bluuebunny/crossref_metadata_2025 huggingface dataset:
Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphatase
Authors: Oda K , Cheng J , Saku T , Takami N , Sohda M , Misumi Y , Ikehara Y , Millán J L
Published: August 2015

Placental alkaline phosphatase (PLAP) is initially synthesized as a precursor (proPLAP) with a C-terminal extension. We constructed a recombinant cDNA which encodes a chimeric protein (alpha GL-PLAP) comprising rat alpha 2u-globulin (alpha GL) and the C-terminal extension of PLAP. Two molecular species (25 kDa and 22 kDa) were expressed in the COS-1 cell transfected with the cDNA for alpha GL-PLAP. Only the 22 kDa form was labelled with both [3H]stearic acid and [3H]ethanolamine. Upon digestion with phosphatidylinositol-specific phospholipase C the 22 kDa form was released into the medium, indicating that this form is anchored on the cell surface via glycosylphosphatidylinositol (GPI). A specific IgG raised against a C-terminal nonapeptide of proPLAP precipitated the 25 kDa form but not the 22 kDa form, suggesting that the 25 kDa form is a precursor retaining the C-terminal propeptide. When a mutant alpha GL-PLAP, in which the aspartic acid residue is replaced with tryptophan at a putative cleavage/attachment site, was expressed in COS-1 cells, the 25 kDa precursor was the only form found inside the cell and retained in the endoplasmic reticulum, as judged by immunofluorescence microscopy. In vitro translation programmed with mRNAs coding for the wild-type and mutant forms of alpha GL-PLAP demonstrated that the C-terminal propeptide was cleaved from the wild-type chimeric protein, but not from the mutant one. This gave rise to the 22 kDa form attached with a GPI anchor, suggesting that GPI is covalently linked to the aspartic acid residue (Asp159) of alpha GL-PLAP. Taken together, these results indicate that the C-terminal propeptide of PLAP functions as a signal to render alpha GL a GPI-linked membrane protein in vitro and in vivo in cultured cells, and that the chimeric protein constructed in this study may be useful for elucidating the mechanism underlying the cleavage of the propeptide and attachment of GPI, which occur in the endoplasmic reticulum.
Inhibition of the differentiation of human myeloid cell lines by redox changes induced through glutathione depletion
Authors: Esposito F , Agosti V , Morrone G , Morra F , Cuomo C , Russo T , Venuta S , Cimino F
Published: August 2015

We have investigated the effect of redox changes in vivo on the differentiation of two human myeloid cell lines, HL-60 and KG-1. The glutathione-depleting agent diethyl maleate (DEM) prevented the development of differentiated features in response to phorbol esters, including adherence of the cells to plastic surfaces and repression of the myeloperoxidase and CD34 genes. Moreover, DEM abolished phorbol 12-myristate 13-acetate-induced activation of the transcription factors AP-1 and Egr-1, suggesting that inhibition of differentiation may be due, at least in part, to redox modifications of these proteins.
Caffeine inhibits cytosolic calcium oscillations induced by noradrenaline and vasopressin in rat hepatocytes
Authors: Combettes L , Berthon B , Claret M
Published: August 2015

The effects of caffeine on agonist-induced changes in intracellular Ca2+ concentration ([Ca2+]i) were studied in single fura 2-loaded cells and suspensions of rat hepatocytes. In single cells, caffeine (5-10 mM) inhibited [Ca2+]i oscillations induced both by noradrenaline (0.1 microM) and by vasopressin (0.1 nM). Caffeine shifted the dose-response curves of the [Ca2+]i rise induced by vasopressin (0.5 to 2 nM) and noradrenaline (from 80 to 580 nM) in suspensions of liver cells loaded with quin2. This inhibitory effect of caffeine was not due to inhibition of phosphodiesterase enzymes and elevation of cyclic AMP levels, because application of 3-isobutyl-1-methylxanthine, forskolin or 8-bromo cyclic AMP had no inhibitory effect on the intracellular Ca2+ rise induced by inositol 1,4,5-trisphosphate (InsP3)-dependent agonists. We demonstrate that the inhibitory effect of caffeine may result from at least three actions of caffeine: (1) inhibition of receptor-stimulated InsP3 formation; (2) inhibition of agonist-stimulated Ca2+ influx; and (3) direct inhibition of the InsP3-sensitive Ca(2+)-release channel.
Drosophila melanogaster alcohol dehydrogenase: product-inhibition studies
Authors: Winberg J O , McKinley-McKee J S
Published: August 2015

The Drosophila melanogaster alleloenzymes AdhS and AdhF have been studied with respect to product inhibition by using the two substrate couples propan-2-ol/acetone and ethanol/acetaldehyde together with the coenzyme couple NAD+/NADH. With both substrate couples the reaction was consistent with an ordered Bi Bi mechanism. The substrates added to the enzyme in a compulsory order, with coenzyme as the leading substrate, to give two interconverting ternary complexes. The second ternary complex broke down with release of products in an obligatory order, with the aldehyde/ketone leaving first. Both the acetaldehyde and acetone products formed binary complexes with the enzyme that affected NAD+ binding. However, only an enzyme-acetone complex seemed to affect NADH binding and hence the reverse reaction. The inhibitory pattern with acetaldehyde as product was also affected by the formation of a ternary enzyme-NAD(+)-acetaldehyde complex, which broke down to acetic acid and NADH. The product-inhibition pattern shown in the present work is different from that published for Drosophila Adh previously and this discrepancy can not be explained by the use of different variants of Drosophila Adh.
Characterization of two membrane-associated β-glucosidases from maize (Zea mays L.) coleoptiles
Authors: Feldwisch J , Vente A , Zettl R , Bako L , Campos N , Palme K
Published: August 2015

