bluuebunny/crossref_metadata_2025 huggingface dataset:
Epitope mapping of a monoclonal antibody to human glutathione transferase P1–1 the binding of which is inhibited by glutathione
Authors: TAKAHATA Takenori , TSUCHIDA Shigeki , OOMURA Masashi , MATSUMOTO Takashi , AZUMI Junichi , HATAYAMA Ichiro , HAYAKARI Makoto , KIMURA Junya , KAKIZAKI Ikuko , KANO Hiroko , SATOH Kimihiko , SATO Kiyomi
Published: August 2015

Although the three-dimensional structure of human glutathione transferase (GST) P1Ő1 crystallized with a GSH analogue has been reported, its structure in the non-complexed form has not been determined. Four monoclonal antibodies to GST P1Ő1 were produced to facilitate structural analysis. Of these, one, clone d-1 of IgG2a isotype, dose-dependently inhibited the activity of GST P1Ő1 but did not affect the activities of either GST A1Ő1 or M1Ő1. On immunoblotting, the antibody reacted strongly with GST P1Ő1 and weakly with rat GST-P and mouse GST-II, indicating cross-reactivity with Pi-class forms but preferential reactivity with GST P1Ő1. When GST P1Ő1 and the antibody were incubated in the presence of 60 ƁM GSH, no inhibition of activity was found, whereas 1-chloro-2,4-dinitrobenzene had no effect at concentrations up to 10 ƁM. The binding of GST P1Ő1 to antibody adsorbed to Protein AŐSepharose was also prevented by both 0.1 mM GSH and N-ethylmaleimide treatment. Trypsin digests of GST P1Ő1 were resolved by HPLC and a peptide that reacted with the antibody was detected by absorption experiments. N-Terminal amino acid sequencing revealed the peptide to be in the C-terminal portion of the enzyme, stretching from amino acid residues 198 to 208. A synthetic peptide of this sequence also absorbed the antibody. These results suggest that both GSH bound to the active site and N-ethylmaleimide bound to the cysteine residue repress antibody binding to the C-terminal region. Thus this antibody may be useful for examining the steric configuration of the C-terminal and other regions of GST P1Ő1 in the absence of GSH.
The human fertilin α gene is non-functional: implications for its proposed role in fertilization
Authors: JURY Jennifer A. , FRAYNE Jan , HALL Len
Published: August 2015

In the guinea-pig, the α subunit of the fertilin complex, a heterodimeric surface membrane glycoprotein found on the head region of spermatozoa, has previously been proposed to mediate membrane fusion with the oolemma plasma membrane during fertilization. Here we describe experiments which indicate that the only fertilin α-like gene in humans is an expressed, but non-functional, pseudogene, possibly derived by genetic recombination between the two fertilin α genes found in some primates. This finding clearly raises questions about the importance and/or role of fertilin α in mammalian fertilization.
Differential regulation of extracellular signal-regulated protein kinase 1 and Jun N-terminal kinase 1 by Ca2+ and protein kinase C in endothelin-stimulated Rat-1 cells
Authors: CADWALLADER Karen , BELTMAN Jerlyn , McCORMICK Frank , COOK Simon
Published: August 2015

The extracellular signal-regulated protein kinase (ERK) and Jun N-terminal kinase (JNK) signalling cascades transduce signals from the cell cytoplasm to the nucleus, where they regulate gene expression. The activation of ERK1 by lysophosphatidic acid (LPA) and endothelin 1 (Et-1) was compared in Rat-1 cells. Both stimulated DNA synthesis to a similar degree but, in contrast with LPA, Et-1 did not stimulate sustained ERK1 activation, a signal that is thought to be important for the proliferation of fibroblasts. Et-1, but not LPA, was able to activate JNK1; pharmacological analysis revealed that the same EtA receptor mediates DNA synthesis, ERK1 and JNK1 activation. However, activation of JNK1 required higher concentrations of Et-1 than was required for stimulation of ERK1 or DNA synthesis. Signalling to ERK1 and JNK1 was partly inhibited by pertussis toxin, suggesting that both pathways are regulated in part by Gi or Go proteins. Activation of JNK1 by Et-1 lagged behind ERK1 activation but was not dependent on it because PD98059, an inhibitor of mitogen-activated protein kinase (or ERK) kinase, was without effect on JNK1 activation. In contrast with recent studies, activation of protein kinase C (PKC) or Ca2+ fluxes inhibited activation of JNK1 but not ERK1; furthermore inhibition of PKC or sequestration of Ca2+ potentiated JNK1 activation by Et-1 but not by anisomycin, and again had little effect on ERK1 activation. These results demonstrate that the same G-protein-coupled receptor can activate both the ERK and JNK signal pathways but the two kinase cascades seem to be separate, parallel pathways that are differentially regulated by PKC and Ca2+. The results are discussed in terms of the role of ERK and JNK in proliferative signalling.
Accumulation of glucosaminyl(acyl)phosphatidylinositol in an S3 HeLa subline expressing normal dolicholphosphomannose synthase activity
Authors: SEVLEVER Daniel , SCHIEMANN Dylan , GUIDUBALDI John , MEDOF M. Edward , ROSENBERRY Terrone L.
Published: August 2015

