bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of Drosophila trpl cRNA in Xenopus laevis oocytes leads to the appearance of a Ca2+ channel activated by Ca2+ and calmodulin, and by guanosine 5′[γ-thio]triphosphate
Authors: LAN Ling , BAWDEN Michael J. , AULD Amanda M. , BARRITT Greg J.
Published: August 2015

The effects of expression of the Drosophila melanogaster Trpl protein, which is thought to encode a putative Ca2+ channel [Phillips, Bull and Kelly (1992) Neuron 8, 631–642], on divalent cation inflow in Xenopus laevis oocytes were investigated. The addition of extracellular Ca2+ ([Ca2+]o) to oocytes injected with trpl cRNA and to mock-injected controls, both loaded with the fluorescent Ca2+ indicator fluo-3, induced a rapid initial and a slower sustained rate of increase in fluorescence, which were designated the initial and sustained rates of Ca2+ inflow respectively. Compared with mock-injected oocytes, trpl-cRNA-injected oocytes exhibited a higher resting cytoplasmic free Ca2+ concentration ([Ca2+]i), and higher initial and sustained rates of Ca2+ inflow in the basal (no agonist) states. The basal rate of Ca2+ inflow in trpl-cRNA-injected oocytes increased with (1) an increase in the time elapsed between injection of trpl cRNA and the measurement of Ca2+ inflow, (2) an increase in the amount of trpl cRNA injected and (3) an increase in [Ca2+]o. Gd3+ inhibited the trpl cRNA-induced basal rate of Ca2+ inflow, with a concentration of approx. 5 μM Gd3+ giving half-maximal inhibition. Expression of trpl cRNA also caused an increase in the basal rate of Mn2+ inflow. The increases in resting [Ca2+]i and in the basal rate of Ca2+ inflow induced by expression of trpl cRNA were inhibited by the calmodulin inhibitors W13, calmodazolium and peptide (281–309) of (Ca2+ and calmodulin)-dependent protein kinase II. A low concentration of exogenous calmodulin (introduced by microinjection) activated, and a higher concentration inhibited, the trpl cRNA-induced increase in basal rate of Ca2+ inflow. The action of the high concentration of exogenous calmodulin was reversed by W13 and calmodazolium. When rates of Ca2+ inflow in trpl-cRNA-injected oocytes were compared with those in mock-injected oocytes, the guanosine 5´-[β-thio]diphosphate-stimulated rate was greater, the onset of thapsigargin-stimulated initial rate somewhat delayed and the inositol 1,4,5-trisphosphate-stimulated initial rate markedly inhibited. It is concluded that (1) the divalent cation channel activity of the Drosophila Trpl protein can be detected in Xenopus oocytes; (2) in the environment of the Xenopus oocyte the Trpl channel admits some Mn2+ as well as Ca2+, is activated by cytoplasmic free Ca2+ (through endogenous calmodulin) and by a trimeric GTP-binding regulatory protein, but does not appear to be activated by depletion of Ca2+ in the endoplasmic reticulum; and (3) expression of the Trpl protein inhibits the process by which the release of Ca2+ from intracellular stores activates endogenous store-activated Ca2+ channels.
Cloning and functional expression of glycosyltransferases from parasitic protozoans by heterologous complementation in yeast: the dolichol phosphate mannose synthase from Trypanosoma brucei brucei
Authors: MAZHARI-TABRIZI Ramin , ECKERT Volker , BLANK Michaela , MÜLLER Rolf , MUMBERG Dominik , FUNK Martin , SCHWARZ Ralph T.
Published: August 2015

The gene for the enzyme dolichol phosphate mannose (Dol-P-Man) synthase from the parasitic protozoan Trypanosoma brucei brucei (T. brucei) was cloned by screening a T. brucei cDNA library and then sequenced. The library was constructed in a yeast expression vector and the positive clone was identified by complementation of a temperature-sensitive defect in the yeast strain DPM 1-6 [Orlean, Albright and Robbins (1988) J. Biol. Chem. 263, 17499–17507]. The insert of this clone displayed an open reading frame of 801 nucleotides coding for a putative protein of 267 amino acids. The deduced protein sequence showed an identity of 49% and a similarity of 69% with the published yeast sequence. Additional features of the T. brucei sequence are the presence of a putative signal sequence, a C-terminal transmembrane domain, a consensus sequence for phosphorylation by cAMP-dependent protein kinase and a stretch of five nucleotides immediately upstream from the putative initiation codon that could function as a prokaryotic ribosome binding site. A consensus sequence for dolichol binding (FI/VXF/YXXIPFXF/Y) found in the yeast protein could not be detected in the putative transmembrane domain of the T. brucei sequence. Biochemical characterization of the recombinant protein showed that it is functionally expressed in the yeast strain DPM 1-6 and Escherichia coli. In both constructs Dol-P-Man synthesis was shown in a cell-free system. Synthesis was stimulated by exogenous dolichol phosphate and inhibited by amphomycin. These results confirm that we have cloned the T. brucei Dol-P-Man synthase by heterologous complementation in yeast, an approach that might be applicable for other glycosyltransferases from various sources.
P2u purinoceptor regulation of mucin secretion in SPOC1 cells, a goblet cell line from the airways
Authors: ABDULLAH Lubna H. , DAVIS Shannon W. , BURCH Lauranell , YAMAUCHI Mitsuo , RANDELL Scott H. , NETTESHEIM Paul , DAVIS C. William
Published: August 2015

