bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative biosynthesis, covalent post-translational modifications and efficiency of prosegment cleavage of the prohormone convertases PC1 and PC2: glycosylation, sulphation and identification of the intracellular site of prosegment cleavage of PC1 and PC2
Authors: Benjannet S , Rondeau N , Paquet L , Boudreault A , Lazure C , Chrétien M , Seidah N G
Published: August 2015

We present herein the pulse-chase analysis of the biosynthesis of the prohormone convertases PC1 and PC2 in the endocrine GH4C1 cells infected with vaccinia virus recombinants expressing these convertases. Characterization of the pulse-labelled enzymes demonstrated that pro-PC1 (88 kDa) is cleaved into PC1 (83 kDa) and pro-PC2 (75 kDa) into PC2 (68 kDa). Secretion of glycosylated and sulphated PC1 (84 kDa) occurs about 30 min after the onset of biosynthesis, whereas glycosylated and sulphated PC2 (68 kDa) is detected in the medium after between 1 and 2 h. Furthermore, in the case of pro-PC2 only, we observed that a fraction of this precursor escapes glycosylation. A small proportion (about 5%) of the intracellular glycosylated pro-PC2 (75 kDa) is sulphated, and it is this glycosylated and sulphated precursor that is cleaved into the secretable 68 kDa form of PC2. Major differences in the carbohydrate structures of PC1 and PC2 are demonstrated by the resistance of the secreted PC1 to endoglycosidase H digestion and sensitivity of the secreted PC2 to this enzyme. Inhibition of N-glycosylation with tunicamycin caused a dramatic intracellular degradation of these convertases within the endoplasmic reticulum, with the net effect of a reduction in the available activity of PC1 and PC2. These results emphasize the importance of N-glycosylation in the folding and stability of PC1 and PC2. Pulse-labelling experiments in uninfected mouse beta TC3 and rat Rin m5F insulinoma cells, which endogenously synthesize PC2, showed that, as in infected GH4C1 cells, pro-PC2 predominates intracellularly. In order to define the site of prosegment cleavage, pulse-chase analysis was performed at low temperature (15 degrees C) or after treatment of GH4C1 cells with either brefeldin A or carbonyl cyanide m-chlorophenylhydrazone. These results demonstrated that the onset of the conversions of pro-PC1 into PC1 and non-glycosylated pro-PC2 into PC2 (65 kDa) occur in a pre-Golgi compartment, presumably within the endoplasmic reticulum. In contrast, pulse labelling in the presence of Na(2)35SO4 demonstrated that the processing of glycosylated and sulphated pro-PC2 occurs within the Golgi apparatus. In order to test the possibility that zymogen processing is performed by furin, we co-expressed this convertase with either pro-PC1 or pro-PC2. The data demonstrated the inability of furin to cleave either proenzyme.
Greater selective uptake by Hep G2 cells of high-density lipoprotein cholesteryl ester hydroperoxides than of unoxidized cholesteryl esters
Authors: Sattler W , Stocker R
Published: August 2015

We have observed recently that high-density lipoproteins (HDL) are the predominant carriers of cholesteryl ester hydroperoxides (CEOOH), the major class of lipid hydroperoxides detectable at nanomolar concentrations in the plasma of healthy fasting humans. The present study investigates the effect of such very low levels of CEOOH in apolipoprotein E-free HDL3 on lipoprotein particle metabolism and ‘selective uptake’ of its CE by human Hep G2 cells. Minimal oxidation with aqueous peroxyl radicals had a negligible effect on the binding, internalization and degradation of 125I-labelled HDL3. In contrast, with an increasing degree of radical-mediated oxidation of labelled HDL3, [3H]cholesteryl linoleate ([3H]Ch18:2) was taken up at an increasingly greater rate than were 125I-apoproteins. When [3H]cholesteryl linoleate hydroperoxide ([3H]Ch18:2-OOH was incorporated into unoxidized HDL3 by exchange from donor liposomes, it was taken up at a more than 8-fold higher rate than was incorporated [3H]Ch18:2. The same degree of preferential uptake of oxidized CE was observed when HDL3 was used that was doubly labelled with [3H]Ch18:2-OOH and cholesteryl [14C]oleate ([14C]Ch18:1). In both situations, uptake of [3H]Ch18:2-OOH exceeded that of 125I-apolipoprotein A-I some 40-fold. This increased selective uptake of [3H]Ch18:2-OOH from very mildly oxidized HDL3 was accompanied by a parallel increase in the intracellular levels of labelled free cholesterol. In contrast, lipid hydroperoxides were not detectable within Hep G2 cells, suggesting efficient detoxification of CEOOH by these cells. Neither the increased selective uptake of Ch18:2-OOHs nor the levels of intracellular free cholesterol were influenced by the presence of 50 microM chloroquine, suggesting extralysosomal hydrolysis of oxidized CEs. These results show that the selective uptake of HDL CEOOH by Hep G2 cells is more efficient than that of unoxidized CE, and support a protective role for rapid selective uptake in the removal of circulating HDL CEOOH.
Time course of the uridylylation and adenylylation states in the glutamine synthetase bicyclic cascade
Authors: Varón-Castellanos R , Havsteen B H , García-Moreno M , Valero-Ruiz E , Molina-Alarcón M , García-Cánovas F
Published: August 2015

A kinetic analysis of the glutamine synthetase bicyclic cascade is presented. It includes the dependence on time from the onset of the reaction of both the uridylylation of Shapiro's regulatory protein and the adenylylation of the glutamine synthetase. The transient phase equations obtained allow an estimation of the time elapsed until the states of uridylylation and adenylylation reach their steady-states, and therefore an evaluation of the effective sensitivity of the system. The contribution of the uridylylation cycle to the adenylylation cycle has been studied, and an equation relating the state of adenylylation at any time to the state of uridylylation at the same instant has been derived.
Heparin-affinity patterns and composition of extracellular superoxide dismutase in human plasma and tissues
Authors: Sandström J , Karlsson K , Edlund T , Marklund S L
Published: August 2015