Glucosaminyl(acyl)phosphatidylinositol [GlcN(acyl)PI], the third intermediate in the mammalian glycosylphosphatidylinositol (GPI) anchor pathway, is undetectable in most cells. This intermediate was previously shown to accumulate, however, in murine lymphoma mutant E and in yeast mutant dpm1,both of which lack dolicholphosphomannose synthase activity. Here we report that a mammalian HeLa S3 subline, denoted D, produces large amounts of GlcN(acyl)PI. The level of GlcN(acyl)PI in this subline is twice that in the murine lymphoma mutant E and 4 times that in the parental S3 line. This HeLa D subline differs from the previously reported mutants that accumulate GlcN(acyl)PI because no defects in the synthesis or utilization of dolicholphosphomannose were found. Kinetic analysis indicated that in this HeLa subline there is an increased rate of synthesis of GlcN(acyl)PI, whereas the rate of metabolism for this GPI is comparable to that in wild-type cells. Furthermore, HeLa D cells accumulate GlcN(acyl)PI without a block in the synthesis of the downstream mannosylated GPI anchor precursors and GPI-anchored proteins. These findings might be relevant for understanding the regulation of the GPI pathway.
Epoxyethylglycyl peptides as inhibitors of oligosaccharyltransferase: double-labelling of the active site
Authors: BAUSE Ernst , WESEMANN Marko , BARTOSCHEK Achim , BREUER Wilhelm
Published: August 2015

Pig liver oligosaccharyltransferase (OST) is inactivated irreversibly by a hexapeptide in which threonine has been substituted by epoxyethylglycine in the Asn-Xaa-Thr glycosylation triplet. Incubation of the enzyme in the presence of Dol-PP-linked [14C]oligosaccharides and the N-3,5-dinitrobenzoylated epoxy derivative leads to the double-labelling of two subunits (48 and 66 kDa) of the oligomeric OST complex, both of which are involved in the catalytic activity. Labelling of both subunits was blocked competitively by the acceptor peptide N-benzoyl-Asn-Gly-Thr-NHCH3 and by the OST inhibitor N-benzoyl-α,γ-diaminobutyric acid-Gly-Thr-NHCH3, but not by an analogue derived from the epoxy-inhibitor by replacing asparagine with glutamine. Our data clearly show that double-labelling is an active-site-directed modification, involving inhibitor glycosylation at asparagine and covalent attachment of the glycosylated inhibitor, via the epoxy group, to the enzyme. Double-labelling of OST can occur as the result of either a consecutive or a syn-catalytic reaction sequence. The latter mechanism, during the course of which OST catalyses its own ‘suicide’ inactivation, is more likely, as suggested by indirect experimental evidence. The syn-catalytic mechanism corresponds with our current view of the functional role of the acceptor site Thr/Ser acting as a hydrogen-bond acceptor, not a donor, during transglycosylation [Bause, Breuer and Peters (1995) Biochem. J. 312, 979Ő985].
Sphingosylphosphocholine modulates the ryanodine receptor/calcium-release channel of cardiac sarcoplasmic reticulum membranes
Authors: BETTO Romeo , TERESI Alessandra , TURCATO Federica , SALVIATI Giovanni , SABBADINI Roger A. , KROWN Kevin , GLEMBOTSKI Chris C. , KINDMAN L. Allen , DETTBARN Christine , PEREON Yann , YASUI Kenji , PALADE Philip T.
Published: August 2015

Sphingosylphosphocholine (SPC) modulates Ca2+ release from isolated cardiac sarcoplasmic reticulum membranes; 50 ƁM SPC induces the release of 70Ő80% of the accumulated calcium. SPC releases calcium from cardiac sarcoplasmic reticulum through the ryanodine receptor, since the release is inhibited by the ryanodine receptor channel antagonists ryanodine, Ruthenium Red and sphingosine. In intact cardiac myocytes, even in the absence of extracellular calcium, SPC causes a rise in diastolic Ca2+, which is greatly reduced when the sarcoplasmic reticulum is depleted of Ca2+ by prior thapsigargin treatment. SPC action on the ryanodine receptor is Ca2+-dependent. SPC shifts to the left the Ca2+-dependence of [3H]ryanodine binding, but only at high pCa values, suggesting that SPC might increase the sensitivity to calcium of the Ca2+-induced Ca2+-release mechanism. At high calcium concentrations (pCa 4.0 or lower), where [3H]ryanodine binding is maximally stimulated, no effect of SPC is observed. We conclude that SPC releases calcium from cardiac sarcoplasmic reticulum membranes by activating the ryanodine receptor and possibly another intracellular Ca2+-release channel, the sphingolipid Ca2+-release-mediating protein of endoplasmic reticulum (SCaMPER) [Mao, Kim, Almenoff, Rudner, Kearney and Kindman (1996) Proc. Natl. Acad. Sci. U.S.A 93, 1993Ő1996], which we have identified for the first time in cardiac tissue.
Effects of modifications near the 2-, 3- and 4-fold symmetry axes on human ferritin renaturation
Authors: SANTAMBROGIO Paolo , PINTO Patrizia , LEVI Sonia , COZZI Anna , ROVIDA Ermanna , ALBERTINI Alberto , ARTYMIUK Peter , HARRISON Pauline M. , AROSIO Paolo
Published: August 2015

Ferritin is a protein of 24 subunits which assemble into a shell with 432 point symmetry. It can be denatured reversibly in acidic guanidine hydrochloride, with the formation of poorly populated renaturation intermediates. In order to increase the accumulation of intermediates and to study the mechanism of ferritin renaturation, we analysed variants of the human ferritin H-chain altered at the N-terminus (Δ1–13), near the 4-fold axis (Leu-169→Arg), the 3-fold axis (Asp-131→Ile+Glu-134→Phe) or the 2-fold axis (Ile-85→Cys). We also carried out specific chemical modifications of Cys-130 (near the 3-fold axis) and Cys-85 (near the 2-fold axis). Renaturation of the modified ferritins yielded assembly intermediates that differed in size and physical properties. Alterations of residues around the 2-, 4- and 3-fold axes produced subunit monomers, dimers and higher oligomers respectively. All these intermediates could be induced to assemble into ferritin 24-mers by concentrating them or by co-renaturing them with wild-type H-ferritin. The results support the hypothesis that the symmetric subunit dimers are the building blocks of ferritin assembly, and are consistent with a reassembly pathway involving the coalescence of dimers, probably around the 4-fold axis, followed by stepwise addition of dimers until the 24-mer cage is completed. In addition they show that assembly interactions are responsible for the large hysteresis of folding and unfolding plots. The implications of the studies for in vivoheteropolymer formation in vertebrates, which have two types of ferritin chain (H and L), are discussed.
Expression, purification and kinetic characterization of wild-type human ornithine transcarbamylase and a recurrent mutant that produces ‘late onset’ hyperammonaemia
Authors: MORIZONO Hiroki , TUCHMAN Mendel , RAJAGOPAL Basavapatna S. , McCANN Mark T. , LISTROM Chad D. , YUAN Xiaoling , VENUGOPAL Divakaramenon , BARANY George , ALLEWELL Norma M.
Published: August 2015

