bluuebunny/crossref_metadata_2025 huggingface dataset:
Co-expression of the α subunit of human prolyl 4-hydroxylase with BiP polypeptide in insect cells leads to the formation of soluble and insoluble complexes. Soluble α-subunit-BiP complexes have no prolyl 4-hydroxylase activity
Authors: Veijola J , Pihlajaniemi T , Kivirikko K
Published: August 2015

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyses the post-translational formation of 4-hydroxyproline in collagens. The vertebrate enzymes are α2β2 tetramers, their β subunit being identical to protein disulphide isomerase (PDI). The function of the PDI-β subunit in prolyl 4-hydroxylases is not fully understood, but it seems to be that of keeping the highly insoluble α subunits in solution. We report here that expression of the α subunit of human type I prolyl 4-hydroxylase in insect cells together with BiP polypeptide leads to the formation of both soluble and insoluble α-subunit–BiP complexes. Formation of the soluble complexes was evident from (1) a marked increase in the amount of the α subunit in the soluble fraction of the cell homogenates when expressed together with BiP, (2) immunoprecipitation experiments and (3) demonstration of the presence of some of the complexes by polyacrylamide gel electrophoresis under non-denaturing conditions. Formation of the insoluble complexes was suggested by an increase in the amount of BiP in the insoluble fraction when expressed together with the α subunit. Nevertheless the soluble α-subunit–BiP complexes had no prolyl 4-hydroxylase activity. This indicates that the function of the PDI-β subunit in the prolyl 4-hydroxylase tetramer is not only that of keeping the α subunits in solution but appears to be more specific, probably that of keeping them in a catalytically active, non-aggregated conformation.
Purification and characterization of a recombinant human Theta-class glutathione transferase (GSTT2-2)
Authors: TAN Kian-Leong , BOARD Philip G.
Published: August 2015

A cDNA encoding the human Theta-class glutathione transferase GSTT2-2 was expressed in Escherichia coli as a ubiquitin fusion protein. The co-translational removal of the ubiquitin by a cloned ubiquitin-specific protease, Ubp1, generates enzymically active GSTT2-2 without any additional N-terminal residues. The recombinant isoenzyme was purified to apparent homogeneity by DEAE anion-exchange, gel filtration, dye ligand chromatography and high resolution anion-exchange chromatography on Mono Q FPLC. The recombinant enzyme had significant activity with a range of substrates, including cumene hydroperoxide and 1-menapthyl sulphate. The activity of GSTT2-2 with a range of secondary lipid peroxidation products such as the trans,trans-alka-2,4-dienals and trans-alk-2-enals, as well as its glutathione peroxidase activity with organic hydroperoxides, suggest that it may play a significant role in protection against the products of lipid peroxidation.
Oxytocin receptor couples to the 80 kDa Ghα family protein in human myometrium
Authors: Jin BAEK Kwang , KWON Nyoun Soo , LEE Hee Sung , KIM Myoung Soo , MURALIDHAR Pallaki , IM Mie-Jae
Published: August 2015

One of the primary functions of the oxytocin receptor is to modulate intracellular calcium levels in myometrium. The oxytocin receptor has been purified and cloned. Although it has been suggested that oxytocin receptor couples with a GTP-binding regulatory protein (G-protein), the identity of this G-protein remains unclear. To elucidate the mechanism of oxytocin receptor signalling, we used the oxytocin–receptor–G-protein ternary complex preparation from human myometrium, and evaluated oxytocin-mediated activation of [35S]guanosine 5´-[γ-thio]triphosphate ([35S]GTP[S]) binding and [α-32P]GTP photoaffinity labelling to a G-protein. Binding of [35S]GTP[S] and the intensity of the [α-32P]GTP photoaffinity labelled protein resulting from activation of the oxytocin receptor were significantly attenuated by the selective oxytocin antagonist, desGlyNH2d(CH2)5[Tyr(Me)2,Thr4]OVT. Furthermore, the molecular mass of the specific GTP-binding protein was ~80 kDa; homologous with the Ghα family, the new class of GTP-binding proteins first identified in rat liver that couples to the α1B-adrenoceptor. Consistent with these observations, in co-immunoprecipitation and co-immunoadsorption of the oxytocin receptor in the ternary complex preparation by anti-Gh7α antibody, the Ghα family protein tightly coupled to the oxytocin receptor. These findings demonstrate that oxytocin receptor couples with ~80 kDa Ghα in signal mediation.
Identification of a new isoform of cell—cell adhesion molecule 105 (C-CAM), C-CAM4: a secretory protein with only one Ig domain
Authors: EARLEY Karen , LUO Weiping , QIU Yuhong , THOMPSON Nancy L. , CHOU Janice , HIXSON Douglas C. , LIN Sue-Hwa
Published: August 2015

A series of Southern blot hybridization experiments using probes derived from different regions of the rat liver cell–cell adhesion molecule 105 (C-CAM) cDNA revealed the presence of a 9.6 kb EcoRI genomic fragment that seemed to encode a unique C-CAM isoform. An RNase protection study showed that this C-CAM transcript was expressed in placenta, spleen, lung and large intestine. In contrast, the other C-CAM isoforms, C-CAM1 and C-CAM2, are expressed in liver and small intestine. This result also suggests that the new isoform, which we named C-CAM4, was indeed encoded by a new C-CAM gene. A rat placenta cDNA library was then screened and the full-length cDNA coding for C-CAM4 was isolated. The deduced protein contained 142 amino acids and had a calculated molecular mass of 15 kDa. C-CAM4 was composed of a leader sequence and the first V-like Ig domain typical of C-CAM-family proteins. However, C-CAM4 lacked the C-like Ig domains, the transmembrane domain, and the cytoplasmic domain found in other C-CAM isoforms. Thus, C-CAM4 is different from the other known C-CAMs in that it is a secreted protein. We have previously shown that the first Ig domain of C-CAM1 is crucial for its adhesion function. The V-like Ig domain of C-CAM4 had 92% and 89% sequence identity with the corresponding regions of C-CAM1 and C-CAM2 respectively. Together these results suggest that C-CAM4 may play a role in regulating the function of other C-CAM family proteins.
Phosphorylation of a membrane-intercalated proteoglycan, syndecan-2, expressed in a stroma-inducing clone from a mouse Lewis lung carcinoma
Authors: ITANO Naoki , OGURI Kayoko , NAGAYASU Yuko , KUSANO Yuri , NAKANISHI Hayao , DAVID Guido , OKAYAMA OKAYAMA
Published: August 2015

