bluuebunny/crossref_metadata_2025 huggingface dataset:
Peroxisomal fatty acid oxidation and inhibitors of the mitochondrial carnitine palmitoyltransferase I in isolated rat hepatocytes
Authors: Skorin C , Necochea C , Johow V , Soto U , Grau A M , Bremer J , Leighton F
Published: August 2015

Fatty acid oxidation was studied in the presence of inhibitors of carnitine palmitoyltransferase I (CPT I), in normal and in peroxisome-proliferated rat hepatocytes. The oxidation decreased in mitochondria, as expected, but in peroxisomes it increased. These two effects were seen, in variable proportions, with (+)-decanoylcarnitine, 2-tetradecylglycidic acid (TDGA) and etomoxir. The decrease in mitochondrial oxidation (ketogenesis) affected saturated fatty acids with 12 or more carbon atoms, whereas the increase in peroxisomal oxidation (H2O2 production) affected saturated fatty acids with 8 or more carbon atoms. The peroxisomal increase was sensitive to chlorpromazine, a peroxisomal inhibitor. To study possible mechanisms, palmitoyl-, octanoyl- and acetyl-carnitine acyltransferase activities were measured, in homogenates and in subcellular fractions from control and TDGA-treated cells. The palmitoylcarnitine acyltransferase was inhibited, as expected, but the octanoyltransferase activity also decreased. The CoA derivative of TDGA was synthesized and tentatively identified as being responsible for inhibition of the octanoylcarnitine acyltransferase. These results show that inhibitors of the mitochondrial CPT I may also inhibit the peroxisomal octanoyl transferase; they also support the hypothesis that the octanoyltransferase has the capacity to control or regulate peroxisomal fatty acid oxidation.
The gene for a novel protein, a member of the protein disulphide isomerase/form I phosphoinositide-specific phospholipase C family, is amplified in hydroxyurea-resistant cells
Authors: Chaudhuri M M , Tonin P N , Lewis W H , Srinivasan P R
Published: August 2015

Cell lines selected in multiple steps for increasing resistance to hydroxyurea have been shown to have corresponding increases in ribonucleotide reductase activity. We have isolated a number of cDNA clones from a cDNA library constructed from a highly hydroxyurea-resistant hamster cell line, 600H, in which the activity of ribonucleotide reductase is elevated more than 80-fold. These clones correspond to genomic DNA sequences amplified in the 600H cell line compared with the V79 parental line. One of these cDNA clones, termed P5, codes for a 50 kDa protein detected by in vitro translation of poly(A)+ RNA isolated by hybridization/selection. The cDNA sequence contains a single open reading frame of 1317 nucleotides which encodes a polypeptide of 439 amino acids. The amino acid sequence deduced from the cDNA insert contains two copies of the 11-amino-acid sequence Val-Glu-Phe-Tyr-Ala-Pro-Trp-Cys-Gly-His-Cys. Duplicate copies of this sequence also occur in the active site of rat and human protein disulphide isomerase (also known as the beta-subunit of human prolyl 4-hydroxylase, tri-iodothyronine-binding protein) and in Form I phosphoinositide-specific phospholipase C, indicating that P5 falls into this newly defined superfamily of proteins. Genomic sequences similar to the cDNA clone are amplified 10-20-fold in hamster cells selected for resistance to increasing concentrations of hydroxyurea, a phenomenon observed earlier with cDNA clones for the M2 subunit of ribonucleotide reductase and ornithine decarboxylase. RNA blots probed with P5 cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells.
Global ischaemia induces a biphasic response of the mitochondrial respiratory chain. Anoxic pre-perfusion protects against ischaemic damage
Authors: Veitch K , Hombroeckx A , Caucheteux D , Pouleur H , Hue L
Published: August 2015

Studies of Langendorff-perfused rat hearts have revealed a biphasic response of the mitochondrial respiratory chain to global ischaemia. The initial effect is a 30-40% increase in the rate of glutamate/malate oxidation after 10 min of ischaemia, owing to an increase in the capacity for NADH oxidation. This effect is followed by a progressive decrease in these oxidative activities as the ischaemia is prolonged, apparently owing to damage to Complex I at a site subsequent to the NADH dehydrogenase component. This damage is exacerbated by reperfusion, which causes a further decrease in Complex I activity and also decreases the activities of the other complexes, most notably of Complex III. Perfusion for up to 1 h with anoxic buffer produced only the increase in NADH oxidase activity, and neither anoxia alone, nor anoxia and reperfusion, caused loss of Complex I activity. Perfusing for 3-10 min with anoxic buffer before 1 h of global ischaemia had a significant protective effect against the ischaemia-induced damage to Complex I.
The interplay of temperature and protons in the modulation of oxygen binding by squid blood
Authors: Giardina B , Condò S G , Brix O
Published: August 2015

An extensive set of data relating to the binding of oxygen by haemocyanin from the squid Todarodes sagittatus has been collected under various experimental conditions. The results obtained show that, within the range of physiological pH, the concentration of protons affects mainly the high-affinity state of the molecule without significantly affecting the low-affinity state. As far as the effect of temperature is concerned, the data show a characteristic feature which is very similar to that previously described in the case of haemoglobins from Arctic mammals such as reindeer (Rangifer tarandus) and musk ox. (Ovibos moschatus). The shape of the oxygen equilibrium curve shows strong temperature-dependence, since the overall heat of the binding of oxygen to the low-affinity state of the molecule is strongly exothermic and that to the high-affinity state is very close to zero. The results provide an outline of the intramolecular compromise that, through the interplay of temperature and protons, optimizes the loading and unloading of oxygen under the various environmental conditions experienced by this species of squid.
Modified kinetics of platelet-derived growth factor-induced Ca2+ increases in NIH-3T3 cells overexpressing phospholipase Cγ1
Authors: Renard D C , Bolton M M , Rhee S G , Margolis B L , Zilberstein A , Schlessinger J , Thomas A P
Published: August 2015

