bluuebunny/crossref_metadata_2025 huggingface dataset:
Post-translational processing of the inter-α-trypsin inhibitor in the human hepatoma HepG2 cell line
Authors: Héron A , Bourguignon J , Callé A , Borghi H , Sesboüé R , Diarra-Mehrpour M , Martin J P
Published: August 2015

In human hepatoma HepG2 cells, the serum inter-alpha-trypsin inhibitor (ITI)-like protein is synthesized from two protein precursors, the heavy chain (H) H2 and the light chain (L). Both of them carry sulphate groups involved in the chondroitin sulphate glycosaminoglycan (GAG) linkage, as demonstrated by [35S]sulphate labelling, chondroitinase digestion and inhibition with beta-D-xyloside, an artificial GAG acceptor. While inhibition of N-glycosylation prevented neither the maturation nor the secretion of the ITI-related entities, brefeldin A induced the accumulation of H and L precursors in the cells, therefore blocking subsequent association and maturation of the precursors before their secretion. The enzyme system involved in the ester linkage between H and L chains is localized in the trans-Golgi network since no ITI-like protein could be obtained in the presence of monensin; instead free heavy-chain protein forms and bikunin were secreted in culture supernatants. The ITI-like protein synthesized by HepG2 cells is therefore composed of two heavy chains HC2 linked to two bikunin chains by chondroitin sulphate bridges, although the GAG linkage between HC2 chains is presumably different. Further, a different maturation route leading to restricted heavy-chain forms, Hm and Hd, could be shown.
Distinctive inhibition of the lysosomal targeting of lysozyme and cathepsin D by drugs affecting pH gradients and protein kinase C
Authors: Radons J , Biewusch U , Grässel S , Geuze H J , Hasilik A
Published: August 2015

Morphological and biochemical evidence indicates that in several cell types, lysozyme is found in both lysosomes and the medium. Here we report that in calcitriol-treated human promonocytes U937, in which approx. two-thirds of the synthesized lysozyme is secreted, most of the intracellular lysozyme co-localizes with cathepsin D in lysosomal organelles. In the presence of NH4Cl the lysosomal targeting of procathepsin D, but not that of lysozyme, is inhibited. In the presence of 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA; ‘TPA’), the lysosomal packaging of lysozyme is almost completely inhibited, while that of procathepsin D is only partially so. However, the inhibition of the lysosomal targeting of procathepsin D by NH4Cl and 4 beta-PMA is additive. The targeting of lysozyme is partially inhibited in the presence of R-59022, an inhibitor of diacylglycerol kinase, whereas it is not affected by 4 alpha-phorbol 12-myristate 13-acetate, an isomer of 4 beta-PMA that does not activate protein kinase C. It is concluded that in U937 cells both carbohydrate-dependent and -independent recognition contributes to the lysosomal targeting of soluble proteins. We suggest that the carbohydrate-independent traffic of proteins to lysosomal compartments is controlled by a signalling pathway involving protein kinase C.
Characteristics and regulation of proline transport in cultured glioblastoma cells
Authors: Zafra F , Aragón C , Giménez C
Published: August 2015

L-Proline transport in C6 glioblastoma cells takes place mainly via a saturable Na(+)-dependent mechanism. The uptake process can be discriminated into two components, system A and system ASC. A minor proportion of L-proline transport is carried out by the ASC system, which appears to be constitutively expressed by the cell, but most is by system A which shows adaptive responses to amino acid deprivation and sensitivity to N-methyl-alpha-aminoisobutyric acid. The transport system is inhibited by proline derivatives, such as methyl and benzyl esters, and also hydroxyproline, and is stereospecific. Incubation of glioblastoma cells with phorbol 12-myristate 13-acetate led to concentration- and time-dependent decreases in L-proline transport. This effect could be mimicked by exogenous phospholipase C. Proline transport is significantly stimulated in the presence of Ca(2+)-mobilization agents and strongly inhibited in the absence of Ca2+. The present data suggest a complex regulation of L-proline transport by different kinases in glioblastoma cells.
Formation of the l-cysteine-glyoxylate adduct is the mechanism by which l-cysteine decreases oxalate production from glycollate in rat hepatocytes
Authors: Baker P W , Bais R , Rofe A M
Published: August 2015

Formation of thiazolidine-2,4-dicarboxylic acid, the L-cysteine-glyoxylate adduct, is the putative mechanism by which L-cysteine reduces hepatic oxalate production from glycollate [Bais, Rofe and Conyers (1991) J. Urol. 145, 1302-1305]. This was investigated in isolated rat hepatocytes by the simultaneous measurement of both adduct and oxalate formation. Different diastereoisomeric ratios of cis- and trans-adduct were prepared and characterized to provide both standard material for the enzymic analysis of adduct in hepatocyte supernatants and to investigate the stability and configuration of the adduct under physiological conditions. In the absence of L-cysteine, hepatocytes produced oxalate from 2 mM glycollate at a rate of 822 +/- 42 nmol/30 min per 10(7) cells. The addition of L-cysteine to the incubation medium at 1.0, 2.5 and 5.0 mM lowered oxalate production by 14 +/- 2, 25 +/- 3 (P < 0.05) and 38 +/- 3% (P < 0.01) respectively. These reductions were accompanied by almost stoichiometric increases in the levels of the adduct: 162 +/- 6, 264 +/- 27 and 363 +/- 30 nmol/30 min per 10(7) cells. Adduct formation is therefore confirmed as the primary mechanism by which L-cysteine decreases oxalate production from glycollate. As urinary oxalate excretion is a prime risk factor in the formation of calcium oxalate stones, any reduction in endogenous oxalate production is of clinical significance in the prevention of this formation.
The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf
Authors: Cross D A E , Alessi D R , Vandenheede J R , McDowell H E , Hundal H S , Cohen P
Published: August 2015