We previously reported that a mouse Lewis lung carcinoma-derived stroma-inducing clone, P29, highly expresses a syndecan-like proteoglycan exhibiting specific binding to fibronectin, a major constituent of the interstitial matrix formed by the induced stromal cells, via its heparan sulphate chains [Itano, Oguri, Nakanishi and Okayama (1993) J. Biochem. (Tokyo) 114, 862–873]. On metabolic labelling of the proteoglycan with [32P]Pi, followed by identification of the radiolabelled material using glycanases, almost all the isotope was found to have been incorporated into a core portion of molecular mass 48 kDa, which was generated by digestion with heparan sulphate lyase I plus chondroitin ABC lyase. Immunoblotting of the core protein with a monoclonal antibody, F58-6G12, demonstrated that the proteoglycan was mouse syndecan-2. CsCl-density-gradient centrifugation after mild treatment of liposome-intercalated 32P-labelled syndecan-2 with trypsin resulted in clear separation of the radioactivity into a bottom fraction containing all the glycosaminoglycans (accounting for 40% of the total radioactivity) and a top fraction containing liposome-associated peptides (60%). The former isotope was shown to be linked covalently to both heparan sulphate and chondroitin sulphate chains, probably at their bridge regions. The latter was mostly attributed to phosphoserine, the one and only phosphorylated amino acid released on acid hydrolysis of this proteoglycan, strongly suggesting that the phosphorylation occurs at a specific serine residue(s) in the cytoplasmic domain of the core protein.
Degradation of pyrene-labelled phospholipids by lysosomal phospholipases in vitro. Dependence of degradation on the length and position of the labelled and unlabelled acyl chains
Authors: Lusa S , Myllarniemi M , Volmonen K , Vauhkonen M , Somerharju P
Published: August 2015

The hydrolysis of pyrenylacyl phosphatidylcholines (PyrnPCs) (n indicates the number of aliphatic carbons in the pyrene-chain) by crude lysosomal phospholipases in vitro was investigated. PyrnPCs consist of several sets in which the length of the pyrene-labelled or the unlabelled acyl chain, linked to the sn-1 or sn-2 position, was systematically varied. Lysophosphatidylcholine and fatty acid were the only fluorescent breakdown products detected, thus indicating that PyrnPCs were degraded by A-type phospholipases and lysophospholipases. Of these, mainly A1-type phospholipases appear to be involved, as determined from the relative amounts of labelled fatty acid and lysolipid released from the positional isomers. Based on the effects of the length and position of the pyrene-labelled and unlabelled chains it is suggested that (1) the lysosomal A-type phospholipases acting on PyrnPCs recognize the carboxy-terminal part of the lipid acyl chains and (2) the relevant part of the binding site is relatively narrow. Thus phospholipids with added bulk in the corresponding region, such as those that are peroxidized and polymerized, may not be good substrates for the lysosomal phospholipases mentioned. The impaired hydrolysis of the most hydrophobic PyrnPCs indicates that lysosomal phospholipases may not be able to penetrate significantly into the substrate interphase, but upward movement of the lipid may be required for efficient hydrolysis. Finally, the rate of hydrolysis of many pyrenyl derivatives was found to be comparable to that of a natural phosphatidylcholine species, both in micelles and in lipoprotein particles, indicating that these derivatives can be used as faithful reporters of lysosomal degradation of natural lipids in vivo and in vitro.
Marked alteration of proteoglycan metabolism in cholesterol-enriched human arterial smooth muscle cells
Authors: VIJAYAGOPAL Parakat , FIGUEROA Julio E. , GUO Qi , FONTENOT Jason D. , TAO Zhuo
Published: August 2015

To elucidate the correlation between vascular cholesterol metabolism and proteoglycan (PrGl) biosynthesis, we investigated PrGl synthesis in human aortic smooth muscle cells (SMCs) after cholesterol enrichment with cationized low-density lipoproteins (LDL). Compared with normal SMCs, total PrGl synthesis by cholesterol-enriched cells decreased 2.4-fold (11874±530 d.p.m. per 105 cells compared with 4890±385 d.p.m. per 105 cells). This was the net result of a 6.9-fold reduction in medium PrGl (11000±490 d.p.m. per 105 cells compared with 1580± 246 d.p.m. per 105 cells) and a 3.8-fold increase in cellular PrGl over controls (874±27 d.p.m. per 105 cells compared with 3310±193 d.p.m. per 105 cells). Prior incubation of SMCs with native LDL had no effect on PrGl synthesis by these cells. The decrease in PrGl synthesis in cholesterol-enriched cells correlated with a 90% and 20% reduction in the steady-state level of mRNA for biglycan and decorin respectively, and a virtual elimination of the steady-state level of mRNA for versican over controls. Despite the down-regulation of PrGl synthesis, cholesterol-loaded cells produced a 2-fold increase in a PrGl subfraction with high affinity for LDL. Compared with the corresponding PrGl subfraction from normal cells, that from the cholesterol-enriched cells exhibited increased charge density and a higher molecular mass and contained relatively larger proportions of chondroitin 6-sulphate and dermatan sulphate. These results show that PrGl metabolism is dramatically altered in cholesterol-enriched human SMCs.
Isolation of InsP4 and InsP6 binding proteins from human platelets: InsP4 promotes Ca2+ efflux from inside-out plasma membrane vesicles containing 104 kDa GAP1IP4BP protein
Authors: O'ROURKE Flavia , MATTHEWS Eileen , FEINSTEIN Maurice B.
Published: August 2015

