bluuebunny/crossref_metadata_2025 huggingface dataset:
A new alternative transcript encodes a 60 kDa truncated form of integrin β3
Authors: Djaffar I , Chen Y P , Creminon C , Maclouf J , Cieutat A M , Gayet O , Rosa J P
Published: August 2015

A cDNA for integrin beta 3 isolated from a human erythroleukaemia (HEL) cell library contained a 340 bp insert at position 1281. This mRNA, termed beta 3c, results from the use of a cryptic AG donor splice site in intron 8 of the beta 3 gene, and is different from a previously described alternative beta 3 mRNA. The predicted open reading frame of beta 3C stops at a TAG stop codon 69 bp downstream from position 1281. It starts with the signal peptide and the 404 N-terminal extracellular residues of beta 3, encompassing the ligand binding sites, followed by 23 C-terminal intron-derived residues, corresponding to a truncated form of beta 3 lacking the cysteine-rich, transmembrane and cytoplasmic domains. Expression of beta 3C mRNA was demonstrated in human platelets, megakaryocytes, endothelial cells and HEL cells by reverse transcriptase/PCR. The beta 3C transcript was also demonstrated in the mouse, suggesting its conservation through evolution. Finally, a 60 kDa polypeptide corresponding to the beta 3C alternative transcript was demonstrated in platelets by Western blotting using a polyclonal antibody raised against a synthetic peptide designed from the beta 3C intronic sequence. Taken together, these results suggest a biological role for beta 3C, the first alternative transcript showing an altered extracellular domain of a beta integrin.
Gene expression of GLUT3 glucose transporter regulated by glucose in vivo in mouse brain and in vitro in neuronal cell cultures from rat embryos
Authors: Nagamatsu S , Sawa H , Inoue N , Nakamichi Y , Takeshima H , Hoshino T
Published: August 2015

This study was designed to determine whether glucose regulates the gene expression of glucose transporter GLUT3 in neurons. We examined the regulation of GLUT3 mRNA by glucose in vivo in mouse brain and in vitro by using neuronal cultures from rat embryos. Hypoglycaemia (< 30 mg/dl), produced by 72 h of starvation, increased GLUT3 mRNA in mouse brain by 2-fold. Hybridization studies in situ demonstrated that hypoglycaemia-induced increases in GLUT3 mRNA expression were observed selectively in brain regions including the hippocampus, dentate gyrus, cerebral cortex and piriform cortex, but not the cerebellum. Primary neuronal cultures from rat embryos deprived of glucose for 48 h also showed an increase (4-fold over control) in GLUT3 mRNA, indicating that glucose can directly regulate expression of GLUT3 mRNA. In contrast with hypoglycaemia, hyperglycaemia produced by streptozotocin did not alter the expression of GLUT3 mRNA. We also confirmed previous findings that hypoglycaemia increases GLUT1 mRNA expression in brain. The increase in GLUT1 expression was probably limited to the blood-brain barrier in vivo, since GLUT1 mRNA could not be detected in neurons of the mouse cerebrum. Thus we conclude that up-regulation of neuronal GLUT3 in response to glucose starvation represents a protective mechanism against energy depletion in neurons.
Actions of the creatine analogue β-guanidinopropionic acid on rat heart mitochondria
Authors: Clark J F , Khuchua Z , Kuznetsov A V , Vassil'eva E , Boehm E , Radda G K , Saks V
Published: August 2015

The action of the creatine analogue beta-guanidinopropionic acid (beta-GPA) was examined in rat heart mitochondria and in isolated cardiomyocytes or fibres which were permeabilized with the non-ionic detergent saponin to determine the kinetics of mitochondrial creatine kinase for beta-GPA. Fibres and myocytes were subjected to increasing [ADP] in the presence and absence of beta-GPA or creatine, whereas isolated mitochondria received a similar protocol with increasing [ATP]. In isolated mitochondria given ATP, there was a stimulation of respiration by creatine, but no significant stimulation of respiration by beta-GPA. Further studies on fibres from control and beta-GPA-fed rats also found that beta-GPA is not utilized by the mitochondria, as evidenced by a lack of beta-GPA-stimulated respiration (Km for ADP = 142 +/- 23 microM) compared with control (Km for ADP from 161 +/- 23 microM), but no significant change in Vmax. Therefore the rat heart mitochondria are not responsive to beta-GPA as compared with creatine. Interestingly, the fibres from beta-GPA-fed rats had no creatine- or beta-GPA-stimulated respiration (Km for ADP = 57.3 +/- 7.2 microM for control, 54.2 +/- 7.2 microM with creatine, and 53.5 +/- 7.8 microM with beta-GPA). The mitochondria prepared from the hearts of rats exposed for 10 weeks to 1% beta-GPA in their diet had a significant decrease in Vmax. and a significant decrease in Km for ADP. Thus the hearts from beta-GPA-fed animals may be pathologic, due to a disruption of the creatine kinase energy circuit.
Glucose oxidation and low-density lipoprotein-induced macrophage ceroid accumulation: possible implications for diabetic atherosclerosis
Authors: Hunt J V , Bottoms M A , Clare K , Skamarauskas J T , Mitchinson M J
Published: August 2015