We isolated membrane vesicles from maize (Zea mays L.) coleoptiles and identified in these vesicles a 58 kDa (pm58) and a 60 kDa (pm60) protein by photoaffinity labelling with 5-azido-[7-3H]indole-3-acetic acid ([3H]N3IAA). Photoaffinity labelling was effectively competed for by auxins as well as by flavonoids. The labelled proteins were solubilized by Triton X-114 from the vesicles and partially purified. Microsequence analysis revealed that pm60 is a beta-glucosidase. This was confirmed by biochemical and immunological analysis. We show that pm60 has a beta-D-glucoside glucohydrolase (EC 3.2.1.21) activity. It uses p-nitro-phenyl beta-D-glucopyranoside (PNPG) as a substrate, with a pH optimum of 5.0. The Km for PNPG is 0.652 mM and the Vmax. 6.24 mumol.min-1.mg-1. The beta-glucosidase activity of pm60 was competitively inhibited by IAA and 1-naphthylacetic acid as well as by gluconolactam and glucose. N-terminal amino-acid-sequence analysis of pm58 revealed similarity to pm60, suggesting that both proteins are encoded by different members of a gene family.
Human GTP cyclohydrolase I: only one out of three cDNA isoforms gives rise to the active enzyme
Authors: Gütlich M , Jaeger E , Rücknagel K P , Werner T , Rödl W , Ziegler I , Bacher A
Published: August 2015

GTP cyclohydrolase I catalyses the first and rate-limiting step of tetrahydrobiopterin biosynthesis. Its expression is regulated by interferon-gamma or kit ligand in a tissue-specific manner. Three different cDNA forms have been reported for human GTP cyclohydrolase I [Togari, Ichinose, Matsumoto, Fujita and Nagatsu (1992) Biochem. Biophys. Res. Commun. 187, 359-365]. We have isolated, from a human liver cDNA library, two clones which contained inserts identical with two of the cDNAs reported by Togari et al. (1992). The three open reading frames corresponding to all reported cDNA sequences were expressed in Escherichia coli. Only the recombinant protein corresponding to the longest reading frame catalysed the conversion of GTP into dihydroneopterin triphosphate. The proteins corresponding to the shorter reading frames failed to catalyse not only the generation of dihydroneopterin triphosphate but also the release of formate from GTP, an intermediate step of the reaction. Recombinant human GTP cyclohydrolase I showed sigmoidal substrate kinetics and maximum activity at 60 degrees C. These findings are well in line with the published properties of the enzyme isolated from rat liver. The data indicate that cytokine-mediated induction of GTP cyclohydrolase I is not due to the expression of enzyme isoforms.
Glucocorticoids down-regulate β1-adrenergic-receptor expression by suppressing transcription of the receptor gene
Authors: Kiely J , Hadcock J R , Bahouth S W , Malbon C C
Published: August 2015

The expression of beta 2-adrenergic receptors is up-regulated by glucocorticoids. In contrast, beta 1-adrenergic receptors display glucocorticoid-induced down-regulation. In rat C6 glioma cells, which express both of these subtypes of beta-adrenergic receptors, the synthetic glucocorticoid dexamethasone stimulates no change in the total beta-adrenergic receptor content, but rather shifts the beta 1:beta 2 ratio from 80:20 to 50:50. Radioligand binding and immunoblotting demonstrate a sharp decline in beta 1-adrenergic receptor expression. Metabolic labelling of cells with [35S]-methionine in tandem with immunoprecipitation by beta 1-adrenergic-receptor-specific antibodies reveals a sharp decline in the synthesis of the receptor within 48 h for cells challenged with glucocorticoid. Steady-state levels of beta 1-adrenergic-receptor mRNA declined from 0.47 to 0.26 amol/microgram of total cellular RNA within 2 h of dexamethasone challenge, as measured by DNA-excess solution hybridization. The stability of receptor mRNA was not influenced by glucocorticoid; the half-lives of the beta 1- and beta 2-subtype mRNAs were 1.7 and 1.5 h respectively. Nuclear run-on assays revealed the basis for the down-regulation of receptor expression, i.e. a sharp decline in the relative rate of transcription for the beta 1-adrenergic-receptor gene in nuclei from dexamethasone-treated as compared with vehicle-treated cells. These data demonstrate transcriptional suppression as a molecular explanation for glucocorticoid-induced down-regulation of beta 1-adrenergic receptors.
A novel adipokinetic octapeptide found in the damselflies Pseudagrion inconspicuum and Ischnura senegalensis
Authors: Janssens M P , Kellner R , Gde G
Published: August 2015

A member of the adipokinetic hormone family of peptide was identified in the damselflies Pseudagrion inconspicuum and Ischnura senegalensis using a heterologous (in migratory locusts and American cockroaches) and a homologous (in P. inconspicuum) bioassay. After isolation of the peptide by reversed-phase h.p.i.c. of corpora cardiaca, its structure was determined by automated Edman degradation and matrix-assisted laser-desorption ionization m.s. The sequence of a blocked uncharged octapeptide was established: pGlu-Val-Asn-Phe-Thr-Pro-Gly-TrpNH2. One corpus cardiacum of P. inconspicuum contains about 2.4 pmol of this novel peptide. The synthetic peptide was chromatographically indistinguishable from the natural compound, and on injection in low quantities into the damselfly P. inconspicuum, haemolymph levels of lipids were increased. It is concluded that the novel peptide may be involved in controlling lipid mobilization during flight. Hence the peptide is designated Psi-AKH, Pseudagrion inconspicuum adipokinetic hormone.
2-Chloro-2′-deoxyadenosine monophosphate residues in DNA enhance susceptibility to 3′ → 5′ exonucleases
Authors: Hentosh P , Grippo P
Published: August 2015

