bluuebunny/crossref_metadata_2025 huggingface dataset:
Protective effect of lipoproteins containing apoprotein A‐I on Cu2+ ‐ catalyzed oxidation of human low density lipoprotein
Authors: Ohta Takao , Takata Kyoko , Horiuchi Seikoh , Morino Yoshimasa , Matsuda Ichiro
Published: July 2002

Two apoprotein A‐I (apoA‐I)‐containing lipoproteins, one containing apoA‐I and apoA‐II (LpA‐I/A‐II) and the other containing only apoA‐I (LpA‐I), were examined for their effect on Cu2+‐mediated oxidation of low density lipoprotein (LDL). The presence of LpA‐I or LpA‐I/A‐II prevented LDL oxidation when assessed by the electrophoretic mobility, apoprotein B fragmentation and amounts of thiobarbituric acid‐reactive substances. The protection of LDL oxidation by these lipoproteins was effective for up to 6 h, with LpA‐I being more active than LpA‐I/A‐II. Results from these in vitro model experiments raise a possibility that LpA‐I mayplay a role in protecting LDL from Cu2+ ‐mediated oxidation.
The interactions of desferrioxamine and hydroxypyridone compounds with haemoglobin and erythrocytes
Authors: Rice-Evans Catherine , Baysal Erol , Singh Surrinder , Jones Shelley-Anne , Jones J.Gareth
Published: July 2002

Hydroxypyridone; Desferrioxamine; Haemoglobin; Iron chelator; Erythrocyte membrane
Angiotensin II stimulates angiotensinogen synthesis in hepatocytes by a pertussis toxin‐sensitive mechanism
Authors: Klett C. , Muller F. , Gierschik P. , Hackenthal E.
Published: July 2002

The role of intracellular messengers in the stimulatory effect of angiotensin II on angiotensinogen synthesis and secretion in hepatocytes was examined. Angiotensinogen secretion was not influenced by modulators of intracellular calcium (calmidazolium, A 23187, Bay K 8644, methoxamine). In contrast, agents decreasing intracellular cAMP (angiotensin II, guanfacine) stimulated, and those increasing cAMP (isoproterenol, glucagon, forskolin) depressed angiotensinogen secretion. An inverse relationship was also observed between cAMP and angiotensinogen mRNA. Pretreatment of hepatocytes with pertussis toxin abolished the stimulation by angiotensin II. It is concluded that angiotensin II‐induced stimulation of angiotensinogen synthesis is initiated by inhibition of adenylate cyclase.
Primary structure of scorpion anti‐insect toxins isolated from the venom of Leiurus quinquestriatus quinquestriatus
Authors: Kopeyan Charles , Mansuelle Pascal , Sampieri François , Brando Thérèse , Mostafa Bahraoui El , Rochat Hervé , Granier Claude
Published: July 2002

The amino acid sequences of insect‐selective scorpion toxins, purified from the venom of Leiurus quinquestriatus quinquestriatus, have been determined by automatic phenyl isothiocyanate degradation of the S‐carboxymethylated proteins and derived proteolytic peptides. The excitatory toxin Lqq IT1 and Lqq IT1' (70 residues) show the shift of one half‐cystine from an external position, which is characteristic of anti‐mammal toxins, to an internal sequence position. Lqq IT2, (61 residues) displays the half‐cystine residue in position 12, common to the sequence of all known antimammal toxins; it induces flaccid paralysis on insects but is non‐toxic for the mouse. Lqq IT2, structurally defines a new type of anti‐insect toxins from scorpion venoms. CD spectra and immunological data are in agreement with this finding.
Complete purification of two identical Na+ ‐pump inhibitors isolated from bovine hypothalamus and hypophysis
Authors: Illescas Manuel , Ricote Mercedes , Mendez Enrique , G-Robles Rafael , Sancho Jose
Published: July 2002

We have completely purified, in parallel, a low molecular weight, non‐peptidic, non‐lipidic, Na+,K+‐ATPase inhibitory factor from bovine hypothalamic and pituitary tissues. In the final purification step we obtain, from both tissues, a single, homogeneous peak, with a maximal absorbance at 247 nm. This factor, at physiological concentrations of potassium (5–25 mM), inhibits in a dose‐response manner Na+,K+‐ATPase and displaces ouabain from its receptor at the enzyme structure. The factor isolated from both tissues is identical, being the specific activity per weight of tissue higher in hypophysis. No factor was found in cerebral cortex, used as tissue control.
A novel and sensitive assay for phospholipase D in intact cells
Authors: Randall Roger W. , Bonser Robert W. , Thompson Neil T. , Garland Lawrence G.
Published: July 2002

A novel and sensitive assay for phospholipase D (PLD) that measures the incorporation of high specific activity [3H]butan‐1‐ol into [3H]phosphatidylbutanol has been developed. The assay has been used to measure PLD activation in human neutrophils and platelets. Both the chemotactic peptide fMet‐Leu‐Phe and opsonised‐zymosan stimulated PLD in the human neutrophil. In the platelet, PLD was stimulated by thrombin and collagen but responses were small and only occurred at high agonist concentrations. This assay has a number of advantages over existing techniques and should be valuable for investigating PLD activation in a variety of isolated cells and possibly intact tissues.
Characterization of mammalian heart annexins with special reference to CaBP33 (annexin V)
Authors: Pula Grazia , Bianchi Roberta , Ceccarelli Paolo , Giambanco Ileana , Donato Rosario
Published: July 2002

