bluuebunny/crossref_metadata_2025_split huggingface dataset:
Studies on recombinant Acetobacter xylinum α-phosphoglucomutase
Authors: KVAM Catrine , OLSVIK Ellen Sofie , MCKINLEY-MCKEE John , SAETHER Olav 
Published: August 2015

The phosphoglucomutase (PGM) from Acetobacter xylinum,which had been cloned and expressed in Escherichia coli,has been studied. After expression, the enzyme was purified from the E. coliin a three-step process consisting of (NH4)2SO4 precipitation, gel filtration and anion-exchange chromatography. The purified enzyme gave one band on gel electrophoresis and was judged essentially free of impurities, although it was unstable when diluted without the addition of 15 μM BSA. The isoelectric point for A. xylinumPGM was 4.8 and the molar absorbance was 3.9×104 M-1·cm-1. The enzyme was reasonably heat-stable below 50 °C and was stable throughout the pH 5.5–7.4 range, but was 70% inactivated at pH 10.0 and completely inactivated after standing for 10 min at pH 3.0 or at pH 12.4. When isolated, the recombinant enzyme was fully active without the addition of extra Mg2+. The Km for glucose 1-phosphate was much higher than that of other PGM species reported, which accords with the production of extracellular cellulose in A. xylinum.Glucose 1,6-diphosphate is not considered to be a substrate or coenzyme but an activating cofactor like Mg2+. The following kinetic constants were determined: Vmax 81.1 units/mg; kcat and the turnover rate 135 s-1; Km (glucose 1,6-diphosphate) 0.2 μM; Km (glucose 1-phosphate) 2.6 mM; kcat/Km (glucose 1-phosphate) 5.2×104 M-1·s-1. The recombinant enzyme is considered to follow a characteristic substituted enzyme or Ping Pong reaction mechanism.
Short-term treatment with oleoyl-oestrone in liposomes (Merlin-2) strongly reduces the expression of the ob gene in young rats
Authors: SANCHIS Daniel , ADÁN Cristina , ARDÉVOL Anna , DEL MAR GRASA Maria , CABOT Cristina , BALADA Francisca , VILÀ Ruth , ESTRUCH Jordi , PUERTA Marisa , FERNÁNDEZ-LÓPEZ José-Antonio , REMESAR Xavier , ALEMANY Marià 
Published: August 2015

Young female rats of 160–180 g were implanted with osmotic minipumps releasing 3.0 μmol/day per kg of oleoyl-oestrone in liposomes (Merlin-2) into the bloodstream for up to 14 days. Merlin-2 induced a loss of appetite in the first days, later recovered, and a decrease in body weight of 7%, which contrasts with the 15% increase in controls during the 2-week period. Neither plasma glucose nor urea was affected by treatment, but liver glycogen increased by 50% in 14 days. Insulin decreased slightly with Merlin-2 treatment. Plasma corticotropin (ACTH) and corticosterone showed a transient increase by day 6 of treatment. The expression of the ob gene in adipose tissue fell during the period studied to practically nil on day 14; circulating leptin levels decreased more than 70% from day 1 to day 14. Oestrone levels increased from 0.3 nM (controls) to a maintained 40–60 nM level for the rest of the experiment. Oleoyl-oestrone levels first increased 4-fold, to decrease again to the initial levels on day 10, increasing later to 100-fold on day 14. The three phases observed in food intake, weight loss and oleoyl-oestrone levels match fairly well, which supports the direct involvement of oleoyl-oestrone in body-weight control. However, the control of oleoyl-oestrone levels seems to be mediated in part by corticosterone. The practical disappearance of leptin synthesis coincides with the massive accumulation of oleoyl-oestrone in plasma. The results presented suggest the involvement of oleoyl-oestrone in the main mechanisms of control of body weight and its regulation by glucocorticoids and leptin.
Re-activation of Clostridium symbiosum glutamate dehydrogenase from subunits denatured by urea
Authors: AGHAJANIAN Suren , ENGEL Paul C. 
Published: August 2015