The exposure of proteins to high concentrations of glucose in vitro is widely considered a relevant model of the functional degeneration of tissue occurring in diabetes mellitus. In particular, the enhanced atherosclerosis in diabetes is often discussed in terms of glycation of low-density lipoprotein (LDL), the non-enzymic attachment of glucose to apolipoprotein amino groups. However, glucose can undergo transition-metal-catalysed oxidation under near-physiological conditions in vitro, producing oxidants that possess a reactivity similar to the hydroxyl radical. These oxidants can fragment protein, hydroxylate benzoic acid and induce lipid peroxidation in human LDL. In this study, glycation of LDL in vitro is accompanied by such oxidative processes. However, the oxidation of LDL varies with glucose concentration in a manner which does not parallel changes in protein glycation. Glycation increases in proportion to glucose concentration, whereas in our studies maximal oxidation occurs at a glucose concentration of approx. 25 mM. The modification of LDL resulting from exposure to glucose alters macrophage ceroid accumulation, a process which occurs in the human atherosclerotic plaque. The accumulation of ceroid in macrophages is shown to be related to LDL oxidation rather than LDL glycation, per se, as it too occurs at a maximum of approx. 25 mM. Oxidative sequelae of protein glycation appear to be a major factor in LDL-macrophage interactions, at least with respect to ceroid accumulation. Our observations are discussed in the context of the observed increase in the severity of atherosclerosis in diabetes.
Human glutathione S-transferase theta (GSTT1): cDNA cloning and the characterization of a genetic polymorphism
Authors: Pemble S , Schroeder K R , Spencer S R , Meyer D J , Hallier E , Bolt H M , Ketterer B , Taylor J B
Published: August 2015

In humans, glutathione-dependent conjugation of halomethanes is polymorphic, with 60% of the population classed as conjugators and 40% as non-conjugators. We report the characterization of the genetic polymorphism causing the phenotypic difference. We have isolated a cDNA that encodes a human class Theta GST (GSTT1) and which shares 82% sequence identity with rat class Theta GST5-5. From PCR and Southern blot analyses, it is shown that the GSTT1 gene is absent from 38% of the population. The presence or absence of the GSTT1 gene is coincident with the conjugator (GSST1+) and non-conjugator (GSTT1-) phenotypes respectively. The GSTT1+ phenotype can catalyse the glutathione conjugation of dichloromethane, a metabolic pathway which has been shown to be mutagenic in Salmonella typhimurium mutagenicity tester strains and is believed to be responsible for carcinogenicity of dichloromethane in the mouse. In humans, the enzyme is found in the erythrocyte and this may act as a detoxification sink. Characterization of the GSTT1 polymorphism will thus enable a more accurate assessment of human health risk from synthetic halomethanes and other industrial chemicals.
The expression of functional link protein in a baculovirus system: analysis of mutants lacking the A, B and B' domains
Authors: Grover J , Roughley P J
Published: August 2015

Functional recombinant human link protein has been produced using a baculovirus expression system. In addition to the intact link protein, three mutant forms have also been expressed. Each mutant bears a deletion equivalent to the protein encoded by one exon in the gene. These deletions represent the A domain, which is thought to be responsible for interaction with aggrecan, and the B or B' domains, which are associated with the interaction with hyaluronate. Such deletions split codons spanning exon boundaries, but maintain the reading frame of the protein and result in the correct amino acid being present at the splice junction. All the recombinant proteins appear as two components upon SDS/PAGe, though the abundance of the two forms does vary between preparations, as a result of variable substitution by N-linked oligosaccharides. The recombinant intact link protein was able to interact with both hyaluronate and aggrecan, showing that the baculovirus system is able to produce functional molecules. All of the recombinant mutant link proteins were also able to interact with hyaluronate, indicating that both the B and B' domains can function independently. The recombinant mutant link proteins were also able to interact with aggrecan, with the exception of the mutant lacking the A domain, confirming that this ability resides entirely within this domain.
Taurolithocholate and taurolithocholate 3-sulphate exert different effects on cytosolic free Ca2+ concentration in rat hepatocytes
Authors: Marrero I , Sanchez-Bueno A , Cobbold P H , Dixon C J
Published: August 2015

Single rat hepatocytes show repetitive oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) when stimulated by agonists acting through the phosphoinositide signalling pathway. We have studied the effect of a natural bile acid, taurolithocholate (TLC), and its sulphated form, taurolithocholate 3-sulphate (TLC-S), on [Ca2+]i in single isolated rat hepatocytes. Although these bile acids are believed to act through a common mechanism to permeabilize the intracellular Ca2+ pool, the [Ca2+]i responses induced by the two compounds were different. Whereas TLC induced a sustained elevation of [Ca2+]i, TLC-S evoked repetitive [Ca2+]i oscillations. In addition, we show that ryanodine, which blocks the Ca(2+)-induced Ca2+ release (‘CICR’) mechanism, blocked TLC-S-induced oscillations in 50% of hepatocytes, but did not affect the TLC-induced rise in [Ca2+]i.
Recombinant expression of the fdxD gene of Rhodobacter capsulatus and characterization of its product, a [2Fe-2S] ferredoxin
Authors: Armengaud J , Meyer C , Jouanneau Y
Published: August 2015

A gene called fdxD that could potentially code for a ferredoxin has recently been identified upstream of the nitrogenase structural genes in Rhodobacter capsulatus [Willison, Pierrard and Hübner (1993) Gene 133, 39-46]. In the present study, the fdxD gene product has been overproduced in Escherichia coli in a soluble form. The recombinant protein, pink in colour, was purified to homogeneity, and biochemically characterized as a new ferredoxin. It represents the fifth ferredoxin so far identified in R. capsulatus and was designated FdV. Its N-terminal sequence is identical with that of the native ferredoxin isolated from R. capsulatus. U.v-visible-absorption spectra as well as results of c.d. and e.p.r. spectroscopy demonstrated that the fdxD product contained a [2Fe-2S] cluster correctly assembled and incorporated into the polypeptide. Although similar to plant-type ferredoxins, FdV appeared poorly competent in the photo-reduction of NADP+. On the basis of in vitro assays, FdV cannot serve as an electron donor for nitrogenase. The lack of reactivity of FdV in either of these assays may primarily be due to its relatively high mid-point redox potential (E'o = -220 mV, pH 7.5).
A caffeine- and ryanodine-sensitive intracellular Ca2+ store can act as a Ca2+ source and a Ca2+ sink in PC12 cells
Authors: Barry V A , Cheek T R
Published: August 2015

