bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the intracellular signalling pathways that underlie growth-factor-stimulated glucose transport in Xenopus oocytes: evidence for ras- and rho-dependent pathways of phosphatidylinositol 3-kinase activation
Authors: THOMSON Fiona J. , JESS Thomas J. , MOYES Colin , PLEVIN Robin , GOULD Gwyn W.
Published: August 2015

The stimulation of glucose transport is one of the early cellular responses to growth factors and is essential for cell proliferation, yet the molecular processes that underlie this response are poorly defined. The aim of this study was to characterize the role of the low-molecular-mass G-proteins, Ras and Rho, and their downstream targets, Raf protein kinase and phosphatidylinositol 3-kinase, in the regulation of glucose transport in Xenopusoocytes by two distinct growth-factor receptors: the insulin-like growth factor I (IGF-I) tyrosine kinase receptor and the heterotrimeric G-protein-coupled lysophosphatidic acid (LPA) receptor. Microinjection of a neutralizing anti-Ras antibody partially blocked IGF-I-stimulated deoxyglucose uptake but was without effect on LPA-stimulated deoxyglucose uptake. In contrast, microinjection of the C3 coenzyme of botulinum toxin, which selectively ADP-ribosylates and inactivates Rho, inhibited LPA-stimulated, but not IGF-I-stimulated, deoxyglucose uptake. Similarly, LPA- but not IGF-I-stimulated deoxyglucose uptake was attenuated in oocytes expressing a dominant negative rhoconstruct. Cells expressing a dominant negative mutant of Raf protein kinase exhibited markedly reduced sensitivity to both LPA and IGF-I, consistent with a role for endogenous Raf in glucose uptake by both growth factors. Furthermore, expression of a constitutively activated form of raf-1 resulted in a growth-factor-independent increase in deoxyglucose uptake. Measurements of phosphatidylinositol 3-kinase activity in microinjected cells support the hypothesis that the IGF-I receptor stimulates glucose transport by a Ras-dependent activation of phosphatidylinositol 3-kinase, whereas the G-protein-coupled LPA receptor controls this response by a pathway that involves Rho-dependent activation of a distinct phosphatidylinositol 3-kinase. Thus we provide evidence for clear differences in the signalling pathways that control glucose transport by G-protein-coupled and tyrosine kinase growth-factor receptors. Furthermore this is the first demonstration that active Rho is involved in the signalling pathways that regulate glucose uptake in response to some growth factors.
Decreased secretion of very-low-density lipoprotein triacylglycerol and apolipoprotein B is associated with decreased intracellular triacylglycerol lipolysis in hepatocytes derived from rats fed orotic acid or n–3 fatty acids
Authors: HEBBACHI Abdel-Malek , SEELAENDER Marilia C. L. , BAKER Paul W. , GIBBONS Geoffrey F.
Published: August 2015

Hepatocytes from rats fed a chow (control) diet or from rats fed a chow diet supplemented with either orotic acid (OA; 1%, w/w) or fish oil (FO; 20%, v/w) were maintained in culture for periods up to 48 h. During the first 24 h period, the low rates of output of very-low-density lipoprotein (VLDL)-associated triacylglycerol (TAG) and apolipoprotein B (apoB) in hepatocytes from the FO- and OA-fed animals were associated with significantly lower rates of intracellular TAG lipolysis and re-esterification. Most of the VLDL TAG secreted was mobilized via lipolysis of the intracellular TAG pool, but the proportion of VLDL TAG secreted via this route in cells from the FO-fed and OA-fed animals was decreased compared with that in the control-fed animals' cells. In the presence of exogenous oleate the inhibitory effect of OA feeding on VLDL apoB and TAG secretion persisted in the derived hepatocytes for up to 48 h following isolation. However, when oleate was absent no inhibitory effect on the secretion of TAG and apoB was observed between 24 and 48 h. Under these conditions the rate of intracellular TAG turnover returned to normal. The initial inhibitory effect of FO feeding on VLDL TAG and apoB secretion did not persist in the derived hepatocytes between 24 h and 48 h of culture in the presence of exogenous oleate. Although intracellular TAG lipolysis and VLDL TAG and apoB secretion rates appear to be positively correlated, a causal relationship has not been conclusively established.
Conformational state of a 25-mer peptide from the cyclophilin-binding loop of the HIV type 1 capsid protein
Authors: REIMER Ulf , DREWELLO Mario , JAKOB Mario , FISCHER Gunter , SCHUTKOWSKI Mike
Published: August 2015

Recently a 25-residue part of Gag polyprotein from HIV type 1 (HIV-1) was reported to bind to the cytosolic 18 kDa cyclophilin (Cyp18) with an IC50 value of 180 μM. This peptide corresponds to the Cyp18-binding domain of HIV-1 Gag. A replacement of Gly with Ala in the cyclophilin-binding loop of HIV-1 Gag polyprotein results in the prevention of the packaging of Cyp18 into virions. We found only two conformers of this peptide among 16 possible expected conformers, owing to cis/trans isomerization of four peptidyl-prolyl bonds. Although this finding implicates the existence of a stabilizing structure, we were not able to detect secondary structure formation by 1H-NMR and CD spectroscopy. We characterized the peptide as a substrate for Cyp18 by two-dimensional exchange 1H-NMR spectroscopy. Surprisingly, we found similar binding characteristics for a peptide corresponding to 25-mer peptide containing the above-mentioned Gly to Ala substitution.
Different kynurenine pathway enzymes limit quinolinic acid formation by various human cell types
Authors: HEYES Melvyn P. , CHEN Cai Y. , MAJOR Eugene O. , SAITO Kuniaki
Published: August 2015