In a study of the re-activation of urea-denatured clostridial glutamate dehydrogenase (GDH) the maximum re-activation achieved without any added ligands was about 6%, but with NAD+ and 2-oxoglutarate in combination about 70%. NAD+ alone was also effective but 2-oxoglutarate was not, in striking contrast with the opposite pattern for protection of this enzyme against unfolding in urea [Aghajanian, Martin and Engel (1995) Biochem. J. 311, 905–910]. The extent of re-activation was not increased by raising the incubation temperature to 37 °C and was independent of the time of enzyme denaturation. CD and fluorimetric studies showed that dilution of denatured enzyme into potassium phosphate buffer led to rapid (half-time &lt; 3–5 s) formation of ‘structured’ intermediates with secondary structure similar to that of native enzyme. These intermediate molecules were inactive, behaved as monomers on a size-exclusion column, and were unable to associate to give the native hexameric structure. Addition of NAD+ facilitated isomerization of these ‘structured’ monomers into a form(s) capable of re-activation. A side effect in the refolding process was non-specific aggregation, depending on final enzyme concentration. The hexamer fraction from re-activated samples, however, showed the same specific activity as native enzyme. The portion of the enzyme that is not lost through aggregation thus appears to regain the native structure fully. Detailed time-course studies showed that re-activation follows second-order kinetics, suggesting that formation of a dimer may be the rate-limiting step. The possible mechanism for the unfolding and refolding processes of clostridial GDH and effects of coenzyme and substrate on these are discussed in relation to the known crystal structure.
Synthetic peptides containing a BXBXXXB(B) motif activate phospholipase C-β1
Authors: PIIPER Albrecht , STRYJEK-KAMINSKA Danuta , ILLENBERGER Daria , KLENGEL Rolf , SCHMIDT Jürgen M. , GIERSCHIK Peter , ZEUZEM Stefan 
Published: August 2015

We have recently shown that synthetic peptides of the effector domain of the low-molecular-mass GTP-binding protein Rab3 stimulate inositol 1,4,5-trisphosphate production in various permeabilized cells. To investigate the mechanism of the peptide-induced activation of phospholipase C (PLC) and to identify the PLC isoenzyme(s) targeted by these peptides, isolated pancreatic acinar membranes and cytosol were preincubated with anti-PLC antibodies before examination of PLC activity in response to the Rab3B/D effector-domain peptide (VSTVGIDFKVKTVYRH, peptide P1). Western blot analysis revealed the presence of PLC-β1, -β3, -γ1 and -δ1 in membrane and cytosolic fractions. P1 stimulated PLC activity in both membrane and cytosolic fractions. Anti-(PLC-β1) antibody inhibited P1-induced PLC activity in both subcellular fractions almost completely. Moreover, P1-induced amylase release in digitonin-permeabilized pancreatic acini was also inhibited. Other immunoneutralizing anti-PLC antibodies had no effect, suggesting that P1 activates PLC-β1 but not PLC-β3, -γ1 or -δ1. P1 also activated recombinant PLC-β1, indicating direct activation of PLC-β1 by Rab3 effector-domain peptides. To investigate further the structure–function relationship of the peptides, truncated peptides of P1 were tested for their ability to activate PLC in isolated pancreatic acinar membranes and to stimulate amylase release from digitonin-permeabilized pancreatic acini. Peptides containing a BXBXXXB(B) motif (where B represents a basic residue and X any residue) [KVKTVYRH (EC50 of 1 nM to stimulate amylase release) ≈ TVGIDFKVKTVYRH &gt; TVGIDFKVKTVYR] were potent stimulators of amylase release and PLC activity, whereas deletion of the C-terminus (VSTVGIDF), of the two basic C-terminal amino acid residues (VSTVGIDFKVKTVY and KVKTVY), or destruction of the BXB motif (VKTVYR) resulted in inactive peptides. In conclusion, the results of the present study show that short peptides containing a BXBXXXB motif represent promising pharmacological agents to activate the PLC-β1 isoenzyme.
Modulation of mitochondrial electrical potential: a candidate mechanism for drug resistance in African trypanosomes
Authors: WILKES Jonathan M. , MULUGETA Wubet , WELLS Clive , PEREGRINE Andrew S. 
Published: August 2015