A low-density membrane fraction from human platelets contained the plasma membrane marker glycoprotein Ib (GpIb) and selective binding sites for InsP4 and InsP6. It was separated from the bulk of InsP3-receptor-containing membranes, but was heterogeneous, probably also containing surface-connected canalicular system and some lighter elements of the internal dense tubule system. After loading with calcium oxalate and re-centrifugation on Percoll gradients, this mixed fraction was subfractionated into light membranes containing all of the GpIb, high-affinity InsP4 binding sites (KD = 18 nM) and phosphate-stimulated Ca2+ transport activity. InsP4 (EC50 0.6 μM), but not InsP3 or InsP6, released up to 35% of the accumulated Ca2+ from these vesicles, which were shown to be inside-out plasma membrane vesicles by a biotinylation labelling technique and selective removal of right-side-out plasma membrane vesicles with streptavidin–agarose. Most of the InsP4, and all of the InsP6, binding was present in the much denser calcium oxalate-loaded subfractions, which were free of GpIb. InsP6 binding activity was chromatographically purified as a 116 kDa protein (KD for InsP6 = 5.9 nM), with an amino acid content and two internal peptide sequences identical to those of 116 kDa vinculin. A 104 kDa InsP4 binding protein (KD for InsP4 = 12 nM), probably identical to GAP1IP4BP described by Cullen, Hsuan, Truong, Letcher, Jackson, Dawson and Irvine [(1995) Nature (London) 376, 527–530], was also isolated. This InsP4 receptor may mediate Ca2+ influx in platelets that occurs subsequent to receptor-stimulated production of InsP3 and unloading of internal Ca2+ stores.
Sphingolipid metabolites differentially regulate extracellular signal-regulated kinase and stress-activated protein kinase cascades
Authors: CORONEOS Emmaneul , WANG Yizheng , PANUSKA James R. , TEMPLETON Dennis J. , KESTER Mark
Published: August 2015

The mitogen-activated protein kinase (MAPK) signalling pathway serves to translocate information from activated plasma-membrane receptors to initiate nuclear transcriptional events. This cascade has recently been subdivided into two analogous pathways: the extracellular signal-regulated kinase (ERK) cascade, which preferentially signals mitogenesis, and the stress-activated protein kinase (SAPK) cascade, which is linked to growth arrest and/or cellular inflammation. In concurrent experiments utilizing rat glomerular mesangial cells (MCs), we demonstrate that growth factors or sphingosine activate ERK but not SAPK. In contrast, inflammatory cytokines or cell-permeable ceramide analogues activate SAPK but not ERK. Ceramide, but not sphingosine, induces interleukin-6 secretion, a marker of an inflamed phenotype. Moreover, ceramide can suppress growth factor- or sphingosine-induced ERK activation as well as proliferation. These studies implicate sphingolipid metabolites as opposing regulators of cell proliferation and inflammation through activation of separate kinase cascades.
Recombinant bovine conglutinin, lacking the N-terminal and collagenous domains, has less conglutination activity but is able to inhibit haemagglutination by influenza A virus
Authors: EDA Souji , SUZUKI Yasuhiko , KASE Tetsuo , KAWAI Takao , OHTANI Katsuki , SAKAMOTO Takashi , KURIMURA Takashi , WAKAMIYA WAKAMIYA
Published: August 2015

Conglutinin is a bovine serum protein which was first described as a vertebrate lectin. This protein belongs to the family of C-type lectins. These lectins are composed of four characteristic domains: (1) an N-terminal cysteine-rich domain, (2) a collagen-like domain, (3) a neck domain and (4) a carbohydrate recognition domain (CRD). Recently lectins have been shown to function as immunoglobulin-independent defence molecules due to a complement-mediated mechanism or opsonization. Our previous study showed that bovine conglutinin can inhibit haemagglutination by influenza A viruses and act by directly neutralizing them due to its lectin properties. In order to elucidate the biological role of the collagen-like domain, a recombinant partial conglutinin lacking this collagen-like domain was produced in an Escherichia coli system and its biological activities were examined. A 497 bp sequence, consisting of a short collagen region (two repeats of G-X-Y amino acid sequences), the neck domain, and the CRD of conglutinin cDNA, was amplified by the reverse-transcriptase PCR technique. The cDNA was transferred to a bacterial expression vector system (pRSET-A) and stable transfectants with a high level of conglutinin production were obtained. SDS/PAGE and Western blotting analyses showed a recombinant fusion protein of 27 kDa. Results of a cross-linking study and gel-filtration assay indicated that the recombinant conglutinin can form a trimeric structure and that it has sugar binding activity and specificity similar to that of native conglutinin. The recombinant conglutinin was also found to inhibit haemagglutination caused by influenza A virus as well as to possess less conglutination activity. These results suggest that in order for conglutinin to inhibit haemagglutination caused by the influenza virus, as well as to have sugar binding activity or to form trimers, it does not require the N-terminal and collagenous domains; however, they are essential for full conglutination activity.
Lysophosphatidic acid stimulates glucose transport in Xenopus oocytes via a phosphatidylinositol 3′-kinase with distinct properties
Authors: THOMSON Fiona J. , MOYES Colin , SCOTT Pamela H. , PLEVIN Robin , GOULD Gwyn W.
Published: August 2015