Porcine heart was observed to express annexins V (CaBP33) and VI in large amounts, and annexins III and IV in much smaller amounts. Annexin V (CaBP33) in porcine heart was examined in detail by immunochemistry. Homogenization and further processing of heart in the presence of EGTA resulted in the recovery of annexin V (CaBP33) in the cytosolic fraction and in an EGTA‐resistant, Triton X‐100‐soluble fraction from cardiac membranes. Including Ca2+ in the homogenization medium resulted in a significant decrease in the annexin V (CaBP33) content of the cytosolic fraction with concomitant increase in the content of this protein in myofibrils, mitochrondria, the sarcoplasmic reticulum and the sarcolemma. The amount of annexin V (CaBP33) in each of these subfractions depended on the free Ca2+ concentration in the homogenizing medium. At the lowest free Ca2+ concentration tested, 0.8 μM, only the sarcolemma appeared to contain bound annexin V (CaBP33). Membrane‐bound annexins V (CaBP33) and VI partitioned in two fractions, one EGTA‐resistant and Triton X‐100‐extractable, and one Triton X‐100‐resistant and EGTA‐extractable. Altogether, these data suggest that annexins V and VI are involved in the regulation of membrane‐related processes.
The α2 B adrenergic receptor of undifferentiated neuroblastoma × glioma hybrid NG108‐15 cells, interacts directly with the guanine nucleotide binding protein, Gi2
Authors: McClue Steven J. , Milligan Graeme
Published: July 2002

In membranes of undifferentiated neuroblastoma × glioma hybrid cell line NG108‐15, the apparent specific binding of [3H]yohimbine measured in the presence of 1 μM noradrenaline, was increased substantially by the presence of the poorly hydrolysed analogue of QTP, guanylyl‐imidodiphosphate (Gpp[NH]p) or by preincubation of membranes with antibodies against the C‐terminal decapeptide of the α subunit of the G‐protein Gi2. Such an effect was not produced by antibodies against the equivalent region of Goα Gi3α or Gsα or from non‐immune serum. By contrast, total specific binding of [3H]yohimbine was not modified by co‐incubation with Gpp[NH]p or by preincubation with the antibodies from any of the anti‐G protein antisera. These results demonstrate a direct interaction of the α2B adrenergic receptor of NG108‐15 cells with Gi2.
An extension of secondary structure prediction towards the production of tertiary structure
Authors: Garratt Richard C. , Thornton Janet M. , Taylor Willie R.
Published: July 2002

Secondary structure prediction parameters and optimised decision constants for use with the method of Garnier et al. [(1978) J. Mol. Biol. 120, 97–120] have been derived for two new and distinct substates of β‐structure. These we term internal and external on the basis of their hydrogen bonding patterns. The profiles of the amino acids for several of the parameters are considerably different in the two substates. Predictions using the new parameters attempt to distinguish the strands at the core of the β‐sheet from those at its edges an so restrict the possible topologies in tertiary structure prediction. The potential application of this parameters is illustrated for the class of β/α proteins.
The transthyretin cDNA sequence is normal in transthyretin‐derived senile systemic amyloidosis
Authors: Christmanson L. , Betsholtz C. , Gustavsson Å. , Johansson B. , Sletten K. , Westermark P.
Published: July 2002

A variety of mutations leading to amino acid substitutions have been described in the transthyretin gene in association with different familial amyloidoses and have been implicated to be involved in the pathogenesis of amyloid deposits. However, there has been disagreement whether or not a transthyretin mutation is present in the most common form of transthyretin‐derived amyloid, namely senile systemic amyloidosis. Therefore, the cDNA sequence of liver transthyretin was determined in a 91‐year‐old patient with typical senile systemic amyloidosis. This sequence was completely normal and lacked any variation. We conclude that in senile systemic amyloidosis factors other than the presence of a sequentially variant transthyretin must determine the amyloid fibril formation.
Human melanotransferrin (p97) has only one functional iron‐binding site
Authors: Baker Edward N. , Baker Heather M. , Smith Clyde A. , Stebbins Mark R. , Kahn Maria , Hellström Karl Erik , Hellström Ingegerd
Published: July 2002

The iron‐binding properties of melanotransferrin, the tumour‐associated antigen also known as p97, have been investigated by UV/visible and fluorescence spectroscopy, amino acid sequence comparison, and modelling. These show that, in contrast to other transferrins, melanotransferrin binds only one Fe3+ ion per molecule. The binding properties of its N‐terminal site are similar to other transferrins, but its C‐terminal site does not bind iron at all. The differences can be related to specific amino acid changes in the C‐terminal site.
Phosphorylation of smg p21B/rap1B p21 by cyclic GMP‐dependent protein kinase
Authors: Miura Yasushi , Kaibuchi Kozo , Itoh Takahito , Corbin Jackie D. , Francis Sharron H. , Takai Yoshimi
Published: July 2002

smg p21B/rap1B p21, a member of ras p21‐like small GTP‐binding protein superfamily, has been shown to be phosphorylated by cyclic AMP‐dependent protein kinase (protein kinase A). We show here that this protein was also phosphorylated by cyclic GMP‐dependent protein kinase (protein kinase G) in a cell‐free system. The same serine residue (Ser179) in the C‐terminal region was phosphorylated by both protein kinases G and A. The K
m and V
max values of smg p21B for protein kinase G were 5 × 10−7 M and 4 × 10−9 mol/min/mg, and those values for protein kinase A were 1 × 10−7 M and 3 × 10−8 mol/min/mg.
7α‐Hydroxylation of 26‐hydroxycholesterol, 3β‐hydroxy‐5‐cholestenoic acid and 3β‐hydroxy‐5‐cholenoic acid by cytochrome P‐450 in pig liver microsomes
Authors: Toll Anders , Shoda Junichi , Axelson Magnus , Sjövall Jan , Wikvall Kjell
Published: July 2002

