M
Published: August 2015

We examined the role of phosphatidylcholine-specific phospholipase D (PC-PLD) in the IgE-dependent activation of protein kinase C (PKC) in RBL 2H3 cells (a model for mast-cell function). Cells were sensitized with mouse monoclonal anti-trinitrophenol (TNP) IgE (0.5 micrograms/ml) and were then triggered with an optimal concentration (10 ng/ml) of TNP-ovalbumin conjugate (TNP-OVA). This resulted in an immediate biphasic increase in the production of 1,2-diacylglycerol (DAG) and activation of PKC. The initial increase in DAG production reached a peak within 30 s, and the second phase reached a plateau within 5 min after stimulation. TNP-OVA-induced PC-PLD activation followed the initial increase in DAG formation in response to IgE-receptor cross-bridging, but coincided with the second peak. Phosphatidic acid (PA), derived from the PC-PLD pathway, is metabolized to DAG by the action of PA phosphohydrolase (PAPase). Propranolol (0.3 mM), which inhibits PAPase, blocked the IgE-dependent increase in DAG, activation of PKC, and subsequently degranulation. The PKC inhibitor staurosporine (0.1 microM) inhibited the second, but not first, peak of DAG accumulation, reversed PKC translocation after 10 min and inhibited subsequent mediator release. Taken together, these results demonstrate that PC-PLD does not initiate, but may play a latent role in, IgE-dependent DAG production, PKC activation and mediator release from RBL 2H3 cells.
Production and interferon-γ-mediated regulation of complement component C2 and factors B and D by the astroglioma cell line U105-MG
Authors: Barnum S R , Ishii Y , Agrawal A , Volanakis J E
Published: August 2015

In this paper, we demonstrate the synthesis of the complement component C2 and factors B and D by the human astroglioma cell line U105-MG. All three components were structurally and antigenically similar to their serum counterparts, as determined by biosynthetic labelling studies or Western blot analysis. Northern blot analysis demonstrated that the mRNAs of all three components had the same apparent sizes as the equivalent mRNAs from hepatocyte and monocyte cell lines. Interestingly, U105-MG cells produce two C2 transcripts with sizes of approximately 2.8 and 2.3 kb. Interferon-gamma (IFN-gamma) enhanced the expression of C2 and factor B mRNA and protein in a dose- and time-dependent fashion, while factor D expression was refractory to IFN-gamma. IFN-gamma appeared to predominantly enhance the expression of the large (2.8 kb) C2 transcript. Kinetic studies demonstrated peak C2 and factor B expression in 48 h in response to IFN-gamma, similar to the acute-phase response of factor B in serum. These data are the first to demonstrate the synthesis of C2 and factor D by astroglioma cells. Combined with previous reports documenting the synthesis of C3 by astrocytes, our data suggest that endogenous synthesis of complement proteins, and particularly of alternative pathway activation components (C3, factors B and D), may play an important role in host defence in the central nervous system.
19F n.m.r. studies of conformational changes accompanying cyclic AMP binding to 3-fluorophenylalanine-containing cyclic AMP receptor protein from Escherichia coli
Authors: Hinds M G , King R W , Feeney J
Published: August 2015

A fluorine-containing analogue of the cyclic AMP (cAMP) receptor protein (CRP) from Escherichia coli was prepared by biosynthetic incorporation of 3-fluorophenylalanine (3-F-Phe). 19F n.m.r. studies on this protein have provided direct evidence for cAMP-induced conformational changes not only within the cAMP-binding domain but also within the hinge region connecting the cAMP-binding domain to the DNA-binding headpiece. At 313 K, the 19F n.m.r. spectrum of [3-F-Phe]CRP showed five signals corresponding to the five phenylalanine residues as expected for a symmetrical dimer. Proteolysis of [3-F-Phe]CRP with subtilisin produced a fragment (the alpha-fragment) containing the cAMP-binding domain. The alpha-fragment contains all the phenylalanines except for Phe-136, a residue located in the hinge region. By comparing the 19F spectra of [3-F-Phe]CRP and its alpha-fragment, the signal for Phe-136 was assigned. The chemical shifts of the corresponding signals in the two spectra are similar, indicating that the alpha-fragment retains the structure it has in the intact protein. The largest cAMP-induced shift was observed for the signal from Phe-136 providing direct evidence for a conformational change in the hinge region. However, whereas binding of a single cAMP molecule to a CRP dimer is known to be sufficient to activate the DNA binding, the n.m.r. data indicate that the hinge region does not have the same conformation in both subunits when only one cAMP molecule is bound.
Evolutionary conservation of the dystrophin central rod domain
Authors: Sherratt T G , Vulliamy T , Strong P N
Published: August 2015

