bluuebunny/crossref_metadata_2025 huggingface dataset:
Preferential antagonism of the interactions of the integrin αIIbβ3 with immobilized glycoprotein ligands by snake-venom RGD (Arg-Gly-Asp) proteins. Evidence supporting a functional role for the amino acid residues flanking the tripeptide RGD in determining the inhibitory properties of snake-venom RGD proteins
Authors: Lu X , Williams J A , Deadman J J , Salmon G P , Kakkar V V , Wilkinson J M , Baruch D , Authi K S , Rahman S
Published: August 2015

The inhibitory properties of a panel of snake-venom-derived RGD (Arg-Gly-Asp) proteins, including the disintegrins kistrin, elegantin and albolabrin, and the neurotoxin homologue dendroaspin, were investigated in a platelet-adhesion assay using three immobilized ligands of the glycoprotein IIb-IIIa complex (alpha IIb beta 3), namely fibrinogen, fibronectin and von Willebrand factor (vWF). The snake-venom proteins preferentially inhibited the adhesion of ADP-treated platelets to one or more of the immobilized ligands. Kistrin and dendroaspin exhibited similar inhibitory characteristics, abrogating platelet adhesion to fibrinogen and vWF at nanomolar concentrations, but poorly inhibiting adhesion to fibronectin. Kistrin and dendroaspin share little overall amino-acid-sequence identity, but a considerable level of sequence similarity exists around the RGD tripeptide. Synthetic cyclic peptides corresponding to these regions of kistrin and dendroaspin inhibited platelet adhesion to both fibrinogen and fibronectin with approximately equal potency, but were 100-fold weaker antagonists of the interactions of the alpha IIb beta 3 complex with fibrinogen than their parent proteins. The disintegrins elegantin and albolabrin, which share approx. 60% overall amino-acid-sequence similarity with kistrin but have different residues around the RGD tripeptide, exhibited different antagonistic preferences. Elegantin inhibited platelet adhesion to immobilized vWF and fibronectin, but was significantly less effective at disrupting adhesion to fibrinogen. Albolabrin selectively inhibited platelet adhesion to immobilized vWF and was less effective with fibrinogen and fibronectin as adhesive ligands. In contrast with the behaviour of these venom proteins, the adhesion of ADP-treated platelets to immobilized fibrinogen, fibronectin and vWF was inhibited non-selectively by a range of monoclonal antibodies with specificity for the alpha IIb beta 3 complex. These observations, therefore, define antagonistic preferences in this panel of venom proteins towards the interactions of the alpha IIb beta 3 complex with three immobilized glycoprotein ligands.
Thyroid hormone treatment alters phospholipid composition and membrane fluidity of rat brain mitochondria
Authors: Bangur C S , Howland J L , Katyare S S
Published: August 2015

We examined effects of graded doses of thyroid hormones 3,3′, 5-tri-iodo-L-thyronine (T3) and L-thyroxine (T4) on the lipid composition of rat brain mitochondria. Neither hormone significantly affected the mitochondrial cholesterol or total phospholipid content, but did increase phosphatidylethanolamine (PE) at the expense of phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylcholine (PC). The phosphatidic acid (PA) content was also elevated, suggesting enhanced phospholipid turnover. Changes in sphingomyelin (SPM) and diphosphatidylglycerol (DPG) were minimal. Mitochondrial membrane fluidity also increased after thyroid-hormone treatment, and the increase was closely correlated with PC/PE and SPM/PE molar ratios.
Purification and characterization of recombinant pea-seed ferritins expressed in Escherichia coli: influence of N-terminus deletions on protein solubility and core formation in vitro
Authors: Van Wuytswinkel O , Savino G , Briat J F
Published: August 2015

Plant ferritin subunits are synthesized as precursor molecules; the transit peptide (TP) in their NH2 extremity, responsible for plastid targeting, is cleaved during translocation to this compartment. In addition, the N-terminus of the mature subunit contains a plant-specific sequence named extension peptide (EP) [Ragland, Briat, Gagnon, Laulhère, Massenet, and Theil, E.C. (1990) J. Biol. Chem. 265, 18339-18344], the function of which is unknown. A novel pea-seed ferritin cDNA, with a consensus ferroxidase centre conserved within H-type animal ferritins has been characterized. This pea-seed ferritin cDNA has been engineered using oligonucleotide-directed mutagenesis to produce DNA fragments (1) corresponding to the wild-type (WT) ferritin precursor, (2) with the TP deleted, (3) with both the TP and the plant specific EP sequences deleted and (4) containing the TP but with the EP deleted. These four DNA fragments have been cloned in an Escherichia coli expression vector to produce the corresponding recombinant pea-seed ferritins. Expression at 37 degrees C led to the accumulation of recombinant pea-seed ferritins in inclusion bodies, whatever the construct introduced in E. coli. Expression at 25 degrees C in the presence of sorbitol and betaine allowed soluble proteins to accumulate when constructs with the TP deleted were used; under this condition, E. coli cells transformed with constructs containing the TP were unable to accumulate recombinant protein. Recombinant ferritins purified from inclusion bodies were found to be assembled only when the TP was deleted; however assembled ferritin under this condition had a ferroxidase activity undetectable at acid pH. On the other hand, soluble recombinant ferritins with the TP deleted and expressed at 25 degrees C were purified as 24-mers containing an average of 40-50 iron atoms per molecule. Despite the conservation in the plant ferritin subunit of a consensus ferroxidase centre, the iron uptake activity in vitro at pH 6.8 was found to be lower than that of the recombinant human H-ferritin, though it was much more active than the recombinant human L-ferritin. The recombinant ferritin with both the TP and the EP deleted (r delta TP/EP) assembled correctly as a 24-mer; it has slightly higher ferroxidase activity and decreased solubility compared with the wild-type protein with the TP deleted (r delta TP). In addition, on denaturation by urea followed by renaturation by dialysis the r delta TP/EP protein showed a 25% increase in core-formation in vitro compared with the r delta TP protein.(ABSTRACT TRUNCATED AT 400 WORDS)
Comparative studies on bile flow and biliary lipid excretion after bile-acid loading in normal and partially hepatectomized rats
Authors: Hoshino M , Hirano A , Hayakawa T , Kamiya Y , Ohiwa T , Tanaka A , Kumai T , Katagiri K , Miyaji M , Takeuchi T
Published: August 2015

