bluuebunny/crossref_metadata_2025 huggingface dataset:
The respiratory-chain poison antimycin A promotes the formation of DNA single-strand breaks and reduces toxicity in U937 cells exposed to t-butylhydroperoxide
Authors: GUIDARELLI Andrea , BRAMBILLA Liliana , ROTA Cristina , TOMASI Aldo , CATTABENI Flaminio , CANTONI Orazio
Published: August 2015

Antimycin A at levels that abolish oxygen consumption had a slight, although statistically significant, inhibitory effect on the toxicity elicited by t-butylhydroperoxide in U937 cells. The protective effect was observed after 6 h of post-treatment incubation, but was no longer apparent after 24 h. Unexpectedly, these events were associated with a marked accumulation of DNA single-strand breaks produced by low levels of t-butylhydroperoxide. Both an oxygen- and a carbon-centred radical were found to arise during treatment with t-butylhydroperoxide, and their formation was significantly lowered by antimycin A. Thus inhibition of electron transport at the level of complex III appears (a) to decrease the formation of toxic species which mediate, at least partially, the lethal effects elicited by t-butylhydroperoxide, and (b) to enhance the formation of DNA-damaging species generated at low concentrations of t-butylhydroperoxide. Rotenone and cyanide, which respectively inhibit complexes I and IV, did not affect DNA damage elicited by t-butylhydroperoxide. These results suggest that DNA single-strand breaks do not mediate the toxicity of t-butylhydroperoxide, and that specific mitochondrial functions might modulate the formation of toxic and of DNA-damaging species generated by organic hydroperoxides.
Activation by phosphorylation of phosphofructokinase from the annelid Lumbricus terrestris and comparison of phosphorylated sites in invertebrate phosphofructokinases
Authors: SCHALOSKE Ralph , BIETHINGER Michael , FOTHERGILL-GILMORE Linda A. , HOFER Hans Werner
Published: August 2015

Purified phosphofructokinase from the earthworm Lumbricus terrestris was phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase from the same organism to an extent of approx. 0.5 mol/mol of subunit. Activation of the enzyme occurred in parallel to the incorporation of covalently bound phosphate and was reversed by the action of the catalytic subunit of protein phosphatase 2A. Phosphorylation decreased the co-operativity of fructose 6-phosphate saturation in the presence of inhibitory concentrations of ATP, and increased the apparent Vmax obtained with saturating concentrations of the activators 5´-AMP and fructose 2,6-bisphosphate. The phosphorylated sites of phosphofructokinase from L. terrestris and from two molluscs (Helix pomatia and Mytilus edulis) were sequenced and shown to exhibit distinct similarity to sequences located near to the N-terminus of nematode phosphofructokinases [Klein, Olson, Favreau, Wintertowed, Hatzenbuhler, Shea, Nulf and Geary (1991) Mol. Biochem. Parasitol. 48, 17–26].
Ca2+ signalling mechanisms of the β2 integrin on neutrophils: involvement of phospholipase Cγ2 and Ins(1,4,5)P3
Authors: HELLBERG Carina , MOLONY Leslie , ZHENG Limin , ANDERSSON Tommy
Published: August 2015

Engagement of β2 integrins triggers a tyrosine kinase-dependent intracellular mobilization and influx of Ca2+ in human neutrophils. However, the transduction pathway involved in generating this Ca2+ signal is obscure. In the present study we identified phospholipase Cγ2 (PLCγ2) as one of the major proteins that was phosphorylated on tyrosine in response to β2 integrin activation. This β2 integrin-induced phosphorylation of PLCγ2 occurred in parallel with an increased accumulation of Ins(1,4,5)P3. The relevance of these observations for the β2 integrin-induced Ca2+ signal was investigated using an inhibitor of PLC signalling pathways, 1-(6-{[17β-3-methoxyoestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione (U73122). U73122 dose-dependently (IC50, approx. 0.15 μM) inhibited both the β2 integrin-induced release of Ca2+ from intracellular stores and the subsequent influx of Ca2+ across the plasma membrane. These effects were not observed with the inactive analogue 1-(6-{[17β-3-methoxyoestra-1,3,5(10)-trien-17-yl]amino}hexyl)-pyrrolidine-2,5-dione (U73343). To gain further support for an involvement of PLC-induced Ins(1,4,5)P3 formation in the β2 integrin-induced Ca2+ signal, we searched for the molecular event(s) underlying the effects of U73122. Our experiments revealed that U73122 had no effect on either β2 integrin-induced tyrosine phosphorylation of PLCγ2 (or any of the other proteins) or on the formation of Ins(1,4,5)P3, but it reduced the Ins(1,4,5)P3-induced release of 45Ca2+ from intracellular stores of electropermeabilized cells. Taken together, the present data suggest that the β2 integrin-induced Ca2+ signal in human neutrophils is generated through activation of a PLCγ2-dependent pathway.
Interaction of dibutyltin-3-hydroxyflavone bromide with the 16 kDa proteolipid indicates the disposition of proton translocation sites of the vacuolar ATPase
Authors: HUGHES Glen , HARRISON Michael A. , KIM Yong-In , GRIFFITHS David E. , FINBOW Malcolm E. , FINDLAY John B. C.
Published: August 2015