Pig liver microsomes were found to catalyze the 7α‐hydroxylation of several potential bile acid precursors besides cholesterol. 26‐Hydroxycholesterol, 3β‐hydroxy‐5‐cholestenoic acid and 3β‐hydroxy‐5‐cholenoic acid were all efficiently converted into the 7α‐hydroxylated products. Two cytochrome P‐450 fractions showing 7α‐hydroxylase activity could be isolated. One fraction catalyzed 7α‐hydroxylation of 26‐hydroxycholesterol. 3β‐hydroxy‐5‐cholestenoic acid and 3β‐hydroxy‐5‐cholenoic acid but was inactive towards cholesterol. The other fraction catalyzed 7α‐hydroxylation of cholesterol in addition to the other substrates. 26‐Hydroxycholesterol in equimolar concentration did not inhibit the cholesterol 7α‐hydroxylase activity of this fraction. It is concluded that liver microsomes contain a cytochrome P‐450 catalyzing 7α‐hydroxylation of 26‐hydroxycholesterol, 3β‐hydroxy‐5‐cholestenoic acid and 3β‐hydroxy‐5‐cholenoic acid. The results indicate that this cytochrome P‐450 is different from that catalyzing 7α‐hydroxylation of cholesterol.
Membrane interactions of mastoparan analogues related to their differential effects on protein kinase C, Na,K‐ATPase and HL60 cells
Authors: Raynor Robert L. , Kim Young-Sook , Zheng Bin , Vogler William R. , Kuo J.F.
Published: July 2002

Membrane interactions of tetradecapeptide toxin mastoparan (MP) and analogues (MP‐3, MP‐X and polistes MP), as indicated by inhibition of various enzymatic and cellular activities, were investigated. MP‐3 was found to be the least active in inhibiting protein kinase C (PKC; activated by phosphatidylserine vesicles, synaptosomal membranes or phorbol ester), synaptosomal membrane Na.K‐ATPase and proliferation and viability or leukemia HL60 cells. MP‐3, however, was as active as others in inhibiting PKC activated by arachidonate monomers and phorbol ester binding. The unique properties or MP‐3, the [des‐lle‐Asn2]‐ analogue or MP, might be related to its low functional amphiphilicity compared to others and useful in further delineating biological activities associated with or regulated by membranes.
Stimulation of human nitric oxide synthase by tetrahydrobiopterin and selective binding of the cofactor
Authors: Klatt Peter , Heinzel Burghard , Mayer Bernd , Ambach Edda , Werner-Felmayer Gabriele , Wachter Helmut , Werner Ernst R.
Published: July 2002

To check the stimulatory potency of the tetrahydro forms of the two major pteridines occurring in human tissues, neopterin and biopterin, NO synthase was purified 6000‐fold from human cerebellum. Tetrahydrobiopterin stimulated the activity up to 4.5‐fold in a concentration dependent manner with a maximum above 1 μM, whereas tetrahydroneopterin was completely inactive in concentrations up to 100 μM. Tetrahydrobiopterin, but not neopterin derivatives, were copurified with the NO synthase activity. Our results demonstrate that human cerebellum contains a tetrahydrobiopterin dependent NO synthase activity.
Cloning and disruption of the gene encoding yeast mitochondrial chaperonin 10, the homolog of E. coli groES
Authors: Rospert Sabine , Junne Tina , Glick Benjamin S. , Schatz Gottfried
Published: July 2002

The mitochondrial chaperonin system consists of chaperonin 60 (also termed hsp60), which is homologous to E. coli groEL, and chaperonin 10, which is homologous to E. coli groES. In yeast, chaperonin 60 function has been shown to be essential for viability. We report here that the same is true for chaperonin 10. We have cloned, sequenced and disrupted the nuclear chaperonin 10 gene CPN10 from Saccharomyces cerevisiae. This gene encodes a protein of 11,372 Da that is imported into the mitochondrial matrix without detectable cleavage. Haploid cells lacking a functional copy of CPN10 fail to grow at temperatures between 23 and 37° C.
The genomic region encoding toxin gamma from the scorpion Tityus serrulatus contains an intron
Authors: Becerril B. , Corona M. , Mejía M.C. , Martin B.M. , Lucas S. , Bolivar F. , Possani L.D.
Published: July 2002

The gene encoding toxin gamma from the scorpion, Tityus serrulatus, was amplified by PCR from genomic DNA employing synthetic oligonucleotides designed from the reported cDNA sequence. The nucleotide sequence of this gene reveals the presence of an intron of 475 base pairs (bp) which interrupts the region that encodes the signal peptide of the precursor toxin. A comparison of the intron boundary sequences of the gamma toxin gene with ones from other arachnid genes is also presented.
Presence of lipocortins I and IV, but not II and VI, in human platelets
Authors: Eldering Joyce A. , Kocher Markus , Clemetson Jeannine M. , Clemetson Kenneth J. , Frey Felix J. , Frey Brigitte M.
Published: July 2002

The present investigation revealed the presence of lipocortins I and IV, but not lipocortins II and VI, in human platelets. Lipocortin I was found in the Triton‐soluble fraction of both resting and thrombin‐activated platelets and was not covalently bound to skeletal components. Without detergents, when resting platelets were lysed and fractionated in the absence of Ca2+, lipocortin I was found only in the cytosolic fraction, whereas, in the presence of Ca2+, lipocortin I was associated only with the crude particulate and not with the membrane nor the cytosolic fractions.
Evidence for release of free glucose from muscle during amylin‐induced glycogenolysis in rats
Authors: Young Andrew A. , Carlo Pam , Smith Pam , Wolfe-Lopez Deborah , Pittner Richard , Wang Ming-Wei , Rink Timothy
Published: July 2002

Amylin, a 37 amino acid product of pancreatic β‐cells, inhibits insulin‐stimulated radioglucose incorporation into muscle glycogen. In the current study, we exercised rats and then prelabelled the glycogen pool by infusing [3‐3H]glucose along with glucose and insulin. Subsequent amylin administration increased the rate of appearance of tritiated water 6.4‐fold, consistent with stimulation of glycogenolysis and passage of the released moieties through the hexose → triose step of glycolysis. Further, there was an increase in plasma [3‐3H]glucose after amylin, consistent with the release of free glucose previously sequestered in muscle glycogen. Calcitonin gene‐releated peptide (8–37), an amylin antagonist, prevented these actions.
Effects of estradiol on very low density lipoprotein receptor mRNA levels in rabbit heart
Authors: Masuzaki Hiroaki , Jingami Hisato , Yamamoto Tokuo , Nakao Kazuwa
Published: July 2002