The SPOC1 cell, a novel goblet cell line derived from rat trachea, was tested for its ability to exhibit regulated mucin secretion in response to purinergic (P2) agonists. High-molecular-mass glycoconjugates (HMMGs) purified by CsCl-density-gradient centrifugation had a buoyant density of 1.45 g/ml. The purified HMMG material exhibited a single major band with an apparent molecular mass of greater than 1000 kDa in SDS/ polyacrylamide gels stained with silver or blotted and stained with soya-bean agglutinin. [3H]HMMG was resistant to proteoglycan-degrading enzymes, but was susceptible to neuraminidase. The HMMG was approx. 91% carbohydrate by weight, and the glycosides were O-linked. The HMMG amino acid composition was enriched in Ser and Thr (sum 27%). Thus SPOC1-cell HMMG possess the characteristics of mucin. Mucin secretion by SPOC1 cells, grown on permeable supports and perfused luminally, was stimulated by ATP, UTP and adenosine 5′-[γ-thio]triphosphate (100 μM) 4–5-fold over a baseline of 4 ng/min. The three dose–effect relations were nearly identical (K0.5 ~4 μM). SPOC1 cells grown on plastic and rat tracheal epithelial primary cells responded similarly to ATP and/or UTP. SPOC1 cells failed to respond to other purinergic agonists, either luminally or serosally, and consequently seem to possess an apical membrane P2u purinoceptor. SPOC1-cell total RNA was probed for P2u purinoceptor mRNA. Using conserved primers for both reverse transcriptase and PCR, a single band of the predicted size was observed, which had a nucleotide base sequence identical with the rat P2u purinoceptor mRNA. Thus SPOC1 cells secrete mucin under the control of a P2u purinoceptor; they should prove useful in dissecting the associated cellular regulatory pathways.
Processing of chromogranins in chromaffin cell culture: effects of reserpine and α-methyl-p-tyrosine
Authors: WOLKERSDORFER Martin , LASLOP Andrea , LAZURE Claude , FISCHER-COLBRIE Reiner , WINKLER Hans
Published: August 2015

Bovine chromaffin cell cultures were treated with either reserpine or α-methyl-p-tyrosine for up to 10 days. Afterwards the cells were harvested and the degree of proteolytic processing of secretogranin II, chromogranin A and chromogranin B was determined by immunoblotting and HPLC followed by RIA. There was a significant increase in the proteolysis of all three chromogranins after 4–6 days in the presence of reserpine. The small peptides formed in the presence of reserpine in vitro are also produced in vivo. A similar effect was observed with α-methyl-p-tyrosine, an inhibitor of tyrosine hydroxylase, but the response took up to 10 days to develop. Both drugs decreased catecholamine levels but reserpine was more effective, reaching a high degree of depletion after 4 days. In addition, experiments in vitro indicate that low millimolar amounts of either adrenaline (IC50 5.2 mM) or noradrenaline (IC50 2.4 mM) can significantly impair the proteolytic activity of recombinant murine prohormone convertase 1 when assayed with synthetic fluorogenic and/or peptidyl substrates. We conclude that a lowering of catecholamine levels in chromaffin granules leads to a concomitant increase in proteolytic processing of all secretory peptides. Apparently within chromaffin granules the endoproteases are inhibited by catecholamines and thus their removal leads to increased proteolysis.
Expression of extracellular matrix molecules in human mesangial cells in response to prolonged hyperglycaemia
Authors: WAHAB Nadia Abdel , HARPER Katherine , MASON Roger M.
Published: August 2015

Post-mitotic cultures of human mesangial cells were maintained in media containing 4–30 mM D-glucose for up to 28 days. Changes in mRNA and protein levels for specific macromolecules occurred between 7 and 14 days after initiating hyperglycaemic conditions. Slot blot analysis showed 2–3-fold increases in mRNAs for collagen type I, fibronectin, versican and perlecan, whereas mRNA for decorin was increased by up to 20-fold. Levels of mRNAs for biglycan and syndecan were unaffected by hyperglycaemic culture. Reverse transcriptase PCR (RT–PCR) confirmed that decorin mRNA levels are greatly elevated and also showed increased transcription of the TGF-β1 gene in hyperglycaemic cultures. Western analysis and ELISA indicated accumulations of collagen types I and III, laminin and fibronectin in the cell layers and media of hyperglycaemic cultures with increasing time. Type IV collagen did not accumulate in either compartment of hyperglycaemic mesangial cell cultures. Collagen types I, III, and fibronectin did not accumulate in the cell layers of hyperglycaemic human dermal fibroblasts, indicating a cell-specific response in mesangial cultures. Decorin and versican, but not biglycan, were increased in the hyperglycaemic mesangial cell culture media. There were no apparent changes in core proteins for decorin and biglycan in fibroblast media. Transforming growth factor β1 (TGF-β1) in hyperglycaemic mesangial cell cultures increased 5-fold after 7 days, but decreased thereafter to only approx. 2-fold after 28 days. The changes in TGF-β1 mRNA, as detected by RT–PCR, and protein followed one another closely.
Purification and characterization of cysteine-S-conjugate N-acetyltransferase from pig kidney
Authors: AIGNER Achim , JÄGER Martina , PASTERNACK Ralf , WEBER Peter , WIENKE Dirk , WOLF Sabine
Published: August 2015