The tetrameric extracellular superoxide dismutase (EC-SOD) in human tissues and plasma has previously been found to be heterogenous with regard to heparin affinity and could be divided into at least three classes: A, lacking heparin affinity; B, with weak affinity; and C, with strong affinity. Using rigorous extraction conditions and an extensive set of anti-proteolytic agents, tissue EC-SOD is now shown to be almost exclusively of native homotetrameric C-class. Plasma EC-SOD on the other hand is shown to be mainly composed of a complex mixture of heterotetramers with modifications probably residing in the C-terminal heparin-binding domain. Proteolytic truncations appear to be a major cause of this heterogeneity. The findings suggest that, since 99% of the EC-SOD in the human body exists in the extravascular space of tissue, EC-SOD is primarily synthesized in tissues and secreted as homotetrameric native EC-SOD C. This tissue EC-SOD C should exist almost completely sequestered by heparin sulphate proteoglycans. C-terminal modifications subsequently occurring in the EC-SOD C would weaken the binding to heparan sulphate proteoglycan, facilitate entrance to the vasculature through capillaries and lymph flow, and finally result in the heterogeneous plasma EC-SOD pattern. With the new extraction and analysis procedure, the tissue content of EC-SOD is found to be higher than previously reported. It is found, for example, when compared with Mn-SOD, to be higher in umbilical cord and uterus, about equal in placenta and testis and as high as that of CuZn-SOD in umbilical cord. The findings suggest that the protection level against superoxide radicals provided by EC-SOD in the tissue interstitial space, given the small distribution volume, is not much less prominent than that bestowed on the intracellular space by CuZn-SOD and Mn-SOD.
Modulation of transferrin receptor mRNA by transferrin-gallium in human myeloid HL60 and lymphoid CCRF-CEM leukaemic cells
Authors: Ul-Haq R , Chitambar C R
Published: August 2015

Gallium binds to the iron transport protein transferrin (Tf), is incorporated into cells through transferrin receptors (TfR) and inhibits iron-dependent DNA synthesis. Since cellular TfR expression is tightly regulated by the availability of iron, we investigated the effects of transferrin-gallium (Tf-Ga) on TfR mRNA levels in myeloid HL60 and lymphoid CCRF-CEM cells. In HL60 cells, Tf-Ga increased TfR mRNA levels in a dose-dependent fashion. This increase in TfR mRNA was blocked by Tf-Fe and by cycloheximide. Analysis of the rate of mRNA decay in the presence of actinomycin D revealed that the half-life of TfR mRNA was increased in HL60 cells incubated with Tf-Ga. The rate of transcription of TfR mRNA was not increased by Tf-Ga. In contrast with HL60 cells, CCRF-CEM cells displayed a decrease in the level of TfR mRNA after incubation with Tf-Ga. Tf-Ga inhibited iron uptake in both HL60 and CCRF-CEM cells but increased the level of TfR mRNA only in HL60 cells, suggesting that the Tf-Ga induction of TfR mRNA was not solely due to inhibition of cellular iron uptake. At growth-inhibitory concentrations, Tf-Ga increased the TfR mRNA level in HL60 cells but decreased it in CCRF-CEM cells. Our studies suggest that in HL60 cells, gallium regulates TfR expression at the post-transcriptional level by mechanisms which require de novo protein synthesis and involve interaction with iron. The divergent effects of Tf-Ga on TfR mRNA in myeloid HL60 and lymphoid CCRF-CEM cells suggest that differences exist in the regulation of TfR expression between these two cell types.
S-adenosyl-l-methionine decarboxylase of Acanthamoeba castellanii (Neff): purification and properties
Authors: Hugo E R , Byers T J
Published: August 2015

S-Adenosyl-L-methionine decarboxylase (AdoMetDC) has been purified to near homogeneity from the Neff strain of Acanthamoeba castellanii. The holoenzyme molecular mass is 88.8 kDa, including two copies each of a 32.8 kDa alpha-subunit and a 10-15 kDa beta-subunit. The alpha-subunit contains the active site. It has an N-terminal pyruvoyl group, and the first 19 amino acids are 63 and 74% identical with comparable sequences from yeast and mammals, respectively. The apparent Km for S-adenosylmethionine (AdoMet) in the presence of 2 mM putrescine was 30.0 microM. The enzyme was stimulated 2-fold by putrescine, but was unaffected by spermidine. It was inhibited by the following anti-metabolites, listed with their Ki values: Berenil (0.17 microM), pentamidine (19.4 microM), propamidine (334 microM), hydroxystilbamidine (357 microM), methylglyoxal bis(guanylhydrazone) (604 microM) and ethidium bromide (1.3 mM). Activity of the enzyme fell to undetectable levels during cell differentiation (encystment).
Differential responses of intestinal glucose transporter mRNA transcripts to levels of dietary sugars
Authors: Miyamoto K , Hase K , Takagi T , Fujii T , Taketani Y , Minami H , Oka T , Nakabou Y
Published: August 2015

