bluuebunny/crossref_metadata_2025 huggingface dataset:
The GLUT5 hexose transporter is also localized to the basolateral membrane of the human jejunum
Authors: Blakemore S J , Aledo J C , James J , Campbell F C , Lucocq J M , Hundal H S
Published: August 2015

The intestine is a major site of expression of the human GLUT5 hexose transporter, which is thought to be localized exclusively to the brush border membrane (BBM) where its major role is likely to be in the absorption of fructose. In this study we present novel biochemical and morphological evidence showing that the GLUT5 transporter is also expressed in the basolateral membrane (BLM) of the human intestine. BBM and BLM were isolated by fractionation of human jejunum. BBM were enriched with alkaline phosphatase activity by over 9-fold relative to a crude jejunal homogenate and contained immunoreactive sucrase-isomaltase and GLUT5 proteins. By contrast the BBM fraction was substantially depleted of immunoreactive a1 subunits of the Na,K-ATPase and GLUT2 glucose transporters which were abundantly present in the BLM fraction. This BLM fraction was enriched by over 11-fold in potassium-stimulated phosphatase activity relative to the crude homogenate; BLM also reacted to immunological probes for GLUT5 but showed no observable reactivity with antibodies directed against sucrase-isomaltase. Quantitative immunoblotting revealed that the BBM and BLM contained near equal amounts of GLUT5 per mg of membrane protein. Immunogold localization of GLUT5 on ultrathin sections of human jejunum showed that GLUT5 was present in both apical BBM and BLM. This gold labelling was absent when antiserum was pre-incubated with the antigenic peptide corresponding to a specific C-terminal sequence of human GLUT5. Quantitative analyses of the number of gold particles per unit length of BBM and BLM indicated that the mean density of gold labelling was marginally greater in the BBM (0.399 gold particles/micrometer) than in the BLM (0.293 gold particle/micrometer). The localization of GLUT5 in the BLM of the human jejunum may suggest that it specifically participates in the transfer of fructose across the basal membrane of the enterocyte.
Hepatic zinc in metallothionein-null mice following zinc challenge: in vivo and in vitro studies
Authors: Coyle P , Philcox J C , Rofe A M
Published: August 2015

Hepatic zinc uptake and accumulation were compared in freshly isolated and cultured hepatocytes prepared from control (MT+/+) and metallothionein (MT)-null (MT-/-) mice. In freshly isolated hepatocytes, rapid (10-15 min) exchange of 65Zn was proportional to the Zn concentration in the medium and occurred to the same extent in hepatocytes from MT+/+ and MT-/- mice. In 24 h culture experiments with MT+/+ and MT-/- hepatocytes it was shown that approx. 40% of newly acquired cell-associated Zn was attached to the cell surface and not internalized. In MT+/+ and MT-/- hepatocyte cultures, internalized Zn (intZn) increased in proportion to extracellular Zn. Zn accumulation in MT-/- hepatocytes was only 60% that of MT+/+ cells. Addition of 1 microM dexamethasone (Dex) and recombinant mouse interleukin-6 (IL-6; 100 units/ml) increased MT accumulation by 8.6-fold in MT+/+ hepatocytes (at 50 microM Zn) and there was an associated parallel increase in intZn. Dex and IL-6 did not increase intZn in the MT-/- hepatocytes. At 16 h after an intraperitoneal injection of 5 micrograms/g Zn, plasma and urine Zn concentrations were 69 +/- 10 microM and 86 +/- 25 microM respectively in MT-/- mice (n = 10) and 27 +/- 1 microM and 23 +/- 4 microM respectively in MT+/+ controls (n = 9) (P < 0.001, plasma; P < 0.05, urine). Hepatic cytosolic Zn concentrations doubled in MT+/+ mice and increased by a significant 15% in MT-/- mice. There was no increase in hepatic Zn (dry wt.) concentrations or in total hepatic Zn, demonstrating that the increase in cytosolic Zn in MT-/- mice was due to hepatic water loss rather than net Zn uptake. It appears that even at extreme plasma concentrations of Zn, little if any accumulates within the liver when there is no MT available for its sequestration. That this is not fully demonstrated in vitro is probably due to nature of cell culture, where organ architecture is lost and the external protein binding milieu is less complex.
Transcription activation at Escherichia coli promoters dependent on the cyclic AMP receptor protein: effects of binding sequences for the RNA polymerase α-subunit
Authors: Savery N J , Rhodius V A , Wing H J , Busby S J W
Published: August 2015

Transcription activation at two semi-synthetic Escherichia coli promoters, CC(-41.5) and CC(-72.5), is dependent on the cyclic AMP receptor protein (CRP) that binds to sites centred 41.5 and 72.5 bp upstream from the respective transcription startpoints. An UP-element that can bind the C-terminal domain of the RNA polymerase (RNAP) alpha-subunit was cloned upstream of the DNA site for CRP at CC(-41.5) and downstream of the DNA site for CRP at CC(-72.5). In both cases CRP-dependent promoter activity was increased by the UP-element, but CRP-independent activity was not increased. DNase I footprinting was exploited to investigate the juxtaposition of bound CRP and RNAP alpha-subunits. In both cases, CRP and RNAP alpha-subunits occupy their cognate binding sites in ternary CRP-RNAP promoter complexes. RNAP alpha-subunits can occupy the UP-element in the absence of CRP, but this is not sufficient for open complex formation. The positive effects of binding RNAP alpha-subunits upstream of the DNA site for CRP at -41.5 are suppressed if the UP-element is incorrectly positioned.
Different properties of the mitochondrial and cytosolic hexokinases in maize roots
Authors: Galina A , Reis M , Albuquerque M C , Puyou A G , Puyou M T G , de Meis L
Published: August 2015