Lysophosphatidic acid (LPA) stimulated the transport of deoxyglucose into oocytes isolated from Xenopus laevis. This stimulation was accounted for entirely by an increase in the Vmax for transport. Various LPAs with different acyl groups in the sn-1 position and phosphatidic acid stimulated deoxyglucose (deGlc) transport in these cells with a rank order potency of 1-oleoyl-LPA > 1-palmitoyl-LPA > phosphatidic acid = 1-stearoyl-LPA > 1-myristoyl-LPA. The phosphatidylinositol 3´-kinase inhibitor LY294002 completely blocked LPA-stimulated deoxyglucose uptake (IC50 ~2 μM). In marked contrast, wortmannin, which can completely block both insulin-like growth factor-I (IGF-I)-stimulated deGlc uptake in oocytes and phosphatidylinositol 3´-kinase activation at concentrations as low as 20 nM [Gould, Jess, Andrews, Herbst, Plevin and Gibbs (1994) J. Biol. Chem. 269, 26622–26625], was a relatively poor inhibitor of LPA-stimulated deGlc transport, even at concentrations as high as 100 nM. We further show that LPA stimulates phosphatidylinositol 3´-kinase activity(s) that can phosphorylate both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate, and that this stimulation is inhibited by LY294002 but is relatively insensitive to wortmannin, again in marked contrast to IGF-I-stimulated phosphatidylinositol 3´-kinase activity. Antibodies against the p85 regulatory subunit of phosphatidylinositol 3´-kinase or antiphosphotyrosine antibodies immunoprecipitated IGF-I-stimulated but not LPA-stimulated phosphatidylinositol 3´-kinase activity. We conclude that LPA stimulates glucose uptake in Xenopus oocytes by a mechanism that may involve activation of a form of phosphatidylinositol 3´-kinase that is distinguished from other isoforms by its resistance to wortmannin and by its substrate specificity. Since the LPA-activated form of phosphatidylinositol 3´-kinase is pharmacologically and immunologically distinct from that which is involved in IGF-I-stimulated glucose transport in these cells, we suggest that distinct isoforms of this enzyme are able to function with the same biological effect, at least in the regulation of sugar transport.
Reversibility of biotin-binding by selective modification of tyrosine in avidin
Authors: MORAG Ely , BAYER Edward A. , WILCHEK Meir
Published: August 2015

The tight interaction between the vitamin biotin and the protein avidin is so strong (Ka ~1015 M-1) that conditions which are usually sufficient for protein denaturation fail to dissociate the avidin–biotin complex. In order to form a reversible interaction between the two biomolecules, we have modified the binding-site tyrosine by nitration, thus reducing the pKa of the phenol group which forms a crucial hydrogen bond with the ureido group of biotin. At relatively low pH values (4–5), the resultant modified forms of avidin bind biotin with a very high association constant (> 109 M-1). The modified avidins are thus capable of supporting stable, long-term binding of biotin or biotinylated macromolecules. The latter molecules can be detached by increasing the pH of the medium or by introduction of excess levels of biotin at neutral pH. These findings demonstrate the importance of a single hydrogen bond for strong biotin binding. The new derivatives of avidin should be useful for applications whereby a reversible interaction between the four biotin-binding sites and biotin is desired, thus increasing the versatility of the avidin–biotin system for biotechnological application.
The catalase-peroxidase of Synechococcus PCC 7942: purification, nucleotide sequence analysis and expression in Escherichia coli
Authors: MUTSUDA Michinori , ISHIKAWA Takahiro , TAKEDA Toru , SHIGEOKA Shigeru
Published: August 2015

Synechococcus PCC 7942, a cyanobacterium, possesses catalase–peroxidase as the sole hydrogen peroxide-scavenging system. The enzyme has been purified to electrophoretic homogenenity from the cells. The native enzyme had a molecular mass of 150 kDa and was composed of two identical subunits of molecular mass 79 kDa. The apparent Km value of the catalase activity for H2O2 was 4.2±0.27 mM and the kcat value was 2.6×104 s-1. The enzyme contained high catalase activity and an appreciable peroxidase activity with o-dianisidine and pyrogallol. The catalase activity was not inhibited by 3-amino-1,2,4-triazole but by KCN and NaN3 (apparent Ki values 19.3±0.84 and 20.2±0.95 μM respectively). The enzyme showed an absorption spectrum of typical protohaem and contained one protohaem molecule per dimer. The gene encoding catalase–peroxidase was cloned from the chromosomal DNA of Synechococcus PCC 7942. A 2160 bp open reading frame (ORF), coding a catalase–peroxidase of 720 amino acid residues (approx. 79.9 kDa), was observed. The deduced amino acid sequence coincided with that of the N-terminus of the purified enzyme and showed a remarkable similarity to those of a family of catalase–peroxidases of prokaryotic cells. Escherichia coli BL21(DE3)plysS, harbouring a recombinant plasmid containing the catalase–peroxidase gene, produced a large amount of proteins that co-migrated on SDS/PAGE with the native enzyme. The recombinant enzyme showed the same ratio of catalase activity to peroxidase activity with o-dianisidine and the same Km for H2O2 as the native enzyme.
Co-operative interactions of oligonucleosomal DNA with the H1e histone variant and its poly(ADP-ribosyl)ated isoform
Authors: D'ERME Maria , ZARDO Giuseppe , REALE Anna , CAIAFA Paola
Published: August 2015