Microsomal cysteine-S-conjugate N-acetyltransferase catalyses the N-acetylation of various S-substituted cysteines in liver and kidney. We describe here the purification and more detailed characterization of this enzyme catalysing the final reaction of mercapturic acid biosynthesis, and thus playing a crucial role in the detoxicating metabolism of many xenobiotics. The solubilization of cysteine-S-conjugate N-acetyltransferase by deoxy-BIGCHAP [N,N´-bis-(3-d-gluconamidopropyl)deoxycholamide] was the prerequisite for partial purification by means of anion-exchange chromatography. The molecular mass of the enzyme was determined by gel filtration. A polyclonal antiserum was raised against the excised protein band from SDS/PAGE and purified antibodies were used for the complete purification of native cysteine-S-conjugate N-acetyltransferase by immunoaffinity chromatography. A dimeric form of the enzyme was sometimes detected on SDS/PAGE, depending on the degree of purification. For further characterization of cysteine-S-conjugate N-acetyltransferase, the stability of catalytic activity, the pH optimum and Km values were determined. The inhibitory effects of various agents were tested, revealing a substantial, yet not complete, loss of cysteine-S-conjugate N-acetyltransferase activity after treatment with cysteine proteinase inhibitors or probenecid under various conditions.
Tetrameric malate dehydrogenase from a thermophilic Bacillus: cloning, sequence and overexpression of the gene encoding the enzyme and isolation and characterization of the recombinant enzyme
Authors: WYNNE Samantha A. , NICHOLLS David J. , SCAWEN Michael D. , SUNDARAM Trichur K.
Published: August 2015

The gene encoding the tetrameric malate dehydrogenase (MDH) in a thermophilic Bacillus species (BI) has been cloned in an Escherichia coli plasmid. The nucleotide sequence of the gene, the first to be elucidated for a tetrameric MDH, shows the MDH subunit to contain 312 amino acids and have a molecular mass of 33648 Da, which confirms the experimentally determined value of about 35 kDa. Like the genomic DNA of BI, the MDH gene is relatively AT-rich; this contrasts with the generally GC-rich nature of the DNA of thermophilic Bacillus species. Comparison of amino acid sequences reveals that BI MDH bears greater structural similarity to lactate dehydrogenases (LDHs) than to other (dimeric) MDHs. MDHs and LDHs resemble each other in catalytic mechanism and several other respects. However, whereas MDHs in the majority of organisms are dimers, the tetrameric structure is favoured among LDHs. The stronger structural resemblance that BI MDH has to LDHs than to the dimeric MDHs provides some explanation as to why Bacillus MDH, unlike most other MDHs, is tetrameric. A 1 kb fragment containing the BI MDH gene, produced in a PCR, has been cloned into a high-expression E. coli plasmid vector. BI MDH synthesized from this clone constitutes about 47% of the total protein in cell extracts of the E. coli strain carrying the clone. MDH purified from BI and that purified from the E. coli strain carrying the MDH gene clone appear to be identical proteins by several criteria. A number of characteristics of the MDH have been elucidated, including the molecular masses of the native enzyme and the subunit, N-terminal amino acid sequence, isoelectric point, pH optimum for activity, thermostability, stability to pH, urea and guanidinium chloride and several kinetic parameters. Whereas the MDH is a stable tetramer in the pH range 5–7, it appears to be converted into a stable dimer at pH 3.5. This suggests that the dimer is a stable intermediate in the dissociation of the tetramer to monomers at low pH.
Biosynthesis of lipophosphoglycan from Leishmania major: solubilization and characterization of a (β 1-3)-galactosyltransferase
Authors: NG Ken , HANDMAN Emmanuela , BACIC Antony
Published: August 2015

Lipophosphoglycan (LPG) is the major cell surface molecule of promastigotes of all Leishmaniaspecies. It is comprised of three domains: a conserved glycosylphosphatidylinositol anchor linked to a repeating phosphorylated disaccharide (P2; PO4-6Galβ1-4Manα1-) backbone and capped with a neutral oligosaccharide. In Leishmania majorthe backbone is substituted at the C(O)3 of the Galpresidue with side chains containing Galp, Glcpand Arapresidues whereas in Leishmania donovanithe backbone is unsubstituted. We report the solubilization of a (β1-3)galactosyltranferase [(β1-3)GalT] from a L. majormicrosomal preparation using Triton X-100. Solubilization occurs with a 10-fold stimulation of enzyme activity. This (β1-3)GalT specifically transfers Gal residues from UDP-Gal to exogenously added L. donovaniLPG acceptor. Depolymerization of the [14C]Gal-labelled LPG product with mild acid and analysis by high-performance anion-exchange chromatography detected only the phosphotrisaccharide (P3; PO4-6([14C]Galβ1-3-4Manα1-) found in L. majorLPG. This contrasts with the activity of the membrane-bound enzyme which also synthesizes the larger phosphosaccharide units [Ng, Handman and Bacic (1994) Glycobiology 4, 845–853]. This suggests that more than one (β1-3)GalT is involved in the addition of these Gal units and that the solubilized activity is the (β1-3)GalT that adds the first βGal residue to the acceptor. The (β1-3)GalT was partially purified by lectin-affinity chromatography and used to establish the Km values for UDP-Gal (445 μM) and L. donovaniacceptor (280 μM as P2 molar equivalent) in kinetic assays. Inhibition studies with various glycosides and mono- and di-saccharides established the P2 repeating unit as the minimum acceptor structure recognized by (β1-3)GalT. The detergent-solubilized (β1-3)GalT was reversibly inactivated by millimolar concentrations of univalent anionic salts. The (β1-3)GalT had an absolute requirement for Mn2+ and also required Mg2+ for optimum activity; Mg2+ cannot substitute for Mn2+, which is loosely bound to β(1-3)GalT and is probably involved in the correct folding of the enzyme. The (β1-3)GalT was unaffected by Ca2+ ions, but were irreversibly inactivated by micromolar levels of transition metal ions (Cu2+ > Zn2+ > Ni2 > Co2+). The (β1-3)GalT activity was also inhibited by diethyl pyrocarbonate, but not by N-ethylmaleimide or iodoacetamide, suggesting that active-site histidine residues, rather than cysteine residue(s), are important for enzyme activity.
Protein kinase C in rod outer segments: effects of phosphorylation of the phosphodiesterase inhibitory subunit
Authors: UDOVICHENKO Igor P. , CUNNICK Jess , GONZALEZ Karen , YAKHNIN Alexander , TAKEMOTO Dolores J.
Published: August 2015