After tissue homogenization, 43% of the total hexokinase activity found in maize radicles was recovered in the mitochondrial fraction and 35% was soluble, in the cytosol. The maize submitochondrial particles obtained after mitochondrial sonication retained a high hexokinase activity. The mitochondrial respiration (state 4 rate) was activated by glucose. This activation was blocked by carboxyatractyloside (0.5 mM) and by oligomycin (2 micrograms/ml). The affinities for ATP and glucose of both soluble and membrane-bound maize hexokinases are similar to those of yeast hexokinase. The Km for ATP of these different forms of hexokinase varied between 0.15 and 0.37 mM, and the Km for glucose between 0.05 and 0.13 mM. A major difference between the two maize hexokinase forms is that only the mitochondrial enzyme was strongly inhibited by ADP (Ki 0.04 mM). The soluble forms of hexokinase found both in the cytosol of maize radicles and in yeast are not inhibited by ADP. In a previous report [de Meis, Grieco and Galina (1992) FEBS Lett. 308, 197-201] it was shown that the mitochondrial F1-F0-ATPase can use glucose 6-phosphate and yeast hexokinase as an ATP regenerating system. We now show that the membrane-bound hexokinase and glucose 6-phosphate can also serve as an ATP regenerating system for the mitochondria of maize radicles provided that the ADP concentration is kept below 0.05 mM. Higher ADP concentrations inhibit the reverse reaction of the mitochondrial hexokinase.
Role of the N-terminal region in covalent modification of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: comparison of phosphorylation and ADP-ribosylation
Authors: Rosa J L , Pérez J X , Ventura F , Tauler A , Gil J , Shimoyama M , Pilkis S J , Bartrons R
Published: August 2015

The effect of cyclic AMP (cAMP)-dependent phosphorylation and ADP-ribosylation on the activities of the rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2), was investigated in order to determine the role of the N-terminus in covalent modification of the enzyme. The bifunctional enzyme was demonstrated to be a substrate in vitro for arginine-specific ADP-ribosyltransferase: 2 mol of ADP-ribose was incorporated per mol of subunit. The Km values for NAD+ and PFK-2/FBPase-2 were 14 microM and 0.4 microM respectively. A synthetic peptide (Val-Leu-Gln-Arg-Arg-Arg-Gly-Ser-Ser-Ile-Pro-Gln) corresponding to the site phosphorylated by cAMP-dependent protein kinase was ADP-ribosylated on all three arginine residues. Analysis of ADP-ribosylation of analogue peptides containing only two arginine residues, with the third replaced by alanine, revealed that ADP-ribosylation occurred predominantly on the two most C-terminal arginine residues. Sequencing of the ADP-ribosylated native enzyme also demonstrated that the preferred sites were at Arg-29 and Arg-30, which are just N-terminal to Ser-32, whose phosphorylation is catalysed by cAMP-dependent protein kinase (PKA). ADP-ribosylation was independent of the phosphorylation state of the enzyme. Furthermore, ADP-ribosylation of the enzyme decreased its recognition by liver-specific anti-bifunctional-enzyme antibodies directed to its unique N-terminal region. ADP-ribosylation of PFK-2/FBPase-2 blocked its phosphorylation by PKA, and decreased its PFK-2 activity, but did not alter FBPase-2 activity. In contrast, cAMP-dependent phosphorylation inhibited the kinase and activated the bisphosphatase. These results demonstrate that ADP-ribosylation of arginine residues just N-terminal to the site phosphorylated by PKA modulate PFK-2 activity by an electrostatic and/or steric mechanism which does not involved uncoupling of N- and C-terminal interactions as seen with cAMP-dependent phosphorylation.
Rat thimet oligopeptidase: large-scale expression in Escherichia coli and characterization of the recombinant enzyme
Authors: McKie N , Dando P M , Brown M A , Barrett A J
Published: August 2015

The coding sequence for rat testis thimet oligopeptidase (TOP) (EC 3.4.24.15) was placed under the control of the T7 polymerase/promoter system. Cultures of Escherichia coli transfected with the resulting plasmid expressed the enzyme as a soluble cytoplasmic protein. Medium-scale cultures allowed isolation of the enzyme in quantities of tens of milligrams. The availability of the recombinant enzyme permitted the determination of such chemical properties as epsilon 280 (48,960), zinc content (2 atom/molecule) and available thiol content (8-10/molecule) for TOP. The recombinant enzyme showed the catalytic activities previously reported for the naturally occurring enzyme, so that we can now conclude with confidence that these are all due to TOP and there is no need to postulate the existence of separate ‘Pz-peptidase’ or ‘endo-oligopeptidase A’ enzymes.
Steady-state kinetics and chemical mechanism of octopus hepatopancreatic glutathione transferase
Authors: Tang S S , Chang G G
Published: August 2015

The kinetic mechanism of glutathione S-transferase (GST) from Octopus vulgaris hepatopancreas was investigated by steady-state analysis. Initial-velocity studies showed an intersecting pattern, which suggests a sequential kinetic mechanism for the enzyme. Product-inhibition patterns by chloride and the conjugate product were all non-competitive with respect to glutathione or 1-chloro-2,4-dinitrobenzene (CDNB), which indicates that the octopus digestive gland GST conforms to a steady-state sequential random Bi Bi kinetic mechanism. Dead-end inhibition patterns indicate that ethacrynic acid ([2,3-dichloro-4-(2-methyl-enebutyryl) phenoxy]acetic acid) binds at the hydrophobic H-site, norophthalmic acid (gamma-glutamylalanylglycine) binds at the glutathione G-site, and glutathione-ethacrynate conjugate occupied both H- and G-sites of the enzyme. The chemical mechanism of the enzyme was examined by pH and kinetic solvent-isotope effects. At pH (and p2H) = 8.011, in which kcat. was independent of pH or p2H, the solvent isotope effects on V and V/KmGSH were near unity, in the range 1.069-1.175. An inverse isotope effect was observed for V/KmCDNB (0.597), presumably resulting from the hydrogen-bonding of enzyme-bound glutathione, which has pKa of 6.83 +/- 0.04, a value lower by 2.34 pH units than the pKa of glutathione in aqueous solution. This lowering of the pKa value for the sulphydryl group of the bound glutathione was presumably due to interaction with the active site Tyr7, which had a pKa value of 8.46 +/- 0.09 that was raised to 9.63 +/- 0.08 in the presence of glutathione thiolate. Subsequent chemical reaction involves attacking of thiolate anion at the electrophilic substrate with the formation of a negatively charged Meisenheimer complex, which is the rate-limiting step of the reaction.
Purification and characterization of citrate synthase isoenzymes from Pseudomonas aeruginosa
Authors: Mitchell C G , Anderson S C K , el-Mansi E M T
Published: August 2015

