bluuebunny/crossref_metadata_2025_split huggingface dataset:
A specific distal promoter controls γ-glutamyl transpeptidase gene expression in undifferentiated rat transformed liver cells
Authors: NOMURA Seiji , LAHUNA Olivier , SUZUKI Takanobu , BROUILLET Arthur , CHOBERT Marie Noêle , LAPERCHE Yannick 
Published: August 2015

In rat undifferentiated hepatoma cells, the γ-glutamyl transpeptidase (GGT) gene is transcribed into a 2.3 and a 2.6 kb mRNA which, in contrast with other rat GGT transcripts, are not detected in more differentiated liver cells or adult tissues. Analysis of the cDNA sequences obtained from H5 hepatoma cells reveals that these two transcripts differ from other GGT mRNAs by a 312-nt unique untranslated leader sequence; this sequence maps on the gene in a single exon 10 kb upstream from the GGT promoter IV transcription start site. We established that the 2.6 kb mRNA V-1 and the 2.3 kb GGT mRNA V-2 derive, by alternate splicing, from a primary transcript initiated on a distal promoter on the rat GGT gene. This gene appears to be transcribed from five promoters, and the specific expression of this new distal promoter in undifferentiated hepatoma cells requires binding of activator protein-1 and hepatic nuclear factor 3 specific transcription factors to a composite cis-element in the proximal region of the promoter. The distal GGT promoter, specifically expressed in undifferentiated liver cells, might reflect the expression of that gene in liver precursor cells before they differentiate in the hepatocytic or biliary lineage.
Isolation, cloning and characterization of a low-molecular-mass purine nucleoside- and nucleotide-binding protein
Authors: GILMOUR Jeffrey , LIANG Nianci , LOWENSTEIN John M. 
Published: August 2015

A purine nucleoside- and nucleotide-binding protein has been isolated from extracts of rat and rabbit heart, calf aortic smooth muscle and rat liver using an affinity column containing adenosine bound through the N6-position. The protein, which was eluted by adenosine, was cloned and expressed in Escherichia coli. The deduced amino acid sequence has a calculated Mr of 13693 (p13.7). The expressed protein has properties identical with the protein isolated from heart and liver, including an anomalous, apparent Mr of 15300, observed on gel electrophoresis. Gel filtration shows it to be a dimer. p13.7 differs by only three amino acids out of 125 from protein kinase C inhibitor 1 [Pearson, DeWald, Mathews, Mozier, Zürcher-Neely, Heinrikson, Morris, McCubbin, McDonald, Fraser et al. (1990) J. Biol. Chem. 265, 4583–4591]. However, we have not been able to demonstrate inhibition of protein kinase C by physiological concentrations of p13.7, regardless of whether it was isolated from tissue extracts or expressed in E. coli. p13.7 is a member of the histidine triad motif family of proteins [Séraphin (1992) J. DNA Sequencing Mapping 3, 177–179]. The affinity of p13.7 for a number of different purine nucleosides and nucleotides, as measured by fluorescence titration and gel filtration, falls within the range 5–50 μM. On the basis of these properties and its crystal structure [Brenner, Garrison, Gilmour, Peisach, Ringe, Petsko and Lowenstein (1997) Nature Struct. Biol. 4, 231–238], we have coined the acronym HINT (histidine triad nucleotide-binding motif) to describe the family of proteins of which p13.7 is a member. Other proteins that bind to the affinity column have been identified as malate and lactate dehydrogenases, cAMP-binding proteins, adenosine kinase and S-adenosylhomocysteine hydrolase.
Association of purified skeletal-muscle AMP deaminase with a histidine–proline-rich-glycoprotein-like molecule
Authors: RANIERI-RAGGI Maria , MONTALI Umberto , RONCA Francesca , SABBATINI Antonietta , BROWN Paul E. , MOIR Arthur J. G. , RAGGI Antonio 
Published: August 2015