Bloodstream forms of four populations of the livestock pathogen Trypanosoma congolense, isolated from different natural infections, have been shown to exhibit a wide range of sensitivities to the trypanocide isometamidium chloride (Samorin®). In mice the 50% curative doses (CD50) for Samorin range from 0.007 to 20 mg/kg body weight. Uptake of isometamidium chloride demonstrated Michaelis–Menten-type kinetics in all the populations, with Km values in the range 0.35–0.87 μM, and Vmax varied from 17 to 216 pmol/min per 108 cells. The magnitude of Vmax was correlated with sensitivity to the drug. In contrast, no correlation was observed between Km values and drug sensitivity. Pulse–chase experiments indicated two compartments for accumulation of drug. The first consists of freely diffusible drug that is invariant between populations; the other consists of retained isometamidium, which is of variable magnitude between the populations and is correlated with drug sensitivity. Autoradiography and fluorescence microscopy demonstrated initial, rapid accumulation of the drug within the mitochondrion, specifically the kinetoplast. In a drug-sensitive population of T. congolense, agents affecting mitochondrial function were shown to produce dose-dependent inhibition of mitochondrial membrane potential (ΔΨmito), as measured by the accumulation of the lipophilic cations [3H]methyltriphenylphosphonium iodide or rhodamine 123. The agents also produced parallel inhibition of isometamidium uptake, suggesting an involvement of ΔΨmito in the accumulation of the drug. When characterized in each of the four populations, the spontaneous ΔΨmito was shown to be characteristic of each population and was correlated with Vmax for drug uptake and sensitivity to the drug in vitro and in vivo. We therefore conclude that in T. congolenseΔΨmito is an important determinant of the rate and accumulation of the trypanocide isometamidium chloride. Populations of this trypanosome species vary with respect to ΔΨmito, which is correlated with sensitivity to isometamidium. We suggest that when exposed to drug, the selection of such populations represents a novel mechanism of drug resistance in protozoan parasites.
cDNA cloning reveals two mouse β5 integrin transcripts distinct in cytoplasmic domains as a result of alternative splicing
Authors: ZHANG Haifeng , TAN Suet M. , LU Jinhua 
Published: August 2015

The integrin β5 subunit has only been found to form a heterodimer with subunit αv which acts as a vitronectin receptor. Integrin αvβ5 has been implicated in cell migration and growth factor-induced angiogenesis. In the present study, a mouse liver cDNA library was screened using a human β5 cDNA fragment obtained by reverse transcriptase PCR (RT-PCR). Three of the clones (MB5, MB15 and MB17) overlapped to give an open reading frame, called β5A, which is homologous to the human β5 subunit. The sequence of another clone (MB26), called β5B, was identical with β5A, except for a deletion of 29 bp near the 3´ end of the open reading frame. The 29 bp deletion resulted in an open-reading-frame shift and a completely different C-terminal sequence in β5B. β5A and β5B were shown, by RT-PCR, to be co-expressed in most mouse tissues tested, although β5B mRNA was detected at much lower levels than β5A. β5A and β5B mRNAs were also detected in the mouse monocytic cell line, J774, and in isolated mouse peritoneal macrophages. Adhesion of peritoneal macrophages has been shown to up-regulate the expression of both β5A and β5B mRNAs. The 29 bp sequence begins with a putative intron-splicing donor site (GTGAT …). A 3´ fragment of the mouse integrin β5 gene was cloned by PCR and sequenced showing that the 29 bp sequence was also immediately followed by an intron. Therefore, the 29 bp sequence was apparently expressed as part of the β5A mRNA but was spliced out as part of the downstream intron in β5B. Since the cytoplasmic domains of the integrin β subunits are important in cytoskeleton attachment and signalling, the two alternatively spliced β5 isoforms may have distinct roles in cell adhesion and other cellular functions.
Cystathionine γ-synthase from Arabidopsis thaliana: purification and biochemical characterization of the recombinant enzyme overexpressed in Escherichia coli
Authors: RAVANEL Stéphane , GAKIÈRE Bertrand , JOB Dominique , DOUCE Roland 
Published: August 2015

