bluuebunny/crossref_metadata_2025_split huggingface dataset:
Fructose-induced increase in intracellular free Mg2+ ion concentration in rat hepatocytes: relation with the enzymes of glycogen metabolism
Authors: GAUSSIN Vinciane , GAILLY Philippe , GILLIS Jean-Marie , HUE Louis 
Published: August 2015

In rat hepatocytes subjected to a fructose load, ATP content decreased from 3.8 to 2.6 μmol/g of cells. Under these conditions, the intracellular free Mg2+ ion concentration, as measured with mag-fura 2, increased from 0.25 to 0.43 μmol/g of cells and 0.35 μmol of Mg2+ ions were released per g of cells in the extracellular medium. Therefore the increase in the intracellular free Mg2+ ion concentration was less than expected from the decrease in ATP, indicating that approx. 80% of the Mg2+ ions released from MgATP2- were buffered inside the cells. When this buffer capacity was challenged with an extra Mg2+ ion load by blocking the fructose-induced Mg2+ efflux, again approx. 80% of the extra Mg2+ ion load was buffered. The remaining 20% appearing as free Mg2+ ions in fructose-treated hepatocytes could act as second messenger for enzymes having a Km for Mg2+ in the millimolar range. Fructose activated glycogen synthase and glycogen phosphorylase, although both the time course and the dose-dependence of activation were different. This was reflected in a stimulation of glycogen synthesis with concentrations of fructose below 5 mM. Indeed, activation of glycogen synthase reached a maximum at 30 min of incubation and was observed with small (5 mM or less) concentrations of fructose, whereas the activation of glycogen phosphorylase was almost immediate (within 5 min) and maximal with large doses of fructose. The fructose-induced activation of glycogen phosphorylase, but not that of glycogen synthase, could be related to an increase in free Mg2+ ion concentration.
Biochemical characterization and pharmacological properties of a phospholipase A2 myotoxin inhibitor from the plasma of the snake Bothrops asper
Authors: LIZANO Sergio , LOMONTE Bruno , FOX Jay W. , GUTIÉRREZ José Maréa 
Published: August 2015

A protein that neutralizes the biological activities of basic phospholipase A2 (PLA2) myotoxin isoforms from the venom of the snake Bothrops asper was isolated from its blood by affinity chromatography with Sepharose-immobilized myotoxins. Biochemical characterization of this B. asper myotoxin inhibitor protein (BaMIP) indicated a subunit molecular mass of 23–25 kDa, an isoelectric point of 4, and glycosylation. Gel-filtration studies revealed a molecular mass of 120 kDa, suggesting that BaMIP possesses an oligomeric structure composed of five 23–25 kDa subunits. Functional studies indicated that BaMIP inhibits the PLA2 activity of B. asper basic myotoxins I and III, as well as the myotoxicity and edema-forming activity in vivo and cytolytic activity in vitro towards cultured endothelial cells, of all four myotoxin isoforms (I–IV) tested. Sequence analysis of the first 63 amino acid residues from the N-terminus of BaMIP indicated more than 65% sequence similarity to the PLA2 inhibitors isolated from the blood of the crotalid snakes Trimeresurus flavoviridis and Agkistrodon blomhoffii siniticus. These inhibitors also share sequences similar to the carbohydrate-recognition domains of human and rabbit cellular PLA2 receptors, suggesting a common domain evolution among snake plasma PLA2 inhibitors and mammalian PLA2 receptors. Despite this similarity, this is the first description of a natural anti-myotoxic factor from snake blood.
Reduction of equilibrative nitrobenzylthioinosine-sensitive nucleoside transporter in tamoxifen-treated MCF-7 cells: an oestrogen-reversible phenomenon
Authors: GOH Lay-Beng , LEE Chee-Wee 
Published: August 2015