Dystrophin cDNA fragments encoding the C-terminal repeats of the central rod region have been expressed as fusion proteins. The polyclonal antisera raised to the purified fusion proteins have been characterized and neither antiserum cross-reacted with dystrophin-related protein. Antisera detected dystrophin with molecular mass close to that of the human in all terrestrial vertebrates and amphibia studied. Experiments with antisera to the N-terminal region of the dystrophin rod confirmed that epitopes to the rod region were conserved during this evolutionary period and the length of this domain remained unaltered.
Characterization of the binding of plasminogen activators to plasma membranes from human liver
Authors: Nguyen G , Self S J , Camani C , Kruithof E K O
Published: August 2015

The binding of tissue-type plasminogen activator (t-PA) to membranes prepared from human liver was investigated, and a specific, saturable, high-affinity binding site (Kd = 3.4 nM) was identified. The binding of t-PA to liver membranes was not affected by an excess of D-mannose or D-galactose, or by active urokinase (u-PA), whereas binding of t-PA to membranes prepared from human HepG2 hepatoma cells was inhibited by u-PA. HepG2-membrane-bound t-PA was fully complexed to PA inhibitor 1 (PAI-1), whereas liver-membrane-bound t-PA was not complexed. Gel filtration on Sephacryl S300 of membrane proteins solubilized in deoxycholate revealed that high-affinity t-PA binding activity elutes at an apparent molecular mass of 40 kDa. Monoclonal antibodies specific for the growth factor and the kringle 2 domains inhibited the binding of t-PA to liver membranes and the catabolism of t-PA by rat hepatoma cells. Human liver membranes also bound u-PA; binding was inhibited by pro-u-PA, the N-terminal fragment of u-PA, but not by the 33 kDa form of u-PA or by t-PA. Our results show that human liver membranes contain a specific 40 kDa binding protein for t-PA that is different from the PAI-1-dependent receptor described on HepG2 cells and the mannose receptor isolated from human liver.
The Caenorhabditis elegans unc-13 gene product is a phospholipid-dependent high-affinity phorbol ester receptor
Authors: Ahmed S , Maruyama I N , Kozma R , Lee J , Brenner S , Lim L
Published: August 2015

The Caenorhabditis elegans unc-13 mutant is a member of a class of mutants that have un-coordinated movement. Mutations of the unc-13 gene cause diverse defects in C. elegans, including abnormal neuronal connections and modified synaptic transmission in the nervous system. unc-13 cDNA encodes a protein (UNC-13) of 1734 amino acid residues with a predicted molecular mass of 198 kDa and sequence identity to the C1/C2 regions but not to the catalytic domain of the ubiquitously expressed protein kinase C family [Maruyama & Brenner (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5729-5733]. To characterize the phorbol ester binding site of the UNC-13 protein, cDNA encoding the C1/C2-like regions (amino acid residues 546-940) was expressed in Escherichia coli and the 43 kDa recombinant protein was purified. Phorbol ester binding to the 43 kDa protein was zinc- and phospholipid-dependent, stereospecific and of high affinity (Kd 67 nM). UNC-13 specific antisera detected a protein of approx. 190 kDa in wild-type (N2) but not in mutant (e1019) C. elegans cell extracts. We conclude that UNC-13 represents a novel class of phorbol ester receptor.
Characterization of three osteogenesis imperfecta collagen α 1(I) glycine to serine mutations demonstrating a position-dependent gradient of phenotypic severity
Authors: Bateman J F , Moeller I , Hannagan M , Chan D , Cole W G
Published: August 2015

