bluuebunny/crossref_metadata_2025 huggingface dataset:
Different properties of monomer and heterodimer forms of phosphatidylinositol 3-kinases
Authors: Shibasaki F , Fukui Y , Takenawa T
Published: August 2015

Phosphatidylinositol (PI) 3-kinase plays an important role in the signalling of cell growth. We previously purified two types of PI 3-kinase from bovine thymus, a monomer from (PI 3-kinase I) and a heterodimer form (PI 3-kinase II) [Shibasaki, Homma and Takenawa (1991) J. Biol. Chem. 266, 8108-8114]. Here we examine the properties of these purified PI 3-kinases. Both PI 3-kinases were inhibited strongly by quercetin and isoquercetin. The inhibition of PI 3-kinase I and PI 3-kinase II by quercetin appears to be non-competitive, with apparent Ki values of 4 microM and 2.5 microM respectively. PI 3-kinase II, but not PI 3-kinase I, co-immunoprecipitates with pp60v-src and polyoma middle T (mT)/pp60c-src, even under conditions where the PI 3-kinases are not phosphorylated, suggesting that non-phosphorylated PI 3-kinase recognizes autophosphorylated pp60v-src. PI 3-kinase II is phosphorylated by pp60v-src and binds to it. Anti-p85 (85 kDa subunit of PI 3-kinase II) antibody precipitates not only PI 3-kinase II but also co-immunoprecipitates pp60v-src in src-transformed cells, suggesting that PI 3-kinase II binds to pp60v-src in vivo. These data suggest that the two PI 3-kinases may be regulated independently.
Regulation of cytosolic 3-hydroxy-3-methylglutaryl-CoA synthase mRNA levels by l-tri-iodothyronine
Authors: Royo T , Haro D , Hegardt F G
Published: August 2015

In hypophysectomized and thyroidectomized rats, cytosolic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase activity, immunoreactive protein and mRNA levels were all considerably decreased. Administration of L-tri-iodothyronine (T3) resulted in a large increase in all three in hypophysectomized rats and in only a 2-fold increase (reaching the values of control rats) in thyroidectomized rats.
Eicosanoid/thromboxane A2-independent and -dependent generation of lysoplasmenylethanolamine via phospholipase A2 in collagen-stimulated human platelets
Authors: Turini M E , Holub B J
Published: August 2015

Collagen-induced human platelet stimulation is dependent on the release of arachidonic acid (AA) from membrane phospholipid and the formation of thromboxane A2 (TxA2) for TxA2-induced platelet activation. Since plasmenylethanolamine represents the single major phospholipid reservoir of AA in resting human platelets, we assessed its hydrolysis via phospholipase A2 upon platelet stimulation with low levels of collagen by determining the generation of [3H]lysoplasmenylethanolamine via eicosanoid/TxA2-independent and -dependent processes. Ethanolamine phospholipids in platelets were prelabelled with [3H]ethanolamine before stimulation with either collagen or the TxA2 mimetic U46619, in the presence or absence of BW755C, a dual inhibitor of the cyclooxygenase/lipoxygenase activities, or GR32191B, a TxA2-receptor antagonist. Collagen stimulation promoted a marked generation of [3H]lysoplasmenylethanolamine, which was only moderately decreased when TxA2 synthesis or TxA2 receptors were blocked by BW755C or GR32191B respectively. The moderate rise in [3H]lysoplasmenylethanolamine formation with U46619 as the agonist was only slightly affected by BW755C and blocked by GR32191B. Evidence for eicosanoid/TxA2-independent and -dependent generation of [3H]lysophosphatidylethanolamine was also obtained. A significant quantitative loss of AA from plasmenylethanolamine was also demonstrated in collagen-stimulated platelets. The present findings indicate the activation of plasmenylethanolamine cleavage via phospholipase A2 in collagen-stimulated human platelets, which, to a considerable extent, does not depend on eicosanoid/TxA2 synthesis. This may represent an important source of releasable AA for TxA2 generation and the promotion of further liberation of AA and phospholipid-mediated signalling pathways.
Generation of inositol phosphates, diacylglycerol and calcium fluxes in myoblasts treated with 1,25-dihydroxyvitamin D3
Authors: Morelli S , de Boland A R , Boland R L
Published: August 2015

We have examined the effects of the seco-steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on membrane phosphoinositide metabolism, protein kinase C (PKC) activation and influx of extracellular Ca2+ in chick-embryo muscle-cell (myoblast) cultures. At physiological concentrations, the hormone induces a rapid (15 s) and transient release of inositol triphosphate (InsP3) and diacylglycerol (DAG). InsP3 release is maximal at 60 s (80% above controls) and then declines. The effects of 1,25(OH)2D3 on InsP3 production exhibited specificity, as 25-hydroxy-vitamin D3 and 24,25-dihydroxy-vitamin D3 did not alter myoblast InsP3 levels. The stimulation of DAG is biphasic, with peaks at 60 s (+105%) and 5 min (+700%). The second phase of DAG release is not associated with changes in InsP3. 1,25(OH)2D3 induces a rapid (within 60 s) accumulation of InsP2, and its effect on InsP is delayed (120 s). The hormone rapidly activates myoblast PKC, with maximal translocation of activity from the cytosol to the cell membrane occurring at 60 s. Myoblast 45Ca uptake significantly increases within 30 s of exposure to 1,25(OH)2D3. The response is time- (0.5-10 min) and dose- (1 pM-10 nM) dependent. The effects of the hormone are mimicked by the Ca(2+)-channel agonist Bay K 8644 and are effectively suppressed by nifedipine and extracellular EGTA. The results suggest that the rapid non-genomic actions of 1,25(OH)2D3 in myoblasts involve second-messenger systems associated with the generation of InsP3 and DAG and regulation of Ca2+ fluxes through voltage-operated channels.
1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene, an extremely potent modulator of mouse hepatic cytochrome P-450 gene expression
Authors: Smith G , Henderson C J , Parker M G , White R , Bars R G , Wolf C R
Published: August 2015

