bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of heterologous phosphofructokinase genes in yeast
Authors: Heinisch Jürgen J.
Published: July 2002

Genes encoding phosphofructokinases (PFK) from Escherichia coli and from the human muscle were expressed in PFK‐deficient strains of Saccharomyces cerevisiae under the control of an inducible GAL1 promoter. They restored PFK activity under inducing conditions and complemented the galactose‐negative growth phenotype of the recipient strains. The PFK enzymes expressed appear to be stable in yeast. The human muscle enzyme crossreacts with specific antibodies and shows the expected subunit size. As expected, its activity can be activated by fructose‐2,6‐bisphosphate, in contrast to the bacterial enzyme.
Site‐directed mutagenesis of AMP‐binding residues in adenylate kinase Alteration of substrate specificity
Authors: Okajima Toshihide , Tanizawa Katsuyuki , Fukui Toshio
Published: July 2002

Adenylate kinase is highly specific for AMP as phosphoryl acceptor. We have found that the replacement of Thr39 by Ala in the chicken muscle enzyme, alone or together with the replacement of Leu66 by Ile, caused remarkable increases in CMP and UMP activities with a concomitant decrease in AMP activity; therefore, the resulting mutant enzymes show CMP and UMP activities/AMP activity ratios much higher than the wild‐type enzyme. The mutant enzyme in which Ala is substituted for Thr39 has a V
max value for CMP comparable to that of CMP‐UMP kinase.
Expression of mRNAs encoding the α1 and the β1 subunits of Na+,K+‐ATPase in human lymphocytes activated with phytohaemagglutinine
Authors: Vereninov A.A. , Marakhova I.I. , Osipov V.V. , Toropova F.V.
Published: July 2002

Increase in Na+,K+‐ATPase mRNAs was detected in activated lymphocytes by the RT‐PCR method. α1 subunit mRNA gradualy increased with time and by 36 h was 2.4 times higher than at the start. Increase in the β1 mRNA was transient reaching a maximum in the 8 h probe and declining to the initial level in the 24 and 36 h probes. The elevation of Na+,K−‐ATPase mRNAs does not underlie a cycloheximide‐inhibited increase in cation pumping peculiar to the prereplicative period as can be judged from the fact that Act D fails to eliminate PHA‐induced enhancement of pump fluxes.
Electrogenicity at the secondary quinone acceptor site of cyanobacterial photosystem II
Authors: Mamedov Mahir D. , Beshta Oleg E. , Samuilov Vitaly D. , Semenov Alexey Yu.
Published: July 2002

Flash‐induced generation of the electric potential difference (ΔΨ) by a direct electrometrical method was studied in Anacystis nidulans photosystem II‐containing proteoliposomes associated with a phospholipid‐impregnated collodion film. Besides a rapid phase of ΔΨ generation corresponding to charge separation between P680 and QA, an additional electrogenic phase with a characteristic time of 0.27 ms at pH 7.0 was observed after the second laser flash. The maximal amplitude of this phase was approx. 4% of that related to the P680+Q−
A formation. The sensitivity of this phase to DCMU, the flash‐number dependence of its amplitude as well as the amplitude and the rate constant pH‐dependences, indicate that it is due to the dismutation of Q−
A and Q−
B and to subsequent protonation of a doubly reduced plastoquinone Q2−
B.
Cloning of an isoform of mouse TGF‐β type II receptor gene
Authors: Suzuki Atsushi , Shioda Noriko , Maeda Toshihiko , Tada Masazumi , Ueno Naoto
Published: July 2002

A variant of transforming growth factor‐β type II receptor (TGF‐βRII) cDNA was isolated from a mouse brain cDNA library. The predicted receptor is identical to previously reported mouse TGF‐βRII except that the isoform has an insertion sequence of 25 amino acids in the predicted ligand‐binding domain. By the use of reverse transcription‐polymerase chain reaction (RT‐PCR), transcripts for both isoforms were detected in all tissues and developing embryos examined. The isoform transiently expressed in COS cells showed a similar ligand‐binding specificity to authentic TGF‐βRII. These results suggest that the mouse TGF‐βRII gene generates multiple isoforms, possibly by alternative splicing, as reported for activin type IIB receptor; and an isoform which has the extra sequence in the ligand‐binding domain is also involved in the TGF‐β signal transduction.
Impaired copper binding by the H46R mutant of human Cu,Zn superoxide dismutase, involved in amyotrophic lateral sclerosis
Authors: Carrì M.Teresa , Battistoni Andrea , Polizio Francesca , Desideri Alessandro , Rotilio Giuseppe
Published: July 2002

