bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional expression in yeast and characterization of a clofibrate-inducible plant cytochrome P-450 (CYP94A1) involved in cutin monomers synthesis
Authors: TIJET Nathalie , HELVIG Christian , PINOT Franck , BOUQUIN Renaud Le , LESOT Agnès , DURST Francis , SALAÜN Jean-Pierre , BENVENISTE Irène 
Published: August 2015

The chemical tagging of a cytochrome P-450-dependent lauric acid ω-hydroxylase from clofibrate-treated Vicia sativa seedlings with [1-14C]11-dodecynoic acid allowed the isolation of a full-length cDNA designated CYP94A1. We describe here the functional expression of this novel P-450 in two Saccharomyces cerevisiae strains overproducing their own NADPH-cytochrome P-450 reductase or a reductase from Arabidopsis thaliana. The results show a much higher efficiency of the yeast strain overproducing the plant reductase compared with the yeast strain overproducing its own reductase for expressing CYP94A1. The methyl end of saturated (from C-10 to C-16) and unsaturated (C18:1, C18:2 and C18:3) fatty acids was mainly oxidized by CYP94A1. Both E/Zand Z/E configurations of 9,12-octadecadienoic acids were ω-hydroxylated. Lauric, myristic and linolenic acids were oxidized with the highest turnover rate (24 min-1). The strong regioselectivity of CYP94A1 was clearly shifted with sulphur-containing substrates, since both 9- and 11-thia laurate analogues were sulphoxidized. Similar to animal ω-hydroxylases, this plant enzyme was strongly induced by clofibrate treatment. Rapid CYP94A1 transcript accumulation was detected less than 20 min after exposure of seedlings to the hypolipidaemic drug. The involvement of CYP94A1 in the synthesis of cutin monomers and fatty acid detoxification is discussed.
Reaction of soluble penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus with β-lactams and acyclic substrates: kinetics in homogeneous solution
Authors: GRAVES-WOODWARD Karen , PRATT R. F. 
Published: August 2015

The kinetics of reaction of solubilized penicillin-binding protein 2a (sPBP2a) of methicillin-resistant Staphylococcus aureus with a variety of β-lactams and acyclic species was studied in homogeneous aqueous solution at 37 °C in 25 mM Hepes buffer, pH 7.0, containing 1 M NaCl. Under these conditions, but not at lower salt concentrations, protein precipitation did not occur either during or after the reaction. The reactions of β-lactams in general could be monitored by competition with a chromophoric β-lactam, nitrocefin, or directly in certain cases by protein fluorescence. Rate constants for reaction of a wide variety of β-lactams are reported. The interactions are characterized by a slow second-order acylation reaction followed by a slower deacylation. For example, the rate constants for benzylpenicillin were 12 M-1·s-1 and 3×10-5 s-1 respectively. The acylation is slow in comparison with those of normal non-resistant high-molecular-mass penicillin-binding proteins. sPBP2a also seemed to catalyse the slow hydrolysis of a variety of acyclic depsipeptides but not that of a d-Ala-d-Ala peptide. The reactions with certain depsipeptides also led to protein precipitation. These reactions were, however, not affected by prior blockage of the β-lactam-binding site by benzylpenicillin and thus might take place elsewhere on the enzyme. Two classes of potential transition- state analogue inhibitors, phosphonate monoesters and boronates, seemed to have little effect on the rate of reaction of sPBP2a with nitrocefin and therefore seem to have little affinity for the β-lactam-binding/d,d-peptidase site.
Identification of a human homologue of the vesicle-associated membrane protein (VAMP)-associated protein of 33 kDa (VAP-33): a broadly expressed protein that binds to VAMP
Authors: WEIR M. Lynn , KLIP Amira , TRIMBLE William S. 
Published: August 2015

We report the identification of a human homologue of the vesicle-associated membrane protein (VAMP)-associated protein (hVAP-33) that has been implicated in neuronal exocytosis in Aplysia californica. This hVAP-33 shared 50% amino acid identity with the A. californica form and had similar length, structural organization and VAMP-binding abilities. However, in contrast with the neuron-specific expression seen in A. californica, hVAP-33 was broadly expressed, suggesting possible roles in vesicle fusion in both neuronal and non-neuronal cells.
The nucleotide sequence reported in this paper can be found in GenBank's database using accession number AF057358.
Induction of stress-activated protein kinases/c-Jun N-terminal kinases by the p55 tumour necrosis factor receptor does not require sphingomyelinases
Authors: ADAM Dieter , RUFF Andrea , STRELOW Astrid , WIEGMANN Katja , KRÖNKE Martin 
Published: August 2015