The organotin complex dibutyltin-3-hydroxyflavone bromide [Bu2Sn(of)Br] has been shown to bind to the 16 kDa proteolipid of Nephrops norvegicus, either in the form of the native protein or after heterologous expression in Saccharomyces and assembly into a hybrid vacuolar H+-ATPase. Titration of Bu2Sn(of)Br against the 16 kDa proteolipid results in a marked fluorescence enhancement, consistent with binding to a single affinity site on the protein. Vacuolar ATPase-dependent ATP hydrolysis was also inhibited by Bu2Sn(of)Br, with the inhibition constant correlating well with dissociation constants determined for binding of Bu2Sn(of)Br complex to the proteolipid. The fluorescence enhancement produced by interaction of probe with proteolipid can be back-titrated by dicyclohexylcarbodiimide (DCCD), which covalently modifies Glu140 on helix-4 of the polypeptide. Expression of a mutant proteolipid in which Glu140 was changed to a glycine resulted in assembly of a vacuolar ATPase which was inactive in proton pumping and which had reduced ATPase activity. Co-expression studies with this mutant and wild-type proteolipids suggest that proton pumping can only occur in a vacuolar ATPase containing exclusively wild-type proteolipid. The fluorescent enhancement or affinity of Bu2Sn(of)Br for the mutant proteolipid was not significantly altered, with the organotin complex having no effect on residual ATPase activity. Interaction of the probe with mutant proteolipid was unaffected by DCCD. These data suggest an overlap in the binding sites for organotin and DCCD, and have implications for the organization and structure of proton-translocating pathways in the vacuolar H+-ATPase.
Labelling the Ca2+-ATPase of skeletal-muscle sarcoplasmic reticulum with the cross-linker o-phthalaldehyde
Authors: KHAN Yamin M. , WICTOME Matthew , EAST Malcolm , LEE Anthony G. J.
Published: August 2015

The Ca2+-ATPase in the sarcoplasmic reticulum of skeletal muscle reacts with o-phthalaldehyde (OPA) to form a fluorescent isoindole product. The stoichiometry of labelling of the ATPase is 9 nmol of isoindole/mg of ATPase, corresponding to a 1:1 molar ratio of isoindole:ATPase. There is no evidence for any intermolecular cross-linking. Isoindole formation is faster in the presence of methylamine, but the stoichiometry of labelling is unchanged, whereas in the presence of 2-mercaptoethanol the level of labelling is much higher. It is concluded that OPA reacts with a single Cys residue (defining the specificity of the reaction) in a fast step, subsequent reaction with a Lys residue to form the isoindole being rate-controlling. Labelling the ATPase with OPA in the absence of methylamine leads to total loss of ATPase activity, whereas in the presence of methylamine, the decrease in ATPase activity on reaction is small. We conclude that the loss of ATPase activity probably follows from formation of the intramolecular cross-link rather than from the initial modification of the Cys residue. Reaction with OPA is not affected by the presence of ATP, ADP or Ca2+, so that the reactive Cys is not part of a ligand-binding site. The fluorescence emission spectrum of the labelled ATPase indicates a hydrophobic environment for the isoindole ring.
Effect of multiple cytokines plus bacterial endotoxin on glucose and nitric oxide production by cultured hepatocytes
Authors: CEPPI Enrico D. , SMITH Fiona S. , TITHERADGE Michael A.
Published: August 2015

Treatment of cultured hepatocytes with a combination of cytokines, including tumour necrosis factor-α, interferon-γ and interleukin-1β, plus lipopolysaccharide resulted in a time-dependent induction of nitric oxide (NO) synthase (as measured by NO2-+NO3- production) and inhibition of hepatic gluconeogenesis and glycogen breakdown. The inhibition of glucose release was comparable with that observed following treatment of rats with lipopolysaccharide or treatment of isolated hepatocytes with artificial NO donors. In addition, this effect was also evident with all substrates tested that enter the gluconeogenic pathway below the level of phosphoenolpyruvate carboxykinase, suggesting that this combination of cytokines may underlie the inhibition of gluconeogenesis observed in endotoxic shock. The maximal inhibition of glucose output required the presence of all the cytokines plus lipopolysaccharide, whereas the induction of NO synthase was independent of the lipopolysaccharide when the cytokines were employed. Inclusion of interferon-γ was essential to obtain a maximal response for either parameter. Inclusion of 1 mM NG-monomethyl-l-arginine in the incubation abolished the increase in NO2-+NO3- observed with the complete cytokine mixture and various combinations; however, it failed to prevent the inhibition in glucose output, indicating that mechanisms other than NO underlie the cytokine-induced inhibition of glucose release.
An uncleaved glycosylphosphatidylinositol signal mediates Ca2+-sensitive protein degradation
Authors: PAULY Peter C. , KLEIN Claudette
Published: August 2015

Inv-gp80 is a chimeric protein which contains a signal for the attachment of a glycosylphosphatidylinositol (GPI) anchor. When expressed in Dictyostelium discoideum, this protein fails to become GPI anchored and is retained within the cell as an integral membrane protein. We have compared the subcellular localization and degradation of Inv-gp80 with that of its intracellular but soluble counterpart, Inv-gp80sc. Inv-gp80sc lacks the hydrophobic C-terminal 22 amino acids of Inv-gp80. The N-linked oligosaccharides of both Inv-gp80 and Inv-gp80sc remained sensitive to endoglycosidase H, and both proteins co-fractionated with endoplasmic reticulum marker enzymes on Percoll gradients. Under normal conditions, Inv-gp80 displayed a half-life (t½) of 90 min, while Inv-gp80sc displayed a t½ of 120 min. The degradation of both proteins required ATP, was inhibited by tosyl phenylalanylchloromethane (Tos-Phe-CH2Cl) and was insensitive to inhibitors of lysosomal function. While depletion of Ca2+ from the endoplasmic reticulum had no effect on the degradation of Inv-gp80sc, it stimulated the degradation of Inv-gp80. When the GPI anchor signal sequence of Inv-gp80 was replaced with the transmembrane domain of the interleukin-2 receptor, the degradation of the protein was no longer influenced by Ca2+ fluxes. The data suggest that while the GPI anchor sequence of Inv-gp80 does not contain determinants regulating the degradation of the protein under basal conditions, it targets Inv-gp80 for rapid degradation under conditions where Ca2+ is depleted from the endoplasmic reticulum.
Structural changes in subdomain 2 of G-actin observed by fluorescence spectroscopy
Authors: MORACZEWSKA Joanna , STRZELECKA-GOŁASZEWSKA Hanna , MOENS Pierre D. J. , dos REMEDIOS Cristobal G.
Published: August 2015