2-Chloro-2′-deoxyadenosine triphosphate, a purine nucleotide analogue and potent antileukaemic agent, was incorporated into double-stranded 36-mers in place of dATP to investigate the effects of 2-chloroadenine (ClAde) on DNA polymerase-associated 3′-->5′ exonuclease activity. ClAde residues within one strand of duplex DNA did not inhibit exonuclease activity; on the contrary, ClAde-containing minus strands were digested to a greater extent than was control DNA in the absence of deoxyribonucleoside triphosphates by Escherichia coli Klenow fragment, yeast DNA polymerase II and T4 DNA polymerase. After a 30 min incubation with 5 units of Klenow fragment, approximately 65% of control DNA remained in DNA fragments of 26 bases or larger compared with only approximately 25% of ClAde-substituted substrates. Unsubstituted plus strands opposite a ClAde-containing strand were likewise digested more quickly by 3′-->5′ exonuclease, but only in the vicinity of the ClAde sites. Approx. 63% of the plus strands from ClAde-containing oligomers were less than 24 bases in length after a 25 min digestion period with Klenow fragment compared with only approximately 32% of control DNA. Such results indicate that, unlike other base modifications such as pyrimidine dimers, methoxy psoralen adducts and certain nucleoside analogues, all of which inhibit or decrease the rate of strand degradation by 3′-->5′ exonucleases, incorporated ClAde enhances strand degradation of duplex DNA.
Phorbol ester selectively stimulates the phospholipase D-mediated hydrolysis of phosphatidylethanolamine in multidrug-resistant MCF-7 human breast carcinoma cells
Authors: Kiss Z , Tomono M , Anderson W B
Published: August 2015

The phospholipase D (PLD)-mediated synthesis of phosphatidylethanol (PtdEtOH) and the hydrolysis of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho) were examined in drug-sensitive and multidrug-resistant lines of MCF-7 human breast carcinoma cells. In drug-sensitive (MCF-7/WT) cells, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) failed to enhance either the synthesis of PtdEtOH or the hydrolysis of either phospholipid. In the drug-resistant (MCF-7/MDR) cells, 100 nM PMA greatly enhanced both the synthesis of PtdEtOH (approximately 21-fold) and the hydrolysis of PtdEtn (approximately 29-fold), but had no effect on the hydrolysis of PtdCho. The PLD activators sphingosine and H2O2 were found to elicit only a slight (1.28-1.4-fold) stimulatory effect on PtdCho hydrolysis in both the MCF-7/WT and MCF-7/MDR cell types, and had only a small effect on PtdEtn hydrolysis in the MCF-7/WT cells as well. However, these agents significantly (approximately 2.6-3.5-fold) stimulated PtdEtn hydrolysis in the MCF-7/MDR cells. These data indicate that MCF-7/MDR cells contain a PtdEtn-specific PLD activity which can be selectively stimulated by PMA, sphingosine and H2O2.
Shear-stress-induced von Willebrand factor binding to platelets causes the activation of tyrosine kinase(s)
Authors: Razdan K , Hellums J D , Kroll M H
Published: August 2015

Pathological arterial blood flow generates fluid shear stresses that directly cause platelet aggregation. The mechanism of shear-induced platelet aggregation is incompletely understood, but involves von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib and GP IIb-IIIa, leading to the transmembrane influx of Ca2+ and the activation of protein kinase C. To investigate this further, shear-stress-induced protein tyrosine phosphorylation (PTP) of washed platelets was studied in a cone-plate viscometer. A time- and shear-stress-dependent tyrosine phosphorylation of substrates with approx. M(r) 29,000-31,000, 36,000, 50,000, 58,000, 64,000, 76,000, 85,000 and 105,000 was observed. PTP in response to a threshold shear stress of 0.3 mN/cm2 (30 dyn/cm2) was enhanced in most cases by exogenous purified human vWF, and PTP in response to a pathological shear stress of 0.9 mN/cm2 (90 dyn/cm2) was inhibited in some cases by inhibiting vWF binding to GP Ib or GP IIb-IIIa, or by inhibiting Ca2+ responses with extracellular EGTA. Shear-induced PTP of a substrate of M(r) approximately 31,000 appeared to be independent of GP Ib, and PTP of a substrate(s) of M(r) approximately 29,000 was shear-stress-dependent but independent of extracellular Ca2+. Cytochalasin D, which inhibits GP Ib-cytoskeleton interactions, inhibits the PTP of a substrate of M(r) approximately 76,000. These results suggest that tyrosine phosphorylation may be involved in transmembrane signalling that mediates platelet adhesion and aggregation in response to pathological shear stresses generated at sites of arterial vaso-occlusion.
Rat liver contains a limited number of binding sites for hepatic lipase
Authors: Schoonderwoerd K , Verhoeven A J , Jansen H
Published: August 2015

The binding of hepatic lipase to rat liver was studied in an ex vivo perfusion model. The livers were perfused with media containing partially purified rat hepatic lipase or bovine milk lipoprotein lipase. The activity of the enzymes was determined in the perfusion media before and after passage through the liver. During perfusion with a hepatic-lipase-containing medium the lipase activity in the medium did not change, indicating that there was no net binding of lipase by the liver. In contrast, more than 80% of the lipoprotein lipase was removed from the medium. This lipoprotein lipase activity could be recovered into the perfusion medium completely by heparin perfusion of the liver. If livers, first depleted of hepatic lipase by heparin, were subsequent perfused with a hepatic-lipase-containing medium, 90 +/- 24 m-units of the lipase activity was bound per g of liver (up to 1000 m-units/total liver). However, heparin treatment of the liver decreases the ability of the liver to re-bind hepatic lipase by 80%. Perfusion of rat livers with 0.3 M NaCl released 60% of the lipase activity into the medium. Upon subsequent perfusion of these livers with hepatic-lipase-containing media, 541 +/- 164 m-units of hepatic lipase could be bound per g of liver (up to 5000 m-units/total liver). The binding of hepatic lipase was also studied in livers of corticotropin (ACTH)-pre-treated rats. In these rats also, hepatic lipase bound only to livers which had been pre-perfused with heparin or 0.3 M NaCl. After heparin pre-perfusion, 88 +/- 12 m-units of hepatic lipase could be bound per g of liver, similar to that with livers of control rats not treated with ACTH. After prior salt perfusion, however, the capacity of the livers of ACTH-pre-treated rats to bind hepatic lipase was 212 +/- 60 m-units/g of liver. This is less than in livers of control rats (541 +/- 164 m-units/g of liver). These results indicate that in rat liver the binding of hepatic lipase is heterogeneous in character and consists of heparin-resistant and heparin-sensitive components. The hepatic-lipase binding capacity of the liver is saturable and fully utilized under various conditions. The heparin-sensitive binding capacity is lowered in ACTH-treated rats, whereas the heparin-resistant binding is unaffected. We postulate that the functional hepatic lipase activity can be regulated by changes in the binding capacity of the liver.
Structure of the human cyclo-oxygenase-2 gene
Authors: Appleby S B , Ristimäki A , Neilson K , Narko K , Hla T
Published: August 2015