Type I collagen alpha 1(I) glycine to serine substitutions, resulting from G-to-A mutations, were defined in three cases of osteogenesis imperfecta (OI). The Gly substitutions displayed a gradient of phenotypic severity according to the location of the mutation in the collagen triple helix. The most C-terminal of these, Gly565 to Ser, led to the lethal perinatal (type II) form of OI, whereas the more N-terminal mutations, Gly415 and Gly352 to Ser, led to severe OI (type III/IV) and moderate OI (type IVB) respectively. These data support the notion that glycine substitutions towards the C-terminus of the alpha 1(I) or alpha 2(I) chains will be more clinically severe than those towards the N-terminus. This results from the more disruptive effect of the mutations at the C-terminus on helix initiation and C- and N-terminal helix directional propagation. This generalization must be modified by considering the nature of the glycine substitution and the surrounding amino acid sequence, since the helix is composed of subdomains of differing stability which will affect the ability of helix re-nucleation and propagation.
Toxic dark effects of protoporphyrin on the cytochrome P-450 system in rat liver microsomes
Authors: Williams M , Van der Zee J , Van Steveninck J
Published: August 2015

In erythropoietic protoporphyria, accumulation of protoporphyrin has been found in various tissues and liver cirrhosis occurs frequently in this disease, probably due to toxic dark effects of protoporphyrin. We have studied the effect of porphyrins on various enzymic functions in rat liver microsomes. Incubation of microsomes with protoporphyrin resulted in a concentration-dependent inhibition of the oxidation of 7-ethoxycoumarin and aminopyrine by the cytochrome P-450 system. Kinetic analysis showed a decrease in Vmax., whereas the Km was not affected (non-competitive inhibition). Furthermore, reduction of cytochrome c by the NADPH-cytochrome P-450 reductase and by the NADH-cytochrome b5 reductase was inhibited. However, the activity of the reductases was only affected when the microsomes were pre-incubated with protoporphyrin, and it was found that the inhibition was dependent on the duration of the pre-incubation. Kinetic analysis again revealed non-competitive inhibition. When these experiments were repeated with uroporphyrin, no inhibition could be observed. With Stern-Volmer plots it was demonstrated that this was most likely caused by the localization of the porphyrins: protoporphyrin is localized in the membrane, whereas uroporphyrin remains in solution. From these results it is concluded that accumulation of protoporphyrin in the liver may markedly affect the cytochrome P-450 system and thus its detoxification function.
Immunological detection of degradation intermediates of skeletal-muscle glycogen phosphorylase in vitro and in vivo
Authors: Cookson E J , Flannery A V , Cidlowski J A , Beynon R J
Published: August 2015

Over 95% of the pyridoxal phosphate (PLP) in skeletal is bound to one protein, glycogen phosphorylase. This, and the fact that phosphorylase constitutes approx. 5% of the soluble protein in skeletal muscle, introduce the possibility that PLP might be used as a specific label to identify degradation intermediates of the enzyme. In this investigation, we have developed immunological methods, using a monoclonal antibody to PLP and polyclonal antibodies to phosphorylase, to detect degradation intermediates in vitro and in vivo. We have identified a family of degradation intermediates of glycogen phosphorylase in the high-speed-supernatant fraction of mouse skeletal muscle. These peptides react with both types of antibodies and are in the size and concentration range expected for degradation intermediates in a model in which the committed step is followed by rapid clearance of the products. Changes in amounts of degradation intermediates are examined in physiological or pathological conditions in which the rate of degradation of phosphorylase is altered.
Expression and characterization of the p85 subunit of the phosphatidylinositol 3-kinase complex and a related p85β protein by using the baculovirus expression system
Authors: Gout I , Dhand R , Panayotou G , Fry M J , Hiles I , Otsu M , Waterfield M D
Published: August 2015