The VLDL receptor, a newly identified lipoprotein receptor, has a domain structure homologous with the LDL receptor but has one additional cysteine rich repeat in the ligand binding domain. To study the regulation of the VLDL receptor in vivo, we administered 17 α‐ethinyl estradiol to rabbits and examined its effects on VLDL receptor mRNA levels in the heart, one of the organs with highly expressed VLDL receptors, by RNA blotting. The ventricular level of VLDL receptor mRNA increased dramatically after estradiol administration. We could not detect VLDL receptor mRNA in the liver even after estradiol administration. We have confirmed the enhanced expression of liver LDL receptor mRNA by estradiol, however, only weak expression of the LDL receptor was detected in the ventricle of the rabbit heart. These results suggest that estradiol exerts its effect on the VLDL receptor gene expression in the heart.
Effect of intracellular Ca2+ concentration on endothelin‐1 secretion
Authors: Brunner Friedrich , Stessel Heike , Simecek Sabine , Graier Wolfgang , Kukovetz Walter R.
Published: July 2002

The role of intracellular free Ca2+ concentration ([Ca2+]i in cellular regulation of endothelin‐(ET‐1) secretion was investigated in cultured porcine aortic endothelial cells of first passage. Intracellular Ca2+ concentration were adjusted between 50 nM and 1 μM using EGTA and thapsigargin, respectively. ET‐1 secretion was maximal at [Ca2+]i of 190–470 nM, and reduced at low (50 and 110 nM) and high (470 nM) [Ca2+]i. The Ca2+ ionophores A23187 and ionomycin (each 1 μM), both of which raise [Ca2+]i above 1 μM, also potently inhibited ET‐1 secretion under basal and stimulated conditions. The A23187‐induced reduction in ET‐1 secretion was not affected by N
G‐nitro‐l‐arginine (0.1 mM). Our results provide evidence that basal ET‐1 secretion is regulated by Ca2+ and that Ca2+ ionophores reduce ET‐1 secretion due to the inhibitory effect of high [Ca2+]i.
Collagen stimulates tyrosine phosphorylation of phospholipase C‐γ2 but not phospholipase C‐γ1 in human platelets
Authors: Blake Robert A. , Schieven Gary L. , Watson Steve P.
Published: July 2002

Collagen is an important primary stimulus of platelets during the process of hemostasis. As with many other platelet stimuli, collagen signal transduction involves the hydrolysis of inositol phospholipids; however, the mechanism which underlies this event is not well understood. Neither the collagen receptor nor the isoform of phospholipase C that is activated have been identified. We report that collagen‐activation of platelets induces tyrosine phosphorylation of phospholipase C‐γ2 but not phospholipase C‐γ1. We also show that the platelet low affinity Fc receptor (FcγRII), which mediates activation of platelets by immune complexes, and wheat germ agglutinin, which binds non‐specifically to glycoprotein, stimulate phospholipase C‐γ2 tyrosine phosphorylation. In contrast, we could not detect phospholipase C‐γ2 tyrosine phosphorylation in platelets stimulated by either thrombin or a stable thromboxane A2 analogue, U46619.
Tumor necrosis factor enhances low density lipoprotein oxidative modification by monocytes and endothelial cells
Authors: Maziere Cécile , Auclair Martine , Maziere Jean-Claude
Published: July 2002

The effect of tumor necrosis factor on the oxidative modification of LDL by U937 human monocytes or murine endothelial cells was studied by determination of the lipid peroxidation product content and the electrophoretic mobility of the particle. In the range of concentrations from 2.5 to 10 , the cytokine induced a dose‐dependent increase in cellular‐induced oxidation of LDL. This effect was accompanied by a stimulation of LDL degradation by J774 macrophage‐like cells. Concurrently, the TNF‐treated cells secreted superoxide anion with a higher rate. Since LDL oxidation is believed to be an inportant feature in the formation of the atherosclerotic plaque, the described effects of TNF might be of importance in long‐term exposure to this cytokine during inflammation.
Molecular cloning of the canine angiotensin II receptor
Authors: Burns Louise , Clark Kenneth L. , Bradley John , Robertson Mark J. , Clark Adrian J.L.
Published: July 2002

Canine aortic strip studies revealed insensitivity of angiotensin II (AII)‐induced aortic contraction to inhibition by the non‐peptide antagonist DuP753 (pK
B = 6.7 ± 0.1). In order to determine the origin of this phenomenon we cloned the canine homologue of the AT1 AII receptor. Expression of this cDNA in COS‐7 cells indicated a low affinity of DuP753 for the cloned receptor (K
D = 92 nM). The predicted amino acid sequence is highly homologous to other mammalian AT1 receptors; sequence comparisons suggest the pharmacological difference may be the result of a threonine residue in position 163 in the IVth transmembrane domain.
Expression of the catalytic domain of cyclic GMP‐dependent protein kinase in a baculovirus system
Authors: Boerth Nancy J. , Lincoln Thomas M.
Published: July 2002

The Type I cGMP‐dependent protein kinase catalytic domain (residues 336‐671 from the Iα isofonn) has been expressed as a cGMP independent kinase in a baculovirus system. Using peptide substrates, the protein retains similar substrate specificity as the native holoenzyme. The recombinant catalytic domain catalyzes the phosphorylation of histone, but does not display the inhibition using non‐substrate histones which has been described for the holoenzyme. The catalytic domain is an active kinase in mammalian cells also since vascular smooth muscle cells transfected with the cDNA encoding the catalytic domain display altered morphology. The catalytic domain of G‐kinase may be a useful tool for delineating the role of cGMP‐mediated protein phosphorylation in cell systems.
Immunological changes in a case of Marchiafava Bignami disease: response to amineptine treatment
Authors: Humbert T , Bureau JP , Chabrand P
Published: July 2002