Glycogen synthase kinase-3 (GSK3) is inactivated in vitro by p70 S6 kinase or MAP kinase-activated protein kinase-1 beta (MAPKAP kinase-1 beta; also known as Rsk-2). Here we show that GSK3 isoforms are inhibited by 40% within minutes after stimulation of the rat skeletal-muscle cell line L6 with insulin-like growth factor-1 (IGF-1) or insulin. GSK3 was similarly inhibited in rabbit skeletal muscle after an intravenous injection of insulin. Inhibition resulted from increased phosphorylation of GSK3, probably at a serine/threonine residue(s), because it was reversed by incubation with protein phosphatase-2A. Rapamycin blocked the activation of p70 S6 kinase by IGF-1 in L6 cells, but had no effect on the inhibition of GSK3 or the activation of MAPKAP kinase-1 beta. In contrast, wortmannin, a potent inhibitor of PtdIns 3-kinase, prevented the inactivation of GSK3 and the activation of MAPKAP kinase-1 beta and p70 S6 kinase by IGF-1 or insulin. Wortmannin also blocked the activation of p74raf-1. MAP kinase kinase and p42 MAP kinase, but not the formation of GTP-Ras by IGF-1. The results suggest that the stimulation of glycogen synthase by insulin/IGF-1 in skeletal muscle involves the MAP-KAP kinase-1-catalysed inhibition of GSK3, as well as the previously described activation of the glycogen-associated form of protein phosphatase-1.
Pancreastatin activates protein kinase C by stimulating the formation of 1,2-diacylglycerol in rat hepatocytes
Authors: Sánchez-Margalet V , Lucas M , Goberna R
Published: August 2015

We describe here the stimulation by pancreastatin of 1,2-diacylglycerol production and protein kinase C activity in liver plasma membrane and isolated hepatocytes. The dose-dependency for the stimulation of both processes was similar to the recently described pattern of glucose output and cytosolic Ca2+ transients produced by pancreastatin. The time course of diacylglycerol production at 30 degrees C showed a rapid increase within 5 min, reaching a maximum at 10 min. Protein kinase C from hepatocytes was dependent on Ca2+ and phosphatidylserine. Neither the pancreastatin-stimulated diacylglycerol production nor the activation of protein kinase C was affected by pretreatment with pertussis toxin. However, the presence of GTP partially inhibited this pancreastatin stimulation of 1,2-diacylglycerol in a dose-dependent manner, although GTP alone stimulates diacylglycerol accumulation. This inhibitory effect of GTP on pancreastatin stimulation of diacylglycerol synthesis was completely abolished by the pretreatment with pertussis toxin. In conclusion, this study provides evidence that pancreastatin stimulates the formation of 1,2-diacylglycerol by a pertussis-toxin-independent mechanism, which may be responsible for the pancreastatin activation of protein kinase C.
Heterogeneity of vacuolar H+-ATPase: differential expression of two human subunit B isoforms
Authors: van Hille B , Richener H , Schmid P , Puettner I , Green J R , Bilbe G
Published: August 2015

The catalytic domain of the vacuolar proton ATPase is composed of a hexamer of three A subunits and three B subunits. Here we describe the cloning and characterization of a cDNA isoform of subunit B, HO57, from an osteoclastoma cDNA library. HO57 is represented by three species of mRNA of 1.6, 2.6 and 2.8 kb and is expressed at low levels in a range of human tissues, but at significantly higher levels in brain, kidney and osteoclastoma, and is probably an ubiquitously expressed isoform. In contrast, the kidney-specific isoform has an mRNA of 2 kb and is specifically expressed at high levels only in kidney and, at a lower level, in placenta. Thus the HO57 isoform is integral to the vacuolar ATPase found in the general secretory system of all cells as well as in vacuolar-ATPase-rich sources such as neurones and osteoclasts, whereas both the kidney-specific isoform and HO57 are highly expressed in the kidney. Furthermore, we show by in situ hybridization that HO57 is the only isoform that is exclusively and highly expressed by osteoclasts.
Thrombin induces endothelial cell growth via both a proteolytic and a non-proteolytic pathway
Authors: Herbert J M , Dupuy E , Laplace M C , Zini J M , Bar Shavit R , Tobelem G
Published: August 2015

