bluuebunny/crossref_metadata_2025_split huggingface dataset:
A 13C-NMR study of the role of Asn-155 in stabilizing the oxyanion of a subtilisin tetrahedral adduct
Authors: O'CONNELL Timothy P. , DAY Regina M. , TORCHILIN Ekaterina V. , BACHOVCHIN William W. , MALTHOUSE J. Paul G. 
Published: August 2015

By removing one of the hydrogen-bond donors in the oxyanion hole of subtilisin BPN, we have been able to determine how it affects the catalytic efficiency of the enzyme and the pKa of the oxyanion formed in a choloromethane inhibitor derivative. Variant 8397 of subtilisin BPN contains five mutations which enhance its stability. Site-directed mutagenesis was used to prepare the N155A mutant of this variant. The catalytic efficiencies of wild-type and variant 8397 are similar, but replacing Asn-155 with alanine reduces catalytic efficiency approx. 300-fold. All three forms of subtilisin were alkylated using benzyloxycarbonylglycylglycyl[2-13C]phenylalanylchloromethane and examined by 13C-NMR. A single signal due to the 13C-enriched carbon was detected in all the derivatives and it was assigned to the hemiketal carbon of a tetrahedral adduct formed between the hydroxy group of Ser-221 and the inhibitor. This signal had chemical shifts in the range 98.3–103.6 p.p.m., depending on the pH. The titration shift of 4.7–4.8 p.p.m. was assigned to oxyanion formation. The oxyanion pKa values in the wild-type and 8397 variants were 6.92 and 7.00 respectively. In the N155A mutant of the 8397 variant the oxyanion pKa increased to 8.09. We explain why such a small increase is observed and we conclude that it is the interaction between the oxyanion and the imidazolium cation of the active-site histidine that is the main factor responsible for lowering the oxyanion pKa.
Molecular cloning, chromosomal assignment and tissue-specific expression of a murine α(1,2)fucosyltransferase expressed in thymic and epididymal epithelial cells
Authors: DOMINO Steven E. , HIRAIWA Nozomu , LOWE John B. 
Published: August 2015

Terminal Fucα(1-2)Galβ epitopes have been proposed to play significant roles in cell–cell interactions in development, cell adhesion, and malignant transformation. To begin to investigate the regulation and function of α(1-2)fucosylated epitopes in an animal model, we have isolated and characterized a mouse genomic DNA segment encoding a protein orthologous to the human H blood group locus α(1,2)fucosyltransferase (FUT1). This segment maintains an open reading frame encoding 376 amino acids sharing 75% sequence identity with the enzyme encoded by human FUT1, and 55% sequence identity with the enzyme encoded by the human Secretor blood group locus (FUT2). Expression of the open reading frame in COS-7 cells yields an α(1,2)fucosyltransferase activity with a Km of 7.6 mM for phenyl-β-d-galactoside. Southern blotting and interspecific backcross analyses indicate that this murine locus represents a single copy sequence mapping to a novel locus 2.1 centimorgans from the Klk1 locus, in a region of homology between mouse chromosome 7 and the human FUT1 locus on the long arm of chromosome 19. Mouse FUT1 yields a 2.8 kb mRNA transcript identifiable in many organs, including thymus, lung, stomach, pancreas, small intestine, colon, uterus and epididymis. Hybridization analyses in situ localize expression of FUT1 transcripts to thymic medullary and epididymal epithelial cells, implying that this gene determines the expression of cell surface Fucα(1-2)Galβ epitopes in these tissues.
ATP-dependent transport of bilirubin glucuronides by the multidrug resistance protein MRP1 and its hepatocyte canalicular isoform MRP2
Authors: JEDLITSCHKY Gabriele , LEIER Inka , BUCHHOLZ Ulrike , HUMMEL-EISENBEISS Johanna , BURCHELL Brian , KEPPLER Dietrich 
Published: August 2015