Dietary sugars are known to stimulate intestinal glucose transport activity, but the specific signals involved are unknown. The Na(+)-dependent glucose co-transporter (SGLT1), the liver-type facilitative glucose transporter (GLUT2) and the intestinal-type facilitative glucose transporter (GLUT5) are all expressed in rat jejunum [Miyamoto, Hase, Taketani, Minami, Oka, Nakabou and Hagihira (1991) Biochem. Biophys. Res. Commun. 181, 1110-1117]. In the present study we have investigated the effects of dietary sugars on these glucose transporter genes. A high-glucose diet stimulated glucose transport activity and increased the levels of SGLT1 and GLUT2 mRNAs in rat jejunum. 3-O-Methylglucose, D-galactose, D-fructose, D-mannose and D-xylose can mimic the regulatory effect of glucose on the SGLT1 mRNA level in rat jejunum. However, only D-galactose and D-fructose increased the levels of GLUT2 mRNA. The GLUT5 mRNA level was increased significantly only by D-fructose. Our results suggest that the increase in intestinal transport activity in rats caused by dietary glucose is due to an increase in the levels of SGLT1 and GLUT2 mRNAs, and that these increases in mRNA may be caused by an enhancement of the transcriptional rate. Furthermore, for expression of the SGLT1 gene, the signal need not be a metabolizable or transportable substrate whereas, for expression of the GLUT2 gene, metabolism of the substrate in the liver may be necessary for signalling. Only D-fructose is an effective signal for expression of the GLUT5 gene.
Degradation of rat C-reactive protein by macrophages
Authors: Nagpurkar A , Hunt D , Yang C Y , Mookerjea S
Published: August 2015

Rat C-reactive protein (CRP) is a serum glycoprotein belonging to the ‘pentraxin’ family of proteins. In this study we have shown the specific binding of 125I-CRP to rat peritoneal macrophages at 4 degrees C. This binding was dependent upon incubation time, CRP and cell concentrations, and was not inhibited by either phosphorylcholine or human IgG. At 37 degrees C, the surface-bound 125I-CRP was internalized and degraded. The degradation of 125I-CRP was measured by the formation of 125I-labelled trichloroacetic-acid-soluble CRP peptides by either precipitation assays or by h.p.l.c. of the incubation medium using a gel-filtration column. Since chloroquine and leupeptin inhibited CRP degradation, it was concluded that degradation of CRP occurred in the lysosomal compartment of the macrophage. There was an absolute requirement for the presence of bivalent cations (Ca2+ and Mg2+) in the incubation medium for the binding and degradation of CRP, which could be inhibited by EDTA but not by phosphorylcholine or human IgG. H.p.l.c. analysis of the medium obtained from incubation of macrophages with 125I-CRP revealed the presence of 125I-labelled low-M(r) peptides, the formation of which was dependent upon incubation time.
Purification and characterization of a phytase (myo-inositol-hexakisphosphate phosphohydrolase) accumulated in maize (Zea mays) seedlings during germination
Authors: Laboure A M , Gagnon J , Lescure A M
Published: August 2015

Phytase (myo-inositol-hexakisphosphate phosphohydrolase, EC 3.1.3.8) has been purified from 5-7-day-old maize (Zea mays) seedlings, using a four-step purification procedure. The native protein has a molecular mass of about 76 kDa and is built up from two 38 kDa subunits. The pH and temperature optima of the purified enzyme were respectively 4.8 and 55 degrees C. The apparent Km for phytate was estimated to be 117 microM. Like other acidic phytases, the maize seedling enzyme exhibited a broad affinity for various phosphorylated substrates and especially for penta- and tri-phosphate esters of myo-inositol. The amino acid composition of the h.p.l.c.-purified protein indicated a high hydrophobicity (44% non-polar amino acids). Rabbit antibodies were produced in response to maize seedling phytase. Western-blot analyses clearly demonstrate that the increase of phytase activity observed during the first 7 days of germination corresponded to an accumulation of the protein in maize seedlings. Phytase accumulated essentially in the shoots (mesocotyl plus coleoptiles.
Expression of two different forms of cDNA for thromboxane synthase in insect cells and site-directed mutagenesis of a critical cysteine residue
Authors: Xia Z , Shen R F , Baek S J , Tai H H
Published: August 2015

cDNA coding for human placental thromboxane synthase (EC 5.3.99.5) was amplified by PCR from a human placental cDNA library and sequenced. This cDNA and a shorter cDNA isolated from a human lung cDNA library with a deletion of 163 bp near the 3′ end were expressed in Spodoptera frugiperda (Sf9) insect cells using a baculovirus expression system. The cDNA from human placenta was expressed as an active enzyme (60 kDa) with a specific activity higher than those reported from other cell types, whereas the shorter cDNA was expressed in an inactive form (52 kDa). The active recombinant enzyme appeared to be unglycosylated as the molecular mass and the enzyme activity were not altered in the presence of tunicamycin. Site-directed mutagenesis was performed to convert a cysteine at position 480 in thromboxane synthase to a serine. This cysteine is found to be highly conserved in related cytochrome P-450 enzymes. The mutant enzyme was found to be inactive, although Western blot, immunoprecipitation and SDS/PAGE analysis indicated that the mutant enzyme was expressed at a level comparable with the wild-type enzyme. These results suggest that Cys-480 is essential for the enzyme catalytic activity and that the short-form cDNA may be a non-functional transcript.
Expression in Escherichia coli of the flavin and the haem domains of Hansenula anomala flavocytochrome b2 (flavodehydrogenase and b2 core) and characterization of the recombinant proteins¶
Authors: Silvestrini M C , Tegoni M , Célerier J , Desbois A , Gervais M
Published: August 2015

The flavin and haem domains of Hansenula anomala flavocytochrome b2 have been independently expressed in Escherichia coli. The flavin domain activity, studied only in the total cellular extract, owing to its instability, has characteristics very similar to those of the flavin domain obtained by proteolysis. The haem domain (r-core) has been purified to homogeneity and characterized in detail from spectroscopic and functional points of view. Spectral differences with respect to the domain produced by proteolysis (p-core) were found using resonance Raman and c.d. spectroscopy and have been interpreted in terms of changes in haem-protein interactions. However, this structural difference is functionally silent, since the r-core is able to reduce cytochrome c with the same efficiency as the proteolytic domain.
Expression of a rat neurotensin receptor in Escherichia coli
Authors: Grisshammer R , Duckworth R , Henderson R
Published: August 2015

