bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of solution composition on microtubule dynamic instability
Authors: Schilstra M J , Bayley P M , Martin S R
Published: August 2015

The exchange of tubulin dimer into steady-state microtubules was studied over a range of solution conditions, in order to assess the effects of various common buffer components on the dynamic instability of microtubules. In comparison with standard buffer conditions (100 mM-Pipes buffer, pH 6.5, containing 0.1 mM-EGTA, 1.8 mM-MgC12 and 1 M-glycerol), the rate and extent of exchange, and thus of dynamic instability, are suppressed by increasing the concentration of glycerol above 2 M. Exchange is enhanced by the addition of further Mg2+ (up to 17 mM) or by the addition of Ca2+ (up to 0.4 mM). Phosphate ion (150 mM) has relatively little effect on the dynamic behaviour of microtubules, as judged by the exchange method. The findings are interpreted within the framework of the Lateral Cap model for microtubule dynamic instability, in terms of the effects of these changes on the intrinsic rate constants of the system. By contrast, the extent of tubulin exchange depends selectively on the value of the dissociation rate constant for tubulin-GDP. A decrease in the extent of exchange, and hence in dynamic activity, is associated with a decreased value for this rate constant, and vice versa. The results also show good agreement of predictions of the model in treating the observed variations in the dynamic properties of individual microtubules, induced by different solution conditions.
Polymerase and hydrolase activities of Bacillus subtilis levansucrase can be separately modulated by site-directed mutagenesis
Authors: Chambert R , Petit-Glatron M F
Published: August 2015

The levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) structural gene from a Bacillus subtilis mutant strain displaying a low polymerase activity was sequenced. Only one missense mutation changing Arg331 to His was responsible for this modified catalytic property. From this allele we created new mutations by directed mutagenesis, which modified the charge and polarity of site 331. Examination of the kinetics of the purified levansucrase variants revealed that transfructosylation activities are affected differently by the substitution chosen. His331→Arg completely restored the properties of the wild-type enzyme. The most striking feature of the other variants, namely Lys331, Ser331 and Leu331, was that they lost the ability of the wild-type enzyme to synthesize levan from sucrose alone. They were only capable of catalysing the first step of levan chain elongation, which is the formation of the trisaccharide ketose. The variant His331→Lys presented a higher kcat. for sucrose hydrolysis than the wild-type, and only this hydrolase activity was preserved in a solvent/water mixture in which the wild-type acted as a true polymerase. The two other substitutions reduced the efficiency of transfructosylation activities of the enzyme via the decrease of the rate of fructosyl-enzyme intermediate formation. For all variants, the sucrose affinity was slightly affected. This strong modulation of the enzyme specificities from a single amino acid substitution led us to postulate the hypothesis that bacterial levansucrases and plant fructosyltransferases involved in fructan synthesis may possess a common ancestral form.
The kinetics of substrate-induced inactivation
Authors: Waley S G
Published: August 2015

The kinetics of a branched-pathway mechanism for a simple enzymic reaction were studied. In this mechanism there is reversible formation of an inactive form of the second complex along the pathway. This substrate-induced inactivation typically results in the progress curve showing a burst. Three parameters can be obtained from the progress curve: the initial rate, the final rate and the rate constant characterizing the transient. The rate constant for the conversion of the inactive form of the complex into the active form can be obtained either from these parameters or by measuring the regain of enzymic activity. The partition ratio can also be obtained from the three parameters; this is the ratio of the rate of conversion of complex into product to the rate of conversion of complex into inactive form. Simulations give guidance to the conditions required for accurate determinations of the rate constants.
Human liver N-acetylgalactosamine 6-sulphatase. Purification and characterization
Authors: Bielicki J , Hopwood J J
Published: August 2015

Human N-acetylgalactosamine 6-sulphatase (EC 3.1.6.14), which is involved in the lysosomal degradation of the glycosaminoglycans keratan sulphate and chondroitin 6-sulphate, was purified more than 130,000-fold in 2.8% yield from liver by an eight-step column procedure. One major form was identified with a pI of 5.7 and a native molecular mass of 62 kDa by gel filtration. When analysed by SDS/PAGE, dithioerythritol-reduced enzyme contained polypeptides of molecular masses 57 kDa, 39 kDa and 19 kDa, whereas non-reduced enzyme contained a major polypeptide of molecular mass 70 kDa. It is proposed that active enzyme contains either the 57 kDa polypeptide or disulphide-linked 39 kDa and 19 kDa polypeptides. Minor amounts of other enzyme forms separated during the chromatofocusing step and the Blue A-agarose step were not further characterized. Purified N-acetylgalactosamine 6-sulphatase was inactive towards 4-methylumbelliferyl sulphate, but was active, with pH optima of 3.5-4.0, towards 6-sulphated oligosaccharide substrates. Km values of 12.5 and 50 microM and Vmax. values of 1.5 and 0.09 mumol/min per mg were determined with oligosaccharide substrates derived from chondroitin 6-sulphate and keratan sulphate respectively. Sulphate, phosphate and chloride ions were inhibitors of enzyme activity towards both substrates, with 50 microM-Na2SO4 giving 50% inhibition towards the chondroitin 6-sulphate trisaccharide substrate.
Stimulation of synthesis de novo of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase in human promyelocytic leukaemia (HL-60) cells by phorbol ester
Authors: Xun C Q , Tian Z G , Tai H H
Published: August 2015