Ornithine Transcarbamylase Deficiency, an X-linked disorder, is the most common cause of inherited urea cycle disorders. Approx. 90 mutations that produce reduced levels of ornithine transcarbamylase (OTCase) activity have been identified in patients [Tuchman (1993) Hum. Mutat. 2, 174–178; Tuchman and Plante (1995) Hum. Mutat. 5, 293–295]. A model of the three-dimensional structure of OTCase, developed on the basis of its homology to the catalytic subunit of Escherichia coli aspartate transcarbamylase (ATCase) [Tuchman, Morizono, Reish, Yuan and Allewell (1995) J. Med. Genet. 32, 680–688], and in good agreement with the crystal structure of Pseudomonas aeruginosa OTCase [Villeret, Tricot, Stalon and Dideberg (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10762–10766], indicates that many mutations that produce severe clinical symptoms are at the active site or buried in the interior of the protein. However, one of the few recurrent mutations, R277W, an alteration that produces a milder phenotype of ornithine transcarbamylase deficiency, is located in the model in a loop remote from the active site that is analogous to a similar loop (the 240's loop, a flexible loop of the catalytic chain of Escherichia coliaspartate transcarbamylase, comprised of residues 230–250) of ATCase. Human wild-type OTCase and the R277W mutant have been cloned and overexpressed in E. coli and a rapid and efficient purification method utilizing the bisubstrate analogue, NΔ-(phosphonacetyl)-l-ornithine, has been developed and used to purify both proteins. Gel chromatography indicates both are trimeric. The pH dependence of the kinetic parameters of the wild-type enzyme is similar to that of E. coli OTCase [Kuo, Herzberg and Lipscomb (1985) Biochemistry 24, 4754–4761], suggesting that its catalytic mechanism is similar, although its maximal activity is approx. 10-fold less. Compared with the wild-type, the R277W mutant has nearly 70-fold lower affinity for l-ornithine, shows no substrate inhibition, and its thermal stability is reduced by 5 °C. Its reduced affinity for l-ornithine, which in turn results in lower activity at physiological concentrations of ornithine, as well as its reduced stability, may contribute to the clinical effects that it produces.
Comparison of long-chain fatty acyl-CoA synthetases from rabbit heart and liver: substrate preferences and effects of Mg2+
Authors: WEIS Margaret T. , BERCUTE Andrea
Published: August 2015

Rabbit heart has a single, non-specific, fatty acyl-CoA synthetase (HP1) which is dependent on Mg2+, apart from the requirement for MgATP2-. Two long-chain fatty acyl-CoA synthetase activities (LP1 and LP2) can be resolved by hydroxyapatite chromatography of liver preparations; the Mg2+ requirement for these enzymes is undefined. These experiments were done to define the Mg2+ requirements of the liver enzymes and to compare them with the heart enzyme. For all three sources of enzyme and for arachidonic, oleic and palmitic acid substrates, the overall velocity of the reaction increased as [Mg2+] increased. Depending on the substrate and the source of enzyme, the increase in overall velocity could be attributed to changes in affinity or maximal velocity or both. The substrate preference of the HP1 enzyme for arachidonic acid (AA) was fifth or sixth of eight substrates regardless of the concentration of Mg2+. In contrast, increasing [Mg2+] shifted the relative substrate preference of both liver enzymes for AA. At low [Mg2+], AA was ranked seventh or eighth (least preferred) of eight substrates, whereas at high [Mg2+], AA was ranked as fifth or sixth. Hill plots of competition studies were consistent with Mg2+-induced positive co-operativity in LP1, but not in HP1 or LP2. Although enzymes from the three sources exhibit substantial kinetic differences, it is uncertain whether they are three different enzymes.
Biosynthesis, release and degradation of the novel endogenous cannabimimetic metabolite 2-arachidonoylglycerol in mouse neuroblastoma cells
Authors: BISOGNO Tiziana , SEPE Nunzio , MELCK Dominique , MAURELLI Stefano , PETROCELLIS Luciano De , MARZO Vincenzo Di
Published: August 2015