Substantial increases in the tryptophan–kynurenine pathway metabolites, L-kynurenine and the neurotoxin quinolinic acid, occur in human brain, blood and systemic tissues during immune activation. Studies in vitrohave shown that not all human cells are capable of synthesizing quinolinate. To investigate further the mechanisms that limit L-kynurenine and quinolinate production, the activities of kynurenine pathway enzymes and the ability of different human cells to convert pathway intermediates into quinolinate were compared. Stimulation with interferon γ substantially increased indoleamine 2,3-dioxygenase activity and L-kynurenine production in primary peripheral blood macrophages and fetal brains (astrocytes and neurons), as well as cell lines derived from macrophage/monocytes (THP-1), U373MG astrocytoma, SKHEP1 liver and lung (MRC-9). High activities of kynurenine 3-hydroxylase, kynureninase or 3-hydroxyanthranilate 3,4-dioxygenase were found in interferon-γ-stimulated macrophages, THP-1 cells and SKHEP1 cells, and these cells made large amounts of quinolinate when supplied with L-tryptophan, L-kynurenine, 3-hydroxykynurenine or 3-hydroxyanthranilate. Quinolinate production by human fetal brain cultures and U373MG cells was restricted by the low activities of kynurenine 3-hydroxylase, kynureninase and 3-hydroxyanthranilate 3,4-dioxygenase, and only small amounts of quinolinate were synthesized when cultures were supplied with L-tryptophan or 3-hydroxyanthranilate. In MRC-9 cells, quinolinate was produced only from 3-hydroxykynurenine and 3-hydroxyanthranilate, consistent with their low kynurenine 3-hydroxylase activity. The results are consistent with the notion that indoleamine 2,3-dioxygenase is an important regulatory enzyme in the production of L-kynurenine and quinolinate. Kynurenine 3-hydroxylase and, in some cells, kynureninase and 3-hydroxyanthranilate 3,4-dioxygenase are important determinants of whether a cell can make quinolinate.
‘GPEET’ procyclin is the major surface protein of procyclic culture forms of Trypanosoma brucei brucei strain 427
Authors: BÜTIKOFER Peter , RUEPP Stefan , BOSCHUNG Monika , RODITI Isabel
Published: August 2015

The surface of Trypanosoma brucei brucei insect forms is covered by an invariant protein coat consisting of procyclins. There are six or seven procyclin genes that encode unusual proteins with extensive tandem repeat units of glutamic acid (E) and proline (P) (referred to as EP repeats), and two genes that encode proteins with internal pentapeptide (GPEET) repeats. Although the EP forms of procyclins have been isolated and characterized by several laboratories, evidence for GPEET procyclin has largely been confined to the expression of its mRNA. To characterize GPEET procyclin further, we isolated the protein from T. b. brucei strain 427. We found that label from [3H]myristic acid and [3H]ethanolamine was incorporated into GPEET procyclin and we demonstrated the protein's covalent modification with a glycosylphosphatidylinositol anchor. The major form of GPEET procyclin showed an apparent molecular mass of 22–32 kDa, was susceptible to proteolytic treatment and was found to be phosphorylated. Surprisingly, our results show that GPEET procyclin represents the major form of procyclin in T. b. brucei 427 culture forms and that the ratio of EP to GPEET procyclin can vary considerably between different cell lines.
Biosynthesis of the labdane diterpene marrubiin in Marrubium vulgare via a non-mevalonate pathway
Authors: KNÖSS Werner , REUTER Bernd , ZAPP Josef
Published: August 2015

The biosynthesis of the furanic labdane diterpene marrubiin has been studied in plantlets and shoot cultures of Marrubium vulgare(Lamiaceae). The use of [2-14C]acetate, [2-14C]pyruvate, [2-14C]mevalonic acid and [U-14C]glucose incorporation experiments showed that the labelling of sterols in etiolated shoot cultures of M. vulgare was in accordance with their biosynthesis via the acetate–mevalonate pathway. In contrast, the incorporation rates of these precursors into the diterpene marrubiin could not be explained by biosynthesis of this compound via the acetate–mevalonate pathway. Cultivation of etiolated shoot cultures of M. vulgare on medium containing [1-13C]glucose and subsequent 13C-NMR spectroscopy of marrubiin led to the conclusion that the biosynthesis of marrubiin follows a non-mevalonate pathway. All isoprenic units of 13C-labelled marrubiin were enriched in those carbons that correspond to positions 1 and 5 of a putative precursor isopentenyl diphosphate. This labelling pattern from [1-13C]glucose is consistent with an alternative pathway via trioses, which has already been shown to occur in Eubacteria and Gymnospermae. The labdane skeleton is a precursor of many other skeletal types of diterpenes. Therefore it becomes obvious that in connection with the few known examples of a non-mevalonate pathway to isoprenoids the formation of some isoprenoids in plants via a non-mevalonate pathway might be quite common.
Inhibition of intracellular proteolytic processing of soluble proproteins by an engineered α2-macroglobulin containing a furin recognition sequence in the bait region
Authors: ROMPAEY Luc VAN , AYOUBI Torik , VAN DE VEN Wim , MARYNEN Peter
Published: August 2015