Human promyelocytic leukaemia (HL-60) cells were employed to study the induction of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), the key enzyme in controlling prostaglandin inactivation. Phorbol 12-myristate 13-acetate (PMA) stimulated 15-PGDH activity in a time- and concentration-dependent manner. Dimethyl sulphoxide (DMSO) also stimulated the enzyme activity, although a much delayed stimulation was observed. Western blot studies indicated that PMA increased significantly a 28 kDa immunoreactive protein characteristic of 15-PGDH. L-[35S]Methionine labelling of the PMA-treated cells showed a similar enhancement over the control cells. These studies indicate that PMA induced synthesis of 15-PGDH. Stimulation of 15-PGDH activity by PMA or DMSO appears to be mediated by protein kinase C activation, since an inactive analogue of PMA failed to induce the effect, and both staurosporine and H-7 blocked the stimulation. Stimulation by PMA was optimal at 10 nM and less effective at higher concentrations. Western blot studies indicated that a similar, if not greater, amount of enzyme protein was induced at high concentrations of PMA, suggesting that enzyme inactivation might be occurring. Possible enzyme inactivation by protein kinase C activation was further examined by incubating DMSO-treated cells with a high concentration of PMA (50 nM). Time-dependent inactivation of 15-PGDH within the first 1 h was observed and this inactivation was partially blocked by staurosporine and H-7. Pulse-chase experiments indicated that 15-PGDH had a rapid turnover rate (t 1/2 = 47 min), and PMA shortened the half-life of the enzyme (t 1/2 = 33 min), suggesting that PMA might have an additional effect on 15-PGDH degradation. The rapid turnover of 15-PGDH indicates that the enzyme activity depends on continued enzyme synthesis, and this could be susceptible to hormone and drug control mechanisms.
Isolation and identification of a novel cellular protein that potently activates Ca2+/phospholipid-dependent protein kinase (protein kinase C)
Authors: Goueli S A
Published: August 2015

A novel heat-stable cellular protein that significantly stimulated Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) was identified. It is ubiquitous and has a molecular mass of over 150 kDa, and was shown to be specific for PKC. It activates PKC in the absence and presence of phospholipids; however, its maximal stimulatory potential was achieved when optimal amounts of phospholipids were also present in the reaction medium. Thus, in comparison with classical cofactors such as phospholipid, where activation of PKC mainly involves interaction with the regulatory domain of PKC, the mechanism of activation of PKC by the activator appears to involve several binding sites.
A vitronectin-receptor-related molecule in human placental brush border membranes
Authors: Vanderpuye O A , Labarrere C A , McIntyre J A
Published: August 2015

The heterodimeric vitronectin receptor (VNR) and platelet glycoprotein IIb/IIIa (GPIIb/IIIa) are two members of the integrin family of cell adhesion receptors that share the same beta subunit (GPIIIa). These proteins are involved in binding to vitronectin, fibrinogen and fibronectin and in cytoskeleton-membrane interactions. The present study shows that the human placental syncytiotrophoblast brush border membrane contains a heterodimer of subunit Mr values of 140,000 and 90,000 (non-reduced) or 125,000 and 100,000 (reduced). This protein was recognized by a monoclonal antibody to GPIIIa, rabbit antisera to the VNR and a human alloantiserum to GPIIIa. Brush border VNR-related protein bound to an immobilized peptide containing the Arg-Gly-Asp sequence and, less avidly, to immobilized fibrinogen. Only a small fraction of brush border VNR was associated with a cytoskeleton fraction. Membrane-bound brush border GPIIIa was distinct from that of platelets in its resistance to digestion by trypsin and Staphylococcus aureus V8 protease, and had a slightly lower mobility on SDS/PAGE. In addition, lectin-binding studies indicate glycosylation differences between microvillar and platelet GPIIIa heterodimers. Thus, although placental syncytiotrophoblast expresses a beta 3 integrin in its apical brush border, differences in protease sensitivity and carbohydrate content suggest that it may lack or mask certain antigenic determinants. This may be beneficial in avoiding harmful maternal alloantibody responses during pregnancy. Immunohistology showed that the VNR was present in syncytiotrophoblast apical but not basal plasma membranes, and was absent from other forms of trophoblast. The brush border VNR could function in localizing Arg-Gly-Asp-sequence-containing plasma proteins to the materno-trophoblastic interface.
T-kininogenase activity of the rat submandibular gland is predominantly due to the kallikrein-like serine protease antigen γ
Authors: Berg T , Wassdal I , Mindroiu T , Sletten K , Scicli G , Carretero O A , Scicli A G
Published: August 2015