We have investigated the effects of TCPOBOP (1,4-bis[2-(3,5- dichloropyridyloxy)]benzene), a potent cytochrome P-450-inducing agent [Poland, Mak, Glover, Boatman, Ebetino and Kende (1980) Mol. Pharmacol. 18, 571-580], on cytochrome P-450 isoenzyme expression in the mouse. Hepatic cytochrome P-450s from several distinct gene families were strikingly induced by a single dose of 75 micrograms of the compound. Northern-blot analysis demonstrated that this induction was almost certainly due to transcriptional activation of the cytochrome P-450 genes. The potency of this inductive effect was further reflected in the finding that cytochrome P-450 levels were still increased 12 weeks after a single injection of 75 micrograms of this compound. Interestingly, the mRNA levels of certain other genes, including those of metallothionein and the mouse major urinary proteins, were also induced. In view of the similarity in the effects of TCPOBOP and the synthetic glucocorticoid dexamethasone on mouse hepatic gene expression, we determined whether TCPOBOP acts through the glucocorticoid receptor. This did not, however, appear to be the case. Experiments with hypophysectomized animals demonstrated that TCPOBOP action was not regulated indirectly via the pituitary. In addition, induction of mouse Cyp2b protein by TCPOBOP in a primary culture of mouse hepatocytes suggests that the compound has a direct action on mouse liver. The above findings demonstrate that TCPOBOP is one of the most potent modulators of cytochrome P-450 gene expression described to date. It is not inconceivable that a single dose of this compound may alter hepatic gene expression for the majority of the lifespan of a mouse.
Identification of monomeric α-macroglobulin proteinase inhibitors in birds, reptiles, amphibians and mammals, and purification and characterization of a monomeric α-macroglobulin proteinase inhibitor from the American bullfrog Rana catesbiana
Authors: Rubenstein D S , Thøgersen I B , Pizzo S V , Enghild J J
Published: August 2015

The alpha-macroglobulins are classified as broad-spectrum inhibitors because of their ability to entrap proteinases of different specificities and catalytic class. Tetrameric and dimeric alpha-macroglobulins have been identified in a wide variety of organisms including those as primitive as the mollusc Octopus vulgaris; however, monomeric alpha-macroglobulin proteinase inhibitors have been previously identified only in rodents. The monomeric alpha-macroglobulin proteinase inhibitors are believed to be analogous to the evolutionary precursor of the multimeric members of this family exemplified by the tetrameric human alpha 2-macroglobulin. Until now, monomeric alpha-macroglobulin proteinase inhibitors have only been identified in rodents and have therefore been considered an evolutionary anomaly. However, in this report we have utilized several sensitive assays to screen various plasmas and sera for the presence of monomeric alpha-macroglobulins, and our results suggest that monomeric alpha-macroglobulin proteinase inhibitors are present in organisms belonging to the avian, reptilian, amphibian and mammalian classes of the chordate phylum. This indicates that these proteins are more widespread than previously recognized and that their presence in rodents is not an anomaly. To demonstrate further that the identified proteins were indeed monomeric alpha-macroglobulin proteinase inhibitors, we purified the monomeric alpha-macroglobulin from the American bullfrog Rana catesbeiana. We conclude that this protein is a monomer of 180 kDa on the basis of its behaviour on (i) pore-limit gel electrophoresis, (ii) non-reducing and reducing SDS/PAGE and (iii) gel-filtration chromatography. In addition, we demonstrate that this protein is an alpha-macroglobulin proteinase inhibitor by virtue of (i) its ability to inhibit proteinases of different catalytic class, (ii) the presence of a putative internal beta-cysteinyl-gamma-glutamyl thioester and (iii) an inhibitory mechanism characterized by steric protection of the proteinase active site and by sensitivity to small primary amines. The frog monomeric alpha-macroglobulin is structurally and functionally similar to the well-characterized monomeric alpha-macroglobulin proteinase inhibitor rat alpha 1-inhibitor-3.
Effects of chronic 5′-{[(Z)-4-amino-2-butenyl]methylamino)-5′-deoxy- adenosine (AbeAdo) treatment on polyamine and eIF-5A metabolism in AbeAdo-sensitive and -resistant L1210 murine leukaemia cells
Authors: Byers T L , Wiest L , Wechter R S , Pegg A E
Published: August 2015

We have previously reported that prolonged chronic exposure to the S-adenosyl-L-methionine decarboxylase (AdoMetDC) inhibitor, 5′-([(Z)-4-amino-2-butenyl]methylamino)-5′-deoxy-adenosine (MDL 73811, AbeAdo), leads to cytostasis of L1210 cells [Byers, Ganem and Pegg (1992) Biochem. J. 287, 717-724]. Further studies to investigate the mechanism by which these effects are brought about were carried out by comparing an L1210-derived cell line (R20) that is resistant to AbeAdo with the parent cells. The R20 cells were derived by two rounds of AbeAdo-induced cytostasis followed by rescue with exogenous polyamines. Cytostasis was induced in L1210 cells treated for 12 days with 10 microM AbeAdo; however, exposure to up to 40 microM AbeAdo did not induce cytostasis in R20 cells. Putrescine levels were elevated and spermine levels were depleted in both treated L1210 and treated R20 cells. Spermidine was depleted in treated L1210 cells but was only partly reduced in treated R20 cells. AdoMetDC activity was below the limit of detection in treated L1210 cells but, although greatly reduced, could be measured in the treated R20 cells. The resistance of the R20 cells to the effects of AbeAdo on cell growth and spermidine depletion correlated with reduced AbeAdo accumulation by R20 cells. In the absence of spermidine synthesis, unhypusinated eukaryotic translation initiation factor 5A (eIF-5A) accumulated in AbeAdo-treated L1210 cells. There was no detectable accumulation of unhypusinated eIF-5A in R20 cells. Unhypusinated eIF-5A accumulated during AbeAdo treatment was depleted in L1210 cells rescued by exogenous spermidine. These findings are consistent with the hypothesis that AbeAdo-induced cytostasis is due to the loss of hypusinated eIF-5A. However, spermine was able to rescue AbeAdo-treated L1210 cells without significantly reducing the unhypusinated eIF-5A accumulated during AbeAdo treatment, suggesting that only a small amount of the unmodified protein must be hypusinated to restore cell growth.
Relationship between phosphorylation and translocation to the plasma membrane of p47phox and p67phox and activation of the NADPH oxidase in normal and Ca2+-depleted human neutrophils
Authors: Dusi S , Della Bianca V , Grzeskowiak M , Rossi F
Published: August 2015