The monoacylglycerol 2-arachidonoylglycerol (2-AG) has been recently suggested as a possible endogenous agonist at cannabinoid receptors both in brain and peripheral tissues. Here we report that a widely used model for neuronal cells, mouse N18TG2 neuroblastoma cells, which contain the CB1 cannabinoid receptor, also biosynthesize, release and degrade 2-AG. Stimulation with ionomycin (1–5 μM) of intact cells prelabelled with [3H]arachidonic acid ([3H]AA) led to the formation of high levels of a radioactive component with the same chromatographic behaviour as synthetic standards of 2-AG in TLC and HPLC analyses. The amounts of this metabolite were negligible in unstimulated cells, and greatly decreased in cells stimulated in the presence of the Ca2+-chelating agent EGTA. The purified component was further characterized as 2-AG by: (1) digestion with Rhizopus arrhizus lipase, which yielded radiolabelled AA; (2) gas chromatographic–MS analyses; and (3) TLC analyses on borate-impregnated plates. Approx. 20% of the 2-AG produced by stimulated cells was found to be released into the incubation medium when this contained 0.1% BSA. Subcellular fractions of N18TG2 cells were shown to contain enzymic activity or activities catalysing the hydrolysis of synthetic [3H]2-AG to [3H]AA. Cell homogenates were also found to convert synthetic [3H]sn-1-acyl-2-arachidonoylglycerols (AcAGs) into [3H]2-AG, suggesting that 2-AG might be derived from AcAG hydrolysis. When compared with ionomycin stimulation, treatment of cells with exogenous phospholipase C, but not with phospholipase D or A2, led to a much higher formation of 2-AG and AcAGs. However, treatment of cells with phospholipase A2 10 min before ionomycin stimulation caused a 2.5–3-fold potentiation of 2-AG and AcAG levels with respect to ionomycin alone, whereas preincubation with the phospholipase C inhibitor neomycin sulphate did not inhibit the effect of ionomycin on 2-AG and AcAG levels. These results suggest that the Ca2+-induced formation of 2-AG proceeds through the intermediacy of AcAGs but not necessarily through phospholipase C activation. By showing for the first time the existence of molecular mechanisms for the inactivation and the Ca2+-dependent biosynthesis and release of 2-AG in neuronal cells, the present paper supports the hypothesis that this cannabimimetic monoacylglycerol might be a physiological neuromodulator.
The role of NAD+ as a signal during nitrogenase switch-off in Rhodospirillum rubrum
Authors: NORÉN Agneta , SOLIMAN Abdelhamid , NORDLUND Stefan
Published: August 2015

The role of NAD+ in the metabolic regulation of nitrogenase, the ‘switch-off’ effect, in Rhodospirillum rubrum has been studied. We now show that the decrease in nitrogenase activity upon addition of NAD+ to R. rubrum is due to modification of dinitrogenase reductase. There was no effect when NAD+ was added to a mutant of R. rubrumdevoid of dinitrogenase reductase ADP-ribosyltransferase, indicating that NAD+ ‘switch-off’ is an effect of the same regulatory system as ammonium ‘switch-off’. We also show that oxaloacetate and α-ketoglutarate function as ‘switch-off’ effectors. On the other hand β-hydroxybutyrate has the opposite effect by shortening the ‘switch-off’ period. Furthermore, by using an inhibitor of glutamate synthase the role of this enzyme in ‘switch-off’ was investigated. The results are discussed in relation to our proposal that changes in the concentration of NAD+ are involved in initiating ‘switch-off’.
Structure and organization of amplicons containing the E4 esterase genes responsible for insecticide resistance in the aphid Myzus persicae (Sulzer)
Authors: FIELD Linda M. , DEVONSHIRE Alan L.
Published: August 2015

Insecticide resistance in the aphid Myzus persicaeresults primarily from the amplification of genes encoding the insecticide-detoxifying esterase, E4. Here we report the analysis of flanking DNA co-amplified with the E4 gene. The 5´ end of this gene has an untranslated leader sequence interspersed by two introns, and the promoter region lacks TATA and CAAT boxes. The DNA breakpoint involved in the generation of the amplification is just upstream (approx. 250 bp) of the putative E4 transcription start site; thus the E4 gene is very close to the 5´ end of the approx. 24 kb amplicon. PCR primers specific to the ‘novel joint’ generated during the amplification have been used to show that a wide range of aphid clones have the same amplicons, arranged as a series of head-to-tail direct repeats. Long-distance mapping has revealed the structure of these repeats. This has important implications for understanding both the generation of the amplified genes and the origin and spread of insecticide resistance in M. persicae.
Intracellular targeting and homotetramer formation of a truncated inositol 1,4,5-trisphosphate receptor–green fluorescent protein chimera in Xenopus laevis oocytes: evidence for the involvement of the transmembrane spanning domain in endoplasmic reticulum targeting and homotetramer complex formation
Authors: SAYERS Lee G. , MIYAWAKI Atsushi , MUTO Akira , TAKESHITA Hiroshi , YAMAMOTO Akitsugu , MICHIKAWA Takayuki , FURUICHI Teiichi , MIKOSHIBA Katsuhiko
Published: August 2015

In an attempt to define structural regions of the type I inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor [Ins(1,4,5)P3R] involved in its intracellular targeting to the endoplasmic reticulum (ER), we have employed the use of green fluorescent protein (GFP) to monitor the localization of a truncated Ins(1,4,5)P3R mutant containing just the putative transmembrane spanning domain and the C-terminal cytoplasmic domain [amino acids 2216-2749; termed inositol trisphosphate receptor(ES)]. We expressed a chimeric GFP-Ins(1,4,5)P3R(ES) fusion protein in Xenopus laevisoocytes, and used fluorescence confocal microscopy to monitor its intracellular localization. Fluorescence confocal microscopy data showed an intense fluorescence in the perinuclear region and in a reticular-network under the animal pole of the oocyte, consistent with the targeting of expressed GFP-Ins(1,4,5)P3R(ES) to perinuclear ER and ER under the animal pole. These findings are consistent with the intracellular localization of the endogenous Xenopus Ins(1,4,5)P3R shown previously. Furthermore, electron microscopy data indicate that expressed GFP-Ins(1,4,5)P3R(ES) is in fact targeted to the ER. Sodium carbonate extraction of microsomal membranes and cross-linking experiments indicate that the expressed chimeric protein is in fact membrane anchored and able to form a homotetrameric complex. Our data provides evidence that Ins(1,4,5)P3R(ES) constitutes the membrane spanning domain of the Ins(1,4,5)P3R and is able to mediate homotetramer formation, without the need for the large N-terminal cytoplasmic domain. Furthermore, the localization of GFP-Ins(1,4,5)P3R(ES) on the ER indicates that an ER retention/targeting signal is contained within the transmembrane spanning domain of the inositol trisphosphate receptor.
NMR study of the galactomannans of Trichophyton mentagrophytes and Trichophyton rubrum
Authors: IKUTA Kyoko , SHIBATA Nobuyuki , BLAKE John S. , DAHL Mark V. , NELSON Robert D. , HISAMICHI Kanehiko , KOBAYASHI Hidemitsu , SUZUKI Shigeo , OKAWA Yoshio
Published: August 2015