Several point mutations in the gene coding for human Cu,Zn superoxide dismutase have been reported as being responsible for familial amyotrophic lateral sclerosis (FALS). However, no direct demonstration has been provided for a correlation between total superoxide dismutase activity and severity of the FALS pathology. In order to get a better insight into the mechanism (s) underlying the FALS phenotype, we have investigated the activity and the copper binding properties of the single mutant H46R, which is associated with a Japanese form of FALS. We have shown that this mutant is structurally stable but lacks significant enzyme activity and has impaired capability of binding catalytic copper. The mutant protein can be fully reconsituted with copper in vitro but its ESR spectrum displays an axial shape quite different from that of the wild‐type.
Site‐specific mutagenesis of the reaction centre from Rhodobacter sphaeroides studied by Fourier transform Raman spectroscopy: mutations at tyrosine M210 do not affect the electronic structure of the primary donor
Authors: Jones Michael R. , Heer-Dawson Manuel , Mattioli Tony A. , Hunter C.Neil , Robert Bruno
Published: July 2002

The effects of mutation of residue tyrosine M210 on the primary donor bacteriochlorophylls have been investigated by near infrared FT‐Raman spectroscopy in reaction centres purified from an antenna‐deficient strain of Rhodobacter sphaeroides. We find that mutation at the M210 position does not significantly perturb the distribution of the unpaired electron over the pair of bacteriochlorophyll molecules which constitute the primary donor radical cation. We conclude, therefore, that the effects of mutation of tyrosine M210 on the rate and asymmetry of primary electron transfer in reaction centres cannot be ascribed to a change in the electronic structure of the primary donor.
Structure of partially‐activated E. coli heat‐labile enterotoxin (LT) at 2.6 Å resolution
Authors: Merritt Ethan A. , Pronk Sylvia E. , Sixma Titia K. , Kalk Kor H. , van Zanten Ben A.M. , Hol Wim G.J.
Published: July 2002

Biological toxicity of E. coli heat‐labile enterotoxin and the closely related cholera toxin requires that the assembled toxin be activated by proteolytic cleavage of the A subunit and reduction of a disulfide bond internal to the A subunit. The structural role served by this reduction and cleavage is not known, however. We have crystallographically determined the structure of the E. coli heat‐labile enterotoxin AB5 hexamer in which the A subunit has been cleaved by trypsin between residues 192 and 195. The toxin is thus partially activated, in that it has been cleaved but the disulfide bond has not been reduced. The structure of the A subunit in the cleaved toxin is substantially the same as that previously observed for the uncleaved AB5 structure, suggesting that although such cleavage is required for biological activity of the toxin it does not by itself cause a conformational change.
Reaction of human α2‐antiplasmin and plasmin Stopped‐flow fluorescence kinetics
Authors: Christensen Ulla , Bangert Kristian , Thorsen Sixtus
Published: July 2002

The interaction of human plasmin with human α2‐antiplasmin was measured in the presence and absence of lysine‐binding ligands using the corresponding active site fluorescence changes. The stopped‐flow method allows for direct determination of reliable values of the second order rate constant for the fast association step of plasmin and α
2‐antiplasmin in the absence of another interacting compound, e.g. a plasmin substrate. At pH 7.4,25°C, k
1= 2.2 X 107 M
−1 s
−1 was obtained. Substantial reductions in k
1 were seen in the presence of trans‐4‐(aminomethyl)cyclohexane‐1‐carboxylic acid at concentrations corresponding to lysine‐binding site interactions at kringle 4 of plasmin; at saturation the rate constant is reduced 20‐fold, whereas the effect of saturation of kringle 1 is only a 2‐fold reduction. It is thus found that the interaction of α
2‐antiplasmin with the lysine‐binding site of kringle 1 is of little importance compared with that of kringle 4 in regulating the inhibition reaction of plasmin with α
2‐antiplasmin. Similar results were recently obtained for the bovine plasmin‐bovine α
2‐antiplasmin reaction (Christensen et al. (1995) Biochem. J. 305, 97–102).
Genomic simple repetitive DNAs are targets for differential binding of nuclear proteins
Authors: Epplen Jörg T. , Kyas Andreas , Mäueler Winfried
Published: July 2002