H1 histone somatic variants from L929 mouse fibroblasts were purified by reverse-phase HPLC. We analysed the ability of each H1 histone variant to allow the H1–H1 interactions that are essential for the formation of the higher levels of chromatin structure, and we investigated the role played by the poly(ADP-ribosyl)ation process. Cross-linking analysis showed that H1e is the only somatic variant which, when bound to DNA, is able to produce H1–H1 polymers; the size of polymers was decreased when H1e was enriched in its poly(ADP-ribosyl)ated isoform. Measurement of the methyl-accepting ability in native nuclei compared with nuclei in which poly(ADP-ribosyl)ation was induced showed that the poly(ADP-ribosyl)ated H1 histone had not been removed from linker regions, in spite of its different interaction with DNA.
Dimerization of truncated melittin analogues results in cytolytic peptides
Authors: RIVETT Donald E. , KIRKPATRICK Alan , HEWISH Dean R. , REILLY Wayne , WERKMEISTER Jerome A.
Published: August 2015

A synthetic peptide with the sequence of the first 20 residues of melittin and terminating with an additional cysteine amide was found to have cytolytic activity similar to that of melittin. It was apparent from MS data that the cysteine-terminating peptides had formed disulphide dimers. A peptide in which the thiol was blocked by iodoacetate showed no activity, whereas the same peptide blocked by acetamidomethyl showed activity marginally less haemolytic than that of melittin. Cytolytic activity of melittin analogues comprising the full 26 residues could be obtained with wide sequence permutations providing that a general amphipathic helical structure was preserved. In contrast, the activity of the dimers was dependent not only on retention of an amphipathic helix but also on certain individual residues and a free positive charge. A free N-terminus was essential for haemolytic activity. In addition, a lysine or arginine residue at position 7 and a proline at position 14 were found to be necessary for activity, although it was apparent that additional residues are important for retention of the full lytic potential.
Regulation of proliferation of LLC-MK2 cells by nucleosides and nucleotides: the role of ecto-enzymes
Authors: LEMMENS Raf , VANDUFFEL Luc , TEUCHY Henri , CULIC Ognjen
Published: August 2015

1. Using the incorporation of [methyl-3H]thymidine as a proliferation marker, the effects of various nucleosides and nucleotides on endothelial LLC-MK2 cells were studied. We found that ATP, ADP, AMP and adenosine in concentrations of 10 μM or higher stimulate the proliferation of these cells. 2. Inhibition of ecto-ATPase (EC 3.6.1.15), 5´-nucleotidase (EC 3.1.3.5) or alkaline phosphatase (EC 3.1.3.1) significantly diminished the stimulatory effect of ATP, indicating that the effect is primarily caused by adenosine and not by adenine nucleotides. Also, the effect depends only on extracellular nucleosides, since inhibition of nucleoside uptake by dipyridamole has no influence on proliferation. 3. Other purine nucleotides and nucleosides (ITP, GTP, inosine and guanosine) also stimulate cell proliferation, while pyrimidine nucleotides and nucleosides (CTP, UTP, cytidine and uridine) inhibit proliferation. Furthermore, the simultaneous presence of adenosine and any of the other purine nucleosides is not entirely additive in its effect on cell proliferation. At the same time any pyrimidine nucleoside, when added together with adenosine, has the same inhibitory effect as the pyrimidine nucleoside alone. 4. Apparently these proliferative effects are neither caused by any pharmacologically known P1-purinoceptor, nor are they mediated by cyclic AMP, cyclic GMP, or D-myo-inositol 1,4,5-trisphosphate as second messenger, nor by extracellular Ca2+. 5. Therefore, we conclude that various purine and pyrimidine nucleosides can influence the proliferation of LLC-MK2 cells by acting on putative purinergic and pyrimidinergic receptors not previously described.
Differential distribution of ferulic acid to the major plasma constituents in relation to its potential as an antioxidant
Authors: CASTELLUCCIO Cinzia , BOLWELL G. Paul , GERRISH Christopher , RICE-EVANS Catherine
Published: August 2015

The hydroxycinnamates, intermediates in the phenylpropanoid synthetic pathway, are effective in enhancing the resistance of low-density lipoprotein (LDL) to oxidation in the order caffeic acid > ferulic acid > p-coumaric acid. It is unclear whether the mode of action of ferulic acid as an antioxidant is based on its activities in the aqueous or the lipophilic phase. Partitioning of 14C-labelled ferulic acid into plasma and its components, LDL and the albumin-rich fractions, has been studied under conditions of maximum aqueous solubility. The majority of ferulic acid associates with the albumin-rich fraction of the plasma, although a proportion is also found to partition between the LDL and aqueous phases; however, ferulic acid does not associate with the lipid portion of the LDL particle, suggesting that it exerts its antioxidant properties from the aqueous phase. This is of particular interest since the results demonstrate that ferulic acid is a more effective antioxidant against LDL oxidation than the hydrophilic antioxidant ascorbic acid.
Effects of the type of divalent cation, Ca2+ or Mg2+, bound at the high-affinity site and of the ionic composition of the solution on the structure of F-actin
Authors: STRZELECKA-GOLASZEWSKA Hanna , WOZNIAK Aleksandra , HULT Thomas , LINDBERG Uno
Published: August 2015