The effects of platelet-derived growth factor (PDGF) on cytosolic free Ca2+ concentration ([Ca2+]i) and inositol phosphates were studied in NIH-3T3 fibroblasts transfected with cDNA for phospholipase C gamma 1 (PLC gamma 1) to yield a 7-fold overexpression of this enzyme, compared with cells containing normal levels of PLC gamma 1. In a study published recently [Margolis, Zilberstein, Franks, Felder, Kremer, Ullrich, Rhee, Skorecki & Schlessinger (1990) Science 248, 607-610] it was reported that this overexpression of PLC gamma 1 caused a specific potentiation of the inositol phosphate response to PDGF, but this was not associated with an enhancement of the [Ca2+]i response. In the present study, measurements of the time course and isomeric profile of PDGF-induced inositol phosphate formation demonstrated that the initial rate of Ins(1,4,5)P3 formation was also enhanced in the PLC gamma 1-overexpressing cells, yielding a 10-fold greater increase at 1 min compared with the parental NIH-3T3 cells. By contrast, bradykinin-induced phosphoinositide metabolism was unchanged in PLC gamma 1-transfected cells. Measurements of [Ca2+]i in cell populations and single cells showed a significant latent period following PDGF addition prior to the [Ca2+]i increases in both cell lines, which decreased in a dose-dependent manner with increasing PDGF concentration. The duration of the latent period was decreased and the maximal rate of [Ca2+]i rise was increased in the PLC gamma 1-overexpressing cells at all doses of PDGF examined. In single-cell measurements these cells also responded to PDGF with a greater peak amplitude of [Ca2+]i. Both intracellular Ca2+ mobilization and Ca2+ influx across the plasma membrane were enhanced in the PLC gamma 1-overexpressing cells. There was no difference between the two cell lines in either the latency or the magnitude of the [Ca2+]i increases induced by bradykinin. These data provide further evidence that PLC gamma 1 is responsible for the PDGF-induced stimulation of Ins(1,4,5)P3 formation. Moreover, in contrast to earlier conclusions, the modified kinetics of the [Ca2+]i changes in PLC gamma 1-overexpressing cells suggest that Ins(1,4,5)P3 does play a predominant second messenger role in the PDGF-induced [Ca2+]i increases. The data also indicate that the latent period may be a function of the time required to reach a threshold level of Ins(1,4,5)P3, rather than an intrinsic property of the PDGF receptor.
Protein turnover, growth and proliferation in CHO cells. Variation within and between mutant classes for salvage pathway enzymes
Authors: Gunn J M , Brancheau M R
Published: August 2015

We have examined the clonal variation in rates of amino acid transport, protein synthesis, protein degradation, growth and proliferation for CHO cells with mutations in the purine and pyrimidine salvage pathways. First we compared three clonal cell lines, each with a different mutation, with the heterozygous parental line AT3-2. Overall, the correlation between rates of protein turnover, growth and proliferation was excellent. The slower growth and proliferation of one mutant, AB3 (TK-, APRT-), is explained by a low intrinsic rate of protein synthesis coupled with a smaller response in rates of amino acid transport, protein synthesis and protein degradation to insulin, serum and dexamethasone. Secondly, we compared seven aza-adenine-resistant and 14 thioguanine-resistant mutants of AT3-2 and found significant differences in control and insulin-stimulated rates of protein turnover both within and between mutant populations. A significant difference between the populations was unexpected because each individual cell line was cloned from a spontaneous pre-existing mutation in AT3-2, and each population should have the same average rate. Remarkably, all 24 mutants had lower rates of protein synthesis than AT3-2. We cannot explain the data solely in terms of mutations in the salvage pathways. Rather, we propose that the mutant survivors have randomly down-regulated the intrinsically fixed growth factor-regulated pathways of protein turnover, resulting in a broad spectrum of lower metabolic rates.
Vasoconstrictor agonists activate G-protein-dependent receptor-operated calcium channels in pig aortic microsomes
Authors: Blayney L M , Gapper P W , Newby A C
Published: August 2015

Receptor-operated Ca2+ channels were characterized by their ability to decrease steady-state ATP-dependent Ca2+ accumulation into pig aortic microsomes. The vasoconstrictor agents noradrenaline, angiotensin II and adenosine 5′-[alpha beta-methylene]triphosphate (pp[CH2]pA) all decreased Ca2+ accumulation only when sonicated into vesicles (to allow access to receptor sites) and in the presence of guanosine 5′-[beta gamma-imido]triphosphate to activate transducing G-proteins. The effect of noradrenaline was inhibited by the alpha 2 antagonist yohimbine, but not by the alpha 1 antagonist prazosin. The effect of none of the agonists was reversed by diltiazem. SK&F 96365 (an inhibitor of receptor-mediated Ca2+ influx into intact cells) reversed the effect of noradrenaline, but not that of pp[CH2]pA, which suggests that at least two receptor-operated channels may be present in this preparation.
The orientation of the three haems of the ‘in situ’ ubiquinol oxidase, cytochrome bd, of Escherichia coli
Authors: Ingledew W J , Rothery R A , Gennis R B , Salerno J C
Published: August 2015