This study was performed to investigate sequential changes in bile secretion and biliary lipids after taurocholic acid (TCA) loading of regenerating rat liver. TCA was administered intravenously at stepwise-increasing doses to groups of non-operated control and partially hepatectomized rats, 24, 72 and 168 h after surgery. Bile flow, bile-acid output (BAO) and phospholipid output (PLO) (expressed per gram of liver) in partially hepatectomized rats increased more than in the controls. Using an isolated perfusion rat-liver system, TCA infusion was also carried out on groups of non-operated control and hepatectomized rats 72 h after operation. Again bile flow, BAO and PLO (expressed per gram of liver) were significantly higher in the partial hepatectomy case, mirroring the results obtained in vivo. When horseradish peroxidase (HRP) was pulse-loaded in isolated perfusion preparations, the second peak of biliary HRP secretion in hepatectomized rats was significantly higher than in controls. We conclude that increased bile-acid flow in partially hepatectomized rats is dependent upon acceleration of vesicular transport accompanying or following proliferation in regenerating livers.
Cloning, sequencing and heterologous expression of a cDNA encoding pigeon liver carnitine acetyltransferase
Authors: Johnson T M , Kocher H P , Anderson R C , Nemecek G M
Published: August 2015

Two overlapping cDNA clones encoding pigeon liver carnitine acetyltransferase (EC 2.3.1.7) (CAT) were isolated from a pigeon liver lambda gt11 cDNA library by gene amplification using oligonucleotide primers based on the N-terminal amino acid sequence of the enzyme. The two clones, which represent the 5′ and 3′ ends of the gene, were spliced together to form a single cDNA construct containing the entire coding sequence for CAT, with an in-frame TGA stop codon 42 bases before the first ATG start site and a 3′-untranslated segment of 1057 bases. The largest open reading frame of 1942 nucleotides predicted a polypeptide of 627 amino acids and a molecular mass of 71.1 kDa. The N-terminus and four internal peptides from the amino acid sequence of pigeon breast muscle CAT were identified in the predicted sequence of the liver cDNA clone. The identity of the CAT cDNA was confirmed by heterologous expression of active recombinant CAT (rCAT) in insect cells using the baculovirus expression system. Western blots of rCAT from infected insect cell lysates and immunodetection with a rabbit anti-CAT polyclonal serum showed an immunoreactive protein band similar in size to native CAT from pigeon breast muscle. Like the native enzyme, rCAT was capable of acylating carnitine with a preference for small-chain acyl-CoAs of carbon chain lengths C2-C4.
Escherichia coli chorismate synthase: a deuterium kinetic-isotope effect under single-turnover and steady-state conditions shows that a flavin intermediate forms before the C-(6proR)-H bond is cleaved
Authors: Bornemann S , Balasubramanian S , Coggins J R , Abell C , Lowe D J , Thorneley R N F
Published: August 2015

We report the observation of a deuterium kinetic isotope effect for the conversion of 5-enolpyruvylshikimate-3-phosphate into chorismate (6proR2HV = 1.13 +/- 0.03) using recombinant chorismate synthase from Escherichia coli. Similar isotope effects were observed for the decay of a spectroscopically characterized flavin intermediate (6proR2Hk = 1.17 +/- 0.04) during single-turnover experiments. The main rate-limiting steps and C-(6proR)-H bond breaking are therefore distinct and both must occur after the formation of the flavin intermediate and either before or concomitant with its decay.
Heterologous expression in Escherichia coli of native and mutant forms of the major intrinsic protein of rat eye lens (MIP26)
Authors: Dilsiz N , Crabbe M J C
Published: August 2015

The complete cDNA of rat eye lens major intrinsic protein (MIP26) was sequenced using the dideoxy chain termination method. The sequence displayed 89% nucleotide identity and 95% identity at the amino acid level with bovine MIP26 [Gorin, Yancey, Cline, Revel and Horwitz (1984) Cell, 39, 49-54]. Both native and mutant cDNAs coding for rat MIP26 were amplified by PCR and subcloned into the pPOW expression vector for expression of Escherichia coli. A membrane signal peptide (PelB) was used for secretion of MIP26 into the cytoplasmic membrane. A hydrophilic octapeptide tail (FLAG) was fused to either the N- or C-terminus of MIP26 to aid monoclonal antibody-mediated identification and purification. Heterologously expressed MIP26 was identified by using a monoclonal antibody corresponding to the FLAG peptide located at the termini of MIP26. Immunofluorescently labelled monoclonal antibody was used to determine the localization of MIP26 in the cytoplasmic membrane. The majority of the protein was integrated into cell plasma membrane. MIP26 was extracted with n-octyl beta-D-glucopyranoside and then purified on an affinity gel column. Rat MIP26 cDNA contains an -Asn-Gly- sequence at the C-terminus, which has been shown in other proteins to be particularly susceptible to spontaneous deamidation [Takemoto and Emmons (1991) Curr. Eye Res. 10, 863-869]. We therefore modified the MIP26 molecule using a site-directed mutagenesis method to generate a mutant MIP26 at the appropriate asparagine residue (Asn244-->Asp) near the C-terminus. The mutation was confirmed by DNA sequencing. The mutant MIP26 protein was also expressed in E. coli and incorporated predominantly into the cytoplasmic membrane.
Expression and alternative splicing of the cytochrome P-450 CYP2A7
Authors: Ding S , Lake B G , Friedberg T , Wolf C R
Published: August 2015