We have investigated the modulation of stimulus-induced changes in intracellular Ca2+ concentration ([Ca2+]i) by a caffeine-and ryanodine-sensitive Ca2+ store in PC12 cells. In populations of fura-2-loaded cells, caffeine cause a concentration-dependent increase in [Ca2+]i that was saturable, reversible and inhibited in a use-dependent fashion by ryanodine. Maximal Ca2+ release occurred with 40 mM caffeine, with an EC50 of 13 mM caffeine and a Hill coefficient (h) of 2.7, indicating that the release mechanism was co-operative. Pretreatment of intact cell populations with increasing concentrations of caffeine in nominally Ca(2+)-free medium inhibited the subsequent Ca2+ response to a maximal concentration of ATP, in a dose-dependent manner. In permeabilized cells, a maximal concentration (40 microM) of InsP3 still released Ca2+ in the presence of a supramaximal concentration (50 mM) of caffeine, whereas caffeine was unable to release Ca2+ after the InsP3-sensitive store had been completely emptied. These data suggest that PC12 cells contain a uniquely InsP3-sensitive Ca2+ store, and a store that is sensitive to both InsP3 and caffeine. Depletion of the caffeine-sensitive Ca2+ store by caffeine and ryanodine pretreatment in intact cells attenuated the Ca2+ response to ATP, but not to 55 mM K+, suggesting that the caffeine-sensitive Ca2+ store acts as a Ca2+ source after ATP stimulation, but not after depolarization with 55 mM K+. Pretreatment of intact cells with ATP and ryanodine resulted in a use-dependent block of both caffeine- and ATP-mediated Ca2+ release, confirming that ATP stimulation of PC12 cells brings about activation of ryanodine receptors. The rate of recovery, but not the magnitude or rate of onset, of the depolarization-induced [Ca2+]i transient was modulated by the state of filling of the caffeine-sensitive Ca2+ store such that recovery was prolonged if the store was either full, or empty and unable to refill. We conclude that the caffeine- and ryanodine-sensitive Ca2+ store can act as a Ca2+ source and a Ca2+ sink in PC12 cells, and that its role may in part be governed by the nature of the stimulating agent.
Agonist regulation of cellular Gs α-subunit levels in neuroblastoma × glioma hybrid NG108-15 cells transfected to express different levels of the human β 2 adrenoceptor
Authors: Adie E J , Milligan G
Published: August 2015

Neuroblastoma x glioma hybrid NG108-15 cells endogenously express at least three receptors which activate adenylate cyclase via the intermediacy of the stimulatory G-protein, Gs. Sustained exposure of the cells to agonists at the IP prostanoid receptor results in a substantial decrease in cellular levels of the alpha-subunit of Gs (Gs alpha) [McKenzie and Milligan (1990) J. Biol. Chem. 265, 17084-17093; Adie, Mullaney, McKenzie and Milligan (1992) Biochem J. 285, 529-536]. By contrast, equivalent treatments of the cells with agonists at either the A2 adenosine receptor or the secretin receptor have no measurable effect on cellular amounts of Gs alpha. To examine whether this is a feature specific to the IP prostanoid receptor or is related to the level of expression of the individual receptors, NG108-15 cells were transfected with a construct containing a human beta 2-adrenoceptor cDNA under the control of the beta-actin promoter. Two clones of these cells were examined in detail, beta N22, which expressed some 4000 fmol/mg of membrane protein, and clone beta N17, which expressed approx. 300 fmol/mg of membrane protein of the receptor. Exposure of beta N22 cells to the beta-adrenergic agonist isoprenaline resulted maximally in some 55% decrease in membrane-associated levels of Gs alpha, without effect on membrane levels of Gi2 alpha, Gi3 alpha, G(o) alpha or Gq alpha/G11 alpha. Dose-response curves to isoprenaline in beta N22 cells indicated that half-maximal down-regulation of Gs alpha was produced by approx. 1 nM agonist. Equivalent exposure of beta N17 cells to isoprenaline did not significantly modify levels of any of the G-protein alpha subunits, including Gs alpha. In beta N22 cells the IP prostanoid receptor was expressed at similar levels to those in wild-type NG108-15 cells, and treatment with iloprost resulted in a similar down-regulation of cellular Gs alpha levels. Iloprost was also effective in causing down-regulation of Gs alpha levels in clone beta N17. Concurrent addition of both isoprenaline and iloprost to clone beta N22 resulted in less than additive down-regulation of Gs alpha. These results demonstrate that the phenomenon of agonist-induced specific G-protein down-regulation is determined by the levels of expression of the receptor.
Expression of the Wilson disease gene is deficient in the Long-Evans Cinnamon rat
Authors: Yamaguchi Y , Heiny M E , Shimizu N , Aoki T , Gitlin J D
Published: August 2015