The bait region of the general protease inhibitor α2-macroglobulin (α2M) was mutated by introducing a recognition sequence of furin. This did not interfere with folding, S-ester formation or tetramerization of the mutant recombinant α2M (rα2M). Mutant rα2M inhibited furin in vitro, by a similar mechanism to that used by plasma α2M to inhibit high-molecular-mass proteases. The mutant α2M was intracellularly active in COS-1 cells in inhibiting the endogenous processing of the soluble substrates for furin (von Willebrand factor, transforming growth factor β1 and a soluble form of the envelope glycoprotein gp160 from HIV-1) but not the membrane-bound form of gp160. The intracellular activity of mutant α2M strongly indicated that α2M attains its native conformation, and thus that the unusual internal S-ester is formed, before α2M passes through the cleavage compartment(s). Our results show for the first time that modulation of the bait region of α2M allows the creation of an inhibitor against membrane-bound proteases. It can be expected that the use of α2M-bait mutants will become important as a technique for the study of various proteolytic processes and for the identification of the proteases involved.
Chicken ovalbumin upstream-promoter transcription factor (COUP-TF) could act as a transcriptional activator or repressor of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene
Authors: RODRÍGUEZ Joan C. , ORTIZ José A. , HEGARDT Fausto G. , HARO Diego
Published: August 2015

The chicken ovalbumin upstream-promoter transcription factor (COUP-TF) has a dual effect on the regulation of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene. COUP-TF could act as a transcriptional activator or repressor of this gene through different DNA sequences. COUP-TF induces expression of a reporter gene linked to the mitochondrial HMG-CoA synthase gene promoter in human hepatoma HepG2 cells, but represses it in a Leydig tumour cell line (R2C); in both these cell lines the expression of the mitochondrial HMG-CoA synthase gene mimics that of liver and testis. The activation is promoted by a fragment of the gene from coordinates -62 to +28, which contains a GC box and a TATA box, and where no COUP-TF binding site was observed by in vitro DNA binding studies. On the other hand, the COUP-TF inhibitory effect is mainly due to repression of peroxisome-proliferator-activated receptor-dependent activation of the gene, interacting with the region from -104 to -92. To our knowledge this work represents the second example of a target gene for COUP-TF I that could be either activated or repressed by the action of this receptor through different DNA sequences of the same gene.
Nitrogenase of Klebsiella pneumoniae: kinetics of formation of the transition-state complex and evidence for an altered conformation of MoFe protein lacking a FeMoco centre
Authors: YOUSAFZAI Faridoon K. , EADY Robert R.
Published: August 2015

We have investigated the kinetics of inactivation of Mo-nitrogenase isolated from Klebsiella pneumoniae when it forms an inhibited putative transition-state complex on incubation with ADP and AlF4-. In the presence of excess Kp2 (Fe protein of the Mo-nitrogenase of K. pneumoniae), the kinetics were found to depend on the Mo content of Kp1 (the MoFe protein of Mo-nitrogenase of K. pneumoniae). The residual nitrogenase activity versus time of incubation using Kp1 preparations containing integral, i.e. one or two Mo atoms per molecule of Kp1, were essentially monophasic, but significantly different rates of inactivation were observed. In contrast, the progress curves for preparations of Kp1 with non-integral Mo content were biphasic, suggesting the presence of two discrete catalytically active species of Kp1. The best fit to the observed data was obtained with a two-exponential expression, the amplitude of which was consistent with the Mo content, provided that the fast phase of the reaction was assigned to a Kp1 species containing one, and the slow phase to a species containing two Mo atoms per α2β2 tetramer. This analysis provides the first evidence for the existence of a catalytically active Kp1 species containing a single Mo atom. These data also indicate that MoFe protein which does not have all FeMoco binding sites occupied has an altered conformation compared with a fully loaded protein, and that the Fe protein reacts with these conformations at different rates to form the stable, but inhibited transition-state complex.
Allosteric control of Zymomonas mobilis glucose-6-phosphate dehydrogenase by phosphoenolpyruvate
Authors: SCOPES Roberts K.
Published: August 2015

The second enzyme of the Entner–Doudoroff glycolytic pathway in Zymomonas mobilis, glucose-6-phosphate dehydrogenase, has been found to be inhibited by phosphoenolpyruvate (PEP). In the presence of PEP levels in the micromolar range, the response of the enzyme to glucose 6-phosphate concentration becomes sigmoidal, with a Hill coefficient up to 2. At low ionic strength in the absence of PEP, the response to glucose 6-phosphate concentration is Michaelis–Menten, but at physiological ionic strength and pH, a Hill coefficient of 1.3 to 1.4 was found even in the absence of PEP. Km values for NAD+ and NADP+ are also ionic-strength-dependent, increasing rapidly as salt concentration increases. Some sigmoidicity was also observed for NAD+ in the presence of PEP at low glucose 6-phosphate concentrations. The results can be interpreted in a Monod–Wyman–Changeux model, in which glucose 6-phosphate binds principally to the R-state, PEP to the T-state, and NAD+ to both states. These observations are clearly physiologically significant, and provide an explanation for the control of the balance between glycolytic throughput and ATP consumption in Z. mobilis.
Novel activity of endothelin-converting enzyme: hydrolysis of bradykinin
Authors: HOANG Mien V. , TURNER Anthony J.
Published: August 2015

Endothelin-converting enzyme (ECE) is the key enzyme in the production of the potent vasoconstrictor endothelin from its inactive precursor big endothelin. To date, no other physiological peptide substrate has been identified for ECE. Here, by using Chinese hamster ovary (CHO) cells transfected with rat ECE-1 cDNA, we have established that ECE can hydrolyse the vasodilator bradykinin. The hydrolysis of bradykinin by ECE is exclusively at the Pro7–Phe8 bond, producing bradykinin-(1–7) and bradykinin-(8–9). Hydrolysis is completely inhibited by 100 μM phosphoramidon and 200 μM EDTA, but only slightly by the specific neprilysin inhibitor thiorphan (100 μM). The ability of ECE to act as a peptidyl dipeptidase rather than an endopeptidase in hydrolysing bradykinin suggests a much broader specificity for the enzyme than previously recognized, which may lead to the design of new and specific inhibitors of ECE and to the identification of other potential physiological substrates.
Δ-1-Piperideine-6-carboxylate dehydrogenase, a new enzyme that forms α-aminoadipate in Streptomyces clavuligerus and other cephamycin C-producing actinomycetes
Authors: de la FUENTE Juan L. , RUMBERO Angel , MARTÍN Juan F. , LIRAS Paloma
Published: August 2015