In order to investigate the relative levels of expression of human cytochrome P-450 (P-450) CYP2A genes and determine how this relates to polymorphism in coumarin hydroxylase activity, cDNA clones for members of the CYP2A gene family were isolated. These clones were CYP2A6, CYP2A7 and an alternatively spliced version of CYP2A7 (CYP2A7AS). The latter clone was missing exon 2, but contained a 10 bp segment of intron 1. Translation of CYP2A7AS resulted in an in-frame deletion of 51 amino acids. The expression of these cDNAs in COS-7 cells showed that both CYP2A6 and CYP2A7 generated a protein of molecular mass 49 kDa, whereas the protein product of CYP2A7AS was about 44 kDa. Only the CYP2A6 had coumarin hydroxylase activity. The relative level of CYP2A7 and CYP2A7AS mRNA was investigated by reverse transcription followed by PCR (RT-PCR) using human liver RNAs and an RNA sample from a human skin fibroblast cell line. In one of five liver RNAs studied, the aberrantly spliced CYP2A7 mRNA was 3-4-fold more abundant than the normal mRNA. The other samples contained very low levels of this mRNA species. Interestingly, CYP2A7AS mRNA was the major CYP2A7 mRNA detected in the fibroblast cell line. In this case only a protein band of 44 kDa was observed by Western-blot analysis. The relative of mRNA encoding CYP2A6 and CYP2A7 was established in seven human liver samples by RT-PCR and found to range between 1:0.5 and 1:3. These data strength the previous findings that alternative splicing is an important factor in determining the levels of many human P-450s and that this may be subject to tissue-specific effects. Whether in this case the protein product has some function remains to be determined.
Cell-shape-dependent modulation of p52(PAI-1) gene expression involves a secondary response pathway
Authors: Higgins P J , Staiano-Coico L , Ryan M P
Published: August 2015

Expression of the rat p52(PAI-1) gene is positively regulated by agents that influence cellular microfilament organization and/or cell-to-substrate adhesion [e.g. cytochalasin D (CD) and sodium n-butyrate (NaB)] [Higgins, Chaudhari and Ryan (1991) Biochem. J. 273, 651-658; Higgins, Ryan and Providence (1994) J. Cell. Physiol. 159, 187-195]. As shape-responsive genes may be subject to inducer-specific controls, the biochemical mechanisms underlying the shape-dependent pathway of p52(PAI-1) gene regulation were examined in v-ras-transformed rat kidney (KNRK) cells. NaB and/or CD effectively stimulated p52(PAI-1) run-off transcription and augmented de novo p52(PAI-1) mRNA and protein synthesis in KNRK cells; induction at both the mRNA and protein levels was inhibited by actinomycin D. Pretreatment with cycloheximide (CX) markedly attenuated NaB- and/or CD-stimulated p52(PAI-1) expression. CX alone, however, induced low levels of p52(PAI-1) mRNA; increased p52(PAI-1) protein synthesis was evident after release of KNRK cells from CX blockade. Such CX-mediated induction was also sensitive to actinomycin D. Full stimulation of p52(PAI-1) expression in KNRK cells in response to the shape modulators NaB and/or CD involves transcriptional activation of the p52(PAI-1) gene, requires de novo RNA synthesis and occurs through a secondary-response (i.e. protein-synthesis-dependent) pathway.
Microcystin-LR induced an inhibition of protein synthesis in isolated rat hepatocytes
Authors: Claeyssens S , Francois A , Chedeville A , Lavoinne A
Published: August 2015

The effect of microcystin-LR, an inhibitor of protein phosphatases PP1 and PP2A, was studied on protein synthesis by measuring the incorporation of labelled amino acid into protein in isolated rat hepatocytes. Microcystin-LR inhibited protein synthesis in the first minutes of the incubation period, and half-maximum effect was obtained at about 60 nM. Such an inhibition was also observed in the presence of different protein phosphatase inhibitors, i.e. okadaic acid, calyculin A and microcystin-RR. This effect was observed in whole hepatocytes, in the supernatant of the post-mitochondrial fraction and in the microsomal fraction. It was independent of a substrate supply and of the labelled amino acid used. Furthermore, this inhibition preceded the previously reported glucose-6-phosphatase activation induced by microcystin-LR [Claeyssens, Chédeville and Lavoinne (1993) FEBS Lett. 315, 7-10].
Effects of CoA and acyl-CoA on Ca2+-permeability of endoplasmic-reticulum membranes from rat liver
Authors: Rich G T , Comerford J G , Graham S , Dawson A P
Published: August 2015