Bilirubin is secreted from the liver into bile mainly as monoglucuronosyl and bisglucuronosyl conjugates. We demonstrate for the first time that ATP-dependent transport of both bilirubin glucuronides is mediated by the multidrug resistance protein (MRP1) as well as by the distinct canalicular (apical) isoform MRP2, also termed cMRP or cMOAT (canalicular multispecific organic anion transporter). In membrane vesicles from MRP1-transfected HeLa cells mono[3H]glucuronosylbilirubin and bis[3H]glucuronosylbilirubin (each at 0.5 μM) were transported with rates of 5.3 and 3.1 pmol/min per mg of protein respectively. Rat hepatocyte canalicular membrane vesicles, which contain Mrp2 (the rat equivalent of MRP2), transported mono[3H]glucuronosylbilirubin and bis[3H]glucuronosylbilirubin at rates of 8.9 and 8.5 pmol/min per mg of protein, whereas membrane vesicles from mutant liver lacking Mrp2 showed no transport of the conjugates. In membrane vesicles from human hepatoma Hep G2 cells, which predominantly expressed MRP2, transport rates were 8.3 and 4.4 pmol/min per mg of protein for monoglucuronosylbilirubin and bisglucuronosylbilirubin respectively. ATP-dependent transport of the glutathione S-conjugate [3H]leukotriene C4, an established high-affinity substrate for MRP1 and MRP2, was inhibited by both bilirubin glucuronides with IC50 values between 0.10 and 0.75 μM. The ratios of leukotriene C4 transport and bilirubin glucuronide transport, determined in the same membrane vesicle preparation, indicated substrate specificity differences between MRP1 and MRP2 with a preference of MRP2 for the glucuronides.
125I-Labelled mapacalcine: a specific tool for a pharmacological approach to a receptor associated with a new calcium channel on mouse intestinal membranes
Authors: VIDALENC Patricia , MOREL Jean-Luc , MIRONNEAU Jean , HUGUES Michel 
Published: August 2015

Mapacalcine is a small protein (Mr = 19041) composed of two homologous chains purified from the marine sponge Cliona vastifica. Recently, we demonstrated that it was able to specifically block a Ca2+ channel which could not be related to already described channels on mouse intestinal myocytes. This Ca2+ current was insensitive to the known peptidic and organic calcium channel blockers. Mapacalcine was ineffective on T-type and L-type Ca2+ currents present on rat portal vein myocytes [Morel, Drobecq, Sautière, Tartar, Mironneau, Qar, Lavie, and Hugues (1997) Mol. Pharmacol. 51, 1042–1052]. We report here the preparation and purification of a monoiodo-derivative of mapa-calcine which retains its biological properties. Binding parameters of mapacalcine to its receptors have been characterized on mouse intestinal membranes. It binds to its receptors with a Kd = 0.8 nM, and a maximal binding capacity of 171 fmol/mg of protein on membrane preparations. Our data show that we have prepared a tool that is usable for pharmacological studies of a receptor associated with a new type of calcium channel for which no ligand was available until now.
Cloning of a functional promoter of the human sodium/iodide-symporter gene
Authors: BEHR Michael , SCHMITT Thomas L. , ESPINOZA Celia R. , LOOS Ulrich 
Published: August 2015

We have cloned and sequenced genomic DNA from a human library extending 1300 bp upstream the 5´-untranslated sequence of the cDNA coding for the sodium/iodide symporter. In transient transfection assays this sequence exhibited promoter activity, which could be confined to nucleotides -443 to -395 relative to the ATG start codon. This minimal promoter, including a putative GC- and TATA- box, was preferentially activated in the rat thyroid cell line FRTL-5, but was also active in non-thyroidal cells, such as COS-7 and Chinese-hamster ovary, albeit to a markedly lower extent.
Subunit interaction of vacuolar H+-pyrophosphatase as determined by high hydrostatic pressure
Authors: YANG Su J. , KO Shu J. , TSAI Yuan R. , JIANG Shih S. , KUO Soong Y. , HUNG Shu H. , PAN Rong L. 
Published: August 2015