Marchiafava-Bignami disease (MBD) is a rare complication of chronic alcoholism. We describe a regressive form of MBD in which a primary decrease in CD4+ cells was improved by amineptine treatment. No significant change in the CD4 : CDS ratio was observed in a few major depressed subjects receiving this drug.
Characterization of the c‐specific promoter of the gene encoding human endothelin‐converting enzyme‐1 (ECE‐1)
Authors: Funke-Kaiser H. , Bolbrinker J. , Theis S. , Lemmer J. , Richter C.-M. , Paul M. , Orzechowski H.-D.
Published: July 2002

Human ECE‐1 is expressed in four isoforms with different tissue distribution and its mRNA and protein levels are altered under certain pathophysiological conditions. To investigate the transcriptional regulation of ECE‐1, we studied the regulatory region of ECE‐1c, the major ECE‐1 isoform. A genomic clone comprising the complete human ECE‐1 gene including the putative ECE‐1c‐specific promoter was obtained. Up to 968 bp upstream of the putative c‐specific translation initiation start codon and several serial deletion mutants were subcloned into a reporter vector and transfected into endothelial (BAEC, EA.hy926, ECV304) and epithelial (MDA MB435S, MCF7) cells, showing very strong promoter activity in comparison to the SV40 promoter and to the previously described ECE‐1a and 1b promoters. Transfection of serial deletion mutants indicated two positive regulatory regions within the promoter (−142/−240 and −240/490) likely involved in binding GATA and ETS transcription factors. RNase protection assay (RPA) and 5′‐RACE revealed multiple transcriptional start sites located at about −110, −140 and −350 bp. Site‐directed mutagenesis demonstrated a crucial role for the E2F cis‐element for basal ECE‐1c promoter activity. Additionally, we found a correlation between isoform‐specific ECE‐1 mRNA levels and corresponding ECE‐1a, 1b, 1c promoter activities.
Desethylamiodarone interferes with the binding of co‐activator GRIP‐1 to the β1‐thyroid hormone receptor
Authors: van Beeren H.C , Bakker O , Wiersinga W.M
Published: July 2002

Ligand binding to the thyroid hormone nuclear receptor β1 (TRβ1) is inhibited by desethylamiodarone (DEA), the major metabolite of the widely used anti‐arrhythmic drug amiodarone. Gene expression of thyroid hormone (triiodothyronine, T3)‐regulated genes can therefore be affected by amiodarone due to less ligand binding to the receptor. Previous studies have indicated the possibility of still other explanations for the inhibitory effects of amiodarone on T3‐dependent gene expression, probably via interference with receptor/co‐activator and co‐repressor complex. The binding site of DEA is postulated to be on the outside surface of the receptor protein overlapping the regions where co‐activator and co‐repressor bind. Here we show the effect of a drug metabolite on the interaction of TRβ1 with the co‐activator GRIP‐1 (glucocorticoid receptor interacting protein‐1). The T3‐dependent binding of GRIP‐1 to the TRβ1 is disrupted by DEA. A DEA dose experiment showed that the drug metabolite acts like an antagonist under ‘normal’ conditions (at 10−7 M T3 and 5×10−6→10−3 M DEA), but as an agonist under extreme conditions (at 0 and 10−9 M T3 and >10−4 M DEA). To our knowledge, these results show for the first time that a metabolite of a drug which was not devised for this purpose can interfere with nuclear receptor/co‐activator interaction.
Functional domains of the ATPase inhibitor protein from bovine heart mitochondria
Authors: Zanotti Franco , Raho Gabriella , Vuolo Rita , Gaballo Antonio , Papa Francesco , Papa Sergio
Published: July 2002

A study is presented of the activity and temperature dependence of the ATPase inhibitor protein (IF1) from bovine heart mitochondria and of synthetic partial IF1 peptides. The results show that the IF1‐(42–58) peptide is the most potent inhibitory domain of IF1.
Functional coupling with Gαq and Gαi1 protein subunits promotes high‐affinity agonist binding to the neurotensin receptor NTS‐1 expressed in Escherichia coli
Authors: Grisshammer Reinhard , Hermans Emmanuel
Published: July 2002

To analyze the coupling of Gα subunits to the rat neurotensin receptor NTS‐1 (NTR), fusion proteins were expressed in Escherichia coli with various Gα subunits covalently linked to the receptor C‐terminus. The presence of Gαq or Gαi/q, in which the six C‐terminal residues of Gαi1 were replaced with those from Gαq, increased the percentage of receptors in the agonist high‐affinity state. This effect was less pronounced for wild‐type Gαi1 and not observed for Gαi/s. Functional coupling of neurotensin receptor to Gα was demonstrated by neurotensin‐induced [35S]GTPγS binding for the Gαq, Gαi/q and Gαi1 subunits, but not for Gαi/s. Our results extend previous findings of the dual coupling of NTR to pertussis toxin‐sensitive and ‐insensitive G‐proteins in Chinese hamster ovary cells with preference for the latter.
Phosphorylation of the C2 subunit of the proteasome in rice (Oryza sativa L.)
Authors: Umeda Masaaki , Manabe Yuzuki , Uchimiya Hirofumi
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Regulation of human renin secretion and gene transcription in Calu‐6 cells
Authors: Germain Stéphane , Philippe Josette , Fuchs Sébastien , Lengronne Armelle , Corvol Pierre , Pinet Florence
Published: July 2002

Calu‐6 cells were characterized for studying the transcriptional regulation of the human renin gene. Analysis of cis‐acting elements of the renin promoter showed the highest activity within the first 582 bp in serum‐free conditions and of the 892 bp in the presence of serum. cAMP activates renin mRNA synthesis parallel to renin production (20‐fold increase) as well renin promoter activity (2‐fold). cAMP response element and the (−77 to −67) element are both necessary for activation of the renin promoter but do not act independently. Functional analysis of Intron A revealed the presence of a silencer specific to renin‐producing cells.
Birch pollen profilin: structural organization and interaction with poly‐(l‐proline) peptides as revealed by NMR
Authors: Domke Tobias , Federau Torsten , Schlüter Kathrin , Giehl Klaudia , Valenta Rudolf , Schomburg Dietmar , Jockusch Brigitte M.
Published: July 2002