The influence of DNase I binding to Ca-ATP-G-actin and of Ca2+/Mg2+ and ATP/ADP exchange on the conformation of G-actin were investigated by measuring the fluorescence of dansyl cadaverine (DC) conjugated to Gln41 in subdomain 2 of the protein. Fluorescence resonance energy transfer (FRET) between this probe and N-[4-(dimethylamino)-3,5-dinitrophenyl]maleimide (DDPM) attached to Cys374 in subdomain 1 was also measured. Contrary to an earlier report [dos Remedios, Kiessling and Hambly (1994) in Synchrotron Radiation in the Biosciences (Chance, B., Deisenhofer, J., Ebashi, S., Goodhead, D. T., Helliwell, J. R., Huxley, H. E., Iizuka, T., Kirz, J., Mitsui, T., Rubenstein, E. et al., eds.), pp. 418–425, Oxford University Press, Oxford], the distance between these probes did not change significantly when DNase I was bound to actin. A small but reproducible increase in the quantum yield and a blue shift of the DC fluorescence maximum were observed when bound Ca2+ was replaced by Mg2+. A large increase (about 70%) in the quantum yield and an approx. 12 nm blue shift of the emission spectrum occurred when ATP in Mg-G-actin was replaced by ADP. These changes were not accompanied by any significant change in the FRET distance between the dansyl donor and DDPM acceptor probes. A substantial change in the fluorescence of DC-actin was observed after proteolytic removal of the last three residues of actin, in accordance with earlier evidence suggesting that there is a conformational coupling between subdomain 2 and the C-terminal segment in subdomain 1 of actin. The results are discussed in relation to recently published data obtained with another fluorescent probe and to earlier observations based on limited cleavage using proteolytic enzymes.
Calcium-dependent ADP-ribosylation of high-mobility-group I (HMGI) proteins
Authors: GIANCOTTI Vincenzo , BANDIERA Antonella , SINDICI Chiara , PERISSIN Laura , CRANE-ROBINSON Colyn
Published: August 2015

Micrococcal nuclease digestion of nuclei from mouse Lewis lung carcinoma cells releases a protein mixture into the supernatant that lacks histone H1 and contains a full complement of high-mobility-group I (HMGI) proteins (i.e. I, Y and I-C). This implies that all three HMGI proteins are localized at the nuclease-sensitive regions of active chromatin. It is also shown that if Ca2+ ions are present in the nuclear incubation buffer (with or without exogenous nuclease), all three HMGI proteins become ADP-ribosylated. We propose that this modification of HMGI family proteins is part of the general poly(ADP-ribosyl)ation that accompanies DNA damage in apoptosis and other processes.
Isolation and characterization of nitrogenase MoFe protein from the mutant strain pHK17 of Klebsiella pneumoniae in which the two bridging cysteine residues of the P-clusters are replaced by the non-coordinating amino acid alanine
Authors: YOUSAFZAI Faridoon K , BUCK Martin , SMITH Barry E.
Published: August 2015

Nitrogenase MoFe protein (Kp1) from the mutant strain pHK17 of Klebsiella pneumoniae has been purified to give three catalytically active fractions. In this mutant, each of the two bridging cysteine ligands to the P-clusters, α-Cys-89 and β-Cys-94, has been replaced by a non-coordinating residue, alanine. SDS/PAGE and earlier native gels showed that the three fractions retained the normal α2β2 tetrameric form of wild-type Kp1; therefore we conclude that in each of the fractions the subunits are folded differently, thus resulting in different surface charges and allowing separation of the fractions on ion-exchange chromatography. Earlier EPR and magnetic CD data had shown that the mutant fractions contain P-clusters, and thus the mutated residues are not as essential for maintaining the integrity of the P-clusters as they appear from the X-ray structure. The specific activity of each of the three fractions was less than that of wild-type Kp1, the most active fraction having only 50% of wild-type activity. No change in substrate specificity or in the relative distribution of electrons to various substrates was found. The relationship between ATP hydrolysis and substrate-reducing activity, the EPR spectra of the S = 3/2 spin state of the iron–molybdenum cofactor (FeMoco) and the pH profile of acetylene-reduction activities of the three fractions did not differ significantly from those exhibited by wild-type Kp1. The specific activities of the three mutant fractions and of wild-type Kp1 were linearly proportional to the intensity of the S = 3/2 EPR signal from the FeMoco centres. This implies that those molecules of the three mutant fractions and the wild-type protein that contain EPR-active FeMoco are fully active, i.e. that the Cys to Ala substitution of the P-cluster ligands does not affect the specific activity of the protein. This in turn implies that the P-clusters are not directly associated with the rate-limiting step in enzyme turnover. We conclude that the lower specific activities of the mutant fractions are observed because the fractions are mixtures of species containing a full complement of FeMoco and P-clusters and species lacking some or all of these clusters. On the basis of the Mo contents and EPR spectroscopy of the mutant fractions, we propose that the loss of the P-clusters causes (i) the physical loss or inhibition of binding of some FeMoco; (ii) the EPR and catalytic inactivation of some FeMoco; and/or (iii) the incorporation of a FeMoco-like species into the FeMoco site of the mutant molecules.
Lecithin hydrophobicity modulates the process of cholesterol crystal nucleation and growth in supersaturated model bile systems
Authors: OCHI Hidenori , TAZUMA Susumu , KAJIYAMA Goro
Published: August 2015