Vacuolar H+-pyrophosphatase (H+-PPase) from etiolated hypocotyls of mung bean (Vigna radiata L.) is a homodimer with a molecular mass of 145 kDa. The vacuolar H+-PPase was subjected to high hydrostatic pressure to investigate its structure and function. The inhibition of H+-PPase activity by high hydrostatic pressure has a pressure-, time- and protein-concentration-dependent manner. The Vmax value of vacuolar H+-PPase was dramatically decreased by pressurization from 293.9 to 70.2 µmol of PPi (pyrophosphate) consumed/h per mg of protein, while the Km value decreased from 0.35 to 0.08 mM, implying that the pressure treatment increased the affinity of PPi to vacuolar H+-PPase but decreased its hydrolysis. The physiological substrate and its analogues enhance high pressure inhibition of vacuolar H+-PPase. The HPLC profile reveals high pressure treatment of H+-PPase provokes the subunit dissociation from an active into inactive form. High hydrostatic pressure also induces the conformational change of vacuolar H+-PPase as determined by spectroscopic techniques. Our results indicate the importance of protein–protein interaction for this novel proton-translocating enzyme. Working models are proposed to interpret the pressure inactivation of vacuolar H+-PPase. We also suggest that association of identical subunits of vacuolar H+-PPase is not random but proceeds in a specific manner.
Peroxynitrite modulates tyrosine phosphorylation and phosphoinositide signalling in human neuroblastoma SH-SY5Y cells: attenuated effects in human 1321N1 astrocytoma cells
Authors: LI Xiaohua , SARNO Patrizia De , SONG Ling , BECKMAN Joseph S. , JOPE Richard S. 
Published: August 2015

Peroxynitrite may contribute to oxidative stress involving neurodegeneration in several disorders, including Alzheimer's disease. As with other reactive oxygen species, peroxynitrite might affect neuronal signalling systems, actions that could contribute to adaptive or deleterious cellular outcomes, but such effects have not previously been studied. To address this issue directly, peroxynitrite (50–500 µM) was administered to human neuroblastoma SH-SY5Y cells to assess its effects on protein tyrosine nitration, phosphoinositide signalling and protein tyrosine phosphorylation. Peroxynitrite rapidly increased the nitrotyrosine immunoreactivity of numerous proteins, primarily in the cytosol. Peroxynitrite inhibited, in a concentration-dependent manner, phosphoinositide hydrolysis stimulated by activation of muscarinic receptors with carbachol and the inhibition was greater after the depletion of cellular glutathione. In comparison, muscarinic receptor-stimulated phosphoinositide hydrolysis in human astrocytoma 1321N1 cells was less vulnerable to inhibition by peroxynitrite either without or with prior depletion of glutathione. There was a large, rapid and reversible increase in the tyrosine phosphorylation of the p120 Src substrate in peroxynitrite-treated SH-SY5Y cells, a response that was potentiated by glutathione depletion; in contrast, peroxynitrite decreased the tyrosine phosphorylation of focal adhesion kinase and paxillin. Tyrosine phosphorylation of p120 in 1321N1 astrocytoma cells was less sensitive to modulation by peroxynitrite. Thus alterations in phosphoinositide signalling and protein tyrosine phosphorylation were greater in neuroblastoma than astrocytoma cells, and modulation of these signalling processes probably contributes to neuronal mechanisms of the response to peroxynitrite.
Proteoglycans in macrophages: characterization and possible role in the cellular uptake of lipoproteins
Authors: HALVORSEN Bente , AAS Une K. , KULSETH Mari Ann , DREVON Christian A. , CHRISTIANSEN Erling N. , KOLSET Svein O. 
Published: August 2015