Two types of citrate synthase (CS) have been purified from Pseudomonas aeruginosa, a ‘large’ form (CSI) and a ‘small’ form (CSII). The M(r)s of the CSI and CSII isoenzymes were determined to be 240,000 +/- 16,000 (mean +/- S.E.M.) and 80,300 +/- 3800 respectively. Chemical cross-linking of the native enzymes with either dimethyl suberimidate or glutaraldehyde followed by electrophoretic analysis by SDS/PAGE showed that CSI is a hexamer and CSII is a dimer. SDS/PAGE showed that CSI and CSII each consist of a single subunit type, of M(r) 42,000 +/- 2000 and M(r) 36,500 +/- 2000 respectively. CSI and CSII were also shown to be distinct kinetically, immunologically and in terms of their regulatory properties. It is suggested that the CS isoenzymes are products of different structural genes.
Octopus S-crystallins with endogenous glutathione S-transferase (GST) activity: sequence comparison and evolutionary relationships with authentic GST enzymes
Authors: Chiou S H , Yu C W , Lin C W , Pan F M , Lu S F , Lee H J , Chang G G
Published: August 2015

S-Crystallin is a major protein present in the lenses of cephalopods (octopus and squid). To facilitate the cloning of this crystallin gene, cDNA was constructed from the poly(A)+ mRNA of octopus lenses, and amplified by PCR for nucleotide sequencing. Sequencing of 10 of 15 positive clones coding for this crystallin revealed three distinct S-crystallin isoforms with 61-64% identity in nucleotide sequences and 42-58% similarity in amino acid sequences when compared with homologous crystallins in squid lenses. These charge-isomeric crystallins also show between 26 and 33% amino acid sequence identity to four major classes of glutathione S-transferase (GST), a major detoxification enzyme present in most mammalian tissues. For further analysis, expression of one of the S-crystallin cDNAs was carried out in the bacterial expression system pQE-30, and the S-crystallin protein produced in Escherichia coli was purified to homogeneity to determine the enzymic properties. We found that the expressed octopus S-crystallin possessed much lower GST activity than the authentic GSTs from other tissues. Sequence comparison and construction of phylogenetic trees for S-crystallins from squid and octopus lenses and various classes of GSTs revealed that S-crystallins represent a multigene family which is structurally related to Alpha-class GSTs and probably derived from the ancestral GST by gene duplication and subsequent multiple mutational substitutions.
Purification and properties of the lipoate protein ligase of Escherichia coli
Authors: Green D E , Morris T W , Green J , Cronan J E , Guest J R
Published: August 2015

Lipoate is an essential component of the 2-oxoacid dehydrogenase complexes and the glycine-cleavage system of Escherichia coli. It is attached to specific lysine residues in the lipoyl domains of the E2p (lipoate acetyltransferase) subunit of the pyruvate dehydrogenase complex by a Mg(2+)- and ATP-dependent lipoate protein ligase (LPL). LPL was purified from wild-type E. coli, where its abundance is extremely low (< 10 molecules per cell) and from a genetically amplified source. The purified enzyme is a monomeric protein (M(r) 38,000) which forms irregular clusters of needle-like crystals. It is stable at -20 degrees C, but slowly oxidizes to an inactive form containing at least one intramolecular disulphide bond at 4 degrees C. The inactive form could be re-activated by reducing agents or by an as-yet unidentified component (reactivation factor) which is resolved from LPL at the final stage of purification. The pI is 5.80, and the Km values for ATP, Mg2+ and DL-lipoate were determined. Selenolipoate and 6-thio-octanoate were alternative but poorer substrates. Lipoylation was reversibly inhibited by the 6- and 8-seleno-octanoates and 8-thio-octanoate, which reacted with the six cysteine thiol groups of LPL. LPL was inactivated by Cu2+ ions in a process that involved the formation of inter- and intra-molecular disulphide bonds. Studies with lplA mutants lacking LPL activity indicated that E. coli possesses another distinct lipoylation system, although no such activity could be detected in vitro.
The biosynthesis of threonine by mammalian cells: expression of a complete bacterial biosynthetic pathway in an animal cell
Authors: Rees W D , Hay S M
Published: August 2015

The coding regions for the Escherichia coli gene for aspartokinase I/homoserine dehydrogenase I (thrA) and the Corynebacterium glutamicum gene for aspartic semialdehyde dehydrogenase (asd) have been subcloned into a Simian Virus 40 (SV40)-based mammalian expression vector. Both enzyme activities are expressed in mouse 3T3 cells after transfer of the corresponding chimaeric gene. The kinetic parameters are similar to those of the native bacterial enzymes, and aspartokinase I/homoserine dehydrogenase I retains its allosteric regulation by threonine. An extract of the cells expressing aspartokinase I/homoserine dehydrogenase I, mixed with one from cells expressing aspartic semialdehyde dehydrogenase, produced homoserine when the mixture was incubated with aspartic acid, ATP and NADPH. The thrA and asd expression cassettes were combined into a single plasmid which, when transfected into 3T3 cells, enabled them to produce homoserine from aspartic acid. Homoserine-producing 3T3 cells were transfected with the plasmid pSVthrB/C (homoserine kinase and threonine synthase) and selected for growth on homoserine. Cell lines isolated from these cells expressed the complete bacterial threonine pathway, were independent of threonine for growth and could be maintained in medium which contained no free threonine. The threonine in the proteins of these cells became enriched in 15N when the culture medium contained [15N]aspartic acid. The production of homoserine and the growth of cells was at a maximum when there was more than 2.5 mM aspartate in the medium. Below this concentration the high Km of aspartokinase limited the flux through the pathway. In the presence of additional aspartic acid the new pathway could sustain a cell cycle time close to that of the same cells cultured in threonine-containing medium.
Purification and characterization of a fatty acid-activated protein kinase (PKN) from rat testis
Authors: Kitagawa M , Mukai H , Shibata H , Ono Y
Published: August 2015