The Escherichia coli cytochrome bd complex incorporates three haems as prosthetic groups. In the ferric form these are a predominantly high-spin chlorin (haem d), a high-spin haem b (b595) and a low-spin haem b (b558). The orientations of these three haems have been determined by e.p.r. studies on oriented multilayer preparations of cytoplasmic membrane fragments. The low-spin haem b (b558) and the high-spin haem d are oriented with their haem planes perpendicular to the membrane plane. The high-spin haem b595 is oriented with its haem plane at approx. 55 degrees to the membrane plane. A minor low-spin component, attributable to a low-spin subpopulation of the haem d, is also oriented with its haem plane perpendicular to the membrane plane.
Importance of the two tryptophan residues in the Streptomyces R61 exocellular dd-peptidase
Authors: Bourguignon-Bellefroid C , Wilkin J M , Joris B , Aplin R T , Houssier C , Prendergast F G , Van Beeumen J , Ghuysen J M , Frère J M
Published: August 2015

Modification of the Streptomyces R61 DD-peptidase by N-bromosuccinimide resulted in a rapid loss of enzyme activity. In consequence, the role of the enzyme's two tryptophan residues was investigated by site-directed mutagenesis. Trp271 was replaced by Leu. The modification yielded a stable enzyme whose structural and catalytic properties were similar to those of the wild-type protein. Thus the Trp271 residue, though almost invariant among the beta-lactamases of classes A and C and the low-Mr penicillin-binding proteins, did not appear to be essential for enzyme activity. Mutations of the Trp233 into Leu and Ser strongly decreased the enzymic activity, the affinity for beta-lactams and the protein stability. Surprisingly, the benzylpenicilloyl-(W233L)enzyme deacylated at least 300-fold more quickly than the corresponding acyl-enzyme formed with the wild-type protein and gave rise to benzylpenicilloate instead of phenylacetylglycine. This mutant DD-peptidase thus behaved as a weak beta-lactamase.
Activation of human neutrophils by mastoparan. Reorganization of the cytoskeleton, formation of phosphatidylinositol 3,4,5-trisphosphate, secretion up-regulation of complement receptor type 3 and superoxide anion production are stimulated by mastoparan
Authors: Norgauer J , Eberle M , Lemke H D , Aktories K
Published: August 2015

In human neutrophils, mastoparan induced rapid F-actin polymerization which was followed by a slow and sustained depolymerization to below the initial F-actin content. Incubation of neutrophils with pertussis toxin inhibited mastoparan-stimulated actin polymerization; however it did not prevent sustained depolymerization of F-actin. Analyses of phospholipids performed in parallel revealed that mastoparan stimulated rapid formation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) and consumption of phosphatidylinositol 4,5-bisphosphate (PIP2). Pertussis toxin treatment blocked mastoparan-induced formation of PIP3. Furthermore, mastoparan stimulated the release of N-acetylglucosaminidase from primary granules. Cytochalasin B enhanced mastoparan-stimulated secretion. Mastoparan triggered superoxide radical production in a cytochalasin B-sensitive manner and induced complement type 3 receptor (CR3) up-regulation.
O-glycosylation in Aspergillus glucoamylase. Conformation and role in binding
Authors: Williamson G , Belshaw N J , Williamson M P
Published: August 2015

Functional peptides have been produced by proteolysis of glucoamylase (glucan 1,4-alpha-glucosidase; EC 3.2.1.3) from Aspergillus niger and purified by affinity chromatography, gel filtration and two ion-exchange-chromatography steps. The peptides correspond to residues 499-616 and 509-616 of the original glucoamylase molecule. Together with G1C (residues 471-616 from glucoamylase 1) [Belshaw & Williamson (1990) FEBS Lett. 269, 350-353], the three peptides all contain the C-terminal domain (residues 509-616) but, in addition, contain different proportions of the O-glycosylated region. The properties of these peptides have been compared to define the function of the O-linked oligosaccharides in this protein. The O-glycosylated region plays only a minor role in binding to hydrogen-bond ordered starch. The difference between the apparent free energy (delta G) for binding between the non-glycosylated C-terminal domain (-26.0 kJ/mol) and the C-terminal domain containing the fully O-glycosylated region (-25.0 kJ/mol) is only 1.0 kJ/mol. Binding to beta-cyclodextrin suggests that even this difference may reflect a small conformational change in the C-terminal domain rather than a direct effect of the O-linked sugars. The c.d. spectrum of the O-glycosylated region is deduced by comparison of the three peptides and is predominantly that of a random-coil structure. Two-dimensional n.m.r. spectra of glucoamylase and of the glycosylated peptide 499-616 show that the binding domain is more mobile than the catalytic domain and that its mobility is further increased on removal of the catalytic domain. The O-glycosylated region is more mobile still, and there is a marked increase in its mobility on removal of the catalytic domain. The O-glycosylated region in the intact protein can therefore be envisaged as a semi-rigid rod. The results show that a major function of O-glycosylation in glucoamylase 1 is to provide an extended peptide backbone and hence a fixed distance in linking the catalytic and binding domains. It does not in itself significantly increase the binding affinity for starch.
Studies on the formation of dipalmitoyl species of phosphatidylcholine and phosphatidylethanolamine in pulmonary type II cells
Authors: Rüstow B , Schlame M , Haupt R , Wilhelm D , Kunze D
Published: August 2015