With the goal of obtaining sufficient quantities of seven-helix G-protein-coupled receptors for structural analysis, we have studied the functional expression of a rat neurotensin receptor cDNA in Escherichia coli with and without a signal sequence and as a fusion with the gene coding for maltose-binding protein. The addition of an N-terminal signal peptide resulted in increased expression levels. In vitro translation at a high level revealed that the codon usage of the rat neurotensin receptor cDNA was not critical for overproduction. Expression of neurotensin receptor cDNA fused to the 3′ end of the gene encoding maltose-binding protein resulted in a 40-fold increase in neurotensin-binding sites. Binding of [3H]neurotensin to intact bacteria or E. coli membranes was saturable, with a dissociation constant, KD, of 0.23 nM (Bmax. = 450 sites/bacterium or 15 pmol/mg of crude membrane protein). The binding properties of all recombinant receptors presented in this study were similar and corresponded to those of the high-affinity binding sites in rat brain. For immunological detection and future purification of neurotensin receptor, a C-terminal pentahistidine/c-myc tail was introduced. Western-blot analysis revealed the association of neurotensin receptor with E. coli membranes.
Purification and characterization of the dissimilatory nitrite reductase from Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015): evidence for the presence of both type 1 and type 2 copper centres
Authors: Abraham Z H L , Lowe D J , Smith B E
Published: August 2015

Dissimilatory nitrite reductase was isolated from extracts of Alcaligenes xylosoxidans subsp. xylosoxidans (N.C.I.M.B. 11015), after activation of crude extracts by the addition of copper(II) sulphate. The enzyme was purified by a combination of (NH4)2SO4 fractionation and cationic-exchange chromatography to 93% homogeneity as judged by SDS/PAGE. SDS/PAGE and spray m.s. showed that the enzyme had a subunit M(r) of 36.5 kDa. The copper content was 3.5 +/- 0.8 Cu atoms/trimer of M(r) 109,500. E.p.r. spectroscopy of nitrite reductase as isolated showed that both type 1 (g parallel = 2.208, A parallel = 6.3 mT) and type 2 (g parallel = 2.298, A parallel = 14.2 mT) Cu centres were present, in contrast with published data [Masuko, Iwasaki, Sakurai, Suzuki and Nakahara (1984) J. Biochem. (Tokyo) 96, 447-454], where only type 1 copper centres were reported. Our preparations had a specific activity of 150-300 mumol of NO2- reduced/min per mg of protein, 6-12-fold higher than reported previously. As isolated, the oxidized form of our preparations of the enzyme showed absorption maxima in the visible region at 460, 593 and 770 nm. The ratio of the absorption bands at 460 nm and 593 nm resulted in this protein having a strong blue colour, in contrast with the green colour of other purified copper-containing nitrite reductases. We conclude that, in contrast with previous reports, this ‘blue’ nitrite reductase requires both type 1 and type 2 copper centres for optimal activity.
Molecular cloning and expression of a rat κ opioid receptor
Authors: Li S , Zhu J , Chen C , Chen Y W , Deriel J K , Ashby B , Liu-Chen L Y
Published: August 2015

At least three types of opioid receptors have been identified in the nervous system. In this paper we report molecular cloning and expression of a rat kappa opioid receptor. PCR was performed on double-stranded cDNA derived from poly(A)+ RNA of the rat striatum with primers similar to those of Libert and co-workers [Libert, Parmentier, Lefort, Dinsart, Van Sande, Maenhaut, Simons, Dumont and Vassart (1989) Science 244, 569-572]. One of the PCR products, which had 65% sequence similarity to the mouse delta opioid receptor, was used to screen a rat striatum cDNA library. Two positive clones were isolated and found to be identical. The clone had a 2.1-kb insert, which was termed RKOR-1. RKOR-1 has an open reading frame of 1140 bp and encodes a 380-amino-acid protein. Hydropathy analysis indicates that RKOR-1 has seven putative transmembrane domains with short intra- and extra-cellular loops. Membranes of Cos-7 cells transfected with RKOR-1 exhibited high specific binding for [3H]diprenorphine ([3H]DIP), a non-selective opioid ligand. Naloxone inhibited [3H]DIP binding with stereospecificity. [3H]DIP binding was potently inhibited by selective kappa opioid ligands, with Ki values in the nanomolar or subnanomolar range, but much less potently inhibited by drugs selective for mu or delta receptors. Thus, RKOR-1 represents an opioid receptor with kappa characteristics.
Stable correction of maple syrup urine disease in cells from a Mennonite patient by retroviral-mediated gene transfer
Authors: Koyata H , Cox R P , Chuang D T
Published: August 2015

We have successfully used retroviral gene transfer to correct the deficiency of the branched-chain alpha-oxo acid dehydrogenase complex in lymphoblasts from a homozygous Mennonite maple syrup urine disease (MSUD) patient. The mutation in Mennonites is a Tyr-393 to Asn substitution in the branched-chain alpha-oxo acid decarboxylase (E1)alpha subunit of the enzyme complex. This promotes improper assembly of mutant E1 alpha with E1 beta subunits, leading to degradation of both polypeptides. For transduction studies, a full-length human E1 alpha CDNA was inserted into the retroviral vector LXSN to produce the recombinant LSN-E1 alpha. High-titre [6 x 10(5) colony-forming units/ml] amphotropic retroviral preparations free of helper viruses were obtained by co-cultivation of infected GP+E86 with PA317 cells. Transduction of MSUD lymphoblasts from the Mennonite patient with LSN-E1 alpha viruses restored the decarboxylation of alpha-oxo[1-14C]isovalerate to the normal level. The normal decarboxylation activity in transduced MSUD cells remained stable without G418 selection during the 14 weeks studied. Southern-blot analysis indicated that the recombinant E1 alpha cDNA was integrated into the host genome. Northern and Western blotting showed that both the normal E1 alpha mRNA and the subunit were properly expressed in transduced MSUD cells. However, the level of E1 beta subunits is lower than that of normal cells, suggesting competition of the recombinant E1 alpha with the mutant form for assembly with E1 beta. The results provide a paradigm for the development of somatic gene therapy for disorders involving mitochondrial multienzyme complexes.
CoA and fatty acyl-CoA derivatives mobilize calcium from a liver reticular pool
Authors: Fulceri R , Gamberucci A , Bellomo G , Giunti R , Benedetti A
Published: August 2015