Cyclo-oxygenase (Cox), a rate-limiting enzyme in the synthesis of prostanoids, is encoded by two genes, Cox-1 and Cox-2, which are differentially expressed and regulated. Human Cox-1 and -2 polypeptides share 61% primary sequence identity. While the expression of Cox-1 is maximal in quiescent cells. Cox-2 expression is induced by growth factors and cytokines. We have screened a human genomic library with a probe from the 5′-untranslated region (UTR) of the human Cox-2 (hCox-2) cDNA and isolated two overlapping genomic clones. We have determined the DNA sequence of 0.8 kb upstream of the transcription start site, 6 kb of protein coding region, which includes 10 exons and 9 introns, as well as 2.5 kb of the 3′-UTR. The structures of the hCox-1 and hCox-2 and the murine TIS10 (Cox-2) genes are highly conserved, with a few exceptions. The 3′-UTRs of the Cox-1 and -2 genes are distinct; for example, the largest exon in the Cox-2 gene encodes the entire 3′-UTR, containing 22 copies of the ‘AUUUA’ RNA instability element. Sequence analysis of the 5′-flanking region has shown several potential transcription regulatory sequences, including a TATA box, a C/EBP motif, two AP-2 sites, three SP1 sites, two NF-kappa B sites, a CRE motif and an Ets-1 site. These efforts serve as a basis for future studies on transcriptional and post-transcriptional mechanisms of Cox-2 gene regulation.
Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases
Authors: Dent P , Chow Y H , Wu J , Morrison D K , Jove R , Sturgill T W
Published: August 2015

Mitogen-activated protein (MAP) kinase kinases (MKKs) are dual-specificity protein kinases which activate p42mapk and p44mapk by phosphorylation of regulatory tyrosine and threonine residues. cDNAs for two isotypes of MKK, MKK1 and MKK2, have been isolated from several species. Here we describe construction of recombinant baculoviruses for high-level expression of histidine-tagged rat MKK1 and MKK2, and procedures for production of nearly homogeneous MKK1 and MKK2 fusion proteins, in both inactive and active forms. Co-infection of Sf9 cells with either MKK1 or MKK2 virus together with recombinant viruses for Raf-1, pp60src (Y527F) and c-Ha-Ras resulted in activations of 250-fold and 150-fold for MKK1 and MKK2 respectively. Specific activities towards kinase-defective p42mapk were of the order of several hundred nanomoles of phosphate transferred/min per mg of MKK protein. The Michaelis constants for both enzymes were approx. 1 microM. Preparations of activated MKK were apparently free of Raf-1 as assessed by Western blotting. Raf-1 phosphorylated MKK1 on one major tryptic phosphopeptide, the phosphorylation of which increased with time. This phosphopeptide contained only phosphoserine and possessed neutral overall charge at pH 1.9 on two-dimensional peptide mapping. Phosphorylation of MKK1 by Raf-1 correlated with activation and reached a plateau of approximately 2 mol/mol.
Effects of aniso-osmolarity and hydroperoxides on intracellular pH in isolated rat hepatocytes as assessed by (2′,7′)-bis(carboxyethyl)-5(6)-carboxyfluorescein and fluorescein isothiocyanate-dextran fluorescence
Authors: Schreiber R , Stoll B , Lang F , Häussinger D
Published: August 2015

Freshly isolated rat hepatocytes were plated for 4-6 h and either loaded with (2′,7)-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) or allowed to endocytose fluorescein isothiocyanate (FITC)-coupled dextran in order to study the effects of aniso-osmotic exposure and oxidative stress on cytosolic (pHcyt) and apparent vesicular pH (pHves) by single-cell fluorescence recordings. In the presence of normo-osmotic (305 mosmol/l) medium pHcyt was 7.23 +/- 0.03 (n = 108), whereas an apparent pH of 6.07 +/- 0.02 (n = 156) was found in the vesicular compartment accessible to endocytosed FITC-dextran. Substitution of 60 mM NaCl against 120 mM raffinose had no effect on pHcyt or apparent pHves, whereas addition of NH4Cl increased both pHcyt and apparent pHves. Hypo-osmotic cell swelling lowered pHcyt, whereas simultaneously apparent pHves increased. These effects were rapidly reversible upon re-institution of normo-osmotic media. Similarly, an increase of apparent pHves was observed when cell swelling was induced by Ba2+, glutamine or histidine. Conversely, hyperosmotic cell shrinkage due to addition of NaCl or raffinose led to a cytosolic alkalinization and a vesicular acidification. Both, H2O2 (0.2 mmol/l) and t-butyl-hydroperoxide (0.2 mmol/l) were without effect on pHcyt, but lowered apparent pHves by about 0.2 pH units. Ba2+ (1 mmol/l) diminished the acidifying effect of the hydroperoxides by about 50%. Pretreatment of the cells with colchicine, but not with lumicolchicine, largely abolished the effects of aniso-osmolarity and hydroperoxides on pHves. The data suggest that hepatocellular hydration affects the proton gradients built up across the membranes of endocytotic FITC-dextran-accessible compartments in a microtubule-dependent way. They further suggest that hydroperoxides induce vesicular acidification in a colchicine- and Ba(2+)-sensitive way. Because hydroperoxides induce Ba(2+)-sensitive cell shrinkage [Hallbrucker, Ritter, Lang, Gerok and Häussinger (1992) Eur. J. Biochem. 211, 449-458], the results are compatible with the view that hydroperoxide-induced cell shrinkage mediates vesicular acidification. It is concluded that modulation of vesicular pH by the hepatocellular hydration state may play a role in triggering some metabolic changes in response to cell swelling/shrinkage.
Human seminal plasma contains somatostatin-64
Authors: Odum L , Johnsen A H
Published: August 2015