MCF-7 cells display both nitrobenzylthioinosine (NBMPR)-sensitive (es) and NBMPR-insensitive (ei) equilibrative, but not the Na+-dependent, nucleoside transport. Transport of uridine by es is more sensitive to inhibition by purine nucleosides, whereas the ei component is more sensitive to nucleosides without an amino side group, such as inosine and thymidine. When exposed to 10 μM tamoxifen for 5 days, MCF-7 cells displayed a 44% decrease in the total number of NBMPR-binding sites [Bmax from 245000±18000 to 136000±25000 sites per cell (mean±S.E.M.; n = 5; P&lt; 0.05)], and a 57% decrease in cell growth with no significant change in binding affinities [Kd from 0.37±0.05 to 0.45±0.08 nM (n = 5; P&gt; 0.05)]. Kinetic studies of [3H]uridine transport showed a decrease in the Vmax of the es component from 21.7±0.3 (n = 8) to 8.4±2.2 μM/s (n = 4; P&lt;0.05), whereas the Vmax of the ei component [from 4.7±0.5 (n =8) to 5.8±1.6 μM/s (n = 4; P&gt; 0.05)] and Km values for both components [es from 460±80 to 630±280 μM (n ⩾ 3; P&gt; 0.05) and ei from 355±115 to 440±220 μM (n ⩾ 4; P&gt; 0.05)] did not change significantly. Oestradiol at 100 nM reversed almost completely the NBMPR-binding site decrease and growth retardation in tamoxifen-treated cells. Thus tamoxifen is shown to cause an oestrogen-reversible decrease of es nucleoside transporters in MCF-7 cells.
Angiotensin-converting enzyme secretase is inhibited by zinc metalloprotease inhibitors and requires its substrate to be inserted in a lipid bilayer
Authors: PARVATHY S. , OPPONG Sylvester Y. , KARRAN Eric H. , BUCKLE Derek R. , TURNER Anthony J. , HOOPER Nigel M. 
Published: August 2015

Mammalian angiotensin-converting enzyme (ACE; EC 3.4.15.1) is one of several proteins that exist in both membrane-bound and soluble forms as a result of a post-translational proteolytic processing event. For ACE we have previously identified a metalloprotease (secretase) responsible for this proteolytic cleavage. The effect of a range of structurally related zinc metalloprotease inhibitors on the activity of the secretase has been examined. Batimastat (BB94) was the most potent inhibitor of the secretase in pig kidney microvillar membranes, displaying an IC50 of 0.47 μM, whereas TAPI-2 was slightly less potent (IC50 18 μM). Removal of the thienothiomethyl substituent adjacent to the hydroxamic acid moiety or the substitution of the P2′ substituent decreased the inhibitory potency of batimastat towards the secretase. Several other non-hydroxamate-based collagenase inhibitors were without inhibitory effect on the secretase, indicating that ACE secretase is a novel zinc metalloprotease that is related to, but distinct from, the matrix metalloproteases. The full-length amphipathic form of ACE was labelled selectively with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine in the membrane-spanning hydrophobic region. Although trypsin was able to cleave the hydrophobic anchoring domain from the bulk of the protein, there was no cleavage of full-length ACE by a Triton X-100-solubilized pig kidney secretase preparation when the substrate was in detergent solution. In contrast, the Triton X-100-solubilized secretase preparation released ACE from pig intestinal microvillar membranes, which lack endogenous secretase activity, and cleaved the purified amphipathic form of ACE when it was incorporated into artificial lipid vesicles. Thus the secretase has an absolute requirement for its substrate to be inserted in a lipid bilayer, a factor that might have implications for the development of cell-free assays for other membrane protein secretases. ACE secretase could be solubilized from the membrane with Triton X-100 and CHAPS, but not with n-octylβ-D-glucopyranoside. Furthermore trypsin could release the secretase from the membrane, implying that like its substrate, ACE, it too is a stalked integral membrane protein.
Functional effects of single amino acid substitutions in the region of Phe113 to Asp138 in the plasminogen activator inhibitor 1 molecule
Authors: SUI Guang-Chao , WIMAN Björn 
Published: August 2015