The present study was performed to determine whether the degree of lecithin hydrophobicity regulates bile metastability and, therefore, affects the process of cholesterol crystallization. Supersaturated model bile (MB) solutions were prepared with an identical composition on a molar basis (taurocholate/lecithin/cholesterol, 73:19.5:7.5; total lipid concentration 9 g/dl) except for the lecithin species; egg yolk phosphatidylcholine, soybean phosphatidylcholine, 1-palmitoyl-2-linoleoyl-sn-phosphatidylcholine, dilinoleoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine. Each MB solution was incubated and sequentially examined. Video-enhanced contrast microscopy demonstrated that the rate of vesicular aggregation and fusion correlated with the degree of lecithin hydrophobicity, and that the rate of cholesterol crystal nucleation correlated with the degree of lecithin hydrophilicity. In MBs containing less hydrophobic lecithin, needle-like crystals developed and transformed into mature plate-like crystals, whereas classical plate-like crystals were consistently observed in MBs composed of hydrophobic lecithin. Laser-diffraction particle size analysis demonstrated that the increase in lecithin hydrophobicity enlarged the vesicle dimension, enhancing its cholesterol-holding capacity. Correlation between vesicular cholesterol packing density and lecithin hydrophobicity suggests that the process of bile cholesterol nucleation and growth is regulated, in part, by acyl chain unsaturation in lecithin. Since the composition of biliary lecithins is responsive to dietary manipulations, this study provides new insights into the prevention of cholesterol gallstones.
Down-regulation of hormone-sensitive lipase in sterol ester-laden J774.2 macrophages
Authors: JEPSON Catherine A , HARRISON Jillian A , KRAEMER Fredric B. , YEAMAN Stephen J.
Published: August 2015

The development of atherosclerotic plaques in arteries is a key step in atherogenesis, with cholesterol ester accumulation in macrophage-derived foam cells being recognized as a major pathogenic event in this process. In this study, the mouse macrophage cell line J774.2 was induced to accumulate intracellular sterol esters by incubation with 25-hydroxycholesterol in the presence of oleic acid. The accumulation of sterol esters in these cells was found to be accompanied by a marked decrease in the activity of the enzyme responsible for their hydrolysis, namely hormone-sensitive lipase (HSL); Western blotting studies revealed a corresponding decrease in the levels of the HSL polypeptide. Similar findings were obtained after incubation with oxidized low-density lipoprotein or very-low-density lipoprotein. These findings suggest that down-regulation of the expression of HSL is important in cholesterol ester accumulation in macrophages.
The SH3 domain of Src tyrosyl protein kinase interacts with the N-terminal splice region of the PDE4A cAMP-specific phosphodiesterase RPDE-6 (RNPDE4A5)
Authors: O'CONNELL Jonathan C. , McCALLUM J. Fraser , McPHEE Ian , WAKEFIELD Jill , HOUSLAY Emma S , WISHART William , BOLGER Graeme , FRAME Margaret , HOUSLAY Miles D.
Published: August 2015

The PDE4A (type IV) cAMP-specific, rolipram-inhibited phosphodiesterase RPDE-6 (RNPDE4A5), when transiently expressed in COS7 cells, could be complexed with the v-Src-SH3 domain expressed as a glutathione S-transferase (GST) fusion protein. RPDE-6 did not interact with GST itself. This complex was not disrupted by treatment with high NaCl concentration together with Triton X-100. Interaction was apparently determined by the N-terminal splice region of RPDE-6, as the PDE4A splice variant RPDE-39, which differs from RPDE-6 at the extreme N-terminus, failed to associate with v-Src-SH3; met26RD1 (where RD1 is rat ‘dunc-like’ PDE), which has the N-terminal splice region deleted, failed to associate with v-Src-SH3, and the association of RPDE-6 and v-Src-SH3 was blocked by a fusion protein formed from the N-terminal splice region. RDPE-6 showed binding to GST fusion proteins of both the intact Src kinase and an SH2–SH3 construct but did not bind to the Src-SH2 domain or to the adaptor protein Grb-2. RPDE-6 could be co-immunoprecipitated from cytosol extracts of transfected cells by using anti-Src antiserum. RPDE-6 exhibited selectivity in binding to the SH3 domains of c-Abl, Crk, Csk, Lck, Lyn, Fyn and v-Src, with binding to the SH3 regions of the Src-related tyrosyl kinases Lyn and Fyn being the most effective. The binding of RPDE-6 to the SH3 domains of Crk, Csk and Lck led to a marked reduction in PDE activity, but no change was apparent in complexes with other species. Endogenous RPDE-6 from brain, but not endogenous RPDE-39 from testis, bound to the Src-SH3 domain. We suggest that the PDE4A splice variant RPDE-6 has a propensity for interaction with selective SH3 domains, in particular those from Src and the Src-related tyrosyl kinases Lyn and Fyn. This interaction seems to be governed by alternative splicing of the PDE4A gene, because RPDE-39, a splice variant that lacks the proline-rich N-terminal splice region of RPDE-6, does not interact with these SH3 domains. It is proposed that the binding site on RPDE-6 for SH3 domains lies within the unique first 102 residues of its N-terminal splice domain, where two motifs representing Class I SH3 binding sites with selectivity for Src kinase SH3 domains can be identified and one motif for a putative Class II SH3 binding site.
Methylamine decreases trafficking and packaging of newly synthesized phosphatidylcholine in lamellar bodies in alveolar type II cells
Authors: CHANDER Avinash , SEN Namita , WU Ai-Min , HIGGINS Stephen , WADSWORTH Sandra , SPITZER Alan R
Published: August 2015