Cystathionine γ-synthase catalyses the first reaction specific for methionine biosynthesis in plants, the γ-replacement of the phosphoryl substituent of O-phosphohomoserine by cysteine. A cDNA encoding cystathionine γ-synthase from Arabidopsis thalianahas been cloned and used to overexpress the enzyme in Escherichia coli.The native recombinant enzyme is a homotetramer composed of 53 kDa subunits, each being tightly associated with one molecule of pyridoxal 5´-phosphate that binds at lysine-379 of the protein precursor. The replacement reaction follows a Ping Pong mechanism with a Vmax of 33.6 units/mg and Km values of 2.5 mM and 460 µM for O-phosphohomoserine and cysteine respectively. The protective effect of O-phosphohomoserine against enzyme inactivation by propargylglycine indicated that the Kd for the substrate is approx. 1/2500 of its Km value. Thus most of these biochemical properties are similar to those previously reported for plant and bacterial cystathionine γ-synthases. However, the plant enzyme differs markedly from its enterobacterial counterparts because it catalyses a very faint γ-elimination of O-phosphohomoserine in the absence of cysteine, this process being about 1/2700 as fast as the γ-replacement reaction and approx. 1/1500 as fast as the γ-elimination catalysed by the E. colienzyme. This huge difference could be attributed to the inability of the A. thalianacystathionine γ-synthase to accumulate a long-wavelength-absorbing species that is characteristic for the efficient γ-elimination reaction catalysed by the enterobacterial enzyme.
Protein heterogeneity of spinach pullulanase results from the coexistence of interconvertible isomeric forms of the monomeric enzyme
Authors: HENKER Anette , SCHINDLER Ilka , RENZ Andreas , BECK Erwin 
Published: August 2015

Purified pullulanase (starch-debranching enzyme, R-enzyme, EC 3.2.1.41) from spinach (Spinacia oleracea L.) chloroplasts separated into at least seven individual enzymically active proteins (isomers, numbered 1–7) on isoelectric focusing or column chromatofocusing. At their isoelectric points (between pH 4.7 and 5.2) these forms were rather stable. At slightly alkaline pH, each converted into the whole set of isomers. PAGE of the purified enzyme under denaturing or non-denaturing conditions resulted in one protein band. When substrate (amylopectin or pullulan) was included in the gel, the native enzyme as well as any of the individual isomers separated into two (sometimes three) bands (‘substrate-induced forms’, numbered I–III) with different specific activities, dissociation constants of the enzyme–substrate complexes and activation energies. Each substrate-induced form produced the whole set of seven isomers on isoelectric focusing. The specific activity of the total enzyme reflected the relative proportions of the substrate-induced forms. To some extent the relative proportions, as determined by crossed immunoelectrophoresis, could be shifted in favour of the more or the less active forms by reduction with dithiothreitol, and gentle oxidation respectively. Activation by dithiothreitol did not alter the mode of action of the enzyme but only increased the velocity of substrate degradation and extended its activity into the pH range of the chloroplast. As a consequence of isomer interconversion, microheterogeneity could serve to regulate pullulanase activity in a biochemical manner that shares some features with allosteric regulation.
Quantification of cathepsins B and L in cells
Authors: XING Ruye , ADDINGTON Adele K. , MASON Robert W. 
Published: August 2015

