bluuebunny/crossref_metadata_2025 huggingface dataset:
Submicromolar La3+ concentrations block the calcium release-activated channel, and impair CD69 and CD25 expression in CD3- or thapsigargin-activated Jurkat cells
Authors: AUSSEL Claude , MARHABA Rachid , PELASSY Claudette , BREITTMAYER Jean-Philippe
Published: August 2015

The calcium release-activated channel (CRAC) opened in Jurkat cells activated either with CD3 monoclonal antibody or the endoplasmic reticulum Ca2+-ATPase blocker, thapsigargin, is blocked by La3+ with an IC50 of 20 nM. Similarly, the entry of Mn2+, used as a surrogate for Ca2+, is also blocked by submicromolar La3+ concentrations. La3+ seems to play its role simply by plugging the CRAC because this ion does not penetrate the cells, as demonstrated by chelation experiments with EGTA. Blocking the Ca2+ influx in activated Jurkat cells results in a lack of expression of CD25, a chain of the interleukin-2 receptor and of CD69, a marker of T-cell activation. By contrast, the very early steps of the T-cell signalling pathway such as the release of Ca2+ from intracellular stores and the subsequent inhibition of phosphatidylserine synthesis are not affected by La3+.
Expression and secretion of recombinant ovine β-lactoglobulin in Saccharomyces cerevisiae and Kluyveromyces lactis
Authors: ROCHA Thales L. , PATERSON Gary , CRIMMINS Kay , BOYD Alan , SAWYER Lindsay , FOTHERGILL-GILMORE Linda A.
Published: August 2015

High expression and secretion of recombinant ovine β-lactoglobulin has been achieved in the yeast Kluyveromyces lactis. The yield of β-lactogobulin is 40–50 mg per litre of culture supernatant and accounts for approx. 72% of the total secreted protein. Constitutive expression is under the control of the Saccharomyces cerevisiae phosphoglycerate kinase promoter from an intronless version of the β-lactoglobulin gene. Secretion is specified by the ovine protein's own signal sequence. This system, coupled to an efficient and novel recovery protocol, allows 30 mg of pure protein to be isolated from a typical 1 litre culture. The protein is virtually indistinguishable from β-lactoglobulin conventionally purified from sheep milk by its behaviour in native PAGE and SDS/PAGE, reactivity to antibodies, CD, fluorescence spectroscopy and N-terminal sequencing. Attempts to achieve a similar expression and secretion system in the yeast S. cerevisiae met with only limited success, although it was found that heat-shock treatment modestly increased the yield up to approx. 3–4 mg per litre of culture supernatant. Site-directed mutagenesis showed that secretion in S. cerevisiae depended upon correct formation of the two disulphide bonds present in β-lactoglobulin.
Investigation of the substrate specificity of cruzipain, the major cysteine proteinase of Trypanosoma cruzi, through the use of cystatin-derived substrates and inhibitors
Authors: SERVEAU Carole , LALMANACH Gilles , JULIANO Maria A. , SCHARFSTEIN Julio , JULIANO Luiz , GAUTHIER Francis
Published: August 2015

A panel of intramolecularly quenched fluorogenic substrates containing the conserved QVVA and LVG inhibitory sequences of cystatin inhibitors was used to describe the specificity of the major cysteine proteinase of Trypanosoma cruzi (cruzipain or cruzain). This approach was based on the observations that: (1) cruzipain is strongly inhibited by chicken cystatin and rat T-kininogen, two representative members of cystatin families 2 and 3; (2) the QVVA- and LVG-containing substrates are specifically hydrolysed by papain-like proteinases; and (3) the cystatin-like motifs are similar to the proteolytically sensitive sequences in cruzipain that separate the pro-region and/or the C-terminal extension from the catalytic domain. Specificity constants (kcat./Km) were determined and compared with those of mammalian cathepsins B and L from rat liver lysosomes. Cruzipain and the mammalian proteinases cleaved cystatin-derived substrates at the same site, but their specificities differed significantly. Increased specificity for cruzipain was obtained by replacing amino acids at critical positions on both sides of the cleavage sites, especially at position P2´. The specificity constants (kcat./Km) obtained for the two substrates with a prolyl residue at P2´ (O-aminobenzoyl-QVVAGP-ethylenediamine 2-4-dinitrophenyl and O-aminobenzoyl-VVGGP-ethylenediamine 2-4-dinitrophenyl) were about 50 times higher for cruzipain than for rat cathepsin L and about 100 times higher than for cathepsin B. Diazomethylketone derivatives, based on the non-prime sequence of cystatin-derived substrates, inhibited cruzipain irreversibly, but their inactivation rate constants were considerably lower than those for mammalian cathepsins B and L, confirming the importance of P´ residues for cruzipain specificity.
Purification of 11 β-hydroxysteroid dehydrogenase type 2 from human placenta utilizing a novel affinity labelling technique
Authors: BROWN Roger W. , CHAPMAN Karen E. , MURAD Parvez , EDWARDS Christopher R. W. , SECKL Jonathan R.
Published: August 2015