Δ-1-Piperideine-6-carboxylate (P6C) dehydrogenase activity, which catalyses the conversion of P6C into α-aminoadipic acid, has been studied in the cephamycin C producer Streptomyces clavuligerus by both spectrophotometric and radiometric assays. The enzyme has been purified 124-fold to electrophoretic homogeneity with a 26% yield. The native protein is a monomer of 56.2 kDa that efficiently uses P6C (apparent Km 14 μM) and NAD+ (apparent Km 115 μM), but not NADP+ or other electron acceptors, as substrates. The enzyme activity was inhibited (by 66%) by its end product NADH at 0.1 mM concentration. It did not show activity towards pyrroline-5-carboxylate and was separated by Blue-Sepharose chromatography from pyrroline-5-carboxylate dehydrogenase, an enzyme involved in the catabolism of proline. P6C dehydrogenase reached maximal activity later than other early enzymes of the cephamycin pathway. The P6C dehydrogenase activity was decreased in ammonium (40 mM)-supplemented cultures, as was that of lysine 6-aminotransferase. P6C dehydrogenase activity was also found in other cephamycin C producers (Streptomyces cattleya and Nocardia lactamdurans) but not in actinomycetes that do not produce β-lactams, suggesting that it is an enzyme specific for cephamycin biosynthesis, involved in the second stage of the two-step conversion of lysine to α-aminoadipic acid.
Expression, purification and characterization of Arabidopsis thaliana acetohydroxyacid synthase
Authors: CHANG Alan K. , DUGGLEBY Ronald G.
Published: August 2015

Acetohydroxyacid synthase (EC 4.1.3.18) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine. The AHAS gene from Arabidopsis thaliana with part of the chloroplast transit sequence removed was cloned into the bacterial expression vector pT7-7 and expressed in the Escherichia coli strain BL21(DE3). The expressed enzyme was purified by an extensive procedure involving (NH4)2SO4 fractionation followed by hydrophobic and anion-exchange chromatography. The purified enzyme appears as a single band on SDS/PAGE with a molecular mass of about 61 kDa. On gel filtration the enzyme is a dimer, migrating as a single peak with molecular masses of 109 and 113 kDa in the absence and presence of FAD respectively. Ion spray MS analysis yielded a mass of 63864 Da. The enzyme has optimum activity in the pH range 6.5–8.5 and exhibits absolute dependence on the three cofactors FAD, Mg2+ and thiamine diphosphate for activity. It displays negatively co-operative kinetics with respect to pyruvate concentration. A model was derived to explain the non-hyperbolic substrate-saturation curve, involving interaction between the active sites of the dimer. The Km for the first active site was found to be 8.01±0.66 mM; the Km for the second active site could not be accurately determined but was estimated to be approx. 100 mM. The enzyme is insensitive to valine, leucine and isoleucine but is strongly inhibited by the sulphonylurea herbicide, chlorsulphuron, and the imidazolinone herbicide, imazapyr. Inhibition by both herbicides exhibits slow-binding kinetics, whereas chlorsulphuron also shows tight-binding inhibition.
Incorporation of copper into lysyl oxidase
Authors: KOSONEN Taru , URIU-HARE Janet Y. , CLEGG Michael S. , KEEN Carl L. , RUCKER Robert B.
Published: August 2015

Lysyl oxidase is a copper-dependent enzyme involved in extracellular processing of collagens and elastin. Although it is known that copper is essential for the functional activity of the enzyme, there is little information on the incorporation of copper. In the present study we examined the insertion of copper into lysyl oxidase using 67Cu in cell-free transcription/translation assays and in normal skin fibroblast culture systems. When a full-length lysyl oxidase cDNA was used as a template for transcription/translation reactions in vitro, unprocessed prolysyl oxidase appeared to bind copper. To examine further the post-translational incorporation of copper into lysyl oxidase, confluent skin fibroblasts were incubated with inhibitors of protein synthesis (cycloheximide, 10 μg/ml), glycosylation (tunicamycin, 10 μg/ml), protein secretion (brefeldin A, 10 μg/ml) and prolysyl oxidase processing (procollagen C-peptidase inhibitor, 2.5 μg/ml) together with 300 μCi of carrier-free 67Cu. It was observed that protein synthesis was a prerequisite for copper incorporation, but inhibition of glycosylation by tunicamycin did not affect the secretion of 67Cu as lysyl oxidase. Brefeldin A inhibited the secretion of 67Cu-labelled lysyl oxidase by 46%, but the intracellular incorporation of copper into lysyl oxidase was not affected. In addition, the inhibition of the extracellular proteolytic processing of prolysyl oxidase to lysyl oxidase had minimal effects on the secretion of protein-bound 67Cu. Our results indicate that, similar to caeruloplasmin processing [Sato and Gitlin (1991) J. Biol. Chem. 266, 5128–5134], copper is inserted into prolysyl oxidase independently of glycosylation.
Multiple muscle-specific regulatory elements are associated with a DNase I hypersensitive site of the cardiac β-myosin heavy-chain gene
Authors: HUANG Weei-Yuarn , CHEN Jin-Jer , SHIH N.-L. , LIEW Choong-Chin
Published: August 2015