Stimulation of neutrophils with different agonists activates a latent multicomponent NADPH oxidase that reduces molecular oxygen to superoxide anion. Evidence has accumulated that phosphorylation of p47phox (the 47 kDa cytosolic phagocyte oxidase factor) and translocation of the two cytosolic components p47phox and p67phox are essential steps in the activation of NADPH oxidase in response to phorbol esters. We analysed the relationships between activation of the NADPH oxidase and phosphorylation and translocation of p47phox and p67phox in normal and Ca(2+)-depleted neutrophils stimulated by the receptor-mediated agonists formyl-methionyl-leucyl-phenylalanine and concanavalin A. The results produced the following conclusions: (1) Translocation of p47phox and p67phox is an essential mechanism for activation of the NADPH oxidase. (2) A continuous translocation of p47phox and p67phox is necessary to maintain the NADPH oxidase in an activated state. (3) Only a fraction of p47phox and p67phox translocated to the plasma membrane is functional for the activation of the oxidase. (4) Translocation is independent of protein kinase C, and is linked to transmembrane signalling involving Ca2+ transients and production of lipidic second messengers. However, under some conditions, such as in Ca(2+)-depleted neutrophils, translocation can also occur independently of signalling pathways involving production of second messengers from hydrolysis of phospholipids and Ca2+ transients. (5) Phosphorylation of p47phox and p67phox can be quantitatively dissociated from translocation, as staurosporine markedly inhibits phosphorylation but not translocation. (6) The activity of NADPH oxidase is not correlated with the amounts of the phosphorylated proteins present in the plasma membrane.
Tumour necrosis factor-induced cytotoxicity is accompanied by intracellular mitogenic signals in ME-180 human cervical carcinoma cells
Authors: Manchester K M , Heston W D W , Donner D B
Published: August 2015

Tumour necrosis factor-alpha (TNF) induced a cytotoxic response in ME-180 human cervical carcinoma cells in vitro. This cytotoxic response was accompanied by a temporal series of intracellular signals that are commonly triggered by a mitogenic stimulus: increased c-fos (20-30 min) and c-myc (40-60 min) expression, increased activity of ornithine decarboxylase (3 h), increased intracellular polyamine content (7 h) and increased thymidine incorporation into DNA (14 h). A cytotoxic response independent of these mitogenic signals could not be explained by an induction of interleukin-6, which is an autocrine cytotoxic agent in some cell types; nor by a biphasic, dose-dependent response in which low concentrations of TNF are mitogenic and higher concentrations are cytotoxic. Conversely, a dependent role of these mitogenic signals was suggested by the absence of a TNF-promoted increase in thymidine incorporation into DNA in an ME-180 clone that is resistant to TNF-induced cytotoxicity. A decrease in the proliferation rate of TNF-sensitive cells induced by either alpha-difluoromethylornithine treatment (resulting in polyamine depletion) or serum starvation rendered the cells insensitive to TNF-induced cytotoxicity, further suggesting a role for mitogenic signals and cell division in TNF-mediated cytotoxicity. However, inhibiting proliferation with cycloheximide resulted in increased sensitivity to TNF, implying that mitogenesis itself was not essential for a cytotoxic response. TNF induced DNA fragmentation in sensitive cells, suggesting that cytotoxicity occurred via apoptosis.
Isolation and characterization of AMP deaminase from mammalian (rabbit) myocardium
Authors: Thakkar J K , Janero D R , Yarwood C , Sharif H , Hreniuk D
Published: August 2015

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) is a ubiquitous enzyme in eukaryotes, which may play a role in ATP catabolism during myocardial ischaemia. We report isolation of AMP deaminase from rabbit myocardium with a 19% recovery and a 650-fold enrichment, using a newly devised protocol involving sequential cation-exchange, gel-permeation and affinity chromatographies. The cardiac AMP deaminase preparation described was electrophoretically and chromatographically homogeneous and contained one unique N-terminal residue (leucine). The isolated enzyme was sensitive to various cations (K+, Mg2+, Ca2+). The pH optimum of purified cardiac AMP deaminase was 6.8, its pI was 6.5, and it displayed substrate-specificity toward 5′-AMP. The subunit molecular mass of rabbit heart AMP deaminase on SDS/PAGE (81 kDa) and the holoenzyme molecular mass as estimated by non-denaturing size-exclusion h.p.l.c. (330 kDa) indicated that the native enzyme was a tetramer. Cardiac AMP deaminase displayed a sigmoidal substrate-saturation curve in the presence of 100 mM KCl. Apparent Michaelis constants were a Km of 5.8 mM AMP and a Vmax. of 11.1 mumol/min per mg of protein. ATP and ADP were positive allosteric effectors of cardiac AMP deaminase: the apparent Km was decreased to 1.7 mM by 1.0 mM ATP. The enzyme was inhibited by GTP, coformycin, coformycin 5′-phosphate, palmitoyl-CoA, inorganic phosphate compounds, and the metal chelator o-phenanthroline. No inhibition either by product nucleotide (IMP) or by nicotinamide nucleotides was detected when these agents were examined at concentrations up to 2.5 mM. We conclude that this enzyme preparation offers a means by which the kinetic mechanism and regulation of mammalian cardiac AMP deaminase may be directly investigated.
Snake venom cardiotoxin can rapidly induce actin polymerization in intact platelets
Authors: Liou R F , Chang W C , Chu S T , Chen Y H
Published: August 2015