The biological meaning of abundant simple repetitive DNA sequences in eukaryote genomes is obscure. Therefore, (GAA)
n
, (GT)
n
, and composite (GT)
n
(GA)
m
blocks were characterized for protein binding in the repeat and flanking sequences of cloned genomic DNA fragments. In gel mobility shift and competition assays the binding of nuclear proteins to the repeats was specific (including some flanking single copy sequences). DNase footprinting revealed the target sequences within and adjacent to the repeats. Chemical modifications (Os04, DAPC) DAmonstrated non‐B DNA structures in the polypurine blocks. The binding of nuclear proteins in and around simple repeat sequences refute biological insignificance of all of these ubiquitously interspersed elements.
Identification and characterisation of a homologue of p64 in rat tissues
Authors: Howell Steven , Duncan Rory R. , Ashley Richard H.
Published: July 2002

Previous work has suggested that the gene encoding p64, a component of a bovine kidney intracellular chloride channel, may be a member of a gene family. We have raised a polyclonal antibody to an E. coli fusion protein which has sequence similarity to p64. Immunoblotting detected a protein in rat brain, kidney, liver and lung. In rat brain, the protein was enriched in cerebellar microsomal membranes. Western blot analyses of denaturing and blue native polyacrylamide gels indicated that the protein is a single non‐disulphide‐linked polypeptide chain with an apparent M
r of 43 kDa that contributes to a native protein complex with an apparent M
r of 130 kDa.
Hydroxy‐ and non‐hydroxy‐galactolipids in developing rat CNS
Authors: De Haas Catharina G.M. , Lopes‐Cardozo Matthijs
Published: July 2002

Rat spinal cord (1–24 weeks postnatal) was analysed by HPLC for various species of galactolipids that accumulate in mammalian myelin during development. Cerebral tissue of the same animals was taken as reference. The levels of the major galactolipids, galactosylceramide (GalCer) and its sulfated analog (SGalCer), increased linearly during the first 2 months after birth. At 3 months, constant levels were reached that were approx. 4‐fold (GalCer) and 2.5‐fold (SGalCer) higher than in cerebral tissue of corresponding age. The accumulation of galactoglycerolipids slightly preceded that of galactosphingolipids. Levels of galacto‐glycerolipids were much lower (4% of galactospingolipids in 3‐ and 2.5% in 6‐month‐old spinal cord on weight basis) and decreased upon CNS maturation. During the first postnatal month, the ratio of non‐hydroxy‐ over hydroxy‐species (NFA/HFA) of cerebral GalCer declined from 2.2 to 0.5 whereas the NFA/HFA ratio for cerebral SGalCer increased from 1.0 to 1.8 in the same period. Through development the hydroxy‐species contributed 56–60% to GalCer and 28–41% to SGalCer in spinal cord, whereas in cerebrum of 24‐week‐old rats 73% of GalCer and 48% of SGalCer was α‐hydroxylated in the ceramide moiety. These data point to different developmental programs with respect to galactolipid metabolism of oligodendrocytes in high‐ (spinal cord) as compared to low‐myelinated (cerebral) areas of rat CNS.
The role of beta-carbolines (harman/norharman) in heroin addicts
Authors: Stohler R , Rommelspacher H , Ladewig D
Published: July 2002

Endogenous substances resulting from interactions between alcohol and possibly opioid metabolites and neurotransmitters (dopamine, indolamines) could be mediators of the pathochemical process towards dependence. Beta-carbolines (harman/norharman) are increased in alcoholics and — according to the presented results — in heroin addicts. Psychopathological disorders such as anxiety or depression do not seem to influence the level of beta-carbolines.
Rehabilitation in the Nordic countries
Authors: Munk-Jørgensen P
Published: July 2002