Binding of 125I-thrombin to human umbilical vein endothelial cells (HUVECs) was specifically displaced by the synthetic tetradecapeptide SFLLRNPNDKYEPF, named thrombin receptor agonist peptide (TRAP), which has recently been described as a peptide mimicking the new N-terminus created by cleavage of the thrombin receptor, and F-14, a tetradecapeptide representing residues 365-378 of the human alpha-thrombin B chain. Binding of 125I-TRAP to HUVECs was time-dependent, reversible and saturable, showing high affinity (KD = 1.5 +/- 0.4 microM) and high binding capacity (Bmax. = 7.1 +/- 0.6 x 10(6) sites/cell) (n = 3). Unlabelled thrombin and TRAP competitively and selectively inhibited the specific binding of 125I-TRAP with IC50 values of 5.8 +/- 0.7 nM and 2.8 +/- 0.4 microM respectively, whereas F-14 remained ineffective at displacing 125I-TRAP from its binding sites, suggesting the presence of at least two different types of thrombin-binding sites on HUVECs. TRAP was a potent mitogen for HUVECs in culture. Both TRAP and alpha-thrombin stimulated the proliferation of HUVECs with half-maximum mitogenic responses between 1 and 10 nM. F-14 also promoted HUVEC growth. The mitogenic effects of F-14 and TRAP were additive. N alpha-(2-Naphthylsulphonylglycyl)-DL-p-amidinophenylalanylpiper idine (NAPAP) and hirudin (two specific inhibitors of the enzyme activity of thrombin) specifically inhibited thrombin-induced HUVEC growth (IC50 values 400 +/- 60 and 52 +/- 8 nM respectively) but remained without effect on the mitogenic effect of TRAP or F-14. This demonstrated that the mitogenic effect of alpha-thrombin for HUVECs was intimately linked to its esterolytic activity but also showed that thrombin can stimulate HUVEC growth via another non-enzymic pathway. This hypothesis was further reinforced by the fact that F-14-induced proliferation of HUVECs remained unaltered by two antibodies directed against TRAP or the cleavage site on the extracellular portion of the thrombin receptor, which both strongly reduced thrombin-induced proliferation of HUVECs. Thrombin-, TRAP- or F-14-induced HUVEC proliferation was strongly inhibited by a neutralizing monoclonal antibody directed against basic fibroblast growth factor (bFGF), suggesting that thrombin regulates the autocrine release of bFGF in HUVECs.
Possible role of protein kinase C-ε isoenzyme in inhibition of interleukin 1β induction of nitric oxide synthase in rat renal mesangial cells
Authors: Mühl H , Pfeilschifter J
Published: August 2015

In cultured glomerular mesangial cells, interleukin 1 beta (IL-1 beta) has been shown to induce a dose- and time-dependent accumulation of nitrite, a stable metabolite of nitric oxide (NO). In parallel, increased levels of mRNA of an inducible macrophage-type of nitric oxide synthase (iNOS) were observed after incubating mesangial cells with IL-1 beta. Here we report that addition of the biologically active phorbol esters, phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), dose-dependently inhibited the IL-1 beta-stimulated increase in iNOS mRNA levels and nitrite production. In contrast, the biologically inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate, had no effect on cytokine induction of iNOS and nitrite formation. Incubation of mesangial cells with PMA or PDBu alone, in the absence of IL-1 beta, did not trigger any iNOS expression. Time-course studies indicated that phorbol ester needs to be added for only 1 h in order to maximally inhibit cytokine-induced nitrite production. Down-regulation of protein kinase C (PKC)-alpha and -delta isoenzymes by 8 h PMA or PDBu treatment before stimulation with IL-1 beta still resulted in full inhibition of iNOS induction. In contrast, a 24 h treatment of mesangial cells with PMA or PDBu, a regimen that also causes depletion of PKC-epsilon, abolished inhibition of IL-1 beta-induced iNOS expression and nitrite production. In addition, the selective PKC inhibitor calphostin C potentiated IL-1 beta induction of iNOS activity. In summary these data suggest that IL-1 beta induction of iNOS expression is tonically suppressed by PKC and the epsilon-isoenzyme is the most likely candidate mediating this effect.
Cellulose hydrolysis by the cellulases from Trichoderma reesei: adsorptions of two cellobiohydrolases, two endocellulases and their core proteins on filter paper and their relation to hydrolysis
Authors: Nidetzky B , Steiner W , Claeyssens M
Published: August 2015

Separate binding of several purified cellulolytic components of Trichoderma reesei on to filter paper was studied and concomitant hydrolysis rates evaluated. Enhancement of mass transfer from the bulk liquid to the solid substrate by agitation has two different effects on adsorption depending on the type of enzyme: (i) the fraction of cellobiohydrolase II (CBH II) and endoglucanase III (EG III) bound at equilibrium is increased, whereas (ii) the rate but not the extent of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) adsorption is affected. The adsorption of CBH I core, a component lacking the cellulose-binding domain (CBD), is, however, not significantly influenced by mass transfer. The CBH I interdomain peptide (present in CBH I core b) does not participate in adsorption but enhances stability. The adsorption of CBH I core proteins is a fully reversible process whereas that of the intact CBH I is not. Thus, the interaction of the CBD with filter paper apparently accounts for the mass-transfer-limited binding rate and also for the irreversible adsorption of intact CBH I. Adsorption isotherms at 50 degrees C indicate very similar relative association constants for the intact cellulases (0.24-0.30 l/g of cellulose), but drastically reduced values for CBH I core proteins (0.03 l/g of cellulose). The specific activities of adsorbed CBH I and of its core proteins are identical and a linear relationship between adsorption and rates of hydrolysis is found only for these enzymes. Thus, non-productive binding on to cellulose seems evident in the case of CBH II and EG III but not CBH I.
β3-adrenoceptor agonist-induced down-regulation of Gsα and functional desensitization in a Chinese hamster ovary cell line expressing a β3-adrenoceptor refractory to down-regulation
Authors: Chambers J , Park J , Cronk D , Chapman C , Kennedy F R , Wilson S , Milligan G
Published: August 2015