We have studied the effects of CoA and palmitoyl-CoA on Ca2+ movements and GTP-dependent vesicle fusion in rat liver microsomes. (1) Inhibition of membrane fusion by CoA depends on esterification of CoA to long-chain acyl-CoA using endogenous non-esterified fatty acids. (2) Binding of long-chain acyl-CoA to microsomal membranes is inhibited by BSA, which also relieves inhibition of membrane fusion. (3) Under conditions where acyl-CoA binding is inhibited, CoA causes increased Ca2+ accumulation, apparently by decreasing the Ca2+ leak rate. (4) Conversely, palmitoyl-CoA, in the presence of BSA, causes Ca2+ efflux. (5) The decrease in Ca(2+)-permeability caused by CoA does not depend on the presence of ATP or GTP, and is irreversible in the short term. (6) Using 14C-labelled CoA we show that CoA derivatives can be formed from endogenous components of microsomal membranes in the absence of ATP. (7) The results are interpreted in terms of a Ca(2+)-permeability which is controlled by CoA and/or long-chain acyl-CoA esters.
Effects of granulocyte-macrophage colony-stimulating factor and tumour necrosis factor-α on tyrosine phosphorylation and activation of mitogen-activated protein kinases in human neutrophils
Authors: Waterman W H , Sha'afi R I
Published: August 2015

The present study was undertaken to determine the identities and characteristics of proteins with molecular masses between 40 and 44 kDa whose tyrosine phosphorylation increases in human neutrophils following stimulation of these cells with tumour necrosis factor alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Immunoblotting results demonstrate that addition of GM-CSF to human neutrophils increases the tyrosine phosphorylation of two proteins with molecular masses of 42 and 44 kDa. However, the addition of TNF-alpha to neutrophils induces a time- and dose-dependent increase in tyrosine phosphorylation of a 40 kDa protein. Immunoprecipitation using specific mitogen-activated protein kinase (MAPK) isoform antibodies and an antibody which recognizes phosphotyrosine-containing proteins demonstrated that the 42 and 44 kDa proteins are isoforms of MAPKs. Utilizing an in situ gel kinase activity assay, GM-CSF increases the kinase activity of the 42 and 44 kDa proteins. Moreover, using immunoprecipitated p42 and p44 MAPK isoforms in this gel assay revealed activity associated with the p42 and p44 MAPK isoforms. Using the same in situ assay, TNF-alpha induces an increase in kinase activity of a 40-42 kDa protein. However, the 40 kDa protein whose phosphorylation on tyrosine residues increased in human neutrophils following stimulation with TNF-alpha is not a member of the known MAPK family, demonstrating the divergences in pathways utilized by GM-CSF and TNF-alpha. This 40 kDa protein may be related to the recently identified protein that becomes phosphorylated on tyrosine residues upon stimulation of the human epidermal carcinoma cell line KB by interleukin-1. In these cells the p40 protein is part of a protein kinase cascade which results in the phosphorylation of the small heat shock protein, hsp27.
The mechanism of inhibition of DNA (cytosine-5-)-methyltransferases by 5-azacytosine is likely to involve methyl transfer to the inhibitor
Authors: Gabbara S , Bhagwat A S
Published: August 2015

The mechanism of inhibition of DNA (cytosine-5-)-methyltransferases by the mechanism-based inhibitor 5-azacytosine has remained unclear, mainly because of the unavailability of a substrate in which the inhibitor, but not normal cytosine, is present at the target site. We synthesized an oligonucleotide duplex containing a single target site for the EcoRII methyltransferase, in which the target base is 5-azacytosine. This substrate formed a stable covalent complex with EcoRII methyltransferase in the absence and in the presence of the cofactor S-adenosylmethionine. The complex formed in the presence of the cofactor was resistant to SDS and moderate heat treatment, and a methyl group was incorporated into the complex. Enzyme titration and kinetic studies of inhibition suggest that methyl transfer to the complex occurred only during the first turnover of the reaction. These results suggest that, when the enzyme binds to 5-azacytosine in the presence of the cofactor, a methyl group is transferred to the N-5 position of the base, resulting in the inactivation of the enzyme.
Temperature regulation of the Tetrahymena mimbres glycosylphosphatidylinositol-anchored protein lipid composition
Authors: Ko Y G , Hung C Y , Thompson G A
Published: August 2015

By incubating Tetrahymena mimbres cells with [3H]myristic acid, [3H]ethanolamine, [3H]inositol, and [3H]mannose, proteins having apparent molecular masses of 23 and 63 kDa were identified as the cells' principal glycosylphosphatidylinositol (GPI)-anchored proteins. These proteins accounted for as much as 2-5% of the whole cell proteins, with the higher levels being recovered from non-growing cells. The two proteins, gpi 23 and gpi 63, were purified to near homogeneity through Triton X-114/water partitioning followed by preparative SDS/PAGE. The lipid components of the GPI anchors were determined by chemical and enzymic hydrolysis. Both proteins were anchored by ceramides, with the principal long-chain base being C18 sphinganine containing an O-methyl group at the 3 position. O-Methylation was shown not to be an artifact of hydrolysis. When T. mimbres was cultured at 15 degrees C, the ceramide fatty acid component of the GPI anchors was principally palmitic acid (75% in gpi 23 and 76% in gpi 63). GPI anchors from 28 degrees C-grown cells contained mainly stearic acid (79% in gpi 23 and 70% in gpi 63). Temperature change had little effect on the long-chain-base composition. The direction of temperature-induced lipid change in the protein-bound anchors was the same as found in the inositolphosphorylceramide putative precursors of the protein anchors described in the accompanying paper [Hung, Ko and Thompson (1995) Biochem. J. 307, 107-113], but the detailed fatty acid compositions of the precursors and the protein-bound lipids were quite different. The precise metabolic regulation of anchor lipid chain length supports the concept that composition of the lipid anchor is important in the function and/or metabolism of the anchored protein.
Epidermal growth factor-stimulated parathyroid hormone-related protein expression involves increased gene transcription and mRNA stability
Authors: Heath J K , Southby J , Fukumoto S , O'Keeffe L M , Martin T J , Gillespie M T
Published: August 2015