Lung lamellar bodies, the storage organelles for lung surfactant phosphatidylcholine (PC), maintain an acidic pH that can be increased with weak bases. This study investigates the effect of a weak base, methylamine, on the pH in lamellar bodies and on the trafficking and packaging of newly synthesized PC in lamellar bodies. Methylamine increased the pH of isolated lung lamellar bodies and of lamellar bodies in intact cells. Metabolic labelling of isolated type II cells with [methyl-3H]choline showed that although methylamine (2.5–10 mM) did not alter the labelling of cellular or microsomal PC and disaturated PC, it decreased the labelling of the PC and disaturated PC in lamellar bodies. The packaging of PC in lamellar bodies (the specific activities ratio between the PC in lamellar bodies and the microsomal PC) also decreased in a time- and concentration-dependent manner. The cellular synthesis of PC or its packaging into lamellar bodies was unaltered by brefeldin A, suggesting that the Golgi was not involved in PC packaging. Although methylamine also increased surfactant secretion, the inhibition of PC packaging in lamellar bodies seems unrelated to the secretagogue effect, (1) on the basis of metabolic consequences of increased secretion and (2) because ATP, another secretagogue, did not inhibit PC packaging. Methylamine seems to inhibit PC packaging by inhibiting trafficking of PC to lipid-rich light subcellular fractions. Together our results suggest that the trafficking of surfactant PC into lamellar bodies might be sensitive to changes in the pH of lamellar bodies.
Biotin-labelled peptidyl diazomethane inhibitors derived from the substrate-like sequence of cystatin: targeting of the active site of cruzipain, the major cysteine proteinase of Trypanosoma cruzi
Authors: LALMANACH Gilles , MAYER Roger , SERVEAU Carole , SCHARFSTEIN Julio , GAUTHIER Francis
Published: August 2015

Biotin-labelled peptidyl diazomethane inhibitors of cysteine proteinases, based on the N-terminal substrate-like segment of human cystatin C, a natural inhibitor of cysteine proteinases, were synthesized. These synthetic derivatives were tested as irreversible inhibitors of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, to compare the kinetics of the inhibition of the parasite proteinase with that of the mammalian cathepsins B and L. The accessibility of the active sites of these proteinases to these probes was also investigated. The inhibition of cruzipain by Biot-LVG-CHN2 (where Biot represents biotinyl and L,V and G are single-letter amino acid residue abbreviations) and Biot-Ahx-LVG-CHN2 (where Ahx represents 6-aminohexanoic acid) was similar to that of unlabelled inhibitor. Biotin labelling of the inhibitor slowed the inhibition of both cathepsin B and cathepsin L. Adding a spacer arm (Ahx) between the biotin and the peptide moiety of the derivative increased the inhibition of cathepsin B but not that of cathepsin L. The discrimination provided by this spacer is probably due to differences in the topologies of the binding sites of proteinases, a feature that can be exploited to improve targeting of individual cysteine proteinases. Analysis of the blotted proteinases revealed marked differences in the accessibility of extravidin–peroxidase conjugate to the proteinase-bound biotinylated inhibitor. Cruzipain molecules exposed to Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 were readily identified, but the reaction was much stronger when the enzyme was treated with the spacer-containing inhibitor. In contrast with the parasite enzyme, rat cathepsin B and cathepsin L treated with either Biot-LVG-CHN2 or Biot-Ahx-LVG-CHN2 produced no detectable bands. Papain, the archetype of this family of proteinases, was poorly labelled with Biot-LVG-CHN2, but strong staining was obtained with Biot-Ahx-LVG-CHN2. These findings suggest that optimized biotinylated diazomethanes might considerably improve their selectivity for the T. cruzi target enzyme.
Phosphodiesterase 4 in macrophages: relationship between cAMP accumulation, suppression of cAMP hydrolysis and inhibition of [3H]R-(—)-rolipram binding by selective inhibitors
Authors: KELLY John J , BARNES Peter J , GIEMBYCZ Mark A
Published: August 2015

A perplexing phenomenon identified in several tissues is the lack of correlation between inhibition of phosphodiesterase 4 (PDE4) and certain functional responses such as smooth muscle relaxation, gastric acid secretion and cAMP accumulation. Interpretation of these data is complicated further by the finding that function correlates with the ability of PDE4 inhibitors to displace [3H]rolipram [4-(3-cyclopentenyloxy-4-methoxyphenyl)-2-pyrrolidone] from a high-affinity site in rat brain that is apparently distinct from the catalytic centre of the enzyme. We have investigated this discrepancy by using guinea pig macrophages as a source of PDE4 and have confirmed that the ability of a limited range of structurally dissimilar PDE inhibitors (Org 20241, nitraquazone and the enantiomers of rolipram and benafentrine) to increase cAMP content did not correlate with their potency as inhibitors of partly purified PDE4, whereas a significant linear and rank order correlation was found when cAMP accumulation was related to the displacement of [3H]R-(-)-rolipram from a specific site identified in macrophage lysates. An explanation for these data emerged from the finding that the IC50 values and rank order of potency of these compounds for inhibition of partly purified PDE4 and the native (membrane-bound) form of the same enzyme were distinct. Similarly, no correlation was found when membrane-bound PDE4 was compared with the same enzyme that had been solubilized with Triton X-100. These unexpected results were attributable to a selective decrease in the potency of those inhibitors [nitraquazone, R-(-)- and S-(+)-rolipram] that interacted preferentially with the rolipram binding site. Indeed, if membrane-bound PDE4 was used as the enzyme preparation, excellent linear and rank order correlations between inhibition of cAMP hydrolysis, displacement of [3H]R-(-)-rolipram and cAMP accumulation were found, which improved further in the presence of the vanadyl (Vo)/2.GSH complex. Moreover, using Vo/2.GSH-treated membranes, the IC50 values of nitraquazone and the enantiomers of rolipram for the inhibition of PDE4 approached their affinity for the rolipram binding site. Collectively, these data suggest that the rolipram binding site and the catalytic domain on CPPDE4 might represent part of the same entity. In addition, these results support the concept that PDE4 can exist in different conformational states [Barnett, Manning, Cieslinski, Burman, Christensen and Torphy (1995) J. Pharmcol. Exp. Ther. 273, 674–679] and provide evidence that the cAMP content in macrophages is regulated primarily by a conformer of PDE4 for which rolipram has nanomolar affinity.
Determination of the primary target for isoniazid in mycobacterial mycolic acid biosynthesis with Mycobacterium aurum A+
Authors: WHEELER Paul R , ANDERSON Paul M
Published: August 2015