Endogenous content of and incorporation of labelled glycerol into alkenylacyl-, alkylacyl- and diacyl-glycerol, -glycerol-3-phosphocholine and -glycero-3-phosphoethanolamine of pulmonary type II cells were measured. On prolonged incubation of type II cells with labelled glycerol, the proportion of label incorporated into the diacyl subclass of these glycerolipids increased and the proportion of label incorporated into the ether lipids declined. Endogenous phosphatidylcholine (PtdCho) of type II cells contained 38.4% of the dipalmitoyl species, but endogenous phosphatidylethanolamine (PtdEtn) only 2.5%. In contrast, similar proportions of labelled glycerol were incorporated into dipalmitoyl-PtdCho and -PtdEtn after short-time incubation but, with prolonged incubation time the proportion of labelled dipalmitoyl-PtdCho increased from 11.3 to 18.8%, whereas that of dipalmitoyl-PtdEtn did not change significantly. Type II cell membranes were found to exhibit cofactor-independent and CoA-mediated transacylations of [1-14C]palmitoyl-lyso-PtdCho and -lyso-PtdEtn. The distribution of label among the palmitic acid-containing species of PtdCho and PtdEtn formed by both transacylation activities was determined. Cofactor-independent and CoA-mediated transacylation showed a strong selectivity for palmitate and arachidonate and a strong discrimination against oleate. The amount (nmol) of dipalmitoyl-PtdEtn formed by both transacylation activities after short-time incubation (2 min) decreased with prolonged incubation time (60 min). In contrast, the nmol of dipalmitoyl-PtdCho formed by cofactor-independent transacylation remains nearly the same after short-time and longer incubation. The nmol of dipalmitoyl-PtdCho formed by CoA-mediated transacylation increased strongly in the same time interval. Beside synthesis de novo via the CDP-choline pathway and reacylation of lyso-PtdCho with palmitoyl-CoA, the CoA-mediated transacylation of lyso-PtdCho may be an effective pathway for the formation of dipalmitoyl-PtdCho in pulmonary type II cells.
Natural polyamines stimulate G-proteins
Authors: Bueb J L , Da Silva A , Mousli M , Landry Y
Published: August 2015

The natural polyamines spermine and spermidine, the biosynthetic precursor putrescine and their analogues cadaverine and tyramine stimulate the GTPase activity of purified GTP-binding proteins (Go/Gi) from calf brain reconstituted into phospholipid vesicles. The order of potency was spermine greater than spermidine greater than putrescine = cadaverine greater than tyramine. The physiological relevance of this observation was assessed, showing the same order of potency of polyamines in the stimulation of peritoneal and tracheal rat mast cells. The activation of rat mast cells by polyamines was inhibited by benzalkonium chloride or by a 2 h pretreatment of the cells with pertussis toxin. The increase in inositol phosphates evoked by polyamines was also inhibited by pertussis toxin. Therefore we propose that intracellular polyamines might control the basal level of second messengers and modulate extracellular signals transduced through G-protein-coupled receptors.
Kinetic analysis of the control through inhibition of autocatalytic zymogen activation
Authors: Manjabacas M C , Valero E , García-Moreno M , García-Cánovas F , Rodríguez J N , Varón R
Published: August 2015

A global kinetic analysis of a model of an autocatalytic zymogen-activation process in which an irreversible inhibitor competes with the zymogen for the active site of the proteinase is presented. Processes like the one here described are of great physiological interest because they are involved in the enzyme regulation of the gastrointestinal-tract enzymes, in blood coagulation, in fibrinolysis and in the complement system. The kinetic equations of both the transient phase and the steady state are derived for this mechanism. In addition, we have introduced a new parameter related to the kinetic behaviour of the system which allows us to predict whether the inhibition route or the activation route prevails in the steady state of the system. Finally, we extend the kinetic equations derived to different particular cases of the system studied.
A study of Ca2+-heparin complex-formation by polarimetry
Authors: Grant D , Long W F , Moffat C F , Williamson F B
Published: August 2015

Possible inflexions in isothermal binding plots derived from equilibrium-dialysis measurements and in equivalent plots obtained by polarimetric measurements accord with the possibility that discrete Ca(2+)-heparin-water complexes/phases may exist, the nature and proportions of which depend on the conditions under which the interaction occurs. Analysis of the plots obtained by polarimetric study of chemically modified heparins suggests, for individual substituents groups, an order of importance of carboxylate greater than N-sulphonate greater than N-acetyl greater than O-sulphate for the Ca(2+)-heparin interaction occurring at [Ca2+]/[heparin disaccharide] ratios of less than 0.5. At higher ratios, transitions occur that eventually lead to the formation of a complex in which the stoichiometry of association is 1 Ca2+ ion/heparin disaccharide unit.
Human serum amyloid A protein. Complete amino acid sequence of a new variant
Authors: Beach C M , De Beer M C , Sipe J D , Loose L D , De Beer F C
Published: August 2015

Serum amyloid A protein (SAA), an acute-phase reactant and apolipoprotein of high-density lipoprotein, is a polymorphic protein with six reported isoforms. These are the products of three genes, i.e., cDNA pA1, cDNA pSAA82 and genomic DNA SAAg9, the last two being allelic variants at a single locus. We have identified an individual with additional novel SAA isoforms on isoelectric-focusing analysis. By using 3-bromo-3-methyl-2-(2′-nitrophenylsulphenyl)-indolenine (BNPS-skatole) cleavage of the protein at tryptophan residues we obtained the complete amino acid sequence of a novel isoform. Additional cleavage by endoproteinase Asp-N allowed verification of the tryptophan residues and complete amino acid sequence of both isoforms. The suitability of this approach to the rapid sequencing of SAA was demonstrated. Sequence analysis and quantification suggest that these isoforms are the result of the first confirmed allelic variation at the SAA1 locus. We designate the protein products of this allele SAA1 beta (pI 6.1) and SAA1 beta des-Arg (pI 5.6).
Mechanisms of fructose-induced hypertriglyceridaemia in the rat. Activation of hepatic pyruvate dehydrogenase through inhibition of pyruvate dehydrogenase kinase
Authors: Park O J , Cesar D , Faix D , Wu K , Shackleton C H L , Hellerstein M K
Published: August 2015