Epidermal growth factor (EGF) produced rapid and striking effects on parathyroid hormone-related protein (PTHrP) gene expression in the immortalized human keratinocyte cell line, HaCaT. Steady-state levels of PTHrP mRNA and secreted PTHrP were increased 10-fold by maximally effective concentrations of EGF. EGF increased both PTHrP gene transcription and PTHrP mRNA stability. Nuclear run-on assays demonstrated a 4-fold increase in transcriptional rate in EGF-stimulated cells while transient transfection analysis indicated that the action of EGF on transcription involved both the GC-rich promoter, P2, and the downstream TATA promoter, P3, but apparently not the upstream TATA promoter, P1. In experiments where EGF treatment produced more stable PTHrP transcripts, the half-life of c-fos mRNA was unaltered, suggesting a relatively specific effect of EGF. Moreover, only those species of PTHrP mRNA containing two of the alternative 3′ exons (exons VII and VIII) were stable, those containing exon IX were not. Reverse-transcription PCR demonstrated that EGF produced differential increases in the abundance of PTHrP mRNA species initiated by the three PTHrP promoters. The major effect was seen on the abundance of transcripts initiated by P1 and P2, with less marked regulation of P3-initiated transcripts. Thus EGF regulation of PTHrP gene expression in HaCaT cells is multifactorial and the combination of its actions at the 5′ and 3′ ends of the gene favours the accumulation of subpopulations of PTHrP mRNA containing exons I, VII and VIII.
Regulation of J6 gene expression by transcription factor GATA-4
Authors: Bielinska M , Wilson D B
Published: August 2015

Retinoic acid-induced differentiation of mouse F9 embryonal carcinoma cells into primitive endoderm is accompanied by increased transcription of the gene for J6, a heat shock protein implicated in collagen biosynthesis. In this paper we present evidence that transcription factor GATA-4, a retinoic acid-inducible GATA-binding protein, is involved in the regulation of J6 gene expression in F9 cells. Northern-blot analysis indicates that transcripts encoding GATA-4 and J6 increase in parallel during retinoic acid-induced differentiation of F9 cells. Gel-shift experiments and antibody binding studies demonstrate that: (1) GATA-4 is the major GATA-binding protein activity in differentiated F9 cells, and (2) GATA-4 binds to consensus GATA motifs in the retinoic acid-responsive portion of the J6 promoter. Co-transfection studies using NIH 3T3 cells show that GATA-4 is a potent trans-activator of the J6 promoter. These lines of evidence suggest that expression of J6 in F9 cells is regulated by GATA-4. We speculate that transcription factor GATA-4 may also control other genes involved in extracellular matrix formation in the yolk sac.
Phospholipase D-induced phosphatidate production in intact small arteries during noradrenaline stimulation: involvement of both G-protein and tyrosine-phosphorylation-linked pathways
Authors: Ward D T , Ohanian J , Heagerty A M , Ohanian V
Published: August 2015

To investigate membrane lipid metabolism during smooth-muscle activation, the role of phospholipase D (PLD) in the production of phosphatidate (PA) was studied in rat small arteries stimulated with noradrenaline. Incubation with [3H]myristate preferentially labelled phosphatidylcholine (PtdCho), and in the presence of 0.5% ethanol [3H]phosphatidylethanol ([3H]PEt) was formed, demonstrating PLD activity. Noradrenaline (NA) stimulation resulted in an increase in PtdCho derived [3H]PA and [3H]PEt formation, indicating PLD activation. Stimulation of [14C]choline release confirmed PLD-mediated hydrolysis of PtdCho. Propranolol, an inhibitor of PA phosphohydrolase, increased [3H]PA levels in non-stimulated tissue and decreased the rate of degradation of both [3H]PA and [3H]PEt, implying that this is an active route for PA metabolism in small arteries. However, [3H]diacylglycerol levels were not increased during NA stimulation. Fluoroaluminate increased [3H]PEt formation and [14C]choline release, whereas high K+ in the presence of alpha 1-adrenoceptor blockade did not. Pervanadate increased phosphotyrosine levels in small arteries, and markedly stimulated [3H]PEt formation and [14C]choline release. The combination of pervanadate and NA stimulation resulted in a dramatic increase in [3H]PEt formation, which was greater than the sum of the individual responses to the two agonists. Pervanadate and fluoroaluminate in combination appeared to give an additive response, whereas high K+ did not alter the pervanadate-induced formation of [3H]PEt. Phosphotyrosine levels were increased by NA in the presence of tyrosine phosphatase inhibitors. This effect was blocked by genistein, a tyrosine kinase inhibitor. These data demonstrate that in NA-stimulated small arteries PLD-induced PtdCho hydrolysis contributes to accumulation of PA, but not of diacylglycerol. Furthermore, regulation of PLD activity appears to require G-protein and tyrosine-phosphorylation-linked pathways.
The mechanism for the ATP-induced uncoupling of respiration in mitochondria of the yeast Saccharomyces cerevisiae
Authors: Prieto S , Bouillaud F , Rial E
Published: August 2015