Long-Evans Cinnamon rats develop a necrotizing hepatitis characterized by excessive hepatic copper accumulation, defective holoceruloplasmin biosynthesis and impaired biliary copper excretion. To elucidate the molecular basis of this defect, a cDNA clone encoding the rat Wilson disease gene was isolated and used to examine gene expression in selected tissues from normal and Long-Evans Cinnamon rats. Although this cDNA readily detects Wilson transcripts in liver and other tissues from normal rats, such transcripts are entirely absent from tissues derived from the Long-Evans Cinnamon rat strain. These data therefore identify the Long-Evans Cinnamon rat as the first bona fide animal model of Wilson disease and suggest that this rat strain may be a valuable resource in the study of this genetic disorder.
One- and two-electron reduction of 2-methyl-1,4-naphthoquinone bioreductive alkylating agents: kinetic studies, free-radical production, thiol oxidation and DNA-strand-break formation
Authors: Giulivi C , Cadenas E
Published: August 2015

The one- and two-electron enzymic reduction of the bioreductive alkylating agents 2-methylmethoxynaphthoquinone (quinone I) and 2-chloromethylnaphthoquinone (quinone II) was studied with purified NADPH-cytochrome P-450 reductase and DT-diaphorase respectively, and characterized in terms of kinetic constants, oxyradical production, thiol oxidation and DNA-strand-break formation. The catalytic-centre activity values indicated that DT-diaphorase catalysed the reduction of quinone I far more efficiently than NADPH-cytochrome P-450 reductase, although the Km values of the two enzymes for this quinone were similar (1.2-3.0 microM). The one-electron-transfer flavoenzyme also catalysed the reduction of quinone II, but the behaviour of DT-diaphorase towards this quinone did not permit calculation of kinetic constants. A salient feature of the redox transitions caused by the one- and two-electron catalysis of these quinones was the different contributions of disproportionation and autoxidation reactions respectively. In the former case, about 26% of NADPH consumed was accounted for in terms of autoxidation (as H2O2 formation), whereas in the latter, the autoxidation component accounted for most (98%) of the NADPH consumed. This difference was abrogated by superoxide dismutase, which enhanced autoxidation during NADPH-cytochrome P-450 catalysis to a maximal value. E.s.r. analysis indicated the formation of superoxide radicals, the signal of which was suppressed by superoxide dismutase and unaffected by catalase. The one- and two-electron reduction of these quinones in the presence of GSH was accompanied by formation of thiyl radicals. Although superoxide dismutase suppressed the thiol radical e.s.r. signal in both instances, the enzyme enhanced GSSG accumulation during NADPH-cytochrome P-450 catalysis of quinone I, whereas it inhibited GSSG formation during reduction of the quinone by DT-diaphorase. One- and two-electron reduction of quinone I led to calf thymus DNA-strand-break formation, a process that (a) was substantially decreased in experiments performed with dialysed DNA and in the presence of desferal and (b) was partially sensitive to superoxide dismutase and/or catalase. These findings are rationalized in terms of the occurrence of metal ions ligated to DNA, protecting against the toxic effects of superoxide radicals generated during enzymic reduction of quinones.
Natural substances (acetogenins) from the family Annonaceae are powerful inhibitors of mitochondrial NADH dehydrogenase (Complex I)
Authors: Degli Esposti M , Ghelli A , Ratta M , Cortes D , Estornell E
Published: August 2015

Natural products from the plants of the family Annonaceae, collectively called Annonaceous acetogenins, are very potent inhibitors of the NADH-ubiquinone reductase (Complex I) activity of mammalian mitochondria. The properties of five of such acetogenins are compared with those of rotenone and piericidin, classical potent inhibitors of Complex I. Rolliniastatin-1 and rolliniastatin-2 are more powerful than piericidin in terms of both their inhibitory constant and the protein-dependence of their titre in bovine submitochondrial particles. These acetogenins could be considered therefore the most potent inhibitors of mammalian Complex I. Squamocin and otivarin also have an inhibitory constant lower than that of piericidin, but display a larger protein-dependence of the titre. Squamocin and otivarin, contrary to the other acetogenins, behave qualitatively like rotenone. Rolliniastatin-2 shows unique properties as its interaction, although mutually exclusive to that of piericidin, appears to be mutually non-exclusive to that of rotenone. It is the first time that a potent inhibitor of Complex I is found not to overlap the active site of rotenone.
Analysis of the optical absorption and magnetic-circular-dichroism spectra of peanut peroxidase: electronic structure of a peroxidase with biochemical properties similar to those of horseradish peroxidase
Authors: Rodríguez Marañón M J , Mercier D , van Huystee R B , Stillman M J
Published: August 2015

The electronic structures of the cationic isoenzyme of peanut peroxidase, horseradish peroxidase (isoenzyme C) and bovine liver catalase are compared through analysis of their optical absorption and magnetic c.d. (m.c.d.) spectral properties. The spectral data for the native resting states and compounds I and II of peanut peroxidase (PeP) are reported. The absorption and m.c.d. data for the native PeP exhibit bands characteristic of the high-spin ferric haem. The absorption spectrum of PeP compound I closely resembles that observed for the HRP compound I species. The m.c.d. data for PeP I clearly identifies that ring oxidation has occurred. One-electron reduction forms the PeP compound II species. The absorption and m.c.d. spectra recorded for PeP II exhibit the well-resolved spectral characteristics previously observed for both HRP compound II and catalase compound II. The spectral data of PeP with HRP and catalase are compared. The data clearly indicate that the m.c.d. spectral patterns of both plant peroxidases (PeP and HRP) are very similar and, therefore, the electronic structures of their resting states, and as well their primary and secondary compounds, must be similar. The m.c.d. data suggest that, while the compound I species of PeP and HRP belong to one electronic class, catalase compound I belongs to a different class. These data emphasize how the ground states of these two classes of oxidized haem, may be characterized as predominantly 2A2u (PeP I and HRP I) or 2A1u (catalase I). Peanut peroxidase is the second plant peroxidase for which the electronic structure of the compound I intermediate has been studied using the m.c.d. technique. The similarities with horseradish peroxidase allow us to suggest that plant peroxidases may operate by the same general mechanism, in spite of the low degree of sequence similarity between their polypeptide chains.
In vivo inhibition of β-glucosidase and β-mannosidase activity in rats by 2-deoxy-2-fluoro-β-glycosyl fluorides and recovery of activity in vivo and in vitro
Authors: McCarter J D , Adam M J , Hartman N G , Withers S G
Published: August 2015