Thirteen amino acid substitutions have been introduced within the stretch Phe113 to Asp138 in the plasminogen activator inhibitor 1 (PAI-1) molecule by site-directed mutagenesis. The different proteins and wild-type (wt) PAI-1 have been overexpressed in Escherichia coliand purified by chromatography on heparin–Sepharose and on anhydrotrypsin–agarose. The PAI-1 variants have been characterized by their reactivity with tissue plasminogen activator (tPA), interactions with vitronectin or heparin, and stability. Most PAI-1 variants, except for Asp125 → Lys, Phe126 → Ser and Arg133 → Asp, displayed a high spontaneous inhibitory activity towards tPA, which did not change greatly on reactivation with 4 M guanidinium chloride, followed by dialysis at pH 5.5. The variants Asp125 → Lys and Arg133 → Asp became much more active after reactivation and they were also more rapidly transformed to inactive forms (t½ 22–31 min) at physiological pH and temperature than the other variants. However, in the presence of vitronectin they were both almost equally stable (t½ 2.3 h) as wtPAI-1 (t½ 3.0 h). The mutant Glu130 → Lys showed an increased stability, both in the absence and in the presence of vitronectin compared with wtPAI-1. Nevertheless a similar affinity between all the active PAI-1 variants and vitronectin was observed. Further, all mutants, including the three mutants with low activity, were to a large extent adsorbed on anhydrotrypsin–agarose and were eluted in a similar fashion. In accordance with these data, the three variants with a low activity were all to a large extent cleaved as a result of their reaction with tPA, suggesting that they occurred predominantly in the substrate conformation. Our results do not support the presence of a binding site for vitronectin in this part of the molecule, but rather that it might be involved in controlling the active PAI-1 to substrate transition. Partly, this region of the PAI-1 molecule (Arg115 to Arg118) seems also to be involved in the binding of heparin to PAI-1.
Pyrazole-inducible proteins in DBA/2 mouse liver bind with high affinity to the 3′-untranslated regions of the mRNAs of coumarin hydroxylase (CYP2A5) and c-jun
Authors: THULKE-GROSS Monika , HERGENHAHN Manfred , TILLOY-ELLUL Anne , LANG Matti , BARTSCH Helmut 
Published: August 2015

An important mechanism in the up-regulation of cytochrome P-450 2A5 (CYP2A5, coumarin hydroxylase, Coh) is the stabilization of the corresponding mRNA; some evidence suggests that proteins binding to CYP2A5 mRNA may be involved in this stabilization. Here we report that pyrazole, a well known inducer of CYP2A5 and stabilizer of its message, enhances the binding of a set of proteins to 32P-labelled 3´-untranslated region (3´UTR) of CYP2A5 to give 32P-labelled bands of apparent molecular mass 37/39, 45/48 and 70/72 kDa after UV cross-linking/RNase cleavage; in addition, we found different proteins binding to other parts of CYP2A5 mRNA. The 70/72 kDa bands are also formed with the 3´UTR of c-jun. The inducible proteins are found in different cellular subfractions at different concentrations, with a maximum of five-fold induction of binding activity in microsomes. When a gel-mobility-shift assay was combined with UV cross-linking to resolve different pyrazole-inducible RNA–protein complexes into single RNA-binding protein bands, the smallest complex contained a double band of 37/39 kDa, 45/48 kDa bands, 70/72 kDa bands, and additional weaker bands at higher molecular masses (around 120 kDa). This composition was found also for all other complexes detected by gel-mobility-shift assay; occasionally, bands at higher molecular masses were also observed. The proteins of the smallest complex might therefore represent a core with which other proteins interact to build up larger complexes. Binding of proteins 37/39 kDa and 70/72 kDa was located to a 20-base loop and adjacent sequences in a 70 nt AU-rich region of the 3´UTR of the CYP2A5. Based on our previous evidence, this 70-nt sequence may play an important role in the stabilization and processing of the message.
Purification and partial characterization of a peroxidase from plant cell cultures of Cassia didymobotrya and biotransformation studies
Authors: VITALI Alberto , BOTTA Bruno , MONACHE Giuliano DELLE , ZAPPITELLI Sabrina , RICCIARDI Paola , MELINO Sonia , PETRUZZELLI Raffaele , GIARDINA Bruno 
Published: August 2015