A method for quantifying active cysteine proteinases in mammalian cells has been developed using an active-site-directed inhibitor. Fluoren-9-ylmethoxycarbonyl(di-iodotyrosylalanyl)-diazomethane (Fmoc-[I2]Tyr-Ala-CHN2) was prepared and shown to react irreversibly with cathepsins B and L, but not with cathepsin S. The non- and mono-iodo forms of the inhibitor reacted with all three enzymes. These results demonstrate that, unlike cathepsins B and L, cathepsin S has a restricted S2-binding site that cannot accommodate the bulky di-iodotyrosine. Fmoc-[I2]Tyr-Ala-CHN2 was able to penetrate cells and react with active enzymes within the cells. A radiolabelled form of the inhibitor was synthesized and the concentration of functional inhibitor was established by titration with papain. This inhibitor was used to quantify active cysteine proteinases in cultured cells. Active cathepsin B was found to be expressed by all of the cells studied, consistently with a housekeeping role for this enzyme. Active forms of cathepsin L were also expressed by all of the cells, but in different quantities. Two additional proteins were labelled in some of the cells, and these may represent other non-characterized proteinases. Higher levels of active cathepsins B and L, and an unidentified protein of Mr 39000, were found in breast tumour cells that are invasive, compared with those that are not invasive. From the data obtained, it can be calculated that the concentrations of both active cathepsins B and L in lysosomes can be as high as 1 mM, each constituting up to 20% of total protein in the organelle. This new technique provides a more direct procedure for determining the proteolytic potential of cellular lysosomes.
Transcript heterogeneity of the human reduced folate carrier results from the use of multiple promoters and variable splicing of alternative upstream exons
Authors: ZHANG Long , WONG So C. , MATHERLY Larry H. 
Published: August 2015

We previously identified three separate cDNAs (KS6, KS32 and KS43) for the human reduced folate carrier (RFC) with unique 5´ untranslated regions (5´ UTRs) [Wong, Proefke, Bhushan and Matherly (1995) J. Biol. Chem. 270, 17468–17475]. Multiple RFC transcripts were confirmed in CCRF-CEM cells and transport-up-regulated K562.4CF cells by 5´ rapid amplification of cDNA ends (5´ RACE) and/or primer extension analysis. Two groups of 5´ RACE clones were identified, one containing a variable length sequence identical with the KS43 cDNA 5´ UTR, and another consisting of variants of the KS32 5´ UTR, apparently generated by alternative splicing. The 5´ UTR for the KS6 cDNA was not detected. A single band was detected on Southern blots of CCRF-CEM genomic DNA probed with a 326 bp genomic fragment common to all three cDNA species. The unique 5´ UTRs for the KS43 and KS32 transcripts were localized to separate non-coding exons (exons 1 and 2 respectively), upstream from a large (approx. 3.42 kb) intron; the KS6 5´UTR also mapped to exon 1. Exons 1 and 2 were contiguous with 996 and 342 bp GC-rich 5´ flanking regions (designated Pro43 and Pro32 respectively) that contained multiple SP1 and AP2 but no TATA or CAAT boxes. Both Pro43 and Pro32 exhibited strong promoter activities when cloned in front of a luciferase reporter gene and transfected into HT1080 and K562 cells. By an analysis of promoter deletion mutants we identified two 89 bp tandem repeats that seemed to increase Pro32 activity, and a 240 bp distal sequence that repressed Pro43 activity. Taken together, our results show that multiple human RFC transcripts are encoded by a single gene locus and that the heterogeneous 5´ UTRs result from multiple transcriptional starts and variable splicing of alternative non-coding exons transcribed from separate promoters.
Nuclear factor 1 regulates the distal silencer of the human PIT1/GHF1 gene
Authors: RAJAS Fabienne , DELHASE Mireille , HOYA Miguel de La , VERDOOD Peggy , CASTRILLO José-Luis , HOOGHE-PETERS Elisabeth L. 
Published: August 2015