Around 90% of chronic dermatophyte infections are caused by the fungi Trichophyton mentagrophytes and Trichophyton rubrum. One of the causes of the chronic infection resides in the immunosuppressive effects of the cell-wall components of these organisms. Therefore we have attempted to identify the chemical structure of galactomannan, one of the major cell-wall components. The cell-wall polysaccharides secreted by T. mentagrophytes and T. rubrum were isolated from the culture medium and fractionated into three subfractions by DEAE-Sephadex chromatography. Analysis of each subfraction by NMR indicated that there are two kinds of polysaccharides present, i.e. mannan and galactomannan. The mannan has a linear backbone consisting of α1,6-linked mannose units, with α1,2-linked mannose units as side chains. The core mannan moiety of the galactomannan was analysed by a sequential NMR assignment method after removing the galactofuranose units by acid treatment. The result indicates that the mannan moiety has a linear repeating structure of α1,2-linked mannotetraose units connected by an α1,6 linkage. The H-1 signals of the two intermediary α1,2-linked mannoses of the tetraose unit showed a significant upfield shift (ΔΔ = 0.05-0.08 p.p.m.), due to the steric effect of an α1,6-linked mannose unit. The attachment point of the galactofuranose units was determined at C-3 of the core mannan by the assignment of the downfield-shifted 13C signals of the galactomannan compared with those of the acid-modified product. In these galactomannans there were no polygalactofuranosyl chains which have been found in Penicillium charlesii and Aspergillus fumigatus.
pH-dependent DNA cleavage in permeabilized human fibroblasts
Authors: CZENE Stefan , TIBÄCK Mona , HARMS-RINGDAHL Mats
Published: August 2015

In several cell types, apoptosis is associated with intracellular acidification and activation of a pH-dependent endonuclease. We have examined the effect of acidic pH on the DNA of permeabilized human fibroblasts, and observed cleavage of DNA into high-molecular-mass fragments. This pH-dependent DNA breakage was modulated by temperature, the presence of histones and diethyl pyrocarbonate. Superoxide dismutase and chelators with high affinity for Cu prevented DNA fragmentation, whereas catalase, DMSO and Desferal (desferrioxamine mesylate) offered no protection. Fragmentation of DNA into high-molecular-mass fragments, which is occasionally observed as an early phase of apoptosis, is thought to result from the activation of endonuclease(s). Our results suggest that such fragmentation also occurs through induction of copper-mediated site-specific DNA damage that is enhanced by intracellular acidification.
Structure of a truncated human surfactant protein D is less effective in agglutinating bacteria than the native structure and fails to inhibit haemagglutination by influenza A virus
Authors: EDA Souji , SUZUKI Yasuhiko , KAWAI Takao , OHTANI Katsuki , KASE Tetsuo , FUJINAGA Yousuke , SAKAMOTO Takashi , KURIMURA Takashi , WAKAMIYA Nobutaka
Published: August 2015

Surfactant protein D (SP-D) is a lung-specific protein that is synthesized and secreted by lung epithelial cells and is believed to play an important role in lung host defence. This protein belongs to the C-type lectin family, which is characterized by an N-terminal cysteine-rich domain, a collagen-like domain, a neck domain and a carbohydrate recognition domain (CRD). To elucidate the biological actions of this animal lectin against such pathogens as micro-organisms, the biological activities of a recombinant partial SP-D lacking a collagen-like domain were examined. A recombinant human SP-D, consisting of a short collagen region (two repeats of Gly-Xaa-Yaa amino acid sequences), the neck domain and the CRD, was expressed in Escherichia coli. The recombinant SP-D was purified on a nickel column and then on a maltose-agarose column. This protein can form a trimeric structure owing to the neck domain and exhibits sugar-binding activity and specificity similar to those of native human SP-D. The recombinant SP-D caused dose-dependent and calcium-dependent agglutination of E. coli Y1088. The agglutination titre (the concentration required to achieve a 50% decrease in light transmission by agglutination) of recombinant SP-D was approx. 6-fold that of native SP-D. As for conglutination, the recombinant trimeric conglutinin required 8–16-fold higher concentrations than the native counterpart. In haemagglutination inhibition (HI) of influenza A virus, although native and recombinant conglutinin showed similar levels of HI activity, the recombinant SP-D was unable to inhibit haemagglutination, even at a concentration approx. 120-fold that of the native SP-D. The lectin precipitation and lectin blot assays showed that the truncated SP-D could bind to influenza A virus as well as native SP-D did. These results indicate that the agglutination activity of trimeric collectins can be largely retained, and furthermore that the oligomeric structure with several hands at opposite sites can enhance agglutination activity. The difference in HI activity against influenza A virus between native and recombinant SP-D suggests that SP-D uses a different mechanism from that of conglutinin to inhibit viral haemagglutination.
Role of the 5′ enhancer of the glutamine synthetase gene in its organ-specific expression
Authors: LIE-VENEMA Heleen , DE BOER Piet A. J. , MOORMAN Antoon F. M. , LAMERS Wouter H.
Published: August 2015