Chinese hamster ovary (CHO) cells transfected to express human beta 2- or beta 3-adrenoceptors (beta 2-CHO and beta 3-CHO cells) were exposed to the beta-adrenoceptor agonist isoprenaline at various concentrations and for differing times. Sustained exposure of the beta 2-CHO but not beta 3-CHO cells to isoprenaline resulted in a time- and concentration-dependent down-regulation of the receptor as measured by a reduction in specific binding of [125I]cyanopindolol. Such maintained exposure of cells expressing either receptor to the agonist produced a marked down-regulation of immunologically detectable levels of the alpha subunit of the stimulatory guanine-nucleotide-binding protein Gs. This effect was specific for Gs because levels of both G12 alpha and Gq alpha/G11 alpha were unaltered by isoprenaline treatment of both beta 2-CHO and beta 3-CHO cells. The effect of isoprenaline on Gs alpha down-regulation was some 30-fold more potent in the beta 2-CHO than in the beta 3-CHO cells. Time courses of isoprenaline-induced down-regulation of Gs alpha were not different, however, in the two cell lines. Isoprenaline treatment of the beta 3-CHO cells produced a desensitization of agonist-mediated regulation of adenylyl cyclase, manifested by a 4-fold reduction in the potency and a 30% reduction in maximal effect of the agonist, whereas desensitization of the beta 2-CHO cells was considerably greater (25-fold reduction in potency and 70% reduction in maximal effect). These results demonstrate that agonist-induced down-regulation of the G-protein which interacts with a receptor can be produced by both beta 2- and beta 3-adrenoceptors. Despite apparent concurrence of down-regulation of receptors and G-proteins in other systems [e.g. Adie, Mullaney, McKenzie and Milligan (1992) Biochem. J. 285, 529-536], agonist-induced receptor down-regulation does not appear to be a prerequisite for down-regulation of the G-protein. Furthermore, the results suggest that agonist-induced down-regulation of a G-protein may be sufficient, in the absence of receptor regulation, to induce some agonist desensitization of effector function.
Divalent cations stabilize the conformation of plasma cell membrane glycoprotein PC-1 (alkaline phosphodiesterase I)
Authors: Belli S I , Sali A , Goding J W
Published: August 2015

The plasma cell-membrane glycoprotein PC-1 is an ectoenzyme with alkaline phosphodiesterase I/5′-nucleotide phosphodiesterase (EC 3.1.4.1) and nucleotide pyrophosphatase (EC 3.6.1.9) activities. It contains sequence motifs which closely match the consensus EF-hand (helix-loop-helix) Ca(2+)-binding regions of parvalbumin, troponin-C and calmodulin, and its enzymic activity is increased in the presence of divalent cations and decreased in the presence of chelating agents. We have undertaken experiments to determine whether divalent cations affect the conformation of the PC-1 protein, as assessed by their effect on thermal stability, resistance to proteolysis and binding of polyclonal antibodies to the whole native protein and monoclonal antibodies to a putative Ca(2+)-binding region. Divalent cations were found to protect solubilized PC-1 against thermal denaturation and proteolysis. They also stabilized PC-1 on intact cells; this form was much more resistant to proteolysis than Triton X-100 solubilized PC-1. Ca2+, Mg2+ and Zn2+ ions were equally effective. Monoclonal antibodies to the bacterially expressed C-terminal EF-hand homology region only bound to mammalian PC-1 in the absence of Ca2+. In contrast, the great majority of polyclonal antibodies to native PC-1 bound regardless of whether Ca2+ was present or not, but with increased binding when Ca2+ was present. These results provide evidence that divalent cations bind to PC-1 and stabilize its conformation.
Purification and characterization of a calcium-independent acidic phospholipase A2 from rat lung
Authors: Wang R , Dodia C R , Jain M K , Fisher A B
Published: August 2015

Several phospholipase A2 (PLA2) activities have been identified in rat lung homogenate and shown to be important in metabolism of lung phospholipids. One PLA2 activity is Ca(2+)-independent, active in vitro at pH 4, and inhibited by a substrate analogue, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33). Purification of this rat lung PLA2 by approx. 550-fold was carried out by sequential column chromatographies using DE-52, Sephacryl-100, heparin-Sepharose, and phenyl-Sepharose columns. The purified activity had an acidic pH optimum, was Ca(2+)-independent, was inhibited by MJ33 in a dose-dependent manner (50% inhibition at 3 mol%), was unaffected by treatment with p-bromophenacyl bromide or mercaptoethanol, and had a unique N-terminal amino acid sequence. The apparent molecular mass was 15 kDa on gel electrophoresis and activity was recovered in part by renaturation of protein from the gel. The properties of this enzyme suggest a new class of PLA2.
Transforming growth factor-β1 inhibits casein secretion from differentiating mammary-gland explants but not from lactating mammary cells
Authors: Sudlow A W , Wilde C J , Burgoyne R D
Published: August 2015

Transforming growth factor-beta (TGF beta) is important in the maturation and function of the mammary gland and is present in milk. We have examined whether, in addition to inhibiting lactogenesis, TGF beta exerts acute regulatory effects on lactating mammary cells. The isoform TGF beta 1 at 5 and 50 ng/ml suppressed the onset of lactation and the subsequent production of beta-casein by differentiating mouse mammary explants from pregnant mice. By contrast, it did not inhibit protein synthesis or secretion from acini isolated from lactating-mouse mammary gland or protein secretion from explants from lactating mice. These data indicate that TGF beta inhibits the onset of casein secretion, but is not an acute regulator of casein synthesis or secretion from differentiated lactating mammary cells.
Antigen and thapsigargin promote influx of Ca2+ in rat basophilic RBL-2H3 cells by ostensibly similar mechanisms that allow filling of inositol 1,4,5-trisphosphate-sensitive and mitochondrial Ca2+ stores
Authors: Ali H , Maeyama K , Sagi-Eisenberg R , Beaven M A
Published: August 2015