Denaturation of rabbit skeletal-muscle AMP deaminase in acidic medium followed by chromatography on DEAE-cellulose in 8 M urea at pH 8.0 allows separation of two main peptide components of similar apparent molecular mass (75–80 kDa) that we tentatively assume correspond to two different enzyme subunits. Whereas the amino acid composition of one of the two peptides is in good agreement with that derived from the nucleotide sequence of the known rat and human AMPD1 cDNAs, the second component shows much higher contents of proline, glycine and histidine. N-Terminal sequence analysis of the fragments liberated by limited proteolysis with trypsin of the novel peptide reveals a striking similarity to the fragments produced by plasmin cleavage of the rabbit plasma protein called histidine–proline-rich glycoprotein (HPRG). However, some divergence is observed between the sequence of one of the fragments liberated from AMP deaminase by a more extensive trypsinization and rabbit plasma HPRG in the region containing residues 472–477. A fragment with a blocked N-terminus, which was found among those liberated by proteolysis with pepsin of either whole AMP deaminase or the novel component of the enzyme, shows an amino acid composition quite different from that of the N-terminus of the known subunit of AMP deaminase. By coupling this observation with the detection in freshly prepared AMP deaminase of a low yield of the sequence (LTPTDX) corresponding to that of HPRG N-terminus, it can be deduced that in comparison with HPRG, the putative HPRG-like component of AMP deaminase contains an additional fragment with a blocked N-terminus, which is liberated by a proteolytic process during purification of the enzyme. The implications of the association to rabbit skeletal-muscle AMP deaminase of a HPRG-like protein species are discussed.
NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis: Isolation, characterization and biochemical properties of the enzyme
Authors: NEUHAUSER Wilfried , HALTRICH Dietmar , KULBE Klaus D. , NIDETZKY Bernd 
Published: August 2015

During growth on D-xylose the yeast Candida tenuis produces one aldose reductase that is active with both NADPH and NADH as coenzyme. This enzyme has been isolated by dye ligand and anion-exchange chromatography in yields of 76%. Aldose reductase consists of a single 43 kDa polypeptide with an isoelectric point of 4.70. Initial velocity, product inhibition and binding studies are consistent with a compulsory-ordered, ternary-complex mechanism with coenzyme binding first and leaving last. The catalytic efficiency (kcat/Km) in D-xylose reduction at pH 7 is more than 60-fold higher than that in xylitol oxidation and reflects significant differences in the corresponding catalytic centre activities as well as apparent substrate-binding constants. The enzyme prefers NADP(H) approx. 2-fold to NAD(H), which is largely due to better apparent binding of the phosphorylated form of the coenzyme. NADP+ is a potent competitive inhibitor of the NADH-linked aldehyde reduction (Ki 1.5 μM), whereas NAD+ is not. Unlike mammalian aldose reductase, the enzyme from C. tenuisis not subject to oxidation-induced activation. Evidence of an essential lysine residue located in or near the coenzyme binding site has been obtained from chemical modification of aldose reductase with pyridoxal 5′-phosphate. The results are discussed in the context of a comparison of the enzymic properties of yeast and mammalian aldose reductase.
Lysosomal α-mannosidases of mouse tissues: characteristics of the isoenzymes, and cloning and expression of a full-length cDNA
Authors: BECCARI T. , APPOLLONI M. G. , COSTANZI E. , STINCHI S. , STIRLING J. L. , DELLA FAZIA M. A. , SERVILLO G. , VIOLA M. P. , ORLACCHIO A. 
Published: August 2015