In mammals, glutamine synthetase (GS) is expressed in a large number of organs, but the precise regulation of its expression is still obscure. Therefore a detailed analysis of the activity of the upstream regulatory element of the GS gene in the transcriptional regulation of its expression was carried out in transgenic mice carrying the chloramphenicol acetyltransferase (CAT) gene under the control of the upstream regulatory region of the GS gene. CAT and GS mRNA expression were compared in liver, epididymis, lung, adipocytes, testis, kidney, skeletal muscle and gastrointestinal tract, both quantitatively by ribonuclease-protection analysis and topographically by in situ hybridization. It was found that the upstream regulatory region is active with respect both to the level and to the topography of GS gene expression in liver, epididymis, gastrointestinal tract (stomach, small intestine and colon) and skeletal muscle. On the other hand, in the kidney, brain, adipocytes, spleen, lung and testis, GS gene expression is not or only partly regulated by the 5´ enhancer. A second enhancer, identified within the first intron, may regulate GS expression in the latter organs. Furthermore, CAT expression in the brain did not co-localize with that of GS, showing that the 5´ regulatory region of the GS gene does not direct its expression to the astrocytes.
Enhanced expression of glucose-6-phosphate dehydrogenase in human cells sustaining oxidative stress
Authors: URSINI Matilde V. , PARRELLA Angelina , ROSA Graziella , SALZANO Salvatore , MARTINI Giuseppe
Published: August 2015

Recent reports have demonstrated that glucose-6-phosphate dehydrogenase (G6PD) activity in mammalian cells is necessary in order to ensure cell survival when damage is produced by reactive oxygen intermediates. In this paper we demonstrate that oxidative stress, caused by agents acting at different steps in the biochemical pathway controlling the intracellular redox status, determines the increase in G6PD-specific activity in human cell lines of different tissue origins. The intracellular level of G6PD-specific mRNA also increases, with kinetics compatible with the induction of new enzyme synthesis. We carried out experiments in which cells were exposed to oxidative stress in the presence of inhibitors of protein or RNA synthesis. These demonstrated that increased G6PD expression is mainly due to an increased rate of transcription, with a minor but significant contribution of regulatory mechanisms acting at post-transcriptional levels. These results provide new information on the defence systems that eukaryotic cells possess in order to prevent damage caused by potentially harmful oxygen derivatives.
Protein kinase C isoenzymes in airway smooth muscle
Authors: WEBB Benjamin L. J. , LINDSAY Mark A. , BARNES Peter J. , GIEMBYCZ Mark A.
Published: August 2015

The protein kinase C (PKC) isoenzymes expressed by bovine tracheal smooth muscle (BTSM) were identified at the protein and mRNA levels. Western immunoblot analyses reliably identified PKCα, PKCβI and PKCβII. In some experiments immunoreactive bands corresponding to PKCδ, PKCϵ and PKCθ were also labelled, whereas the γ, η and ζ isoforms of PKC were never detected. Reverse transcriptase PCR of RNA extracted from BTSM using oligonucleotide primer pairs designed to recognize unique sequences in the PKC genes for which protein was absent or not reproducibly identified by immunoblotting, amplified cDNA fragments that corresponded to the predicted sizes of PKCδ, PKCϵ and PKCζ, which was confirmed by Southern blotting. Anion-exchange chromatography of the soluble fraction of BTSM following homogenization in Ca2+-free buffer resolved two major peaks of activity. Using ϵ-peptide as the substrate, the first peak of activity was dependent upon Ca2+ and 4β-PDBu (PDBu = phorbol 12,13-dibutyrate), and represented a mixture of PKCs α, βI and βII. In contrast, the second peak of activity, which eluted at much higher ionic strength, also appeared to comprise a combination of conventional PKCs that were arbitrarily denoted PKCα′, PKCβI′ and PKCβII′. However, these novel enzymes were cofactor-independent and did not bind [3H]PDBu, but were equally sensitive to the PKC inhibitor GF 109203X compared with bona fide conventional PKCs, and migrated on SDS/polyacrylamide gels as 81 kDa polypeptides. Taken together, these data suggest that PKCs α′, βI′ and βII′ represent modified, but not proteolysed, forms of their respective native enzymes that retain antibody immunoreactivity and sensitivity to PKC inhibitors, but have lost their sensitivity to Ca2+ and PDBu when ϵ-peptide is used as the substrate.
Recombinant fish neurotrophin-6 is a heparin-binding glycoprotein: implications for a role in axonal guidance
Authors: LI Xiaoling , FRANZ Juliane , LOTTSPEICH Friedrich , GÖTZ Rudolf
Published: August 2015

Neurotrophin-6 (NT-6) was identified in the teleost fish Xiphophorus as a new member of the neurotrophin gene family. NT-6 binds specifically the glycosaminoglycan heparin. In this study NT-6 was expressed in a stably transfected mammalian cell line, and in insect cells via a recombinant baculovirus. It was purified to homogeneity and characterized by MS and N-terminal sequencing. NT-6 from both expression systems was proteolytically processed at one of two protease cleavage motifs and was found to be glycosylated. It supported the survival of embryonic chick sensory neurons; half-maximal survival was observed at 100 ng/ml. Furthermore, NT-6 elicited neurite outgrowth in explanted embryonic dorsal root ganglia. Addition of heparin into the medium did not potentiate the activity of NT-6 in survival assays. However, when a sensory ganglion explant was cultured in a collagen gel matrix assay adjacent to a heparin bead coated with NT-6, neurite outgrowth directed towards the bead was observed. This indicated that NT-6 was slowly released from the heparin bead generating a concentration gradient of NT-6 instrumental for axonal guidance in vitro. Thus the interaction of NT-6 with heparin might not be required for the activation of the cellular receptor for NT-6 on responsive cells but rather may serve to control, in vivo, the distribution of NT-6.
Regulatory domains of the A-Myb transcription factor and its interaction with the CBP/p300 adaptor molecules
Authors: FACCHINETTI Valeria , LOFFARELLI Livio , SCHREEK Sabine , OELGESCHLÄGER Michael , LÜSCHER Bernhard , INTRONA Martino , GOLAY Josée
Published: August 2015