The action of Taiwan cobra (Naja naja atra) venom cardiotoxin on rabbit platelets at 37 degrees C was characterized by observing cytoskeletal alterations and cell lysis. At a concentration of 21.4 microM the toxin produced no cell lysis within 30 s, and less than 5% of the total lactate dehydrogenase activity of intact cells was detected in the suspending medium after the interaction had proceeded for 3 min. The extent of cell lysis was proportional to toxin concentration and interaction time. Before cell lysis, the toxin caused rapid incorporation of actin monomers into cross-linked actin filaments. The actin incorporation could be inhibited by either the presence of cytochalasin B or increased CaCl2 concentration in the suspending medium. However, addition of indomethacin did not influence the toxin-induced cytoskeletal change.
Characterization of three alleles causing aspartylglycosaminuria: two from a British family and one from an American patient
Authors: Park H , Vettese M B , Fensom A H , Fisher K J , Aronson N N
Published: August 2015

Aspartylglycosaminuria (AGU) is a lysosomal storage disease principally occurring in Finland that results from mutations in the structural gene for glycosylasparaginase (AGU). This work characterizes the inheritance of two previously reported AGU mutations in a British patient [Ikonen, Aula, Grön, Tollersrud, Halila, Manninen, Syvänen and Peltonen (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 11222-11226]. Use of the PCR determined the glycosylasparaginase cDNA sequence from both parents of the British patient and his AGU-affected brother. The father of the British AGU-affected siblings was found to be a heterozygote carrier for a C-->T point mutation which causes an Ala-->Val amino-acid substitution, while the mother was heterozygous for a 7 bp deletion that results in premature translational termination. The brother of the previously studied patient was similarly shown to be a compound heterozygote. Expression in COS-1 cells revealed the paternal Ala-->Val amino-acid substitution destroyed glycosylasparaginase catalytic activity, prevented transport of the mutant protein to the lysosome, and prevented maturation of the enzyme precursor to its native subunit structure. The Ala-->Val mutation therefore affects glycosylasparaginase in a manner similar to the Finnish AGU Cys-->Ser substitution, further supporting a linkage of glycosylasparaginase catalytic activity to its lysosomal transport and subunit processing [Fisher and Aronson (1991) J. Biol. Chem. 266, 12105-12113]. In addition, a 5 bp deletion mutation from an American patient with AGU has been characterized. The deleted sequence occurs at the beginning of the glycosylasparaginase coding sequence, resulting in an extremely truncated polypeptide. The American 5 bp deletion and the British maternal 7 bp deletion possibly decrease mRNA stability.
Deduced amino acid sequence, gene structure and chromosomal location of a novel human class Mu glutathione S-transferase, GSTM4
Authors: Zhong S , Spurr N K , Hayes J D , Wolf C R
Published: August 2015

The Mu-Class glutathione S-transferases (GSTs) are subject to marked inter-individual variation in man, owing to the fact that 40-50% of the population fail to express M1 subunits. Mu-Class GST from two lymphoblastoid cell lines (expressing M1 subunits and the other ‘nulled’ for M1) have been studied. Both cell lines were found to express a Mu-Class GST that has not been described previously. The cDNA encoding this novel transferase, designated ‘GSTM4’ has been isolated and the enzyme shown to be comprised of 218 amino acids (including the initiator methionine residue) with an M(r) of approx. 25.5 kDa. Molecular cloning demonstrated that the lymphoblastoid cell line which expressed GSTM1 possessed the b allelic variant (i.e. that with an asparagine residue at position 173). The genes for GSTM4 and GSTM1b have been cloned and found to contain seven introns and eight exons. The coding region of the GSTM4 gene, including the seven introns, encompasses 5.0 kb, whereas the same region of GSTM1b is 5.5 kb; the difference in the size of the two genes is due to the length of intron 7. DNA sequencing allowed a GSTM4-gene-specific oligo-primer to be designed which has been utilized in a PCR-based assay to determine that the GSTM4 gene is located on chromosome 1.
Conformational changes at the active site of creatine kinase at low concentrations of guanidinium chloride
Authors: Zhou H M , Zhang X H , Yin Y , Tsou C L
Published: August 2015

It has been previously reported that, during denaturation of creatine kinase by guanidinium chloride (GdmCl) or urea [Tsou (1986), Trends Biochem. Sci. 11, 427-429], inactivation occurs before noticeable conformational change can be detected, and it is suggested that the conformation at the active site is more easily perturbed and hence more flexible than the molecule as a whole. In this study, the thiol and amino groups at or near the active site of creatine kinase are labelled with o-phthalaldehyde to form a fluorescent probe. Both the emission intensity and anisotropy decrease during denaturation indicating exposure of this probe and increased mobility of the active site. The above conformational changes take place together with enzyme inactivation at lower GdmCl concentrations than required to bring about intrinsic fluorescence changes of the enzyme. At the same GdmCl concentration, the rate of exposure of the probe is comparable with that of inactivation and is several orders of magnitude faster than that for the unfolding of the molecule as a whole.
Biosynthesis, surface expression and function of the fibronectin receptor after rat liver cell transformation to tumorigenicity
Authors: Decastel M , Doyennette-Moyne M A , Gouet E , Aubery M , Codogno P
Published: August 2015