The effect of CoA and fatty acyl-CoA esters on Ca2+ fluxes has been studied in isolated liver microsomes and in digitonin-permeabilized hepatocytes. When microsomes were loaded with increasing concentrations of Ca2+ (6-29 nmol/mg of protein), the extent to which CoA and palmitoyl-CoA released Ca2+ increased. At 23 nmol of Ca2+/mg of protein, half-maximal [CoA] and [palmitoyl-CoA] were 35 and 50 microM respectively. Under conditions of minimal Ca2+ loading, net release of Ca2+ was absent, but Ca2+ translocation from a CoA-sensitive to a CoA-insensitive pool took place. The effect of CoA required the presence of fatty acids, probably to form fatty acyl esters. In permeabilized hepatocytes, the pool(s) mobilized by CoA (or by palmitoyl-CoA) appeared to be different from that mobilized by Ins(1,4,5)P3.
A fibroblast protein binds the 3′-untranslated region of pro-α1(I) collagen mRNA
Authors: Määttä A , Penttinen R P K
Published: August 2015

Post-transcriptional regulation of the expression of the pro alpha 1(I) chain of type I collagen (COL1A1) was studied by analysing cytoplasmic RNA-binding proteins and by transient transfections with collagen minigene plasmids. In this paper we present evidence for a factor from NIH 3T3 cells and human skin fibroblasts that interacts with the conserved 3′-untranslated region (UTR) of the shorter 4.8 kb mRNA species of the COL1A1 gene. The specificity of the interaction was confirmed by using (i) unlabelled specific and non-specific competitor RNAs and (ii) oligodeoxyribonucleotides annealed to the probe or used as single-stranded competitors. An antisense oligonucleotide annealed to the RNA probe near its 3′-terminus [20-42 nucleotides upstream of the first polyadenylation signal of the alpha 1(I) collagen mRNA] inhibited the binding, whereas other sense or antisense oligonucleotides had no effect on the interaction. The binding was sensitive to alkylation of free SH groups but not to phosphatase treatment of the extracts. In u.v. cross-linking analysis this factor migrated as a single polypeptide chain of about 67 kDa, and was named alpha 1-RBF67 (type I collagen alpha 1 chain RNA-binding factor). Dexamethasone treatment of fibroblasts, which is known to accelerate the turnover of COL1A1 mRNA, decreased the alpha 1-RBF67 activity markedly as evaluated by gel-retardation and u.v. cross-linking assays. Transient transfections with plasmids carrying the alpha 1(I) collagen promoter and 3′-UTR sequences demonstrated that the 3′-UTR participates in the response to dexamethasone. Thus the loss of alpha 1-RBF67 activity might be associated with decreased alpha 1(I) collagen mRNA levels after dexamethasone treatment.
The presence and subcellular distribution of sterol carrier protein 2 in embryonic-chick tissues
Authors: Reinhart M P , Avart S J , Dobson T O , Foglia T A
Published: August 2015

Transport of lipids from the yolk to the tissues of the chick embryo is slow during the first 2 weeks of development, but increases abruptly during the last week. Evidence suggests that the lipid traverses the cytoplasm of the yolk-sac membrane before secretion as lipoprotein into the fetal circulation. Little is known about the cytoplasmic transport of lipid in avian systems, but recently the presence of sterol carrier protein 2 (SCP2) was reported in chicken liver. Here we examine the cells of yolk-sac membrane, liver and small intestine for the presence of this protein as a function of the time of embryonic development. The quantity of SCP2 present in the embryonic cells did not appear to correlate with the rate of lipid flux in these tissues. The abrupt appearance of a high-molecular-mass form of SCP2 was detected in small intestine shortly before hatching, but the significance of this protein is not clear. The presence of SCP2 in these tissues was also confirmed by immunocytochemical techniques. Similarly to SCP2 of mammalian cells, avian SCP2 is localized in both peroxisome-like structures and mitochondria. To a lesser extent it is associated with the endoplasmic reticulum.
Reactive oxygen species mediate phorbol ester-regulated tyrosine phosphorylation and phospholipase A2 activation: potentiation by vanadate
Authors: Zor U , Ferber E , Gergely P , Szücs K , Dombrádi V , Goldman R
Published: August 2015

We have previously shown that vanadate potentiates the activating effect of phorbol ester (TPA) on cellular phospholipase A2 (PLA2) in a pathway dependent on the formation of reactive oxygen species (ROS). Here we evaluate the chain of enzymes (protein kinases and phosphatases) that participate in this process. Treatment of macrophages with vanadate plus TPA led to activation of protein kinase C (PKC) and NADPH oxidase (O2- generation in intact cells), massive cellular protein tyrosine phosphorylation, suppression of protein tyrosine phosphatase (PTP) activity and a sustained activation of protein tyrosine kinase (PTK) and myelin basic protein kinase activity (the latter three enzyme activities were assessed in cell lysates). Inhibition of ROS formation by diphenyleneiodonium (DPI) prevented PTP inhibition, PTK activation and protein tyrosine phosphorylation by vanadate plus TPA. Vanadate plus H2O2 mimicked the effect of vanadate plus TPA on PKC activation, cellular protein tyrosine phosphorylation, PTP and PTK, but their effects were resistant to DPI. Suppression of PKC activity (down-regulation; selective inhibitors) prevented the above-mentioned effects of vanadate plus TPA, but not of vanadate plus H2O2. Collectively, the results show that ROS formation induced by TPA in association with vanadate is essential in the modulation of protein tyrosine phosphorylation and PLA2 activity.
Kinetic mechanism of activation by cardiolipin (diphosphatidylglycerol) of the rat liver multicatalytic proteinase
Authors: Ruiz de Mena I , Mahillo E , Arribas J , Castaño J G
Published: August 2015