2-Deoxy-2-fluoro-beta-glucosyl and -beta-mannosyl fluorides administered to rats in a single dose (10 mg/kg) inhibited beta-glucosidase and beta-mannosidase activity respectively after 1 h in brain, spleen, liver and kidney tissues. This inhibition, presumably caused by accumulation of 2-deoxy-2-fluoroglycosyl-enzyme intermediates, indicates that intact 2-deoxy-2-fluoroglycosyl fluorides are distributed to these organs and, in the case of brain, that they cross the blood/brain barrier. beta-Glucosidase activity recovered completely or partially in brain, spleen, liver and kidney by 20-48 h. beta-Mannosidase activity partially recovered in all tissues by 48 h. beta-Galactosidase activity in brain and kidney was not significantly affected by administration of either the gluco or manno compounds at this dosage, indicating that these inhibitors are directed towards specific glycosidases. Observation of similar relatively rapid rates of beta-glycosidase re-activation in vivo and in tissue homogenates in vitro at 37 degrees C suggests that hydrolysis or transglycosylation of 2-deoxy-2-fluoroglycosyl-enzymes, not protein synthesis, are the primary mechanisms involved in the recovery of glycosidase activity inhibited by this class of compounds in vivo.
Kinetic mechanism of adenosine 5′-phosphosulphate kinase from rat chondrosarcoma
Authors: Lyle S , Geller D H , Ng K , Stanczak J , Westley J , Schwartz N B
Published: August 2015

Biosynthesis of the activated sulphate donor adenosine 3′-phosphate 5′-phosphosulphate (PAPS) involves the sequential action of two enzyme activities. ATP-sulphurylase catalyses the formation of APS (adenosine 5′-phosphosulphate) from ATP and free sulphate, and APS is then phosphorylated by APS kinase to produce PAPS. Initial-velocity patterns for rat chondrosarcoma APS kinase indicate a single-displacement formal mechanism with KmAPS 76 nM and KmATP = 24 microM. Inhibition studies using analogues of substrates and products were carried out to determine the reaction mechanism. An analogue of PAPS, adenosine 3′-phosphate 5′-[beta-methylene]phosphosulphate, exhibited competitive inhibition with APS and non-competitive inhibition with ATP. An analogue of APS, adenosine 5′-[beta-methylene]phosphosulphate was also competitive with APS and non-competitive with ATP. Adenosine 5′-[beta gamma-imido]triphosphate showed competitive inhibition with respect to ATP and produced mixed-type inhibition, with a pronounced intercept effect and a small slope effect, with respect to APS. These results are in accord with the formulation of the predominant pathway as a steady-state ordered mechanism with APS as the leading substrate and PAPS as the final product released.
Maitotoxin activates cation channels distinct from the receptor-activated non-selective cation channels of HL-60 cells
Authors: Musgrave I F , Seifert R , Schultz G
Published: August 2015

We investigated whether maitotoxin activates non-selective cation channels, as was recently proposed [Soergel, Yasumoto, Daly and Gusovsky (1992) Mol. Pharmacol. 41, 487-493]. Stimulation of dibutyryl cyclic AMP-differentiated HL-60 cells with the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 0.1 microM), the Ca(2+)-ATPase inhibitor thapsigargin (0.1 microM) or maitotoxin (25 ng/ml) resulted in an increase in cytoplasmic free calcium concentration ([Ca2+]i). Unlike fMLP and thapsigargin, maitotoxin produced no increase in [Ca2+]i in the absence of extracellular Ca2+. The increase in [Ca2+]i induced by fMLP was blocked by pretreatment with pertussis toxin (100 ng/ml for 24 h) but not that induced by maitotoxin. Similarly, the increase in [Ca2+]i produced by fMLP but not that produced by maitotoxin was inhibited by pretreatment with phorbol myristate acetate (100 ng/ml). Both fMLP- and maitotoxin-induced increases in [Ca2+]i were blocked by 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenylethyl)-1H-imid azole hydrochloride (SKF 96365) in a concentration-dependent manner. However, the maitotoxin-induced increase in [Ca2+]i was more sensitive to inhibition by SKF 96365 than the fMLP-induced increase. fMLP-induced increases in [Ca2+]i were blocked by cations with Gd3+ being more effective than Cd2+, whereas for maitotoxin Cd2+ was more effective than Gd3+. Both fMLP and thapsigargin stimulated quenching of Fura-2 fluorescence in the presence of extracellular Mn2+, whereas maitotoxin produced no Mn2+ quenching. Taken together these results suggest that maitotoxin does not stimulate the nonselective cation channel activated by fMLP, but instead activates Ca2+ influx by a different mechanism.
Isolation of cDNAs from Brassica napus encoding the biotin-binding and transcarboxylase domains of acetyl-CoA carboxylase: assignment of the domain structure in a full-length Arabidopsis thaliana genomic clone
Authors: Elborough K M , Swinhoe R , Winz R , Kroon J T , Farnsworth L , Fawcett T , Martinez-Rivas J M , Slabas A R
Published: August 2015

