bluuebunny/crossref_metadata_2025 huggingface dataset:
Different roles of protein kinase C-β and -δ in arachidonic acid cascade, superoxide formation and phosphoinositide hydrolysis
Authors: Duyster J , Schwende H , Fitzke E , Hidaka H , Dieter P
Published: August 2015

In contrast with protein kinase C (PKC)-beta, PKC-delta is exclusively detectable in the membrane fraction of liver macrophages. After long-term treatment with phorbol 12-myristate 13-acetate (PMA) PKC-beta is depleted faster (within 3 h) than PKC-delta (> 7h). Simultaneously, pretreatment with PMA for 3 h inhibits the PMA- and zymosan-induced generation of superoxide and the PMA-induced formation of prostaglandin (PG) E2, whereas a preincubation of more than 7 h is required to affect the zymosan-induced release of PGE2 and inositol phosphates. These results support an involvement of PKC-beta in the PMA-induced activation of the arachidonic acid cascade and in superoxide formation and imply an involvement of PKC-delta in zymosan-induced phosphoinositide hydrolysis and PGE2 formation. Two phorbol ester derivates, sapintoxin A (SAPA) and 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA), which have been previously reported to activate preferentially PLC-beta but not PKC-delta in vitro [Ryves, Evans, Olivier, Parker and Evans (1992) FEBS Lett. 288, 5-9], induce the formation of PGE2 and superoxide, down-regulate PKC-delta and potentiate inositol phosphate formation in parallel SAPA, but not DOPPA, down-regulates PKC-beta and inhibits the PMA-induced formation of eicosanoids and superoxide.
Stimulation of platelet-activating factor synthesis by progesterone and A23187 in human spermatozoa
Authors: Baldi E , Falsetti C , Krausz C , Gervasi G , Carloni V , Casano R , Forti G
Published: August 2015

The presence of platelet-activating factor (PAF) has been demonstrated recently in mammalian spermatozoa, together with evidence for a role of this phospholipid in enhancing sperm motility and fertilizing ability. To investigate whether PAF synthesis and release occurs in human spermatozoa following incubation with stimuli that induce acrosome reaction, spermatozoa were incubated with progesterone and A23187, two known inducers of the exocytotic event. PAF synthesis (remodelling pathway) was assessed by [3H]acetate incorporation into PAF. Treatment of spermatozoa with progesterone and A23187 resulted in an increase of [3H]acetate incorporation into PAF. Most of the newly synthesized [3H]PAF formed in response to acrosome reaction was found in the supernatant, suggesting a release of the phospholipid from spermatozoa. PAF-like material extracted from human spermatozoa was able to induce aggregation of rabbit platelets and showed identical retention time and the same ion m/e values as authentic PAF when analysed with g.c.-m.s. Lyso-PAF:acetyl-CoA acetyltransferase (EC 2.3.1.67) activity in human spermatozoa was also studied and showed similar kinetic parameters to those described for other cell systems. Stimulation of spermatozoa with progesterone and A23187 induced an increase of [3H]arachidonic acid release, suggesting an activation of phospholipase A. In conclusion, our results demonstrated increased production and release of PAF in human sperm following stimulation with progesterone and A23187 and suggest a role for this phospholipid in the activation of spermatozoa.
A computer program for the algebraic determination of control coefficients in Metabolic Control Analysis
Authors: Thomas S , Fell D A
Published: August 2015

A computer program (MetaCon) is described for the evaluation of flux control, concentration control and branch-point distribution control coefficients of a metabolic pathway. Requiring only the reaction scheme as input, the program produces algebraic expressions for the control coefficients in terms of elasticity coefficients, metabolite concentrations and pathway fluxes. Any of these variables can be substituted by numeric or simple algebraic expressions; the expressions will then be automatically rearranged in terms of the remaining unknown variables. When all variables have been substituted, numeric values will be obtained for the control coefficients. The program is a computerized implementation of the matrix method for the determination of control coefficients. The features of MetaCon are compared with those of other programs available to workers in Metabolic Control Analysis. Potential benefits of, and methods of using, MetaCon are discussed. The mathematical background and validity of the matrix method rules are discussed, and the algorithm used by MetaCon is described. The matrix method is shown to be a specific case of a previously described general formalism for calculating control coefficients.
Glutathione analogue sorbents selectively bind glutathione S-transferase isoenzymes
Authors: Castro V M , Kelley M K , Engqvist-Goldstein Å , Kauvar L M
Published: August 2015

Novel affinity sorbents for glutathione S-transferases (GSTs) were created by binding glutathione (GSH) analogues to Sepharose 6B. The GSH molecule was modified at the glycine moiety and at the group attached to the sulphur of cysteine. When tested by affinity chromatography in a flow-through microplate format, several of these sorbents selectively bound GST isoenzymes. gamma E-C(Hx)-phi G (glutathione with a hexyl moiety bound to cysteine and phenylglycine substituted for glycine) specifically bound rat GST 7-7, the Pi-class isoenzyme, from liver, kidney and small intestine. gamma E-C(Bz)-beta A (benzyl bound to cysteine and beta-alanine substituted for glycine) was highly selective for rat subunits 3 and 4, which are Mu-class isoenzymes. By allowing purification of the isoenzymes under mild conditions that preserve activity, the novel sorbents should be useful in characterizing the biological roles of GSTs in both normal animal and cancer tissues.
Changes in rates of glucose utilization and regulation of glucose disposal by fast-twitch skeletal muscles in late pregnancy
Authors: Holness M J , Sugden M C
Published: August 2015