F-actins containing either Ca2+ or Mg2+ at the single high-affinity site for a divalent cation differ in their dynamic properties [Carlier (1991) J. Biol. Chem. 266, 1–4]. In an attempt to obtain information on the structural basis of this difference, we probed the conformation of specific sites in the subunits of Mg- and Ca-F-actin with limited proteolysis by subtilisin and trypsin. The influence of the kind of polymerizing salt was also investigated. At high proteinase concentrations required for digestion of actin in the polymer form, subtilisin gives a complex fragmentation pattern. In addition to the earlier known cleavage between Met47 and Gly48 in the DNAse-I-binding loop, cleavage of F-actin between Ser234 and Ser235 in subdomain 4 has recently been reported [Vahdat, Miller, Phillips, Muhlrad and Reisler (1995) FEBS Lett. 365, 149–151]. Here we show that actually a larger segment, comprising residues 227–235, is removed and the bond between Leu67 and Lys68 in subdomain 2 is split in both G- and F-actin, and that the differences in the fragmentation patterns of the G- and F-forms are accounted for by the protection of the bond 47–48 in F-actin. The subtilisin and trypsin cleavage sites in segment 61–69, subtilisin sites in segment 227–235 and trypsin sites between Lys373 and Cys374 were less accessible in Mg-F-actin than in Ca-F-actin. These are intramolecular effects, as similar changes were observed on Ca2+/Mg2+ replacement in G-actin. The cation-dependent effects, in particular those on segment 61–69, were however less pronounced in F-actin than in G-actin. The results suggest that substitution of Mg2+ for Ca2+, and KCl-induced polymerization of CaATP-G-actin, bring about a similar change in the conformation of subdomain 2 of the monomer. The presence of Mg2+ at the high-affinity site also resulted in an increased protection of the bond 47–48. This latter appears to be an intermolecular effect because it is specific for F-actin. The susceptibility to subtilisin and trypsin was also strongly influenced by the kind and concentration of polymerizing salt. The digestion patterns suggest that the exposure and/or flexibility of the regions containing the cleavage sites diminish with enhancement of the ionic strength of the solution. The results are discussed in terms of the current models of F-actin.
Phosphorylation of a cAMP-specific phosphodiesterase (HSPDE4B2B) by mitogen-activated protein kinase
Authors: LENHARD James M. , KASSEL Daniel B. , ROCQUE Warren J. , HAMACHER Lawrence , HOLMES William D. , PATEL Indravadan , HOFFMAN Christine , LUTHER Michael
Published: August 2015

A cAMP-specific phosphodiesterase, HSPDE4B2B, was found to be phosphorylated when expressed in Sf 9 cells or yeast. Deletion of amino acids 81–151 and 529–564 had no effect on the phosphorylation of HSPDE4B2B. Mass spectrometric analysis of purified HSPDE4B2B(1–564), HSPDE4B2B(81–564) and HSPDE4B2B(152–528) showed that phosphorylation occurred predominantly on Ser487 and Ser489. The stoicheiometry of phosphorylation was 1.2:1 (Ser487:Ser487, 489). There was no evidence by MS for a non-phosphorylated form of HSPDE4B2B(81–564) or HSPDE4B2B(152–528) when expressed in Sf 9 cells. There was no detectable phosphorylation of purified HSPDE4B2B(152–528) expressed in Escherichia coli. Radiolabelling experiments with 32P revealed that phosphorylation of HSPDE4B2B(152–528) expressed in Sf 9 cells was abolished when Ser487 and Ser489 were mutated to alanines. Analysis of the amino acid sequence revealed that Ser487 and Ser489 of HSPDE4B2B conform to the consensus motifs for phosphorylation by mitogen-activated protein kinase (MAP kinase) and casein kinase II respectively. Kinetic experiments in vitro showed that MAP kinase-phosphorylated E. coli expressed and purified HSPDE4B2B(151–528) with a Km of 63 μM and a Vmax of 3.0 μmol/min per mg. In comparison, MAP kinase phosphorylated myelin basic protein with a Km of 26.0 μM and a Vmax of 5.5 μmol/min per mg under the same conditions. Using MS and mutational analysis we found that MAP kinase-phosphorylated E. coli expressed HSPDE4B2B(152–528) exclusively at Ser487. These results suggest that phosphodiesterases could contribute to the cross-talk between the cAMP and MAP kinase signalling pathways.
Demonstration that 1-trans-epoxysuccinyl-l-leucylamido-(4-guanidino) butane (E-64) is one of the most effective low Mr inhibitors of trypsin-catalysed hydrolysis. Characterization by kinetic analysis and by energy minimization and molecular dynamics simulation of the E-64-β-trypsin complex
Authors: SREEDHARAN Suneal K. , VERMA Chandra , CAVES Leo S. D. , BROCKLEHURST Simon M. , GHARBIA Saheer E. , SHAH Haroun N. , BROCKLEHURST Keith
Published: August 2015

1-trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64) was shown to inhibit β-trypsin by a reversible competitive mechanism; this contrasts with the widely held view that E-64 is a class-specific inhibitor of the cysteine proteinases and reports in the literature that it does not inhibit a number of other enzymes including, notably, trypsin. The Ki value (3×10-5 M) determined by kinetic analysis of the hydrolysis of Nα-benzoyl-L-arginine 4-nitroanilide in Tris/HCl buffer, pH 7.4, at 25 °C, I = 0.1, catalysed by β-trypsin is comparable with those for the inhibition of trypsin by benzamidine and 4-aminobenzamidine, which are widely regarded as the most effective low Mr inhibitors of this enzyme. Computer modelling of the β-trypsin–E-64 adsorptive complex, by energy minimization, molecular dynamics simulation and Poisson–Boltzmann electrostatic-potential calculations, was used to define the probable binding mode of E-64; the ligand lies parallel to the active-centre cleft, anchored principally by the dominant electrostatic interaction of the guanidinium cation at one end of the E-64 molecule with the carboxylate anion of Asp-171 (β-trypsin numbering from Ile-1) in the S1-subsite, and by the interaction of the carboxylate substituent on C-2 of the epoxide ring at the other end of the molecule with Lys-43; the epoxide ring of E-64 is remote from the catalytic site serine hydroxy group. The possibility that E-64 might bind to the cysteine proteinases clostripain (from Clostridium histolyticum) and α-gingivain (one of the extracellular enzymes from Porphyromonas gingivalis) in a manner analogous to that deduced for the β-trypsin–E-64 complex is discussed.
Recombinant human tumour necrosis factor-α suppresses synthesis, activity and secretion of lipoprotein lipase in cultures of a human osteosarcoma cell line
Authors: SAKAYAMA Kenshi , MASUNO Hiroshi , OKUMURA Hideo , SHIBATA Taihoh , OKUDA Hiromichi
Published: August 2015