Since the beginning of the 1980s the five Nordic countries: Denmark, Finland, Iceland, Norway and Sweden have gone through a process of decentralization. This process has taken place at a different pace and according to slightly varying decentralization models, but in all five of the countries the reduction in capacity of psychiatric hospitals has leveled out, whereas the establishing of decentralized treatment functions are not yet sufficiently developed. Furthermore, all five countries focus on rehabilitation of the most severe mentally ill. Examples of different models from the Nordic countries are mentioned.
Covalent binding of ATPγS to the nucleotide‐binding site in S14C‐actin
Authors: Schüler Herwig , Schutt Clarence E. , Lindberg Uno , Karlsson Roger
Published: July 2002

We have recently reported on the characterization of β‐actin carrying the mutation S14C in one of the phosphate‐binding loops. The present paper describes the attachment of the adenosine 5′‐[gamma‐thio]‐triphosphate (ATPγS) to actin containing this mutation. Treatment of S14C‐actin with ATPγS blocked further nucleotide exchange and raised the thermal stability of the protein, suggesting the formation of a covalent bond between the sulfhydryl on the terminal phosphate of ATPγS and cysteine‐14 of the mutant actin. The affinity of the derivatized G‐actin for DNase I as compared to wild‐type ATP‐actin was lowered to a similar extent as that of ADP.AlF4‐actin. The derivatized actin polymerized slower than ATP‐actin but faster than ADP‐actin. Under these conditions the bound ATPγS was hydrolyzed, suggesting the formation of a state corresponding to the transient ADP.Pi‐state. ATPγS‐actin interacted normally with profilin, whereas the interaction with actin depolymerizing factor (ADF) was disturbed, as judged on the effects of these proteins on actin polymerization.
Adrenaline potentiates PI 3‐kinase in platelets stimulated with thrombin and SFRLLN: role of secreted ADP
Authors: Selheim Frode , Frøyset Ann Kristin , Strand Ingrid , Vassbotn Flemming S , Holmsen Holm
Published: July 2002

Adrenaline significantly potentiated late thrombin‐ and SFRLLN‐induced PtdIns(3,4)P2 production. Furthermore, the potentiating effect of adrenaline on thrombin‐induced PtdIns(3,4)P2 production was independent on secreted ADP, whereas, the effect of adrenaline on SFRLLN‐induced PtdIns(3,4)P2 production was completely dependent of secreted ADP. However, the ADP‐dependent accumulation of PtdIns(3,4)P2 was not required for irreversible platelet aggregation induced by SFRLLN in the presence of adrenaline. It is concluded that adrenaline can replace secreted ADP to potentiate PtdIns(3,4)P2 production in thrombin‐stimulated but not in SFRLLN‐stimulated platelets, thus demonstrating a qualitative difference between platelet stimulation by thrombin and the thrombin receptor activating peptide SFRLLN.
The structure of glutamate transporters shows channel‐like features
Authors: Slotboom Dirk Jan , Konings Wil N , Lolkema Juke S
Published: July 2002

Neuronal and glial glutamate transporters remove the excitatory neurotransmitter glutamate from the synaptic cleft and thus prevent neurotoxicity. The proteins belong to a large family of secondary transporters, which includes transporters from a variety of bacterial, archaeal and eukaryotic organisms. The transporters consist of eight membrane‐spanning α‐helices and two pore‐loop structures, which are unique among secondary transporters but may resemble pore‐loops found in ion channels. Another distinctive structural feature is the presence of a highly amphipathic membrane‐spanning α‐helix that provides a hydrophilic path through the membrane. The unusual structural features of the transporters are discussed in relation to their function.
O‐Glycosylation of G‐protein‐coupled receptor, octopus rhodopsin
Authors: Nakagawa Masashi , Miyamoto Takayuki , Kusakabe Rie , Takasaki Seiichi , Takao Toshifumi , Shichida Yoshinori , Tsuda Motoyuki
Published: July 2002