Here we report the characterization of 12 kb genomic DNA upstream of the human PIT1/GHF1 promoter. Different regions involved in the modulation of human PIT1/GHF1 gene expression were defined by transient transfection studies. Two regions, one proximal (-7.1/-2.3) and one distal (-11.8/-10.9), presented an enhancer activity in pituitary cells when placed upstream of the SV40 promoter. The 0.9 kb distal region was analysed further and found to decrease the basal transcriptional activity of the human PIT1/GHF1 minimal promoter, indicating that this region behaves as a silencer for its own promoter. Three Pit-1/GHF-1-binding sites and two ubiquitous nuclear factor 1 (NF-1)-binding sites were identified by DNase I footprinting in the distal regulatory region. Deletion analysis indicated that NF-1 or NF-1-related protein(s) participate in the down-regulation of human PIT1/GHF1 gene expression by interacting with an NF-1-binding site within the distal regulatory region.
The nucleotide sequence data reported will appear in DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession number X97489.
Sp3 regulates Fas expression in lung epithelial cells
Authors: PANG Huiling , MIRANDA Kathleen , FINE Alan 
Published: August 2015

By transducing an apoptotic signal in immune effector cells, Fas has been directly implicated in the control of immunological activity. Expression and functional results, however, have also suggested a role for Fas in regulating cell turnover in specific epithelial populations. To characterize factors responsible for Fas expression in epithelial cells, approximately 3 kb of the 5´ flanking region of the mouse Fas gene was isolated. By rapid amplification of cDNA ends and primer extension, transcriptional start sites were identified within 50 bp upstream of the translation start site. Transient transfection of promoter–luciferase constructs in a mouse lung epithelial cell line, MLE-15, localized promoter activity to the first 77 bp of upstream sequence. By using a 60 bp DNA probe (-18 to -77) in electrophoretic mobility-shift assays, three shifted complexes were found. Incubation with excess cold Sp1 oligonucleotide or an anti-Sp3 antibody inhibited complex formation. Site-directed mutagenesis of the Sp1 site resulted in 60–70% loss of promoter activity. In Drosophila SL-2 cells, promoter activity was markedly increased by co-transfection of an Sp3 expression construct. These results show that the Sp3 protein is involved in regulating Fas gene expression in lung epithelial cells.
The survival of ingested lactoferrin in the gastrointestinal tract of adult mice
Authors: KUWATA Hidefumi , YIP Tai-Tung , YAMAUCHI Koji , TERAGUCHI Susumu , HAYASAWA Hirotoshi , TOMITA Mamoru , HUTCHENS T. William 
Published: August 2015

Lactoferrin is an 80 kDa major protein component of mammalian colostral whey. The antimicrobial active centre of lactoferrin, lactoferricin (Lfcin), may also be an important determinant of the interaction between lactoferrin and specific receptors on lymphocytes. We have documented the survival in vivo of ingested lactoferrin in the gastrointestinal tract of adult mice by surface-enhanced laser desorption/ionization affinity MS. Various kinds of degraded lactoferrin fragments were detected as molecular-ion peaks corresponding to Lfcin after being captured by an affinity capture device, hydrolysis in situ and laser desorption/ionization. No evident molecular-ion peaks of Lfcin were observed upon analysis of faeces from mice fed commercial milk, whereas lactoferrin fragments containing the Lfcin region were detected at concentrations in the order of at least pmol/g in the faeces of mice fed milk enriched with lactoferrin at 40 mg/ml. These results suggest that ingested lactoferrin would survive transit through the gastrointestinal tract as partially degraded forms containing the receptor-binding region(s) as well as the antimicrobial active centre.