We have recently reported that ATP induces an uncoupling pathway in Saccharomyces cerevisiae mitochondria [Prieto, Bouillaud, Ricquier and Rial (1992) Eur. J. Biochem. 208, 487-491]. The presence of this pathway would explain the reported low efficiency of oxidative phosphorylation in S. cerevisiae, and may represent one of the postulated energy-dissipating mechanisms present in these yeasts. In this paper we demonstrate that ATP exerts its action in two steps: first, at low ATP/Pi ratios, it increases the respiratory-chain activity, probably by altering the kinetic properties of cytochrome c oxidase. Second, at higher ATP/Pi ratios, an increase in membrane permeability leads to a collapse in membrane potential. The ATP effect on cytochrome c oxidase corroborates a recent report showing that ATP interacts specifically with yeast cytochrome oxidase, stimulating its activity [Taanman and Capaldi (1993) J. Biol. Chem. 268, 18754-18761].
Volume-activated chloride currents are not correlated with P-glycoprotein expression
Authors: De Greef C , Sehrer J , Viana F , van Acker K , Eggermont J , Mertens L , Raeymaekers L , Droogmans G , Nilius B
Published: August 2015

It has been proposed that P-glycoprotein, the product of the human MDR1 gene, may function not only as a drug transporter but, depending on the conditions, as a volume-activated Cl- channel [Valverde, Diaz, Sepúlveda, Gill, Hyde and Higgins (1992) Nature (London) 355, 830-833; Gill, Hyde, Higgins, Valverde, Mintenig and Sepúlveda (1992) Cell 71, 23-32]. To verify this hypothesis, we have compared volume-activated Cl- currents with the level of MDR1 mRNA and its protein product in the human KB3 (epitheloid lung cancer) and HeLa cell lines. The related MDR2 was also included to find out whether it could account for observed discrepancies between Cl- current and MDR1 expression. A 40% decrease in osmolarity evoked a Cl- current in both cell types (at +80 mV: 50.3 +/- 4.3 pA/pF in KB3, n = 13; 28.2 +/- 3.3 pA/pF in HeLa, n = 16). The blocking of this current in both cell types by 5-nitro-2-(3-phenylpropylamino)-benzoic acid and by 1,9-dideoxyforskolin is similar to that of the presumed P-glycoprotein associated Cl- channel. As measured by reverse-transcriptase polymerase chain reaction, KB3 cells expressed only an extremely small amount of the messengers for MDR1 and MDR2. The signal observed for MDR1 in HeLa cells was at least an order of magnitude more intense than in KB3 cells, while MDR2 mRNA was undetectable. A clear difference in MDR1 expression between KB3 and HeLa was also observed at the protein level. These data are difficult to reconcile with the hypothesis that in HeLa and KB3 cells MDR1- or MDR2- encoded P-glycoproteins are associated with volume-activated Cl- channels.
Increased expression of procoagulant activity on the surface of human platelets exposed to heavy-metal compounds
Authors: Goodwin C A , Wheeler-Jones C P D , Namiranian S , Bokkala S , Kakkar V V , Authi K S , Scully M F
Published: August 2015

One of the essential roles for platelets in haemostasis is in the potentiation of blood clotting due to the contribution of anionic phospholipid from the surface of the cells, as an essential cofactor to the proteolytic reactions of coagulation (platelet procoagulant activity). Only a limited number of agonists are known to initiate platelet procoagulant activity. In this study the rate of thrombin formation on the platelet surface was observed to increase in a dose-dependent manner upon treatment of washed platelets with heavy-metal compounds. Unlike the immediate increase observed upon treatment of platelets with calcium ionophore, A23187, the change due to these agents was progressive, approaching a maximum after 10 min. The maximum-fold acceleration of the rate of thrombin formation compared with control platelets was calculated for HgCl2 (56-fold), AgNO3 (42-fold) phenylmercuriacetate (24-fold) and thimerosal (14-fold), compared with 70-fold observed for calcium ionophore. The increase in procoagulant activity due to HgCl2 coincided with a large increase in intracellular calcium and phosphorylation of 22 and 45 kDa proteins. It is considered that the mechanism responsible for the increase in procoagulant activity is exposure of anionic phospholipids. This was detected by a 2-fold increase in the binding of 125I-annexin V upon addition of HgCl2, compared with resting platelets (3-fold on treatment of platelets with calcium ionophore). In contrast to the generation of activity by A23187 and other known agonists of this reaction, heavy-metal compounds appeared to cause little or no release of microparticles from the platelet surface. Since HgCl2 did not cause aggregation of platelets or significant release of serotinin, these findings may give further support to the need for exposure and ligation of glycoprotein IIb:IIIa for vesiculization to occur. Treatment of platelets with heavy metals may constitute a new approach to investigating the early changes in the cell membrane which lead to increased expression of anionic phospholipid.
The calmodulin-binding domain in the mouse type 1 inositol 1,4,5-trisphosphate receptor
Authors: Yamada M , Miyawaki A , Saito K , Nakajima T , Yamamoto-Hino M , Ryo Y , Furuichi T , Mikoshiba K
Published: August 2015

We determined the amino acid sequence responsible for the calmodulin (CaM)-binding ability of mouse type 1 Ins(1,4,5)P3 receptor (IP3R1). We expressed various parts of IP3R1 from deleted cDNA and examined their CaM-binding ability. It was shown that the sequence stretching from Lys-1564 to Arg-1585 is necessary for the binding. The full-length IP3R1 with replacement of Trp-1576 by Ala lost its CaM-binding ability. Antibody against residues 1564-1585 of IP3R1 inhibited cerebellar IP3R1 from binding CaM. The fluorescence spectrum of the peptide that corresponds to residues 1564-1585 shifted when Ca(2+)-CaM was added. From the change in the fluorescence spectrum, we estimated the dissociation constant (KD) between the peptide and CaM to be 0.7 microM. The submicromolar value of KD suggests an actual interaction between CaM and IP3R1 within cells. The CaM-binding ability of other types of IP3Rs was also examined. A part of the type 2IP3R, including the region showing sequence identity with the CaM-binding domain of IP3R1, also bound CaM, while the expressed full-length type 3 IP3R did not.
Activation and labelling of the purified skeletal muscle ryanodine receptor by an oxidized ATP analogue
Authors: Hohenegger M , Herrmann-Frank A , Richter M , Lehmann-Horn F
Published: August 2015