An acidic peroxidase (EC 1.11.1.7) produced by cell suspension cultures of Cassia didymobotrya(wild senna) was purified from culture medium collected on the 29th day. The enzyme was shown to be a glycoprotein with a pI of 3.5, a molecular mass of approx. 43 kDa by SDS/PAGE and 50 kDa by gel filtration. The N-terminal sequence was very similar to those of other plant peroxidases. The peroxidase was characterized by a high specificity towards coniferyl alcohol and other natural phenolics such as guaiacol and ferulic and caffeic acids. These findings suggest that the enzyme is involved in lignification processes of the cell wall. Moreover, the enzyme was able to catalyse the oxidation of 4,3´,4´-trihydroxychalcone and 4,3´,4´-trihydroxy-3-methoxychalcone to the corresponding 3,3´-biflavanones, as mixtures of racemic and mesoforms.
Characterization of the leupeptin-inactivating enzyme from Streptomyces exfoliatus SMF13 which produces leupeptin
Authors: KIM In Seop , KIM Young Bae , LEE Kye Joon 
Published: August 2015

Leupeptin-inactivating enzyme (LIE) was purified from Streptomyces exfoliatusSMF13 by ammoniumm sulphate fractionation of cell-free culture broth, ultrafiltration, anion-exchange chromatography on DEAE–Sephadex A-50 and gel filtration chromatography on Sephadex G-75. The molecular mass of the purified enzyme was measured as 34700 Da and the N-terminal amino acid sequence was APTPPDIPLANVPA. Acetyl-leucine, leucine and argininal were identified as the products of leupeptin inactivated by the LIE, indicating that leupeptin is inactivated by hydrolysis of peptide bond between leucine and leucine and between leucine and argininal of leupeptin (acetyl-leucine-leucine-argininal). Synthetic-peptide substrates specificity of LIE showed that LIE has absolute specificity for peptide bonds with leucine in the P1 position, suggesting that LIE is a leucine-specific protease. The optimum pH and temperature were pH 9.0 and 45 °C, respectively. LIE activity was inhibited by metalloprotease inhibitors such as EDTA, EGTA, o-phenanthroline and bestatin, but activated by Mg2+ and Ca2+, suggesting that the enzyme is a metalloprotease. Aerial-mycelium growth and aerial spore formation of S. exfoliatus SMF13 were inhibited by the addition of bestatin, an inhibitor of LIE. The inhibition of morphological differentiation was due to the inhibition of trypsin-like protease (TLP) activity, which is essential for aerial-mycelium formation and is inhibited specifically by remaining leupeptin that was not inactivated. These results show that LIEs play a role in controlling the amount of leupeptin during colony development. Therefore, it is suggested that the physiological function of LIE is to inactivate leupeptin when or where TLP activity is required for aerial-mycelium formation.
Periplasmic nitrate-reducing system of the phototrophic bacterium Rhodobacter sphaeroides DSM 158: transcriptional and mutational analysis of the napKEFDABC gene cluster
Authors: REYES Francisca , GAVIRA Mónica , CASTILLO Francisco , MORENO-VIVIÁN Conrado 
Published: August 2015