The effect of recombinant human tumour necrosis factor-α (TNF-α) on synthesis, activity and secretion of lipoprotein lipase (LPL) was examined using a human osteosarcoma cell line, osteosarcoma Takase (OST). Treatment of OST cells with TNF-α decreased LPL synthesis, resulting in a decrease in expression of activity and secretion of LPL. When OST cells were incubated with glycerol tri[1-14C]palmitate, TNF-α decreased dose- and time-dependently the production of 14CO2 and the amounts of radioactivity incorporated into cellular triacylglycerol and phospholipid. The similar reduction of synthesis and activity of LPL as suppression of CO2 production and cellular lipid synthesis indicated that the suppression of 14CO2 production and 14C-labelled lipid synthesis was secondary. TNF-α also suppressed expression of proliferating cell nuclear antigen, indicating that it had an anti-proliferative activity on OST cells. The findings suggest that one cause of the anti-proliferative activity of TNF-α is the suppression of the LPL-mediated supply of non-esterified fatty acids as an energy source for growth.
Evidence for a non-capacitative Ca2+ entry during [Ca2+] oscillations
Authors: SHUTTLEWORTH Trevor J. , THOMPSON Jill L.
Published: August 2015

Current models for the agonist-induced activation of Ca2+ entry from the extracellular medium in non-excitable cells generally emphasize a capacitative mechanism whereby Ca2+ entry is activated simply as a result of the emptying of intracellular Ca2+ stores, without any direct involvement of inositol phosphates. To date, the activation and control of Ca2+ entry have generally been studied under conditions where the agonist-sensitive stores undergo a profound and sustained depletion. However, responses under more normal physiological conditions typically involve the cyclical release and refilling of the stores associated with oscillations in [Ca2+], and the nature and control of entry under these conditions has received relatively little attention. In this study, using isolated cells from the exocrine avian nasal gland as a model system, we show that: (a) the agonist-enhanced rate of Mn2+ quench is independent of the cyclical emptying and refilling of the agonist-sensitive Ca2+ pool during oscillations; (b) the Ca2+ entry pathway is maintained in an activated state for extended periods following inhibition of oscillations under conditions in which agonist-sensitive stores can be shown to be full; (c) no Ca2+ entry could be detected in oscillating cells in experiments that followed a definitive protocol for the demonstration of capacitative entry; and (d) on initial exposure to low agonist concentrations, activation of Ca2+ entry preceded any detectable release of Ca2+ from the stores. We conclude that the essential characteristics of the control of Ca2+ entry during oscillations are incompatible with current capacitative models.
Protein kinase C isoforms ɛ, η, δ and ζ in murine adipocytes: expression, subcellular localization and tissue-specific regulation in insulin-resistant states
Authors: FREVERT Ernst U. , KAHN Barbara B.
Published: August 2015

The Ca2+-insensitive protein kinase C (PKC) isoforms ε, η, δ and ζ are possible direct downstream targets of phosphatidylinositol 3-kinase (PI3-K), and might therefore be involved in insulin signalling. Although isoform-specific changes in PKC expression have been reported for skeletal muscle and liver in insulin-resistant states, little is known about these isoforms in adipocytes. Therefore we studied (1) expression and subcellular localization of these isoforms in murine adipocytes, (2) translocation of specific isoforms to membranes in response to treatment with insulin and phorbol 12-myristate 13-acetate (PMA) and (3) regulation of expression in insulin-resistant states. The PKC isoforms ε, η, δ and ζ are expressed in adipocytes. Immunoreactivity for all isoforms is higher in the membranes than in the cytosol, but subcellular fractionation by differential centrifugation shows an isoform-specific distribution within the membrane fractions. PMA treatment of adipocytes induces translocation of PKC-ε and -δ from the cytosol to the membrane fractions. Insulin treatment does not alter the subcellular distribution of any of the isoforms. 3T3-L1 adipocytes express PKC-ε and -ζ, and PKC-ε expression increases with differentiation from preadipocytes to adipocytes. PKC-ε expression decreases in an adipose-specific and age/obesity-dependent manner in two insulin-resistant models, the brown-adipose-tissue-deficient mouse and db/db mouse compared with control mice. We conclude that, although none of the isoforms investigated seems to be activated by insulin, the decrease in PKC-ε expression might contribute to metabolic alterations in adipocytes associated with insulin resistance and obesity.
Effect of antisense oligonucleotides on the expression of hepatocellular bile acid and organic anion uptake systems in Xenopus laevis oocytes
Authors: HAGENBUCH Bruno , SCHARSCHMIDT Bruce F. , MEIER Peter J.
Published: August 2015

A Na+-dependent bile acid (Na+/taurocholate co-transporting polypeptide; Ntcp) and a Na+-independent bromosulphophthalein (BSP)/bile acid uptake system (organic-anion-transporting polypeptide; oatp) have been cloned from rat liver by using functional expression cloning in Xenopus laevis oocytes. To evaluate the extent to which these cloned transporters could account for overall hepatic bile acid and BSP uptake, we used antisense oligonucleotides to inhibit the expression of Ntcp and oatp in Xenopus laevis oocytes injected with total rat liver mRNA. An Ntcp-specific antisense oligonucleotide co-injected with total rat liver mRNA blocked the expression of Na+-dependent taurocholate uptake by approx. 95%. In contrast, an oatp-specific antisense oligonucleotide when co-injected with total rat liver mRNA had no effect on the expression of Na+-dependent taurocholate uptake, but it blocked Na+-independent uptake of taurocholate by approx. 80% and of BSP by 50%. Assuming similar expression of hepatocellular bile acid and organic anion transporters in Xenopus laevis oocytes, these results indicate that Ntcp and oatp respectively represent the major, if not the only, Na+-dependent and Na+-independent taurocholate uptake systems in rat liver. By contrast, the cloned oatp accounts for only half of BSP transport, suggesting that there must be additional, non-bile acid transporting organic anion uptake systems in rat liver.
The denaturation and degradation of stable enzymes at high temperatures
Authors: DANIEL Roy M. , DINES Mark , PETACH Helen H.
Published: August 2015