T-kininogen, the major kininogen in rat plasma, releases Ile-Ser-bradykinin (T-kinin) when incubated with trypsin, but is not a substrate for tissue kallikrein. Enzymes able to release T-kinins from T-kininogen have been found in the rat submandibular gland, but precise identification of these enzymes and their possible relationship to kallikrein-like enzymes has not been established. We studied T-kininogenase activity in fractionated submandibular gland homogenate. The main T-kininogen catalytic enzyme was purified and characterized, and found to be identical to antigen gamma, a kallikrein-like enzyme which we have previously characterized. Of other identified kallikrein-like enzymes only tonin showed weak T-kininogenase activity, which was about 0.25% of that of antigen gamma. No other T-kininogen catalytic enzymes were observed. Antigen gamma released a kinin which was identified as T-kinin by reverse-phase h.p.l.c. The T-kininogenase activity of antigen gamma had a Km of 29 +/- 4 microM and a kcat/Km of 140 M-1.s-1, and was comparable with its high and low molecular mass-kininogenase activity (7.4 and 10 micrograms of kinin/h per mg respectively). In contrast, tissue kallikrein released 0.2 and 42,200 micrograms of kinin/h per mg respectively. Thus antigen gamma is a weak kininogenase. The isoelectric point of antigen gamma, but not its molecular mass, differed from that of other kallikrein-like enzymes. Isoelectrofocusing in flat-bed gels combined with immunostaining was therefore a convenient method for identification. The kallikrein-like nature of antigen gamma was demonstrated by its immunological similarity to tissue kallikrein and tonin and by 91% and 87% amino acid sequence similarity with tonin and kallikrein respectively (67 amino acids sequenced). Complete identity was also not observed with other sequenced kallikrein genes, mRNAs or proteins.
Characterization of the interaction between p100, a novel G-protein-related protein, and rat liver endosomes
Authors: Traub L M , Shai E , Sagi-Eisenberg R
Published: August 2015

p100 is a recently identified 100 kDa protein which shares a putative receptor-binding sequence with the signal transducing G-proteins Gt and Gi. In liver, p100 immunoreactivity is distributed between the cytosolic and the microsomal fractions [Traub, Evans & Sagi-Eisenberg (1990) Biochem. J. 272, 453-458; Udrisar & Rodbell (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6321-6325]. More specifically, we have localized the membrane-associated form of p100 to an endosomal subfraction of rat liver microsomes. In this study we have investigated the nature of the interaction between p100 and microsomal membranes. p100 was located on the cytoplasmic surface of the microsomal vesicles, and could be released by treatment with 0.5 M-NaCl or 0.5 M-Tris/HCl, pH 7.0. However, p100 was not released by non-ionic detergents, such as Triton X-100. Binding of p100 to the membrane was reversible, as both membrane-released and cytosolic p100 could re-bind stripped (Tris-washed) microsomes. Soluble p100 could not, however, bind to untreated microsomes. Binding to stripped microsomes approached saturation and was inhibited by up to 60% by either heat treatment or mild trypsin treatment of the vesicles. This implies that the interaction between p100 and the microsomal vesicles involves the direct binding of p100 to vesicular proteins. This binding was regulated by both adenine and guanine nucleotides. As p100 contains a region similar to the C-terminal decapeptide of alpha i, (the alpha-subunit of Gi) and has a localization that is restricted to an endosomal subfraction, we propose that cytosolic p100 may bind to cytoplasmically exposed domains of internalized receptors. Thus, like the adaptins, p100 may be involved in the process of sorting and receptor trafficking through the endosomal compartment of the cells.
Synthesis and secretion of a cobalamin-binding protein by HT 29 cell line
Authors: Schohn H , Guéant J L , Girr M , Nexø E , Baricault L , Zweibaum A , Nicolas J P
Published: August 2015

An HT 29 cell line derived from human colonic carcinoma was shown to synthesize and release a cobalamin-binding protein. The cobalamin-binding protein was classified as transcobalamin (TC). By gel filtration on Sephacryl S200 HR, we observed that the secreted protein bound to cobalamin had the same size as plasma transcobalamin. Like transcobalamin, the cobalamin-binding protein bound cobalamin but not cobinamide. Purification of the cobalamin-binding protein was performed by heparin-Sepharose affinity chromatography and by Sephacryl S200 gel filtration. The molecular mass of the purified protein was estimated at 44 kDa by SDS/PAGE. The isoelectric point was determined to be 6.4. The purified cobalamin-binding protein reacted with an antiserum produced against human transcobalamin. A 44 kDa band was also identified by SDS/PAGE of an immunoprecipitated homogenate from HT 29 cells labelled with [35S]methionine and in a Western blot of cell homogenates. The secretion of the cobalamin-binding protein was maximal between 10 and 12 days of cell culture and was inhibited by cycloheximide.
The plasma membrane ferrireductase activity of Saccharomyces cerevisiae is partially controlled by cyclic AMP
Authors: Lesuisse E , Horion B , Labbe P , Hilger F
Published: August 2015

The plasma-membrane-bound ferrireductase activity of ras1 and ras2 mutants of Saccharomyces cerevisiae is not induced in response to iron limitation. This phenotype was suppressed by the bcy1 mutation in ras2 but not in ras1 mutants. The cellular haem content of ras-1-bearing strains decreased dramatically when cells were grown in semi-synthetic medium (low yeast extract content), which could account for their very low ferrireductase activity. The ferrireductase activity of cdc25 and cdc35 mutants dropped when the cells were shifted to a non-permissive temperature. This drop was prevented in the double mutant cdc35 sra5 by adding cyclic AMP to the growth medium. We propose that ferrireductase activity is under the control of a cyclic AMP-dependent protein phosphorylation.
Labelling in vivo and chirality of griseofulvin-derived N-alkylated protoporphyrins
Authors: De Matteis F , Gibbs A H , Martin S R , Milek R L B
Published: August 2015