The A-Myb transcription factor belongs to the Myb family of oncoproteins and is likely to be involved in the regulation of proliferation and/or differentiation of normal B cells and Burkitt's lymphoma cells. To characterize in detail the domains of A-Myb that regulate its function, we have generated a series of deletion mutants and have investigated their trans-activation potential as well as their DNA-binding activity. Our results have allowed us to delineate the trans-activation domain as well as two separate regulatory regions. The boundaries of the trans-activation domain (amino acid residues 218–319) are centred on a sequence rich in charged amino acids (residues 259–281). A region (residues 320–482) localized immediately downstream of the trans-activation domain and containing a newly identified conserved stretch of 48 residues markedly inhibits specific DNA binding. Finally the last 110 residues of A-Myb (residues 643–752), which include a sequence conserved in all mammalian myb genes (region III), negatively regulate the maximal trans-activation potential of A-Myb. We have also investigated the functional interaction between A-Myb and the nuclear adaptor molecule CBP [cAMP response element-binding protein (CREB)-binding protein]. We demonstrate that CBP synergizes with A-Myb in a dose-dependent fashion, and that this co-operative effect can be inhibited by E1A and can also be observed with the CBP homologue p300. We show that this functional synergism requires the presence of the A-Myb charged sequence and that it involves physical interaction between A-Myb and the CREB-binding domain of CBP.
Comparison of the ligand-binding properties of native and copper-less cytochromes bo from Escherichia coli
Authors: MOODY John A. , MITCHELL Roy , JEAL Alan E. , RICH Peter R.
Published: August 2015

The binding of four anionic ligands, cyanide, fluoride, azide and formate, to cytochrome bo purified from Escherichia coli cells grown with a copper supplement (+Cu cyt.bo) is described. Membrane-bound cytochrome bo that lacks the copper component, CuB, of its active site can be prepared from cells grown under conditions where the availability of copper is limited by the presence of a CuI chelator, 2,2′-bicinchinonic acid. The ligand-binding properties of this copper-less enzyme (-Cu cyt.bo) are compared with those of +Cu cyt.bo. As judged from near-UV/visible spectroscopic changes, cyanide forms a low-spin complex with +Cu cyt.bo, whereas azide, fluoride and formate form high-spin complexes. The pH-dependences of binding suggest that for all four of these anionic ligands, both the rates of binding and the binding affinities are primarily dependent on the concentration of their protonated forms. -Cu cyt.bo, which shows less than 15% of the duroquinol oxidase activity of +Cu cyt.bo, binds cyanide, azide and fluoride, but with greatly decreased affinity (< 1/30, 1/2000 and 1/2500 respectively at pH 5.5 compared with +Cu cyt.bo). The complex of azide with -Cu cyt.bo still seems to be high-spin and azide binding to -Cu cyt.bo is still pH-dependent, although less so than azide binding to +Cu cyt.bo.
Plasmodium falciparum CTP:phosphocholine cytidylyltransferase expressed in Escherichia coli: purification, characterization and lipid regulation
Authors: YEO Hye-Jeong , LARVOR Marie-Pierre , ANCELIN Marie-Laure , VIAL Henri J.
Published: August 2015

The Plasmodium falciparum CTP:phosphocholine cytidylyltransferase (PfCCT) has been isolated from an overexpressing strain of Escherichia coli. The plasmid pETPfCCT mediated the overexpression of the full-length polypeptide directly. The recombinant protein corresponded to 6–9% of the total cellular proteins and was found essentially in the insoluble membrane fraction. Urea at 6 M was used to solubilize the recombinant protein from the insoluble fraction. The CCT activity was restored upon the removal of urea, and the protein was subsequently purified to homogeneity on a Q-Sepharose column. Approx. 1.4 mg of pure enzyme was obtained from a 250 ml culture of E. coli. Biochemical properties, including in vitro substrate specificity and enzymic characterization, were assessed. The lipid regulation of the recombinant plasmodial CCT activity was characterized for the first time. The Km values were 0.49±0.03 mM (mean±S.E.M.) for phosphocholine and 10.9±0.5 mM for CTP in the presence of lipid activators (oleic acid/egg phosphatidylcholine vesicles), whereas the Km values were 0.66±0.07 mM for phosphocholine and 28.9±0.8 mM for CTP in the absence of lipid activators. The PfCCT activity was stimulated to the same extent in response to egg phosphatidylcholine vesicles containing anionic lipids, such as oleic acid, cardiolipin and phosphatidylglycerol, and was insensitive or slightly sensitive to PC vesicles containing neutral lipids, such as diacylglycerol and monoacylglycerol. Furthermore, the stimulated enzyme activity by oleic acid was antagonized by the cationic aminolipid sphingosine. These lipid-dependence properties place the parasite enzyme intermediately between the mammalian enzymes and the yeast enzyme.
Protein involvement in the fusion between the equatorial segment of acrosome-reacted human spermatozoa and liposomes
Authors: ARTS Eugène G. J. M. , WIJCHMAN Janny G. , JAGER Siemen , HOEKSTRA Dick
Published: August 2015