One independent and two overlapping rape cDNA clones have been isolated from a rape embryo library. We have shown that they encode a 2.3 kb and a 2.5 kb stretch of the full-length acetyl-CoA carboxylase (ACCase) cDNA, corresponding to the biotin-binding and transcarboxylase domains respectively. Using the cDNA in Northern-blot analysis we have shown that the mRNA for ACCase has a higher level of expression in rape seed than in rape leaf and has a full length of 7.5 kb. The level of expression during rape embryogenesis was compared with both oil deposition and expression of two fatty acid synthetase components enoyl-(acyl-carrier-protein) reductase and 3-oxoacyl-(acyl-carrier-protein) reductase. Levels of ACCase mRNA were shown to peak at 29 days after anthesis during embryonic development, similarly to enoyl-(acyl-carrier-protein) reductase and 3-oxoacyl-(acyl-carrier-protein) reductase mRNA. In addition, a full-length genomic clone (19 kb) of Arabidopsis ACCase has been isolated and partially sequenced. Analysis of the clone has allowed the first plant ACCase activity domains (biotin carboxylase-biotin binding-transcarboxylase) to be ordered and assigned. Southern-blot analysis using the Arabidopsis clone indicates that ACCase is a single-copy gene in Arabidopsis but is encoded by a small gene family in rape.
Characterization of a metalloprotease from ovine chromaffin granules which cleaves a proenkephalin fragment (BAM12P) at a single arginine residue
Authors: Tezapsidis N , Parish D C
Published: August 2015

A metalloprotease has been identified in ovine chromaffin granules which cleaves the proenkephalin fragment BAM12P to produce adrenorphin-Gly. This cleavage occurs at a single arginine residue and is an intermediate step in the production of the opiate adrenorphin in vivo. The identity of the product was confirmed by reverse-phase and ion-exchange chromatography. The adrenorphin-Gly-generating enzyme (AGE) was determined by chromatofocusing to have a pI value of 5.2 and bound strongly to a metal-chelate affinity column. After purification by gel-filtration and ion-exchange chromatography AGE was free of contaminating activities, as cleavage of radiolabelled BAM12P generated a single product as judged by reverse-phase and ion-exchange chromatography. The enzyme has a molecular mass of approx. 45 kDa and a pH optimum of 8.6 in Mops, Taps and Hepes buffers, but was inhibited by phosphate buffers. It was inhibited by micromolar concentrations of copper and zinc ions, but not by millimolar concentrations of calcium or manganese ions. The addition of BAM22P, dynorphin 1-13 or dynorphin 1-8 to the incubation mixture inhibited the cleavage of radiolabelled BAM12P. The cleavage was also inhibited by the presence of catecholamines at concentrations similar to those found within the chromaffin granule. This may explain the known effect of reserpine on chromaffin cells of reducing catecholamine levels and simultaneously increasing adrenorphin levels. It may also indicate a function for AGE and adrenorphin as reporters of intragranular conditions.
Multi-site phosphorylation of the inhibitory guanine nucleotide regulatory protein Gi-2 occurs in intact rat hepatocytes
Authors: Morris N J , Bushfield M , Lavan B E , Houslay M D
Published: August 2015

A phosphorylated form of alpha-Gi-2 (the alpha-subunit of Gi-2), immunoprecipitated from hepatocytes under basal conditions, migrated as a single species of pI approximately 5.7, the labelling of which increased approximately 2-fold in cells challenged with either vasopressin or phorbol 12-myristate 13-acetate (PMA); agents which activate protein kinase C. In contrast, treatment of hepatocytes with 8-bromo-cyclic AMP produced a more acidic species of phosphorylated alpha-Gi-2 having a pI of approximately 5.4 and whose labelling was increased approximately 3-fold. Trypsin digestion of labelled alpha-Gi-2 isolated from hepatocytes under basal conditions identified, on two-dimensional peptide analyses, three positively charged phosphoserine-containing peptides (C1, C2 and C3), with only peptides C1 and C2 being evident upon less extensive digestion with trypsin. These are suggested to reflect a single site of phosphorylation, with proteolysis by trypsin being incomplete, and where C2 is larger than C1, which is larger than C3. An identical pattern of tryptic phosphopeptides was seen in hepatocytes treated with either vasopressin or PMA, although labelling of this group of peptides was increased by approximately 2-fold compared with the basal state. In contrast, treatment of hepatocytes with glucagon, 8-bromo-cyclic AMP or forskolin not only resulted in increased labelling of the ‘basal’ sites approximately 3-fold, but identified a novel positively charged tryptic phosphoserine-containing peptide (AN). All four tryptic peptides were susceptible to proteolysis by V8 protease. Treatment of labelled alpha-Gi-2 from basal and PMA-treated cells produced a pattern of peptides which was identical with those found when the tryptic phosphopeptide was treated with V8 protease. We tentatively suggest that, on alpha-Gi-2, Ser144 is phosphorylated through the action of protein kinase C and Ser207 is phosphorylated upon elevation of the intracellular concentrations of cyclic AMP.
Cloning of cDNA encoding the bifunctional dehydroquinase.shikimate dehydrogenase of aromatic-amino-acid biosynthesis in Nicotiana tabacum
Authors: Bonner C A , Jensen R A
Published: August 2015

Nicotiana tabacum cDNA encoding a bifunctional protein having catalytic domains for dehydroquinase and shikimate dehydrogenase was cloned and sequenced. Complementation of Escherichia coli aroD and aroE auxotrophs was successful. Amino acid sequencing located the N-terminus of the mature protein. The two catalytic domains exhibited greater amino acid identity with prokaryote homologues than with yeast and fungal homologues.
A synthetic peptide deduced from the sequence in the cross-region of laminin A chain mediates neurite outgrowth, cell attachment and heparin binding
Authors: Tashiro K , Nagata I , Yamashita N , Okazaki K , Ogomori K , Tashiro N , Anai M
Published: August 2015