The murine macrophage cell line J774 was incubated with [35S]sulphate. The cell-associated 35S-labelled macromolecules were shown to be proteoglycans and glycosaminoglycans in similar amounts. The possible presence of cell-surface proteoglycans was investigated by incubating [35S]sulphate-labelled cells with trypsin for 15 min. The released material contained approx. 70% free glycosaminoglycan chains and 30% proteoglycans. The latter component was demonstrated by HNO2 treatment to contain heparan sulphate. In the total cell fraction not treated with trypsin a small but significant portion was shown to be chondroitin sulphate proteoglycan. The cell-associated glycosaminoglycans contained both chondroitin sulphate and heparan sulphate. To investigate possible biological functions of cell-surface proteoglycans in macrophages, cells were incubated with NaClO3 to inhibit sulphation of proteoglycans and β-d-xyloside to abrogate proteoglycan expression. The uptake of oxidized 125I-tyraminylcellobiose-labelled low-density lipoprotein (125I-TC-LDL) was typically two to three times higher than that of native 125I-TC-LDL in untreated J774 cells. The cellular uptake at 37 °C of native 125I-TC-LDL was decreased 25% after both NaClO3 and xyloside treatment, whereas the uptake of oxidized 125I-TC-LDL was decreased 35% after both types of treatment. The mRNA levels for the scavenger receptor A-II and the LDL receptor were not affected by NaClO3 or xyloside treatment. Furthermore, fluid-phase endocytosis, measured as uptake of horseradish peroxidase, and receptor-mediated endocytosis, measured as uptake of 125I-TC-ovalbumin, were not affected by NaClO3 treatment of J774 cells. Removal of cell-surface chondroitin sulphate with chondroitinase ABC decreased only the binding of native 125I-TC-LDL, whereas removal of heparan sulphate with heparitinase decreased the binding of both oxidized and native 125I-TC-LDL. Addition of lipoprotein lipase increased the uptake of oxidized 125I-TC-LDL 1.7 times and the uptake of native 125I-TC-LDL 2.1 times. The binding of the former was more sensitive to NaClO3 treatment than the latter. The results presented support the notion that some of the uptake pathways for lipoproteins in the foam-cell-forming macrophages depend on the presence of cell-surface heparan sulphate and chondroitin sulphate.
Pseudomonas cellulose-binding domains mediate their effects by increasing enzyme substrate proximity
Authors: BOLAM David N. , CIRUELA Antonio , McQUEEN-MASON Simon , SIMPSON Peter , WILLIAMSON Michael P. , RIXON Jane E. , BORASTON Alisdair , HAZLEWOOD Geoffrey P. , GILBERT Harry J. 
Published: August 2015

To investigate the mode of action of cellulose-binding domains (CBDs), the Type II CBD from Pseudomonas fluorescenssubsp. cellulosaxylanase A (XYLACBD) and cellulase E (CELECBD) were expressed as individual entities or fused to the catalytic domain of a Clostridium thermocellumendoglucanase (EGE). The two CBDs exhibited similar Ka values for bacterial microcrystalline cellulose (CELECBD, 1.62×106 M-1; XYLACBD, 1.83×106 M-1) and acid-swollen cellulose (CELECBD, 1.66×106 M-1; XYLACBD, 1.73×106 M-1). NMR spectra of XYLACBD titrated with cello-oligosaccharides showed that the environment of three tryptophan residues was affected when the CBD bound cellohexaose, cellopentaose or cellotetraose. The Ka values of the XYLACBD for C6, C5 and C4 cello-oligosaccharides were estimated to be 3.3×102, 1.4×102 and 4.0×101 M-1 respectively, suggesting that the CBD can accommodate at least six glucose molecules and has a much higher affinity for insoluble cellulose than soluble oligosaccharides. Fusion of either the CELECBD or XYLACBD to the catalytic domain of EGE potentiated the activity of the enzyme against insoluble forms of cellulose but not against carboxymethylcellulose. The increase in cellulase activity was not observed when the CBDs were incubated with the catalytic domain of either EGE or XYLA, with insoluble cellulose and a cellulose/hemicellulose complex respectively as the substrates. PseudomonasCBDs did not induce the extension of isolated plant cell walls nor weaken cellulose paper strips in the same way as a class of plant cell wall proteins called expansins. The XYLACBD and CELECBD did not release small particles from the surface of cotton. The significance of these results in relation to the mode of action of Type II CBDs is discussed.
Metabolic Control Analysis of the bc1 complex of Saccharomyces cerevisiae: effect on cytochrome c oxidase, respiration and growth rate
Authors: BOUMANS Hans , BERDEN Jan A. , GRIVELL Leslie A. , DAM Karel van 
Published: August 2015