In single, Fura 2-loaded RBL-2H3 cells, antigen and thapsigargin depleted the same intracellular pool of Ca2+ in the absence of external Ca2+; provision of external Ca2+ induced immediate increases in levels of free Ca2+ ([Ca2+]i). These increases were dependent on the presence of external Ca2+ and, presumably, on influx of Ca2+ across the cell membrane. Both stimulants enhanced intracellular accumulation of 45Ca2+ through ostensibly similar mechanisms because accumulation was blocked to similar extents by various multivalent cations or by depolarization with K+. Because thapsigargin blocked reuptake of Ca2+ into inositol 1,4,5-trisphosphate sensitive stores, uptake occurred independently of the refilling of these stores. Uptake was dependent instead on sequestration of 45Ca2+ in a pool of high capacity that was insensitive to thapsigargin, caffeine, GTP and inositol 1,4,5-trisphosphate but sensitive to ionomycin and mitochondrial inhibitors. The existence of an inositol 1,4,5-trisphosphate-insensitive pool was also apparent in permeabilized cells; at 0.1 microM [Ca2+]i, uptake of 45Ca2+ was largely confined (> 80%) to the inositol 1,4,5-trisphosphate-sensitive pool, but at 2 microM [Ca2+]i uptake was largely (> 60%) into the inositol 1,4,5-trisphosphate-insensitive pool. Provision of mitochondrial inhibitors along with thapsigargin to block uptake into both pools, did not impair the thapsigargin-induced increase in [Ca2+]i or influx of Ca2+, as indicated by changes in Fura 2 fluorescence, but did block the intracellular accumulation of 45Ca2+. The studies illustrate the utility of simultaneous measurements of [Ca2+]i and 45Ca2+ uptake for a full accounting of Ca2+ homoeostasis as exemplified by the ability to distinguish between influx and mitochondrial uptake of Ca2+.
Mitochondrial metabolism of a hydroperoxide to free radicals in human endothelial cells: an electron spin resonance spin-trapping investigation
Authors: O'Donnell V , Burkitt M J
Published: August 2015

Oxidative damage to the vascular endothelium may be an important event in the promotion of atherosclerosis. Several lines of evidence suggest that lipid hydroperoxides may be responsible for the induction of such damage. Hydroperoxides cause loss of endothelial cell integrity, increase the permeability of the endothelium to macromolecules, and compromise its ability to control vascular tone via the secretion of vasoactive molecules in response to receptor stimulation. The molecular mechanisms responsible for these effects are, however, poorly understood. In this paper, we describe an e.s.r. spin-trapping investigation into the metabolism of the model hydroperoxide compound tert-butylhydroperoxide to reactive free radicals in intact human endothelial cells. The hydroperoxide is shown to undergo a single electron reduction to form free radicals. Experiments with metabolic poisons indicate that the mitochondrial electron-transport chain is the source of electrons for this reduction. The metal-ion-chelating agent desferrioxamine was found to prevent cell killing by tert-butylhydroperoxide, but did not affect free radical formation, suggesting that free metal ions may serve to promote free-radical chain reactions involved in cell killing following the initial conversion of the hydroperoxide to free radicals by mitochondria. These processes may well be responsible for many of the reported effects of hydroperoxides on endothelial cell integrity and function.
Purification and characterization of thyroid transcription factor 2
Authors: Civitareale D , Saiardi A , Falasca P
Published: August 2015

Thyroid transcription factor 2 binds to the promoters of both thyroglobulin and thyroperoxidase genes, two markers of thyroid tissue differentiation, and its binding modulates the activity of both promoters. In this paper we describe the purification of thyroid transcription factor 2 essentially to homogeneity and demonstrate that it is a thyroid-specific DNA-binding protein. Furthermore, we provide a biochemical characterization suggesting that thyroid transcription factor 2 binds to DNA as a dimer and that it is a zinc-finger DNA-binding protein regulated in vitro by the redox state.
Human kininogens interact with M protein, a bacterial surface protein and virulence determinant
Authors: Ben Nasr A , Herwald H , Müller-Esterl W , Björck L
Published: August 2015

Streptococcus pyogenes, the most significant streptococcal species in clinical medicine, expresses surface proteins with affinity for several human plasma proteins. Here we report that kininogens, the precursors to the vasoactive kinins, bind to the surface of S. pyogenes. M protein, a surface molecule and a major virulence factor-in these bacteria, occurs in > 80 different serotypes. Among 49 strains of S. pyogenes, all of different M serotypes, 41 bound radiolabelled kininogens, whereas 6 M protein-negative mutant strains showed no affinity. M protein of most serotypes bind fibrinogen, and among the 55 strains tested, binding of kininogens was closely correlated to fibrinogen binding (r = 0.88, P < 0.0001). Western blotting, slot binding and enzyme immunoassay experiments demonstrated that M proteins isolated from S. pyogenes of three different M protein serotypes (M1, M6 and M46) bound kininogens. The affinity between kininogens and M1 protein was determined to be 5 x 10(7) M-1 and < or = 10(6) M-1 for high molecular weight (H-kininogen) and low molecular weight kininogen, respectively. The kininogen binding site was tentatively mapped to the N-terminal portion of M1 protein, and this site does not overlap the specific and separate binding sites for albumin, IgG and fibrinogen using monoclonal antibodies to, and synthetic peptides of, the kininogen sequence, the major M protein-binding site(s) was mapped to the C-terminal portion of the H-kininogen light chain. We anticipate that the kininogen-M protein interaction contributes to the host-parasite relationship in S. pyogenes infections.
Phorbol ester stimulates choline uptake in Swiss 3T3 fibroblasts following introduction of the gene encoding protein kinase Cα
Authors: Slack B E , Breu J , Livneh E , Eldar H , Wurtman R J
Published: August 2015