Zajdela hepatoma cells are poorly-adherent cells derived from an undifferentiated tumour and transplanted into rat. We compared the biosynthesis, structure and function of the fibronectin receptor in normal rat hepatocytes with that in Zajdela hepatoma cells. The rat hepatocyte fibronectin receptor has been isolated. It is composed of two subunits: alpha 5 (molecular mass 155 kDa) and beta 1 (molecular mass 115 kDa). However, its biosynthesis has not yet been described. Using polyclonal antibodies raised against each of the subunits of the receptor, we observed that the alpha 5-subunit was synthesized as a 155-kDa polypeptide in normal rat hepatocytes and Zajdela hepatoma cells. In contrast, the molecular mass of the beta 1-subunit was 130 kDa in Zajdela hepatoma cells versus 115 kDa in normal rat hepatocytes. Pulse-chase experiments showed that the apparent transition time from the 100-kDa beta 1-precursor to the 130-kDa mature form was abnormally prolonged in Zajdela hepatoma cells since the latter was not detected until 24 h, while the transition from the 100-kDa precursor to the 115-kDa mature form began within 3 h in normal rat hepatocytes. Digestion of both the normal rat hepatocytes and Zajdela hepatoma cells 100-kDa beta 1-precursors with endo-beta-N-acetylglucosaminidase H and peptide N-glycosidase yielded products from 100 kDa to 84 kDa and 82 kDa, respectively, as judged by SDS/PAGE, suggesting that the same polypeptide chain is synthesized in normal rat hepatocytes and in Zajdela hepatoma cells. Incubation of the mature normal rat hepatocyte beta 1-subunit with peptide N-glycosidase reduced its molecular mass from 115 kDa to 82 kDa, as judged by SDS/PAGE, while the molecular mass of the abnormal mature Zajdela hepatoma cell beta 1-subunit decreased from 130 to 110 kDa. Thus, in addition to alterations in the Asn-linked oligosaccharide processing, ‘ascitic growth’ induced other post-translational modifications in the Zajdela hepatoma cell beta 1-subunit. Furthermore, both the abnormal mature 130-kDa and precursor 100-kDa beta 1-subunits were detected on the surface of Zajdela hepatoma cells, associated with the alpha 5-subunit. The relationship between these structural alterations in the fibronectin receptor and the impaired Zajdela hepatoma cell binding to soluble fibronectin or to a coated fibronectin matrix that was observed in this study is discussed.
Active transport of l-proline in the protozoan parasite Trypanosoma brucei brucei
Authors: L'Hostis C , Geindre M , Deshusses J
Published: August 2015

The characteristics of L-proline transport in the procyclic form of Trypanosoma brucei were studied by using L-[14C]proline and a quick separation technique by centrifugation through an oil mixture. L-Proline uptake displayed typical Michaelis-Menten kinetics, with a Km of 19 microM and a maximum transport velocity of 17 nmol/min per 10(8) cells at 27 degrees C. The maximum concentration gradient factor obtained after 1 min of incubation was 270-fold in 0.02 mM proline. Cells permeabilized with 80 microM digitonin were still able to accumulate 14C label, but to a lower extent. The temperature-dependence of proline uptake gave an apparent activation energy of 74.9 kJ.mol-1. In competition studies with a 10-fold excess of structural analogues, L-alanine, L-cysteine and L-azetidine-2-carboxylate were found to inhibit L-proline uptake. Variation of pH or addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (‘CCCP’) did not affect proline transport, showing that it is not driven by a protonmotive force. The absence of Na+, with or without monensin, did not affect proline transport. The absence of K+ and the addition of the Na+,K(+)-ATPase inhibitor ouabain had no significant effect on proline uptake activity. The thiol-modifying reagent iodoacetate (10 mM) decreased proline uptake by half. KCN (1 mM) inhibited proline uptake to a lesser extent, and the degree of inhibition was proportional to the intracellular ATP concentration. Preliminary experiments on proline transport in plasma-membrane vesicles of the cells, using a filtration technique, showed an uptake of proline (0.67 nmol/mg of protein) by the vesicles, but only in the presence of intravesicular ATP. The results thus obtained suggest that the proline carrier system in T. brucei is ATP-driven and independent of Na+, K+ or H+ co-transport.
Klebsiella pneumoniae nitrogenase MoFe protein: chymotryptic proteolysis affects function by limited cleavage of the β-chain and provides high-specific-activity MoFe protein
Authors: Fisher K , Lower D J , Pau R N
Published: August 2015

Proteinase treatment with chymotrypsin has been used to probe the structure of native Klebsiella pneumoniae nitrogenase MoFe protein (Kp1). Reaction with chymotrypsin did not bleach Kp1, suggesting that it did not destroy the metal centres, and the Mo and Fe contents of Kp1 were unchanged. High ratios of chymotrypsin to Kp1 (1:1 by mass) cleaved the beta-chain of Kp1 to give 44 and 14 kDa polypeptides, which N-terminal amino acid sequence analysis showed to be derived from cleavage at residue beta-Phe124. A mutant MoFe protein, Kp1Met-124, in which beta-Phe124 is replaced by methionine, was not cleaved by chymotrypsin. Under non-denaturing conditions, the ‘nicked’ beta-chain of the wild-type protein remained associated with the alpha-chain. The alpha-chain was not cleaved by the proteinase treatment. Fission of the wild-type beta-chain was accompanied by loss of enzyme activity, loss of intensity of the g = 3.7 e.p.r. signal derived from dithionite-reduced FeMoco and by changes in the visible spectrum. The e.p.r. spectra of potassium ferricyanide-oxidized native and digested Kp1 show differences in the signals between g = 1.6 and 2.0. After prolonged treatment, the final specific activity of Kp1 was about 25 +/- 5% of the initial activity. This corresponded to 25 +/- 5% of the beta-chain which was resistant to proteolytic action. Brief treatment of Kp1 with a lower concentration of chymotrypsin (chymotrypsin/Kp1 ratio = 1:10 by mass, for 10 min) preferentially cleaved high-molecular-mass polypeptides that routinely contaminate preparations of Kp1 prepared by standard procedures. Treatment with chymotrypsin followed by gel filtration to remove the proteinase and cleaved protein fragments can therefore be used to increase significantly the specific activity of Kp1 preparations and remove contaminating activities, such as the ATPase activity of myokinase.
Role of tryptophan-388 of GLUT1 glucose transporter in glucose-transport activity and photoaffinity-labelling with forskolin
Authors: Katagiri H , Asano T , Ishihara H , Lin J L , Inukai K , Shanahan M F , Tsukuda K , Kikuchi M , Yazaki Y , Oka Y
Published: August 2015