Glucose utilization indices (GUI) were measured in vivo in conjunction with active pyruvate dehydrogenase complex (PDH(a) and glycogen synthase (GS) activities in fast-twitch skeletal muscles [extensor digitorum longus (EDL), tibialis anterior and gastrocnemius] of late-pregnant rats and age-matched virgin control rats in the fed state, after 24 h starvation and at 2 h after re-feeding with standard laboratory chow ad libitum after 24 h starvation. As demonstrated previously [Holness and Sugden (1990) Biochem. J 277, 429-433], GUI values of fast-twitch skeletal muscles of virgin rats were low in the fed ad libitum and the 24 h-starved states, but dramatically increased after subsequent chow re-feeding. GUI values of fast-twitch skeletal muscles of late-pregnant rats were also low in the fed and starved states and were increased by re-feeding, but the increase in GUI values elicited by re-feeding was greatly attenuated. PDHa activities in EDL, tibialis anterior and gastrocnemius in the fed state were unaffected by late pregnancy, and skeletal-muscle PDHa activities were decreased after 24 h of starvation in both groups. Whereas re-feeding of virgin rats with standard diet for 2 h restored PDHa activities in fast-twitch skeletal muscles to values for rats continuously fed ad libitum, PDHa activities in fast-twitch skeletal muscles of late-pregnant rats, although increased in response to re-feeding, remained considerably less than the corresponding fed ad libitum values after 2 h of re-feeding. In contrast, neither skeletal-muscle GS re-activation nor rates of skeletal-muscle glycogen deposition after re-feeding were markedly affected by late pregnancy. The results are discussed in relation to the specific targeting of individual pathways of glucose disposal in fast-twitch skeletal muscles during re-feeding in late pregnancy.
The effect of thiamin on the activation of thiamin pyrophosphate-dependent 2-oxoglutarate decarboxylase in Euglena gracilis
Authors: Shigeoka S , Nakano Y
Published: August 2015

The effect of thiamin on thiamin pyrophosphate-dependent 2-oxoglutarate (2-OG) decarboxylase activity in Euglena gracilis was investigated. The total activity of 2-OG decarboxylase in thiamin-sufficient cells in 3 times that in thiamin-deficient cells. The addition of thiamin to thiamin-deficient cells causes the total enzyme and holoenzyme activities to increase and reach similar levels to that in thiamin-sufficient cells. Cycloheximide and chloramphenicol, inhibitors of protein synthesis, have no effect on the total enzyme activity. Immunochemical titration and determination of 2-OG decarboxylase mRNA by using an antibody directed against Euglena 2-OG decarboxylase indicate that the increase in the holoenzyme activity of 2-OG decarboxylase is due to activation of pre-existing protein and does not require synthesis of new proteins in thiamin-deficient cells. During the period of the increase in the total activity, the apoenzyme increases and reaches a temporary peak in 2 h. Immunoblot analysis demonstrates that the precursor form (a 65 kDa subunit) of 2-OG decarboxylase in thiamin-deficient cells is more abundant than that in thiamin-sufficient cells and the increase in the apoenzyme by addition of thiamin results from the conversion of the precursor form into the mature form (a 62 kDa subunit).
Separation and partial characterization of proteinases with substrate specificity for basic amino acids from human MOLT-4 T lymphocytes: identification of those inhibited by variable-loop-V3 peptides of HIV-1 (human immunodeficiency virus-1) envelope glycoprotein
Authors: Harvima I T , Harvima R J , Nilsson G , Ivanoff L , Schwartz L B
Published: August 2015

The V3 loop of the HIV (human immunodeficiency virus)-1 envelope glycoprotein gp120 likely plays a role in HIV-1 infectivity. Although the amino acid sequence of the V3 loop is hypervariable, it contains a conserved region, Gly-Pro-Gly-Arg, that shows similarity to the active-site Gly-Pro-Cys-Arg sequence of inter-alpha-trypsin and trypstatin proteinase inhibitors. The purpose of the present work was to identify proteinases recognizing substrates with basic amino acids in the P1 substrate site that are present in MOLT-4 cells, a human CD4-positive T helper lymphocyte cell line, and to characterize these enzymes in terms of substrate, pH and ionic-strength preferences, size and susceptibility to various inhibitors, including 24- and 36-amino-acid-long V3 loop peptides. Extraction of MOLT-4 cells at low ionic strength solubilized nearly all of the trypsin-like activity, which was separable into five peaks of activity by chromatography on Mono-Q: Peaks 1, 2a, 2b, 3 and 4. All showed a neutral pH optimum, and all except Peak 4 showed optimal activity at high ionic strength. Peak 1 preferred Tos-Gly-Pro-Arg, p-nitroanilide (-pNA) substrate; Peaks 2-4 preferred benzyloxycarbonyl-Val-Leu-Gly-Arg-pNA. Peak 1, a zinc-dependent enzyme with serine and histidine in the active site, exhibited an M(r) of 75,000 on Superose 12 and was poorly inhibited by V3 loop peptides. Peak 2 contained two overlapping peaks, called 2a and 2b, that exhibited properties of zinc-dependent metalloproteinases. Gel filtration of Peak 2 activities revealed a major peak of activity at 81 kDa and a shoulder centred at 240 kDa. Each was modestly inhibited by V3 loop peptides. Peak 3, a zinc-dependent proteinase, exhibited a molecular mass of 100 kDa by gel filtration and was particularly sensitive to inhibition by V3 loop peptides. Peak 4 exhibited a molecular mass of 1100 kDa by gel filtration and was not inhibited by V3 loop peptides. None of these enzymes could be classified as mast-cell tryptase, and material in MOLT-4 cells cross-reactive with anti-(human tryptase) antibodies was not detected. Whether any of the MOLT-4 proteinases described in this study play a role in HIV-1 infectivity remains to be examined.
Cholesterol esters selectively delivered in vivo by high-density-lipoprotein subclass LpA-I to rat liver are processed faster into bile acids than are LpA-I/A-II-derived cholesterol esters
Authors: Pieters M N , Castro G R , Schouten D , Duchateau P , Fruchart J C , Van Berkel T J C
Published: August 2015