The effect of phospholipids on the trypsin-like, chymotrypsin-like and peptidylglutamyl-peptide-hydrolysing activities of the so-called latent form of the rat liver multicatalytic proteinase was studied, assaying them with the following substrates: N-Cbz-ARR-4MNA (N-Cbz, N-benzyloxycarbonyl; 4MNA, 4-methoxy-beta-naphthylamide), N-Suc-LLVY-MCA (N-Suc, N-succinyl; MCA, methylcoumarin) and N-Cbz-LLE-beta-NA (beta-NA, beta-naphthylamide) respectively (amino acids are shown as their one-letter symbol). For the most part neither lysophospholipids nor phospholipids at 20 micrograms/ml have any effect on the activity of the enzyme (assayed at 50 microM peptide), except for phosphatidylserine, which activates 2-fold the hydrolysis of N-Suc-LLVY-MCA, and phosphatidylinositol, which inhibits by 20% the hydrolysis of N-Cbz-LLE-beta-NA. By contrast, cardiolipin (diphosphatidylglycerol) is a strong activator of the hydrolysis of N-Suc-LLVY-MCA (60-fold) and N-Cbz-LLE-beta-NA (30-fold), with half-maximal activation at concentrations of 0.15 micrograms/ml and 1.5 micrograms/ml respectively. The activation of N-Suc-LLVY-MCA hydrolysis is due to an increase of the affinity of the enzyme for the peptide and to an increase in the Vmax. (30-fold). The activation of N-Cbz-LLE-beta-NA hydrolysis is explained by suppressing the co-operativity for this substrate, producing hyperbolic kinetics with a Km of 60 microM and a 15-fold increase in the Vmax. of the enzyme. This activation by cardiolipin was completely suppressed by micromolar concentrations of fluophenazine, a drug known to inhibit other phospholipid-regulated process. Cardiolipin activation and the known activation by SDS are additive, either at suboptimal or optimal concentrations of both activators. Cardiolipin also activates the in vitro degradation of some proteins from metabolically labelled total cellular extracts by the latent multicatalytic proteinase. These results clearly show that cardiolipin is a natural positive modulator of the peptidase and proteolytic activities of the multicatalytic proteinase, probably acting through a binding site different from that of SDS.
Inhibition of glutathione synthesis increases the toxicity of oxidized low-density lipoprotein to human monocytes and macrophages
Authors: Gotoh N , Graham A , Nikl E , Darley-Usmar V M
Published: August 2015

Macrophages are thought to play an important role in the pathogenesis of atherosclerosis by mediating the oxidation of low-density lipoprotein (LDL). However, it is known that these cells show elevated glutathione levels after exposure to oxidized LDL. Here we demonstrate that this increase in the level of intracellular glutathione is due to synthesis de novo stimulated by oxidized LDL. Furthermore, inhibition of glutathione synthesis renders oxidized LDL cytotoxic to both monocytes and macrophages at a concentration well tolerated by untreated cells. The stimulation of cholesterol esterification in macrophages by low, non-toxic, concentrations of oxidized LDL is enhanced under conditions where glutathione synthesis is inhibited. These results suggest that the glutathione status of macrophages in the artery wall could be important in both controlling foam-cell formation and the detoxification of oxidized LDL.
In vitro translation of androgen receptor cRNA results in an activated androgen receptor protein
Authors: Kuiper G G J M , de Ruiter P E , Trapman J , Jenster G , Brinkmann A O
Published: August 2015

Translation of androgen receptor (AR) cRNA in a reticulocyte lysate and subsequent analysis of the translation products by SDS/PAGE showed a protein with an apparent molecular mass of 108 kDa. Scatchard-plot analysis revealed a single binding component with high affinity for R1881 (Kd = 0.3 nM). All AR molecules synthesized specifically bound steroid. No evidence for AR phosphorylation during in vitro synthesis was found. When AR was labelled with [3H]R1881 and analysed on sucrose-density gradients, a complex of approx. 6 S was observed. The complex was shifted to a higher sedimentation coefficient after incubation with a monoclonal AR antibody directed against an epitope in the DNA-binding domain. In the presence as well as the absence of hormone, AR molecules were able to bind to DNA-cellulose without an activation step. Gel retardation assays revealed that the AR forms complexes with a DNA element containing glucocorticoid-responsive element/androgen-responsive element sequences. Receptor-DNA interactions were stabilized by different polyclonal antibodies directed against either the N- or C-terminal part of the AR and were abolished by an antibody directed against the DNA-binding domain of the receptor. In conclusion, translation of AR cRNA in vitro yields an activated AR protein which binds steroid with high affinity. It is proposed that AR antibodies enhance AR-DNA binding by stabilizing AR dimers when bound to DNA.
Involvement of human plasma angiotensin I-converting enzyme in the degradation of the haemoregulatory peptide N-acetyl-seryl-aspartyl-lysyl-proline
Authors: Rieger K J , Saez-Servent N , Papet M P , Wdzieczak-Bakala J , Morgat J L , Thierry J , Voelter W , Lenfant M
Published: August 2015