The secondary structure of birch pollen profilin, a potent human allergen, was elucidated by multidimensional nuclear magnetic resonance (NMR), as a prerequisite to study the interaction of this profilin with ligands for its poly‐(l‐proline) (PLP)‐binding site. The chemical shifts of the 15N‐labeled backbone amide groups were used to monitor complex formation with various PLP peptides. Titration with deca‐l‐proline (P10) yielded a K
D of 0.2 mM. P8 was the shortest PLP to provoke a significant reaction. (GP5)3G bound significantly, confirming the interaction between profilins and the protein VASP containing this motif. Birch profilin interacted also with GP6GP5, found in the cyclase‐associated protein (CAP), a suspected profilin ligand.
Human serotonin 5‐HT7 receptor: cloning and pharmacological characterisation of two receptor variants
Authors: Stam Nico J , Roesink Carolien , Dijcks Fred , Garritsen Anja , van Herpen Anne , Olijve Wiebe
Published: July 2002

Two splice variants of the 5‐HT7 receptor were identified in human brain that differ in the lengths of their intracellular carboxy terminal tail. Identification of the variants of this receptor is of particular interest since the 5‐HT7 receptor is known to have a high affinity for a number of antidepressants and is localized in brain regions thought to be implicated in depression. The two isoforms are expressed in roughly equal amounts in various regions of the human brain. When expressed in NIH‐3T3 cells, both variants encode functional 5‐HT7 receptors, positively coupled to adenylyl cyclase. We suggest that both variants are derived from a single gene by alternative mRNA splicing. Furthermore, our results from Southern blot analysis studies suggest that additional 5‐HT7 receptor genes may exist in human.
Protein oxidation, tyrosine nitration, and inactivation of sarcoplasmic reticulum Ca2+‐ATPase in low‐frequency stimulated rabbit muscle
Authors: Klebl Bert M , Ayoub Ahmed T , Pette Dirk
Published: July 2002

Sustained contractile activity by chronic low‐frequency stimulation in rabbit fast‐twitch muscle causes a partial (40–50%) inactivation of the sarcoplasmic reticulum (SR) Ca2+‐ATPase and, with prolonged stimulation, a SERCA1a to SERCA2a transition. To investigate the underlying mechanism of the inactivation which precedes the isoform transition, we analyzed SR from 4‐day stimulated muscles for Ca2+‐ATPase activity, lipid peroxidation, SH and carbonyl groups, and nitrotyrosine. At unaltered SH group and malondialdehyde contents, carbonyl groups were elevated 50% in the SR from stimulated muscles. Immunoblotting with anti‐dinitrophenyl and anti‐nitrotyrosine antibodies revealed strong labeling of the Ca2+‐ATPase, suggesting the inactivation of the enzyme to result from protein oxidation and peroxynitrite‐mediated tyrosine nitration.
Sec/SRP‐independent insertion of two thylakoid membrane proteins bearing cleavable signal peptides
Authors: Kim Soo Jung , Robinson Colin , Mant Alexandra
Published: July 2002

Two imported thylakoid membrane proteins, PSII‐X and PSII‐W, are synthesised with cleavable N‐terminal signal peptides that closely resemble those of Sec‐dependent lumenal proteins. In this report we have reconstituted the insertion of pre‐PSII‐X and pre‐PSII‐W into isolated thylakoids. We show that insertion does not require either nucleoside triphosphates or stromal extracts, both of which are required for Sec‐ and signal recognition particle (SRP)‐dependent targeting mechanisms. Insertion is furthermore unaffected by protease treatments that destroy the known protein translocation apparatus in the thylakoid membrane. We conclude that these membrane proteins are inserted by an unusual Sec/SRP‐independent mechanism that probably resembles that used by CFoII, and we discuss possible parallels with the biogenesis of phage M13 procoat.
Altered expression of α‐actin, smooth muscle myosin heavy chain‐1 and calponin in cultured smooth muscle cells by oxidized low density lipoproteins
Authors: Damiani Elisabetta , Sugiyama Tomoyasu , Shimamura Kyoko , Greci Lucedio , Matsuda Yuzuru
Published: July 2002

The expression of the contractile proteins, α‐actin, smooth muscle myosin heavy chain‐1 (SM1) and calponin present in smooth muscle cells (SMC) in the presence of oxidized low density lipoproteins (oxLDL) was investigated in two different cell cultures: the mouse smooth muscle cell line SVSC and rat smooth muscle cells (RSMC). Exposure of the cells to 187 μg protein/ml oxLDL for 24 h reduced the expression of all three contractile proteins in both cell cultures when compared to cells incubated in the presence of native LDL. This investigation of the response of SMC contractile proteins to oxLDL may provide further insights into the mechanisms by which oxidatively modified LDL is atherogenic and suggests that oxLDL may contribute to the regulation of the expression of the genes responsible for the synthesis of smooth muscle cell contractile proteins.
Activation of two types of brain glutamate dehydrogenase isoproteins by gabapentin
Authors: Cho Sung-Woo , Cho Eun Hee , Choi Soo Young
Published: July 2002