High-density lipoprotein (HDL) subclass LpA-I has been reported to promote cholesterol efflux from mouse adipose cells in vitro, whereas subclass LpA-I/A-II has no effect. To investigate whether the apolipoprotein composition of HDL plays a role in the selective delivery of cholesterol esters to the liver in vivo, we labelled HDL in its cholesterol ester moiety and separated [3H]cholesterol oleate-labelled HDL into subclasses LpA-I and LpA-I/A-II by immuno-affinity chromatography. Serum decay and liver association of LpA-I and LpA-I/A-II were compared for the apoprotein and cholesterol ester moieties. Both LpA-I and LpA-I/A-II selectively delivered cholesterol esters to the liver with similar kinetics. The kinetics of biliary secretion of processed cholesterol esters, initially associated with LpA-I or LpA-I/A-II, were studied in rats equipped with permanent catheters in bile, duodenum and heart. For both LpA-I and LpA-I/A-II, liver association was coupled to bile acid synthesis, with an increase in secretion rate during the night. During the first night period, the biliary secretion of LpA-I-derived radio-activity was significantly greater than for LpA-I/A-II. The data indicate that with both LpA-I and LpA-I/A-II selective delivery of cholesterol esters from HDL to the liver occurs, but that cholesterol esters delivered by LpA-I are more efficiently coupled to bile acid synthesis.
Phosphorylation and nucleotide-dependent dephosphorylation of hepatic polypeptides related to the plasma cell differentiation antigen PC-1
Authors: Uriarte M , Stalmans W , Hickman S , Bollen M
Published: August 2015

A glycoprotein fraction was isolated from rat liver membranes by affinity chromatography on immobilized wheat-germ lectin. Incubation of this fraction with MgATP or MgGTP resulted in a sequential phosphorylation and dephosphorylation of a complex of three polypeptides (118, 128 and 197 kDa on SDS/PAGE) with N-linked sialyloligosaccharides. Each polypeptide was recognized by polyclonal antibodies against recombinant plasma cell differentiation antigen PC-1. The relationship of the 118 kDa and 128 kDa polypeptides with PC-1 was confirmed by observations that they are linked by disulphide bonds into a larger protein, and that they are exclusively phosphorylated on Thr residues. Phosphorylation of p118, p128 and p197 only occurred after a lag period (up to 90 min at 30 degrees C), which lasted until most of the ATP had been converted to adenosine and Pi, with ADP and AMP as intermediate products. The length of the latency period increased with the concentration of initially added ATP (5-1000 microM) and could be prolonged by a second addition of similar concentrations of ATP, ADP, AMP and various nucleotide analogues. Most potent were the alpha beta-methylene derivatives of ADP and ATP. Adenosine was poorly effective. AMP, ADP, and perhaps ATP, emerge as the direct determinants of the latency. After further purification of the lectin-purified membrane fraction on anion-exchange and molecular-sieve columns, the complex of p118, p128 and p197 was still capable of autophosphorylation and dephosphorylation. The dephosphorylation was not affected by classical inhibitors (NaF, okadaic acid, EDTA, EGTA, phenylalanine). It was stimulated about 20-fold by various adenine nucleotides and analogues, with the same order of efficiency as noted for the induction of the latency. A similar stimulation of dephosphorylation was caused by 0.5 mM Na3VO4, which also prevented the phosphorylation of the three polypeptides. The likely explanation for the latency that precedes the phosphorylation of the membrane proteins is that the action of a protein kinase is initially offset by nucleotide-stimulated dephosphorylation.
Phosphoenolpyruvate carboxylase from Streptomyces coelicolor A3(2): purification of the enzyme, cloning of the ppc gene and over-expression of the protein in a streptomycete
Authors: Bramwell H , Nimmo H G , Hunter I S , Coggins J R
Published: August 2015

Phosphoenolpyruvate carboxylase [PEPC; orthophosphate:oxaloacetate carboxy-lyase (phosphorylating); EC 4.1.1.31] is a major anaplerotic enzyme in the polyketide producer Streptomyces coelicolor A3(2). PEPC was purified from S. coelicolor and the amino-acid sequences of four tryptic peptides were determined. Synthetic oligonucleotides based on the sequences of two of the peptides hybridized to the same bands in various restriction-enzyme digests of S. coelicolor genomic DNA. This hybridization allowed molecular cloning of an 8 kb BamHI fragment of genomic DNA. Partial DNA sequencing of this fragment showed that it could encode amino acid sequences similar to those of PEPC from other microorganisms. A BamHI/PstI fragment was subcloned into the streptomycete high-copy-number plasmid vector pIJ486 and transferred into Streptomyces lividans. The resulting strain over-expressed PEPC activity 21-fold and also over-expressed a protein with a subunit of 100,000 M(r), the same as that of purified S. coelicolor PEPC.
Molecular cloning and expression of an intracellular serpin: an elastase inhibitor from horse leucocytes
Authors: Kordula T , Dubin A , Schooltink H , Koj A , Heinrich P C , Rose-John S
Published: August 2015