The inhibitory subunit (PDEγ) of the cGMP phosphodiesterase (PDEαβγ2) in rod outer segments (ROS) realizes its regulatory role in phototransduction by inhibition of PDEαβ catalytic activity. The photoreceptor G-protein, transducin, serves as a transducer from the receptor (rhodopsin) to the effector (PDE) and eliminates the inhibitory effect of PDEγ by direct interaction with PDEγ. Our previous study [Udovichenko, Cunnick, Gonzalez and Takemoto (1994) J. Biol. Chem. 269, 9850–9856] has shown that PDEγ is a substrate for protein kinase C (PKC) from ROS and that phosphorylation by PKC increases the ability of PDEγ to inhibit PDEαβ catalytic activity. Here we report that transducin is less effective in activation of PDEαβ(γp)2 (a complex of PDEαβ with phosphorylated PDEγ, PDEγp) than PDEαβγ2. PDEγp also increases the rate constant of GTP hydrolysis of transducin (from 0.16 s-1 for non-phosphorylated PDEγ to 0.21 s-1 for PDEγp). These data suggest that phosphorylation of the inhibitory subunit of PDE by PKC may regulate the visual transduction cascade by decreasing the photoresponse.
Characterization of the effects of Ca2+ depletion on the synthesis, phosphorylation and secretion of caseins in lactating mammary epithelial cells
Authors: DUNCAN Jennifer S. , BURGOYNE Robert D.
Published: August 2015

We have examined the effects of depleting lumenal Ca2+ on the synthesis, phosphorylation and secretion of caseins in lactating mouse mammary cells by using inhibitors of the endoplasmic reticulum Ca2+-ATPase or the ionophore ionomycin in the absence of external Ca2+. Treatment with these drugs resulted in a transient increase in the cytosolic Ca2+ concentration due to Ca2+ mobilization. Protein synthesis over a 1 h period was substantially inhibited by Ca2+ depletion, but in a pulse–chase protocol secretion of pre-synthesized proteins was unaffected by Ca2+ depletion. Analysis of polysome profiles showed that Ca2+ depletion resulted in a loss in polysomes, consistent with an inhibition of initiation of protein synthesis. Neither treatment with Ca2+-ATPase inhibitors to deplete endoplasmic reticulum Ca2+ nor treatment with ionomycin/EGTA had any effect on an early phase of phosphorylation of α- or β/γ-caseins, but Ca2+ depletion resulted in a decrease in a late phase of casein phosphorylation. These results indicate that lumenal Ca2+ is required to maintain protein synthesis in lactating mammary cells but is not required for protein secretion, and that Ca2+ accumulation in the Golgi cisternae is required for a late but not for an early phase of casein phosphorylation.
Alternative processing of the sarco/endoplasmic reticulum Ca2+-ATPase transcripts during muscle differentiation is a specifically regulated process
Authors: VAN DEN BOSCH Ludo , MERTENS Luc , CAVALOC Yvon , PETERSON Martha , WUYTACK Frank , EGGERMONT Jan
Published: August 2015

Expression of the muscle-specific 2a isoform of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) requires activation of an otherwise inefficient splice process at the 3´-end of the primary gene transcript. We provide evidence that SERCA2 splicing is a specifically regulated process, rather than the result of an increase in general splice efficiency or a decrease in polyadenylation efficiency at the 5´-most polyadenylation site. This is indicated by the fact that changes in general splice and polyadenylation efficiency, as observed during B-cell maturation, did not affect SERCA2 splicing. Furthermore, expression and overexpression studies did not support the hypothesis that changes in the level of the alternative splice factor ASF/SF2 or other arginine and serine rich proteins are sufficient to obtain the regulation of muscle- and neuronal-specific splicing.
Calcineurin is essential for DNA synthesis in Swiss 3T3 fibroblasts
Authors: TOMONO Masahiro , TOYOSHIMA Kotaro , ITO Motohiro , AMANO Hisao
Published: August 2015

DNA synthesis was measured 16 h after stimulation of Swiss 3T3 fibroblasts in the resting phase with various growth factors (platelet-derived growth factor, fibroblast growth factor, lysophosphatidic acid and thrombin). When extracellular Ca2+ was chelated by EGTA, or when the influx of Ca2+ from outside to inside the cell was blocked by cobalt, DNA synthesis was completely inhibited. As there was no effect whatsover on DNA synthesis when Ca2+ was chelated, or when the influx of Ca2+ was blocked up to the first 4 h after growth stimulation, it was concluded that, at an early stage, Ca2+ influx from outside to inside the cell is not related to the transition from the G1 to the S phase. A Ca2+/calmodulin-dependent protein kinase II inhibitor (KN-62) had no effect on DNA synthesis. However, cyclosporin A and FK-506, which are inhibitors of Ca2+/calmodulin-dependent protein phosphatase 2B (calcineurin), markedly inhibited DNA synthesis stimulated by all of the growth factors. These results indicate that calcineurin plays a role, not only in activation of T-cells of the immune system in the initial phase, but also in DNA synthesis in fibroblasts. It was concluded that Ca2+ influx from outside to inside the cell during the mid-to-late G1 phase, followed by calcineurin activation, is essential as a mechanism of growth signal transduction.
Differential expression of fibromodulin mRNA associated with tendon fibril growth: isolation and characterization of a chicken fibromodulin cDNA
Authors: NURMINSKAYA Maria V. , BIRK David E.
Published: August 2015