PtdIns 3-kinase associates with certain activated protein-tyrosine kinase receptors and with the pp60c-src/polyoma middle-T complex, suggesting that the enzyme is involved in growth regulation. The purified PtdIns 3-kinase appears to have two subunits, of 85 kDa and 110 kDa. Structural analysis at protein and cDNA levels revealed two forms of the 85 kDa subunit, one which associates with PtdIns 3-kinase activity termed p85 alpha, and a protein of unknown function, p85 beta. Both 85 kDa proteins contain src-homology regions 2 and 3 (SH2 and SH3), but lack enzymic activity, suggesting that they may be regulatory subunits of PtdIns 3-kinase. To probe their structure and function further, p85 alpha and p85 beta have been expressed and purified in large amounts from insect cells by using baculovirus vectors. Specific antisera detect p85 alpha, but not p85 beta, associated with PtdIns 3-kinase activity in various cell types. Co-expression studies in insect cells have shown that p85 alpha and p85 beta are substrates for the protein-tyrosine kinases of epidermal growth factor, colony-stimulating factor 1 and c-erbB2 receptors and the src family kinase p59c-fyn. Both p85 alpha and p85 beta form tight complexes with these protein-tyrosine kinases as measured by immunoprecipitation and kinase assays in vitro. The specificity of binding of free p85 is less restricted than that of p85 in the active PtdIns 3-kinase complex with the 110 kDa protein. The relevance of these results to growth-factor-induced PtdIns 3-kinase activation is discussed.
A non-glycosylated extracellular superoxide dismutase variant
Authors: Edlund A , Edlund T , Hjalmarsson K , Marklund S L , Sandström J , Strömqvist M , Tibell L
Published: August 2015

The secretory tetrameric extracellular superoxide dismutase (EC-SOD) is the only glycosylated SOD isoenzyme. The importance of the carbohydrate moiety for the properties of the enzyme is unknown. An expression vector defining nonglycosylated EC-SOD (ngEC-SOD) was constructed by mutagenesis of the codon for Asn-89 into a codon for Gln. The vector was transfected into Chinese hamster ovary DXB-11 cells and ngEC-SOD was isolated to 70% purity from the culture media of selected clones. The absence of glycosylation was established by the lack of affinity for various lectins, the absence of staining with the periodic acid-Schiff reagent, the change in mobility and composition of the tryptic peptide containing the mutated glycosylation site, and the reduction in apparent molecular mass upon SDS/PAGE and size-exclusion chromatography. The tetrameric state was retained. The heparin affinity, a fundamental and distinguishing property of EC-SOD, was found to be slightly increased. The enzymic activity was essentially retained. The major difference from native glycosylated enzyme in physical properties was a marked reduction in solubility. Like glycosylated EC-SOD, ngEC-SOD was, after intravenous injection into rabbits, rapidly sequestered by the vessel endothelium, and was promptly released into plasma after injection of heparin. The only difference from glycosylated EC-SOD in this behaviour, was a slightly more rapid elimination of the mutant enzyme from the vasculature. It is concluded that no specific biological role for the EC-SOD carbohydrate moiety could be revealed.
Monoclonal antibodies for dystrophin analysis. Epitope mapping and improved binding to SDS-treated muscle sections
Authors: Nguyen T M , Ginjaar I B , van Ommen G J B , Morris G E
Published: August 2015

A group of 44 monoclonal antibodies (mAbs) raised against the central helical rod (25 mAbs) and C-terminal (19 mAbs) regions of dystrophin were prepared using trpE recombinant fusion proteins as immunogens. Some mAbs cross-react with the structurally related proteins, alpha-actinin and utrophin. Epitope mapping revealed uneven distribution of mAb-binding sites, no mAbs being produced against the C-terminal end of the helical fragment or the cysteine-rich region of the C-terminal dystrophin fragment. The failure of these large regions of the recombinant immunogens to elicit anti-dystrophin antibodies may be because of their inability to fold into the correct dystrophin-like conformation. The mAbs were selected for their ability to recognize 427 kDa dystrophin on Western blots after SDS/PAGE, and/or for immunostaining of the membrane in frozen muscle sections. Although some mAbs obtained by Western-blot screening failed to bind native dystrophin in frozen muscle sections, successful binding could be obtained after SDS or urea treatment of the tissue section to expose the epitopes. This increases the range of mAbs available for detection of dystrophin deletions in muscular dystrophy and evaluation of myoblast therapy.
A comparison of the effects of calponin on smooth and skeletal muscle actomyosin systems in the presence and absence of caldesmon
Authors: Winder S J , Sutherland C , Walsh M P
Published: August 2015