Lysosomal α-D-mannosidase from mouse tissues was separated into its constituent isoenzymes by DEAE-cellulose chromatography. Forms corresponding to the human isoenzymes B and A were present in testis, brain, spleen and kidney, whereas in epididymis and liver only the B form was present. Murine α-mannosidases A and B are glycoproteins and have pH optima, thermal stabilities and molecular masses similar to those of the human isoenzymes. A full-length cDNA (3.1 kb) containing the complete coding sequence for α-mannosidase was isolated from a mouse macrophage cDNA library. Comparison of the deduced amino acid sequences of human and mouse α-mannosidases showed that they had 75% identity and 83% similarity. Expression of this cDNA in COS cells showed that both the A and the B isoenzymes can arise from a single transcript. Northern blotting analysis showed a 10-fold range in the abundance of α-mannosidase mRNA in mouse tissues, with the highest levels found in epididymis, and the lowest in liver.
Purification and characterization of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine synthetase from Penicillium chrysogenum
Authors: THEILGAARD Hanne B.Aa. , KRISTIANSEN Klaus N. , HENRIKSEN Claus M. , NIELSEN Jens 
Published: August 2015

δ-(L-α-Aminoadipyl)-L-cysteinyl-d-valine synthetase (ACVS) from Penicillium chrysogenum was purified to homogeneity by a combination of (NH4)2SO4 precipitation, protamine sulphate treatment, ion-exchange chromatography, gel filtration and hydrophobic interaction chromatography. The molecular mass of ACVS was estimated with native gradient gel electrophoresis and SDS/PAGE. The native enzyme consisted of a single polymer chain with an estimated molecular mass of 470 kDa. The denatured enzyme had an estimated molecular mass of 440 kDa. The influence of different reaction parameters such as substrates, cofactors and pH on the activity of the purified ACVS was investigated. The Km values for the three precursor substrates L-α-aminoadipic acid, L-cysteine and L-valine were determined as 45, 80 and 80 μM respectively, and the optimal assay concentration of ATP was found to be 5 mM (with 20 mM MgCl2). The dimer of the reaction product bis-δ-(L-α-aminoadipyl)-l-cysteinyl-D-valine (bisACV) gave feedback inhibition of the purified ACVS; the inhibition parameter KbisACV was determined as 1.4 mM. Furthermore dithiothreitol was shown to inhibit the purified ACVS. From the addition of a glucose pulse to a steady-state glucose-limited continuous culture of P. chrysogenum it was found that there is glucose repression of the synthesis of ACVS and that there must be a constant turnover of ACVS owing to synthesis and degradation.
Prolonged activation of phospholipase D in Chinese hamster ovary cells expressing platelet-activating-factor receptor lacking cytoplasmic C-terminal tail
Authors: LIU Bo , NAKASHIMA Shigeru , ADACHI Takahito , ITO Yuzuru , TAKANO Tomoko , SHIMIZU Takao , NOZAWA Yoshinori 
Published: August 2015

The mechanism and role of phospholipase D (PLD) activation by platelet-activating factor (PAF) were examined with Chinese hamster ovary cells stably expressing wild-type PAF receptor (WT-H cells) and truncated PAF receptor lacking the C-terminal cytoplasmic tail (D-H cells). Treatment of D-H cells with PAF resulted in the rapid formation of Ins(1,4,5)P3, which was followed by a sustained phase for more than 10 min. In these cells, PAF-induced PLD activation lasted for more than 20 min. In contrast, PLD activation in WT-H cells was transient. PAF stimulation caused the biphasic formation of 1,2-diacylglycerol (DG) in both types of cell. The first phase was rapid and transient, coinciding with the Ins(1,4,5)P3 peak. The second sustained phase of DG formation was attenuated by butanol, which produces phosphatidylbutanol at the expense of phosphatidic acid (PA) by transphosphatidylation activity of PLD, and by propranolol, a selective inhibitor for PA phosphohydrolase catalysing the conversion of PA into DG. The DG level returned nearly to basal at 20 min after PAF stimulation in WT-H cells, whereas in D-H cells the elevated DG level was sustained for more than 20 min. The profile of translocation of protein kinase Cα (PKCα) to membrane was similar to that of DG formation. In WT-H cells, PKCα was transiently associated with membranes and then returned to the cytosol. However, in D-H cells PKCα was rapidly translocated to and remained in membranes for more than 20 min. Butanol suppressed this sustained translocation of PKCα. Furthermore the mRNA levels of c-fos and c-jun by PAF in WT-H cells were much lower than those in D-H cells. Propranolol and butanol at concentrations that inhibited the formation of DG suppressed the PAF-induced mRNA expression of c-fos and c-jun. Taken together, the prolonged PLD activation in D-H cells confirmed a primary role for phospholipase C/PKC in PLD activation by PAF. Furthermore the results obtained here suggest that sustained PLD activation in turn leads to chronic activation and membrane translocation of PKCα, which might play an important role in the expression of c-fos and c-jun.
Rapid stimulation of amyloid precursor protein release by epidermal growth factor: role of protein kinase C
Authors: SLACK Barbara E. , BREU Jeffrey , MUCHNICKI Lisa , WURTMAN Richard J. 
Published: August 2015