A 450 bp cDNA fragment similar to that encoding bovine fibromodulin was isolated using a screening procedure to isolate genes differentially expressed between the pre- and post-growth phases of fibril growth in the developing chicken embryo metatarsal tendon. Using this fragment, a 2.4 kb cDNA clone for chicken fibromodulin was isolated from a λZAP library, and the 5´ rapid amplification of cDNA ends technique was employed to clone the 5´ end of the fibromodulin cDNA. The full-length cDNA contained an open reading frame coding for a 380-amino-acid protein. There was ∼80% similarity with human, rat and bovine fibromodulins, which confirmed its identity as fibromodulin. Structural features of the deduced sequence include an 18-amino-acid signal peptide, cysteine residues in conserved positions in the N- and C-terminal regions, and a central leucine-rich domain containing eleven repeats of the sequence LXXLXLXXNXL/I. Features unique to chicken fibromodulin include an additional glycosylation site as well as a decreased number of tyrosine residues that could be sulphated, and therefore potential changes in the charge of the molecule. In addition, there was little similarity among the untranslated regions. When compared with chicken decorin and lumican, fibromodulin showed greater similarity to the other keratan sulphate-containing proteoglycan, lumican. Northern blot analysis revealed a 6–8-fold increase in the fibromodulin mRNA level from day 14 to day 19 of development. In the chicken tendon, collagen fibril growth is a process characterized by a precipitous increase in length during a short developmental period. The necessary changes would require the expression of different genes regulating fibril formation and growth, and interactions between fibromodulin and collagen fibrils may participate in the regulation of collagen fibril growth and matrix assembly.
Nucleoside uptake in rat liver parenchymal cells
Authors: MERCADER Joan , GOMEZ-ANGELATS Mireia , SANTO Belén del , CASADO Javier , FELIPE Antonio F. , PASTOR-ANGLADA Marçal
Published: August 2015

Rat liver parenchymal cells express Na+-dependent and Na+-independent nucleoside transport activity. The Na+-dependent component shows kinetic properties and substrate specificity similar to those reported for plasma membrane vesicles [Ruiz-Montasell, Casado, Felipe and Pastor-Anglada (1992) J. Membr. Biol. 128, 227–233]. This transport activity shows apparent Km values for uridine in the range 8–13 μM and a Vmax of 246 pmol of uridine per 3 min per 106 cells. Most nucleosides, including the analogue formycin B, cis-inhibit Na+-dependent uridine transport, although thymidine and cytidine are poor inhibitors. Inosine and adenosine inhibit Na+-dependent uridine uptake in a dose-dependent manner, reaching total inhibition. Guanosine also inhibits Na+-dependent uridine uptake, although there is some residual transport activity (35% of the control values) that is resistant to high concentrations of guanosine but may be inhibited by low concentrations of adenosine. The transport activity that is inhibited by high concentrations of thymidine is similar to the guanosine-resistant fraction. These observations are consistent with the presence of at least two Na+-dependent transport systems. Na+-dependent uridine uptake is sensitive to N-ethylmaleimide treatment, but Na+-independent transport is not. Nitrobenzylthioinosine (NBTI) stimulates Na+-dependent uridine uptake. The NBTI effect involves a change in Vmax, it is rapid, dose-dependent, does not need preincubation and can be abolished by depleting the Na+ transmembrane electrochemical gradient. Na+-independent uridine transport seems to be insensitive to NBTI. Under the same experimental conditions, NBTI effectively blocks most of the Na+-independent uridine uptake in hepatoma cells. Thus the stimulatory effect of NBTI on the concentrative nucleoside transporter of liver parenchymal cells cannot be explained by inhibition of nucleoside efflux.
Spectrophotometric titration with cobalt(III) for the determination of accurate absorption coefficients of transferrins
Authors: HE Qing-Yu , MASON Anne B , WOODWORTH Robert C
Published: August 2015

A rapid and sensitive technique, involving difference spectral titration with cobalt(III), to measure the ϵ values of chicken ovotransferrin, human serum transferrin, the N-lobe of human transferrin and several single point mutants is reported. The resulting ϵ values were compared with the values calculated from the equation proposed by Pace, Vajdos, Fee, Grimsley and Gray [(1995) Protein Sci. 4, 2411–2423]. The titrations with cobalt feature sharp break-points and do not destroy the protein samples. The choice of buffer was found to be important, depending on the metal-binding avidity of the proteins. Cobalt titration should prove useful for studying the comparative metal-binding properties of transferrin and mutants of transferrin being generated by recombinant technology.
Phorbol esters stimulate phosphatidylinositol 3,4,5-trisphosphate production in 3T3-L1 adipocytes: implications for stimulation of glucose transport
Authors: NAVÉ Barbara T. , SIDDLE Kenneth , SHEPHERD Peter R
Published: August 2015

The effects of insulin and phorbol 12-myristate 13-acetate (PMA) on the levels of cellular phosphoinositides were investigated in 3T3-L1 adipocytes. Stimulation for 4 min with PMA (1 µM) or insulin (10 nM) increased levels of PtdIns(3,4,5)P3 approx. 2-fold and 6-fold respectively. PMA also had a small effect on the cellular levels of PtdIns4P, whereas insulin had no effect on PtdIns4P levels; levels of PtdIns(4,5)P2 and PtdIns3P were not significantly affected by either agent. Insulin increased the levels of the p85α subunit of phosphoinositide (PI) 3-kinase associated with membranes, whereas PMA decreased levels of membrane-associated p85α. PMA did not increase PI 3-kinase activity in anti-phosphotyrosine or anti-p85 immunoprecipitates. The stimulation of glucose transport by insulin or PMA was blocked by 100 nM wortmannin or 10 ng/ml LY294002, indicating that PI 3-kinase is essential for stimulation by both agents. In summary, these results demonstrate: (1) that PMA and insulin stimulate PtdIns(3,4,5)P3 production by distinct mechanisms in 3T3-L1 adipocytes, and (2) that stimulation of PtdIns(3,4,5)P3 production by PMA is likely to be important in signalling pathways leading from PMA stimulation to end-point responses such as glucose transport.
Inhibition of mitogen-activated protein kinase kinase does not impair primary activation of human platelets
Authors: BÖRSCH-HAUBOLD Angelika G. , KRAMER Ruth M. , WATSON Steve P
Published: August 2015