1. The effects of purified diets containing 70% glucose or 70% fructose on the activation state of hepatic pyruvate dehydrogenase (PDHa), activity of mitochondrial PDH kinase, plasma triacylglycerols (TG) and hepatic lipogenesis de novo in rats were measured. 2. Plasma TG were significantly increased in the fructose-fed compared with the glucose-fed group (125 +/- 45 mg/dl versus 57 +/- 19 mg/dl; P less than 0.002) after 3-5 weeks on the diet despite less daily food intake. 3. Hepatic PDHa in fructose-fed rats was 144% of the value in glucose-fed rats (15.4 +/- 1.2% versus 10.7 +/- 0.5%; P less than 0.002), whereas cardiac muscle PDHa was not different (45.5 +/- 6.6% versus 41.0 +/- 7.8%). 4. Intrinsic hepatic PDH kinase activity was decreased to 34% of glucose-fed values by fructose feeding (-k = 3.56 +/- 0.39 versus 10.41 +/- 1.85 min-1; P less than 0.005). 5. The fractional contribution to very-low-density-lipoprotein palmitate from hepatic lipogenesis de novo, measured by a stable-isotope mass-spectrometric method, was 10.49 +/- 2.42% (n = 8) in fructose-fed rats versus 5.55 +/- 1.38% (n = 9) in glucose-fed rats (P less than 0.05), and 2.66 +/- 2.39% (n = 3) in chow-fed rats (P less than 0.05 versus fructose-fed group). The absolute contribution to circulating TG from lipogenesis de novo was also significantly higher in the fructose-fed than in the glucose-fed group (14.9 +/- 5.1 mg/dl versus 2.9 +/- 0.6 mg/dl; P less than 0.05) 6. Portal insulin concentrations were significantly higher in the fructose-fed rats (206 +/- 49 mu-units/ml versus 81 +/- 15 mu-units/ml; P less than 0.05). 7. In conclusion, dietary fructose appears to have a specific activating effect on hepatic PDH, mediated at least in part by inhibition of PDH kinase. These results are consistent with increased flux through hepatic PDH and synthesis of new fat, not just increased re-esterification of non-esterified fatty acids.
Interleukin-2-dependent phosphorylation of the retinoblastoma-susceptibility-gene product p110-115RB in human T-cells
Authors: Evans G A , Wahl L M , Farrar W L
Published: August 2015

The state of phosphorylation of the retinoblastoma-susceptibility gene product, p110-115RB, is thought to have fundamental importance in controlling the progression of the cell through the cell cycle. We have studied RB phosphorylation in human T-cells in the context of T-cell activation, stimulated by phytohaemagglutinin (PHA) and interleukin-2 (IL-2). We show that, of the signals associated with T-cell activation, only signals that directly lead to movement into S phase of the cell cycle are capable of stimulating RB phosphorylation. Cyclosporin A (CsA), a potent inhibitor of IL-2 synthesis and cellular proliferation, blocked RB phosphorylation, and this was recovered with exogenous IL-2, indicating a direct involvement of IL-2 in controlling RB phosphorylation. We found that PHA did not stimulate RB phosphorylation within 10 h of treatment, but IL-2 could effectively stimulate RB phosphorylation within 2 h, and this approached a maximum within 8-10 h of IL-2 treatment. Further, by using actinomycin D to inhibit new gene transcription following IL-2 stimulation, we found that early-cell-cycle phosphorylation of RB required IL-2-stimulated gene transcription. From these data we conclude that, in human T-cells, RB phosphorylation is not directly associated with T-cell receptor-mediated events, but requires the interaction of IL-2 and new gene transcription following IL-2 stimulation.
Evidence for two cDNA clones encoding human GM2-activator protein
Authors: Nagarajan S , Chen H C , Li S C , Li Y T , Lockyer J M
Published: August 2015

Two cDNAs encoding GM2 activator, pGM2A (648 bp) and GAP (1093 bp), were isolated from human placenta lambda gt11 libraries. The DNA sequence of pGM2A from 1 to 302 was almost identical with GAP, but diverged from 303-648. PCR was used to demonstrate the presence of both species of GM2 activator in placental RNA. Both cDNAs hybridized to mRNAs of approximately 2.3 kb and to identical single bands on genomic Southern blots.
Determination of Flux Control Coefficients from transient metabolite concentrations
Authors: Delgado J , Liao J C
Published: August 2015

Flux Control Coefficients have been used in the analysis of metabolic regulation for quantifying the effect of an enzyme on the overall steady-state flux. However, the experimental determination of these coefficients is very time-consuming, involving either determining the individual enzyme kinetics or perturbing the enzyme activity by genetic or other means. We developed a methodology that enables the determination of the Flux Control Coefficients from transient metabolite concentrations without knowing kinetic parameters. The transient states can be generated by changing the incubation conditions or adding the initial substrate. This approach is suitable for investigating metabolic regulation in vivo or multiple enzyme systems in vitro. It is particularly helpful if used in conjunction with n.m.r. measurements. The approach is based on a relationship between transient metabolite concentrations and the Flux Control Coefficients. The methodology has been improved from our previous results, and it is illustrated by three examples with simple pathway topologies.
Amino acid sequence of band-3 protein from rainbow trout erythrocytes derived from cDNA
Authors: Hübner S , Michel F , Rudloff V , Appelhans H
Published: August 2015