We have tested the periodate-oxidized ATP analogue 2′,3′-dialdehyde adenosine triphosphate (oATP) as a ligand for the skeletal muscle ryanodine receptor/Ca(2+)-release channel. Ca2+ efflux from passively loaded heavy sarcoplasmic reticulum vesicles of skeletal muscle is biphasic. oATP stimulates the initial phase of Ca2+ release in a concentration-dependent manner (EC50 160 microM), and the efflux proceeds with a half-time in the range 100-200 ms. This oATP-modulated initial rapid Ca2+ release was specifically inhibited by millimolar concentrations of Mg2+ and micromolar concentrations of Ruthenium Red, indicating that the effect of oATP was mediated via the ryanodine receptor. The purified Ca(2+)-release channel was incorporated into planar lipid bilayers, and single-channel recordings were carried out to verify a direct interaction of oATP with the ryanodine receptor. Addition of oATP to the cytoplasmic side activated the channel with an EC50 of 76 microM, which is roughly 30-fold higher than the apparent affinity of ATP. The oATP-induced increase in the open probability of the ryanodine receptor displays a steep concentration-response curve with a Hill coefficient of approximately 2, which suggests a co-operativity of the ATP binding sites in the tetrameric protein. oATP binds to the ryanodine receptor in a quasi-irreversible manner via Schiff base formation between the aldehyde groups of oATP and amino groups in the nucleotide binding pocket. This allows for the covalent specific incorporation of [alpha-32P]oATP by borhydride reduction. A typical adenine nucleotide binding site cannot be identified in the primary sequence of the ryanodine receptor. Our results demonstrate that oATP can be used to probe the structure and function of the nucleotide binding pocket of the ryanodine receptor and presumably of other ATP-regulated ion channels.
Effects of glucose refeeding and glibenclamide treatment on glucokinase and GLUT2 gene expression in pancreatic B-cells and liver from rats
Authors: Tiedge M , Lenzen S
Published: August 2015

The mutual role of glucose and insulin in the regulation of glucokinase and GLUT2 glucose transporter gene expression in pancreatic B-cells and liver has been studied in vivo in the rat. Glucokinase mRNA was quantified by competitive reverse-transcriptase PCR analysis, and GLUT2 mRNA by Northern-blot analysis in total RNA fractions. As in the liver, glucokinase mRNA decreased by 64% in pancreatic B-cells after starvation for 2 days and was induced 3-fold by short-term treatment (1 h) of the rats with oral glucose (4 g/kg body wt.). In contrast the sulphonylurea compound glibenclamide (0.1 mg/kg body wt.) did not significantly stimulate glucokinase gene expression in pancreatic B-cells. But glibenclamide caused a 4-fold increase of glucokinase mRNA in liver which was abolished by concomitant administration of diazoxide, a drug which antagonizes glibenclamide stimulated insulin secretion. GLUT2 gene expression was decreased by 50% in pancreatic B-cells and liver after starvation of the rats for 2 days. Neither short-term treatment (1 h) with glucose nor glibenclamide resulted in a significant increase of GLUT2 gene expression in pancreatic B-cells and liver. The results suggest that it is glucose which stimulates glucokinase gene expression in pancreatic B-cells whereas the transcriptional regulation of the glucokinase gene in liver is directed by insulin.
Ethanol potentiates interleukin-1 β-stimulated inducible nitric oxide synthase expression in cultured vascular smooth muscle cells
Authors: Durante W , Cheng K , Sunahara R K , Schafer A I
Published: August 2015

Experiments were performed to examine the effect of ethanol on the production of nitric oxide from interleukin-1 beta (IL-1 beta)-treated cultured rat aortic smooth muscle cells. Incubation of vascular smooth muscle cells with IL-1 beta resulted in the release of nitrite and in the intracellular accumulation of L-citrulline. In parallel with this, IL-1 beta increased inducible nitric oxide synthase (iNOS) mRNA and protein. Ethanol (6.5-650 mM) potentiated the IL-1 beta-mediated stimulation of iNOS mRNA production, the appearance of iNOS protein and the generation of nitrite and L-citrulline from smooth muscle cells in a concentration-dependent manner. In the absence of IL-1 beta, ethanol failed to induce iNOS expression. These results demonstrate that pharmacologically relevant concentrations of ethanol enhance the IL-1 beta-induced expression of the iNOS gene in vascular smooth muscle. The ability of ethanol to augment the release of the platelet inhibitor and vasodilator nitric oxide may, in part, contribute to the beneficial cardiovascular effects associated with moderate alcohol consumption.
Energetics of carbohydrate binding by a 14 kDa S-type mammalian lectin
Authors: Ramkumar R , Surolia A , Podder S K
Published: August 2015