GLUT1 glucose-transporter cDNA was modified to substitute leucine for Trp-388 and transfected into Chinese hamster ovary cells using the expression vector termed pMTHneo. This tryptophan residue is conserved among most of the facilitative glucose-transporter isoforms and has been proposed to be the photolabelling site of forskolin, a competitive inhibitor of glucose transport. In addition, this residue is located on membrane-spanning helix 10 which is suggested to contain the dynamic segment of the transporter. The mutated glucose transporter was expressed and inserted into the plasma membrane in a fashion similar to the wild-type. Unexpectedly, this mutation did not abolish photolabelling with forskolin. However, the mutation induced a marked decrease in 2-deoxyglucose uptake with a 4-fold decrease in turnover number and a 1.25-fold increase in Km compared with the wild-type GLUT1. A similar decrease in zero-trans influx activity was also observed for 3-O-methylglucose. In contrast, no apparent decrease was observed in zero trans efflux activity for 3-O-methylglucose. The mutation decreased the turnover number of the glucose transporter in equilibrium exchange influx for 3-O-methylglucose by 33% without any change in Km. These results indicate that (1) Trp-388 is not the photolabelling site for forskolin, if we assume that the labelling occurs at a single site and (2) Trp-388 is more likely to be involved in interconversion between the inward-facing and outward-facing conformers of GLUT1 than binding of glucose, and thus, substitution of leucine for Trp-388 in this dynamic segment would decrease the rate of alternating conformation, which would preferentially affect the influx activity.
Inactivation of penicillin acylase from Kluyvera citrophila by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline: a case of time-dependent non-covalent enzyme inhibition
Authors: Martín J , Mancheño J M , Arche R
Published: August 2015

Penicillin acylase (PA) from Kluyvera citrophila was inhibited by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a specific carboxy-group-reactive reagent. Enzyme activity progressively decreased to a residual value depending on EEDQ concentration. Neither enzymic nor non-enzymic decomposition of EEDQ is concomitant with PA inactivation. Moreover, enzyme re-activation is achieved by chromatographic removal of EEDQ, pH increase or displacement of the reagent with penicillin G. It was then concluded that PA inactivation is due to an equilibrium reaction. The kinetics of enzyme inactivation was analysed by fitting data to theoretical equations derived in accordance with this mechanism. Corrections for re-activation during the enzyme assay were a necessary introduction. The pH-dependence of the rate constant for EEDQ hydrolysis either alone or in the presence of enzyme was studied by u.v. spectroscopy. It turned out to be coincident with the pH-dependence of the forward and reverse rate constants for the inactivation process. It is suggested that previous protonation of the EEDQ molecule is required for these reactions to occur. The thermodynamic values associated with the overall reaction showed little change. Finally it is proposed that the inactivation of PA by EEDQ proceeds through a two-step reaction. The initial and rapid reversible binding is followed by a slow, time-dependent, non-covalent, reversible inactivating step. The expected behaviour in the case of enzyme modification by covalent activation of carboxy residues is also reviewed.
The interaction of phospholipid bilayers with pig heart AMP deaminase: Fourier-transform infrared spectroscopic and kinetic studies
Authors: Tanfani F , Kossowska E , Purzycka-Preis J , Zydowo M M , Wozniak M , Tartaglini E , Bertoli E
Published: August 2015

The interaction of pig heart AMP deaminase with different chemical species of phosphatidylcholine and with natural plasma membranes has been investigated. Phospholipids added to the system either as natural biological membranes (plasma membrane vesicles) or in the form of liposomes containing unsaturated phosphatidylcholine considerably enhanced AMP deaminase activity. The secondary structure of pig heart AMP deaminase in the absence and in the presence of dioleoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine liposomes was investigated by Fourier-transform infrared spectroscopy. Quantitative analysis of the amide I band showed that the enzyme contains 45% beta-sheets, 28% alpha-helix, 16% turns and 11% non-ordered structure. In the presence of dioleoyl phosphatidylcholine liposomes, the beta/alpha content ratio decreased; this decrease was dependent on the amount of lipid added. This phenomenon was not observed in the case of dipalmitoyl phosphatidylcholine liposomes. These data suggest a possible role for membrane phospholipids in the regulation of AMP deaminase activity.
Klebsiella pneumoniae nitrogenase: pre-steady-state absorbance changes show that redox changes occur in the MoFe protein that depend on substrate and component protein ratio; a role for P-centres in reducing dinitrogen?
Authors: Lowe D J , Fisher K , Thorneley R N F
Published: August 2015