Prosomatostatin is processed into two biologically active peptides, somatostatin (SS)-14 and SS-28. SS-14 is found in many tissues, whereas SS-28 is most prominent in the intestine. Human seminal plasma contained high concentrations (median = 3 nmol/l, range 0.6-76 nmol/l, n = 17) of SS with a higher M(r) than that of SS-28 as determined by gel chromatography. After isolation the SS immunoreactivity was identified as SS-64 based on the N-terminal amino acid sequence Leu-Ala-Glu-Leu-Leu-Ser-Glu-Pro-Asn-Gln-, and M(r) of 7242 determined by m.s. and on reactivity with two antibodies raised against SS-14. SS-64 displaced the binding of [125I-Tyr1]SS-14 to rat cerebrocortical membranes as effectively as SS-14 (IC50 = 1.6 mol/l). The concentration of SS-64 in seminal plasma from vasectomized men was significant higher (P < 0.005) than in normal men (median = 25.4 nmol/l, range 1.5-156 nmol/l, n = 21), suggesting that the synthesis of SS-64 takes place in the male accessory sex glands.
The effect of calcium-store depletion and refilling with various bivalent cations on tyrosine phosphorylation and Mn2+ entry in fura-2-loaded human platelets
Authors: Jenner S , Farndale R W , Sage S O
Published: August 2015

To investigate the possible involvement of tyrosine phosphorylation in the process of store-regulated Ca2+ entry, ionomycin (in the presence of EGTA) was used to deplete the intracellular Ca2+ stores of fura-2-loaded human platelets, and the effect of refilling with Ca2+, Ba2+ or Sr2+ evaluated. Depletion of the intracellular Ca2+ stores resulted in an increase in protein tyrosine phosporylation. This increase is reversed when the stores were refilled in Ca2+ or Sr2+, but not Ba2+. Refilling of the stores with Ca2+ or Sr2+, but not Ba2+, suppressed Mn2+ entry. These findings support the hypothesis that tyrosine phosphorylation plays a role in mediating store-regulated Ca2+ entry in human platelets and provides evidence for tyrosine phosphatase activity regulated by the Ca2+ content of the intracellular stores.
NF-κB/Rel family members are physically associated phosphoproteins
Authors: Li C C H , Korner M , Ferris D K , Chen E , Dai R M , Longo D L
Published: August 2015

We performed radioimmunoprecipitation followed by serial immunoblots to show that, in the unstimulated Jurkat T cell line, the NF-kappa B/Rel family proteins, p80-c-Rel, p105-NF-kappa B, p65-NF-kappa B, p50-NF-kappa B and p36-I kappa B alpha, can be detected as complexes using antisera against c-Rel, p105-NF-kappa B or p65-NF-kappa B. p36-I kappa B alpha and p105, both known inhibitors of NF-kappa B function, can physically associate with NF-kappa B/Rel family members, but not with each other. In vivo and in vitro phosphorylation experiments demonstrated that NF-kappa B/Rel family members, including p105, c-Rel, p50, p65 (for the first time for p50 and p65) and p36-I kappa B alpha are also phosphoproteins. Phosphoserine and phosphothreonine residues were identified in these proteins isolated from unstimulated Jurkat cells. Both unphosphorylated and hyperphosphorylated forms of p36-I kappa B alpha were found in the complexes, suggesting that hyperphosphorylated I kappa B alpha is still capable of associating with the NF-kappa B/Rel family members. After stimulation with phorbol 12-myristate 13-acetate and phytohaemagglutinin for 10 min, p105-NF-kappa B and p50-NF-kappa B, but not p36-I kappa B, were highly phosphorylated. Phosphopeptide mapping of p105 showed that phorbol ester/phytohaemagglutinin stimulation may change p105 phosphorylation qualitatively.
Ca2+ influx in platelets: activation by thrombin and by the depletion of the stores. Effect of cyclic nucleotides
Authors: Doni M G , Cavallini L , Alexandre A
Published: August 2015

In aspirin-treated platelets the thrombin-induced increase of cytosolic Ca2+ ([Ca2+]i) associated with the release from the intracellular stores is followed by a decrease to the baseline which is largely dependent on the re-uptake into the stores. This is shown by the further increase of [Ca2+]i upon inhibition of the endomembrane Ca(2+)-ATPase with thapsigargin. The re-uptake of Ca2+ into the stores is accelerated by sodium nitroprusside (SNP) or prostacyclin (PGI2). In all cases, after store depletion with thapsigargin the influx of external Ca2+ is maximal. After a thrombin-induced cycle of Ca(2+)-release re-uptake the stores are partly full: in these conditions the addition of external Ca2+ elicits a significant increment of [Ca2+]i and a further filling of the stores. Both are strongly reduced if Ca2+ addition is preceded by SNP or PGI2. Similar results are obtained also if (by supplementing and then cheleting Ca2+) the stores are as full as in native platelets at the moment of adding Ca2+. The thrombin-activated Ca2+ influx is reversed by hirudin. A PGI2- and SNP-sensitive Mn2+ influx is observed if Mn2+ is added in place of Ca2+. It is concluded that thrombin activates a cyclic nucleotide-sensitive Ca2+ (and Mn2+) influx pathway dependent on the occupancy of the thrombin receptor and independent of the filling state of the stores. In the absence of thrombin, thapsigargin releases Ca2+ relatively rapidly from a fraction of the stores; the remaining deposits are discharged much more slowly. This may indicate that platelets contain two distinct classes of agonist-sensitive stores. The addition of external Ca2+ (or Mn2+) at short or long incubation times with thapsigargin monitors the influx of Ca2+ activated by the depletion of one or both types of stores. The depletion of each type of store activates Ca2+ (Mn2+) influx. This type of cation influx is not inhibited by the cyclic nucleotides.
Glucose modulates the binding activity of the β-cell transcription factor IUF1 in a phosphorylation-dependent manner
Authors: MacFarlane W M , Read M L , Gilligan M , Bujalska I , Docherty K
Published: August 2015