Using nuclei isolated from neonatal cardiomyocytes, we have mapped the DNase I hypersensitive sites (DHSs) residing within the 5ʹ-upstream regions of the hamster cardiac myosin heavy-chain (MyHC) gene. Two cardiac-specific DHSs within the 5 kb upstream region of the cardiac MyHC gene were identified. One of the DHSs was mapped to the -2.3 kb (β-2.3 kb) region and the other to the proximal promoter region. We further localized the β-2.3 kb site to a range of 250 bp. Multiple, conserved, muscle regulatory motifs were found within the β-2.3 kb site, consisting of three E-boxes, one AP-2 site, one CArG motif, one CT/ACCC box and one myocyte-specific enhancer factor-2 site. This cluster of regulatory elements is strikingly similar to a cluster found in the enhancer of the mouse muscle creatine kinase gene (-1256 to -1050). The specific interaction of the motifs within the β-2.3 kb site and the cardiac nuclear proteins was demonstrated using gel mobility-shift assays and footprinting analysis. In addition, transfection analysis revealed a significant increase in chloramphenicol acetyltransferase activity when the β-2.3 kb site was linked to a heterologous promoter. These results suggest that previously undefined regulatory elements of the β-MyHC gene may be associated with the β-2.3 kb site.
Evidence for redox regulation of the transcription factor NtcA, acting both as an activator and a repressor, in the cyanobacterium Anabaena PCC 7120
Authors: JIANG Fanyi , MANNERVIK Bengt , BERGMAN Birgitta
Published: August 2015

NtcA has been identified as a nitrogen-responsive regulatory protein required for nitrogen assimilation and heterocyst differentiation in cyanobacteria. It is proposed that NtcA functions through the formation of DNA-protein complexes with its specific target sequence within the promoter regions of the regulated genes. In vitro, NtcA of Anabaena PCC 7120 binds to upstream regions of the genes whose products are involved in nitrogen assimilation, but also to the upstream region of rbcLS (carbon-fixation gene), xisA (encoding a site-specific recombinase expressed during heterocyst differentiation) and ntcA (encoding NtcA itself). However, the mechanism by which NtcA serves as a critical regulator for such diverse processes is not understood. With the use of electrophoretic mobility shift assays, NtcA from Anabaena PCC 7120 was here shown to interact with the promoter sequence of the gor gene, encoding glutathione reductase, thereby providing a novel example of NtcA's acting as a repressor, previously found only for the rbcLS gene. Furthermore we demonstrate that the binding of DNA by NtcA is regulated in vitro by a redox-dependent mechanism involving cysteine residues of the NtcA protein. These findings suggest that NtcA is a transcriptional regulator that responds not only to the nitrogen status but also to the cellular redox status, a function that might be particularly significant during heterocyst differentiation.
Galactosamine in walls of slow-growing mycobacteria
Authors: DRAPER Philip , KHOO Kay-Hooi , CHATTERJEE Delphi , DELL Anne , MORRIS R. Howard
Published: August 2015

Galactosamine was found consistently as a minor component of the envelope of five species of slow-growing mycobacteria, including all the major human pathogens, but not three rapid-growing species. The amino sugar was a component of the arabinogalactan of the cell wall skeleton, and occurred at the level of about one residue per arabinogalactan chain. Its amino group was in the free, un-N-acetylated state. Examination of oligosaccharides released by partial acid hydrolysis of arabinogalactan by fast atom bombardment-MS and gas chromatography-MS identified a series of oligoarabinans, each possessing one GalN unit, linked to position 2 of arabinose. It is proposed that the GalN residues occur as stub branches of 1 → 5-linked arabinose chains in the arabinogalactan. Possible functions of GalN are discussed.
A mutant phosphofructokinase produces a futile cycle during gluconeogenesis in Escherichia coli
Authors: TORRES C. Juan , GUIXÉ Victoria , BABUL Jorge
Published: August 2015

Strains of Escherichia coli bearing different forms of phosphofructokinase were used to assess the occurrence of futile cycling in cell resuspensions supplied with glycerol as gluconeogenic carbon source. A model was used to simulate results of different kinds of experiments for different levels of futile cycle. The main predictions of the model were experimentally confirmed in a strain with a mutant phosphofructokinase-2 (phosphofructokinase-2*) which is not inhibited by MgATP. The intracellular fructose 1,6-bisphosphate concentration reaches significantly higher levels in the mutant-bearing strain than in strains with either phosphofructokinase-1 or -2. Also, this strain showed a higher rate and level of in vivo radioactive labelling of fructose 1,6-bisphosphate, from a trace of [U-14C]glucose supplied during gluconeogenesis, indicating higher kinase activity in these conditions. Cell resuspensions of the mutant-bearing strain produced higher levels of radioactively labelled CO2 when supplied with [U-14C]glycerol as the only carbon source. Simultaneously, fewer glycerol carbons were incorporated into HClO4-insoluble macromolecules. Finally, radioactive CO2 output was measured in resuspensions supplied with glycerol as the major carbon source with traces of either [1-14C]glucose or [6-14C]glucose. It was found that, whereas in the strains with either of the wild-type phosphofructokinase isoenzymes, radioactive CO2 output from [1-14C]glucose was higher than with [6-14C]glucose, the reverse is found for the strain with phosphofructokinase-2*. This result also agrees with the corresponding prediction of the model. Using the radioactivity flux rates predicted by the model, an explanation linking the futile cycle to the differential labelling of CO2 is advanced. Finally, on the basis of these results it is proposed that strains bearing phosphofructokinase-2* sustain higher rates of futile cycling during gluconeogenesis than strains bearing either of the wild-type isoforms of phosphofructokinase. The kinetic equations and parameter values used for the model simulations are given in Supplementary Publication SUP 50183 (8 pages), which has been deposited at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1997) 321, 8.
Suppression of smooth-muscle α-actin expression by platelet-derived growth factor in vascular smooth-muscle cells involves Ras and cytosolic phospholipase A2
Authors: LI Xiaomei , PUTTEN Vicki VAN , ZARINETCHI Fariba , NICKS E. Michael , THALER Seth , HEASLEY E. Lynn , NEMENOFF A. Raphael
Published: August 2015