The degradation of N-Ac-Ser-Asp-Lys-Pro (AcSDKP), a negative regulator controlling the proliferation of the haematopoietic stem cell, by enzymes present in human plasma, has been investigated. Radiolabelled AcSD[4-3H]KP ([3H]AcSDKP, 1 mM) was completely metabolized in human plasma with a half-life of 80 min, leading exclusively to the formation of radiolabelled lysine. The cleavage of AcSDKP was insensitive to classical proteinase inhibitors including leupeptin, but sensitive to metalloprotease inhibitors. The degradation was completely blocked by specific inhibitors of angiotensin I-converting enzyme (ACE; kininase II; peptidyldipeptide hydrolase, EC 3.4.15.1), showing that the first step of the hydrolysis was indeed due to ACE. In dialysed plasma, the hydrolysis proceeded at only 17% of the maximal rate, whereas addition of 20 mM NaCl led to the recovery of the initial rate observed with normal plasma. Hydrolysis of AcSDKP by commercial rabbit lung ACE generated the C-terminal dipeptide Lys-Pro. Thus, ACE cleaves AcSDKP by a dipeptidyl carboxypeptidase activity. In fact the formation of Lys-Pro was observed when AcSDKP was incubated in human plasma in the presence of HgCl2. These results suggest that ACE is involved in the first limiting step of AcSDKP degradation in human plasma. The second step seems to be under the control of a leupeptin- and E-64-insensitive, HgCl2-sensitive plasmatic enzyme.
Structure-activity relationships in the inhibition of serine β-lactamases by phosphonic acid derivatives
Authors: Rahil J , Pratt R F
Published: August 2015

A new series of phosphonyl derivatives has been prepared and tested for inhibition of serine (classes A and C) beta-lactamases. The results were compared with those previously acquired with aryl phosphonate monoesters and with alkaline hydrolysis rates. A methyl p-nitrophenyl phosphate monoanion was markedly poorer as an inhibitor of the class C beta-lactamase of Enterobacter cloacae P99 than a comparable p-nitrophenyl phosphonate. Phosphonyl fluorides, thiophenyl esters, N-phenylphosphonamidates and a p-nitrophenyl thionophosphonate were, in general, comparable with p-nitrophenyl phosphonates in inhibitory power. The incorporation of a specific amino side chain led to an increase in the rates of inhibition of around 10(4)-fold. Apparently unresponsive to the addition of the side chain to the enzyme was N-phenyl methylphosphonamidate, where binding of the side chain may interfere with access of the leaving group to a proton which is necessary to active-site phosphonylation and inhibition. Typical class A beta-lactamases were significantly more refractory than the class C enzyme to all of these reagents.
Platelet adhesion to collagen via the α2β1 integrin under arterial flow conditions causes rapid tyrosine phosphorylation of pp125FAK
Authors: Polanowska-Grabowska R , Geanacopoulos M , Gear A R L
Published: August 2015

Adhesion of human platelets to collagen under arterial flow conditions mediated by the alpha 2 beta 1 integrin increased tyrosine phosphorylation of several proteins, one of which was the focal adhesion tyrosine kinase, pp125FAK. Tyrosine phosphorylation of pp125FAK did not occur in non-adherent flowing platelets or in platelets attached to poly(L-lysine). Neither adhesion nor tyrosine phosphorylation was affected by pretreatment of platelets with GRGDSP peptide or by anti-alpha IIb beta 3 monoclonal antibody P2. Adherent platelets retained their discoid shape, suggesting that induction of pp125FAK precedes platelet spreading. The tyrosine kinase inhibitor erbstatin decreased tyrosine phosphorylation in non-stimulated platelets and blocked platelet adhesion. These results suggest that pp125FAK plays an important role in platelet adhesion to collagen via the alpha 2 beta 1 integrin.
p40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains
Authors: Wientjes F B , Hsuan J J , Totty N F , Segal A W
Published: August 2015

The NADPH oxidase generates superoxide in phagocytic cells. It is important for immunity and its deficiency leads to chronic granulomatous disease (CGD). It consists of a membrane-bound flavocytochrome b that lies dormant until activated by the translocation to the plasma membrane of cytosolic proteins, p47phox (phox for phagocyte oxidase), p67phox and p21rac, a small GTP-binding protein. We show here that a novel component, p40phox, forms an activation complex with p47phox and p67phox with which it translocates to the membrane to associate with the flavocytochrome b. cDNA cloning and amino acid analysis revealed that p40phox has an src homology 3 (SH3) domain and a large region of sequence similarity with the N-terminus of p47phox. The primary association of p40phox appears to be with p67phox, and it is present in reduced amounts in patients with CGD lacking p67phox.
Arthrobacter
d-xylose isomerase: chemical modification of carboxy groups and protein engineering of pH optimum
Authors: Siddiqui K S , Loviny-Anderton T , Rangarajan M , Hartley B S
Published: August 2015

To try to lower the pH optimum, the carboxy groups of Arthrobacter D-xylose isomerase were coupled to glycinamide using a water-soluble carbodi-imide. In conditions that substituted all of the 59 carboxy groups in the denatured monomer, a maximum of 30 groups/monomer reacted in the native enzyme, whether in presence or absence of ligands, and the enzyme remained fully active and tetrameric throughout the coupling reaction. Purification by f.p.l.c. ion-exchange chromatography gave broad symmetrical peaks with increased pI, suggesting that the modified enzymes are essentially homogeneous. However, they are less stable than native enzyme in 8 M urea or on heating (‘melting points’ of 59 degrees versus 73 degrees C for the apoenzymes and 67 degrees versus 81.5 degrees C for the Mg(2+)-enzymes). Kinetic studies of the D-fructose isomerase activity at 30 degrees C showed that the glycinamidylated enzyme had unaltered activation constant for Mg2+, and Km was also similar to that of the native enzyme at pH 7.3, but increased rapidly at higher pH rather than remaining constant. Vmax. was constant from pH 6.2 to 8.0, suggesting a reduced pKa for His-219, which controls Vmax. in the native enzyme (normally 6.0). Three mutants were constructed by protein engineering with a view to reducing the pH optimum of enzyme activity. Two of these, Glu140→Lys and Asp189→Lys, could be detected in crude extracts of Escherichia coli by SDS/PAGE, but could not be purified, whereas mutant Trp136→Glu was produced as a tetramer in amounts similar to the wild-type enzyme. However, it did not show any enzyme activity and was less stable in 0-9 M urea gradient PAGE.
Catabolism of hirudin and thrombin-hirudin complexes in the rat
Authors: Bichler J , Baynes J W , Thorpe S R
Published: August 2015