Horse blood leucocytes contain an elastase inhibitor (HLEI) belonging to the serpin family. Poly(A)+RNA isolated from these cells was used to construct a cDNA library in lambda gt10, which was first screened with a synthetic degenerate oligonucleotide probe corresponding to the amino acid sequence of the reactive centre of the inhibitor. Three clones were obtained covering the entire coding region of the protein. Sequencing of these clones showed identity with the amino acid sequence obtained from Edman degradation of the elastase inhibitor. The coding sequence of the HLEI cDNA was cloned into the bacterial expression vector pKK233-2 and expressed in Escherichia coli cells. Transformed bacteria expressed significant amounts of the protein, which was immunoprecipitated with a specific anti-HLEI antiserum. Furthermore, HLEI expressed in bacteria inhibited the activity of elastase but not trypsin.
An echistatin C-terminal peptide activates GPIIbIIIa binding to fibrinogen, fibronectin, vitronectin and collagen type I and type IV
Authors: Wright P S , Saudek V , Owen T J , Harbeson S L , Bitonti A J
Published: August 2015

Integrin binding to proteins often involves recognition of domains containing the arginine-glycine-aspartate (RGD) motif. Different binding affinities and specificities of the integrin-ligand protein interactions involve additional protein domains. The n.m.r. structure of the snake-venom protein echistatin suggested that the C-terminal portion of the molecule might be important, in addition to the RGD domain, in binding to the integrin glycoprotein IIbIIIa (GPIIbIIIa) [Saudek, Atkinson and Pelton (1991) Biochem. 30, 7369-7372]. The synthetic C-terminal peptide, echistatin-(40-49), PRNPHKGPAT, (1) inhibited binding of GPIIbIIIa to immobilized echistatin (IC50 3-6 mM), but did not inhibit binding of GPIIbIIIa to immobilized fibrinogen (up to 5 mM peptide), (2) activated GPIIbIIIa binding to fibronectin and vitronectin, usual ligands for the activated integrin, (3) activated binding of GPIIbIIIa to collagen type I and type IV, proteins not usually regarded as ligands for the integrin, and (4) stimulated 125I-fibrinogen binding by human platelets. These findings argue for an interaction of this non-RGD domain in echistatin with GPIIbIIIa, leading to activation of the integrin and extension of the ligand specificity to include immobilized collagen.
The erythrocyte calcium pump is inhibited by non-enzymic glycation: studies in situ and with the purified enzyme
Authors: González Flecha F L , Castello P R , Caride A J , Gagliardino J J , Rossi J P
Published: August 2015

In a previous paper we demonstrated that incubation of either intact erythrocytes or erythrocytes membranes with glucose decreases the activity of the membrane Ca(2+)-ATPase [González Flecha, Bermúdez, Cédola, Gagliardino and Rossi (1990) Diabetes 39, 707-711]. The aim of the present work was to obtain information about the mechanism of this inhibition. For this purpose, experiments were carried out with purified Ca(2+)-ATPase, inside-out vesicles and membranes from human erythrocytes. Incubation of the purified Ca(2+)-ATPase with glucose led to a decay in the enzyme activity of up to 50% of the control activity under the conditions used. The decrease in ATPase activity was concomitant with labelling by [6-3H]glucose of the purified Ca2+ pump; the kinetic properties of both processes were almost identical, suggesting that inhibition is a consequence of the incorporation of glucose into the Ca(2+)-ATPase molecule. In inside-out vesicles, glucose also promoted inhibition of Ca(2+)-ATPase activity as well as of active Ca2+ transport. Arabinose, xylose, mannose, ribose, fructose and glucose 6-phosphate (but not mannitol) were also able to inactive the ATPase. The activation energy for both the decrease in ATPase activity by glucose and the labelling of the pump with [6-3H]glucose was about 65 kJ/mol. Furthermore, inorganic phosphate enhanced the inactivation of the Ca(2+)-ATPase by glucose. This evidence strongly suggests that inhibition is a non-enzymically catalysed process. Inactivation of the Ca(2+)-ATPase by glucose was enhanced by reductive alkylation with sodium borohydride. Aminoguanidine, an inhibitor of the formation of the advanced end products of glycosylation, did not prevent the deleterious effect of glucose on the enzyme activity. Therefore it is concluded that inactivation of the Ca2+ pump is a consequence of the glycation of this protein.
Endothelins stimulate tyrosine phosphorylation and activity of p42/mitogen-activated protein kinase in astrocytes
Authors: Cazaubon S , Parker P J , Strosberg A D , Couraud P O
Published: August 2015

Endothelins (ET-1, -2, -3) display pleiotropic activities, by signalling through G-protein-coupled membrane receptors. We show here that ET-1 and ET-3 stimulate within minutes the tyrosine phosphorylation of a 42 kDa protein (p42) in primary cultures of mouse embryo astrocytes, but not in any of two subclones of rat astrocytoma C6 cells. This effect, measured by anti-phosphotyrosine immunoblotting of cell extracts, was also observed in response to bradykinin, platelet-derived growth factor, the phorbol ester phorbol 12-myristate 13-acetate and the G-protein activator fluoroaluminate. Pretreatment of cells with pertussis toxin, which inactivates Gi/G(o) proteins, did not affect these responses. However, down-regulation of protein kinase C completely blocked the response to phorbol ester and fluoroaluminate and at least partially impaired the ET-1-stimulated phosphorylation of p42. We have identified p42 as p42mapk, a mitogen-activated protein (MAP) kinase, on the basis of the following data: by sequential immunoblotting with antiphosphotyrosine and anti-MAP kinase antibodies, (i) similar kinetics are observed for p42 phosphorylation and the decrease in p42mapk electrophoretic mobility, likely corresponding to its tyrosine/threonine phosphorylation [de Vries-Smits, Boudewijn, Burgering, Leevers, Marshall and Bos (1992) Nature (London) 357, 602-604]; (ii) p42 and the shifted form of p42mapk co-migrate on SDS/PAGE; (iii) the myelin-basic-protein kinase activity of p42mapk is stimulated by ET-1, in parallel with the tyrosine phosphorylation of p42. In conclusion, these findings strongly suggest that endothelins can stimulate the tyrosine phosphorylation and activation of p42mapk in astrocytes, via pertussis-toxin-insensitive G protein and protein kinase C-dependent and -independent pathways.
A type I collagen reporter gene construct for protein engineering studies. Functional equivalence of transfected reporter COL1A1 and endogenous gene products during biosynthesis and in vitro extracellular matrix accumulation
Authors: Lamandé S R , Bateman J F
Published: August 2015