Synthetic peptides from the cross-region of the laminin A chain were prepared and tested for their biological activities, especially for neurite outgrowth. A synthetic 8-mer peptide (designated LMA-5) in the cross-region of the laminin A chain was found to promote neurite outgrowth in PC12 cells and cerebellar microexplant cultures. Furthermore, this peptide mediated cell attachment and heparin binding. In addition, an antibody against peptide LMA-5 inhibited laminin- and LMA-5-mediated cell attachment. This antibody also inhibited more than half of the LMA-5-promoted neurite outgrowth in cerebellar microexplant cultures. These data suggest that peptide LMA-5 is one of the active sites in laminin that regulate cell behaviour including neurite outgrowth, cell attachment and heparin binding.
Acetylation with succinimidyl acetate affects both the catalytic site and the regulation of the erythrocyte Ca2+ pump
Authors: Donnet C , Caride A J , Fernández H N , Rossi J P F C
Published: August 2015

Acetylation of lysine residues of the erythrocyte Ca2+ pump using succinimidyl acetate (SA) led to its complete inactivation. In the absence of any of the major activators of the pump (namely calmodulin and acidic phospholipids), ATP fully protected the pump from inactivation by SA, with a K0.5 of 13 microM. This value is very close to the Km of the high-affinity site for ATP, thus suggesting that the residue(s) involved is(are) near or at the catalytic site of the Ca(2+)-ATPase. Furthermore, the presence of 500 microM ATP prevented the acetylation of about two residues per molecule of enzyme. Acetylation by SA also prevented the activation of the Ca2+ pump by calmodulin, acidic phospholipids or controlled trypsin proteolysis. This effect of SA treatment was not avoided by the presence of ATP in the preincubation medium, indicating a second set of modified residues. The fact that the three modes of activation were cancelled in a similar fashion by SA suggests that, although acting via different mechanisms, they share at least a common step in which SA-sensitive lysine residues may participate. Moreover, modification of the pump by SA plus ATP decreased the KCa when the activity was measured in both the absence and presence of calmodulin, suggesting that the residue(s) modified in this case is(are) involved directly in the regulation of the affinity for Ca2+.
Purification of acetaldehyde dehydrogenase and alcohol dehydrogenases from Thermoanaerobacter ethanolicus 39E and characterization of the secondary-alcohol dehydrogenase (2° Adh) as a bifunctional alcohol dehydrogenase-acetyl-CoA reductive thioesterase
Authors: Burdette D , Zeikus J G
Published: August 2015

The purification and characterization of three enzymes involved in ethanol formation from acetyl-CoA in Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E) is described. The secondary-alcohol dehydrogenase (2 degrees Adh) was determined to be a homotetramer of 40 kDa subunits (SDS/PAGE) with a molecular mass of 160 kDa. The 2 degrees Adh had a lower catalytic efficiency for the oxidation of 1 degree alcohols, including ethanol, than for the oxidation of secondary (2 degrees) alcohols or the reduction of ketones or aldehydes. This enzyme possesses a significant acetyl-CoA reductive thioesterase activity as determined by NADPH oxidation, thiol formation and ethanol production. The primary-alcohol dehydrogenase (1 degree Adh) was determined to be a homotetramer of 41.5 kDa (SDS/PAGE) subunits with a molecular mass of 170 kDa. The 1 degree Adh used both NAD(H) and NADP(H) and displayed higher catalytic efficiencies for NADP(+)-dependent ethanol oxidation and NADH-dependent acetaldehyde (identical to ethanal) reduction than for NADPH-dependent acetaldehyde reduction or NAD(+)-dependent ethanol oxidation. The NAD(H)-linked acetaldehyde dehydrogenase was a homotetramer (360 kDa) of identical subunits (100 kDa) that readily catalysed thioester cleavage and condensation. The 1 degree Adh was expressed at 5-20% of the level of the 2 degrees Adh throughout the growth cycle on glucose. The results suggest that the 2 degrees Adh primarily functions in ethanol production from acetyl-CoA and acetaldehyde, whereas the 1 degree Adh functions in ethanol consumption for nicotinamide-cofactor recycling.
Purification of enzymes of the kallikrein gene family (rK8 and rK9) from the rat prostate
Authors: Schøyen H , Wassdal I , Toft K , Almendingen M , Berg T
Published: August 2015

The rat kallikrein family consists of multiple closely related proteins. A method for demonstration and identification of kallikrein-like proteins has been developed based on their differences in isoelectric point and their immunological similarity. The method, which involved separation in flat-bed isoelectro-focusing gels (pH range 3-9) and detection by immunoblotting using polyclonal antiserum against one of the family members, has been used in the present study to detect kallikrein-like proteins in the rat prostate. Nine immunoreactive kallikrein-like protein bands were detected with pI ranging from 5.30 to 8.35. Of these, six were completely purified and three were partially purified. Two proteins (pI 5.30 and 6.75-6.90) corresponded to protein bands in gels of rat submandibular-gland extracts, and were identified by partial amino acid sequence analysis as rK8 and rK9 respectively. In addition, sequence analysis revealed complete sequence similarity between rK9 and the immunoreactive prostate proteins with pI 7.15, 7.25, 7.50 and 8.27. On the basis of this finding and immunological and biochemical characterization, we concluded that all the kallikrein-like proteins detected, except for rK8, represented isoenzymes of rK9. The molecular masses of the prostate rK9 isoenzymes (24,600-29,300 Da) were close to that of submandibular-gland rK9 (24,600 Da), although differences were observed after reduction with mercaptoethanol. The prostate rK9 isoenzymes were, like submandibular-gland rK9, inhibited by soya-bean trypsin inhibitor but not by aprotinin, and were classified as serine proteases as they were inhibited by phenylmethanesulphonyl fluoride. rK8 (28,700 Da) showed no activity with any of the substrates tested, and its inhibitory profile could therefore not be studied. No other enzymes of the kallikrein family were found in the rat prostate.
Carrier-mediated transport of intact UDP-glucuronic acid into the lumen of endoplasmic-reticulum-derived vesicles from rat liver
Authors: Bossuyt X , Blanckaert N
Published: August 2015