The pre-steady-state absorbance changes that occur during the first 0.6 s of reaction of the nitrogenase of Klebsiella pneumoniae can be simulated by associating redox changes with the different states of the MoFe protein described by our published kinetic model for nitrogenase [Lowe and Thorneley (1984) Biochem. J. 224, 877-886]. When the substrate is changed, from H+ to C2H2 (acetylene) or N2, or the nitrogenase component protein ratio is altered, these pre-steady-state absorbance changes are affected in a manner that is quantitatively predicted by our model. The results, together with parallel e.p.r. studies, are interpreted as showing that the P-clusters become oxidized when the MoFe protein is in the state where bound N2 is irreversibly committed to being reduced and is protonated to the hydrazido(2-) level.
Glycogenolytic and haemodynamic responses to bovine serum albumin in isolated perfused livers from sensitized rats
Authors: Buxton D B , Halvorsen O , Zhou W , Olson M S
Published: August 2015

Infusion of BSA into isolated perfused livers of rats sensitized by intraperitoneal injection of BSA led to rapid increases in portal-vein pressure, glucose output and the lactate/pyruvate ratio in the effluent perfusate, with concomitant decreases in oxygen consumption and lactate+pyruvate efflux. The responses were attenuated at low (approximately 7 microM) perfusate Ca2+, but were restored on re-addition of normal Ca2+ concentration. Co-infusion of the cyclo-oxygenase inhibitor ibuprofen (50 microM) or of the platelet-activating factor receptor antagonist WEB 2170 (1.2 microM) inhibited haemodynamic responses to BSA (5 micrograms/ml) by 48% and 59% respectively. Responses to BSA were also attenuated by prior infusion of the beta-adrenergic agonist isoprenaline. Glycogen phosphorylase a activity was increased by 26% in livers freeze-clamped 2 min after onset of BSA infusion; tissue prostaglandin E2 content was increased at 2 min, but returned to control levels at 5 min. Homologous desensitization of hepatic responses to BSA was observed, but heterologous desensitization with heat-aggregated IgG did not take place. It is concluded that livers from rats sensitized to antigen respond directly to subsequent antigen administration by vasoconstriction and glycogenolysis, and that autacoid mediators are involved in these responses.
Human inter-α-inhibitor family in inflammation: simultaneous synthesis of positive and negative acute-phase proteins
Authors: Daveau M , Rouet P , Scotte M , Faye L , Hiron M , Lebreton J P , Salier J P
Published: August 2015

The inter-alpha-inhibitor (I alpha I) family encompasses four plasma proteins, namely free bikunin as well as I alpha I, pre-alpha-inhibitor (P alpha I) and inter-alpha-like inhibitor (I alpha LI). Each of the last three proteins is a distinct assembly of one bikunin chain with one or more unique heavy (H) chains designated H1, H2 and H3. The three H chains and the bikunin chain are encoded by four distinct mRNAs. These molecules and chains, as well as the corresponding mRNAs, were quantified in sera and liver biopsies from a series of patients with or without mild or severe acute infection. The decrease or increase observed for a given molecule or chain in the serum was in agreement with a similar change in the corresponding liver mRNA. In acute inflammation the H2 and bikunin chains are down-regulated and the relevant molecules (I alpha I, I alpha LI) behave as negative acute-phase proteins, whereas the H3 chain is up-regulated and the corresponding P alpha I molecule is a positive acute-phase protein. Also, P alpha I displays a higher-than-usual M(r); this is probably due to ligand binding. The H1 gene does not seem to be affected by the inflammatory condition. The quantitative changes in RNA levels seen in vivo were confirmed in vitro in the human hepatoma Hep3B cell line prior to or after induction with the acute-phase mediators interleukin-1 and/or -6. These results provide the first example in humans of positive and negative acute-phase proteins that are encoded by evolutionary related genes.
Developmental changes in mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene expression in rat liver, intestine and kidney
Authors: Thumelin S , Forestier M , Girard J , Pegorier J P
Published: August 2015

The tissue-specific expression of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene was studied in 15-day-old suckling rats. The mRNA and protein were present in liver, intestine and kidney, but were absent from brain, heart, skeletal muscles, brown and white adipose tissues. Kidney-cortex mitochondria from suckling rats were able to produce low amounts of ketone bodies from oleate. Hepatic, intestinal and renal HMG-CoA synthase mRNA levels increased slowly during foetal life and markedly after birth. The postnatal increase in liver HMG-CoA synthase mRNA could be due to the increase in plasma glucagon levels, since it rapidly induced the accumulation of HMG-CoA synthase mRNA in cultured foetal hepatocytes. Hepatic, intestinal and renal HMG-CoA synthase mRNA levels remained elevated throughout the suckling period or in rats weaned on to a high-fat carbohydrate-free diet (HF), but decreased by 50% in the liver and totally disappeared from the intestine and the kidney of rats weaned on to a high-carbohydrate low-fat diet (HC). When HC-weaned rats were fed on a HF-diet for a week, HMG-CoA synthase mRNA was re-induced in the intestine and the kidney. The role of hormones and nutrients in the regulation of HMG-CoA synthase gene expression is discussed.
Stimulation of lipoprotein lipase synthesis by refeeding, insulin and dexamethasone
Authors: Oliver J D , Rogers M P
Published: August 2015

Lipoprotein lipase synthesis in adipose tissue was greater in rats fed ad libitum or refed than in fasted rats. Insulin alone and together with dexamethasone increased lipoprotein lipase synthesis in adipose tissue incubated in vitro. The changes in relative lipoprotein lipase synthesis (immunoprecipitable 35S-labelled lipoprotein lipase as a fraction of general [35S]protein after pulse-labelling with [35S]methionine) indicate that insulin and dexamethasone exert a selective effect on lipoprotein lipase synthesis. There was no evidence for an inverse relationship between lipoprotein lipase synthesis and activity for any of the conditions studied.
Oxidative stress and ageing in Caenorhabditis elegans
Authors: Vanfleteren J R
Published: August 2015