PKN, a novel protein kinase with a catalytic domain homologous to that of the protein kinase C (PKC) family and unique N-terminal leucine-zipper-like sequences, was identified by molecular cloning from a human hippocampus cDNA library [Mukai and Ono (1994) Biochem. Biophys. Res. Commun. 199, 897-904]. Recently we partially purified recombinant PKN from COS7 cells transfected with the cDNA construct encoding human PKN, and demonstrated that the recombinant PKN was activated by unsaturated fatty acids and limited proteolysis [Mukai, Kitagawa, Shibata et al. (1994) Biochem. Biophys. Res. Commun. 204, 348-356]. The present work has focused on the further purification and characterization of PKN from native rat tissue. Immunochemical measurement revealed that PKN was found in every tissue, and was especially abundant in testis, spleen and brain; subcellular fractionation of rat brain showed that half of the PKN was localized in the soluble cytosolic fraction. PKN was purified approx. 8000-fold to apparent homogeneity from the cytosolic fraction of rat testis by DEAE-cellulose chromatography, ammonium sulphate fractionation and chromatography on butyl-Sepharose, heparin-Sepharose, Mono Q and protamine-CH-Sepharose. The enzyme migrates as a band of apparent molecular mass 120 kDa. Using serine-containing peptides based on the pseudosubstrate sequence of PKC-delta as phosphate acceptors, the kinase activity was stimulated several-fold by 40 microM unsaturated fatty acids or by detergents such as 0.04% sodium deoxycholate and 0.004% SDS. In the absence of modifiers, protamine sulphate, myelin basic protein and synthetic peptides based on the pseudosubstrate site of PKCs or ribosomal S6 protein were good substrates for phosphorylation by the kinase. In the presence of 40 microM arachidonic acid the kinase activity of PKN for these phosphate acceptors was increased 2-18-fold. The autophosphorylation activity of purified PKN was partially inhibited by pretreatment with alkaline phosphatase. These properties appear to distinguish PKN from many protein kinases isolated previously.
Nitric oxide rapidly scavenges tyrosine and tryptophan radicals
Authors: Eiserich J P , Butler J , van der Vliet A , Cross C E , Halliwell B
Published: August 2015

By utilizing a pulse-radiolytic technique, we demonstrate for the first time that the rate constant for the reaction of nitric oxide (.NO) with biologically relevant tyrosine and tryptophan radicals (Tyr. and Trp. respectively) in amino acids, peptides and proteins is of the order of (1-2) x 10(9) M-1.s-1. We also show that .NO effectively interferes with electron-transfer processes between tryptophan and tyrosine residues in proteins subjected to pulse radiolysis. The near diffusion-controlled rates of these reactions, coupled with the increasingly recognized role of protein radicals in enzyme catalysis and oxidative damage, suggest that Tyr. and Trp. are likely and important targets for .NO generated in vivo.
Ca2+ storage in Trypanosoma brucei: the influence of cytoplasmic pH and importance of vacuolar acidity
Authors: Scott D A , Moreno S N J , Docampo R
Published: August 2015

The hypothesis that changes in cytosolic pH effect the release from intracellular compartments of stored calcium in Trypanosoma brucei was addressed by the use of procyclic and bloodstream trypomastigotes of T. brucei loaded with the fluorescent reagents 2′,7′-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF) to measure intracellular pH (pHi), or fura 2 to measure intracellular free calcium ([Ca2+]i). Experiments were performed in EGTA-containing buffers, so increases in [Ca2+]i reflected release of stored calcium rather than Ca2+ entry. Nigericin reduced pHi and increased [Ca2+]i in loaded cells, whilst propionate reduced pHi, but did not affect [Ca2+]i, and NH4Cl increased both variables, so there appears to be no correlation between pHi and [Ca2+]i. Treatment of the cells with the calcium ionophore ionomycin under similar conditions (nominal absence of extracellular Ca2+) resulted in an increase of [Ca2+]i which was greatly increased by addition of either NH4Cl, nigericin or the vacuolar H(+)-ATPase inhibitor bafilomycin A1. Similar results were obtained when the order of additions was reversed or when digitonin-permeabilized cells were used with the Ca2+ indicator arsenazo III. The results suggest that more Ca2+ is stored in this acidic compartment in procyclic than in bloodstream forms. Taking into account the relative importance of the ionomycin-releasable and the ionomycin-plus-NH4Cl-releasable Ca2+ pools, it is apparent that a significant amount of the Ca2+ stored in T. brucei trypomastigotes is present in the acidic compartment thus identified.
Structure of Leishmania lipophosphoglycan: inter- and intra-specific polymorphism in Old World species
Authors: McConville M J , Schnur L F , Jaffe C , Schneider P
Published: August 2015