1. We have compared the response to griseofulvin of rats and mice and, in mice, the effect of griseofulvin itself with that of two of its analogues. The severity of protoporphyria shows a correlation with the accumulation of both types of N-alkylated porphyrins previously described after treatment with this drug, namely N-methylproptoporphyrin and the N-griseofulvin protoporphyrin adduct. 2. Both N-alkylporphyrins are chiral, are labelled from 5-amino[4-14C]laevulinate, and their liver accumulation can be inhibited by pretreatment with a suicide substrate of cytochrome P-450, which also prevents porphyria. 3. These findings suggest that cytochrome P-450 is involved in the mechanism of griseofulvin-induced protoporphyria by generating N-methylprotoporphyrin. The N-griseofulvin protoporphyrin adduct may also originate from cytochrome P-450, but more work is necessary to elucidate whether it acts as the precursor for N-methylprotoporphyrin.
Induction of peroxisomal β-oxidation genes by retinoic acid in cultured rat hepatocytes
Authors: Hertz R , Bar-Tana J
Published: August 2015

Retinoic acid is reported here to induce peroxisomal beta-oxidation activities in cultured rat hepatocytes, with a concomitant increase in respective peroxisomal mRNAs. The concentrations of retinoic acid required for inducing liver peroxisomal acyl-CoA oxidase were similar to those required for inducing liver transglutaminase. A putative 5′-flanking response element for retinoic acid may be found within the enhancer region involved in the induction of peroxisomal genes by xenobiotic amphipathic carboxylates.
Macrophages can convert citrulline into arginine
Authors: Wu G , Brosnan J T
Published: August 2015

Rat peritoneal macrophages were incubated in the presence of 0.05-1.0 mM-[14C]citrulline. The synthesis of [14C]arginine from 0.1 mM-[14C]citrulline was about 300 pmol/h per 10(6) cells in macrophages from saline-injected (control) rats. Both arginine synthesis from citrulline and nitrate production (an indicator of NO generation) were increased about 3-fold in the cells from lipopolysaccharide (LPS)-treated animals. The arginine synthesis was very sensitive to extracellular citrulline concentration in the range found in plasma (0.05-0.1 mM). The rate of arginine synthesis from citrulline was inhibited by about 20% by 0.5 mM-L-glutamine in both control and LPS-treated rat cells, but was inhibited by 0.5 mM-L-arginine only in control cells. Our results demonstrate that citrulline, produced by NO synthetase, can be recycled to arginine in macrophages. The citrulline-arginine cycle may contribute to the regulation of intracellular availability of arginine and thus the prolonged production of NO by macrophages.
Deficiencies in DNA replication and cell-cycle progression in polyamine-depleted HeLa cells
Authors: Koza R A , Herbst E J
Published: August 2015

Synchronized HeLa cells depleted of polyamines by alpha-difluoromethylornithine exhibited substantially decreased DNA synthesis, and proliferation ceased after the release of the cells into S phase. Nuclei from these cells synthesized 70-80% less DNA than did nuclei from control cells. Extraction of isolated nuclei with 0.3 M-KCl decreased DNA synthesis by about 60%, which was recovered almost completely in control cell nuclei by reconstitution with the salt extracts of these nuclei. On the other hand, salt extracts of polyamine-depleted nuclei restored only 50% of DNA synthesis in extracted control nuclei. Salt extracts of control cell nuclei contained twice the DNA polymerase alpha activity of polyamine-depleted nuclear extracts. Extracts of cell lysates of both control and polyamine-depleted HeLa cells exhibited similar DNA polymerase alpha activity, suggesting that uptake of the enzyme or its retention by the nuclei of polyamine-depleted cells was decreased. Polyamine-depleted nuclei also showed altered phosphorylation of a 31 kDa protein as compared with control nuclei. Almost normal DNA synthesis, cell proliferation, DNA polymerase alpha activity and nuclear protein phosphorylation were restored in polyamine-depleted cells grown in medium supplemented with 20 microM-spermidine at least 10-12 h before S phase. Cultures in which proliferation was blocked by alpha-difluoromethylornithine did not exhibit synchronous growth after the block was removed. Thus it may be concluded that HeLa cells depleted of polyamines are not inhibited at a single control point in the cell cycle, but are arrested at diverse sites throughout G1 phase.
Purification and characterization of a nutritionally controlled endodeoxyribonuclease from Streptomyces glaucescens
Authors: Aparicio J F , Hardisson C , Sánchez J
Published: August 2015

Streptomyces glaucescens has a DNAase whose synthesis is under nutritional control. We have purified this enzyme to apparent homogeneity by phosphocellulose chromatography followed by heparin-agarose, Cibacron Blue F3-GA-Sepharose and Sephadex G-75 chromatography and MonoQ f.p.l.c. The enzyme had an apparent Mr of 39,600 and a pI of approx. 8.15. The Mr of the native enzyme estimated by gel chromatography was 49,000. The DNAase had a pH optimum of 7.5 and an absolute requirement for bivalent cations in the reaction buffer. It was inhibited by high salt concentrations, chelating agents or phosphate-containing compounds and was stimulated by dimethyl sulphoxide. The activity was greatly diminished unless dithiothreitol or 2-mercaptoethanol was included in the reaction mixture. Reagents such as Hg2+ or iodoacetate strongly inhibited the enzyme. The nuclease hydrolysed both double-stranded and single-stranded DNA, showing greater affinity for double-stranded DNA, and no detectable hydrolysis of RNA. The enzyme produced nicks in double-stranded DNA, generating 3′-hydroxy and 5′-phosphate termini, and degraded circular DNA.
Optimal association-saturation procedure for estimating association and dissociation rate parameters in receptør studies. Application to solubilized A1 adenosine receptors
Authors: Casadó V , Franco R , Mallol J , Lluis C , Canela E I
Published: August 2015