The phototrophic bacterium Rhodobacter sphaeroidesDSM 158 is able to reduce nitrate to nitrite by means of a periplasmic nitrate reductase which is induced by nitrate and is not repressed by ammonium or oxygen. Recently, a 6.8 kb PstI DNA fragment carrying the napABCgenes coding for this periplasmic nitrate-reducing system was cloned [Reyes, Roldán, Klipp, Castillo and Moreno-Vivián (1996) Mol. Microbiol. 19, 1307–1318]. Further sequence and genetic analyses of the DNA region upstream from the napABCgenes reveal the presence of four additional napgenes. All these R. sphaeroidesgenes seem to be organized into a napKEFDABCtranscriptional unit. In addition, a partial open reading frame similar to the Azorhizobium caulinodans yntCgene and the Escherichia coli yjcCand yhjKgenes is present upstream from this napgene cluster. The R. sphaeroides napKgene codes for a putative 6.3 kDa transmembrane protein which is not similar to known proteins and the napEgene codes for a 6.7 kDa transmembrane protein similar to the Thiosphaera pantotrophaNapE. The R. sphaeroides napFgene product is a 16.4 kDa protein with four cysteine clusters that probably bind four [4Fe-4S] centres. This iron–sulphur protein shows similarity to the NapF and NapG proteins of E. coliand Haemophilus influenzae.Finally, the napDgene product is a 9.4 kDa soluble protein which is also found in E. coliand T. pantotropha. The 5´ end of the naptranscript has been determined by primer extension, and a δ70-like promoter has been identified upstream from the napKgene. The same transcriptional start site is found for cells growing aerobically or anaerobically with nitrate. Different mutant strains carrying defined polar and non-polar insertions in each napgene were constructed. Characterization of these mutant strains demonstrates the participation of the napgene products in the periplasmic nitrate reduction in R. sphaeroides.
Molecular cloning, expression and catalytic activity of a human AKR7 member of the aldo–keto reductase superfamily: evidence that the major 2-carboxybenzaldehyde reductase from human liver is a homologue of rat aflatoxin B1-aldehyde reductase
Authors: IRELAND Linda S. , HARRISON David J. , NEAL Gordon E. , HAYES John D. 
Published: August 2015

The masking of charged amino or carboxy groups by N-phthalidylation and O-phthalidylation has been used to improve the absorption of many drugs, including ampicillin and 5-fluorouracil. Following absorption of such prodrugs, the phthalidyl group is hydrolysed to release 2-carboxybenzaldehyde (2-CBA) and the pharmaceutically active compound; in humans, 2-CBA is further metabolized to 2-hydroxymethylbenzoic acid by reduction of the aldehyde group. In the present work, the enzyme responsible for the reduction of 2-CBA in humans is identified as a homologue of rat aflatoxin B1-aldehyde reductase (rAFAR). This novel human aldo–keto reductase (AKR) has been cloned from a liver cDNA library, and together with the rat protein, establishes the AKR7 family of the AKR superfamily. Unlike its rat homologue, human AFAR (hAFAR) appears to be constitutively expressed in human liver, and is widely expressed in extrahepatic tissues. The deduced human and rat protein sequences share 78% identity and 87% similarity. Although the two AKR7 proteins are predicted to possess distinct secondary structural features which distinguish them from the prototypic AKR1 family of AKRs, the catalytic- and NADPH-binding residues appear to be conserved in both families. Certain of the predicted structural features of the AKR7 family members are shared with the AKR6 β-subunits of voltage-gated K+-channels. In addition to reducing the dialdehydic form of aflatoxin B1-8,9-dihydrodiol, hAFAR shows high affinity for the γ-aminobutyric acid metabolite succinic semialdehyde (SSA) which is structurally related to 2-CBA, suggesting that hAFAR could function as both a SSA reductase and a 2-CBA reductase in vivo. This hypothesis is supported in part by the finding that the major peak of 2-CBA reductase activity in human liver co-purifies with hAFAR protein.
The cDNA sequence for human AFAR has been assigned by GenBank the accession number AF026947.
Structural/functional properties of a mammalian multi-component structure containing all major spliceosomal small nuclear ribonucleoprotein particles
Authors: MORAITOU Marina , PATRINOU-GEORGOULA Meropi , GUIALIS Apostolia 
Published: August 2015