In the human insulin gene, three regulatory sequences upstream of the transcription start site at -77 (the CT1 box), -210 (the CT2 box), and -315 (the CT3 box) bind a beta-cell-specific transcription factor, IUF1. Recent studies have mapped a glucose response element to a CT-like sequence in the rat insulin I gene. The present study was therefore undertaken to ascertain the role of IUF1 in glucose-stimulated insulin gene transcription. IUF1-binding activity was measured by electrophoretic mobility shift assay using the CT2 box as probe. When freshly isolated rat islets of Langerhans were incubated in medium containing low concentrations (3 mM) of glucose IUF1 activity fell to undetectable levels within 6 h. In high (20 mM) glucose IUF1 activity remained constant over a 24 h period. The loss of IUF1 activity was reversible. Thus when islets were incubated for 4 h in low glucose and transferred to high glucose, IUF1 levels recovered within 15 min. This effect was dependent on glucose metabolism as it was inhibited by mannoheptulose. Incubation of islets for 4 h in low concentrations of glucose supplemented with phosphatase inhibitors prevented the fall in IUF1 activity. No recovery in IUF1 activity was observed when islets were treated for 4 h with low glucose and then for a further 1 h with low glucose and dibutyryl cyclic AMP, or forskolin, or the phorbol ester phorbol 12-myristate 13-acetate. These results demonstrate that the IUF1-binding activity in islets of Langerhans is modulated by glucose in a phosphorylation-dependent manner, and that protein kinase A or protein kinase C are not involved. Finally, IUF1 was shown to be immunologically related to a recently cloned factor, IPF1, that binds to a CT-like sequence in the rat insulin I gene promoter.
Characterization of proteoglycans associated with mouse splenic AA amyloidosis
Authors: Stenstad T , Magnus J H , Husby G
Published: August 2015

We here report for the first time on the chemical characteristics of proteoglycans associated with mouse splenic reactive AA amyloid. Amyloid was induced in CBA/J mice by two different procedures; conventional casein treatment and by employing Freund's complete adjuvant, accelerated by Trypan Blue. Pulse-labelling was employed at distinct stages during amyloid development, followed by [35S]proteoglycan characterization of organ extracts. Repetitive 35S injections were also administered during the phase where amyloid deposition occurred most rapidly. Proteoglycans were extracted with guanidine in the presence of protease inhibitors and purified. The results showed that the production of proteoglycans is dramatically enhanced during amyloidogenesis, the glycosaminoglycan and proteoglycan accumulation being not only dependent on alterations in proteoglycan catabolism, but rather on increased synthesis. The increment could be demonstrated even at the stage before microscopic detection of amyloid deposits, clearly suggesting that the upregulation of proteoglycan expression precedes amyloid fibril formation. Two major proteoglycans were found to accumulate in advanced splenic amyloid; one a heparan sulphate proteoglycan of approx. 200 kDa with a core protein of 70 kDa, the other a chondroitin sulphate proteoglycan of smaller size. Moreover, free dermatan sulphate chains seemed to specifically accumulate in the organs during amyloid fibrillogenesis. We suggest that free glycosaminoglycans may be a specific feature of amyloidosis and that different proteoglycans and glycosaminoglycans play a role in formation and stabilization of amyloid fibrils in vivo.
SmgGDS stabilizes nucleotide-bound and -free forms of the Rac1 GTP-binding protein and stimulates GTP/GDP exchange through a substituted enzyme mechanism
Authors: Chuang T H , Xu X , Quilliam L A , Bokoch G M
Published: August 2015

The Rac proteins, Rac1 and Rac2, are essential components of the NADPH oxidase system of phagocytes and regulate the actin assembly associated with membrane ruffling. These functions are controlled by the GTP-bound form of Rac. The biochemical interaction between Rac and its only known GDP-dissociation stimulator (termed smgGDS) was characterized. SmgGDS was able to stimulate the incorporation of guanosine 5′-[gamma-thio]-triphosphate GTP[gamma S] into the RhoA, Rac2, Rac1, Rap1A and CDC42Hs GTP-binding proteins, but the activity was greatest toward RhoA and Rac2. Isoprenoid modification of these proteins was not absolutely required for the interaction with smgGDS. Interestingly, the activity of smgGDS toward Rac1 could not be observed in a [3H]GDP/GTP exchange assay under conditions where it stimulated incorporation of GTP[gamma S] into Rac1. We determined that smgGDS prevented the loss of Rac1 activity during the [3H]GDP/GTP exchange assay by demonstrating the ability of smgGDS to inhibit the loss of Rac1 GTP[gamma S]-binding during incubations at 30 degrees C. This stabilizing effect was exactly counterbalanced by the ability of smgGDS to stimulate the release of [3H]GDP from Rac1, thereby producing no net observable effect in the exchange assay. SmgGDS was able to effectively stimulate the release of GDP but not GTP[gamma S] from Rac1. SmgGDS maintains Rac1 in a nucleotide-free form after release of GDP, indicating that the reaction between Rac1 and smgGDS involves a substituted enzyme mechanism.
Regulation of basal adenylate cyclase activity in neuroblastoma × glioma hybrid, NG108-15, cells transfected to express the human β 2 adrenoceptor: evidence for empty receptor stimulation of the adenylate cyclase cascade
Authors: Adie E J , Milligan G
Published: August 2015