A method of obtaining estimates of the maximum binding and association and dissociation rate constants for a receptor-ligand interaction is described. This new procedure, the association-saturation method, is based on an exact mathematical equation which defines the model without simplifications. The method proposed is readily applicable to any system consisting of one ligand and one receptor. With only four determinations, each involving one ligand concentration and an association time, it is possible to determine the number of specific binding sites, the dissociation and association rate constants and the equilibrium dissociation constant with accuracy. No dissociation curve is required, and only one point of the association curve for each ligand concentration is necessary. In addition to the higher confidence in the estimates of the parameter values obtained, this method leads to important savings in radiolabelled compounds and experimental time. The results were compared with those obtained with standard association-dissociation curves and saturation isotherms. The optimum number of replicates of each experimental point to obtain reliable estimates of different parameters is discussed on the basis of simulation studies. The performance of the procedure is analysed by means of association-saturation experiments with the agonist [adenine-2,8-3H,ethyl-2-3H]N6-phenylisopropyladenosine and solubilized A1 adenosine receptors from pig brain cortex.
Purification and characterization by fast-atom-bombardment mass spectrometry of the polymorphonuclear-leucocyte-elastase-generated Aα (1–21) fragment of fibrinogen from human blood after incubation with calcium ionophore A23187
Authors: Dewey R S , Liesch J M , Williams H R , Sugg E E , Dolan C A , Davies P , Mumford R A , Albers-Schönberg G
Published: August 2015

The stimulation of human blood with a Ca2+ ionophore, A23187, leads to activation of polymorphonuclear leucocytes (PMN) with release of small amounts of catalyticaly active elastase, as demonstrated by the formation of a characteristic product, the N-terminal A alpha (1-21) peptide of the Aa subunit of fibrinogen. The identity of the peptide was initially established by radioimmunoassay (r.i.a.) with an antibody raised to A alpha (1-21). We now provide independent confirmation of the formation of A alpha (1-21) by fast-atom-bombardment-m.s. analysis of the fractions separated chromatographically after spiking of plasma samples with peptide labelled with [2H8]Phe at position 8. Identity of the peptides was established on the basis of their chromatographic retention time and by the distinct peaks in the mass spectra of these fractions. The relative intensities of the molecular ions of natural and labelled peptides were measured. On the basis of a comparison of the peaks of similar intensities, the concentration of the natural peptide at the time of spiking was close (79%) to the amount obtained by r.i.a. An additional peptide, des-alanyl-A alpha (2-21), was also seen. The total amount of material measured by r.i.a. could be accounted for by the sum of these two provides. The addition of label and assay by m.s. has provided an independent physical-chemical method for identifying A alpha (1-21) as a characteristic product of PMN elastase release in whole blood, but which is absent in freshly drawn blood.
Stimulus-response uncoupling in the neutrophil. Adenosine A2-receptor occupancy inhibits the sustained, but not the early, events of stimulus transduction in human neutrophils by a mechanism independent of actin-filament formation
Authors: Cronstein B N , Haines K A
Published: August 2015

Generation of superoxide anion (O2-) in response to occupancy of neutrophil chemoattractant receptors requires both early events (‘triggering’) and sustained signals (‘activation’). We have previously demonstrated that occupancy of adenosine A2 receptors inhibits O2- generation by neutrophils. In parallel, adenosine-receptor occupancy promotes association of bound N-formylmethionyl-leucyl-phenylalanine (fMLP) receptors with the cytoskeleton, a process associated with termination of neutrophil activation (stimulus-response uncoupling). We undertook this study to determine whether inhibition of neutrophil function by adenosine-receptor occupancy requires intact actin filaments and to examine the effect of adenosine-receptor occupancy on the stimulated generation of intracellular signals involved in neutrophil triggering and activation. Occupancy of adenosine A2 receptors by 5′-N-ethylcarboxamidoadenosine (NECA, 1 microM) significantly increased (130 +/- 1% of control, P less than 0.001, n = 3) association of [3H]fMLP with cytoskeletal preparations. Cytochalasin B (5 micrograms/ml), an agent which disrupts actin filaments, completely blocked association of [3H]fMLP with cytoskeletal preparations, as previously reported. However, NECA markedly increased association of [3H]fMLP with the cytoskeleton even in the presence of cytochalasin B (P less than 0.0002). Moreover, NECA did not significantly affect either the early (30s) or the late (5 min) formation of actin filaments after stimulation by chemoattractant (fMLP, 0.1-100 nM). Cytochalasin B markedly inhibited actin-filament formation by stimulated neutrophils, and NECA did not reverse the effect of cytochalasin B on actin-filament formation. Adenosine-receptor occupancy did not affect the rapid peak in diacylglycerol generation (less than or equal to 15 s) from either [3H]arachidonate- or [14C]glycerol-labelled phospholipid pools. However, as would be predicted if occupancy of the adenosine receptor was a signal for early termination of cell activation, NECA (1 microM) markedly diminished the slow sustained generation of diacylglycerol. These results suggest that adenosine-A2-receptor occupancy does not affect triggering of the neutrophil, but that occupancy of adenosine receptors is an early signal for the termination of neutrophil activation, i.e. the ‘premature’ finish of signal transduction. Moreover, these data indicate that at least two pathways are available for increasing the association of ligated chemoattractant receptors with the cytoskeleton of neutrophils: F-actin-dependent and -independent.
Guanosine nucleotides regulate hormone binding of insulin receptors
Authors: Mortensen E R , Drachman J , Guidotti G
Published: August 2015