The stimulatory effects of gabapentin on the activities of two types of glutamate dehydrogenase (GDH) isoproteins homogeneously purified from bovine brain have been studied at various conditions. When the effects of different gabapentin concentrations on GDH activities were studied in the direction of reductive amination of 2‐oxoglutarate with NADPH as a coenzyme, a marked activation was observed for both isoproteins, whereas both isoproteins showed activation to a lesser extent with NADH as a coenzyme. Stimulatory effects of gabapentin on GDH activities in the direction of the oxidative deamination of glutamate were also observed, but to a much lesser extent than reductive amination. There were big differences between the two GDH isoproteins in their sensitivity to the action of gabapentin. The largest activation was observed with GDH II when NADPH was used as a coenzyme. Half‐maximal stimulation was reached at around 1.5 mM. Gabapentin relieved the inhibition of GDH isoproteins by GTP and this resulted in an increase in the apparent activation by gabapentin in the presence of GTP. 2‐Oxoglutarate was found to give rise to high substrate inhibition and gabapentin reduced the substrate inhibition in the presence of 0.2 mM NADH. Since there are neurodegenerative disorders in which GDH activity is decreased, the therapeutic modulation of the activity of this enzyme may be clinically useful.
The mechanism of proton transport mediated by mitochondrial uncoupling proteins
Authors: Garlid Keith D. , Jabůrek Martin , Ježek Petr
Published: July 2002

The effort to understand the mechanism of uncoupling by UCP has devolved into two models – the fatty acid protonophore model and the proton buffering model. Evidence for each hypothesis is summarized and evaluated. We also evaluate the obligatory requirement for fatty acids in UCP1‐mediated uncoupling and the question of fatty acid affinity for UCP1. The structural bases of UCP transport function and nucleotide inhibition are discussed in light of recent mutagenesis studies and in relationship to the sequences of newly discovered UCPs.
Substituting selenocysteine for active site cysteine 149 of phosphorylating glyceraldehyde 3‐phosphate dehydrogenase reveals a peroxidase activity
Authors: Boschi-Muller Sandrine , Muller Sabine , Van Dorsselaer Alain , Böck August , Branlant Guy
Published: July 2002

Replacing the essential Cys‐149 by a selenocysteine into the active site of phosphorylating glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus leads to a selenoGAPDH that mimics a selenoperoxidase activity. Saturation kinetics were observed with cumenyl and tert‐butyl hydroperoxides, with a better catalytic efficiency for the aromatic compound. The enzymatic mechanism fits a sequential model where the formation of a ternary complex between the holoselenoenzyme, the 3‐carboxy 4‐nitrobenzenethiol used as the reductant and the hydroperoxide precedes product release. The fact that the selenoGAPDH is NAD‐saturated supports a binding of hydroperoxide and reductant in the substrate binding site. The catalytic efficiency is similar to selenosubtilisins but remains low compared to selenoglutathione peroxidase. This is discussed in relation to what is known from the X‐ray crystal structures of selenoglutathione peroxidase and GAPDHs.
Actin dynamics in lamellipodia of migrating border cells in the Drosophila ovary revealed by a GFP‐actin fusion protein
Authors: Verkhusha Vladislav V , Tsukita Shoichiro , Oda Hiroki
Published: July 2002

Directional migration of border cells in the Drosophila egg chambers is a developmentally regulated event that requires dynamic cellular functions. In this study, the electron microscopic observation of migrating border cells revealed loose actin bundles in forepart lamellipodia and numerous microvilli extending from nurse cells and providing multiple adhesive contacts with border cells. To analyze the dynamics of actin in migrating border cells in vivo, we constructed a green fluorescent protein‐actin fusion protein and induced its expression in Drosophila using the GAL4/UAS system. The green fluorescent protein‐actin was incorporated into the actin bundles and it enabled visualization of the rapid cytoskeletal changes in border cell lamellipodia. During the growth of the lamellipodia, the actin bundles that increased in number and size radiated from the bundle‐organizing center. Quantification of the fluorescence intensity showed that an accumulation of bundle‐associated and spotted green fluorescent protein‐actin signals took place during their centripetal movement. Our results favored a treadmilling model for actin behavior in border cell lamellipodia.
Oxidation of low density lipoprotein and plasma by 15‐lipoxygenase and free radicals
Authors: Yamashita Hiromasa , Nakamura Akio , Noguchi Noriko , Niki Etsuo , Kühn Hartmut
Published: July 2002

It is generally accepted that the oxidation of pentadiene structures of polyunsaturated lipids by lipoxygenase (LOX) is regio‐ and enantio‐specific, while the free radical‐mediated lipid peroxidation gives stereo‐random racemic products. It was confirmed that the oxidation of human low density lipoprotein (LDL) by 15‐LOX from rabbit reticulocytes gave phosphatidylcholine (PC) and cholesteryl ester (CE) hydroperoxides regio‐, stereo‐ and enantio‐specifically. 15‐LOX also oxidized human plasma to give specific PC and CE hydroperoxides in spite of the presence of high concentrations of antioxidants. More CE hydroperoxides were formed than PC hydroperoxides from LDL, but the reverse order was observed for plasma oxidation. The S/R ratio of the hydroperoxides decreased during long time incubation but remained significantly larger than one, while free radical‐mediated oxidation of LDL and plasma gave racemic products.
Ultrasensitive glycogen synthesis inCyanobacteria
Authors: Gómez Casati Diego F , Aon Miguel A , Iglesias Alberto A
Published: July 2002

CyanobacterADPglucose pyrophosphorylase exhibits a ultrasensitive response in activity towards its allosteric effector 3‐phosphoglycerate, elicited by orthophosphate and polyethyleneglycol‐induced molecular crowding. The ultrasensitive response was observed either when the enzyme operates in the zero or first order region for its physiological substrates. The ultrasensitivity exhibited maximal amplification factors of 15–19‐fold with respect to 1% of the maximal system velocity. Only a 2.4–3.8‐fold increase in 3PGA concentration was necessary to augment the flux from 10% to 90% through AGPase as compared with 200‐fold required for the control. The results are discussed in terms of finely tuned regulatory mechanisms of polysaccharide synthesis in oxygenic photosynthetic organisms.
Cell membrane dynamics and the induction of apoptosis by lipid compounds
Authors: Fujimoto Keiji , Iwasaki Chizu , Kawaguchi Haruma , Yasugi Etsuko , Oshima Mieko
Published: July 2002