Mitogen-activated protein kinases (MAPKs), a family of protein serine/threonine kinases regulating cell growth and differentiation, are activated by a dual-specificity kinase through phosphorylation at threonine and tyrosine. We used a recently described selective inhibitor of the p42/p44mapk-activating enzyme, PD 98059 [2-(2´-amino-3´-methoxyphenyl)-oxanaphthalen-4-one], to investigate the role of the p42/p44mapk pathway in human platelets. PD 98059 inhibited p42/p44mapk activation in thrombin-, collagen- and phorbol ester-stimulated platelets, as determined from in-gel renaturation kinase assays, with an IC50 of approx. 5 µM (thrombin stimulation). It also prevented activation of MAPK kinase, which was measured in whole-cell lysates with glutathione S-transferase/p42mapk fusion protein (GST–MAPK) as substrate. Inhibition of p42/p44mapk did not affect platelet responses to thrombin or collagen such as aggregation, 5-hydroxytryptamine release and protein kinase C activation. In addition, PD 98059 did not interfere with release of arachidonic acid, a response mediated by cytosolic phospholipase A2 (cPLA2), or with cPLA2 phosphorylation. This suggests that platelet cPLA2 is not regulated by p42/p44mapk after stimulation with physiological agonists. In contrast, phorbol ester-induced phosphorylation of cPLA2 and potentiation of arachidonic acid release stimulated by Ca2+ ionophore A23187 were inhibited by PD 98059, indicating that p42/p44mapk phosphorylates cPLA2 after activation of protein kinase C by the non-physiological tumour promoter.
Cloning of a novel membrane-linked metalloproteinase from human myeloma cells
Authors: McKIE Norman , DALLAS Donna J , EDWARDS Tracy , APPERLEY Jane F , RUSSELL Graham G. , CROUCHER Peter I
Published: August 2015

We have isolated a novel cDNA from human myeloma cells encoding a member of the reprolysin family of metalloproteinases. Derived amino acid sequence predicts a protein of approx. 76 kDa. The open reading frame predicts the presence of a leader peptide, a pro-peptide with a ‘cysteine switch’, a metalloproteinase domain, a disintegrin-like domain, a cysteine-rich domain, an epidermal growth factor-like domain and a putative transmembrane sequence. Expression of the mRNA for this metalloproteinase has been demonstrated in human myeloma cells.
Changes associated with aging and replicative senescence in the regulation of transcription factor nuclear factor-κB
Authors: HELENIUS Merja , HÄNNINEN Maarit , LEHTINEN Sanna K. , SALMINEN Antero
Published: August 2015

Both the aging of animals and the senescence of cultured cells involve an altered pattern of gene expression, suggesting changes in transcription factor regulation. We studied age-related changes in transcription factors nuclear factor (NF)-κB, activator protein factor-1 (AP-1) and Sp-1 by using electrophoretic mobility shift binding assays; we also analysed changes in the protein components of NF-κB complex with Western blot assays. Nuclear and cytoplasmic extracts were prepared from heart, liver, kidney and brain of young adult and old NMRI mice and Wistar rats as well as from presenescent, senescent and simian virus 40-immortalized human WI-38 fibroblasts. Aging of both mice and rats induced a strong and consistent increase in the nuclear binding activity of NF-κB factor in all tissues studied, whereas those of AP-1 and Sp-1 decreased, e.g. in liver. Protein levels of p50, p52 and p65 components of the NF-κB complex did not show any age-associated changes in the cytoplasmic fraction but in the nuclear fraction the level of p52 strongly increased in heart and liver during aging. The protein levels of inhibitory IκB-α and Bcl-3 components were not affected by aging in any of the tissues studied. Replicative cellular senescence of human WI-38 fibroblasts induced a strong decrease in nuclear NF-κB, AP-1 and Sp-1 binding activities. Protein levels of p50 and p52 components of NF-κB complex were decreased in the nuclear fraction of senescent WI-38 fibroblasts but in the cytoplasm of senescent fibroblasts the level of p65 protein was increased. Cellular senescence also slightly decreased the protein levels of IκB-α and Bcl-3. Transfection assays with NF-κB-enhancer-driven chloramphenicol acetyltransferase reporter gene showed a significant down-regulation of NF-κB promoter activity in senescent WI-38 fibroblasts. Results suggest that the aging process might be regulated differently in tissues and cultured fibroblasts, perhaps reflecting differences between mitotic and post-mitotic cells. In tissues, aging seems to involve specific changes in the regulation of NF-κB components and perhaps also changes in the DNA-binding affinities of the NF-κB complex.
Insulin-induced tyrosine dephosphorylation of paxillin and focal adhesion kinase requires active phosphotyrosine phosphatase 1D
Authors: OUWENS D. Margriet , MIKKERS Harald M. M. , van der ZON Gerard C. M. , STEIN-GERLACH Matthias , ULLRICH Axel , MAASSEN Antonie J.
Published: August 2015