Insulin receptors in turkey erythrocyte and rat adipocyte plasma membranes display non-linear hormone binding by Scatchard analysis. This result is consistent with evidence that the insulin-binding sites are heterogeneous and have at least two affinities for the hormone. Mild reduction of plasma membranes with dithiothreitol, before insulin binding, increased the fraction of hormone binding with high affinity without significantly changing the total number of receptor-binding sites. In the presence of guanosine 5′-[gamma-thio]triphosphate, the amount of receptor with high affinity for insulin in the reduced membranes decreased to that present in the absence of reduction; the effect of the nucleotide was concentration- and temperature-dependent. This decrease in insulin binding was specific for guanine nucleotides.
Two glucose transporter isoforms are sorted differentially and are expressed in distinct cellular compartments
Authors: Shibasaki Y , Asano T , Lin J L , Tsukuda K , Katagiri H , Ishihara H , Yazaki Y , Oka Y
Published: August 2015

Rat GLUT4 (adipocyte/muscle-type glucose transporter) was expressed in two fibroblastic cell lines, Chinese hamster ovary (CHO) cells and 3T3-L1 fibroblasts, under the control of the methallothionein I promoter. Although immunoblotting with a GLUT4-specific anti-peptide antibody demonstrated that the amount of GLUT4 expressed was comparable with that in 3T3-L1 adipocytes and rat adipose tissues, no increase in 2-deoxy-D-glucose uptake was observed in the basal state in fibroblasts. Immunocytochemical studies showed that the expressed GLUT4 appeared to be localized in a specific region in the cytoplasm. These results were in marked contrast to those obtained in CHO cells expressing GLUT1 (HepG2/erythrocyte-type glucose transporter) using the same expression vector. In this case the expressed GLUT1 protein appeared to reside mainly on the plasma membranes, and a significant increase in glucose uptake was observed. Although insulin increased glucose uptake in CHO cells and 3T3-L1 fibroblasts as well as in the cells expressing rat GLUT4, an increment due to insulin above basal values was small, at most 2-fold, and no significant differences were observed in insulin-stimulated glucose uptake between transfected and parental cells. In addition, no apparent differences in the subcellular distribution of expressed GLUT4 were observed between the insulin-stimulated and the basal state. These results indicate that in fibroblastic cell lines GLUT1 and GLUT4 proteins are sorted in a different fashion, and the expression of GLUT4 protein per se is not enough to produce a large insulin-induced increase in glucose transport activity such as that observed in rat adipocytes and 3T3-L1 adipocytes. Thus unidentified aspects of the cellular environment which are present in the adipocytes but not in fibroblastic cell lines may be required for a large insulin-induced increase in glucose transport activity to be observed.
Damage-recognition proteins as a potential indicator of DNA-damage-mediated sensitivity or resistance of human cells to ultraviolet radiation
Authors: Chao C C K
Published: August 2015

We have previously identified damage-recognition proteins that bind to cisplatin[cis-diamminedichloroplatinum(II), a DNA cross-linking agent]- or u.v.-modified DNA in HeLa cells [Chao, Huang, Huang & Lin-Chao (1991) Mol. Cell. Biol. 11, 2075-2080; Chao, Huang, Lee & Lin-Chao (1991) Biochem. J. 277, 875-878]. In the present study we compared damage-recognition proteins in cells expressing different sensitivities to DNA damage. An increase in damage-recognition proteins and an enhancement of plasmid re-activation were detected in HeLa cells resistant to cisplatin and u.v. However, repair-defective cells derived from xeroderma-pigmentosum (a rare skin disease) patients did not express less cisplatin damage-recognition proteins than repair-competent cells, suggesting that damage-recognition-protein expression may not be related to DNA repair. By contrast, cells resistant to DNA damage consistently expressed high levels of u.v.-modified-DNA damage-recognition proteins. The results support the notion that u.v. damage-recognition proteins are different from those that bind to cisplatin. These findings also suggest that the damage-recognition proteins identified could be used as potential indicators of the sensitivity or resistance of cells to u.v.
Neuronal-glial metabolism under depolarizing conditions. A 13C-n.m.r. study
Authors: Badar-Goffer R S , Ben-Yoseph O , Bachelard H S , Morris P G
Published: August 2015

Time courses of incorporation of 13C from 13C-labelled glucose and/or acetate into the individual carbon atoms of amino acids, citrate and lactate in depolarized cerebral tissues were monitored by using 13C-n.m.r. spectroscopy. There was no change in the maximum percentage of 13C enrichments of the amino acids on depolarization, but the maxima were reached more rapidly, indicating that rates of metabolism in both glycolysis and the tricarboxylic acid cycle were accelerated. Although labelling of lactate and of citrate approached the theoretical maximum of 50%, labelling of the amino acids was always below 20%, suggesting that there is a metabolic pool or compartment that is inaccessible to exogenous substrates. Under resting conditions labelling of citrate and of glutamine from [1-13C]glucose was not detected, whereas both were labelled from [2-13C]acetate, which is considered to reflect glial metabolism. In contrast, considerable labelling of these two metabolites from [1-13C]glucose was observed in depolarized tissues, suggesting that the increased metabolism may be due to increased consumption of glucose by glial cells. The labelling patterns on depolarization from [1-13C]glucose alone and from both precursors [( 1-13C]glucose plus [2-13C]acetate) were similar, which also indicates that the changes are due to increased consumption of glucose rather than acetate.
Stimulation of glycolysis as an activation signal in rat peritoneal macrophages. Effect of glucocorticoids on this process
Authors: Bustos R , Sobrino F
Published: August 2015