11β-Hydroxysteroid dehydrogenase type 2 (11β-HSD2) efficiently inactivates potent glucocorticoid hormones (cortisol and corticosterone), leaving aldosterone unmetabolized. Abundant 11β-HSD2 activity in human placenta plays a central role in controlling fetal glucocorticoid exposure, which if excessive is harmful and may predispose to low birth weight and hypertension in adulthood. Similar 11β-HSD2 activity in the distal nephron protects mineralocorticoid receptors from glucocorticoids and appears to be important in normal blood pressure control. We have purified human placental 11β-HSD2 16000-fold, to homogeneity, and determined over 100 residues of the internal amino acid sequence. Purification was assisted by a novel technique allowing highly specific (single spot on two-dimensional electrophoresis) photoaffinity labelling of active 11β-HSD2 in crude tissue extracts by its glucocorticoid substrates. This work reveals that 11β-HSD2 is a member of the short-chain alcohol dehydrogenase superfamily (apparent monomer Mr ~40000). It is a very basic (apparent pI = 9.1) intrinsic membrane protein, requiring as yet undefined membrane constituents for full stability. Affinity chromatography and affinity labelling studies suggest that 11β-HSD2 has a compulsory ordered mechanism, with NAD+ binding first, followed by a conformational change allowing glucocorticoid binding with high affinity.
Fatty acid binding and conformational stability of mutants of human muscle fatty acid-binding protein
Authors: PRINSEN Clemens F. M. , VEERKAMP Jacques H.
Published: August 2015

Human muscle fatty acid-binding protein (M-FABP) is a 15 kDa cytosolic protein which may be involved in fatty acid transfer and modulation of non-esterified fatty acid concentration in heart, skeletal muscle, kidney and many other tissues. Crystallographic studies have suggested the importance of the amino acids Thr-40, Arg-106, Arg-126 and Tyr-128 for the hydrogen bonding network of the fatty acid carboxylate group. Two phenylalanines at 16 and 57 are positioned to interact with the acyl chain of the fatty acid. We prepared 13 mutant proteins by site-directed mutagenesis and tested them for fatty acid binding and stability. Substitution of amino acids Phe-16, Arg-106 or Arg-126 created proteins which showed a large decrease in or complete loss of oleic acid binding. Substitution of Phe-57 by Ser or Val and of Tyr-128 by Phe had no great effect. The stability of the mutant proteins was tested by denaturation studies on the basis of fatty acid binding or tryptophan fluorescence and compared with that of the wild-type M-FABP. There was no direct relationship between fatty acid-binding activity and stability. Less stable mutants (F57S and Y128F) did not show a marked change in fatty acid-binding activity. Substitution of Arg-126 by Gln or Arg-106 by Thr eliminated binding activity, but the former mutant protein showed wild-type stability, in contrast to the latter. The results are in agreement with crystallographic data.
Signalling mechanisms of endothelin-induced mitogenesis and melanogenesis in human melanocytes
Authors: IMOKAWA Genji , YADA Yukihiro , KIMURA Mitsutoshi
Published: August 2015

To understand the signalling mechanisms involved in the dual stimulatory effects of endothelin-1 (ET-1) on DNA synthesis and melanization in cultured human melanocytes, we analysed the biological profile of ET-1 receptor and determined the effects of ET-1 on the protein kinase C, cyclic AMP system and mitogen-activated protein kinase (MAP kinase) in comparison with their relevant simulants. The photoaffinity labelling of ET-1 receptors with Denny–Jaff reagents revealed an ET-1 receptor with a molecular mass of 51 kDa in human melanocytes. The ETA receptor subtype-sensitive antagonist BQ123 (50 nM) or pertussis toxin (100 ng/ml) significantly suppressed the ET-1-induced intracellular calcium mobilization, indicating the presence of pertussis toxin-sensitive G-protein-coupled ETA receptors. An assay of protein kinase C activity revealed that 10 nM ET-1 translocated cytosolic protein kinase C to membrane-bound protein kinase C within 5 min of the start of incubation. In contrast, receptor-mediated melanocyte activation by ET-1 was accompanied by an elevated level of cyclic AMP (4-fold over control) after 10–60 min of incubation, whereas 60 min of incubation of human melanocytes with c-Kit or c-Met ligands such as stem cell factor (10 nM) or basic fibroblast growth factor (10 nM) did not elevate the cyclic AMP level. We have also demonstrated that a specific tyrosine kinase inhibitor, tyrphostin B-42 (10 μM), inhibited the ET-1-induced growth stimulation, suggesting the involvement of the tyrosine kinase pathway in growth stimulation. Consistently, an assay of MAP kinase revealed that ET-1 caused a 10-fold activation of MAP kinase after 5 min of incubation with human melanocytes in a similar way to tyrosine kinase ligands such as stem cell factor and hepatocyte growth factor. Further, the DNA synthesis stimulated by the c-Kit ligand stem cell factor at a concentration of 1 nM was synergistically enhanced by 5 nM ET-1. These results suggest that ET-induced dual cellular events in human melanocytes are closely associated with cross-talk between the protein kinase C and A and tyrosine kinase pathways.
Transgenic mice expressing the human ornithine decarboxylase gene under the control of mouse metallothionein I promoter
Authors: ALHONEN Leena , HEIKKINEN Sami , SINERVIRTA Riitta , HALMEKYTÖ Maria , ALAKUIJALA Pekka , JÄNNE Juhani
Published: August 2015

We have generated a transgenic mouse line harbouring the human ornithine decarboxylase gene under the control of mouse metallothionein I promoter. Even in the absence of an exposure to heavy metals, ornithine decarboxylase was over-expressed in heart, testis, brain, and especially in liver, of the transgenic animals. An exposure of the transgenic mice to zinc further enhanced the enzyme activity to a level which in liver represented up to 8000-fold increase in comparison with non-transgenic animals. The striking stimulation of liver ornithine decarboxylase activity upon treatment of the transgenic mice with zinc was accompanied by a nearly 150-fold increase in the hepatic putrescine content as compared with similarly treated non-transgenic animals. Even though the liver putrescine concentration reached that of spermidine and spermine in the transgenic animals, the contents of the higher polyamines only transiently increased upon zinc administration and then returned to the basal level. These findings once again indicate that mammalian cells possess extremely powerful regulatory machinery to prevent an over-accumulation of spermidine and spermine in non-dividing cells, and that very high tissue putrescine concentrations can be tolerated, at least for periods of a few days, with seemingly no phenotypic changes.
ATP-dependent glutathione disulphide transport mediated by the MRP gene-encoded conjugate export pump
Authors: LEIER Inka , JEDLITSCHKY Gabriele , BUCHHOLZ Ulrike , CENTER Melvin , COLE Susan P. C. , DEELEY Roger G. , KEPPLER Dietrich
Published: August 2015