Insulin stimulation of fibroblasts rapidly induces the tyrosine dephosphorylation of proteins of 68 kDa and 125 kDa, in addition to the tyrosine phosphorylation of the insulin receptor β-chain, insulin receptor substrates 1 and 2, and Shc. Using specific antibodies, the 68 kDa and 125 kDa proteins were identified as paxillin and focal adhesion kinase (pp125FAK) respectively. We have examined whether dephosphorylation of paxillin and pp125FAK requires interaction of the cells with the extracellular matrix. For this, cells were grown on poly(l-lysine) plates, and the tyrosine phosphorylation of pp125FAK and paxillin was increased by addition of lysophosphatidic acid. Under these conditions, insulin still induced the complete dephosphorylation of pp125FAK and paxillin, indicating that this process can occur independently of the interaction of integrins with extracellular matrix proteins. We also studied whether dephosphorylation of pp125FAK and paxillin results from the action of a phosphotyrosine phosphatase. It was found that phenylarsine oxide, a phosphotyrosine phosphatase inhibitor, prevented the insulin-induced dephosphorylation of pp125FAK and paxillin. Furthermore, this insulin-induced dephosphorylation was also impaired in cells expressing a dominant-negative mutant of phosphotyrosine phosphatase 1D (PTP 1D). Thus we have identified paxillin as a target for dephosphorylation by insulin. In addition, we have obtained evidence that the insulin-mediated dephosphorylation of paxillin and pp125FAK requires active PTP 1D.
Glycosylation of multiple extracytosolic loops in Band 3, a model polytopic membrane protein
Authors: TAM Lisa Y , LANDOLT-MARTICORENA Carolina , REITHMEIER Reinhart A. F.
Published: August 2015

N-glycosylated sites in polytopic membrane proteins are usually localized to single extracytosolic (EC) loops containing more than 30 residues [Landolt-Marticorena and Reithmeier (1994) Biochem. J. 302, 253–260]. This may be due to a biosynthetic restriction whereby only a single loop of nascent polypeptide is available to the oligosaccharyl transferase in the lumen of the endoplasmic reticulum. To test this hypothesis, two types of N-glycosylation mutants were constructed using Band 3, a polytopic membrane protein that contains up to 14 transmembrane segments and a single endogenous site of N-glycosylation at Asn-642 in EC loop 4. In the first set of mutants, an additional N-glycosylation acceptor site (Asn-Xaa-Ser/Thr) was constructed by site-directed mutagenesis in EC loop 3, with or without retention of the endogenous site. In the second set of mutants, EC loop 4 was duplicated and inserted into EC loop 2, again with or without retention of the endogenous site. Cell-free translation experiments using reticulocyte lysates showed that microsomes were able to N-glycosylate multiple EC loops in these Band 3 mutants. The acceptor site in EC loop 3 was poorly N-glycosylated, probably due to the suboptimal size (25 residues) of this EC loop. The localization of N-glycosylation sites to single EC loops in multi-span membrane proteins is probably due to the absence of suitably positioned acceptor sites on multiple loops.
Melanotransferrin gene expression in melanoma cells is correlated with high levels of Jun/Fos family transcripts and with the presence of a specific AP1-dependent ternary complex
Authors: ROZÉ-HEUSSE Anne , HOUBIGUIAN Marie-Laure , DEBACKER Cécile , ZAKIN Mario M , DUCHANGE Nathalie
Published: August 2015

The involvement of the transcription factor AP1 in the regulation of melanotransferrin (MTf) gene expression was investigated. MTf, also known as p97, is a tumour-associated antigen that is overproduced in most melanomas. Its gene expression is under the control of an enhancer element containing two AP1 binding sites. By Northern analysis, we demonstrate that MTf mRNA is detected at various levels in melanoma SK-MEL-28 cells and that its greatest expression coincides with the presence of large amounts of jun and fos transcripts. Gel retardation assays revealed that the induction of expression of these proto-oncogenes is correlated with increased AP1 binding activity and that a region of the MTf enhancer is involved in the formation of a ternary AP1-dependent complex, implicating a second nuclear factor whose binding characteristics are similar to those of nuclear factor of activated T cells (NF-AT). In transient expression experiments, the activity resulting from ternary complex formation was high and specific to melanoma cells. These data provide a possible explanation for the mechanisms of AP1 factor family involvement in MTf up-regulation in melanoma cells.
Agonist regulation of adenylate cyclase activity in neuroblastoma × glioma hybrid NG108-15 cells transfected to co-express adenylate cyclase type II and the β2-adrenoceptor. Evidence that adenylate cyclase is the limiting component for receptor-mediated stimulation of adenylate cyclase activity
Authors: MACEWAN David J. , KIM Gun-Do , MILLIGAN Graeme
Published: August 2015

Stable cell lines, derived from NG108-15 cells and transfected to express both the β2-adrenoceptor and adenylate cyclase type II, were produced and examined. The absence of adenylate cyclase type II in the parental cells and its presence in these clones was demonstrated by reverse transcriptase-PCR. Total cellular levels of adenylate cyclase were increased in a number of clones between 3- and 8-fold, as assessed by guanine nucleotide-stimulated specific high-affinity binding of [3H]forskolin to cellular membranes. Basal adenylate cyclase activity was markedly elevated compared with a clone expressing similar levels of the β2-adrenoceptor in the absence of adenylate cyclase type II. Each of NaF, forskolin and guanosine 5´-[β,γ-imido]triphosphate (a poorly hydrolysed analogue of GTP) produced substantially higher levels of adenylate cyclase activity in membranes of the clones positive for expression of adenylate cyclase type II than was achieved with the parental cells. Both isoprenaline, acting at the introduced β2-adrenoceptor, and iloprost, acting at the endogenously expressed IP prostanoid receptor, stimulated adenylate cyclase activity to much higher levels in the clones expressing adenylate cyclase type II compared with the clone lacking this adenylate cyclase; however, the concentration–effect curves for adenylate cyclase stimulation by these two agonists were not different between parental cells and clones overexpressing adenylate cyclase type II. A maximally effective concentration of the β-adrenoceptor partial agonist ephedrine displayed similar intrinsic activity and potency to stimulate adenylate cyclase in membranes of clones both with and without adenylate cyclase type II. Both secretin and 5´-N-ethylcarboxamidoadenosine (acting at an endogenous A2 adenosine receptor) were also able to produce substantially greater maximal activations of adenylate cyclase in the clones expressing excess adenylate cyclase type II, without alterations in agonist intrinsic activity or potency. These results demonstrate that the maximal output of the stimulatory arm of the adenylate cyclase cascade can be increased by increasing total levels of adenylate cyclase in the genetic background of NG108-15 cells.
Transformation of Rat-1 fibroblasts with the v-src oncogene induces inositol 1,4,5-trisphosphate 3-kinase expression
Authors: WOODRING Pamela J , GARRISON James C
Published: August 2015