A type I collagen reporter gene construct, designed to facilitate detailed analysis of the consequences of introduced structural and regulatory mutations on collagen biosynthesis and participation in the extracellular matrix, was produced by site-directed mutagenesis of the mouse COL1A1 gene. The reporter construct, pWTCI-Ile822, carried a single base change which converted the codon for amino acid 822 of the triple helix from methionine to isoleucine. This change allowed the reporter protein, [Ile822]alpha 1(I), to be distinguished from the wild-type alpha 1(I), and quantified, by its altered CNBr cleavage pattern. In mouse Mov13 cells, which synthesize no endogenous pro alpha 1(I), reporter chains associated with endogenous pro alpha 2(I), formed pepsin-stable triple helices and were secreted efficiently from the cell. The thermal stability of wild-type molecules and molecules containing the reporter [Ile822]alpha 1(I) chains was identical. The biosynthetic characteristics of wild-type and reporter chains were directly compared in stably transfected 3T6 cells. These cells did not make a distinction between reporter and endogenous alpha 1(I) chains, which were secreted from the cells at the same rate and were processed and deposited into the 3T6 cell in vitro accumulated extracellular matrix with equal efficiency. These data demonstrate that the helical sequence alteration in the reporter protein is functionally neutral and that the reporter construct, pWTCI-Ile822, is a suitable vector for the analysis of the biochemical effects of site-directed mutations in the putative COL1A1 functional domains.
Non-enzymic glycation of collagen inhibits binding of oxidized low-density lipoprotein
Authors: Kalant N , McCormick S , Parniak M A
Published: August 2015

We have examined the effect of non-enzymic glycation of native soluble collagen, in solution or in gels, on binding of oxidized low-density lipoprotein (LDL). We found the following. (1) Glycation markedly inhibited binding of LDL. This is contrary to results previously reported; the difference may be attributable to the use of detergent- and heat-denatured collagen, covalently bound to agarose beads, in the earlier study. (2) With increased duration of glycation, collagen solution would not gel, and preformed gels dissolved. (3) [14C]Glucose bound to collagen gels dissociated slowly, even at pH 5, suggesting that it was not present as a Schiff's base; in addition, ketoamines, pentosidine and fluorescent advanced glycation products were not detectable in glycated collagen gels, although they accumulated in tendon collagen glycated under the same conditions. It is hypothesized that the difference in glycation effects between gel and tendon may be due to the strength of cross-linking before glycation: the increase in intermolecular distance in collagen fibrils which results from glycation disrupts the fibrils in gels, preventing binding of LDL and formation of glycation-dependent cross-links, whereas the extensive cross-linking in tendon maintains the intermolecular distances within a range which permits formation of glycation cross-links.
A glycosulphatase that removes sulphate from mucus glycoprotein
Authors: Roberton A M , McKenzie C G , Sharfe N , Stubbs L B
Published: August 2015

A novel glycosulphatase has been purified from a mucus glycopeptide-degrading Prevotella from the colon. The purified enzyme removed inorganic [35S]sulphate from 35S-labelled native rat gastric mucus glycoprotein. Desulphation of mucus glycoprotein was initially rapid (19% complete after 10 min) but then plateaued, reaching only 33% after 3 h. Crude periplasmic extracts could remove 79% of the radioactivity as inorganic sulphate. These results suggest that steric hindrance may limit the access of the purified glycosulphatase to the mucus glycoprotein oligosaccharide chains in the absence of glycosidases, and/or that the enzyme may have the wrong specificity for some of the remaining sulphated sugars in the chains. The apparent molecular mass of the enzyme was 111 kDa as judged from gel exclusion chromatography, and it appeared to be composed of two identical subunits. The enzyme was localized in the periplasm of the bacterium, and using pig gastric mucus glycopeptide as a growth substrate markedly increased enzyme levels. Enzymic activity increased at the end of the growth phase. The substrate specificity of the enzyme was tested against low-molecular-mass sulphated molecules. The monosaccharides glucose 6-sulphate and N-acetylglucosamine 6-sulphate were rapidly desulphated, the latter being the major sulphated sugar in some mucus glycoproteins. Lactose 6-sulphate, galactose 6-sulphate, sulphated steroids and unsaturated disaccharide sulphate breakdown products from chondroitin sulphate were not desulphated. Glycosulphatases which can remove sulphate from mucus glycoproteins may play an important role in the degradation of highly sulphated mucus glycoproteins in the digestive tract, and could modify the effectiveness of mucus glycoproteins in mucosal protection.
Common sequence motifs coding for higher-plant and prokaryotic O-acetylserine (thiol)-lyases: bacterial origin of a chloroplast transit peptide?
Authors: Rolland N , Job D , Douce R
Published: August 2015

A comparison of the amino acid sequence of O-acetylserine (thiol)-lyase (EC 4.2.99.8) from Escherichia coli and the isoforms of this enzyme found in the cytosolic and chloroplastic compartments of spinach (Spinacia oleracea) leaf cells allows the essential lysine residue involved in the binding of the pyridoxal 5′-phosphate cofactor to be identified. The results of further sequence comparison of cDNAs coding for these proteins are discussed in the frame of the endosymbiotic theory of chloroplast evolution. The results are compatible with a mechanism in which the chloroplast enzyme originated from the cytosolic enzyme and both plant genes originated from a common prokaryotic ancestor. The comparison also suggests that the 5′-non-coding sequence of the bacterial gene was transferred to the plant cell nucleus and that it has been used to create the N-terminal portions of both plant enzymes, and possibly the transit peptide of the chloroplast enzyme.
Identification of the putative hexose-phosphate translocator of amyloplasts from cauliflower buds
Authors: Batz O , Scheibe R , Neuhaus H E
Published: August 2015