We have previously shown that the multidrug resistance protein (MRP) mediates the ATP-dependent membrane transport of the endogenous glutathione conjugate leukotriene C4 (LTC4) and of structurally related anionic conjugates of lipophilic compounds [Jedlitschky, Leier, Buchholz, Center and Keppler (1994) Cancer Res. 54, 4833–4836; Leier, Jedlitschky, Buchholz, Cole, Deeley and Keppler (1994) J. Biol. Chem. 269, 27807–27810]. We demonstrate in the present study that MRP also mediates the ATP-dependent transport of GSSG, as shown in membrane vesicles from human leukaemia cells overexpressing MRP (HL60/ADR cells) or HeLa cells transfected with an MRP expression vector (HeLa T5 cells) in comparison with the respective parental or control cells. The Km value for ATP-dependent transport of GSSG was 93±26 μM (mean value±S.D., n = 5) in membrane vesicles from HeLa T5 cells. GSH, at a concentration of 100 μM, was not a substrate for any significant ATP-dependent MRP-mediated transport. The transport of GSSG was competitively inhibited by LTC4, by the leukotriene D4 receptor antagonist 3-([{3-(2-[7-chloro-2-quinolinyl]ethenyl)phenyl}-{(3-dimethylamino-3-oxopropyl)-thio}-methyl]thio)propanoic acid (MK571) and by S-decylglutathione, with Ki values of 0.3, 0.6 and 0.7 μM respectively. These studies identify MRP as the membrane glycoprotein which mediates the ATP-dependent export of GSSG from these cells.
Phosphonamidate analogues of dipeptides with carboxypeptidase A and β-lactamase-inhibitory activity: elucidation of the mechanism of β-lactamase inhibition by electrospray mass spectrometry
Authors: PAYNE David J. , BATESON John H. , TOLSON David , GASSON Brian , KHUSHI Teresa , LEDENT Philippe , FRÈRE Jean-Marie
Published: August 2015

A series of phosphonamidate compounds with different P1´ amino acid residues have been shown to be irreversible inactivators of the serine β-lactamase from Enterobacter cloacae P99. The efficiency of inhibition (based on k2/K values) of P99 by these derivatives, ordered in decreasing potency, is: β-phenyl-β-Ala> L-Phe> β-Ala> Gly>D-Phe>D-Pro> D-thiazolidine. The D- and L-Phe compounds also inhibit carboxypeptidase A. The proline and thiazolidine derivatives were phosphonamidate methyl esters, whereas the others were salts of diacids. Electrospray mass spectrometry showed that equimolar mixtures of the P99 enzyme with each of the following derivatives, Gly, D-Phe, L-Phe, β-Ala and β-phenyl-β-Ala, effected efficient adduct formation (70–95% of enzyme modified), illustrating the particularly active nature of some of these compounds. All the primary amino acid derivatives gave a similar mass increment, which suggests the displacement of the variable P1´ part of the molecule. This observation provides evidence that the compounds phosphonylate the active-site serine, with the phosphonamidate bond as the scissile bond and the amino acid as the leaving group. The thiazolidine derivative (phosphonamidate methyl ester) also appeared to work by the same mechanism. The comparable proline derivatives caused lower than expected mass shifts of 227–229, and therefore it is proposed that with these compounds both the amino acid and the phosphonamidate ester methoxy group were displaced at the phosphorus atom during the inhibition process. Therefore, electrospray mass spectrometry has provided both a measure of potency and a rationale for the mechanism of inhibition of P99 by these compounds.
Mitochondrial differentiation during the early development of the brine shrimp Artemia franciscana
Authors: VALLEJO Carmen G. , LÓPEZ Mercedes , OCHOA Pilar , MANZANARES Miguel , GARESSE Rafael
Published: August 2015

During the early development of Artemia there is an increase in mitochondrial enzyme activities of about one order of magnitude, whereas the activities of two cytoplasmic enzymes tested as controls remain unaltered. The mitochondrial enzyme activation correlates with (i) large changes in mitochondrial morphology, (ii) a 5-fold increase in the amount of the H+-ATP synthase β-subunit and (iii) a dramatic increase in the steady-state level of mitochondrial mRNAs, whereas mitochondrial rRNA concentrations remain mostly unchanged. In contrast, the level of mitochondrial DNA does not change significantly during the first 20 h after resumption of development. After hatching, the mitochondrial DNA content increases twice in parallel with one round of cellular division, thus indicating that mitochondrial and nuclear replication are coupled in Artemia postgastrular development. The data presented strongly suggest that mitochondrial maturation in the absence of significant mitochondrial proliferation is responsible for the dramatic increase in mitochondrial function that takes place after resumption of development in Artemia.
Phosphatidylcholine increases the secretion of triacylglycerol-rich lipoproteins by CaCo-2 cells
Authors: MATHUR Satya N. , BORN Ella , MURTHY Shubha , FIELD F. Jeffrey
Published: August 2015