Ceramide has been implicated in the activation of stress-activated protein kinases/c-Jun N-terminal kinases (SAPK/JNK). Binding of tumour necrosis factor (TNF) to its 55 kDa receptor (TR55) leads to the generation of ceramide through activation of either acid or neutral sphingomyelinase (A/N-SMase) as well as to potent activation of SAPK/JNK. We have examined a putative role of both N- and A-SMase in the TR55-dependent activation of SAPK/JNK. The analysis of TR55 deletion mutants expressed in 70Z/3 pre-B cells revealed that activation of SAPK/JNK occurs independently of N-SMase. Although both SAPK/JNK and A-SMase are activated by the death domain of TR55, pharmacological prevention of the TR55-dependent activation of A-SMase, or proteolytic degradation of A-SMase in 70Z/3 cells, did not impair SAPK/JNK activation, indicating that SAPK/JNK are not secondary to A-SMase. In addition, proteolytic degradation of A-SMase also did not affect SAPK/JNK activation by ultraviolet (UV-C) irradiation, arguing against a general role of A-SMase in stress-mediated responses. Furthermore, fibroblasts from Niemann–Pick A patients deficient in A-SMase did not show altered activation of SAPK/JNK in response to either TNF or UV-C. These results suggest that TR55 can activate SAPK/JNK without direct participation of sphingomyelinases or ceramide.
Enzymological characterization of the nuclease domain from the bacterial toxin colicin E9 from Escherichia coli
Authors: POMMER Ansgar J. , WALLIS Russell , MOORE Geoffrey R. , JAMES Richard , KLEANTHOUS Colin 
Published: August 2015

The cytotoxicity of the bacterial toxin colicin E9 is due to a non-specific DNase that penetrates the cytoplasm of the infected organism and causes cell death. We report the first enzymological characterization of the overexpressed and purified 15 kDa DNase domain (E9 DNase) from this class of toxin. CD spectroscopy shows the E9 DNase to be structured in solution, and analytical ultracentrifugation data indicate that the enzyme is a monomer. The nuclease activity of the E9 DNase was compared with the well-studied, non-specific DNase I by using a spectrophotometric assay with calf thymus DNA as the substrate. Both enzymes require divalent metal ions for activity but, unlike DNase I, the E9 DNase is not activated by Ca2+ ions. Somewhat surprisingly, the E9 DNase shows optimal activity and linear kinetics in the presence of transition metals such as Ni2+ and Co2+ but displays non-linear kinetics with metals such as Mg2+ and Ca2+. Conversely, Ni2+ and other transition metals showed poor activity in a plasmid-based nicking assay, yielding significant amounts of linearized plasmid, whereas Mg2+ was very active, with the main intermediate being open-circle DNA. The results suggest that, on entry into bacterial cells, the E9 DNase is likely to exhibit primarily Mg2+-dependent nicking activity against chromosomal DNA, although other metals could also be utilized to introduce both single- and double-strand cleavages.
Detailed characterization of a cyclophilin from the human malaria parasite Plasmodium falciparum
Authors: BERRIMAN Matthew , FAIRLAMB Alan H. 
Published: August 2015

Cyclosporin (Cs) A has pronounced antimalarial activity in vitro and in vivo. In other organisms, the drug is thought to exert its effects either by inhibiting the peptidylprolyl cis/trans isomerase activity of cyclophilin (CyP) or by forming a CyP–CsA complex that inhibits the phosphatase activity of calcineurin. We have cloned and overexpressed in Escherichia coli a gene encoding a CyP from Plasmodium falciparum (PfCyP19) that is located on chromosome 3. The sequence of PfCyP19 shows remarkable sequence identity with human CyPA and, unlike the two previously identified CyPs from P. falciparum, PfCyP19 has no signal peptide or N-terminal sequence extension, suggesting a cytosolic localization. All the residues implicated in the recognition of the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide are conserved, resulting in characteristically high Michaelis–Menten and specificity constants (Km ≫ 120 µM, kcat/Km = 1.2×107 M-1·s-1 respectively). As the first line in the functional characterization of this enzyme, we have assessed its binding affinity for CsA. In accordance with its tryptophan-containing CsA-binding domain, PfCyP19 binds CsA with high affinity (Kd = 13 nM, Ki = 6.9 nM). Twelve CsA analogues were also found to possess Ki values similar to CsA, with the notable exceptions of Val2-Cs (Ki = 218 nM) and Thr2-Cs (Ki = 690 nM). The immunosuppressants rapamycin and FK506 were inactive as inhibitors, consistent with other members of the CyP family of rotamases. The CsA analogues were also assessed as inhibitors of P. falciparum growth in vitro. Although their antimalarial activity did not correlate with inhibition of enzyme activity, residues 3 and 4 of CsA appeared to be important for inhibition of parasite growth and residues 1 and 2 for PfCyP19 inhibition. We propose that a malarial CyP–CsA complex presents residues 3 and 4 as part of an ‘effector surface ’ for recognition by a downstream target, similar to the proposed mechanism for T-cell immunosuppression.
Hepatocyte nuclear factor 6: organization and chromosomal assignment of the rat gene and characterization of its promoter
Authors: RASTEGAR Mojgan , SZPIRER Claude , ROUSSEAU Guy G. , LEMAIGRE Frédéric P. 
Published: August 2015