The amyloid precursor protein (APP) of Alzheimer's disease is a transmembrane protein that is cleaved by an uncharacterized enzyme known as α-secretase within its extracellular/intraluminal domain after the activation of guanine nucleotide-binding protein-coupled receptors linked to phosphoinositide hydrolysis. The secretory process results in the release of large soluble derivatives of APP (APPs), and, when elicited by muscarinic receptor activation, exhibits both protein kinase C (PKC)-dependent and tyrosine phosphorylation-dependent components [Slack, Breu, Petryniak, Srivastava and Wurtman (1995) J. Biol. Chem. 270, 8337–8344]. In this report we examine the regulation of the release of APPs by epidermal growth factor (EGF) receptors, which possess intrinsic tyrosine kinase activity, and are coupled to a variety of effectors including phosphoinositide-specific phospholipase Cγ. In A431 cells, EGF caused time-dependent and dose-dependent increases in the formation of inositol phosphates in cultures prelabelled with myo-[3H]inositol, and in the release of APPs into the culture medium; the two responses exhibited similar time courses and EC50 values for EGF. Concomitant with these effects, there were concentration-dependent (3–300 ng/ml) increases in the phosphorylation of tyrosine residues in several proteins, including the EGF receptor itself. The specific PKC antagonist GF 109203X decreased the effect of EGF by approx. 35% at a concentration that abolished the stimulation of the release of APPs by the PKC activator PMA. Tyrphostin AG 1478, an inhibitor of EGF receptor tyrosine kinase, abolished the EGF-induced release of APPs. These results demonstrate that in A431 cells, activation of the EGF receptor stimulates α-secretase activity by a mechanism that is partly dependent on PKC activity.
The novel endogenous cannabinoid 2-arachidonoylglycerol is inactivated by neuronal- and basophil-like cells: connections with anandamide
Authors: MARZO Vincenzo Di , BISOGNO Tiziana , SUGIURA Takayuki , MELCK Dominique , PETROCELLIS Luciano De 
Published: August 2015