In addition to the N‐glycan that is evidently conserved in G‐protein‐coupled receptors (GPCRs), O‐glycans in the N‐terminus of GPCRs have been suggested. Using a combination of enzymatic and manual Edman degradation in conjunction with G‐protein coupled receptor mass spectrometry, the structure and sites of O‐glycans in octopus rhodopsin are determined. Two N‐acetylgalactosamine residues are O‐linked to Thr4 and Thr5 in the N‐terminus of octopus rhodopsin. Further, we found chicken iodopsin, but not bovine rhodopsin, contains N‐acetylgalactosamine. This is the first direct evidence to determine the structure and sites of O‐glycans in GPCRs.
The shapes and sizes of two domains of tropomodulin, the P‐end‐capping protein of actin‐tropomyosin
Authors: Fujisawa Tetsuro , Kostyukova Alla , Maéda Yuichiro
Published: July 2002

Tropomodulin, the P‐end (slow‐growing end)‐capping protein of the actin‐tropomyosin filament, and its fragment (C20) of the C‐terminal half were studied by synchrotron small‐angle X‐ray scattering, restoring low‐resolution shapes using an ab initio shape‐determining procedure. Tropomodulin is elongated (115 Å long) and consists of two domains, one of 65 Å in length and the other being similar to C20 in shape and size if the long axes of the two are tilted by about 40° relative to each other. We propose a model for tropomodulin in association with tropomyosin and actin: the N‐terminal half of tropomodulin, a rod, binds to the N‐terminus of tropomyosin and the C‐terminal triangle domain protrudes from the P‐end being slightly bent towards the actin subunit at the end, thereby blocking the P‐end.
Differential and opposed transcriptional effects of protein fusions containing the VP16 activation domain
Authors: Matis Christelle , Chomez Patrick , Picard Jacques , Rezsohazy René
Published: July 2002

Overexpression of strong transcriptional activators like herpes simplex virion protein 16 (VP16) may lead to non‐specific inhibition of gene expression as a result of the titration of transcription factors. Here we report that a fusion between the homeoprotein Hoxa2 and the VP16 activation domain inhibits transcription from the strong promoter/enhancers of cytomegalovirus (CMV) and Rous sarcoma virus (RSV). A similar fusion involving a Hoxa2 mutant protein that is defective in DNA binding has no effect on the CMV promoter but increases, rather than inhibits, the RSV promoter activity. This suggests that depending on its ability to bind DNA, the VP16 activator can interact with different sets of cofactors, giving rise to distinct transcriptional effects.
Specific binding sites for rat prepro‐TRH‐(160–169) on C6 glioma and BN1010 clonal neural cells
Authors: Ladram Ali , Montagne Jean-Jacques , Nicolas Pierre , Bulant Marc
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Mechanochemical mechanism for peptidyl free radical generation by amyloid fibrils
Authors: Kay Christopher J
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Thiostrepton binds to malarial plastid rRNA
Authors: Clough Barbara , Strath Malcolm , Preiser Peter , Denny Paul , Wilson Iain (R.J.M.)
Published: July 2002

Binding of the thiazolyl peptide antibiotic thiostrepton to the GTPase domain of 23S rRNA involves a few crucial nucleotides, notably A1067 (E. coli). Small RNA transcripts were prepared corresponding to the GTPase domain of the plastid 23S rRNA and the two forms of cytosolic 28S rRNAs found in the human malaria parasite Plasmodium falciparum, as well as the plastid form of rRNA of the AIDS‐related pathogen Toxoplasma gondii. Binding affinities of the wild type and mutated RNA sequences were as predicted; the malarial plastid sequence had by far the highest affinity, whereas that from toxoplasma did not bind thiostrepton.
Role of modified glutamic acid in the helical structure of conantokin‐T
Authors: Lin C.H , Chen C.S , Hsu K.S , King D.S , Lyu P.C
Published: July 2002