Starch synthesis in amyloplasts isolated from cauliflower buds is strongly inhibited by the addition of micromolar concentrations of 4,4′-di-isothiocyano-2,2′-stilbenedisulphonic acid (DIDS). Using [3H]DIDS it was possible to label specifically a 31.6 kDa membrane protein of the envelope fraction of isolated amyloplasts. The intensity of the radioactive label was decreased in the presence of glucose 6-phosphate or dihydroxyacetone phosphate, indicating that this protein might be the amyloplastic hexosephosphate translocator.
The ELAM ligand fucosyltransferase, ELFT, directs E-selectin binding to a secreted scaffold protein: a method to produce and purify large quantities of specific carbohydrate structures
Authors: Meier W , Leone D R , Miatkowski K , Lobb R , Goelz S E
Published: August 2015

Many biological processes depend on cell surface recognition of receptor-ligand pairs. Some receptors, such as the selectins, recognize specific carbohydrate structures as part of their ligands. The ability to synthesize such ligands for use in the study of cell adhesion mechanisms or as inhibitors of a variety of pathological conditions would be extremely useful. However, the chemical or enzymic in vitro synthesis of carbohydrate-based ligands has thus far been difficult and costly. We have used E-selectin and its carbohydrate ligand as a model system to test if it is possible to express specific carbohydrate structures on a secreted, glycosylated and easily purified scaffold protein and to use this newly modified protein as a functional adhesion molecule. We co-expressed a fucosyltransferase (ELFT) and a secreted immunoglobulin-LFA3 fusion protein (LFA31G) in the same cell to modify the carbohydrate structures on the secreted LFA3IG scaffold protein (we refer to this novel protein as X-LFA3IG). Using glycosidase digestion, lectin binding, carbohydrate composition analysis and antibody-binding assays, we show that approximately 50% of the potential N-linked carbohydrate sites on X-LFA3IG are, indeed, modified and that the modification is the addition of fucose. Furthermore, we show that X-LFA3IG contains epitopes recognized by anti-Slex antibodies, and, using an E-selectin-specific adhesion assay, we demonstrate that X-LFA3IG is a functional ligand for E-selectin. This in vivo approach for generating specific carbohydrate structures could be generalized to produce and purify large quantities of other biologically important carbohydrate structures.
The characteristics, capacity and receptor regulation of inositol uptake in 1321N1 astrocytoma cells
Authors: Batty I H , Michie A , Fennel M , Downes C P
Published: August 2015

The uptake of inositol into 1321N1 astrocytoma cells was studied by measurement of the accumulation of free [3H]inositol within the intracellular pool. Uptake occurs via a saturable transporter with apparent Km for inositol approximately 40 microM and Vmax approximately 180 pmol/min per mg of protein, which permits intracellular inositol concentrations to exceed those of the medium by a factor of approximately 500. At extracellular concentrations up to 500 microM, inositol uptake is highly dependent (> or = 85%) on the presence of Na+ in the medium, and at physiological extracellular inositol concentrations, allows inositol to achieve an intracellular concentration of approximately 20 mM, indicating an active process driven by the Na+ gradient. Despite this, uptake was only minimally impaired or was unaffected by ouabain (1 mM) or dinitrophenol (1 mM). Consistent with a carrier-mediated mechanism, uptake was competitively blocked by phlorhizin (K1 approximately 125 microM). Uptake was also inhibited by carbachol and histamine, which act respectively via muscarinic and H1 receptors in these cells to stimulate phospholipase C. Inhibition by carbachol was dose-dependent (EC50 approximately 3-30 microM) and blocked by atropine. Inhibition by carbachol (1 mM) was non-competitive, resulting from approximately 50% decrease in the Vmax for uptake without affecting the Km and was persistent over 30-90 min. Inhibition by carbachol and histamine was independent of extracellular Ca2+ and was reproduced by phorbol ester, but not by Ca2+ ionophore or stimulation of adenylate cyclase. These results imply that receptors which couple to phospholipase C may mediate inhibition of inositol uptake via protein kinase C. The data are discussed in relation to inositol homoeostasis in resting and stimulated cells.
Expression of precursor and mature carnitine palmitoyltransferase II in Escherichia coli and in vitro: differential behaviour of rat and human isoforms
Authors: Brown N F , Sen A , Soltis D A , Jones B , Foster D W , McGarry J D
Published: August 2015

cDNAs corresponding to the precursor and mature forms of rat carnitine palmitoyltransferase II (CPT II) were found to be readily expressed in Escherichia coli. In both cases, catalytically active immunoreactive protein was produced and became largely membrane-associated. The precursor form of the enzyme was not proteolytically processed. Removal of 126 bp from the 5′ end of the cDNA coding region allowed expression of a truncated CPT II (lacking the N-terminal 17 residues of the mature protein), but this product was inactive. cDNAs encoding the precursor and mature forms of human CPT II resisted direct expression in E. coli. However, the impediment was overcome when the latter cDNA was ligated in-frame 3′ to sequence encoding a glutathione S-transferase. This construct yielded abundant quantities of the corresponding fusion protein, a portion of which was soluble and catalytically active. In vitro transcription and translation of the various cDNAs established that the lower mobility on SDS/PAGE of rat CPT II compared with its human counterpart (despite their identical numbers of amino acids) is an intrinsic property of the primary sequences of the proteins themselves. Also, the human cDNA was found to contain an artifactual termination signal for T3 RNA polymerase that could be bypassed by the T7 polymerase. Thus rat CPT II can be expressed in active form in E. coli with characteristics similar to those of the enzyme in mitochondria, opening the way to future location of active sites within the molecule. An alternative expression system will be needed for similar studies on human CPT II.
Transformation of the aryl hydrocarbon receptor to a DNA-binding form is accompanied by release of the 90 kDa heat-shock protein and increased affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin
Authors: Henry E C , Gasiewicz T A
Published: August 2015

The binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the aryl hydrocarbon receptor (AhR) elicits a sequence of poorly defined molecular events that ultimately yield a heteromeric transformed AhR that is active as a transcription factor. We have previously developed a model of the ligand-initiated transformation of the AhR to the DNA-binding state based on characterization of several forms of the AhR with respect to their physicochemical properties and DNA-binding affinities. The present studies were designed to determine whether, and at what stage, this process of transformation alters the receptor's affinity for TCDD. In rat hepatic cytosol, approx. 10% of the TCDD specifically bound to the AhR rapidly dissociated (t1/2 approximately 1 h), while the remainder was only slowly dissociable (t1/2 approximately 70 h). The isolated DNA-binding forms of the receptor (monomeric and transformed) bound TCDD very tightly (t1/2 > 100 h), whereas TCDD was dissociable from the non-DNA-binding receptor form(s). A lower incubation temperature (0-4 degrees C) and the presence of molybdate partially stabilized the non-DNA-binding fraction of the TCDD.receptor complex and also enhanced TCDD dissociation in crude cytosol. Immunoprecipitation of the different AhR forms with an anti-AhR antibody and immunoblotting with antibody to the 90 kDa heat-shock protein (hsp90) demonstrated that hsp90 was associated with the unoccupied receptor complex as well as with a fraction of the non-DNA-binding TCDD.receptor complex; isolated DNA-binding forms did not contain detectable hsp90. We conclude that while hsp90 remains associated with the AhR, TCDD is readily dissociable; following release of hsp90, however, TCDD becomes very tightly bound, and remains so upon completion of transformation.
Tamoxifen decreases the rate of proliferation of rat vascular smooth-muscle cells in culture by inducing production of transforming growth factor β
Authors: Grainger D J , Weissberg P L , Metcalfe J C
Published: August 2015

Tamoxifen selectively and reversibly decreased the rate of proliferation of adult rat aortic vascular smooth-muscle cells (VSMCs). Half-maximal inhibition of proliferation occurred at 2-5 microM tamoxifen for VSMCs and at > 50 microM for adventitial fibroblasts. The cell cycle time for all the VSMCs in the population was increased from 35 +/- 2 h to 54 +/- 4 h in the presence of 33 microM tamoxifen. Tamoxifen did not affect the time of entry into DNA synthesis, but delayed arrival at mitosis by > 24 h. It therefore extended the duration of the G2-to-M phase of the cell cycle. However, the rate of proliferation of VSMCs was not decreased by tamoxifen (at concentrations up to 50 microM) in the presence of neutralizing antibody to transforming growth factor beta (TGF-beta). The level of mRNA for TGF-beta 1 in VSMCs was strongly induced by 10 microM tamoxifen, and TGF-beta activity in conditioned medium from tamoxifen-treated cells was more than 50-fold higher than from control cells. Tamoxifen therefore extended the G2-to-M phase of the cell cycle in VSMCs by increasing TGF-beta activity in the culture.
13C-n.m.r. of the cyanylated apoflavodoxin and flavodoxin from Clostridium pasteurianum
Authors: Doherty G M , Mayhew S G , Malthouse J P G
Published: August 2015

The thiol group of the flavodoxin from Clostridium pasteurianum has been cyanylated in a single step using [cyanato-13C]2-nitro-5-thiocyanatobenzoic acid. This chemical modification increases the dissociation constant of the apoflavodoxin-FMN complex 10-fold from 0.33 +/- 0.15 nM to 2.9 +/- 1.3 nM. The thiocyanate carbons of the cyanylated cysteine residue of apoflavodoxin and flavodoxin had chemical shift values of 114.7 and 112.3 p.p.m. respectively. From these chemical shifts we conclude that the binding of FMN by the cyanylated apoflavodoxin causes a decrease in the polarity and/or hydrogen bonding capacity of the environment of the thiocyanate group. We compare these results with those obtained from similar studies on the cyanylated apoflavodoxin and flavodoxin from Megasphaera elsdenii and we discuss how FMN binding and cyanylation perturb the structures of apoflavodoxins from Megasphaera elsdenii and Clostridium pasteurianum.
Pancreastatin increases free cytosolic Ca2+ in rat hepatocytes, involving both pertussis-toxin-sensitive and -insensitive mechanisms
Authors: Sánchez-Margalet V , Lucas M , Goberna R
Published: August 2015