The novel endogenous cannabinoid 2-arachidonoylglycerol (2-AG) was rapidly inactivated by intact rat basophilic leukaemia (RBL-2H3) and mouse neuroblastoma (N18TG2) cells through diffusion/hydrolysis/reacylation processes. The hydrolysis of 2-AG was inhibited by typical esterase inhibitors and by more specific blockers of ‘fatty acid amide hydrolase ’ (FAAH), the enzyme catalysing the hydrolysis of the other ‘endocannabinoid ’, anandamide (AEA). No evidence for a facilitated-diffusion process was found. A 2-AG-hydrolysing activity was detected in homogenates from both cell lines, with the highest levels in membrane fractions. It exhibited an optimal pH at 10, and recognized both 2- and 1(3)- isomers of monoarachidonoylglycerol with similar efficiencies. The apparent Km and Vmax values for [3H]2-AG hydrolysis were 91 µM and 29 µM and 2.4 and 1.8 nmol·min-1·mg of protein-1 respectively in N18TG2 and RBL-2H3 cells. [3H]2-AG hydrolysis was inhibited by Cu2+, Zn2+ and p-hydroxymercuribenzoate, and by 2- or 1(3)-monolinoleoyl- and -linolenoyl-glycerols, but not by the oleoyl, palmitoyl and myristoyl congeners. Purified fractions from solubilized membrane proteins catalysed, at pH 9.5, the hydrolysis of 2-AG as well as AEA. Accordingly, AEA as well as FAAH inhibitors, including arachidonoyltrifluoromethyl ketone (ATFMK), blocked [3H]2-AG hydrolysis by N18TG2 and RBL-2H3 membranes, whereas 2-AG inhibited [14C]AEA hydrolysis. FAAH blockade by ATFMK preserved from inactivation the 2-AG synthesized de novo by intact N18TG2 cells stimulated with ionomycin. These data suggest that FAAH may be one of the enzymes deputed to the physiological inactivation of 2-AG, and create intriguing possibilities for the cross-regulation of 2-AG and AEA levels.
Redox regulation of cAMP levels by ascorbate in 1,25-dihydroxy- vitamin D3-induced differentiation of HL-60 cells
Authors: LÓPEZ-LLUCH Guillermo , BURÓN M. Isabel , ALCAÍN Francisco J. , QUESADA Jose M. , NAVAS Placido 
Published: August 2015

1α,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] induces differentiation to monocyte–macrophage lineage of several leukaemic cell lines such as HL-60, U937, M1 and Mono Mac 6. Ascorbate also modulates growth and differentiation of different animal cells in culture. We have previously reported the stimulating effect of ascorbate on 1,25-(OH)2D3-induced HL-60 cell differentiation. We show here that 1,25-(OH)2D3 induces a transient increase in cAMP levels in these cells, and ascorbate significantly increases these cAMP levels. Ascorbate alone does not have any effect. Other cAMP-increasing agents such as isobutylmethylxanthine, forskolin and prostaglandin E2 maintain high levels of cAMP at 48 h of incubation and also enhance differentiation along the monocytic pathway induced by 1,25-(OH)2D3, as revealed by specific differentiation markers, demonstrating the importance of cAMP in the differentiation process. It is also shown that the presence of ascorbate and its free radical (AFR) during 1,25-(OH)2D3-induced differentiation significantly decreases cytoplasmic NADH levels compared with those induced by 1,25-(OH)2D3 in HL-60 cells. The results indicate that NADH is an inhibitor of adenylate cyclase in these cells. AFR is an electron acceptor of the trans-plasma-membrane electron-transport system, and NADH is the electron donor. Through this system, ascorbate and AFR keep levels of NADH low, thereby decreasing its inhibitory effect on adenylate cyclase activity and so increasing cAMP synthesis. We also demonstrate that other ascorbate derivatives, such as ascorbate 2-phosphate and dehydroascorbate, both of which are unable to produce AFR, do not alter intracellular NADH levels during 1,25-(OH)2D3-induced differentiation. Also, ascorbate and AFR increase specific differentiation markers (CD14 and NitroBlue Tetrazolium reduction) but neither ascorbate 2-phosphate nor dehydroascorbate show this enhancing activity. In summary, we propose that the effect of ascorbate on 1,25-(OH)2D3-induced differentiation of HL-60 cells can be explained by redox regulation of the cAMP pathway.
Bovine submaxillary mucin contains multiple domains and tandemly repeated non-identical sequences
Authors: JIANG Weiping , WOITACH Joseph T. , KEIL Ralph L. , BHAVANANDAN Veer P. 
Published: August 2015