Thiosphosphorylated smooth muscle myosin and skeletal muscle myosin, both of which express Ca(2+)-independent actin-activated MgATPase activity, were used to examine the functional effects of calponin and caldesmon separately and together. Separately, calponin and caldesmon inhibited the actin-activated MgATPase activities of thiophosphorylated smooth muscle myosin and skeletal muscle myosin, calponin being significantly more potent in both systems. Calponin-mediated inhibition resulted from the interaction of calponin with actin since it could be reversed by increasing the actin concentration. Caldesmon had no significant influence on the calponin-induced inhibition of the smooth muscle actomyosin ATPase, nor did calponin have a significant effect on caldesmon-induced inhibition. In the skeletal muscle system, however, caldesmon was found to override the inhibitory effect of calponin. This difference probably reflects the lower affinity of skeletal muscle actin for calponin compared with that of smooth muscle actin. Calponin inhibition of skeletal muscle actin-activated myosin MgATPase was not significantly affected by troponin/tropomyosin, suggesting that the thin filament can readily accommodate calponin in addition to the troponin complex, or that calponin may be able to displace troponin. Calponin also inhibited acto-phosphorylated smooth muscle heavy meromyosin and acto-skeletal muscle heavy meromyosin MgATPases. The most appropriate protein preparations for analysis of the regulatory effects of calponin in the actomyosin system therefore would be smooth muscle actin, tropomyosin and thiophosphorylated myosin, and for analysis of the kinetic effects of calponin on the actomyosin ATPase cycle they would be smooth muscle actin, tropomyosin and phosphorylated heavy meromyosin, due to the latter's solubility.
Hepatic cholesterol synthesis and the secretion of newly synthesized cholesterol in bile
Authors: Robins S J , Fasulo J M , Lessard P D , Patton G M
Published: August 2015

To determine the effect of increased hepatic cholesterol synthesis on the secretion of newly synthesized cholesterol in bile, rats were fed with cholestyramine, a bile-acid-binding resin that increases the number of hepatocytes that synthesize cholesterol. Cholesterol synthesis was measured 15 min after [3H]water injection to avoid appreciable exchange between the liver and serum of newly synthesized cholesterol that accumulates in the serum in studies of several hours duration. At 15 min after [3H]water injection, the specific radioactivity of cholesterol in the liver and hepatic microsomes was greatly increased in resin-fed animals compared with controls. However, with resin, the specific radioactivity of newly synthesized cholesterol that was secreted in bile was the same as for controls. At 15 min after [3H]water injection the specific radioactivity of serum cholesterol was minimally increased and not different in resin and control groups. In contrast, in studies that were longer than 60 min, newly synthesized cholesterol in serum was appreciably increased in resin-fed animals, and newly synthesized cholesterol in bile was also greatly increased compared with controls. Thus, when appreciable cholesterol exchange is avoided, an increase in hepatic cholesterol synthesis and the number of hepatocytes that synthesized cholesterol does not result in an increase in newly synthesized cholesterol in bile. Our results suggest that newly synthesized cholesterol is secreted in bile from a fixed subpopulation of hepatocytes. From a comparison of the specific radioactivity of newly synthesized cholesterol in whole liver and bile, it can be estimated that this subpopulation of hepatocytes represents about 20% of the total hepatocyte mass.
Aldehydes from metal ion- and lipoxygenase-induced lipid peroxidation: detection by 1H-n.m.r. spectroscopy
Authors: Lodge J K , Patel S U , Sadler P J
Published: August 2015

The modification of lipoproteins by reactive aldehydes formed via lipid peroxidation is thought to be a key process in the pathogenesis of atherosclerosis. We show that 1H-n.m.r. spectroscopy can readily be used to detect a variety of different aldehydes resulting from peroxidation of liposomes induced by Fenton's reagent or lipoxygenase, and aldehydes arising from copper-induced reactions of low-density lipoprotein. There is a clear contrast between the major aldehydic products arising from metal-ion- and lipoxygenase-induced reactions.