Transformation of Rat-1 fibroblasts with the v-src oncogene leads to a 6- to 8-fold enhancement of the activity of the Ins(1,4,5)P3 3-kinase in cytosolic extracts [Johnson, Wasilenko, Mattingly, Weber and Garrison (1989) Science 246, 121–124]. This study confirms these results using another v-src-transformed Rat-1 cell line (B31 cells) and investigates the molecular mechanism by which pp60v-src activates Ins(1,4,5)P3 3-kinase. The mRNA and protein levels for two rat isoforms of Ins(1,4,5)P3 3-kinase were determined in the v-src-transformed cell line. Both the mRNA and protein levels for isoform A were elevated in v-src-transformed Rat-1 cells while those for isoform B were not significantly affected. Moreover, stable expression of either form of Ins(1,4,5)P3 3-kinase in the B31 v-src-transformed Rat-1 cell line did not result in tyrosine phosphorylation of Ins(1,4,5)P3 3-kinase A or B. These results suggest that at least one mechanism by which the v-src oncogene increases the activity of the Ins(1,4,5)P3 3-kinase in the Rat-1 transformed fibroblast is by increasing the level of expression of Ins(1,4,5)P3 3-kinase A.
Distinct biochemical properties of the native members of the G12 G-protein subfamily. Characterization of Gα12 purified from rat brain
Authors: HARHAMMER Rainer , NÜRNBERG Bernd , HARTENECK Christian , LEOPOLDT Daniela , EXNER Torsten , SCHULTZ Günter
Published: August 2015

G12 and G13 are insufficiently characterized pertussis toxin-insensitive G-proteins. Here, we describe the isolation of Gα12 from rat brain membranes. Gα12 was purified to apparent homogeneity by three steps of conventional chromatography, followed by two cycles of subunit-exchange chromatography on immobilized G subunits. Purified Gα12 bound guanosine 5´-[γ-thio]triphosphate slowly and substoichiometrically. For isolation of functionally active Gα12, it was mandatory to use sucrose monolaurate as a detergent. Comparative studies of both rat-brain-derived members of the G12 subfamily revealed differences in the affinity of Gα12 and Gα13 for Gβγ. Gα12 required a higher Mg2+ concentration for AlF4--induced dissociation from immobilized Gβγ than did Gα13. In addition, the G12 subfamily members differed in their sedimentation velocities, as determined by sucrose-density-gradient centrifugation. Analysis of sedimentation coefficients revealed a higher tendency of G12 to form supramolecular structures in comparison to G13 and other G-proteins. These G12 structures were stabilized by sucrose monolaurate, which in turn may explain the necessity for this detergent for purification of functionally active Gα12. Despite these distinct biochemical characteristics of G12 and G13, both purified G-proteins coupled to a recombinant thromboxane A2 (TXA2) receptor reconstituted into phospholipid vesicles. These data indicate, (1) significant differences in the biochemical properties of native members of the G12 subfamily, and (2) their specific coupling to TXA2 receptors.
Mutagenesis of the active site of the human Theta-class glutathione transferase GSTT2-2: catalysis with different substrates involves different residues
Authors: TAN Kian-Leong , CHELVANAYAGAM Gareth , PARKER Michael W. , BOARD Philip G.
Published: August 2015

The role of serine-11 in the catalytic mechanism of recombinant human GSTT2-2 was examined by site-directed mutagenesis. Amino acid sequence comparison of the Theta-class isoenzymes has identified a conserved serine residue in the N-terminal domain [Wilce, Board, Feil and Parker (1995) EMBO J. 14, 2133–2143]. This conserved serine has been implicated in the activation of the enzyme-bound glutathione [Board, Coggan and Parker (1995) Biochem. J. 311, 247–250]. Mutating the equivalent serine (residue 11) of GSTT2-2 to Ala, Thr or Tyr abolished the catalytic properties of GSTT2-2 with cumene hydroperoxide and ethacrynic acid as second substrate. However, with 1-menaphthyl sulphate (MSu) as the second substrate, the specific activity of the S11A mutant was doubled, while the S11T mutant retained half the wild-type activity and the S11Y mutant was inactive. The role of Ser-11 in catalysis seems to vary with different second substrates. In the substitution reaction with MSu, GSTT2-2 activity appears to depend on the size of the Ser-11 replacement rather than the presence of a side-chain hydroxy group. In addition, the reaction rate appears to be a function of pH, and there is no non-enzymic reaction even at high pH. We demonstrated that a reaction between MSu and an alternative thiol such as L-cysteine or 2-mercaptoethanol can take place in the presence of S-methylglutathione and GSTT2-2. We propose that the catalytic activity of GSTT2-2 with MSu is preceded by a conformational or charge modification to the enzyme upon the binding of glutathione or S-methylglutathione. This is followed by the binding of MSu and the subsequent removal of the sulphate group, giving rise to the carbonium ion of 1-methylnaphthelene as the electrophile that reacts with the nucleophilic species. The reaction mechanism of GSTT2-2 with MSu may represent a novel function of GSTT2-2 as a glutathione-dependent sulphatase.