Circular dichroism (CD) and 2‐dimensional NMR were used to study the solution conformation of conantokin‐T (Con‐T), a small peptide toxin found in the venom of fish‐hunting cone snails, and its Glu‐substituted analog. Con‐T lacks disulfide bonds but contains many γ‐carboxyglutamic acids (Gla), a post‐translationally modified residue. Our results show that Con‐T adopts an α‐helical conformation in aqueous solution even in the absence of calcium. Glu replacements diminish both helicity and function of Con‐T. The helical content of Con‐T is higher than most natural helical peptides of this length in aqueous solution. The sequence of this small toxin incorporates several known elements that stabilize α‐helical structure in peptides. Gla residues form several salt bridges that stabilize helical conformation of Con‐T.
Unfolding of dimeric creatine kinase in urea and guanidine hydrochloride as measured using small angle X‐ray scattering with synchrotron radiation
Authors: Zhou Jun-Mei , Fan Ying-Xin , Kihara Hiroshi , Kimura Kazumoto , Amemiya Yoshiyuki
Published: July 2002

The denaturation of dimeric creatine kinase (CK) induced by urea and guanidine hydrochloride (GuHCl) has been studied by small angle X‐ray scattering (SAXS), which is a direct way to measure the changes in the overall dimensions of a protein molecule. The radii of gyration (R
g) of CK are 29±0.4 Å in the native state and 46±1.5 Å in the unfolded state in either 8 M urea or 3 M GuHCl. The transition curves of urea denaturation derived from the R
g values and the zero angle intensity (I(0)) are similar to that from intrinsic fluorescence, indicating that the changes in the molecular shape, the tertiary structure and the dissociation of the subunits proceed simultaneously. In the case of GuHCl‐induced denaturation, the dramatic increases both in R
g and in I(0) in 0.3–0.5 M GuHCl suggest clearly that soluble aggregates form at low GuHCl concentrations. The aggregates dissociate and the molecule unfolds at higher GuHCl concentrations. The results suggest that the mechanisms of CK denaturation in urea and in GuHCl are somewhat different and the intermediate in GuHCl denaturation can much more easily form soluble aggregates.
Role of the constriction loop in the gating of outer membrane porin PhoE of Escherichia coli
Authors: Eppens Elaine F , Saint Nathalie , Van Gelder Patrick , van Boxtel Ria , Tommassen Jan
Published: July 2002

Porins form voltage‐gated channels in the bacterial outer membrane. These proteins are composed of three identical subunits, each forming a 16‐stranded β‐barrel. In this study, the role in voltage gating of a loop that forms a constriction within the pore was studied. The channel characteristics of mutant PhoE porins, in which the tip of the constriction loop was connected to the barrel wall, were determined. Whereas the properties of several mutant channels were changed, all of these channels could still be closed at high potential, showing that a gross movement of the constriction loop within the channel is not implicated in voltage gating.
Dissociation of tubulin assembly‐inhibiting and aggregation‐promoting activities by a vinblastine derivative
Authors: Rai Sadananda S. , Wolff J.
Published: July 2002

A fluorescent vinblastine analogue, vinblastine‐4′‐anthranilate (Antvin), that binds to the vinca site on tubulin, inhibits tubulin assembly but does not lead to spiral or other large aggregate formation at concentrations up to 1.6 mM. As judged by turbidity, 90° light scattering and fluorescence anisotropy, little aggregation could be detected. This is in marked contrast to vinblastine and suggests that inhibition of assembly and aggregate formation can be dissociated from each other by suitable substitution in vinblastine.
2 Å X‐ray structure of adamalysin II complexed with a peptide phosphonate inhibitor adopting a retro‐binding mode
Authors: Cirilli M. , Gallina C. , Gavuzzo E. , Giordano C. , Gomis-Rüth F.X. , Gorini B. , Kress L.F. , Mazza F. , Paglialunga Paradisi M. , Pochetti G. , Politi V.
Published: July 2002