Phorbol 12-myristate 13-acetate (PMA) stimulated radiolabelled choline uptake and incorporation into phosphatidylcholine (PtdCho) in a time- and concentration-dependent manner in wild-type NIH 3T3 fibroblasts. The accumulation of labelled choline induced by PMA was paralled by an increase in choline mass. The results implicate protein kinase C (PKC) in the regulation of choline uptake. In order to address the PKC-subtype specificity of this response, a study was undertaken in Swiss 3T3 fibroblast cells, which normally express very low levels of PKC alpha. A retroviral expression system was used to introduce the genes for PKC alpha and neomycin resistance (used for selection) into the cells. Two resulting lines expressed PKC alpha at levels that were 20-fold higher than those found in the control (neomycin-resistant) line, or in the wild-type cells. In control Swiss 3T3 fibroblasts, 1 microM PMA elevated choline levels by only 30%, whereas, in Swiss 3T3 cell lines that stably over-expressed PKC alpha, PMA caused a 5-fold enhancement in [14C]choline accumulation. This concentration of PMA significantly increased [14C]PtdCho levels in both control and PKC alpha-over-expressing lines, although the effect in the latter was significantly greater. The effects of PMA were inhibited by the PKC antagonist sphingosine. These results implicate PKC alpha in the regulation of choline accumulation and phospholipid synthesis in fibroblasts. Although additional PKC subtypes appear to participate in the control of PtdCho synthesis in these cells, PMA-stimulated choline uptake in Swiss 3T3 fibroblasts is almost entirely dependent on the presence of PKC alpha.
The effect of histone H1 and DNA methylation on transcription
Authors: Johnson C A , Goddard J P , Adams R L P
Published: August 2015

We have previously shown that DNA methylation acts as a focus for the formation of inactive chromatin in vivo. We have investigated the mechanism further by in vitro transcription of a template containing two tRNA genes and an extensive (G+C)-rich sequence characteristic of a CpG island. The extent of transcription from the unmethylated or fully methylated template was assayed in the presence of varied levels of histone H1. The transcriptional activity of both templates was inhibited by increasing amounts of histone H1, although inhibition with the methylated template occurs at a lower H1:DNA ratio. The H1c variant shows the greatest preferential inhibition of the methylated template. We demonstrated that histone H1 complexed to DNA is one of the factors that inhibits transcription by preventing the formation of initiation complexes, particularly on methylated template, rather than the formation of disordered H1.DNA aggregates.
Monoclonal antibodies that specifically recognize neoepitope sequences generated by ‘aggrecanase’ and matrix metalloproteinase cleavage of aggrecan: application to catabolism in situ and in vitro
Authors: Hughes C E , Caterson B , Fosang A J , Roughley P J , Mort J S
Published: August 2015

Monoclonal antibodies have been prepared that react specifically with the neoepitopes present on proteoglycan degradation products generated from the proteolytic cleavage of aggrecan in the interglobular domain. Antibody BC-3 recognizes the new N-terminus (ARGSV...) on aggrecan degradation products produced by the action of the as yet uncharacterized proteolytic activity, ‘aggrecanase’, and antibody BC-4 recognizes the new C-terminus (...DIPEN) generated by the proteolytic action of matrix metalloproteinases. Specificity for these neoepitope sequences was determined in competitive e.l.i.s.a. using synthetic peptide antigens as inhibitors. Antibody BC-3 was used in the detection of aggrecan degradation products in the culture medium obtained from two different in vitro culture systems: bovine cartilage explants treated with either retinoic acid or interleukin-1, and secondly, rat chondrosarcoma cells treated with retinoic acid. Both interleukin-1 and retinoic acid treatment caused an increase in aggrecan catabolism resulting in an increased release to the medium of specific aggrecan degradation products containing the BC-3 neoepitope generated by the action of ‘aggrecanase'. However, several additional aggrecan catabolites were present that were not immunoreactive with antibody BC-3. In addition, under control conditions, in the bovine cartilage cultures the BC-3 epitope was found on some of these aggrecan catabolites. In contrast, no immune-reactive material was found in the aggrecan degradation products present in control media of rat chondrosarcoma cells cultured in the absence of retinoic acid. Collectively, these results demonstrate that ‘aggrecanase’ activity is not a constitutive event in all cartilage culture systems and also suggest that proteolytic agents other than ‘aggrecanase’ are involved in aggrecan catabolism in normal turnover compared with pathological conditions. Antibody BC-4 was used to demonstrate the identity of the G1 domain of aggrecan following proteolytic cleavage of a purified G1-G2 preparation with collagenase, gelatinase A or stromelysin. The G2 product of this cleavage did not react with antibody BC-3, indicating that, under the experimental conditions used, none of these enzymes exhibited ‘aggrecanase’ activity. It is expected that both of these antibodies will play a pivotal role in detailed studies elucidating molecular mechanisms of aggrecan degradation and they will be particularly useful for the sensitive monitoring of aggrecan degradation products in tissue extracts and body fluids.
Methional derived from 4-methylthio-2-oxobutanoate is a cellular mediator of apoptosis in BAF3 lymphoid cells
Authors: Quash G , Roch A M , Chantepie J , Michal Y , Fournet G , Dumontet C
Published: August 2015