The most abundant surface macromolecule on the promastigote stage of leishmanial parasites is a polymorphic lipophosphoglycan (LPG). We have elucidated the structures of two new LPGs, from Leishmania tropica (LRC-L36) and L. aethiopica (LRC-L495), and investigated the nature of intra-specific polymorphism in the previously characterized LPG of L. major (LRC-L456 and -L580). These molecules contain a phosphoglycan chain, made up of repeating PO4-6Gal beta 1-4Man units and a conserved hexaglycosyl-phosphatidylinositol membrane anchor. Extensive polymorphism occurs in the extent to which the LPG repeat units are substituted with different glycan side chains. The L. tropica LPG is the most complex LPG characterized to date, as most of the repeat units are substituted with more than 19 different glycan side chains. All of these side chains, including the novel major glycans, Arap beta 1-3Glc beta 1- and +/- Arap beta 1-2Glc beta 1-4[+/- Arap beta 1-2]Glc beta 1-, are linked to the C-3 position of the backbone disaccharide galactose. In contrast, the L. aethiopica LPG repeat units are partially substituted (35%) with single alpha-mannose residues that are linked, unusually, to the C-2 position of the mannose in the backbone disaccharide. Polymorphism is also evident in the spectrum of alpha-mannose-containing oligosaccharides that cap the non-reducing terminus of the phosphoglycan chains of these LPGs. Finally, analysis of the L. major LPGs showed that, while some strains contain LPGs which are highly substituted with side chains of beta Gal, Gal beta 1-3Gal beta 1- and Arap beta 1-2Gal beta 1-3Gal beta 1-, the LPGs of other strains (i.e L. major LRC-L456) are essentially unsubstituted. Recent studies have shown that the LPG side chains and cap structures can mediate promastigote attachment to a number of different receptors along the midgut of the sandfly vector. The possible significance of LPG polymorphism on the ability of these parasites to infect a number of different sandfly vectors is discussed.
Effects of fasting on hepatic and peripheral glucose metabolism in conscious rats with near-total fat depletion
Authors: Barzilai N , Massillon D , Rossetti L
Published: August 2015

Experimental diabetes and fasting are both associated with hypoinsulinaemia and share several other metabolic features. We investigated hepatic and peripheral glucose metabolism in young rats after near-total depletion of their fat mass. Conscious rats were fasted for 72 h (n = 13), while 6 h-fasted animals (n = 14) served as controls. Rats were studied either during saline infusion or insulin (18 m-units/kg per min)-clamp studies. In fasting, despite a 2-fold increase in hepatic glucose-6-phosphatase (Glc-6-Pase) Vmax. (from 16 +/- 2 mumol/g of liver per min in control; P < 0.001), the basal hepatic glucose production (HGP) decreased by 47% [from 88 +/- 3 mumol/kg lean body mass (LBM) per min in control; P < 0.01]. The decreased HGP in fasting was associated with a 70% decrease in the hepatic levels of glucose 6-phosphate (Glc-6-P) (from 366 +/- 53 nmol/g wet wt. in control; P < 0.01). Thus Glc-6-Pase activity assayed in the presence of the Glc-6-P levels found in vivo was decreased by 44%. During hyperinsulinaemia, peripheral glucose uptake was decreased by 15% with 3 days of fasting (from 272 +/- 17 mumol/kg LBM per min in control; P < 0.01). This was completely accounted for by a 42% decrease in whole-body glycolysis (P < 0.01), while the rate of glycogen synthesis was unchanged. Thus fasting (after near-total fat depletion) differs from experimental diabetes because: (1) despite markedly increased Glc-6-Pase, HGP is decreased in fasting, due to a marked decrease in the substrate level (Glc-6-P) in vivo; and (2) the impairment in peripheral insulin sensitivity in fasting is due to a decrease in the glycolytic, and not the glycogen-synthetic, pathway.
The purification of a cysteine-dependent NAD+ glycohydrolase activity from bovine erythrocytes and evidence that it exhibits a novel ADP-ribosyltransferase activity
Authors: Saxty B A , van Heyningen S
Published: August 2015

An NAD+:cysteine ADP-ribosyltransferase activity was purified from bovine erythrocytes on the assumption that, like pertussis toxin, the enzyme would exhibit a cysteine-dependent NAD+ glycohydrolase activity. A three-step purification procedure was developed involving (1) precipitation with 40% (NH4)2SO4, (2) binding to a cysteine-Sepharose affinity column, and (3) binding to an NAD+ affinity column. PAGE showed a single band of M(r) 45,000. The enzyme had been purified 47,000-fold and had a specific activity of 1900 nmol nicotinamide released/min per mg. A study of the kinetic properties of this enzyme showed saturation kinetics for cysteine (Km = 4.0 mM). The ability of this enzyme to ADP-ribosylate protein was investigated using re-sealed inverted bovine erythrocyte ghosts. Incubation of the purified enzyme with erythrocyte ghosts and [adenylate-32P]NAD+ led to the enhanced dose-dependent labelling of several proteins, a doublet of high M(r) and proteins of M(r) 60,000, 55,000 and 29,000, identified by autoradiography of separated proteins on SDS/PAGE. The enzyme-catalysed labelling of the major component at M(r) 55,000 was blocked by pre-treatment of the erythrocyte ghosts with N-ethymaleimide, a sulphydryl alkylating agent, and the label was released by mercuric ion, but not by hydroxylamine. These experiments suggested that a cysteine residue on the target protein had been mono-ADP-ribosylated. This supposition was further supported by identification of the mercf1p4ion-released radiolabelled product as ADP-ribose by HPLC, and the observation that free ADP-ribose was unable to modify the membrane target protein directly.
Protochlorophyllide reductase in photosynthetic prokaryotes and its role in chlorophyll synthesis
Authors: Rowe J D , Griffiths W T
Published: August 2015