1. Peritoneal macrophages were prepared from control, Escherichia coli-treated and triamcinolone acetonide-treated rats. Control and E. coli-treated rats produced resident and activated macrophages respectively. Glycolysis in these cells was studied by the fructose 2,6-bisphosphate (Fru-2,6-P2) content, lactate release and 6-phosphofructo-1-kinase (PFK-1) and 6-phosphofructo-2-kinase (PFK-2) activities. 2. In activated macrophages, lactate release and Fru-2,6-P2 content were increased several-fold compared with those in resident cells. Moreover, the response of these parameters to phorbol 12-myristate 13-acetate in activated macrophages was greater than for resident cells. 3. PFK-2 activity was moderately increased (about 3-fold), but PFK-1 activity was increased 5-fold in activated macrophages compared with resident cells. Partially purified preparations of PFK-1 were sensitive to Fru-2,6-P2, with K0.5 about 0.25 microM in both control and activated cells. However, the Vmax. of PFK-1 from activated cells was increased. In addition, AMP stimulated PFK-1, but the kinetic pattern was different from that described for Fru-2,6-P2. Moreover there was no difference in the stimulation by AMP of PFK-1 from resident and activated cells. 4. Fru-2,6-P2 content and lactate release in macrophages from triamcinolone acetonide-treated rats were decreased in both resident and activated cells. Also, the glucocorticoid inhibited PFK-1 and PFK-2 activities in both resident and activated macrophages. PFK-1 from triamcinolone acetonide-treated rats was not stimulated by Fru-2,6-P2, whereas the effect of AMP was unchanged. The effects of glucocorticoid seem to be specific for phagocytic cells, since the glucocorticoid treatment increased PFK-1 and PFK-2 activities in liver.
The ‘natural’ hybrid haemoglobin from mule. Interrelationships with its parent haemoglobins from horse and donkey
Authors: Condò S G , Coletta M , Cicchetti R , Argentin G , Guerrieri P , Marini S , el-Sherbini S , Giardina B
Published: August 2015

The equilibrium O2-binding properties of the hybrid haemoglobin (Hb) present in vivo in erythrocytes from mule and of its parent Hbs from horse and donkey were compared with special reference to the effect of heterotropic ligands such as Cl-, D-glycerate 2,3-bisphosphate (DPG) and inositol hexakisphosphate. All these Hbs display a decreased effect by polyphosphates, confirming that what has been observed for horse Hb [Giardina, Brix, Clementi, Scatena, Nicoletti, Cicchetti, Argentin & Condò (1990) Biochem. J. 266, 897-900] is common to other equine species, at least from a qualitative standpoint. However, different quantitative aspects can be detected, which can be accounted for by a different role for the two types of chain in characterizing the binding free energy for the various heterotropic effectors. In particular, it is shown that the binding mode of DPG and inositol hexakisphosphate displays different features since long-range effects can be observed clearly for inositol hexakisphosphate but not for DPG. In general terms, in spite of a different intrinsic O2 affinity, the modulation of functional properties by third ligands leads these Hbs to behave, under physiological conditions, similarly to human HbA. It might represent an interesting example of how different species with similar functional needs find different ways to produce a similar functional behaviour.
Structural and functional comparison of two human liver dihydrodiol dehydrogenases associated with 3 α-hydroxysteroid dehydrogenase activity
Authors: Deyashiki Y , Taniguchi H , Amano T , Nakayama T , Hara A , Sawada H
Published: August 2015

Two monomeric dihydrodiol dehydrogenases with pI values of 5.4 and 7.6 were co-purified with androsterone dehydrogenase activity to homogeneity from human liver. The two enzymes differed from each other on peptide mapping and in their heat-stabilities; with respect to the latter the dihydrodiol dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase activities of the respective enzymes were similarly inactivated. The pI 5.4 enzyme was equally active towards trans- and cis-benzene dihydrodiols, and towards (S)- and (R)-forms of indan-1-ol and 1,2,3,4-tetrahydronaphth-1-ol and oxidized the 3 alpha-hydroxy group of C19-, C21- and C24-steroids, whereas the pI 7.6 enzyme showed high specificity for trans-benzene dihydrodiol, (S)-forms of the alicyclic alcohols and C19- and C21-steroids. Although the two enzymes reduced various xenobiotic carbonyl compounds and the 3-oxo group of C19- and C21-steroids, and were A-specific in the hydrogen transfer from NADPH, only the pI 5.4 enzyme showed reductase activity towards 7 alpha-hydroxy-5 beta-cholestan-3-one and dehydrolithocholic acid. The affinity of the two enzymes for the steroidal substrates was higher than that for the xenobiotic substrates. The two enzymes also showed different susceptibilities to the inhibition by anti-inflammatory drugs and bile acids. Whereas the pI-5.4 enzyme was highly sensitive to anti-inflammatory steroids, showing mixed-type inhibitions with respect to indan-1-ol and androsterone, the pI 7.6 enzyme was inhibited more potently by non-steroidal anti-inflammatory drugs and bile acids than by the steroidal drugs, and the inhibitions were all competitive. These structural and functional differences suggest that the two enzymes are 3 alpha-hydroxysteroid dehydrogenase isoenzymes.
Effect of diabetes and fasting on GLUT-4 (muscle/fat) glucose-transporter expression in insulin-sensitive tissues. Heterogeneous response in heart, red and white muscle
Authors: Camps M , Castelló A , Muñoz P , Monfar M , Testar X , Palacín M , Zorzano A
Published: August 2015