The target of the potent antituberculosis drug isoniazid was investigated in Mycobacterium aurum A+, against which isoniazid has an MIC (the minimum concentration required to give growth inhibition) of 0.3 µg/ml. Mycolic acid biosynthesis, measured by the incorporation of label from [1-14C]acetate into mycolic acids, was inhibited differentially by isoniazid in cell-wall preparations of M. aurum A+. Thus at an isoniazid concentration of 1 µg/ml, mycolic acid biosynthesis was inhibited by 80% but concomitant biosynthesis of non-hydroxylated fatty acids was inhibited by only 15%. Three lines of evidence identified 24:1 cis-5 elongase as the primary isoniazid target. First, 24:1 cis-5 did not restore isoniazid-inhibited mycolic acid biosynthetic activity in a crude cell-wall preparation, suggesting that the drug acts after the formation of the Δ-5 double bond. Secondly, a 24:1 cis-5 elongase assay in which the product is mycolic acid is completely inhibited by isoniazid. Finally, the only intermediates that accumulate as a result of the addition of isoniazid are acids of 24 carbons. Both 24:0 and 24:1 are observed in a similar ratio whether or not isoniazid is present, even though concomitant mycolic acid biosynthesis is inhibited by isoniazid. These results are consistent with studies of the M. tuberculosis InhA protein by Dessen, Quemard, Blanchard, Jacobs and Sacchettini [(1995) Science 267, 1638–1641].
Vav in natural killer cells is tyrosine phosphorylated upon cross-linking of Fcγ RIIIA and is constitutively associated with a serine/threonine kinase
Authors: XU Xiulong , CHONG Anita S.-F.
Published: August 2015

Cross-linking of FcγRIIIA (CD16) receptor on natural killer (NK) cells induces receptor-associated tyrosine kinase activation and tyrosine phosphorylation of numerous intracellular proteins, including phospholipase C (PLC)-γ1, PLC-γ2 and the associated ζ chain. Here we report that Vav, a proto-oncogene, also became tyrosine phosphorylated upon stimulation of CD16 in interleukin 2-activated NK cells (LAK-NK) as well as in an NK cell line, NK3.3. In addition, we observed that in LAK-NK cells, Vav was associated with a 70 kDa protein that also became tyrosine phosphorylated upon CD16 cross-linking. The association of this 70 kDa protein with Vav was disrupted by ionic detergent treatment. Tyrosine phosphorylation of Vav was inhibited by herbimycin A, a specific tyrosine kinase inhibitor. In vitro kinase assays with Vav immunoprecipitates derived from NK3.3 cells or LAK-NK cells resulted in the appearance of a phosphorylated 58 kDa protein, suggesting the presence of a kinase within the Vav immunoprecipitates. Cross-linking of CD16 did not enhance this Vav-associated kinase activity. Phosphoamino acid analysis of the 58 kDa protein revealed that it was phosphorylated only on serine and threonine residues, indicating that an unidentified serine/threonine kinase is constitutively associated with Vav. These observations suggest that the downstream signalling events regulated by Vav and its associated proteins are complex involving both tyrosine kinases as well as the yet unidentified serine/threonine kinase in NK cells.
Comparison of the mRNA sequences for Pi class glutathione transferases in different hamster species and the corresponding enzyme activities with anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide
Authors: SWEDMARK Stellan , JERNSTRÖM Bengt , JENSSEN Dag
Published: August 2015

Glutathione S-transferase (GST) of class Pi (GST Pi) is known to detoxify the mutagenic and carcinogenic (+)-anti-benzo[a]pyrene-7,8-dihydrodiol 9,10-epoxide [(+)-anti-BPDE] by conjugation with glutathione. Previously, we have shown that Chinese hamster V79 cells contain GST Pi, but seem to lack the capacity to conjugate (+)-anti-BPDE, although these cells do conjugate other substrates with GSH [Romert, Dock, Jenssen and Jernström (1989) Carcinogenesis 10, 1701–1707; Swedmark, Romert, Morgenstern and Jenssen (1992) Carcinogenesis 13, 1719–1723; Swedmark and Jenssen (1994) Gene 139, 251–256]. In the present study we have compared four cell lines derived from different hamster species with respect to GST cDNA sequences and capacity to conjugate (+)- or (-)-anti-BPDE. The cell lines were V79 and Chinese hamster ovary cells (CHO), Armenian hamster lung (AHL) cells and baby hamster kidney (BHK) cells. The sequencing revealed a complete homology between the V79 and CHO cDNA for GST Pi, whereas the corresponding amino acid sequences predicted from the corresponding AHL and BHK cDNAs differed by six and nine amino acids, respectively, from the predicted V79 sequence. None of these changes alone was found to influence the xenobiotic substrate-binding site. The cytosolic fractions from BHK and AHL cells were found to catalyse conjugation of (+)-anti-BPDE with GSH, whereas the corresponding activity in CHO cells was non-detectable. As shown previously, V79 cells were devoid of activity towards (+)-anti-BPDE. All the cell lines studied demonstrated appreciable GST activity towards 1-chloro-2,4-dinitrobenzene, but no activity with (-)-anti-BPDE. The latter result suggests that GST Pi is the sole or predominant GST in these cell lines. This was confirmed by HPLC analysis of purified enzymes obtained by affinity chromatography. However, when the catalytic activities of the pure enzymes were determined, all four different GST Pi enzymes were found to be highly capable of conjugating (+)-anti-BPDE with GSH. This observation indicates the existence of an intracellular factor that selectively inhibits conjugation of (+)-anti-BPDE, but not of 1-chloro-2,4-dinitrobenzene in the V79 and CHO cell lines. This new phenomenon seems to be specific for Chinese hamster, since both these cell lines originate from this species.
Lipid metabolism in Trypanosoma brucei: utilization of myristate and myristoyllysophosphatidylcholine for myristoylation of glycosyl phosphatidylinositols
Authors: WERBOVETZ Karl A , ENGLUND Paul T
Published: August 2015