Now that enzymes are available that are stable above 100 °C it is possible to investigate conformational stability at this temperature, and also the effect of high-temperature degradative reactions in functioning enzymes and the inter-relationship between degradation and denaturation. The conformational stability of proteins depends upon stabilizing forces arising from a large number of weak interactions, which are opposed by an almost equally large destabilizing force due mostly to conformational entropy. The difference between these, the net free energy of stabilization, is relatively small, equivalent to a few interactions. The enhanced stability of very stable proteins can be achieved by an additional stabilizing force which is again equivalent to only a few stabilizing interactions. There is currently no strong evidence that any particular interaction (e.g. hydrogen bonds, hydrophobic interactions) plays a more important role in proteins that are stable at 100 °C than in those stable at 50 °C, or that the structures of very stable proteins are systematically different from those of less stable proteins. The major degradative mechanisms are deamidation of asparagine and glutamine, and succinamide formation at aspartate and glutamate leading to peptide bond hydrolysis. In addition to being temperature-dependent, these reactions are strongly dependent upon the conformational freedom of the susceptible amino acid residues. Evidence is accumulating which suggests that even at 100 °C deamidation and succinamide formation proceed slowly or not at all in conformationally intact (native) enzymes. Whether this is the case at higher temperatures is not yet clear, so it is not known whether denaturation or degradation will set the upper limit of stability for enzymes.
Determination of the ionization state of the active-site histidine in a subtilisin-(chloromethane inhibitor) derivative by 13C-NMR
Authors: O'CONNELL Timothy P. , MALTHOUSE J. Paul G.
Published: August 2015

Subtilisin BPN´ has been alkylated using benzyloxycarbonylglycylglycyl[1-13C]phenylalanylchloromethane. Using difference 13C-NMR spectroscopy a single signal due to the 13C-enriched α-methylene carbon of the subtilisin–(chloromethane inhibitor) derivative was detected. No evidence for the denaturation/autolysis of this derivative was obtained from pH 3.5 to 11.5. However, incubating at pH 12.75 or heating in the presence of SDS at pH 6.9 did denature this derivative. The negative titration shift of the α-methylene carbon of the denatured derivatives confirmed that the inhibitor had alkylated N-3 of the imidazole ring of the active-site histidine. The positive titration shift of 3.96 p.p.m. and the pKa of 7.04 obtained from studying the native subtilisin–(chloromethane inhibitor) derivative are assigned to oxyanion formation. We conclude that the pKa of the alkylated histidine residue in the native subtilisin–(chloromethane inhibitor) derivative must be > 12 and that subtilisin preferentially stabilizes the zwitterionic tetrahedral adduct consisting of the oxyanion and the imidazolium ion of the active-site histidine residue. We show that even before the oxyanion is formed the pKa of the active-site histidine must be much greater than that of the oxyanion in the zwitterionic tetrahedral adduct. We discuss the significance of our results for the catalytic mechanism of the serine proteinases.
Firefly luciferase can use L-luciferin to produce light
Authors: LEMBERT Nicolas
Published: August 2015

l-Luciferin is a competitive inhibitor of firefly luciferase with a Ki between 3 and 4 μM. Furthermore l-luciferin can serve as an alternative substrate for light production. Catalysis of l-luciferin can be observed in the absence of, or at low concentrations of, d-luciferin. The light production from l-luciferin increases slowly (maximal half-time 8 min) to a stable plateau. At low concentrations of enzyme and l-luciferin, maximal light production is about half of that observed at corresponding d-luciferin concentrations. Increasing the concentration of enzyme or l-luciferin reduces the light production relative to that obtained by d-luciferin catalysis. In contrast to the catalysis of d-luciferin the light production from l-luciferin can be effectively stimulated by the addition of PPi provided that luciferase is premixed with inorganic pyrophosphatase (PPi-ase). A flash is emitted if PPi is injected into a mixture of luciferase, l-luciferin, ATP and PPi-ase. The system maintains its responsiveness and emits further flashes of about equal duration and intensity upon repeated additions of PPi. It is proposed that PPi induces a racemization of enzyme-bound l-luciferyl adenylate. The potential usefulness of PPi-dependent intracellular ATP monitoring is discussed. The proposed activation of firefly luciferase by PPi may be part of the regulation of in vivo flashing.
Decreased accumulation and dephosphorylation of the mitosis-specific form of nucleophosmin/B23 in staurosporine-induced chromosome decondensation
Authors: LU Yi-Yi , LAM Chun-Yuen , YUNG Benjamin Yat-Ming
Published: August 2015

Nucleophosmin/B23 is highly phosphorylated by cdc2 kinase during mitosis, and this phosphorylation most probably has a role in initiating and controlling the entry of cells into mitosis [Peter, Nakagawa, Doree, Labbe and Nigg (1990) Cell 60, 791–801]. In the present study, the protein kinase inhibitor staurosporine has been used to examine possible changes in nucleophosmin/B23 at mitosis in HeLa cells. Addition of staurosporine to HeLa cells already arrested at mitosis by nocodazole causes: (i) decreased accumulation of the mitosis-specific form of nucleophosmin/B23, (ii) dephosphorylation of nucleophosmin/B23, (iii) redistribution of nucleophosmin/B23 to the cytosol, and (iv) concomitant decondensation of chromosomes. These results suggest that the mitosis-specific phosphorylated form of nucleophosmin/B23 may play a role in maintaining mitotic chromosomes in their condensed state.