Platelet-derived growth factor (PDGF), which is a potent mitogen for vascular smooth-muscle cells (VSMC), also inhibits the expression of specific smooth-muscle proteins, including smooth-muscle α-actin (SM-α-actin), in these cells. The goal of this study was to identify signalling pathways mediating these distinct effects. In rat aortic VSMC, PDGF caused a rapid activation of Ras and Raf, leading to the activation of mitogen-activated protein kinases (ERKs). Cells stably transfected with constitutively active Ras (H-Ras) expressed low levels of SM-α-actin protein. Arginine vasopressin, which stimulated SM-α-actin promoter activity in wild-type cells or controls (Neo; transfected with a plasmid lacking an insert), failed to do so in cells transiently expressing H-Ras. The effects of Ras on suppression of SM-α-actin expression were not mediated by the Raf/ERK pathway, since cells stably expressing constitutively active Raf (BxB-Raf) had normal levels of SM-α-actin protein, and stimulation of SM-α-actin promoter activity by vasopressin was unaffected in cells transiently expressing BxB-Raf. Furthermore a specific inhibitor of ERK activation had no effect on SM-α-actin expression. Exposure of wild-type VSMC to PDGF, or stable expression of Ras but not Raf, also resulted in constitutive increases in prostaglandin E2 production and cytosolic phospholipase A2 (cPLA2) activity, which was mediated by an increased expression of cPLA2 protein. Transient expression of cPLA2 in wild-type VSMC inhibited the stimulation of SM-α-actin promoter activity by vasopressin. These results suggest that PDGF-induced inhibition of SM-α-actin expression is mediated through a Ras-dependent/Raf independent pathway involving the induction of cPLA2 and eicosanoid production.
Enhanced bradykinin-stimulated phospholipase C activity in murine embryonic stem cells lacking the G-protein αq-subunit
Authors: RICUPERO A. Dennis , POLGAR Peter , TAYLOR Linda , SOWELL O. Margaret , GAO Yunling , BRADWIN Gary , MORTENSEN M. Richard
Published: August 2015

The gene coding for the G-protein αq subunit was interrupted by homologous recombination in murine embryonic stem cells (αq-null ES cells) as detected by Southern analysis and reverse-transcriptase PCR. The bradykinin (BK) B2 receptor was stably transfected into wild-type (WT) αi-2-null and αq-null ES cells. The B2 receptor bound BK with high affinity and mobilized Ca2+. BK also activated phospholipase C (PLC), as determined by total inositol phosphate (IP) accumulation in a Bordetella pertussis toxin- and genistein-insensitive manner. In WT and αi-2-null ES cells, BK increased IP levels approx. 4-fold above baseline. Most interestingly, in αq-null ES cells, BK increased IP accumulation approx. 9-fold above baseline. Re-expression of αq in αq-null ES cells resulted in normalization of the BK-stimulated IP accumulation (4-fold above baseline). These results suggest that the B2 receptor activates PLC through more than one member of the Gq family. Additionally, the absence of αq alters the kinetics of IP generation, which may reflect intrinsic characteristics of individual members of the Gq family or a decreased susceptibility to heterologous regulation in the αq-null ES cells, thus allowing for a more sustained generation of IP.
Differential regulation of nitric oxide synthase mRNA expression by lipopolysaccharide and pro-inflammatory cytokines in fetal hepatocytes treated with cycloheximide
Authors: CASADO Marta , DÍAZ-GUERRA María J. M , BOSCÁ Lisardo , MARTÍN-SANZ Paloma
Published: August 2015

The effect of cycloheximide (CHX) on the mRNA expression of the cytokine-inducible, calcium-independent nitric oxide synthase (iNOS) was investigated in fetal hepatocytes stimulated with lipopolysaccharide (LPS) or pro-inflammatory cytokines. In the presence of CHX the LPS-dependent iNOS mRNA levels were reduced, whereas the response to pro-inflammatory cytokines was enhanced. Because iNOS transcription is highly dependent on the activation of nuclear factor κB (NF-κB), this factor was evaluated by electrophoretic mobility shift assays, and a close correlation between NF-κB activity and iNOS mRNA levels was observed. CHX itself potentiated the degradation of the IκBα and IκBβ inhibitory subunits (IκB is inhibitory κB) of the NF-κB complex, and therefore the loss of LPS-dependent iNOS mRNA expression cannot be attributed to a blockage in the activation of NF-κB. These results suggest the existence of a CHX-sensitive pathway in the expression of iNOS mediated by LPS, a mechanism that is not involved in the response to pro-inflammatory cytokines.
Immortalized, cloned mouse chondrocytic cells (MC615) produce three different matrix proteoglycans with core-protein-specific chondroitin/dermatan sulphate structures1
Authors: KOKENYESI Robert , SILBERT E. Jeremiah
Published: August 2015