Myristate is the exclusive fatty acid species in the glycosyl phosphatidylinositol (GPI) anchor of the Trypanosoma brucei variant surface glycoprotein (VSG). [3H]Myristate can be incorporated into T. brucei GPIs by two distinct processes known as fatty acid remodelling and myristate exchange. Myristoyllysophosphatidylcholine (M-LPC) can also serve as a myristate donor for VSG in trypanosomes [Bowes, Samad, Jiang, Weaver and Mellors (1993) J. Biol. Chem. 268, 13885–13892]. We have studied in detail the myristoylation of GPIs using a [3H]M-LPC substrate. Labelling of VSG and free GPIs by [3H]M-LPC in cultured trypanosomes occurred at the same rate as with [3H]myristate. Concurrent with GPI labelling, there was rapid hydrolysis of [3H]M-LPC to generate extracellular [3H]myristate. Experiments in a trypanosomal cell-free system indicated that GPI labelling by fatty acid remodelling and myristate exchange was also equally efficient with [3H]M-LPC and [3H]myristate. Furthermore, both ATP and CoA are required for the myristoylation of GPIs by [3H]M-LPC. These experiments suggest that GPI myristoylation from M-LPC involves hydrolysis of M-LPC to free myristate. To address the physiological importance of myristate and M-LPC in VSG myristoylation, we radiolabelled trypanosomes in vivo with both substrates in medium containing serum, and found that [3H]myristate labelled VSG and GPIs more efficiently. Thus, VSG myristoylation by free myristate may be favoured in bloodstream trypanosome infections.
Affinity purification and characterization of protein gene product 9.5 (PGP9.5) from retina
Authors: PICCININI Marco , MERIGHI Adalberto , BRUNO Renato , CASCIO Paolo , CURTO Magda , MIOLETTI Silvia Clarissa , RINAUDO Maria T.
Published: August 2015

Protein gene product 9.5 (PGP9.5) is a cytosolic protein that is highly expressed in vertebrate neurons, which is now included in the ubiquitin C-terminal hydrolase subclass (UCH) on the basis of primary-structure homology and hydrolytic activity on the synthetic substrate ubiquitin ethyl ester (UbOEt). Some UCHs show affinity for immobilized ubiquitin, a property exploited to purify them. In this study we show that this property can also be applied to PGP9.5, since a protein has been purified to homogeneity from bovine retina by affinity chromatography on a ubiquitin–Sepharose column that can be identified with: (a) PGP9.5 with respect to molecular mass, primary structure and immunological reactivity; (b) the known UCHs with respect to some catalytic properties, such as hydrolytic activity on UbOEt, (which also characterizes PGP9.5), Km value and reactivity with cysteine and histidine-specific reagents. However, it differs with respect to other properties, e.g. inhibition by UbOEt and a wider pH range of activity.
Aging of phosphylated human acetylcholinesterase: catalytic processes mediated by aromatic and polar residues of the active centre
Authors: SHAFFERMAN Avigdor , ORDENTLICH Arie , BARAK Dov , STEIN Dana , ARIEL Naomi , VELAN Baruch
Published: August 2015

We have examined the effects of 11 substitutions of active centre gorge residues of human acetylcholinesterase (HuAChE) on the rates of phosphonylation by 1,2,2-trimethylpropyl methylphosphonofluoridate (soman) and the aging of the resulting conjugates. The rates of phosphonylation were reduced to as little as one-seventieth, mainly in mutants of the hydrogen-bond network (Glu-202, Glu-450, Tyr-133). These recombinant enzymes as well as the F338A, W86A, W86F and D74N mutant HuAChEs varied in their resistance to aging (15–3300-fold relative to the wild type). The most dramatic resistance to aging was observed for the phosphonyl conjugate of the mutant W86A enzyme (1850–3300-fold relative to the wild type). It is proposed that Trp-86 contributes to the aging process by stabilizing the evolving carbonium ion on the 1,2,2-trimethylpropyl moiety, via charge–π interaction. The rate-enhancing effect of Trp-86 provides a rationale for the unique facility of aging in soman-inhibited cholinesterases, compared with the corresponding conjugates in other serine hydrolases. Replacements of Glu-202 by aspartic acid, glutamine or alanine residues resulted in a similar (1/130–1/300) decrease of the rates of aging. A comparable decrease was also observed for the conjugate of the F338A mutant. These results, and the similar pH dependence of aging rates for the wild-type and E202Q and F338A mutant HuAChEs, indicate that Glu-202 is not involved in proton transfer to the phosphonyl moiety. On the basis of these findings and of molecular modelling we suggest that Glu-202 and Phe-338 contribute to the aging process by stabilizing the imidazolium of the catalytic triad His-447 via charge–charge and charge–π interactions respectively, thereby facilitating an oxonium formation on the phosphonyl moiety.
3′:5′-cyclic guanosine monophosphate (cGMP) potentiates the inositol 1,4,5-trisphosphate-evoked Ca2+ release in guinea-pig hepatocytes
Authors: GUIHARD Gilles , COMBETTES Laurent , CAPIOD Thierry
Published: August 2015