A number of strains varying in steady-state level of assembled bc1 complex were used to test the conclusions from inhibitor titration experiments with isolated mitochondria that, in cells of Saccharomyces cerevisiae grown on non-fermentable carbon sources, the control coefficient of the bc1 complex on the mitochondrial respiratory capacity equals 1 and the respiratory chain consists of supermolecular respiratory units [Boumans, Grivell and Berden (1998) J. Biol. Chem. 273, 4872–4877]. In addition, the control coefficient of mitochondrial respiration on the growth rate was determined. It was found that a reduced level of bc1 complex is accompanied by an almost parallel decrease in steady-state level of cytochrome c oxidase. Since the linear relationship between level of active bc1 complex and respiratory capacity still holds, it is concluded that cytochrome c oxidase has disappeared from respiratory units that are already deficient in the bc1 complex and that the cytochrome c oxidase in a respiratory unit is destabilized when the bc1 complex is deficient. The control coefficient of the bc1 complex, and thus of mitochondrial electron-transfer capacity, on respiration of intact cells (without uncoupler added) is 0.20. Addition of uncoupler results in an increase in the coefficient to 0.36. Thus changing the respiratory state changes the distribution of control, increasing the control coefficient of electron-transfer activity as the respiratory state goes towards State 3u. Rates of growth of the strains on different carbon sources were determined and subsequently fitted to calculate control coefficients of the bc1 complex (and therefore of the respiratory capacity) on growth. Little variation was found between lactate-, ethanol- and glycerol-containing media, control coefficients being around 0.18 at pH 5. At pH 7 the control coefficient increased to 0.57, indicative of a higher dependence of the cell on ATP derived from oxidative phosphorylation. During growth on glucose-containing medium, the bc1 complex has no control on the growth rate, as indicated by the fact that all strains, including a respiratory-deficient strain, grow as fast as the wild-type. However, the presence of respiratory capacity in the wild-type does result in a higher growth yield compared with the respiratory-deficient strain, indicating that, in contrast with what is generally assumed, in S. cerevisiae the ‘Pasteur effect ’ is not restricted to special experimental conditions.
Phospholipid hydroperoxide glutathione peroxidase activity of human glutathione transferases
Authors: HURST Rachel , BAO Yongping , JEMTH Per , MANNERVIK Bengt , WILLIAMSON Gary 
Published: August 2015

Human glutathione transferases (GSTs) from Alpha (A), Mu (M) and Theta (T) classes exhibited glutathione peroxidase activity towards phospholipid hydroperoxide. The specific activities are in the order: GST A1-1 &gt; GST T1-1 &gt; GST M1-1 &gt; GST A2-2 &gt; GST A4-4. Using a specific and sensitive HPLC method, specific activities towards the phospholipid hydroperoxide, 1-palmitoyl-2-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl)-l-3-phosphatidylcholine (PLPC-OOH) were determined to be in the range of 0.8–20 nmol/min per mg of protein. Two human class Pi (P) enzymes (GST P1-1 with Ile or Val at position 105) displayed no activity towards the phospholipid hydroperoxide. Michaelis–Menten kinetics were followed only for glutathione, whereas there was a linear dependence of rate with PLPC-OOH concentration. Unlike the selenium-dependent phospholipid hydroperoxide glutathione peroxidase (Se-PHGPx), the presence of detergent inhibited the activity of GST A1-1 on PLPC-OOH. Also, in contrast with Se-PHGPx, only glutathione could act as the reducing agent for GST A1-1. A GST A1-1 mutant (Arg15Lys), which retains the positive charge between the GSH- and hydrophobic binding sites, exhibited a decreased kcat for PLPC-OOH but not for CDNB, suggesting that the correct topography of the GSH site is more critical for the phospholipid substrate. A Met208Ala mutation, which gives a modified hydrophobic site, decreased the kcat for CDNB and PLPC-OOH by comparable amounts. These results indicate that Alpha, Mu and Theta class human GSTs provide protection against accumulation of cellular phospholipid hydroperoxides.
Functional studies of the rabbit intestinal Na+/glucose carrier (SGLT1) expressed in COS-7 cells: evaluation of the mutant A166C indicates this region is important for Na+-activation of the carrier
Authors: VAYRO Steven , LO Bryan , SILVERMAN Mel 
Published: August 2015