1. DNA sequences hybridizing with the wheat protochlorophyllide reductase gene have been detected during genomic Southern blots of various cyanobacterial DNA samples. No such hybridization was observed with DNA from photosynthetic bacteria. 2. A fragment amplified from Phormidium laminosum DNA has been characterized and shown to be 73% similar to the corresponding wheat sequence. At the protein level the similarity is 91%. When used as a probe for Southern blotting the Phormidium DNA fragment confirmed the authenticity of some of the original signals obtained with the wheat probe. 3. Peptides of molecular mass 36, 30 and 60 kDa are immunodetected by a wheat reductase antibody during Western blotting of Phormidium preparations. These are purported to correspond to the cyanobacterial mature reductase protein, a stable proteolytic fragment and soluble dimeric forms of the latter respectively. 4. Adaptation of Phormidium to growth in red light (delta > 670 nm) or darkness led to no significant changes in the total level of immunodetected peptides or protochlorophyllide within the cells. 5. The specific activity of the reductase in Phormidium membranes has been tentatively estimated as 0.5 unit/mg of protein, a value comparable with that found in preparations from mature chloroplasts of higher plants.
Differential modulation of expression of the two acylphosphatase isoenzymes by thyroid hormone
Authors: Chiarugi P , Raugei G , Marzocchini R , Fiaschi T , Ciccarelli C , Berti A , Ramponi G
Published: August 2015

The modulation of expression of the skeletal muscle and erythrocyte acylphosphatase isoenzymes by thyroid hormone has been investigated. Our results indicate a differential regulation of the two enzymic isoforms by tri-iodothyronine (T3) in K562 cells in culture: an increase in the specific mRNA during T3-stimulation is shown only for the skeletal muscle isoenzyme. A fast and transient T3 induction of the accumulation of the specific mRNA can be observed, reaching a maximum 8 h after hormone treatment and then rapidly decreasing almost to the steady-state level after 24 h. A nuclear run-on assay was performed to explore the mechanisms of this regulation. These studies indicate that T3 induction of skeletal muscle acylphosphatase mRNA is due, at least in part, to a fast and transient increase in the rate of gene transcription, within 4 h after hormone administration. A very rapid decrease is then observed within a further 2 h. T3-dependent accumulation of the mRNA for the skeletal muscle acylphosphatase requires ongoing protein synthesis, as confirmed by inhibition with cycloheximide or puromycin. These findings indicate that the transcriptional regulation of the gene may be indirect.
5-hydroxytryptamine stimulates glucose transport in cardiomyocytes via a monoamine oxidase-dependent reaction
Authors: Fischer Y , Thomas J , Kamp J , Jüngling E , Rose H , Carpéné C , Kammermeier H
Published: August 2015

This study deals with the effect of 5-hydroxytryptamine (5-HT; serotonin) on glucose transport in isolated rat cardiac myocytes. In these cells, 5-HT (10-300 microns), as well as tryptamine, 5-methoxytryptamine and dopamine, elicited a 3-5 fold increase in glucose transport, as compared with control. This effect was maximal after 90 min, and was concomitant with a 1.8- and 1.5-fold increase in the amounts of glucose transporters GLUT1 and GLUT4 at the cell surface of the cardiomyocytes, as determined by using the photoaffinity label 3H-2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis-(D-manno s-4-yl) propyl-2-amine (3H-ATB-BMPA). In contrast, 3-3000 microM of the selective 5-HT receptor agonists 5-carboxyamido-tryptamine, alpha-methyl-serotonin, 2-methyl-serotonin or renzapride failed to stimulate glucose transport. The effect of 5-HT was not affected by (i) the 5-HT receptor antagonists methysergide (1 microM), ketanserin (1 microM), cyproheptadine (1 microM), MDL 72222 (1 microM) or ICS 205-930 (3 microM), nor by (ii) the adrenergic receptor antagonists prazosin (1 microM), yohimbine (1 microM) or propranolol (5 microM), nor by (iii) the dopaminergic antagonists SCH 23390 (1 microM) or haloperidol (1 microM). The monoamine oxidase inhibitors clorgyline (1 microM) and tranylcypromine (1 microM) completely suppressed the effect of 5-HT, whereas the control and insulin-stimulated rates of glucose transport were unaffected. Addition of catalase or glutathione diminished the 5-HT-dependent stimulation of glucose transport by 50%; these two factors are known to favour the degradation of H2O2 (which can be formed during the deamination of amines by monoamine oxidases). Glutathione also depressed the stimulatory action of exogenously added H2O2 (20 microM) by 30%. Furthermore, in cells treated with 5_HT, a time-dependent accumulation of 5-hydroxy-1H-indol-3-ylacetic acid (a product of 5-HT metabolism via monoamine oxidases) was observed, which paralleled the changes in glucose transport. In conclusion, the stimulation of glucose transport by 5-HT in cardiomyocytes is not mediated by a 5-HT1, 5-HT2, 5-HT3 or 5-HT4 receptor, nor by an adrenergic or dopaminergic receptor, but is likely to occur through the degradation of by a monoamine oxidase and concomitant formation of H2O2.
Isolated rat hepatocytes acquire iron from lactoferrin by endocytosis
Authors: McAbee D D
Published: August 2015