1. GLUT-4 glucose-transporter protein and mRNA levels were assessed in heart, red muscle and white muscle, as well as in brown and white adipose tissue from 7-day streptozotocin-induced diabetic and 48 h-fasted rats. 2. In agreement with previous data, white adipose tissue showed a substantial decrease in GLUT-4 mRNA and protein levels in response to both diabetes and fasting. Similarly, GLUT-4 mRNA and protein markedly decreased in brown adipose tissue in both insulinopenic conditions. 3. Under control conditions, the level of expression of GLUT-4 protein content differed substantially in heart, red and white skeletal muscle. Thus GLUT-4 protein was maximal in heart, and red muscle had a greater GLUT-4 content compared with white muscle. In spite of the large differences in GLUT-4 protein content, GLUT-4 mRNA levels were equivalent in heart and red skeletal muscle. 4. In heart, GLUT-4 mRNA decreased to a greater extent than GLUT-4 protein in response to diabetes and fasting. In contrast, red muscle showed a greater decrease in GLUT-4 protein than in mRNA in response to diabetes or fasting, and in fact no decrease in GLUT-4 mRNA content was detectable in fasting. On the other hand, preparations of white skeletal muscle showed a substantial increase in GLUT-4 mRNA under both insulinopenic conditions, and that was concomitant to either a modest decrease in GLUT-4 protein in diabetes or to no change in fasting. 5. These results indicate that (a) the effects of diabetes and fasting are almost identical and lead to changes in GLUT-4 expression that are tissue-specific, (b) white adipose tissue, brown adipose tissue and heart respond similarly to insulin deficiency by decreasing GLUT-4 mRNA to a larger extent than GLUT-4 protein, and (c) red and white skeletal muscle respond to insulinopenic conditions in a heterogeneous manner which is characterized by enhanced GLUT-4 mRNA/protein ratios.
Primary and predicted secondary structures of the Actinomadura R39 extracellular dd-peptidase, a penicillin-binding protein (PBP) related to the Escherichia coli PBP4
Authors: Granier B , Duez C , Lepage S , Englebert S , Dusart J , Dideberg O , Van Beeumen J , Frère J M , Ghuysen J M
Published: August 2015

As derived from gene cloning and sequencing, the 489-amino-acid DD-peptidase/penicillin-binding protein (PBP) produced by Actinomadura R39 has a primary structure very similar to that of the Escherichia coli PBP4 [Mottl, Terpstra & Keck (1991) FEMS Microbiol. Lett. 78, 213-220]. Hydrophobic-cluster analysis of the two proteins shows that, providing that a large 174-amino-acid stretch is excluded from the analysis, the bulk of the two polypeptide chains possesses homologues of the active-site motifs and secondary structures found in the class A beta-lactamase of Streptomyces albus G of known three-dimensional structure. The 174-amino-acid insert occurs at equivalent places in the two PBPs, between helices alpha 2 and alpha 3, away from the active site. Such an insert is unique among the penicilloyl serine transferases. It is proposed that the Actinomadura R39 PBP and E. coli PBP4 form a special class, class C, of low-Mr PBPs/DD-peptidases. A vector has been constructed and introduced by electrotransformation in the original Actinomadura R39 strain, allowing high-level expression and secretion of the DD-peptidase/PBP (250 mg.l-1). The gene encoding the desired protein is processed differently in Actinomadura R39 and Streptomyces lividans. Incorrect processing in Streptomyces lividans leads to a secreted protein which is inert in terms of DD-peptidase activity and penicillin-binding capacity.
Long-chain acyl-CoA ester intermediates of β-oxidation of mono- and di-carboxylic fatty acids by extracts of Corynebacterium sp. strain 7E1C
Authors: Broadway N M , Dickinson F M , Ratledge C
Published: August 2015

beta-Oxidation of palmitate and tetradecanedioic acid was studied in cell-free extracts of the Gram-positive bacterium Corynebacterium sp. strain 7E1C, and the acyl-CoA ester intermediates formed were analysed by h.p.l.c. beta-Oxidation assays displayed a lag phase before a constant rate of NAD+ reduction was obtained. The length of the lag phase was inversely proportional to the number of units of activity added to assays. This is a characteristic feature of a system of consecutive reactions proceeding via free intermediates. During beta-oxidation of palmitate all the saturated acyl-CoAs from C16 to C8 were detected together with trace amounts of unsaturated and 3-hydroxy-intermediates. The time-course of intermediate formation again indicated a precursor-product relationship indicative of free intermediates being formed. When 3-hydroxyacyl-CoA dehydrogenase was inhibited by completely removing NAD+ from assays, the major acyl-CoAs, detected during palmitate beta-oxidation were palmitoyl-CoA, hexadeca-2-enoyl-CoA and 3-hydroxypalmitoyl-CoA. These compounds also displayed a precursor-product relationship. Under normal assay conditions the acyl-CoA dehydrogenase(s) are the probable rate-limiting enzyme(s) of the beta-oxidation spiral. These results indicate that in cell-free extracts of Corynebacterium sp. strain 7E1C, beta-oxidation proceeds via free acyl-CoA intermediates and is at variance with the concept of substrate channelling or of a ‘leaky hose pipe’ model as proposed for mitochondrial beta-oxidation in eukaryotic cells. The significant accumulation of chain-shortened acyl-CoA esters is similar to the situation observed for mammalian peroxisomal beta-oxidation.