We have exploited two mutants of the rabbit intestinal Na+/glucose carrier SGLT1 to explore the structure/function relationship of this Na+/glucose transporter in COS-7 cells. A functional N-terminal myc-epitope-tagged SGLT1 protein was constructed and used to determine the plasma-membrane localization of SGLT1. The kinetic and specificity characteristics of the myc-tagged SGLT1 mutant were identical with those of wild-type SGLT1. Immunogold labelling and electron microscopy confirmed the topology of the N-terminal region to be extracellular. Expression of the SGLT1 A166C mutant in these cells showed diminished levels of Na+-dependent α-methyl-d-glucopyranoside transport activity compared with wild-type SGLT1. For SGLT1 A166C, Vmax was 0.92±0.08 nmol/min per mg of protein and Km was 0.98±0.13 mM; for wild-type SGLT1, Vmax was 1.98±0.47 nmol/min per mg of protein and Km was 0.36±0.16 mM. Significantly, phlorrhizin (phloridzin) binding experiments confirmed equal expression of Na+-dependent high-affinity phlorrhizin binding to COS-7 cells expressing SGLT1 A166C or wild-type SGLT1 (Bmax 1.55±0.18 and 1.69±0.57 pmol/mg of protein respectively); Kd values were 0.46±0.15 and 0.51±0.11 µM for SGLT1 A166C and wild-type SGLT1 respectively. The specificity of sugar interaction was unchanged by the A166C mutation. We conclude that the replacement of an alanine residue by cysteine at position 166 has a profound effect on transporter function, resulting in a decrease in transporter turnover rate by a factor of 2. Taken as a whole the functional changes observed by SGLT1 A166C are most consistent with the mutation having caused an altered Na+ interaction with the transporter.
BeWo choriocarcinoma cells produce laminin 10
Authors: CHURCH Heather J. , APLIN John D. 
Published: August 2015

BeWo is a choriocarcinoma cell line that generates an extracellular matrix (ECM) rich in laminin and is a useful model for human trophoblast. Immunofluorescence with monoclonal antibodies demonstrates that BeWo ECM contains laminin subunits β1 and γ1. Immunoprecipitation from conditioned medium shows that the cells secrete two distinct laminin trimers both containing β1 and γ1 but with α subunits of approx. 400 and 450 kDa. The culture medium also contains a species thought to be β1γ1 dimer. Immunoprecipitation with monoclonal antibody 4C7, previously thought to recognize the α1 subunit, isolates complexes containing only the smaller α subunit. A second complex containing the larger α subunit along with β1, γ1 and a 150 kDa polypeptide is precipitated from 4C7-depleted medium with an anti-(laminin 1) polyclonal antibody. Peptide sequencing demonstrates that the 4C7-reactive species is α5, which is present as two similarly sized polypeptides. mRNA species encoding laminin subunits α1, α5, β1, β2 and γ1 are all present in the cells. These results demonstrate the secretion of a novel laminin isoform, laminin 10, the subunit composition of which is α5β1γ1. Laminin 1 is also produced. No evidence for the secretion of β2-containing laminin isoforms could be derived despite the presence of β2 mRNA. Analysis with reverse transcriptase-mediated PCR also showed the presence of laminin α5 in first-trimester placenta and decidua.