The regulation of lipid synthesis and secretion by phosphatidylcholine was investigated in CaCo-2 cells grown on semipermeable filters. In cells incubated with 1 mM taurocholate and 100–500 μM phosphatidylcholine, cholesteryl ester synthesis was decreased, triacylglycerol synthesis was increased modestly, whereas phospholipid synthesis was unaffected. Acyl-CoA–cholesterol acyltransferase activity was decreased secondary to a decrease in the substrate (cholesterol) supply. The basolateral secretion of newly synthesized triacylglycerol and triacylglycerol mass was significantly increased by phosphatidylcholine, whereas cellular triacylglycerol mass decreased. This effect was not specific for phosphatidylcholine as phosphatidylethanolamine and phosphatidylserine also increased the secretion of newly synthesized triacylglycerols. Dioleoylphosphatidylcholine was as effective as egg phosphatidylcholine in increasing triacylglycerol transport. Dipalmitoylphosphatidylcholine, in contrast, was without effect. Phosphatidylcholine also increased the basolateral secretion of apolipoprotein B (apoB) mass without altering apoB mRNA levels. Disruption of the Golgi apparatus by monensin or brefeldin A prevented the increase in apoB secretion by phosphatidylcholine. Compared with microsomes prepared from control cells, those from cells incubated with phosphatidylcholine contained more newly synthesized apoB. The percentage of new synthesized apoB isolated from the lumen of microsomes (as an estimate of apoB destined for secretion), however, was similar in the two preparations. Thus in CaCo-2 cells incubated with phosphatidylcholine, the transport of apoB and triacylglycerols is increased whereas cholesteryl ester synthesis and secretion are decreased. A normally functioning secretory pathway is required for phosphatidylcholine to increase triacylglycerol-rich lipoprotein secretion.
Properties of chicken erythrocyte histone deacetylase associated with the nuclear matrix
Authors: LI Wei , CHEN Hou Yu , DAVIE James R.
Published: August 2015

Histone H2B is deacetylated more rapidly than H3 and H4 in chicken immature erythrocytes. Histone deacetylase from chicken immature erythrocytes was partially purified, and the histone specificities of the multiple histone deacetylase forms were determined. Ion-exchange (Q-Sepharose) and gel-exclusion (Superdex 200) chromatography of extracts from erythrocyte nuclei showed two forms (HD1 and HD2) of histone deacetylase. HD1, with a molecular mass of about 55 kDa, preferred free H3–H4 relative to H2A–H2B, while HD2, with a molecular mass of approx. 220 kDa, had a slight preference for H3–H4. HD1 and HD2 differed in pH- and ionic-strength-dependence. HD2 dissociated into HD1 when treated with 1.6 M NaCl or when applied to a Q-Sepharose column. The enzymic properties of nuclear-matrix-bound histone deacetylase showed a striking difference from that of HD1 and HD2, particularly in its strong preference for H2A–H2B. Treatment of the nuclear matrix with 1.6 M NaCl and 1% 2-mercaptoethanol solubilized histone deacetylase, which chromatographed as 400 and 220 kDa forms on a Superdex 200 column. The solubilized enzyme retained its histone preference for H2A-H2B. Chromatography of the nuclear-matrix-derived enzyme on Q-Sepharose yielded one peak of enzyme activity with chromatographic properties and histone specificities similar to those of HD1. These results provide support for the active form of the enzyme in situ being a high-molecular-mass complex associated with proteins that are components of the nuclear matrix. Substrate preference of the enzyme is governed by the proteins associated with the histone deacetylase.
Selection of hammerhead ribozymes for optimum cleavage of interleukin 6 mRNA
Authors: HENDRIX Chris , ANNÉ Jozef , JORIS Bernard , VAN AERSCHOT Arthur , HERDEWIJN Piet
Published: August 2015

Four GUC triplets in the coding region of the mRNA of interleukin 6 (IL-6) were examined for their suitability to serve as a target for hammerhead ribozyme-mediated cleavage. This selection procedure was performed with the intention to down-regulate IL-6 production as a potential treatment of those diseases in which IL-6 overexpression is involved. Hammerhead ribozymes and their respective short synthetic substrates (19-mers) were synthesized for these four GUC triplets. Notwithstanding the identical catalytic core sequences, the difference in base composition of the helices involved in substrate binding caused substantial variation in cleavage activity. The cleavage reactions on the 1035 nucleotide IL-6 mRNA transcript revealed that two ribozymes were able to cleave this substrate, showing a decrease in catalytic efficiency to 1/30 and 1/300 of the short substrate. This study indicates that the GUC triplet located at nucleotide 510 of the mRNA of IL-6 is the best site for hammerhead ribozyme-mediated cleavage. We suggest that in future targeting of chemically modified hammerhead ribozymes for cleavage of IL-6 mRNA should be directed at this location.
The conformation of Alzheimer's β peptide determines the rate of amyloid formation and its resistance to proteolysis
Authors: SOTO Claudio , CASTAÑO Eduardo M.
Published: August 2015

Amyloid β-peptide (Aβ) is found in an aggregated poorly soluble form in senile or neuritic plaques deposited in the brain of individuals affected by Alzheimer's disease (AD). In addition soluble Aβ (sAβ) is identified normally circulating in human body fluids. In this study we report that synthetic peptides containing the sequences 1–40 and 1–42 of Aβ, and Aβ analogues bearing amino acid substitutions can adopt two major conformational states in solution: (1) an amyloidogenic conformer (Aβac) with a high content of β-sheet and partly resistant to proteases and (2) a non-amyloidogenic conformer (Aβnac) with a random coil conformation and protease-sensitive. The differences in the fibrillogenesis rate and in the protease resistance among the several Aβ peptides studied depend mainly on the relative propensity for adopting the amyloidogenic conformation, which in the absence of external factors is largely conditioned by the primary structure of the peptide. Aβnac containing the sequence 1–40, 1–42 or bearing amino acid substitutions (Dutch variant of Aβ) was protease-sensitive and unable to form a significant amount of amyloid even at high concentrations or after long incubations. The finding of the simultaneous existence of different Aβ conformers with distinct abilities to form amyloid may help to explain why Aβ is found in both soluble and fibrillar forms in vivo.
Exposure of ligand-binding sites on platelet integrin αIIB/β3 by phosphorylation of the β3 subunit
Authors: van WILLIGEN Gijsbert , HERS Ingeborg , GORTER Gertie , AKKERMAN Jan-Willem N.
Published: August 2015