The thermodynamics of the binding of derivatives of galactose and lactose to a 14 kDa beta-galactoside-binding lectin (L-14) from sheep spleen has been studied in 10 nM phosphate/150 mM NaCl/10 mM beta-mercaptoethanol buffer, pH 7.4, and in the temperature range 285-300 K using titration calorimetry. The single-site binding constants of various sugars for the lectin were in the following order: N-acetyl-lactosamine thiodigalactoside > 4-methylumbelliferyl lactoside > lactose > 4-methylumbelliferyl alpha-D-galactoside > methyl-alpha-galactose > methyl-beta-galactose. Reactions were essentially enthalpically driven with the binding enthalpies ranging from -53.8 kJ/mol for thiodigalactoside at 301 K to -2.2 kJ/mol for galactose at 300 K, indicating that hydrogen-bonding and van der Waals interactions provide the major stabilization for these reactions. However, the binding of 4-methylumbelliferyl-alpha-D-galactose displays relatively favourable entropic contributions, indicating the existence of a non-polar site adjacent to the galactose-binding subsite. From the increments in the enthalpies for the binding of lactose, N-acetyl-lactosamine and thiodigalactoside relative to methyl-beta-galactose, the contribution of glucose binding in the subsite adjacent to that for galactose shows that glucose makes a major contribution to the stability of L-14 disaccharide complexes. Observation of enthalpy-entropy compensation for the recognition of saccharides such as lactose by L-14 and the absence of it for monosaccharides such as galactose, together with the lack of appreciable changes in the heat capacity (delta Cp), indicate that reorganization of water plays an important role in these reactions.
Prephenate dehydratase of the actinomycete Amycolatopsis methanolica: purification and characterization of wild-type and deregulated mutant proteins
Authors: Euverink G J W , Wolters D J , Dijkhuizen L
Published: August 2015

Prephenate dehydratase (PDT) is a key regulatory enzyme in L-phenylalanine biosynthesis in the Gram-positive bacterium Amycolatopsis methanolica. The PDT protein was purified to homogeneity (1957-fold) from wild-type cells with a final yield of 6.5%. It was characterized as a 150 kDa homotetrameric protein with a subunit size of 34 kDa. The first 35 N-terminal amino acids were identified, revealing highest similarity to the PDT proteins from Corynebacterium glutamicum and Bacillus subtilis. Kinetic studies showed that the A. methanolica PDT is allosterically inhibited by phenylalanine and activated by tyrosine. Phenylalanine caused an increase in the S0.5 for prephenate and a decrease in the Vmax. Tyrosine caused a decrease in the S0.5 for prephenate and an increase in the Vmax. Spontaneous o-fluoro- and p-fluoro-DL-phenylalanine-resistant mutants of A. methanolica were isolated. Kinetic studies with the partially purified PDT proteins of strains pFPhe32 and oFPhe84 showed that these mutant proteins had become (partly) insensitive to both phenylalanine inhibition and tyrosine activation.
Plasmodium falciparum-infected erythrocytes utilize a synthetic truncated ceramide precursor for synthesis and secretion of truncated sphingomyelin
Authors: Ansorge I , Jeckel D , Wieland F , Lingelbach K
Published: August 2015

Plasmodium falciparum is an intracellular parasite of human erythrocytes. Parasite development is accompanied by an increase of the phospholipid content of the infected erythrocyte, but it results in a selective decrease of sphingomyelin. We have studied sphingomyelin biosynthesis in infected erythrocytes using as substrate a synthetic radiolabelled ceramide precursor, truncated in both hydrophobic chains. Lysates of infected, unlike those of non-infected, erythrocytes contained sphingomyelin synthase activity, which therefore is of parasite origin. The enzyme activity was associated with a membrane fraction. In contrast to mammalian cells, the parasite did not synthesize detectable levels of glycosphingolipids. In intact infected erythrocytes the ceramide precursor was converted into a correspondingly truncated soluble sphingomyelin which was released into the medium at 37 degrees C. Release of truncated sphingomyelin was inhibited by low temperature (15 degrees C) but not by the fungal metabolite brefeldin A which, however, arrests protein export from the parasite. While membranes of mammalian cells, including the plasma membrane of non-infected erythrocytes, are impermeable to truncated sphingomyelin, the membrane of infected erythrocytes allowed passage of the molecule in both directions. The results obtained with the unicellular eukaryote used here as an experimental model are discussed in comparison with sphingomyelin synthesis and transport in mammalian cells.
Inhibition of proteolysis by cell swelling in the liver requires intact microtubular structures
Authors: vom Dahl S , Stoll B , Gerok W , Häussinger D
Published: August 2015

In the perfused rat liver, proteolysis is inhibited by cell swelling in response to hypo-osmotic media, glutamine and insulin. Colchicine, an inhibitor of microtubules, did not affect cell swelling in response to these agonists. However, the antiproteolytic action of these effectors was largely blunted in the presence of colchicine or the microtubule inhibitors colcemid and taxol. On the other hand, inhibition of proteolysis by phenylalanine, asparagine or NH4Cl, i.e. compounds which exert their antiproteolytic effects by mechanisms distinct from cell swelling, was not sensitive to colchicine. Swelling-induced inhibition of proteolysis was not affected by cytochalasin B. The anti-proteolytic effect of hypo-osmotic cell swelling and insulin was largely abolished in freshly isolated rat hepatocytes; however, it reappeared upon cultivation of the hepatocytes for 6-10 h. The restoration of the sensitivity of proteolysis to cell volume changes was accompanied by a progressive reorganization of microtubule structures, as shown by immunohistochemical staining for tubulin. It is concluded that intact microtubules are required for the control of proteolysis by cell volume, but not for the control of proteolysis by phenylalanine, asparagine or NH4Cl. These findings may explain why others [Meijer, Gustafson, Luiken, Blommaart, Caro, Van Woerkom, Spronk and Boon (1993) Eur. J. Biochem. 215, 449-454] failed to detect an antiproteolytic effect of hypo-osmotic exposure of freshly isolated hepatocytes. This effect, however, which is consistently found in the intact perfused rat liver, also reappeared in isolated hepatocytes when they were allowed to reorganize their microtubular structures in culture.