Uptake and metabolism of UDP-glucuronic acid (UDPGlcA) by rough-endoplasmic-reticulum (RER)-derived vesicles was studied. Analysis of the molecular species, double-labelling experiments and trans-stimulation experiments revealed that initial uptake represented entry into microsomes of predominantly intact UDPGlcA, concomitant with rapid hydrolysis of the internalized nucleotide sugar. The uptake constituted effective translocation from the medium into the lumen of the vesicles. Thus the amount of vesicle-associated label at equilibrium uptake was directly proportional to the volume of the intravesicular space. Permeabilized microsomes were unable to retain UDPGlcA. The microsomal uptake of UDPGlcA met the criteria of bidirectional carrier-mediated translocation. Transport was time- and temperature-dependent, saturable, selective, capable of trans-stimulation, and operational against a concentration gradient. Microsomal uptake was inhibited by N-ethylmaleimide that was presented at the cytosolic side of the endoplasmic-reticulum (ER) membrane. Uptake studies performed in membrane preparations that were highly enriched in RER, smooth ER or Golgi revealed that UDPGlcA was taken up by the ER as well as by the Golgi apparatus. Our findings demonstrate the existence in rat liver ER of a carrier system mediating proper translocation of intact UDPGlcA across the membrane.
Staurosporine inhibits phorbol 12-myristate 13-acetate- and insulin-stimulated translocation of GLUT1 and GLUT4 glucose transporters in rat adipose cells
Authors: Nishimura H , Simpson I A
Published: August 2015

Staurosporine, a widely used protein kinase C inhibitor, completely inhibited both phorbol 12-myristate 13-acetate (PMA)- and insulin-stimulated glucose transport activity in isolated rat adipocytes. The inhibition was non-competitive and was attributed to a blockade of the PMA- and insulin-induced translocation of both GLUT1 and GLUT4 glucose transporters. The PMA-stimulated glucose transport activity was more sensitive to inhibition by staurosporine than was insulin-stimulated transport activity (PMA, IC50 = 1.1 +/- 0.1 microM; insulin, IC50 = 6.4 +/- 0.7 microM; P < 0.05, n = 3). At 1 microM staurosporine the insulin-sensitivity was decreased, i.e. EC50 increased from 0.12 nM to 5.4 nM, but the maximum response to insulin and the time course for stimulation were unaffected. At 6 microM staurosporine the insulin-sensitivity was further decreased, the maximal stimulation was decreased by 25%, and the apparent half-time for stimulation was extended from 2.5 min in control cells to 9.4 min. Staurosporine (30 microM) was able to block insulin's ability to stimulate glucose transport, whether added before or after insulin, by a mechanism that did not alter the rate of GLUT4 internalization. In intact adipose cells, staurosporine (30 microM) induced a slight (30%) decrease in the maximal insulin-induced receptor autophosphorylation and a similar decrease in the tyrosine phosphorylation of pp60 and pp160 (insulin-receptor substrate-1: ‘IRS-1’), but was without effect on insulin binding to its receptor. Conversely, staurosporine induced a concentration-dependent inhibition of the constitutively tyrosine-phosphorylated (pp120) protein and of an insulin-stimulated protein pp53 in the cytosol. The locus of staurosporine's action appears to be distal from the initial insulin-receptor signalling, at a step that regulates the specific translocation of the glucose transporters to the plasma membranes.
Yeast acyl-CoA-binding protein: acyl-CoA-binding affinity and effect on intracellular acyl-CoA pool size
Authors: Knudsen J , Faergeman N J , Skøtt H , Hummel R , Børsting C , Rose T M , Andersen J S , Højrup P , Roepstorff P , Kristiansen K
Published: August 2015

Acyl-CoA-binding protein (ACBP) is a 10 kDa protein characterized in vertebrates. We have isolated two ACBP homologues from the yeast Saccharomyces carlsbergensis, named yeast ACBP types 1 and 2. Both proteins contain 86 amino acid residues and are identical except for four conservative substitutions. In comparison with human ACBP, yeast ACBPs exhibit 48% (type 1) and 49% (type 2) conservation of amino acid residues. The amino acid sequence of S. carlsbergensis ACBP type 1 was found to be identical with the one ACBP present in Saccharomyces cerevisiae. A recombinant form of this protein was expressed in Escherichia coli and S. cerevisiae, purified, and its acyl-CoA-binding properties were characterized by isoelectric focusing and microcalorimetric analyses. The yeast ACBP was found to bind acyl-CoA esters with high affinity (Kd 0.55 x 10(-10) M). Overexpression of yeast ACBP in S. cerevisiae resulted in a significant expansion of the intracellular acyl-CoA pool. Finally, Southern-blotting analysis of the two genes encoding ACBP types 1 and 2 in S. carlsbergensis strongly indicated that this species is a hybrid between S. cerevisiae and Saccharomyces monacensis.