The exposure of ligand-binding sites for adhesive proteins on platelet integrin αIIB/β3 (glycoprotein IIB/IIIA) by platelet-activating factor (PAF) is transient, whereas sites exposed by α-thrombin remain accessible. The same difference is seen in the phosphorylation of the β3 subunit. Inhibition of protein kinases (1 μM staurosporine) and protein kinase C (10 μM bisindolylmaleimide) closes binding sites exposed by both agonists and induces dephosphorylation of β3. Inhibition of Tyr-kinases (20 μM Herbimycin A) has only a slight effect. Inhibition of Ser/Thr-phosphatases (1 μM okadaic acid, 30 s preincubation) changes the transient exposure and β3 phosphorylation by PAF into the ‘permanent’ patterns induced by α-thrombin. Inhibition of Tyr-phosphatases (100 μM vanadate) has little effect. Preincubation with okadaic acid makes exposed binding sites and phosphorylated β3 insensitive to staurosporine, resulting in exposed αIIB/β3 independent of concurrent phosphorylation/dephosphorylation. The stoichiometry of β3 phosphorylation by α-thrombin is 0.80±0.10. Thus, one of the mechanisms that regulates exposure and closure of ligand-binding sites on the αIIB/β3 is phosphorylation/dephosphorylation of a Ser/Thr-residue in the β3 subunit.
Amino acid substitutions enhancing thermostability of Bacillus polymyxa β-glucosidase A
Authors: LOPEZ-CAMACHO Cristina , SALGADO Jesus , LEQUERICA Juan Luis , MADARRO Alejo , BALLESTAR Esteban , FRANCO Luis , POLAINA Julio
Published: August 2015

Mutations enhancing the thermostability of β-glucosidase A of Bacillus polymyxa, a family 1 glycosyl hydrolase, have been obtained after hydroxylamine mutagenesis of a plasmid containing the bglA gene, transformation of Escherichia coli with the mutagenized plasmid, and identification of transformant colonies that showed β-glucosidase activity after a thermal treatment that inactivated the wild-type enzyme. Two additive mutations have been characterized that cause replacement of glutamate at position 96 by lysine and of methionine at position 416 by isoleucine respectively. The thermoresistant mutant enzymes showed increased resistance to other denaturing agents, such as pH and urea, while their kinetic parameters did not change. CD spectra indicated that the E96K replacement caused an increase in α-helix content. The observed effect of the M416I mutation is consistent with the lower content of cysteine and methionine found in family 1 enzymes of thermophilic species compared with similar ones from mesophilic organisms.
Nitric oxide and peroxynitrite exert distinct effects on mitochondrial respiration which are differentially blocked by glutathione or glucose
Authors: LIZASOAIN Ignacio , MORO Maria A. , KNOWLES Richard G. , DARLEY-USMAR Victor , MONCADA Salvador
Published: August 2015

Nitric oxide (NO) and peroxynitrite both inhibit respiration by brain submitochondrial particles, the former reversibly at cytochrome c oxidase, the latter irreversibly at complexes I–III. Both GSH (IC50 = 10 μM) and glucose (IC50 = 8 mM) prevented inhibition of respiration by peroxynitrite (ONOO-), but neither glucose (100 mM) nor GSH (100 μM) affected that by NO. Thus, unless ONOO- is formed within mitochondria it is unlikely to inhibit respiration in cells directly, because of reactions with cellular thiols and carbohydrates. However, the reversible inhibition of respiration at cytochrome c oxidase by NO is likely to occur (e.g. in the brain during ischaemia) and could be responsible for cytotoxicity.
Cellular processing of the nerve growth factor precursor by the mammalian pro-protein convertases
Authors: SEIDAH Nabil G. , BENJANNET Suzanne , PAREEK Sangeeta , SAVARIA Diane , HAMELIN Josée , GOULET Brigitte , LALIBERTÉ Jacynthe , LAZURE Claude , CHRÉTIEN Michel , MURPHY Richard A.
Published: August 2015

In order to define the enzymes responsible for the maturation of the precursor of nerve growth factor (proNGF), its biosynthesis and intracellular processing by the pro-protein convertases furin, PC1, PC2, PACE4, PC5 and the PC5 isoform PC5/6-B were analysed using the vaccinia virus expression system in cells containing a regulated and/or a constitutive secretory pathway. Results demonstrate that in both cell types furin, and to a lesser extent PACE4 and PC5/6-B, are the best candidate proNGF convertases. Furthermore, two processed NGF forms of 16.5 and 13.5 kDa were evident in constitutively secreting cell lines such as LoVo and BSC40 cells, whereas only the 13.5 kDa form was observed in AtT20 cells, which contain secretory granules. Both forms display the same N-terminal sequence as mature NGF, and were also produced following site-directed mutagenesis of the C-terminal Arg-Arg sequence of NGF into Ala-Ala, suggesting that the difference between them is not at the C-terminus. Co-expression of proNGF with furin and either chromogranin B or secretogranin II (but not chromogranin A) in BSC40 cells eliminated the 16.5 kDa form. Data also show that N-glycosylation of the pro-segment of proNGF and trimming of the oligosaccharide chains are necessary for the exit of this precursor from the endoplasmic reticulum and its eventual processing and secretion. Sulphate labelling experiments demonstrated that proNGF is processed into mature NGF following the arrival of the precursor in the trans-Golgi network. This comparative study shows that the three candidate mammalian subtilisin/kexin-like convertases identified process proNGF into NGF and that the nature of the final processed products is dependent on the intracellular environment.
Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32
Authors: SLEE Elizabeth A. , ZHU Huijun , CHOW Sek C. , MacFARLANE Marion , NICHOLSON Donald W. , COHEN Gerald M.
Published: August 2015