Hepatocyte nuclear factor 6 (HNF-6) is the prototype of a family of tissue-specific transcription factors characterized by a bipartite DNA-binding domain consisting of a single cut domain and a novel type of homeodomain. We have previously cloned rat cDNA species coding for two isoforms, HNF-6α (465 residues) and β (491 residues), which differ only by the length of the spacer between the two DNA-binding domains. We have now localized the rat Hnf6 gene to chromosome 8q24–q31 by Southern blotting of DNA from somatic cell hybrids and by fluorescence in situhybridization. Cloning and sequencing of the rat gene showed that the two HNF-6 isoforms are generated by alternative splicing of three exons that are more than 10 kb apart from each other. Exon 1 codes for the N-terminal part and the cut domain, exon 2 codes for the 26 HNF-6β-specific amino acids, and exon 3 codes for the homeodomain and the C-terminal amino acids. The transcription initiation site was mapped by ribonuclease protection and 5´ rapid amplification of cDNA ends. Transfection experiments showed that promoter activity was contained within 0.75 kb upstream of the transcription initiation site. This activity was detected by the transfection of liver-derived HepG2 cells, but not of Rat-1 fibroblasts, suggesting that the promoter is sufficient to confer liver-specific expression.
Identification of Glu-277 as the catalytic nucleophile of Thermoanaerobacterium saccharolyticum β-xylosidase using electrospray MS
Authors: VOCADLO David J. , MACKENZIE Lloyd F. , HE Shouming , ZEIKUS Gregory J. , WITHERS Stephen G. 
Published: August 2015

Thermoanaerobacterium saccharolyticum β-xylosidase is a member of family 39 of the glycosyl hydrolases. This grouping comprises both retaining β-d-xylosidases and α-l-iduronidases. T. saccharolyticum β-xylosidase catalyses the hydrolysis of short xylo-oligosaccharides into free xylose via a covalent xylosyl–enzyme intermediate. Incubation of T. saccharolyticum β-xylosidase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-d-xyloside resulted in time-dependent inactivation of the enzyme (inactivation rate constant ki = 0.089 min-1, dissociation constant for the inactivator Ki = 65 µM) through the accumulation of a covalent 2-deoxy-2-fluoro-α-d-xylosyl–enzyme, as observed by electrospray MS. Removal of excess inactivator and regeneration of the free enzyme through transglycosylation with either xylobiose or thiobenzyl xyloside demonstrated that the covalent intermediate was kinetically competent. Peptic digestion of the 2-deoxy-2-fluoro-α-d-xylosyl–enzyme intermediate and subsequent analysis by electrospray ionization triple-quadrupole MS in the neutral-loss mode indicated the presence of a 2-deoxy-2-fluoro-α-d-xylosyl peptide. Sequence determination of the labelled peptide by tandem MS in the daughter-ion scan mode permitted the identification of Glu-277 (bold and underlined) as the catalytic nucleophile within the sequence IILNSHFPNLPFHITEY.
Regulation of mammalian pyruvate dehydrogenase α subunit gene expression by glucose in HepG2 cells
Authors: TAN Jie , YANG Hsin-Sheng , PATEL Mulchand S. 
Published: August 2015