In this report we present the first complete band-3 cDNA sequence of a poikilothermic lower vertebrate. The primary structure of the anion-exchange protein band 3 (AE1) from rainbow trout erythrocytes was determined by nucleotide sequencing of cDNA clones. The overlapping clones have a total length of 3827 bp with a 5′-terminal untranslated region of 150 bp, a 2754 bp open reading frame and a 3′-untranslated region of 924 bp. Band-3 protein from trout erythrocytes consists of 918 amino acid residues with a calculated molecular mass of 101 827 Da. Comparison of its amino acid sequence revealed a 60-65% identity within the transmembrane spanning sequence of band-3 proteins published so far. An additional insertion of 24 amino acid residues within the membrane-associated domain of trout band-3 protein was identified, which until now was thought to be a general feature only of mammalian band-3-related proteins.
Purification and properties of 3′-nucleotidase of Leishmania donovani
Authors: Gbenle G O , Dwyer D M
Published: August 2015

A surface membrane 3′-nucleotidase from Leishmania donovani promastigotes has been purified to SDS/PAGE homogeneity. The enzyme has apparent subunit molecular mass of 38 kDa, pI 5.8 and a broad pH optimum, 5.5-7.5. EDTA partially inhibited the enzyme activity, which was fully restored by Co2+; Mg2+, Ca2+ or Mn2+ had no effect on the activity. ZnCl2 or dithiothreitol at 1 mM was inhibitory at pH 7.5, but was without effect at pH 5.5, whereas at both pH values 5 mM of either compound inhibited the enzyme. The substrate-specificity of the purified enzyme is restricted to ribonucleoside 3′-phosphates. 3′-AMP and 3′-IMP are the best substrates, whereas ADP, ATP, 2′-deoxyadenosine 3′-phosphate and 5′-AMP are competitive inhibitors of the enzyme. The enzyme showed low latency in intact-cell preparations. The kinetic properties and the surface membrane localization of the enzyme suggest its implication in the formation of nucleosides from 3′-nucleotides of the parasite's host.
‘Pore’ formation is not required for the hydroperoxide-induced Ca2+ release from rat liver mitochondria
Authors: Schlegel J , Schweizer M , Richter C
Published: August 2015

It has recently been suggested by several investigators that the hydroperoxide- and phosphate-induced Ca2+ release from mitochondria occurs through a non-specific ‘pore’ formed in the mitochondrial inner membrane. The aim of the present study was to investigate whether ‘pore’ formation actually is required for Ca2+ release. We find that the t-butyl hydroperoxide (tbh)-induced release is not accompanied by stimulation of sucrose entry into, K+ release from, and swelling of mitochondria provided re-uptake of the released Ca2+ (‘Ca2+ cycling’) is prevented. We conclude that (i) the tbh-induced Ca2+ release from rat liver mitochondria does not require ‘pore’ formation in the mitochondrial inner membrane, (ii) this release occurs via a specific pathway from intact mitochondria, and (iii) a non-specific permeability transition (‘pore’ formation) is likely to be secondary to Ca2+ cycling by mitochondria.
The effect of methyl-lidocaine on the biosynthesis of phospholipids de novo in the isolated hamster heart
Authors: Tardi P G , Man R Y K , Choy P C
Published: August 2015

Methyl-lidocaine is an amphiphilic agent which has been used as an experimental anti-arrhythmic drug. When hamster hearts were perfused with labelled glycerol, the presence of methyl-lidocaine in the perfusate was found to enhance the labelling in phosphatidylserine, phosphatidylinositol, diacylglycerol and triacylglycerol. However, the labelling of phosphatidylcholine and phosphatidylethanolamine was not significantly changed by methyl-lidocaine treatment. Assays in vitro for the enzymes involved in the synthesis of neutral lipids and acidic phospholipids revealed that phosphatidate phosphatase and CTP: phosphatidate cytidylyltransferase activities were stimulated by methyl-lidocaine. The intracellular pool sizes of diacylglycerol and CDP-diacylglycerol were also elevated. We postulate that the enhanced syntheses of the neutral lipids and acidic phospholipids in the methyl-lidocaine-perfused heart were mediated via the direct activation of the key enzymes in the biosynthesis of these lipids de novo.
Differential effects of platelet-derived growth factor, serum and bombesin on phospholipase D-mediated hydrolysis of phosphatidylethanolamine in NIH 3T3 fibroblasts
Authors: Kiss Z
Published: August 2015

In previous studies, activators of protein kinase C, sphingosine, ATP and various oncogenes were each found to enhance phospholipase D-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts. Here I examined possible stimulation of PtdEtn hydrolysis by various growth-stimulatory agents, including serum, bombesin, platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and insulin. Treatment of NIH 3T3 fibroblasts, prelabelled with [14C]Etn or [32P]PtdEtn, with PDGF-BB resulted in enhanced formation of [14C]Etn or [32P]phosphatidic acid from the respective labelled cellular pools of PtdEtn. A maximal effect (approximately 3-fold stimulation) on PtdEtn hydrolysis was obtained with 50 ng of PDGF/ml after 5 min of treatment. Phosphatidylcholine (PtdCho) was also hydrolysed, although less extensively than PtdEtn, in PDGF-stimulated cells. PDGF-stimulate hydrolysis of both PtdEtn and PtdCho was prevented by prolonged (30 h) treatment of cells with 400 nM-phorbol 12-myristate 13-acetate (PMA). Similar to PDGF, fetal calf serum (1-10%) also stimulated PtdEtn hydrolysis. However, in contrast to PDGF, the effect of serum on PtdEtn hydrolysis (i) was not diminished by pretreatment with PMA, and (ii) was synergistic with that of PMA after a 1 h incubation. Compared with PDGF and serum, bombesin had less effect on PtdEtn hydrolysis, while FGF and insulin had no effects at all. In contrast to PDGF or serum, bombesin inhibited the effect of PMA on PtdEtn hydrolysis.
Activation of 6-phosphofructo-2-kinase by pp60v-src is an indirect effect
Authors: Marchand M J , Maisin L , Hue L , Rousseau G G
Published: August 2015