The metabolic fate of the anticoagulant protein, hirudin, and its complex with thrombin are presently unknown. Therefore we have labelled hirudin and human thrombin-hirudin complex with the residualizing label dilactitol-125I-tyramine (*I-DLT) in order to identify their tissue sites of catabolism in the rat. The rapid plasma clearance of hirudin after intravenous injection was unaffected by *I-DLT labelling, and by 2 h 6% or less of the injected dose remained in the blood. The majority (80.3 +/- 4.0%, n = 2) of *I-DLT-hirudin radioactivity recovered in tissues was found in kidney, and kidney was also at least 150 times more active in taking up hirudin, on a weight basis, than any other tissue examined (liver, spleen, skin, muscle, intestine, fat, lung). *I-DLT-hirudin which bound to thrombin was isolated by chromatography on concanavalin A-Sepharose; hirudin itself does not bind to concanavalin A. Radioactivity from thrombin-*I-DLT-hirudin was precipitable by anti-thrombin antibody and *I-DLT-thrombin-hirudin was precipitable by anti-hirudin antibody. By 1 h after injection of labelled thrombin-hirudin complexes, the recoveries of radioactivity from hirudin and thrombin in liver were comparable (38.6 +/- 3.0 and 36.4 +/- 4.1%, n = 3), whereas more radioactivity was recovered in kidney from hirudin than from thrombin (27.6 +/- 8.7 compared with 13.6 +/- 4.5%) and less was recovered in lung (0.4 +/- 0.2 compared with 17.7 +/- 2.9%). We conclude that hirudin is catabolized predominantly in kidney, whereas the thrombin-hirudin complex is catabolized by both liver and kidney.
Desulphation of dextran sulphate during kidney ultrafiltration
Authors: Comper W D , Tay M , Wells X , Dawes J
Published: August 2015

The renal clearance of [3H]dextran sulphate by the isolated perfused rat kidney was associated with desulphation of the molecule, as demonstrated by ion-exchange and affinity chromatography of material resident in both glomeruli and urine samples. This process also occurred in vivo. The molecular size distribution of glomerular dextran sulphate in the perfused kidney was indistinguishable from that in the perfusate, and although urinary material was smaller it remained macromolecular. Sulphatase activity was not detected in urine or in the perfusate of perfused kidneys, but was detected in glomerular and non-glomerular cortex fractions isolated by a sieving procedure. The identification of significant biochemical changes to dextran sulphate demonstrates that it does not function as an inert transport probe, and supports the concept of cellular involvement in the process of renal charge selectivity.
Identification of a binding site on retinal transducin α for the phosphodiesterase inhibitory γ subunit
Authors: Cunnick J , Twamley C , Udovichenko I , Gonzalez K , Takemoto D J
Published: August 2015

Transducin alpha (T alpha) activates retinal rod cyclic GMP phosphodiesterase (PDE) by interacting with and removing the inhibitory PDE gamma subunit. A T alpha-PDE gamma complex can be isolated in vitro, and our previous work [Morrison, Rider and Takemoto (1987) FEBS Lett. 222, 266-270; Morrison, Cunnick, Oppert and Takemoto (1989) J. Biol. Chem. 264, 11671-11681] has identified a region of PDE gamma, residues 24-45, that binds to T alpha. The C-terminal region of PDE gamma is the site that interacts with PDE alpha/beta and inhibits catalytic function. The site on T alpha that binds to the PDE gamma 24-45 region has not been identified. Synthetic peptides (15-mers) which span the bovine T alpha sequence were tested for binding to purified recombinant PDE gamma using a solid-phase assay. The peptides were also tested for ability to activate a PDE complex. We have identified a region, residues 250-275 of T alpha, which shows a high affinity of PDE gamma and for the PDE gamma (24-45) binding peptide. The peptide did not bind to the C-terminal residues 50-87 of PDE gamma. Likewise, a region of T alpha, 1-25 did not exhibit high-affinity binding to PDE gamma or to the 24-45 PDE gamma peptide. Specific binding of the 250-275 peptide to PDE gamma was confirmed by its ability to compete with T alpha binding to PDE gamma, although a higher concentration was required (10x). The T alpha-(250-275) peptide activated a fully inhibited PDE alpha beta gamma 2 complex in a dose-dependent manner. These results suggest that a region on T alpha that recognizes the PDE gamma-binding site is found within residues 250-275 of T alpha.
Effect on ligand binding of arginine mutations in recombinant rat liver fatty acid-binding protein
Authors: Thumser A E , Evans C , Worrall A F , Wilton D C
Published: August 2015

Rat liver fatty acid-binding protein is able to accommodate a wide range of non-polar anions in addition to long-chain fatty acids. The two arginine residues of rat liver fatty acid-binding protein, Arg122 and Arg126, have been mutated and the effect of mutation on ligand binding investigated. No significant decrease in affinity for the fluorescent fatty acid analogue, 11-(5-dimethylaminonaphthalenesulphonyl amino)undecanoic acid, or oleate was observed. However, the apparent affinity for oleoyl-CoA was slightly increased with the mutations Ala122 and Gln122 such that oleoyl-CoA rather than oleate became the preferred ligand for these mutants. Small changes in protein stability were observed with the Arg122 mutations. The lack of notable ionic involvement of the conserved internal residue Arg122 in ligand binding is consistent with the hypothesis that the mode of ligand binding in liver fatty acid-binding protein is markedly different from that of other members of this lipid-binding protein family.