We report the effect of glucose on the expression of the gene encoding the pyruvate dehydrogenase (E1) α subunit (E1α) in human hepatoma (HepG2) cells. Total pyruvate dehydrogenase complex activity as well as the levels of protein and mRNA of the E1α subunit were significantly increased in HepG2 cells cultured in medium containing 16.7 mM glucose compared with 1.0 mM glucose for a period of 4 weeks. The level of E1α mRNA was elevated approx. 2-fold in HepG2 cells cultured for 24 h in medium containing 16.7 mM glucose compared with 1 mM glucose. This effect was specific to glucose and independent of insulin. Nuclear run-on assays and promoter analysis indicate that the glucose-induced increases in the levels of E1α mRNA in HepG2 cells are due to increased transcription of the human E1α (PDHA1) gene. Mutational analysis of the E1α promoter region has identified two regions, from -78 to -73 bp (CCCCTG) and from -8 to -3 bp (GCGGTG), that are responsible for the effect of glucose on promoter activity; the former exhibits a larger effect. These two sequences represent new variations of the carbohydrate-response element that has been identified in other genes. The stimulation of E1α promoter activity by glucose was abolished by okadaic acid at 100 nM but not at 5 nM, suggesting that glucose-mediated regulation of pyruvate dehydrogenase complex E1α gene transcription involves a phosphorylation/dephosphorylation mechanism, possibly involving protein phosphatase-1.
Changes in polyamine catabolism in HL-60 human promyelogenous leukaemic cells in response to etoposide-induced apoptosis
Authors: LINDSAY Gayle S. , WALLACE Heather M. 
Published: August 2015

The topoisomerase II inhibitor etoposide induced apoptosis in HL-60 cells within 4 h of exposure to the drug, as measured by changes in morphology, DNA fragmentation and cytotoxicity assays. Etoposide-induced apoptosis was accompanied by an increase in polyamine efflux from the cells and a decrease in total polyamine content during the first 24 h of exposure to the drug. Although both enzyme activities increased slightly, there were no significant changes in spermidine/spermine N1-acetyltransferase activity or polyamine oxidase activity. After longer exposures (48–72 h), significant induction of spermidine/spermine N1-acetyltransferase activity and loss of polyamine content occurred. These results suggest that polyamine oxidation and the resultant hydrogen peroxide produced may be associated with the initiation of apoptosis, while induction of the acetyltransferase and overall loss of intracellular polyamines may be involved in the final, possibly necrotic, stages of cell death.
Gene structure, expression and chromosomal localization of murine Theta class glutathione transferase mGSTT1-1
Authors: WHITTINGTON Angela T. , VICHAI Vanicha , WEBB Graham C. , BAKER Rohan T. , PEARSON William R. , BOARD Philip G. 
Published: August 2015

We have isolated and characterized a cDNA and partial gene encoding a murine subfamily 1 Theta class glutathione transferase (GST). The cDNA derived from mouse GSTT1 has an open reading frame of 720 bp encoding a peptide of 240 amino acids with a calculated molecular mass of 27356 Da. The encoded protein shares only 51% deduced amino acid sequence identity with mouse GSTT2, but greater than 80% deduced amino acid sequence identity with rat GSTT1 and human GSTT1. Mouse GSTT1-1 was expressed in Escherichia colias an N-terminal 6× histidine-tagged protein and purified using immobilized-metal affinity chromatography on nickel-agarose. The yield of the purified recombinant protein from E. coli cultures was approx. 14 mg/l. Recombinant mouse GSTT1-1 was catalytically active towards 1,2-epoxy-3-(p-nitrophenoxy)propane, 4-nitrobenzyl chloride and dichloromethane. Low activity towards 1-menaphthyl sulphate and 1-chloro-2,4-dinitrobenzene was detected, whereas mouse GSTT1-1 was inactive towards ethacrynic acid. Recombinant mouse GSTT1-1 exhibited glutathione peroxidase activity towards cumene hydroperoxide and t-butyl hydroperoxide, but was inactive towards a range of secondary lipid-peroxidation products, such as the trans-alk-2-enals and trans,trans-alka-2,4-dienals. Mouse GSTT1 mRNA is most abundant in mouse liver and kidney, with some expression in intestinal mucosa. Mouse GSTT1 mRNA is induced in liver by phenobarbital, but not by butylated hydroxyanisole, β-napthoflavone or isosafrole. The structure of mouse GSTT1 is conserved with that of the subfamily 2 Theta class GST genes mouse GSTT2 and rat GSTT2, comprising five exons interrupted by four introns. The mouse GSTT1 gene was found, by in situ hybridization, to be clustered with mouse GSTT2 on chromosome 10 at bands B5–C1. This region is syntenic with the location of the human Theta class GSTs clustered on chromosome 22q11.2. Similarity searches of a mouse-expressed sequence tag database suggest that there may be two additional members of the Theta class that share 70% and 88% protein sequence identity with mouse GSTT1, but less than 55% sequence identity with mouse GSTT2.
Concerted action of cytosolic Ca2+ and protein kinase C in receptor-mediated phospholipase D activation in Chinese hamster ovary cells expressing the cholecystokinin-A receptor
Authors: BOSCH Remko R. , SMEETS Rolf L. L. , SLEUTELS Frank , PATEL Anjana M. P. , EMST-De VRIES Sjenet E. Van , DE PONT Jan Joep H. H. M. , WILLEMS Peter H. G. M. 
Published: August 2015