Freshly isolated rat hepatocytes, loaded with the Ca2+ probe Fluo-3, responded to homologous pancreastatin with a sudden increase in free cytosolic Ca2+ ([Ca2+]i) as well as glucose release. Addition of rat pancreastatin (0.1 microM) to hepatocytes resulted in an increase in [Ca2+]i from 150 nM to 700 nM, which declined back to nearly basal values within 2-3 min. Half-maximal and maximal effects were observed at 0.3 and 100 nM pancreastatin respectively. The increase in [Ca2+]i induced by vasopressin and noradrenaline was very similar in extent (from 150 to 800 nM) to that produced by pancreastatin. Neither the alpha 1-adrenergic blocker prazosin nor the vasopressin antagonist V1 modified the increase in [Ca2+]i induced by pancreastatin. Pig pancreastatin and its 33-49 C-terminal fragment produced about 65 and 75% of the effect of homologous pancreastatin respectively. Glucose production correlated with changes in [Ca2+]i in the same order of potency: vasopressin > rat pancreastatin > pig 33-49 pancreastatin > pig 1-49 pancreastatin. The effect of pancreastatin on [Ca2+]i was decreased by 50% when Ca2+ was omitted from the medium, and totally abolished when hepatocytes were depleted of internal Ca2+ stores by preincubation without Ca2+ and with 2 mM EGTA. When hepatocytes were preincubated for 5 min with PMA, the effects of ATP and noradrenaline were prevented, and those of vasopressin and pancreastatin remained unchanged. The pretreatment of hepatocytes with pertussis toxin diminished the response to pancreastatin and vasopressin. These results suggest that pancreastatin is a new Ca(2+)-mobilizing glycogenolytic hormone acting through a specific receptor which may involve both pertussis-toxin-sensitive and -insensitive GTP-binding regulatory proteins.
Stress induction of the spermidine/spermine N1-acetyltransferase by a post-transcriptional mechanism in mammalian cells
Authors: Gerner E W , Kurtts T A , Fuller D J M , Casero R A
Published: August 2015

Heat shock and diethyldithiocarbamate stimulate polyamine catabolism in animal cells by a mechanism involving the induction of spermidine/spermine N1-acetyltransferase (N1-SSAT) activity. Steady-state levels of RNA encoding this enzyme remain essentially unchanged during periods after these stresses when N1-SSAT activity is increased by 3.5-10-fold or more in three different cell lines of hamster and human origin. Depletion of intracellular spermidine pools by alpha-difluoromethylornithine (DFMO) inhibits stress induction of N1-SSAT activity. Exogenous spermidine can restore stress inducibility of N1-SSAT to DFMO-treated cells, and induce this enzyme activity in non-heat-shocked but polyamine-depleted cells. Acetylation at N1 suppresses the ability of spermidine to induce N1-SSAT activity, relative to this same modification at N8. Fluorinated spermidine analogues, which decrease the pKa values of the amine groups at positions 4 and 8, neither induce nor inhibit N1-SSAT activity in DFMO-treated cells. These data demonstrate that certain stresses induce N1-SSAT by a spermidine-dependent post-transcriptional mechanism. The mode of induction is affected by both the propyl and butyl moieties of spermidine.
The role of the third intracellular loop of the neutrophil N-formyl peptide receptor in G protein coupling
Authors: Prossnitz E R , Quehenberger O , Cochrane C G , Ye R D
Published: August 2015

The G-protein-coupled N-formyl peptide receptor (FPR) contains one of the smallest known third intracellular loops of this class of receptors, consisting of only 15 amino acids. To study the role of this region of the receptor in G protein coupling and signal transduction, we generated a deletion mutant (D3i) in which 10 amino acids of the loop were removed, as well as a series of site-directed mutants containing substitutions of the charged and polar amino acids of this loop. The D3i mutant, expressed at normal levels on the cell surface, displayed a KD for labelled N-formyl-Met-Leu-Phe ([3H]FMLP) of 165 nM. This value compares with a KD for the wild-type FPR of 1.0 nM, or 20 nM in the presence of guanosine 5′-[gamma-thio]triphosphate, which uncouples G proteins from the receptor. These results indicate that D3i contains significant structural defects, beyond the disruption of G protein coupling, that affect ligand binding properties. Ten site-directed mutants generated in the third intracellular loop (T226A, K227E, H229A, K230Q, K235Q, S236A, S236A/S237G, R238G, R241E and S244A) displayed KD values between 0.5 and 1.0 nM, with expression levels between 22% (K227E) and 111% (H229A) of that of wild type receptor. The capacity of the mutants for signal transductions was determined by measuring intracellular Ca2+ mobilization. Eight of the ten mutants displayed EC50 values for FMLP of between 0.07 and 0.9 nM, as compared with 0.12 nM for the wild-type receptor. The two mutants K227E and R238G had EC50 values of 2.7 and 2.9 nM respectively. The increase in EC50 could be accounted for partially by the low levels of receptor expression. All ten mutants gave maximum levels of Ca2+ mobilization similar to that produced by the wild-type FPR. These results contradict the conclusions reached with other G-protein-coupled receptors and indicate that the third intracellular loop of the FPR does not have a critical role in the functional coupling of G proteins.
Biochemical characterization of the molecular interaction between recombinant basic fibroblast growth factor and a recombinant soluble fibroblast growth factor receptor
Authors: Caccia P , Cletini O , Isacchi A , Bergonzoni L , Orsini G
Published: August 2015

The extracellular domain of human fibroblast growth factor receptor (XC-FGF-R) was expressed in Escherichia coli. The protein was purified to homogeneity and the interaction with basic fibroblast growth factor (bFGF), its physiological ligand, was examined. Using resins on which bFGF was reversibly bound, we analysed the characteristics of the binding between XC-FGF-R and immobilized bFGF. We also investigated the stoichiometry of the binding between XC-FGF-R and recombinant human bFGF (rhbFGF) applying non-denaturing gel electrophoresis, chemical cross-linking followed by SDS/PAGE, and gel-filtration chromatography. In cross-linking and gel-filtration chromatography experiments, a 1:1 complex between rhbFGF and XC-FGF-R was observed. The complex was separated from the non-complexed proteins using non-denaturing PAGE in the presence of 0.1% Triton X-100. The band corresponding to the complex was recognized by specific antibodies directed against bFGF and its receptor, blotted on poly(vinylidene difluoride) membranes and submitted to sequence and amino acid analysis. The data obtained from these determinations confirmed the formation of a 1:1 complex between rhbFGF and XC-FGF-R.