Interleukin-1β converting enzyme (ICE)-like proteases, which are synthesized as inactive precursors, play a key role in the induction of apoptosis. We now demonstrate that benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK), an ICE-like protease inhibitor, inhibits apoptosis by preventing the processing of CPP32 to its active form. These results suggest that novel inhibitors of apoptosis can be developed which prevent processing of proforms of ICE-like proteases.
Hormonal regulation of glucose transport in a brown adipose cell preparation isolated from rats that shows a large response to insulin
Authors: OMATSU-KANBE Mariko , ZARNOWSKI Mary Jane , CUSHMAN Samuel W.
Published: August 2015

Isolated brown adipose cells from rats are prepared whose viability is indicated by the expected stimulation of oxygen consumption by noradrenaline and counter-regulation of this oxygen consumption response by insulin. Insulin stimulates 3-O-methyl-D-glucose transport by approx. 15-fold in the absence of adenosine, and adenosine augments this response at least 2-fold. The insulin-stimulated translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane is readily detected by subcellular fractionation and Western blotting, and the appearance of GLUT4 on the cell surface in response to insulin is demonstrated by bis-mannose photolabelling. Isoprenaline also stimulates glucose transport activity but only by approx. 3-fold; this effect is not altered by adenosine. Isoprenaline increases insulin-stimulated glucose transport activity in the absence of adenosine but decreases it in the presence of adenosine. These results demonstrate that although the regulation of glucose transport by insulin in brown adipose cells is qualitatively similar to that in white adipose cells, counter-regulation by adenosine and isoprenaline is at least quantitatively and may be qualitatively different. Isolated brown adipose cells from rats thus represent an excellent model for further examination of the mechanism by which multiple hormone signalling pathways interact to control glucose transport and GLUT4 subcellular trafficking.
Identification of 14-3-3 proteins in human platelets: effects of synthetic peptides on protein kinase C activation
Authors: WHEELER-JONES Caroline P. D. , LEARMONTH Michele P. , MARTIN Harry , AITKEN Alastair
Published: August 2015

The 14-3-3 proteins inhibit protein kinase C (PKC) activity in vitro and contain conserved sequences that resemble the pseudosubstrate domain of PKC and the C-terminus of the annexins. In the present study we have identified the isoforms of 14-3-3 in human platelets and used synthetic peptides derived from the regions with similarity to PKC and annexins to examine the potential role of 14-3-3 in regulating platelet PKC activity. Immunoblotting studies with isoform-specific antisera raised against the acetylated peptides corresponding to the N-termini of 14-3-3 showed that these cells express high levels of the β, γ and ζ 14-3-3 isoforms. In addition, low levels of the ε and η 14-3-3 isoforms were detected. In washed, saponin-permeabilized platelets incubated with [γ-32P]ATP, thrombin- and phorbol 12-myristate 13-acetate (PMA)-induced phosphorylation of several proteins (66, 45, and 20 kDa) was inhibited by preincubation with AS peptide (KNVVGARRSSWRVISSIEQK) based on the pseudosubstrate-like region of the 14-3-3 family. A control peptide of similar size had no effect on PKC-mediated phosphorylation. PMA caused a rapid translocation of PKC activity from the cytosol to the particulate fraction of saponin-permeabilized platelets that was unaffected by either the AS peptide or a peptide derived from the annexin-like 14-3-3 domain (MKGDYYRYLAEVATGDD). These results suggest that isoforms of the 14-3-3 family may play an important physiological role as inhibitors of PKC activity in human platelets but are unlikely to be involved in controlling association of PKC with the membrane.
A 33 kDa serine proteinase from Scedosporium apiospermum
Authors: LARCHER Gérald , CIMON Bernard , SYMOENS Franç;oise , TRONCHIN Guy , CHABASSE Dominique , BOUCHARA Jean-Philippe
Published: August 2015

An extracellular proteinase produced by the filamentous fungus Scedosporium apiospermum has been purified and characterized. Initially, in vitro conditions for enzyme synthesis were investigated. The highest yield of enzyme production was obtained when the fungus was cultivated in modified Czapek–Dox liquid medium supplemented with 0.1% bacteriological peptone and 1% (w/v) glucose as the nitrogen and carbon sources respectively. Purification to homogeneity of the proteinase was accomplished by (NH4)2SO4 precipitation, followed by gel filtration through Sephadex G-75 and finally affinity chromatography through immobilized phenylalanine. Analysis of the purified enzyme by SDS/PAGE revealed a single polypeptide chain with an apparent molecular mass of 33 kDa. Further investigation of its physical and biochemical properties disclosed numerous similarities with those of the previously described serine proteinase of Aspergillus fumigatus. The enzyme was not glycosylated and its pI was 9.3. Proteinase activity was optimum between 37 and 50 °C and at pH 9.0, but remained high within a large range of pH values between 7 and 11. The inhibition profile and N-terminal amino acid sequencing confirmed that this enzyme belongs to the subtilisin family of serine proteinases. In agreement with this, the specific synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide proved to be an excellent substrate for the proteinase, with an estimated Km of 0.35 mM. Like the alkaline proteinase of A. fumigatus, this enzyme was able to degrade human fibrinogen, and thus may act as a mediator of the severe chronic bronchopulmonary inflammation from which cystic fibrosis patients suffer.
Cloning and expression of pig kidney dopa decarboxylase: comparison of the naturally occurring and recombinant enzymes
Authors: MOORE Patrick S. , DOMINICI Paola , BORRI VOLTATTORNI Carla
Published: August 2015