To investigate the induction of apoptosis by some lipid compounds which are a potent inducer of apoptosis, the plasma membrane fluidity of U937 cells was measured using the fluorescent probe, pyrene. The increase of the membrane fluidity was observed immediately after the treatment of cells with lipid inducers. We also found that the trigger of apoptosis was pulled within 30 min after treatment. Data from the dynamic light scattering experiment indicated that lipid inducers were dissolved to form the emulsion. At the very early stage of apoptosis, possibly, the well‐controlled transfer of lipid inducers from the emulsion to the lipid layer of cells can bring about the increase of membrane dynamics which might lead to the induction of apoptosis.
Thermostable aminopeptidase from Pyrococcus horikoshii
Authors: Ando Susumu , Ishikawa Kazuhiko , Ishida Hiroyasu , Kawarabayasi Yutaka , Kikuchi Hisasi , Kosugi Yoshitsugu
Published: July 2002

From the genome sequence data of the thermophilic archaeon Pyrococcus horikoshii, an open reading frame was found which encodes a protein (332 amino acids) homologous with an endoglucanase from Clostridium thermocellum (42% identity), deblocking aminopeptidase from Pyrococcus furiosus (42% identity) and an aminopeptidase from Aeromonas proteolytica (18% identity). This gene was cloned and expressed in Escherichia coli, and the characteristics of the expressed protein were examined. Although endoglucanase activity was not detected, this protein was found to have aminopeptidase activity to cleave the N‐terminal amino acid from a variety of substrates including both N‐blocked and non‐blocked peptides. The enzyme was stable at 90°C, with the optimum temperature over 90°C. The metal ion bound to this enzyme was calcium, but it was not essential for the aminopeptidase activity. Instead, this enzyme required the cobalt ion for activity. This enzyme is expected to be useful for the removal of N
α‐acylated residues in short peptide sequence analysis at high temperatures.
The starch‐binding domain from glucoamylase disrupts the structure of starch
Authors: Southall Stacey M. , Simpson Peter J. , Gilbert Harry J. , Williamson Gary , Williamson Michael P.
Published: July 2002

The full‐length glucoamylase from Aspergillus niger, G1, consists of an N‐terminal catalytic domain followed by a semi‐rigid linker (which together constitute the G2 form) and a C‐terminal starch‐binding domain (SBD). G1 and G2 both liberate glucose from insoluble corn starch, although G2 has a rate 80 times slower than G1. Following pre‐incubation of the starch with SBD, the activity of G1 is uniformly reduced with increasing concentrations of SBD because of competition for binding sites. However, increasing concentrations of SBD produce an initial increase in the catalytic rate of G2, followed by a decrease at higher SBD concentrations. The results show that SBD has two functions: it binds to the starch, but it also disrupts the surface, thereby enhancing the amylolytic rate.
Microinjected glutathione reductase crystals as indicators of the redox status in living cells
Authors: Keese Michael A. , Saffrich Rainer , Dandekar Thomas , Becker Katja , Schirmer R.Heiner
Published: July 2002

The flavoenzyme glutathione reductase catalyses electron transfer reactions between two major intracellular redox buffers, namely the NADPH/NADP+ couple and the 2 glutathione/glutathione disulfide couple. On this account, microcrystals of the enzyme were tested as redox probes of intracellular compartments. For introducing protein crystals into human fibroblasts, different methods (microinjection, particle bombardment and optical tweezers) were explored and compared. When glutathione reductase crystals are present in a cytosolic environment, the transition of the yellow Eox form to the orange‐red 2‐electron reduced charge transfer form, EH2, is observed. Taking into account the midpoint potential of the Eox/EH2 couple, the redox potential of the cytosol was found to be <−270 mV at pH 7.4 and 37°C. As a general conclusion, competent proteins in crystalline – that is signal‐amplifying – form are promising probes for studying intracellular events.
Implication of proteasome in estrogen receptor degradation
Authors: El Khissiin Abdelhamid , Leclercq Guy
Published: July 2002

In MCF‐7 breast cancer cells, estradiol (E2) and pure antiestrogen RU 58668 down‐regulate the estrogen receptor (ER). Interestingly, the protein synthesis inhibitor cycloheximide (CHX) abrogated solely the effect of E2 suggesting a selective difference in the degradation of the receptor induced by estrogenic and antiestrogenic stimulations. A panel of lysosome inhibitors (i.e. bafilomycin, chloroquine, NH4Cl, and monensin), calpain inhibitors (calpastatin and PD 150606) and proteasome inhibitors (lactacystin and proteasome inhibitor I) were tested to assess this hypothesis. Among all inhibitors tested, lactacystin and proteasome inhibitor I were the sole inhibitors to abrogate the elimination of the receptor induced by both E2 and RU 58668; this selective effect was also recorded in cells prelabeled with [3H]tamoxifen aziridine before exposure to these ligands. Hence, differential sensitivity to CHX seems to be linked to the different mechanisms which target proteins for proteasome‐mediated destruction. Moreover, the two tested proteasome inhibitors produced a slight increase of ER concentration in cells not exposed to any ligand, suggesting also the involvement of proteasome in receptor turnover.
MEK is a negative regulator of Stat5b in PDGF‐stimulated cells
Authors: Valgeirsdóttir Sigrı́dur , Ruusala Aino , Heldin Carl-Henrik
Published: July 2002

In this study we show that platelet‐derived growth factor (PDGF)‐induced DNA binding as well as transcriptional activation of Stat5b are markedly increased by inhibition of the MAP (mitogen‐activated protein) kinase kinase MEK. In addition to the previously demonstrated tyrosine phosphorylation, we show that serine and threonine phosphorylation of Stat5b is increased in response to PDGF stimulation. However, inhibition of MEK had no effect on the phosphorylation level of Stat5b or on the nuclear translocation of Stat5b. These observations indicate that MEK is a negative modulator of PDGF‐induced Stat5b activation through a mechanism not involving direct phosphorylation of Stat5b.