4-Methylthio-2-oxobutanoic acid is the direct precursor of methional, which is a potent inducer of apoptosis in a BAF3 murine lymphoid cell line which is interleukin-3 (IL3)-dependent. Cultures treated for 8 h with methional in the presence of IL3 show extensive DNA double-strand breaks on flow cytometric analysis, increases in DNA fragmentation as measured by the amount of non-sedimentable DNA present in the 30,000 g supernatant of cell lysates and the typical laddering pattern of multiples of 180 bp seen upon agarose gel electrophoresis. No such features of apoptosis were found in cells treated with 4-methylthio 2-oxobutanoic acid or propanal, suggesting that the simultaneous presence of the methylthio group on the propanal moiety is essential for apoptosis to take place. Methional is further metabolized in cells by two reactions: oxidation via aldehyde dehydrogenase to (methylthio)propionic acid or beta-hydroxylation to malondialdehyde. The formation of malondialdehyde from methional in vitro by chemical hydroxylation under the conditions of the Fenton reaction provides a mechanism for the beta-hydroxylation which takes place in vivo. During apoptosis induced by IL3 deprivation, the ratio of 2,4-DNPH MDA to 2,4-DNPH methional is 0.94 in cells in IL3- medium compared with 0.54 in cells in IL3+ medium. These results support a role of cellular methional and malondialdehyde in apoptosis.
Characterization of the invertase from Pichia anomala
Authors: Rodriguez J , Perez J A , Ruiz T , Rodriguez L
Published: August 2015

Synthesis of invertase (EC 3.2.1.26) in Pichia anomala is controlled by the carbon source in the culture medium. The enzyme was purified to homogeneity from P. anomala cells fully derepressed for invertase synthesis and shown to be a multimeric glycoprotein composed of identical subunits with an apparent molecular mass of 86.5 kDa. The carbohydrate moiety accounts for approx. 30% of the total mass of the molecule and consists of manno-oligosaccharides N-linked to the polypeptide. Most of the characteristics of the enzyme analysed in this study were similar to those previously reported for other yeast invertases, with the remarkable exception of its thermal sensitivity which appears after 15 min incubation at temperatures above 32 degrees C.
Purification of a phosphatidic-acid-hydrolysing phospholipase A2 from rat brain
Authors: Thomson F J , Clark M A
Published: August 2015

A phosphatidic-acid-hydrolysing phospholipase A2 was purified from rat brain and characterized. This phospholipase A2 was purified by sequential cation, hydrophobic, heparin and gel-filtration chromatography. The purified protein had a mass of approximately 58 kDa as assayed by SDS/PAGE, had a pH optimum of 6.0, and was Ca(2+)-independent. This enzyme was apparently phosphatidic-acid-selective and had little measurable catalytic activity when phosphatidylcholine, phosphatidylethanolamine or diacylglycerol was used as substrate. On the basis of its physical and catalytic properties, we conclude that this phospholipase A2 is unique from those previously purified, and we speculate that it may be important for the production of the bioactive lipid lysophosphatidic acid.
Two sequences flanking the major autophosphorylation site of the insulin receptor are essential for tyrosine kinase activation
Authors: Leconte I , Clauser E
Published: August 2015

The tyrosine kinase domain of the human insulin receptor (IR) contains several short amino acid motifs which are strictly conserved in all protein kinases and two sequence motifs which are specific to the tyrosine kinases (AAR or RAA and P(I)/VK/RWT/M). In the serine/threonine kinases these motifs are replaced by the sequences KPE and GT/SXXY/PX respectively. In the present work, the tyrosine kinase-specific sequences of the IR (1134AAR1136 and 1172PVRWM1176) were replaced using site-directed mutagenesis by sequences which confer a serine kinase specificity on the receptor. Five different IR mutants were expressed in Chinese hamster ovary (CHO) or COS cells and their structural and functional properties compared with those of the wild-type recombinant human IR. These mutants are processed normally and bind insulin with normal affinities. None of the mutants containing a putative serine kinase-specific sequence display detectable autophosphorylation or tyrosine kinase activity in response to insulin, either in vitro or in vivo. These mutants were also unable to phosphorylate serine/threonine kinase substrates after insulin stimulation. Unexpectedly, they showed impaired ATP binding, as studied by an original technique consisting of cross-linking adenosine 5′-([35S]thio)triphosphate to partially purified receptors. Finally, none of the studied mutants transmit the insulin signal necessary to stimulate either DNA or glycogen synthesis. These data provide evidence for the importance of these conserved sequences in the kinase domain for both receptor activation and kinase activity. Furthermore, they demonstrate that the exchange of sequences specific to the catalytic domain of tyrosine kinases for those specific to the serine/threonine kinases is not sufficient to confer serine/threonine specificity on the insulin receptor.
Tyrosine phosphorylation induced by cross-linking of Fc γ-receptor type II in human neutrophils
Authors: Liang L , Huang C K
Published: August 2015

Neutrophils express several receptors for the Fc region of IgG molecules. Specific cross-linking of the type II receptor (Fc gamma RII) can be achieved by treating neutrophils with the Fab fragment of a specific monoclonal antibody IV.3 against the receptor followed by goat anti-mouse IgG F(ab′)2 fragment. Such treatment initiates a number of neutrophil responses including the release of O2-. and increased protein tyrosine phosphorylation. The increase in tyrosine phosphorylation is rapid and transient and correlates with O2-. release. Both responses are inhibited by pretreatment of neutrophils with a protein tyrosine kinase inhibitor, genistein. The increase in protein tyrosine phosphorylation is not inhibited by pretreatment of neutrophils with pertussis toxin or an intracellular Ca2+ chelator, but is enhanced by a phosphoprotein phosphatase inhibitor, okadaic acid. The activity of a neutrophil Ca2+/calmodulin-dependent protein kinase II (CAMPKII) is also stimulated by cross-linking Fc gamma RII. The increase in CAMPKII activity is inhibited by pretreatment with either genistein or Ca2+ chelator. The results suggest that the increase in protein tyrosine phosphorylation induced by cross-linking of Fc gamma RII requires neither pertussis-toxin-sensitive G-proteins nor a rise in intracellular Ca2+ but can be regulated by protein phosphatases. Furthermore, protein tyrosine phosphorylation may be an early signal functionally linked to Fc gamma RII-mediated signal transduction leading to CAMPKII activation and O2-. release in human neutrophils.
Lack of glycosyl-phosphatidylinositol anchoring leads to precursor retention by a unique mechanism in Dictyostelium discoideum
Authors: Pauly P C , Klein C
Published: August 2015