The iron-binding protein lactoferrin (Lf) present in blood is metabolized by the liver. Isolated rat hepatocytes vigorously endocytose bovine Lf via recycling Ca2(+)-dependent binding sites, but the uptake of iron from Lf by hepatocytes has not been examined. In this study, isolated rat hepatocytes were incubated with radiolabelled bovine Lf (125I-Lf, 59Fe-Lf or 125I-59Fe-Lf) at 37 degrees C, then washed at 4 degrees C in the presence of dextran sulphate with either Ca2+ or EGTA to distinguish between total bound and internal radioactivity respectively. Cells internalized 125I-Lf protein and Lf-bound 59Fe at maximal endocytic rates of 1700 and 480 mol.cell-1.s-1 respectively. When Lf was normalized for 59Fe content, these endocytic rates were equivalent and reflected an uptake potential of at least 3400 mol of iron.cell-1.s-1. Cells prebound with 125I-59Fe-Lf to Ca2+(-)dependent sites at 4 degrees C internalized more than 80% of both 125I-Lf protein and Lf-bound 59Fe approx. 6 min after warming to 37 degrees C at similar rates (125I-Lf: k(in) = 0.276 min-1, 59Fe: k(in) = 0.303 min-1). Within 4 h at 37 degrees C, cells had released 25% or less internalized Lf protein in the form of acid-soluble 125I-by-products but retained all the Lf-delivered 59Fe. Hyperosmotic disruption of clathrin-dependent endocytosis blocked the uptake of 125I-Lf and Lf-bound 59Fe. Incubation of cells with 125I-59Fe-Lf and a 100 molar excess of diferric transferrin reduced slightly the endocytosis of 125I-Lf protein and 59Fe accumulation. Treatment of cells with the ferric chelator desferrioxamine did not alter uptake of 125I-Lf protein or Lf-bound 59Fe, but the ferrous chelator bathophenanthroline disulphonate slightly elevated endocytosis of 125I-Lf protein and Lf-bound 59Fe. These findings indicate that Lf does not release its bound iron before endocytosis. It was concluded from this study that hepatocytes take up iron from Lf at high rates by a process that requires endocytosis of Lf-iron complexes.
Human lymphocyte activation is associated with the early and high-level expression of the endogenous lectin CSL at the cell surface
Authors: Zanetta J P , Wantyghem J , Kuchler-Bopp S , Badache A , Aubery M
Published: August 2015

Lymphocytes undergo activation in response to antigens, cytokines, lectins and antibodies interacting with specific cell-surface molecules or through substances influencing signal transduction pathways. This study shows that human T- and B-cells stimulated using phorbol esters or plant lectins express early (2 h using phorbol esters and 24 h using plant lectins) a high level of a polyvalent carbohydrate-binding protein, the cerebellar soluble lectin (CSL), which is in part externalized. The lectin, immunologically related to CDw70, interacts with specific glycoprotein ligands of the lymphocyte surface, including CD3 on T-cells and CD24 on B-cells. Major changes in phosphorylations associated with activation appear as largely CSL-dependent since they are specifically inhibited by anti-CSL Fab fragments. It is suggested that the lectin induces the clustering of specific cell-surface glycoproteins and plays the role of an endogenous amplifier of activation signals.
Polyenoic very-long-chain fatty acids mobilize intracellular calcium from a thapsigargin-insensitive pool in human neutrophils. The relationship between Ca2+ mobilization and superoxide production induced by long- and very-long-chain fatty acids
Authors: Hardy S J , Robinson B S , Ferrante A , Hii C S T , Johnson D W , Poulos A , Murray A W
Published: August 2015

Fatty acids with more than 22 carbon atoms (very-long-chain fatty acids; VLCFAs) are normal cellular components that have been implicated in the pathophysiology of a number of peroxisomal disorders. To date, however, essentially nothing is known regarding their biological activities. Ca2+ mobilization is an important intracellular signalling system for a variety of agonists and cell types. Given that several polyunsaturated long-chain fatty acids mobilize intracellular Ca2+ and that we have postulated that the VLCFAs may be involved in signal transduction, we examined whether the tetraenoic VLCFA induced Ca2+ mobilization in human neutrophils. We report that fatty acid-induced intracellular Ca2+ mobilization declined for fatty acid species of more than 20 carbon atoms, but increased again as the carbon chain length approached 30. This Ca2+ mobilization occurred independently of inositol 1,4,5-triphosphate production and protein kinase C translocation and involved both the release of Ca2+ from the intracellular stores and changes to the influx or efflux of the ion. We further observed that triacontatetraenoic acid [30:4(n-6)] mobilized Ca2+ from a thapsigargin-insensitive intracellular pool distinct from the thapsigargin-sensitive pools affected by arachidonic acid [20:4(n - 6)] or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). 20:4 (n - 6) induced strong superoxide production (chemiluminescence) which was inhibited by thapsigargin pretreatment. In contrast, fatty acid-induced superoxide production progressively declined as the carbon chain length increased beyond 20-22 carbon atoms. Further studies suggested that the thapsigargin-insensitive Ca2+ mobilization elicited by 30:4 (n - 6) was not related to oxyradical formation, while the thapsigargin-sensitive Ca2+ mobilization induced by 20:4 (n - 6) may be involved in the initiation but not necessarily the maintenance of superoxide production. In conclusion, this is the first report to demonstrate a biological activity for the VLCFA and indicates that 30:4 (n - 6) influences second messenger systems in intact cells that differ from those affected by long-chain fatty acids such as 20:4 (n - 6).
Induction of apoptosis by excessive polyamine accumulation in ornithine decarboxylase-overproducing L1210 cells
Authors: Poulin R , Pelletier G , Pegg A E
Published: August 2015

Deregulation of polyamine transport in L1210 cells overexpressing ornithine decarboxylase leads to a lethal accumulation of spermidine. We now provide evidence that over-accumulation of natural and synthetic polyamines, but not putrescine, rapidly induces apoptosis, as shown by hypercondensation of peripheral chromatin and internucleosomal cleavage, followed by nuclear fragmentation. Polyamine oxidation is not responsible for the apoptosis observed. Thus, abnormally high polyamine pools could be an important physiological trigger of apoptosis.
Substitutions of aspartic acid for glycine-220 and of arginine for glycine-664 in the triple helix of the pro α1(I) chain of type I procollagen produce lethal osteogenesis imperfecta and disrupt the ability of collagen fibrils to incorporate crystalline hydroxyapatite
Authors: Culbert A A , Lowe M P , Atkinson M , Byers P H , Wallis G A , Kadler K E
Published: August 2015