Cloned immortalized MC615 mouse chondrocytic cells were used to examine their capability to produce multiple types of matrix proteoglycans. Immunofluorescence staining indicated a uniform expression of aggrecan, biglycan and decorin by all cells. After culture with [35S]sulphate, proteo[35S]glycans secreted by the cells were found to elute in two peaks from a Sepharose CL-4B column. The first peak, at the void volume of the column, contained a large proteoglycan with an estimated average hydrodynamic mass of 103 kDa. The glycosaminoglycan chains of this proteoglycan had an average hydrodynamic size of 17 kDa, estimated by Sepharose CL-6B chromatography, indicating the presence of 30-70 glycosaminoglycan chains per core protein, which was consistent with the characteristics of aggrecan. Biglycan and decorin were immunoisolated from the second Sepharose CL-4B peak, and had average glycosaminoglycan hydrodynamic sizes of approx. 25 kDa and 32 kDa respectively. Glycosaminoglycan chains of the aggrecan, biglycan and decorin were treated with chondroitin ABC lyase, chondroitin AC lyase and chondroitin B lyase to determine the positions of sulphation and the degree of uronic acid epimerization. The aggrecan glycosaminoglycan chains were found to contain a 4-sulphate/6-sulphate ratio of 7:3, with no epimerization of glucuronic acid to iduronic acid. The biglycan glycosaminoglycan chains were found to contain a similar ratio of 4-sulphate/6-sulphate, but with approx. 40-45% of the glucuronic acid epimerized to iduronic acid. The decorin glycosaminoglycan chains were found to contain 4-sulphate but no detectable 6-sulphate, and approx. 30-35% epimerization of the glucuronic acid to iduronic acid. The results, using these cloned cells, indicated that a single MC615 cell is able to make all three proteoglycans with distinctive differences between the glycosaminoglycans of aggrecan, biglycan and decorin. These data indicate that a mechanism must exist for a single MC615 cell to regulate the sizes and fine structures of glycosaminoglycans on simultaneously produced, different proteoglycans in a core-protein-specific manner.
A tyrosine residue essential for catalytic activity in aminopeptidase A
Authors: VAZEUX Gilles , ITURRIOZ Xavier , CORVOL Pierre , LLORENS-CORTÈS Catherine
Published: August 2015

Aminopeptidase A (EC 3.4.11.7; APA) is a 130 kDa membrane-bound zinc enzyme that contains the consensus sequence HEXXH (residues 385-389) conserved among the zinc metalloprotease family. In this motif, both histidine residues and the glutamic residue were shown to be involved respectively in zinc co-ordination and catalytic activity. Treatment of APA with N-acetylimidazole results in a loss of enzymic activity; this is prevented by the competitive aminopeptidase inhibitor amastatin, suggesting the presence of an important tyrosine, lysine or cysteine residue at the active site of APA. A tyrosine residue was previously proposed to be involved in the enzymic activity of aminopeptidase N. Furthermore sequence alignment of mouse APA with other monozinc aminopeptidases indicates the presence of a conserved tyrosine (Tyr-471 in APA). The functional role of Tyr-471 in APA was investigated by replacing this residue with a phenylalanine (Phe-471) or a histidine (His-471) residue by site-directed mutagenesis. Kinetic studies showed that the Km values of both mutants were similar to that of the wild-type enzyme, whereas kcat values were decreased by three orders of magnitude and corresponded to a variation in free energy of the rate-limiting step by 4.0 and 4.2 kcal/mol (0.96 and 1.00 kJ/mol) for the Phe-471 and His-471 mutants respectively. The mutation did not modify the inhibitory potency of a thiol-containing inhibitor that strongly chelates the active-site zinc ion, whereas that of a putative analogue of the transition state presumed to mimic the reaction intermediate was reduced. Taken together, these results strongly suggest that the Tyr-471 hydroxy group participates in catalysis by stabilizing the transition state complex through interaction with the oxyanion.
Major co-localization of the extracellular-matrix degradative enzymes heparanase and gelatinase in tertiary granules of human neutrophils
Authors: MOLLINEDO Faustino , NAKAJIMA Motowo , LLORENS Ana , BARBOSA Enrique , CALLEJO Sagrario , GAJATE Consuelo , FABRA Angels
Published: August 2015

The expression of cell-surface adhesion proteins and the release of extracellular-matrix degradative enzymes constitute crucial processes for the attachment of neutrophils to the endothelium and for the subsequent extravasation of these cells through the endothelial layer. We have analysed in resting human neutrophils the subcellular localization of heparanase, a heparan-sulphate-degrading endoglycosidase that can degrade basement-membrane components, thereby facilitating neutrophil passage into the tissue during an inflammatory reaction. By subcellular fractionation of postnuclear supernatants from resting human neutrophils on continuous sucrose gradients, we have found that heparanase activity was mainly located in gelatinase-containing tertiary granules. Using a specific antibody, the 96-kDa heparanase protein was further located in the gelatinase-rich subcellular fractions. Following immunoblotting and immunoprecipitation analysis in the distinct subcellular fractions, we also found co-localization of heparanase and Mo1 (CD11b/CD18), a leucocyte integrin involved in the attachment of neutrophils to the endothelium, in the fractions enriched in gelatinase-containing tertiary granules. Treatment of human neutrophils with tumour necrosis factor or granulocyte/macrophage colony-stimulating factor induced an increase in the CD11b/CD18 cell-surface expression, as well as the release of both gelatinase (matrix metalloproteinase-9) and heparanase, but not of other granule markers, indicating a major co-localization of gelatinase, heparanase and CD11b/CD18 in the same organelle. Furthermore, confocal laser scanning microscopy using specific antibodies against gelatinase and heparanase revealed a major co-localization of both enzymes in intracellular cytoplasmic granules. The major localization of heparanase and CD11b/CD18 in the gelatinase-containing tertiary granule supports the notion that mobilization of this organelle can regulate extravasation of human neutrophils.
Mutagenesis of Glu403 to Cys in rabbit neutral endopeptidase-24.11 (neprilysin) creates a disulphide-linked homodimer: analogy with endothelin-converting enzyme
Authors: HOANG V. Mien , SANSOM E. Clare , TURNER J. Anthony
Published: August 2015