Mutations in the age-1 gene double both the mean and maximum life span of Caenorhabditis elegans. They also result in an age-specific increase of catalase and Cu/Zn superoxide dismutase activity levels. The higher superoxide dismutase activity levels in age-1 mutants confer hyperresistance to the superoxide-anion-generating drug paraquat. The rate of superoxide anion production by microsome fractions declines linearly with age in age-1(+) worms, but, after an initial decline, is stabilized at a higher level in senescent age-1 mutant nematodes. These results clearly show that oxidative stress resistance and potential life span are correlated in this organism, and they suggest that the natural product of age-1 either directly or indirectly downregulates the activities of several other genes as a function of age.
NADH oxidase activity of rat liver plasma membrane activated by guanine nucleotides
Authors: Morré D J , Davidson M , Geilen C , Lawrence J , Flesher G , Crowe R , Crane F L
Published: August 2015

The activity of a hormone- and growth-factor-stimulated NADH oxidase of the rat liver plasma membrane responds to guanine nucleotides, but in a manner that differs from that of the classic trimeric and low-molecular-mass monomeric G-proteins. In the absence of added bivalent ions, both GTP and GDP as well as guanosine 5′-[gamma-thio]triphosphate (GTP[gamma-S]) but not guanosine 5′[beta-thio]diphosphate (GDP[beta-S]) stimulate the activity over the range 1 microM to 100 microM. Other di- and tri-nucleotides also stimulate, but only at concentrations of 100 microM or higher. Added bivalent ions are not required either for NADH oxidation or guanine nucleotide stimulation. Bivalent ions (Mg2+ > Mn2+ > or = Ca2+) alone stimulate only slightly at low concentrations and then inhibit at high concentrations. The inhibitions are augmented by GDP or GTP [gamma-S] but not by GTP. Although the activity is the same, or less, in the presence of 0.5 mM MgCl2, GTP at 1-100 nM and other nucleotides at 0.1 mM or 1 mM still stimulate in its presence. The NADH oxidase is activated by mastoparan but aluminum fluoride is weakly inhibitory. Cholera and pertussis toxins elicit only marginal responses. Both the Mg2+ and the GDP and GTP[gamma-S] inhibitions (but not the GTP stimulations) shift to higher concentrations when the membrane preparations are first solubilized with Triton X-100. The results suggest a role for guanine nucleotides in the regulation of plasma membrane NADH oxidase, but with properties that differ from those of either trimeric or the low-molecular-mass G proteins thus far described.
Effects of fluorinated inositols on the proliferation of Swiss 3T3 fibroblasts
Authors: Cosulich S C , Offer J , Smith G A , Hesketh R , Metcalfe J C
Published: August 2015

The six monodeoxyfluoro-myo-inositols (nFIns) have previously been synthesized as potential inhibitors of signalling pathways mediated by phosphoinositides and their derivatives. Each of the six nFIns isomers was introduced into Swiss 3T3 fibroblasts by the techniques of microinjection or scrape loading at intracellular concentrations of approx. 2-4 mM. Of the six nFIns analogues, only 3FIns and 5FIns inhibited the serum-stimulated proliferation of 3T3 fibroblasts assayed by cell counting. Proliferation was inhibited to a similar extent by 3FIns or 5FIns, irrespective of which technique was used to introduce the nFIns analogues into the cells. Proliferation of cells 35 h after serum stimulation (i.e. when the first cell cycle was completed in control cells) was inhibited by approx. 50% by both 3FIns and 5FIns, and entry into S phase in the first cell cycle was inhibited to the same extent. This indicated that the nFIns analogues were inhibiting proliferation in the G1 phase of the cell cycle. Proliferation during the second cell cycle (35-60 h after stimulation) was inhibited by 75-85%. The inhibitory nFIns analogues were not toxic to the cells, nor did they affect the cellular ATP/ADP ratio. The effectiveness of the nFIns analogues in inhibiting proliferation was directly correlated with their ability to be incorporated into phosphatidylinositol analogues, suggesting that they may act by modulating phosphoinositide signalling pathways or other functions essential for DNA synthesis.
Herpes simplex virus type 1 uracil-DNA glycosylase: isolation and selective inhibition by novel uracil derivatives
Authors: Focher F , Verri A , Spadari S , Manservigi R , Gambino J , Wright G E
Published: August 2015

We have purified Herpes simplex type 1 (HSV1) uracil-DNA glycosylase from the nuclei of HSV1-infected HeLa cells harvested 8 h post-infection, at which time the induction of the enzyme is a maximum. The enzyme has been shown to be distinct from the host enzyme, isolated from HeLa cells, by its lack of sensitivity to a monoclonal antibody to human uracil-DNA glycosylase. Furthermore, several uracil analogues were synthesized and screened for their capacity to discriminate between the viral and human uracil-DNA glycosylases. Both enzymes were inhibited by 6-(p-alkylanilino)uracils, but the viral enzyme was significantly more sensitive than the HeLa enzyme to most analogues. Substituents providing the best inhibitors of HSV1 uracil-DNA glycosylase were found to be in the order: p-n-butyl < p-n-pentl = p-n-hexyl < p-n-heptyl < p-n-octyl. The most potent HSV1 enzyme inhibitor, 6-(p-n-octylanilino)uracil (OctAU), with an IC50 of 8 microM, was highly selective for the viral enzyme. Short-term [3H]thymidine incorporation into the DNA of HeLa cells in culture was partially inhibited by OctAU, whereas it was unchanged when 6-(p-n-hexylanilino)uracil was present at concentrations that completely inhibited HSV1 uracil-DNA glycosylase activity. These compounds represent the first class of inhibitors that inhibit HSV1 uracil-DNA glycosylase at concentrations in the micromolar range. The results suggest their possible use to evaluate the functional role of HSV1 uracil-DNA glycosylase in viral infections and re-activation in nerve cells.