Receptor-mediated activation of phosphatidylcholine phosphatidohydrolase or phospholipase D (PLD) was studied in Chinese hamster ovary (CHO) cells expressing the cholecystokinin-A (CCK-A) receptor. Cells were labelled with [3H]myristic acid for 24 h and PLD-catalysed [3H]phosphatidylethanol formation was measured in the presence of 1% (v/v) ethanol. Cholecystokinin-(26–33)-peptide amide (CCK8) increased PLD activity both time- and dose-dependently. Maximal activation of protein kinase C (PKC) with 1 µM PMA or sustained elevation of the cytosolic free Ca2+ concentration ([Ca2+]i) with 1 µM thapsigargin increased PLD activity to 50% and 70% of the maximal value obtained with CCK8 respectively. The stimulatory effects of CCK8, PMA and thapsigargin were abolished in cells in which PKC was downregulated or inhibited by chelerythrine. PMA/Ca2+-stimulated PLD activity was absent in a homogenate of PKC-downregulated cells but could be restored upon addition of purified rat brain PKC. CCK8-induced PLD activation was inhibited by 90% in the absence of external Ca2+, demonstrating that receptor-mediated activation of PKC in itself does not significantly add to PLD activation but requires a sustained increase in [Ca2+]i. Taken together, the results presented demonstrate that, in CHO-CCK-A cells, receptor-mediated PLD activation is completely dependent on PKC, but that the extent to which PLD becomes activated depends largely, if not entirely, on the magnitude and duration of the agonist-induced increase in [Ca2+]i.
Transcription factor Sp1 activates involucrin promoter activity in non-epithelial cell types
Authors: BANKS Eric B. , CRISH James F. , ECKERT Richard L. 
Published: August 2015

The gene for human involucrin (hINV) is selectively expressed in stratifying epithelial cells lining external body surfaces. Previously, we characterized the hINV promoter 5´ distal regulatory region (DRR) located between nt -2473 and -2088 upstream of the transcription start site. This region is required for optimal hINV gene expression. The DRR contains weak and strong activator elements. The strong activator comprises AP1- and Sp1-binding sites that combine to drive high-level promoter expression in human keratinocytes. Here we show that the hINV promoter is expressed in a cell-specific manner in vitro and that the DRR contains elements that are partly responsible for cell-type-specific expression of hINV. hINV promoter activity is barely detectable in 3T3 fibroblasts or HEK-293 human embryonic kidney cells. Reporter plasmids containing the full-length promoter or the isolated DRR can, however, be activated in 3T3 and HEK-293 cells by co-transfection with a plasmid encoding the transcription factor Sp1. Consistently with the lower hINV promoter activity, immunoblotting studies indicate that Sp1 protein levels are lower in 3T3 and HEK-293 cells than in human epidermal keratinocytes. Increased Sp1 protein in transfected 3T3 cells and HEK-293 cells correlates with increased promoter activity. In addition, Sp1 transfection activates the expression of the endogenous gene for hINV in HEK-293 cells. These studies suggest that Sp1 might have a role in cell-specific expression of hINV.
Dermatopontin interacts with transforming growth factor β and enhances its biological activity
Authors: OKAMOTO Osamu , FUJIWARA Sakuhei , ABE Mayumi , SATO Yasufumi 
Published: August 2015

Dermatopontin, a recently found low-molecular-mass component of the extracellular matrix, was studied for its interaction with decorin and transforming growth factor β (TGF-β) and its influence on TGF-β bioactivity. Dermatopontin reacted with decorin with an apparent Kd of 100 nM in a solid-phase assay. Dermatopontin inhibited the formation of the decorin–TGF-β1 complex. Decorin also competed with dermatopontin for the binding of this cytokine. The dermatopontin–decorin complex bound 3-fold more TGF-β1 than did each component individually, and binding was inhibited more strongly by decorin preincubated with dermatopontin than by dermatopontin or decorin alone. Dermatopontin augmented the biological activity of TGF-β1, as analysed by the expression of luciferase in mink lung epithelial cells transfected with a plasminogen activator inhibitor–promoter–luciferase construct, although dermatopontin itself did not show apparent induction of luciferase. Dermatopontin showed weak inhibitory activity on the proliferation of mink lung epithelial cells, and it enhanced the growth-inhibitory activity of TGF-β on these cells. Thus dermatopontin increases the cellular response to TGF-β. These findings strongly suggest that dermatopontin modifies the behaviour of TGF-β through interaction with decorin in the microenvironment of the extracellular matrix in vivo.