L-Aromatic amino acid decarboxylase (dopa decarboxylase; DDC) is a pyridoxal 5´-phosphate (PLP)-dependent homodimeric enzyme that catalyses the decarboxylation of L-dopa and other L-aromatic amino acids. To advance structure–function studies with the enzyme, a cDNA that codes for the protein from pig kidney has been cloned by joining a partial cDNA obtained by library screening with a synthetic portion constructed by the annealing and extension of long oligonucleotides. The hybrid cDNA was then expressed in Escherichia coli to produce recombinant protein. During characterization of the recombinant enzyme it was unexpectedly observed that it possesses certain differences from the enzyme purified from pig kidney. Whereas the latter protein binds 1 molecule of PLP per dimer, the recombinant enzyme was found to bind two molecules of coenzyme per dimer. Moreover, the Vmax was twice that of the protein purified from tissue. On addition of substrate, the absorbance changes accompanying transaldimination were likewise 2-fold greater in the recombinant enzyme. Examination of the respective apoenzymes by absorbance, CD and fluorescence spectroscopy revealed distinct differences. The recombinant apoprotein has no significant absorbance at 335 nm, unlike the pig kidney apoenzyme; in the latter case this residual absorbance is associated with a positive dichroic signal. When excited at 335 nm the pig kidney apoenzyme has a pronounced emission maximum at 385 nm, in contrast with its recombinant counterpart, which shows a weak broad emission at about 400 nm. However, the holoenzyme–apoenzyme transition did not markedly alter the respective fluorescence properties of either recombinant or pig kidney DDC when excited at 335 nm. Taken together, these findings indicate that recombinant pig kidney DDC has two active-site PLP molecules and therefore displays structural characteristics typical of PLP-dependent homodimeric enzymes. The natural enzyme contains one active-site PLP molecule whereas the remaining PLP binding site is most probably occupied by an inactive covalently bound coenzyme derivative; some speculations are made about its origin. The coenzyme absorbing bands of recombinant DDC show a modest pH dependence at 335 and 425 nm. A putative working model is presented to explain this behaviour.
Ligand-induced conformational change in the human mineralocorticoid receptor occurs within its hetero-oligomeric structure
Authors: COUETTE Brigitte , FAGART Jérôme , JALAGUIER Stéphan , LOMBES Marc , SOUQUE Anny , RAFESTIN-OBLIN Marie-Edith
Published: August 2015

To determine the first steps involved in the mechanism of action of aldosterone and its antagonists, we analysed the ligand-induced structural changes of the human mineralocorticoid receptor (hMR) translated in vitro. Limited chymotrypsin digestion of the receptor generated a 30 kDa fragment. Following binding of a ligand to hMR, the 30 kDa fragment became resistant to chymotrypsin proteolysis, indicating a change in the receptor conformation. Differences in sensitivity to chymotrypsin of the 30 kDa fragment were observed after binding of agonists and antagonists to hMR, suggesting that these two classes of ligands induced different hMR conformations. Several lines of evidence allowed us to identify the 30 kDa fragment as the subregion encompassing the C-terminal part of the hinge region and the ligand-binding domain (LBD) of hMR (hMR 711–984). (1) The 30 kDa fragment is not recognized by FD4, an antibody directed against the N-terminal region of hMR. (2) Aldosterone remains associated with the 30 kDa fragment after chymotrypsin proteolysis of the aldosterone–hMR complex. (3) A truncated hMR, lacking the last 40 C-terminal amino acids (hMR 1–944), yields a 26 kDa proteolytic fragment. In addition, we showed that the unbound and the aldosterone-bound 30 kDa fragment were both associated with heat-shock protein (hsp) 90, indicating that the ligand-induced conformational change takes place within the hetero-oligomeric structure and that the 711–984 region is sufficient for hsp90–MR interaction. We conclude that the ligand-induced conformational change of the receptor is a crucial step in mineralocorticoid action. It occurs within the LBD, precedes the release of hsp90 from the receptor and is dependent upon the agonist/antagonist nature of the ligand.
Arginine-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leucocytes
Authors: DONNELLY Louise E. , RENDELL Nigel B. , MURRAY Stephen , ALLPORT Jennifer R. , LO Gar , KEFALAS Panagiotis , TAYLOR Graham W. , MacDERMOT John
Published: August 2015

An Arg-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leucocytes (PMNs) was confirmed by the use of diethylamino(benzylidineamino)guanidine (DEA-BAG) as an ADP-ribose acceptor. Two separate HPLC systems were used to separate ADP-ribosyl-DEA-BAG from reaction mixtures, and its presence was confirmed by electrospray mass spectrometry. ADP-ribosyl-DEA-BAG was produced in the presence of PMNs, but not in their absence. Incubation of DEA-BAG with ADP-ribose (0.1–10 mM) did not yield ADP-ribosyl-DEA-BAG, which indicates that ADP-ribosyl-DEA-BAG formed in the presence of PMNs was not simply a product of a reaction between DEA-BAG and free ADP-ribose, due possibly to the hydrolysis of NAD+ by an NAD+ glycohydrolase. The assay of mono(ADP-ribosyl)transferase with agmatine as a substrate was modified for intact PMNs, and the activity was found to be approx. 50-fold lower than that in rabbit cardiac membranes. The Km of the enzyme for NAD+ was 100.1±30.4 μM and the Vmax 1.4±0.2 pmol of ADP-ribosylagmatine/h per 106 cells. The enzyme is likely to be linked to the cell surface via a glycosylphosphatidylinositol anchor, since incubation of intact PMNs with phosphoinositol-specific phospholipase C (PI-PLC) led to a 98% decrease in mono(ADP-ribosyl)transferase activity in the cells. Cell surface proteins were labelled after exposure of intact PMNs to [32P]NAD+. Their molecular masses were 79, 67, 46, 36 and 26 kDa. The time course for labelling was non-linear under these conditions over a period of 4 h. The labelled products were identified as mono(ADP-ribosyl)ated proteins by hydrolysis with snake venom phosphodiesterase to yield 5´-AMP.
Specific binding of the Akt-1 protein kinase to phosphatidylinositol 3,4,5-trisphosphate without subsequent activation
Authors: JAMES Stephen R. , Peter DOWNES C. , GIGG Roy , GROVE Simon J. A. , HOLMES Andrew B. , ALESSI Dario R.
Published: August 2015