Artificial membranes (liposomes) can interact with the equatorial segment (ES) of human spermatozoa, provided that the acrosome reaction (AR) has occurred [Arts, Kuiken, Jager and Hoekstra (1993) Eur. J. Biochem. 217, 1001–1009]. Using fluorescently labelled liposomes, this interaction can be seen as either punctate fluorescence in the ES (lip-ESp), reflecting only bound liposomes, or as diffuse fluorescence in this region (lip-ESd), indicating that the liposomes have fused with the ES membrane. Only equatorial segments that still contain constituents of the acrosomal matrix have the capacity to bind liposomes and eventually to fuse with them. Since the exposure of such intact equatorial segments is the exclusive result of induction of the AR under physiological conditions, these results imply that liposomes can be used for the rapid detection of acrosome-reacted spermatozoa. The lip-ESp and lip-ESd patterns were shown to be reflections of two distinct properties of the ES. Proteolytic treatment after AR completely inhibited the formation of a lip-ESd pattern, whereas formation of the lip-ESp pattern was only marginally inhibited by the proteolytic treatment. The same results were obtained using anti-sperm antibodies, which did not react with acrosome-intact spermatozoa. Proteolytic treatment of spermatozoa before AR induction had no effect on the fusion capacity of the ES after subsequent AR, which implies that the putative fusion protein is not accessible before AR. Thus fusion of liposomes with the ES of human spermatozoa is mediated by a sperm protein(s), whereas the lip-ESp pattern is not likely to represent the liposome-binding stage that precedes the fusion step.
Alternative splicing of ClC-6 (a member of the CIC chloride-channel family) transcripts generates three truncated isoforms one of which, ClC-6c, is kidney-specific
Authors: EGGERMONT Jan , BUYSE Gunnar , VOETS Thomas , TYTGAT Jan , DE SMEDT Humbert , DROOGMANS Guy , NILIUS Bernd
Published: August 2015

ClC-6 is a protein that structurally belongs to the family of ClC-type chloride channels. We now report the identification of three additional ClC-6 isoforms that are truncated because of alternative splicing. We have isolated, from human K562 cells, four types of ClC-6 cDNAs that encode four distinct ClC-6 protein isoforms. ClC-6a (869 amino acids) corresponds to the previously published ClC-6 protein [Brandt and Jentsch (1995) FEBS Lett. 377, 15–20] and it has a canonical ClC structure. However, ClC-6b (320 amino acids), ClC-6c (353 amino acids) and ClC-6d (308 amino acids) are truncated at their C-termini. Hydropathy-plot analysis indicates that the shortened isoforms contain maximally four (ClC-6b and -6d) or seven (ClC-6c) transmembrane domains. Sequence analysis of a human genomic ClC-6 fragment indicates that the cDNA variability arises from alternative splicing at two different positions: the first alternative site consists of an intron flanked by two alternative donor sites and two alternative acceptor sites, the second being due to an exon that is optionally included or excluded. Reverse-transcription-PCR analysis of ClC-6 expression in human cell lines and tissues shows that the majority (83%) of ClC-6 mRNAs consists of ClC-6a or ClC-6c messengers. Furthermore, in a mouse tissue panel, the ClC-6a mRNA has a relatively broad tissue expression pattern, since it could be detected in brain, kidney, testis, skeletal muscle, thymus and pancreas. In contrast, expression of ClC-6c is more restricted, since it was only detected in kidney.
Expression of the murine RanBP1 and Htf9-c genes is regulated from a shared bidirectional promoter during cell cycle progression
Authors: GUARGUAGLINI Giulia , BATTISTONI Alessandra , PITTOGGI Carmine , DI MATTEO Gigliola , DI FIORE Barbara , LAVIA Patrizia
Published: August 2015

The murine Htf9-a/RanBP1and Htf9-c genes are divergently transcribed from a bidirectional promoter. The Htf9-a gene encodes the RanBP1 protein, a major partner of the Ran GTPase. The divergently transcribed Htf9-cgene encodes a protein sharing similarity with yeast and bacterial nucleic acid-modifying enzymes. We report here that both mRNA species produced by the Htf9-associated genes are regulated during the cell cycle progression, peak in S phase and decrease during mitosis. Transient expression experiments with reporter constructs showed that cell cycle expression is controlled at the transcriptional level, because the bidirectional Htf9 promoter is down-regulated in growth-arrested cells, is activated at the G1/S transition and reaches maximal activity in S phase, though with a different efficiency for each orientation. We have delimited specific promoter regions controlling S phase activity in one or both orientations: identified elements contain recognition sites for members belonging to both the E2F and Sp1 families of transcription factors. Together, the results suggest that the sharing of the regulatory region supports co-regulation of the Htf9-a/RanBP1 and Htf9-cgenes in a common window of the cell cycle.
Ca2+-dependent interaction of the growth-associated protein GAP-43 with the synaptic core complex
Authors: HARUTA Tatsumi , TAKAMI Noboru , OHMURA Manami , MISUMI Yoshio , IKEHARA Yukio
Published: August 2015

The synaptic vesicle exocytosis occurs by a highly regulated mechanism: syntaxin and 25 kDa synaptosome-associated protein (SNAP-25) are assembled with vesicle-associated membrane protein (VAMP) to form a synaptic core complex and then synaptotagmin participates as a Ca2+ sensor in the final step of membrane fusion. The 43 kDa growth-associated protein GAP-43 is a nerve-specific protein that is predominantly localized in the axonal growth cones and presynaptic terminal membrane. In the present study we have examined a possible interaction of GAP-43 with components involved in the exocytosis. GAP-43 was found to interact with syntaxin, SNAP-25 and VAMP in rat brain tissues and nerve growth factor-dependently differentiated PC12 cells, but not in undifferentiated PC12 cells. GAP-43 also interacted with synaptotagmin and calmodulin. These interactions of GAP-43 could be detected only when chemical cross-linking of proteins was performed before they were solubilized from the membranes with detergents, in contrast with the interaction of the synaptic core complex, which was detected without cross-linking. Experiments invitro showed that the interaction of GAP-43 with these proteins occurred Ca2+-dependently; its maximum binding with the core complex was observed at 100 μM Ca2+, whereas that of syntaxin with synaptotagmin was at 200 μM Ca2+. These values of Ca2+ concentration are close to that required for the Ca2+-dependent release of neurotransmitters. Furthermore we observed that the interaction invitro of GAP-43 with the synaptic core complex was coupled with protein kinase C-mediated phosphorylation of GAP-43. Taken together, our results suggest a novel function of GAP-43 that is involved in the Ca2+-dependent fusion of synaptic vesicles.