Gp80, a cell-adhesion molecule in Dictyostelium discoideum, is modified by N- and O-linked oligosaccharides, and a glycosylphosphatidylinositol (GPI) anchor. To identify sequences important for the addition of these modifications to gp80, we created a hybrid protein in which the C-terminal 136 amino acids of yeast invertase were replaced by the C-terminal 110 amino acids of gp80. When expressed in D. discoideum, this protein (Inv-gp80) was not GPI-anchored and was retained in a pre-Golgi compartment. Inv-gp80 did, however, display characteristics of a transmembrane protein, suggesting a novel mechanism for its retention. We also expressed a truncated version of the hybrid protein in which the C-terminal 22 amino acids of the Inv-gp80 were deleted. The truncated protein (Inv-gp80stop) was O-glycosylated and secreted. These observations indicate that the hybrid protein is not abnormally folded and demonstrate the importance of the C-terminal 22 amino acids in the retention of Inv-gp80. Together, the data suggest that oligomerization of the protein blocks its GPI anchoring.
The apolipoprotein B3304–3317 peptide as an inhibitor of the lipoprotein (a):apolipoprotein B-containing lipoprotein interaction
Authors: Trieu V N , Olsson U , McConathy W J
Published: August 2015

Lipoprotein (a) [Lp(a)] is a risk factor for coronary artery disease. It is characterized by apolipoprotein (a) [apo(a)] disulphide linked to apolipoprotein B (apoB), by Cys4057 of apo(a) and possibly Cys3734 of apoB. We call this the covalent apo(a):apoB-Lp interaction, to distinguish it from the non-covalent Lp(a):apoB-Lp interaction, mediated by the proline-binding kringle-4-like domain(s) of Lp(a). The Lp(a):apoB-Lp interaction was inhibited by an apoB peptide spanning residues 3304-3317. This peptide was found by a computerized search for sites on apoB similar to the plasminogen's kringle-4-binding site of alpha 2-antiplasmin. It probably constitutes part of the Lp(a)-binding site on apoB because: (1) it corresponds to the alpha 2-antiplasmin minimum binding domain for plasminogen's kringle-4; (2) the competitive nature of inhibition [KI = (1.5 +/- 0.7) x 10(-4) M, n = 5] suggested that it and apoB-Lp bound to Lp(a) by the same mechanism at the same site; and (3) it specifically bound Lp(a) and not apoB-Lp, and the bound Lp(a) was dissociated by inhibitors of the Lp(a):apoB-Lp interaction, 6-aminohexanoic acid and L-proline. Inhibition was independent of its proline residue, suggesting that proline in the context of a peptide is not a ligand for the kringle(s) which mediated the binding of Lp(a) to apoB-Lp.
Regulation of fibroblast procollagen production. Transforming growth factor-β1 induces prostaglandin E2 but not procollagen synthesis via a pertussis toxin-sensitive G-protein
Authors: McAnulty R J , Chambers R C , Laurent G J
Published: August 2015

Transforming growth factor-beta 1 (TGF beta 1) initiates a series of signalling events resulting in diverse cellular responses including stimulation of extracellular matrix protein production. In this study we have investigated the role of pertussis toxin-sensitive G-proteins in mediating the effects of TGF beta 1 on fibroblast procollagen metabolism. TGF beta 1 stimulated human fetal lung fibroblast procollagen synthesis and production in a dose-dependent manner which was maximal at 0.5 ng/ml. TGF beta 1 also decreased the proportion of newly synthesized procollagen degraded intracellularly. Pertussis toxin, a G-protein inhibitor, further stimulated TGF beta 1-induced procollagen synthesis and production, but alone it had no effect on fibroblast procollagen metabolism. Addition of indomethacin also potentiated the TGF beta 1-induced increase in procollagen synthesis and production. The effects of pertussis toxin and indomethacin were not additive. Pertussis toxin and indomethacin did not affect the proportion of newly synthesized procollagen degraded intracellularly, either alone or in combination, by control cells. The TGF beta 1-induced decrease in intracellular procollagen degradation was maintained but not further affected by pertussis toxin or indomethacin. TGF beta 1 increased prostaglandin E2 (PGE2) compared with PGE2 production by control cells. Addition of pertussis toxin or indomethacin blocked the TGF beta 1-induced increase in PGE2 production. The TGF beta 1-induced increase in PGE2 preceded the increase in procollagen production. These results demonstrate that TGF beta 1-induced procollagen synthesis by lung fibroblasts is modulated by production of PGE2. Pertussis toxin and indomethacin block the production of PGE2 and enhance the effect of TGF beta 1 on procollagen synthesis. From these data we conclude that the effects of TGF beta 1 on PGE2 production but not procollagen synthesis are mediated via a receptor linked to a pertussis toxin-sensitive G-protein.