Clones of neuroblastoma x glioma hybrid, NH108-15, cells expressing differing levels of the human beta 2 adrenoceptor were isolated. Two clones were examined in detail, beta N22 which expressed some 4000 fmol/mg of membrane protein and clone beta N17 which expressed approx. 300 fmol/mg of membrane protein of the receptor. In beta N22 cells ‘basal’ adenylate cyclase activity measured in the presence of Mg2+ was significantly greater than that in wild-type NG108-15 or beta N17 cells. Both isoprenaline and iloprost were able to stimulate adenylate cyclase activity in each of beta N22 and beta N17 membranes. However, the EC50 for isoprenaline stimulation of adenylate cyclase in membranes of beta N22 cells (6 nM) was significantly lower than that in membranes of beta N17 cells (80 nM), whereas the EC50 for iloprost stimulation of adenylate cyclase (approx. 25 nM) was the same in the two clones and in parental NG108-15 cells. The high basal adenylate cyclase activity of beta N22 cell membranes was not a reflection of higher levels of expression of the adenylate cyclase catalytic unit, as adenylate cyclase activity measured in the presence of Mn2+ was equivalent in membranes of each of wild-type NG108-15 cells and clones beta N22 and beta N17. Basal adenylate cyclase activity measured in the presence of Mg2+ in clone beta N22 was significantly reduced, however, by the beta-receptor antagonist propranolol, whereas this agent was without effect on basal adenylate cyclase activity in membranes of wild-type NG108-15 cells. These data indicate that the elevated basal adenylate cyclase cascade in NG108-15 cells expressing high levels of the beta 2 adrenoceptor represents empty receptor activation of the signalling cascade.
Kinetic and physical studies of β-lactamase inhibition by a novel penem, BRL 42715
Authors: Farmer T H , Page J W J , Payne D J , Knowles D J C
Published: August 2015

The interactions of Staphylococcus aureus, Bacillus cereus I, TEM, Klebsiella pneumoniae K1 and Enterobacter cloacae P99 beta-lactamases with the novel penem inhibitor BRL 42715 were investigated kinetically and, in some cases, by electrospray mass spectrometry (e.s.m.s.). All the beta-lactamases were rapidly inactivated by BRL 42715, with second-order rate constants ranging from 0.17 to 6.4 microM-1.s-1. The initial stoichiometry of beta-lactamase inhibition was essentially 1:1, with the exception of the K1 enzyme. In this instance about 20 molecules of BRL 42715 were hydrolysed before the enzyme was completely inhibited. Inhibited beta-lactamases did not readily regain activity in the absence of BRL 42715, the half-lives for regeneration of free enzyme ranging from 5 min for the K1 beta-lactamase to over 2 days for the staphylococcal enzyme. Recovery of activity was incomplete for TEM-1, K1 and P99 beta-lactamases, suggesting partitioning of the inhibited enzymes to give a permanently (or at least very stable) inactivated species. Examination of the interactions of the penem with TEM, B. cereus I and P99 beta-lactamases by e.s.m.s. also showed rapid and stoichiometric binding of the inhibitor. In all cases a mass increase of 264 Da over the native enzyme was observed, corresponding to the molecular mass of BRL 42715, showing that no fragmentation of the penem occurred on reaction with the beta-lactamases.
Role of N-glycosylation in the synthesis, dimerization and secretion of human interferon-γ
Authors: Sareneva T , Pirhonen J , Cantell K , Kalkkinen N , Julkunen I
Published: August 2015

Human interferon-gamma (IFN-gamma) is a secretory glycoprotein, which has two potential N-linked glycosylation sites at positions Asn-25 and Asn-97 of its 143 amino acid long mature polypeptide chain. In order to understand the role of glycan residues in the synthesis and secretion of human IFN-gamma, both or either one of the potential N-linked glycosylation sites were mutated to Gln. The mutant and the wild-type (Wt) polypeptides were expressed in insect cells using a baculovirus vector. Elimination of the N-glycosylation site at position Asn-97 (N97Q) resulted in secreted protein yields of 70-90% as compared with the Wt production, whereas only 10-25% (N25Q) and 1-10% (N25Q,N97Q) levels of protein production was observed when the first or both sites were mutated, respectively. Although there was a difference between extracellular levels of produced protein, the kinetics of secretion was similar for all different IFN-gamma molecules. The Wt and the N-glycosylation site mutants were all secreted as dimers. The formation of biologically active dimers was more efficient for IFN-gamma polypeptides that had the intact glycosylation site at Asn-25 as compared with the other two mutant forms of IFN-gamma. The extent of dimerization correlated well with the observed secretion. The specific antiviral activity was of the same order (1 x 10(7) i.u./mg of protein) for the glycosylated IFN-gamma molecules, whereas it was slightly lower (0.5 x 10(7) i.u./mg of protein) for the unglycosylated mutant form.
Differential expression of cyclophilin isoforms during keratinocyte differentiation
Authors: Chatellard-Gruaz D , Saurat J H , Siegenthaler G
Published: August 2015