6-Phosphofructo-2-kinase (PFK-2) catalyses the synthesis of fructose 2,6-bisphosphate (Fru-2,6-P2), a potent stimulator of glycolysis. In chick-embryo fibroblasts, PFK-2 activity and Fru-2,6-P2 concentration increase upon transformation by Rous sarcoma virus. We show here that the increase in PFK-2 activity required more than 2 h after shifting fibroblasts infected with a thermosensitive mutant of Rous sarcoma virus from the restrictive to the permissive temperature. Pretreatment of the cells with actinomycin D prevented this increase in PFK-2 activity, suggesting a requirement for RNA synthesis. However, the increase in PFK-2 activity did not correspond to an increase in immunoprecipitable PFK-2. Moreover, the thermostability of PFK-2 and the affinity of this enzyme for its substrate fructose 6-phosphate were increased upon transformation by Rous sarcoma virus. Staurosporine, an inhibitor of protein kinase C, prevented the increase in PFK-2 activity brought about by the shift to the permissive temperature. This, together with a comparison of the effects of phorbol esters on PFK-2 activity, suggests that pp60v-src stimulates, via protein kinase C, the transcription of a gene whose products is a distinct PFK-2 isoenzyme or a protein that activates PFK-2.
Ceramide glycanase from the earthworm, Lumbricus terrestris
Authors: Carter B Z , Li S C , Li Y T
Published: August 2015

Ceramide glycanase (CGase) is an enzyme that cleaves the linkage between the sugar chain and the ceramide. To make this enzyme readily available, we have developed a simple method for preparing it from the earthworm, Lumbricus terrestris. The method involves Bio-Gel A-0.5m, octyl-Sepharose and p-aminophenylthiogalactoside-agarose column chromatography. By gel filtration, the molecular mass of earthworm CGase was found to be 43.7 kDa. With ganglioside GM1 as substrate, the optimal pH of this enzyme was found to be between pH 3.5 and 4.0. Earthworm CGase hydrolyses glycolipids only in the presence of a detergent. Among various bile salts tested, sodium cholate was found to be the most effective in stimulating the hydrolysis of GM1 by this enzyme. Earthworm CGase released intact glycan chains from various glycosphingolipids in which the glycan chain is linked to the ceramide through a beta-glucosyl linkage. It also detached glycan chains from lactosyldialkylglycerol and alkyl-beta-lactosides.
Involvement of GTP in cell-free activation of neutrophil NADPH oxidase. Studies with GTP analogues
Authors: Klinger E , Aviram I
Published: August 2015

Activation of superoxide-producing NADPH oxidase of neutrophils requires the presence of cell membranes, cytosolic components and arachidonate and is markedly enhanced by non-hydrolysable analogues of guanine nucleotides, i.e. guanosine 5′-[gamma-thio]triphosphate and guanosine 5′[beta gamma-imido]triphosphate (p[NH]ppG). Gel filtration and ultrafiltration of the cytosol decreased the basal activity of NADPH oxidase. Activity could be restored by GTP, suggesting participation of the nucleotide in basal activation. Preincubation of neutrophil cytosol with periodate-oxidized p[NH]ppG (ox-p[NH]ppG) followed by gel filtration resulted in a time-dependent enhancement of basal oxidase activity. The presence of GDP or GTP, but not ATP, during the incubation with ox-p[NH]ppG abolished this enhancement. These data are consistent with a stable association of ox-p[NH]ppG with an oxidase-linked cytosolic protein. SDS/PAGE of neutrophil cytosol preincubated with [3H]ox-p[NH]ppG revealed radioactivity in bands migrating as 100, 70, 47, 34 and 22 kDa proteins. Evidence for covalent labelling of the cytosolic protein p47-phox with [3H]ox-p[NH]ppG is presented. Heterogeneity of cytosolic GTP-binding sites and possible participation of protein p47-phox in functional interaction with GTP analogues during cell-free activation are suggested.
Anti-(14–3–3 protein) antibody inhibits stimulation of noradrenaline (norepinephrine) secretion by chromaffin-cell cytosolic proteins
Authors: Wu Y N , Vu N D , Wagner P D
Published: August 2015

Incubation of digitonin-permeabilized bovine chromaffin cells in the absence of Ca2+ results in a loss of both cytosolic proteins and Ca(2+)-dependent secretion. Addition of these leaked proteins prevents this loss of secretory activity. We have purified a protein from an extract of bovine adrenal medulla which can partially prevent this loss of Ca(2+)-dependent secretion. Antibody against this protein inhibited the ability of leaked chromaffin-cell proteins to prevent the loss of Ca(2+)-dependent secretion. Sequence analysis showed it to have sequence identity with bovine brain 14-3-3 protein. These results demonstrate that 14-3-3 protein makes a significant contribution to the ability of leaked chromaffin-cell proteins to maintain secretory activity.
Characterization of the ileal Na+/bile salt co-transporter in brush border membrane vesicles and functional expression in Xenopus laevis oocytes
Authors: Mullins J G L , Beechey R B , Gould G W , Campbell F C , Shirazi-Beechey S P
Published: August 2015