Neutral endopeptidase-24.11 (NEP; neprilysin; EC 3.4.24.11) and endothelin-converting enzyme (ECE) are related zinc metallopeptidases involved in the processing of biologically active peptides. Only ECE, however, exists as a disulphide-linked homodimer. The covalent linkage in rat ECE is between Cys412 in each subunit, which is equivalent to Glu403 in rabbit NEP. Here we report that directed mutagenesis of Glu403 to cysteine in rabbit NEP creates a disulphide-linked homodimer, as revealed by transient transfection in COS-1 cells and SDS/PAGE of a membrane fraction. Under reducing conditions, both the mutant (E403C) and the wild-type NEP migrate as a polypeptide of 92 kDa. However, under non-reducing conditions, the Mr of the wild type remains unchanged, whereas that of the mutant is doubled. Co-transfection of wild-type ECE and E403C NEP cDNA did not result in the production of a NEP-ECE heterodimer. Comparison of the kinetic constants for wild-type and E403C mutant NEP with either [D-Ala2,Leu5]enkephalin or 3-carboxypropanoyl-alanyl-alanyl-leucine-4-nitroanilide (Suc-Ala-Ala-Leu-NH-Np) as substrate show a decrease of approx. 50% in Vmax/Km for the mutant form. The IC50 value for inhibition of the mutant by phosphoramidon or thiorphan is increased 3-fold and 5-fold respectively. Although NEP and ECE exhibit only about 40% identity and differ substantially in substrate specificity and some other characteristics, these data indicate that they have considerable similarity in three-dimensional structure, allowing dimer formation in the mutant NEP with the disulphide link probably occurring in a hydrophilic surface loop.
Regulation of human γ-glutamylcysteine synthetase: co-ordinate induction of the catalytic and regulatory subunits in HepG2 cells
Authors: GALLOWAY C. Diane , BLAKE G. David , SHEPHERD A. Graeme , McLELLAN I. Lesley
Published: August 2015

We have shown that in HepG2 cells treatment with 75 μM t-butylhydroquinone (tBHQ) results in a 2.5-fold increase in glutathione concentration, as part of an adaptive response to chemical stress. In these cells the elevation in intracellular glutathione level was found to be accompanied by an increase of between 2-fold and 3-fold in the level of the 73 kDa catalytic subunit of γ-glutamylcysteine synthetase (heavy subunit, GCSh) and the 31 kDa regulatory subunit (light subunit, GCSl). Levels of GCSh and GCSl mRNA were increased by up to 5-fold in HepG2 cells in response to tBHQ. To study the transcriptional regulation of GCSl, we subcloned 6.7 kb of the upstream region of the human GCSl gene (GLCLR) from a genomic clone isolated from a P1 lymphoblastoid cell line genomic library. HepG2 cells were transfected with GLCLR promoter reporter constructs and treated with tBHQ. This resulted in an induction of between 1.5-fold and 3.5-fold in reporter activity, indicating that transcriptional regulation of GLCLR is likely to contribute to the induction of GCSl by tBHQ in HepG2 cells. Sequence analysis of the promoter region demonstrated the presence of putative enhancer elements including AP-1 sites and an antioxidant-responsive element, which might be involved in the observed induction of the GLCLR promoter.
Inhibition of nitric oxide synthase expression by PPM-18, a novel anti-inflammatory agent, in vitro and in vivo
Authors: YU M. Sheu , LIN Jeng F. WU Tzong L. , KUO C. Sheng
Published: August 2015

We studied the effect of PPM-18, a chemically synthesized naphthoquinone derivative and also an anti-inflammatory agent, on the lipopolysaccharide (LPS)-activated inducible NO synthase (iNOS) expression in rat alveolar macrophages. Pretreatment of macrophages with PPM-18 (0.1-10 μM) significantly inhibited nitrite production, iNOS protein expression and iNOS mRNA accumulation. PPM-18 did not directly affect the enzymic activities of iNOS and other constitutive NOS forms. The LPS-induced increase in nuclear transcription factor κB (NF-κB) p65 and p50 in nucleus was suppressed by PPM-18 (10 μM). Moreover electrophoretic mobility-shift assays demonstrated that PPM-18 inhibited DNA binding to NF-κB induced by LPS in whole cells but not when added in the nuclear extract, suggesting that PPM-18 did not interfere directly with the binding of NF-κB to DNA and that some events had to be processed before NF-κB could bind DNA. Examination of NF-κB showed that PPM-18 stabilized the NF-κB inhibitor, IκBα, by preventing its degradation from NF-κB. Therefore the stabilization of IκBα might have contributed to the inhibition of NF-κB activation. These results also indicate strongly that NF-κB is involved in the production of NO on stimulation by LPS. PPM-18 significantly decreased the production of tumour necrosis factor α in response to LPS. PPM-18 protects mice against LPS-induced lethal toxicity. These results also indicate that PPM-18 is a potent inhibitor of iNOS expression by blocking the binding of NF-κB to promoter and exerts a beneficial effect in the mouse model of sepsis.