Cyclophilin A, the major intracellular binding protein for the immunosuppressive drug cyclosporin A (CsA), was studied in human keratinocytes during differentiation both in vivo and in vitro. Analysis of cyclophilin by gel-filtration radiobinding-assay with tritiated CsA showed one specific radioactive peak at 17 kDa. By this technique, the levels of cyclophilin (mean 55.23 +/- 8.43 pmol/mg protein) did not significantly differ during keratinocyte differentiation. When the protein extracts from calcium-induced differentiating keratinocytes and normal human skin were analysed by PAGE radiobinding-assay, two specific radioactive CsA-binding peaks were detected. The major peak (RF 0.13) was expressed in all samples (mean 47.32 +/- 17.53 pmol/mg protein) whereas the minor peak (RF 0.23) was dramatically decreased about 6-fold in abnormally differentiated skin (psoriasis) as well as in non-differentiated keratinocytes. At least six [3H]CsA-binding isoforms with pI values ranging from 5.58 to 7.75 were detected by isoelectrofocusing autoradio-blotting-assay in normal human skin; three of them immunoreacted with antibodies to cyclophilin. These results demonstrated the presence of several cyclophilin isoforms in human epidermal cells and an expression which correlated with the differentiation of human keratinocytes both in vivo and in vitro.
Purification and further characterization of peroxisomal trihydroxycoprostanoyl-CoA oxidase from rat liver
Authors: Van Veldhoven P P , Van Rompuy P , Vanhooren J C T , Mannaerts G P
Published: August 2015

The acyl-CoA oxidase, catalysing the peroxisomal desaturation of the CoA-ester of trihydroxycoprostanic acid, a bile acid intermediate, has been purified to homogeneity from rat liver. Its native molecular mass, as determined by gel filtration and native gel electrophoresis, was 120 and 175 kDa respectively, suggesting a homodimeric protein consisting of 68.6 kDa subunits. If isolated in the presence of FAD, the enzyme showed a typical flavoprotein spectrum and contained most likely 2 mol of FAD per mol of enzyme. The cofactor, however, was loosely bound. The enzyme acted exclusively on 2-methyl-branched compounds, including pristanoyl-CoA and 2-methylhexanoyl-CoA if albumin was present. Important parameters to obtain a pure and active enzyme were the following: (1) using chromatographic separations like hydrophobic interaction and metal affinity, which allow the presence of high salt concentrations, conditions which stabilize the oxidase; (2) avoiding dialysis and (NH4)2SO4 precipitation; (3) including, when appropriate, FAD, dithiothreitol and a diol-compound in the solvents; and (4) carefully monitoring the removal of other acyl-CoA oxidases which possess the same native molecular mass and subunit size.
Adenosine A1 receptors mediate chronic ethanol-induced increases in receptor-stimulated cyclic AMP in cultured hepatocytes
Authors: Nagy L E , DeSilva S E F
Published: August 2015

Cellular responses to adenosine depend on the distribution of the two adenosine receptor subclasses. In primary cultures of rat hepatocytes, adenosine receptors were coupled to adenylate cyclase via A1 and A2 receptors which inhibit and stimulate cyclic AMP production respectively. R-(-)-N6-(2-phenylisopropyl)-adenosine (R-PIA), the adenosine A1 receptor-selective agonist, inhibited glucagon-stimulated cyclic AMP production with an IC50 of 19 nM. This inhibition was blocked by the A1-specific antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPDX). 5′-N- Ethylcarboxamidoadenosine (NECA), an agonist which stimulates A2 receptors, increased cyclic AMP production with an EC50 of 0.6 microM. Treatment of primary cultures of rat hepatocytes with 100 mM ethanol for 48 h decreases the quantity and function of the inhibitory guanine-nucleotide regulatory protein (G(i)), resulting in a sensitization of receptor-stimulated cyclic AMP production [Nagy and deSilva (1992) Biochem. J. 286, 681-686]. When cells were cultured with 2 units/ml adenosine deaminase, to degrade extracellular adenosine, ethanol-induced increases in cyclic AMP production were completely prevented. Moreover, the specific A1-receptor antagonist, CPDX, also blocked the chronic effects of ethanol on receptor-stimulated cyclic AMP production. Treatment with adenosine deaminase or CPDX also prevented the decrease in quantity of the alpha subunit protein of G(i) observed in hepatocytes after chronic treatment with ethanol. Taken together, these results suggest that activation of adenosine A1 receptors on primary cultures of hepatocytes is involved in the development of chronic ethanol-induced sensitization of receptor-stimulated cyclic AMP production.
DNA cleavage induced by glycation of Cu, Zn-superoxide dismutase
Authors: Kaneto H , Fujii J , Suzuki K , Kasai H , Kawamori R , Kamada T , Taniguchi N
Published: August 2015

Human Cu,Zn-superoxide dismutase (Cu,Zn-SOD) undergoes site-specific and random fragmentation by non-enzymic glycosylation (glycation). Released Cu2+ from the glycated Cu,Zn-SOD probably facilitates a Fenton reaction to convert H2O2 into hydroxy radical, which then participates in the non-specific fragmentation [Ookawara et al. (1992) J. Biol. Chem. 267, 18505-18510]. In the present study, we investigated the effects of glycated Cu,Zn-SOD on cloned DNA fragments and nuclear DNA and analysed the formation of 8-hydroxydeoxyguanosine (8-OH-dG). Incubation of cloned DNA fragments with Cu,Zn-SOD and reducing sugars resulted in cleavage of the DNA. The extent of the cleavage corresponded to the reducing capacity of the sugar. Metal-chelating reagents, EDTA and bathocuproine, and an H2O2 scavenger, catalase, inhibited the DNA cleavage. Hydroxy radical scavengers and aminoguanidine, an inhibitor of glycation, also inhibited the reaction. Moreover, the glycation of Cu,Zn-SOD caused the substantial formation of 8-OH-dG in DNA. When isolated nuclei were incubated with CuCl2 plus H2O2, nuclear DNA cleavage was observed. Incubation of isolated nuclei with Cu,Zn-SOD that had been pre-incubated with glucose also resulted in nuclear DNA cleavage. These results suggest that hydroxy radical is produced through a Fenton reaction by Cu2+ and H2O2 released from the glycated Cu,Zn-SOD, and participates in nuclear DNA cleavage. This mechanism may partly explain the deterioration of organs under diabetic conditions.