Recent evidence has suggested that activation of phosphoinositide 3-kinase (PI 3-kinase) is required for the activation of Akt-1 by growth factors and insulin. Here we demonstrate by two independent methods that Akt-1 from L6 myotubes binds to PtdIns(3,4,5)P3, PtdIns(3,4)P2 and PtdIns(4,5)P2 when presented against a background of phosphatidylserine (PtdSer) or a 1:1 mixture of PtdSer and phosphatidylcholine (PtdCho). No binding was observed with the lipids PtdIns(3,5)P2, PtdIns4P and PtdIns3P or background lipids. Activated, hyperphosphorylated forms of Akt-1 from insulin-stimulated L6 myotubes bound to PtdIns(3,4,5)P3 in a similar manner as inactive Akt-1. Quantitative analysis using surface plasmon resonance showed that the equilibrium association constant for the binding of Akt-1 to PtdIns(3,4,5)P3 was submicromolar and that PtdIns(3,4)P2 and PtdIns(4,5)P2 bound to Akt-1 with 3- and 6-fold lower affinities respectively. Interaction of Akt-1 with PtdIns(3,4,5)P3 did not activate the protein kinase activity, either before or after incubation with MgATP. A model is presented in which PtdIns(3,4,5)P3 may prime Akt-1 for activation by another protein kinase, perhaps by recruiting it to the plasma membrane.
Tissue-specific and androgen-regulated expression of human prostate-specific transglutaminase
Authors: DUBBINK Hendrikus J. , VERKAIK Nicole S. , FABER Peter W. , TRAPMAN Jan , SCHRÖDER Fritz H. , ROMIJN Johannes C.
Published: August 2015

Transglutaminases (TGases) are calcium-dependent enzymes catalysing the post-translational cross-linking of proteins. In the prostate at least two TGases are present, the ubiquitously expressed tissue-type TGase (TGC), and a prostate-restricted TGase (TGP). This paper deals with the molecular cloning and characterization of the cDNA encoding the human prostate TGase (hTGP). For this purpose we have screened a human prostate cDNA library with a probe from the active-site region of TGC. The largest isolated cDNA contained an open reading frame encoding a protein of 684 amino acids with a predicted molecular mass of 77 kDa as confirmed by in vitro transcription–translation and subsequent SDS/PAGE. The hTGP gene was tissue-specifically expressed in the prostate, yielding an mRNA of approx. 3.5 kb. Furthermore, a 3-fold androgen-induced up-regulation of hTGP mRNA expression has been demonstrated in the recently developed human prostate cancer cell line, PC346C. Other well established human prostate cancer cell lines, LNCaP and PC-3, showed no detectable hTGP mRNA expression on a Northern blot. The gene coding for prostate TGase was assigned to chromosome 3.
Purification, cloning and expression of dehydroascorbic acid-reducing activity from human neutrophils: identification as glutaredoxin
Authors: PARK Jae B. , LEVINE Mark
Published: August 2015

Dehydroascorbic acid-reducing activity in normal human neutrophil lysates was characterized and identified by activity-based purification and measurement of newly synthesized ascorbate by HPLC. The initial reducing activity was non-dialysable and could not be accounted for by the activity of glutathione as a reducing agent. The reducing activity was purified to homogeneity as an 11 kDa protein. The protein had a specific activity of 3 μmol/min per mg of protein and was glutathione dependent. Kinetic experiments showed that the protein had a Km for glutathione of 2.0 mM and a Km for dehydroascorbic acid of 250 μM. Dehydroascorbic acid reduction by the purified protein was pH dependent and was maximal at pH 7.5. Peptide fragments from the purified protein were analysed for amino acid sequence and the protein was identified as glutaredoxin. By using degenerate oligonucleotides based on the amino acid sequence, glutaredoxin was cloned from a human neutrophil library. Expressed purified glutaredoxin displayed reducing activity and kinetics that were indistinguishable from those of native purified enzyme. Several approaches indicated that glutaredoxin was responsible for the most of the protein-mediated dehydroascorbic acid reduction in lysates. From protein purification data, glutaredoxin was responsible for at least 47% of the initial reducing activity. Dehydroascorbic acid reduction was at least 5-fold greater in neutrophil lysates than in myeloid tumour cell lysates, and glutaredoxin was detected in normal neutrophil lysates but not in myeloid tumour cell lysates by Western blotting. Glutaredoxin inhibitors inhibited dehydroascorbic acid reduction in neutrophil lysates as much as 80%. These findings indicate that glutaredoxin plays a major role in dehydroascorbic acid reduction in normal human neutrophil lysates, and represent the first identification of dehydroascorbic acid reductase in human tissue by activity-based purification.