A number of cDNA fragments coding for bovine submaxillary mucin (BSM) were cloned, and the nucleotide sequence of the largest clone, BSM421, was determined. Two peptide sequences determined from the purified apoBSM were found near the N-terminus of the mucin-coding region of BSM421. This clone does not contain a start or stop codon, but its 3´ end overlaps with the 5´ end of a previously isolated clone, λBSM10. The composite sequence of 1589 amino acid residues consists of five distinct protein domains, which are numbered from the C-terminus. The cysteine-rich domain I can be further divided into a von Willebrand factor type C repeat and a cystine knot. Domains III and V consist of similar repeated peptide sequences with an average of 47 residues. Domains II and IV do not contain such sequences but are similar to domains III and V in being rich in serine and threonine, many of which are predicted to be potential O-glycosylation sites. Domain III also contains two sequences that match the ATP/GTP-binding site motif A (P-loop). Only β-strands and no α-helices are predicted for the partial deduced amino acid sequence. Northern analysis of submaxillary gland RNA with the BSM421 probe detected multiple messages of BSM with sizes from 1.1 to over 10 kb. The tandemly repeated, non-identical peptide sequences of approx. 47 residues in domains III and V of BSM differ from the tandemly repeated, identical 81-residue sequences of pig submaxillary mucin (PSM), although both BSM and PSM contain similar C-terminal domains. In contrast, two peptide sequences of ovine submaxillary mucin are highly similar (86% and 65% identical respectively) to the corresponding sequences in domain V of BSM.
pH-dependent inhibition by azide and fluoride of the iron superoxide dismutase from Propionibacterium shermanii
Authors: MEIER Beate , SCHERK Christoph , SCHMIDT Marius , PARAK Fritz 
Published: August 2015

The iron-containing superoxide dismutase from Propionibacterium shermanii shows, in contrast with other iron superoxide dismutases, only a minor inhibition by azide or fluoride (10–100 mM) of up to 23% at pH 7.8. The activity of the protein with Mn bound to the active site was not diminished under the same conditions. The binding constant between azide and the Fe3+ ion was determined as approx. 2 mM and for fluoride approx. 2.3 mM; they are so far comparable to those known for other iron superoxide dismutases. This seems to be a discrepancy because all other iron superoxide dismutases so far known are described as being inhibited by 50–70% by 10 mM azide. However, towards lower pH there was a drastically increased inhibition by both anions. At pH 6.8 about 80% inhibition was exhibited by azide or fluoride at a concentration of 10 mM or higher. In contrast, on increasing the pH, azide or fluoride still bound to the Fe3+ at the active site but their inhibition capacity decreased. This observation implies that both anions bind to the metal at a position that is empty at low pH, whereas at higher pH water or a negatively charged hydroxyl anion is bound. It is likely that the superoxide anion binds to the same position and has to replace the sixth ligand, leading to a diminished catalytic activity of the superoxide dismutase owing to steric and/or electrostatic inhibition of the ligand.
Histones and basic polypeptides activate Ca2+/cation influx in various cell types
Authors: GAMBERUCCI Alessandra , FULCERI Rosella , MARCOLONGO Paola , PRALONG William F. , BENEDETTI Angelo 
Published: August 2015

Histone H2A (1–10 µg/ml) added to Ehrlich ascite cell suspensions promoted: (i) Ca2+ influx, but no apparent intracellular Ca2+ mobilization; (ii) plasma-membrane depolarization and Na+ influx in Ca2+-free medium, which were recovered by Ca2+ readmission; (iii) influx of other cations such as Ba2+, Mn2+, choline+ and N-methyl-d-glucamine+, but not of propidium+, ethidium bromide and Trypan Blue. H2A-induced Ca2+ influx and cell depolarization were: (i) blocked by La3+ and Gd3+, but not by various inhibitors of receptor-activated Ca2+-influx pathways/channels; (ii) mimicked by various basic polypeptides, with Mr &gt; 4000; (iii) prevented or reversed by polyanions such as polyglutamate or heparin; (iv) present in other cell types, such as Jurkat, PC12 and Friend erythroleukaemia cells, but virtually absent from rat hepatocytes and thymocytes. We conclude that cationic proteins/polypeptides, by interacting in a cell-specific manner with the cell surface, can activate in those cells putative non-selective Ca2+ channels and membrane depolarization.