The Na+/bile salt co-transporter of the pig ileal brush border membrane has been expressed in Xenopus laevis oocytes. Injection of pig ileal poly (A)+ RNA into oocytes resulted in the functional expression of an Na(+)-gradient-stimulated taurocholate uptake within 2-5 days. The expressed Na(+)-dependent taurocholate uptake exhibited saturation kinetics (apparent Km of 48 microM), and displayed similar competitive substrate inhibition by taurodeoxycholate as the native brush border Na+/bile salt co-transporter studied in pig ileal brush border membrane vesicles. Interestingly, injection of pig proximal and mid intestinal poly (A)+ RNA into oocytes also resulted in the expression of the Na+/bile salt co-transporter, though the Na(+)-dependent transport of bile salts does not occur in brush border membrane vesicles (BBMV) isolated from pig proximal and mid intestine. This suggests that the mRNA coding for the co-transporter is present in the enterocytes lining the whole length of the small intestine, but that the function is only expressed in the brush border of the distal small intestine. The transport of D-glucose into BBMV, and the transport of methyl-alpha-D-glucopyranoside (a non-metabolizable hexose derivative) into oocytes were used throughout the study as methods of confirming the integrity of vesicles and oocytes.
Changes in keratin expression during fetal and postnatal development of intestinal epithelial cells
Authors: Calnek D , Quaroni A
Published: August 2015

We have investigated keratin expression in fetal, newborn and adult rat intestines by immunofluorescence staining, immunoblotting of two-dimensional gels and Northern blot analysis of total cellular RNAs. Keratin-type intermediate filaments, composed predominantly of keratin no. 19, were observed already in the undifferentiated stratified epithelium present at 15-16 days of gestation. The marked maturation and differentiation of the epithelium taking place at 18-19 days of gestation was characterized by the appearance of the differentiation-specific keratin no. 21 and by a significant increase in the relative amount of keratin no. 8. The keratin pattern typical of adult villus cells became established at the time of birth, and was marked by a considerable increase in the complexity of the keratin-related polypeptides detected on two-dimensional gels, indicative of extensive post-translational modification of all keratins. Starting at 20 days of gestation there was a major increase in the relative abundance of mRNAs coding for keratin nos. 8, 19 and 21; in contrast, the relative amount of keratin no. 18 mRNA reached a peak shortly after birth and declined to very low levels in adult intestine. These results demonstrated marked changes in keratin expression and post-translational processing taking place at key stages of intestinal development. The appearance of keratin no. 21 in coincidence with the formation of an adult-type brush border and terminal web would be consistent with it having an important role in the organization of the intermediate filament network in the apical cytoplasm of the differentiated intestinal cells.
Inositol monophosphatase is a highly conserved enzyme having localized structural similarity to both glycerol 3-phosphate dehydrogenase and haemoglobin
Authors: Wreggett K A
Published: August 2015

The cDNA coding for an inositol monophosphatase in the oocytes of the African clawed frog, Xenopus laevis, has been isolated and sequenced. The predicted primary structure of this enzyme is markedly conserved when it is compared with its mammalian functional homologues; up to 84% of the amino acid residues are identical, and conservative substitutions increase the similarity to 95%, suggesting that this sequence represents the most parsimonious primary structure for the protein to maintain not only catalytic activity but also perhaps the facility to interact with other macromolecules. Two regions of the protein, each of about 11 residues and separated by about 90 residues, have been identified as a consensus found also in glycerol 3-phosphate dehydrogenase (EC 1.1.1.8). One of these regions is also found to be particularly conserved in the alpha-globin of birds and reptiles; birds and some turtles are known to modulate the oxygen affinity of their haemoglobin with inositol polyphosphate in the same way as with 2,3-bisphosphoglycerate in other species. This region is also conserved in the beta-globin of most species, beginning with lysine-82, which is known to participate in the binding of organic phosphates. These regions of the inositol monophosphatase may represent motifs for the binding of its substrate.
A large decrease in heat-shock-induced proteolysis after tryptophan starvation leads to increased expression of phage λ lysozyme cloned in Escherichia coli
Authors: Soumillion P , Fastrez J
Published: August 2015

The R gene coding for phage lambda lysozyme (lambda L), cloned under the control of the PL promoter on a multicopy vector, is expressed in an Escherichia coli strain auxotrophic for tryptophan. Induction by a thermal shift after tryptophan supplementation in a culture initially brought into stationary phase by tryptophan starvation leads to highly increased expression. A thermally unstable mutant protein, difficult to obtain under standard conditions, can be easily produced by post-stationary-phase expression. It is shown that this is due to a drastic decrease in the heat-shock-induced proteolysis normally observed on thermal induction. These data are discussed in relation to our present knowledge of stringent and heat-shock responses.
Characterization of a Zn2+-requiring glycerophosphocholine cholinephosphodiesterase possessing p-nitrophenylphosphocholine phosphodiesterase activity
Authors: Sok D E , Kim M R
Published: August 2015

p-Nitrophenylphosphocholine phosphodiesterase activity was purified 5000-fold from mouse brain by treatment of membranes with Bacillus cereus phospholipase C preparation and sequential chromatographies on concanavalin A-Sepharose and CM-Sephadex columns. The phosphodiesterase (Zn(2+)-requiring) showed Km and Vmax. values of 5.5 microM and 4.2 mumol/min per mg respectively in the hydrolysis of p-nitrophenylphosphocholine, and possessed an optimum pH of 10.5 and a molecular mass of approx. 74 kDa. The purified enzyme was found to convert glycerophosphocholine into glycerol and phosphocholine, with Km and Vmax. of 48 microM and 5 mumol/min per mg respectively. In the hydrolysis of glycerophosphocholine the enzyme also exhibited a Zn2+ requirement and optimal pH at 10.5. Additionally, the p-nitrophenylphosphocholine phosphodiesterase activity was competitively inhibited by glycerophosphocholine, with a Ki value of 50 microM. These observations, together with chromatographic behaviour and heat-denaturation analyses, indicate that both p-nitrophenylphosphocholine phosphodiesterase and glycerophosphocholine cholinephosphodiesterase activities reside in the same protein.