The effect of cGMP on noradrenaline-induced intracellular Ca2+ mobilization was investigated in whole-cell voltage-clamped guinea-pig hepatocytes. Treatment of the cells with 8-Br-cGMP (1–500 µM) resulted in an increase in the sensitivity of the cells to noradrenaline and to inositol 1,4,5-trisphosphate (InsP3) photoreleased from caged InsP3. The positive effect of 8-Br-cGMP on the Ca2+ release evoked by Ca2+-mobilizing agonists or InsP3 was blocked by a protein kinase G (PKG; cGMP-dependent protein kinase) inhibitor, the Rp-8-(4-chlorophenylthio)guanosine 3´:5´-monophosphorothioate. 8-Br-cGMP affected neither the basal InsP3 concentration nor the noradrenaline-induced production of InsP3. In permeabilized hepatocytes, the dose–response curve for InsP3-induced Ca2+ release was shifted to the left in the presence of 8-Br-cGMP. Furthermore, the treatment with 8-Br-cGMP did not affect the Ca2+ content of the InsP3-sensitive Ca2+ stores. These results indicate that intracellular cGMP potentiates the noradrenaline-induced Ca2+ response by enhancing Ca2+ release from the intracellular Ca2+ stores. We suggest that cGMP increases the apparent affinity of InsP3 receptors for InsP3 in guinea-pig hepatocytes probably by phosphorylation via the activation of PKG.
Starch degradation in chloroplasts isolated from C3 or CAM (crassulacean acid metabolism)-induced Mesembryanthemum crystallinum L
Authors: NEUHAUS H. Ekkehard , SCHULTE Norbert
Published: August 2015

C3 or crassulacean acid metabolism (CAM)-induced Mesembryanthemum crystallinum plants perform nocturnal starch degradation which is linear with time. To analyse the composition of metabolites released by isolated leaf chloroplasts during starch degradation we developed a protocol for the purification of starch-containing plastids. Isolated chloroplasts from C3 or CAM-induced M. crystallinum plants are also able to degrade starch. With respect to the endogenous starch content of isolated plastids the rate of starch degradation in these organelles is close to the observed rates of starch degradation in intact leaves. The combined presence of Pi, ATP, and oxaloacetate is identified to be the most positive effector combination to induce starch mobilization. The metabolic flux through the oxidative pentose-phosphate pathway in chloroplasts isolated from CAM-induced M. crystallinum is less than 3.5% compared with other metabolic routes of starch degradation. Here we report that starch-degrading chloroplasts isolated from CAM-induced M. crystallinum plants use exogenously supplied oxaloacetate for the synthesis of malate. The main products of starch degradation exported into the incubation medium by these chloroplasts are glucose 6-phosphate, 3-phosphoglyceric acid, dihydroxyacetone phosphate and glucose. The identification of glucose 6-phosphate as an important metabolite released during starch degradation is in contrast to the observations made on all other types of plastids analysed so far, including chloroplasts isolated from M. crystallinum in the C3 state. Therefore, we analysed the transport properties of isolated chloroplasts from M. crystallinum. Surprisingly, both types of chloroplasts, isolated from either C3 or CAM-induced plants, are able to transport glucose 6-phosphate in counter exchange with endogenous Pi, indicating the presence of a glucose 6-phosphate translocator as recently demonstrated to occur in other types of plastids. The composition of metabolites released and the stimulatory effect of oxaloacetate on the rate of starch degradation are discussed with respect to the acidification observed for CAM leaves during the night.
A novel keratan sulphate domain preferentially expressed on the large aggregating proteoglycan from human articular cartilage is recognized by the monoclonal antibody 3D12/H7
Authors: FISCHER Dagmar-Christiane , HAUBECK Hans-Dieter , EICH Kirsten , KOLBE-BUSCH Susanne , STÖCKER Georg , STUHLSATZ Helmut Wolfgang , GREILING Helmut
Published: August 2015

Monoclonal antibodies (mAbs) were prepared against aggrecan which has been isolated from human articular cartilage and purified by several chromatographic steps. One of these mAbs, the aggrecan-specific mAb 3D12/H7, was selected for further characterization. The data presented indicate that this mAb recognizes a novel domain of keratan sulphate chains from aggrecan: (1) immunochemical staining of aggrecan is abolished by treatment with keratanase/keratanase II, but not with keratanase or chondroitin sulphate lyase AC/ABC; (2) after chemical deglycosylation of aggrecan no staining of the core-protein was observed; (3) different immunochemical reactivity was observed against keratan sulphates from articular cartilage, intervertebral disc and cornea for the mAbs 3D12/H7 and 5D4. For further characterization of the epitope, reduced and 3H-labelled keratan sulphate chains were prepared. In an IEF–gel-shift assay it was shown that the 3H-labelled oligosaccharides obtained after keratanase digestion of reduced and 3H-labelled keratan sulphate chains were recognized by the mAb 3D12/H7. Thus it can be concluded that the mAb 3D12/H7 recognizes an epitope in the linkage region present in, at least some, keratan sulphate chains of the large aggregating proteoglycan from human articular cartilage. Moreover, this domain seems to be expressed preferentially on those keratan sulphate chains which occur in the chondroitin sulphate-rich region of aggrecan, since the antibody does not recognize the keratan sulphate-rich region obtained after combined chondroitinase AC/ABC and trypsin digestion of aggrecan.