We identified two infants with lethal (type II) osteogenesis imperfecta (OI) who were heterozygous for mutations in the COL1A1 gene that resulted in substitutions of aspartic acid for glycine at position 220 and arginine for glycine at position 664 in the product of one COL1A1 allele in each individual. In normal age- and site-matched bone, approximately 70% (by number) of the collagen fibrils were encrusted with plate-like crystallites of hydroxyapatite. In contrast, approximately 5% (by number) of the collagen fibrils in the probands' bone contained crystallites. In contrast with normal bone, the c-axes of hydroxyapatite crystallites were sometimes poorly aligned with the long axis of fibrils obtained from OI bone. Chemical analysis showed that the OI samples contained normal amounts of calcium. The probands' bone samples contained type I collagen, overmodified type I collagen and elevated levels of type III and V collagens. On the basis of biochemical and morphological data, the fibrils in the OI samples were co-polymers of normal and mutant collagen. The results are consistent with a model of fibril mineralization in which the presence of abnormal type I collagen prevents normal collagen in the same fibril from incorporating hydroxyapatite crystallites.
A model for the initial phase of cell/surface interactions based on ligand binding phenomena
Authors: Hubble J , Eisenthal R , Whish W J D
Published: August 2015

The interaction between mammalian cells and solid surfaces plays an important role in a number of biological phenomena. Of particular clinical importance is the migration of cells suspended in blood to the wall of a blood vessel in the event of tissue damage. While the resultant inflammation often represents a desirable response to an external challenge, responses of this type can also lead to adverse consequences. Although the cell migration phenomenon is well known, a plausible mechanism for controlling the critical ‘rolling’ stages of adhesion has yet to be proposed. In this report we suggest how a simple consideration of ligand/receptor binding interactions can be used to explain a switch between a situation where a cell population is almost entirely in free suspension, to one where a significant fraction is attached to the solid surface.
Molecular evidence for the direct involvement of a protein kinase C in developmental and behavioural susceptibility to tumour-promoting phorbol esters in Caenorhabditis elegans
Authors: Tabuse Y , Sano T , Nishiwaki K , Miwa J
Published: August 2015

The nematode Caenorhabditis elegans displays developmental and behavioural sensitivity to tumour-promoting phorbol esters. This sensitivity involves the gene tpa-1, which encodes two protein kinase C isoforms, TPA-1A and TPA-1B. Here we report the molecular nature of the sensitivity in this animal. Characterization of transposon Tc1-induced phorbol ester-resistant mutants has revealed that Tc1 was inserted in a region encoding the kinase domain, resulting in the loss of tpa-1 products. Introduction of a genomic DNA containing the entire wild-type tpa-1 locus into a Tc1-inserted mutant restored the sensitivity to tumour promoters, and tpa-1 products were also produced. These results suggest that the function of wild-type TPA-1 is necessary and sufficient for tumour promoters to cause developmental and behavioural sensitivity in C. elegans.
Translocon-associated protein TRAP δ and a novel TRAP-like protein are coordinately expressed with pro-opiomelanocortin in Xenopus intermediate pituitary
Authors: Holthuis J C M , van Riel M C H M , Martens G J M
Published: August 2015

In the intermediate pituitary gland of Xenopus laevis, the expression levels of the prohormone pro-opiomelanocortin (POMC) can be readily manipulated. When the animal is placed on a black background, the gene for POMC is actively transcribed, whereas on a white background the gene is virtually inactive. In this study, we characterized two genes whose transcript levels in the intermediate pituitary are regulated in coordination with that for POMC. One of these codes for a protein homologous to translocon-associated protein TRAP delta, a subunit of a transmembrane protein complex located at the site where nascent secretory proteins enter the endoplasmic reticulum (ER). Both Xenopus and mice were found to express an alternatively spliced transcript that gives rise to a previously unknown version of the TRAP delta protein. The product of the second gene is a novel and highly conserved protein with structural similarity to glycoprotein gp25L, a constituent of another translocon-associated protein complex. A database search revealed the existence of a novel family of gp25L-related proteins whose members occur throughout the animal kingdom. Together, our data imply that (i) the group of ER proteins surrounding translocating polypeptide chains may be far more complex than previously expected, and (ii) a number of the accessory components of the translocon participate in early steps of prohormone biosynthesis.
Modulation of CD4 lateral mobility in intact cells by an intracellularly applied antibody
Authors: Grebenkämper K , Tosi P F , Lazarte J E , Sneed L , Brüggemann U , Kubitscheck U , Nicolau C , Peters R
Published: August 2015

This study shows that the lateral mobility of CD4, an important plasma-membrane immune receptor, can be modulated by intracellular application of an anti-CD4 antibody. For this purpose, (i) full-length CD4 and a truncated CD4 mutant, lacking a 32-residue-long C-terminal intracellularly exposed domain, were expressed in Spodoptera frugiperda (Sf9) insect cells, (ii) a monoclonal antibody, C6, with specificity for the C-terminal domain was generated, and (iii) a versatile apparatus for fluorescence microphotolysis (FM) studies was constructed. By these means it was found that the commercial anti-CD4 antibody Leu3a-PE, in contrast with several other anti-CD4 antibodies, could be used as a fluorescent label of CD4 without interfering greatly with CD4 mobility. Labelled by Leu3a-PE, full-length CD4 had a lateral diffusion coefficient of D = (4.7 +/- 1.9) x 10(-10) cm2/s and a mobile fraction of fm = 80 +/- 16% (room temperature). Within experimental accuracy the truncated CD4 had the same mobility as full-length CD4. Introduction of the C6 antibody into Sf9 cells by microinjection or by fusion with C6-loaded liposomes decreased the mobility of full-length CD4 (fm = 40%) but not of truncated CD4 (fm = 80%). Treatment of Sf9 cells with phorbol ester also reduced the mobility of full-length CD4 (fm = 50%) but not truncated CD4 (fm = 90%). A calmodulin inhibitor but not a protein kinase C (PKC) inhibitor abolished the phorbol ester effect.