An approx. 40 S multi-component structure, consisting of all major spliceosomal small nuclear ribonucleoprotein particles (snRNP) (U1, U2, U4/U6 and U5) in stable association with a large number of polypeptides, mainly in the range 50–210 kDa, has been reported to exist within rat liver nuclear extracts [Guialis, Moraitou, Patrinou-Georgoula and Dangli (1991) Nucleic Acids Res. 19, 287–296]. Using a new polyclonal antibody recognizing a 63 kDa protein component of the complex, this multi-snRNP assembly was detected within rat liver nuclear extracts as efficiently as with the antibody for the U2 snRNP-specific B´ polypeptide. The 63 kDa protein was found to correspond to the 66 kDa subunit of the splicing factor SF3a, a known integral component of the HeLa 17 S U2 snRNP. Anti-2,2,7-trimethylguanosine affinity chromatography was an easy and efficient way of purifying the multi-snRNP complex from rat liver 40 S heterogeneous nuclear ribonucleoprotein particle (hnRNP)-containing sucrose gradient fractions. By subsequent glycerol-gradient sedimentation, all known snRNP forms active in RNA splicing were identified among its constituents. A complex structurally similar to the rat multi-snRNP was also identified in HeLa nuclear extracts. Preservation of hnRNP–snRNP interactions was observed within HeLa 40 S fractions. Moreover, these fractions were capable of restoring splicing activity when applied in reconstitution studies to supplement a micrococcal nuclease-treated splicing extract.
Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone-sensitive lipase subfamily
Authors: MANCO Giuseppe , ADINOLFI Elena , PISANI Francesca M. , OTTOLINA Gianluca , CARREA Giacomo , ROSSI Mosè 
Published: August 2015

We previously purified a new esterase from the thermoacidophilic eubacterium Bacillus acidocaldarius whose N-terminal sequence corresponds to an open reading frame (ORF3) reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. To compare the biochemical properties of this thermophilic enzyme with those of the homologous mesophilic and psychrophilic members of the HSL group, an overexpression system in Escherichia coli was established. The protein, expressed in soluble and active form at 10 mg/l E. coli culture, was purified to homogeneity and characterized biochemically. The enzyme, a 34 kDa monomeric protein, was demonstrated to be a B´-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-exanoate with Km and kcat values of 11±2 µM (mean±S.D., n = 3) and 6610±880 s-1 (mean±S.D., n = 3) respectively at 70 °C and pH 7.1. In spite of relatively high sequence identity with the mammalian HSLs, the psychrophilic MoraxellaTA144lipase 2 and the human liver arylacetamide deacetylase, no lipase or amidase activity was detected. A series of substrates were tested for enantioselectivity. Substantial enantioselectivity was observed only in the resolution of (±)-3-bromo-5-(hydroxymethyl)-Δ2-isoxazoline, where the (R)-product was obtained with an 84% enantiomeric excess at 36% conversion. The enzyme was also able to synthesize acetyl esters when tested in vinyl acetate and toluene. Inactivation by diethylpyrocarbonate, diethyl-p-nitrophenyl phosphate, di-isopropylphosphofluoridate (DFP) and physostigmine, as well as labelling with [3H]DFP, supported our previous suggestion of a catalytic triad made up of Ser-His-Asp. The activity–stability–temperature relationship is discussed in relation to those of the homologous members of the HSL group.
Prothymosin α is a chromatin-remodelling protein in mammalian cells
Authors: GÓMEZ-MÁRQUEZ Jaime , RODRÍGUEZ Pilar 
Published: August 2015

Prothymosin α (ProTα) is an abundant mammalian acidic nuclear protein whose expression is related to cell proliferation. Here we report that in HL-60 cells overexpressing ProTα, the accessibility of micrococcal nuclease to chromatin is strongly increased. In the DNA ladder generated by the nuclease activity, the sizes of the mononucleosome (146 bp, the DNA fragment that is bound to the histone octamer) and its multimers correspond to nucleosomes lacking histone H1. The percentage of histone-H1-depleted chromatin (active chromatin) is also higher in the cells overexpressing ProTα. On the basis of these and previous findings, we propose a biological role for ProTα in the remodelling of chromatin fibres through its interaction with histone H1.