The search of reprolysin inhibitors offers the possibility of intervention against both matrixins and ADAMs. Here we report the crystal structure of the complex between adamalysin II, a member of the reprolysin family, and a phosphonate inhibitor modeled on an endogenous venom tripeptide. The inhibitor occupies the primed region of the cleavage site adopting a retro‐binding mode. The phosphonate group ligates the zinc ion in an asymmetric bidentate mode and the adjacent Trp indole system partly fills the primary specificity subsite S1′. An adamalysin‐based model of tumor necrosis factor‐α‐converting enzyme (TACE) reveals a smaller S1′ pocket for this enzyme.
Modulation of forskolin‐mediated adenylyl cyclase activation by constitutively active Gs‐coupled receptors
Authors: Alewijnse Astrid E , Smit Martine J , Sol Rodriguez Pena M , Verzijl Dennis , Timmerman Henk , Leurs Rob
Published: July 2002

In transfected CHO cells constitutively active histamine H2 receptors not only increase the basal cAMP level, but also enhance forskolin‐induced cAMP production. The increased forskolin response was inhibited by inverse H2 agonists with potencies similar to those determined at basal levels. The modulation of the forskolin response was also observed after H2 receptor expression in HEK‐293 and Sf9 cells or TSH receptor expression in COS‐7 cells. The enhancement of forskolin‐induced cAMP production seems to be a general characteristic of constitutively active Gs‐coupled receptors and can be very useful to study inverse agonism at wild‐type receptors.
Effective restoration of dystrophin‐associated proteins in vivo by adenovirus‐mediated transfer of truncated dystrophin cDNAs
Authors: Yuasa Katsutoshi , Miyagoe Yuko , Yamamoto Kanji , Nabeshima Yo-ichi , Dickson George , Takeda Shin'ichi
Published: July 2002

A series of truncated dystrophin cDNAs (3.1–4.2 kbp) containing only three, three, two or one rod repeats with hinge 1 and 4 (named ΔDysAX2, AX11, AH3, M3, respectively) or no rod repeat retaining either hinge 1 or 4 (named ΔDysH1, H4, respectively) were constructed. These cDNAs were introduced into skeletal muscle of adult mdx mice using the adenovirus vector with a strong CAG promoter. ΔDysAX2, AX11, AH3 and ΔDysM3 expressed themselves successfully and recovered dystrophin‐associated proteins effectively. Especially 3.7 kbp cDNA for ΔDysM3 offers the possibility of an approach utilizing newly developed virus vectors, such as an adeno‐associated virus vector, toward gene therapy of Duchenne muscular dystrophy.
Different domains of the ORL1 and κ‐opioid receptors are involved in recognition of nociceptin and dynorphin A
Authors: Lapalu Sophie , Moisand Christiane , Butour Jean-Luc , Mollereau Catherine , Meunier Jean-Claude
Published: July 2002

In order to gain further insight into the functional architecture of structurally related G protein‐coupled receptors, the ORL1 (nociceptin) and opioid receptors, we have constructed chimeras of ORL1 and μ‐, δ‐ and κ‐opioid receptors, and compared their binding and functional properties with those of the parent receptors. We find in particular that a ORL1–κ‐opioid (O‐K) hybrid construct has retained high affinity for non‐type‐selective opiate ligands, and has acquired the ability to bind and respond to enkephalins and μ‐ and/or δ‐opioid receptor‐selective enkephalins analogs, thus behaving like a `universal' opioid receptor. Most significantly however, whilst the ORL1 and κ‐opioid receptors display high binding preference (K
D 0.1 vs. 100 nM) for their respective endogenous ligands, nociceptin and dynorphin A, the O‐K chimeric receptor binds both nociceptin and dynorphin A, with high affinity (K
D<1 nM). Together, these data (i) add weight to the hypothesis that the extracellular loops of opioid receptors act as a filter for ligand selection, and (ii) demonstrate that different domains of the ORL1 and κ‐opioid receptors are involved in recognition of their endogenous peptide ligands.
A new opioid peptide predicted from cloned cDNAs from skin of Pachymedusa dacnicolor and Agalychnis annae
Authors: Wechselberger Christian , Severini Cinzia , Kreil Günther , Negri Lucia
Published: July 2002

We have isolated a cDNA encoding a precursor of dermorphin from the skin of Pachymedusa dacnicolor. Besides four copies of this opioid peptide, the deduced sequence also contains the genetic information for a novel peptide Tyr‐Ile‐Phe‐His‐Leu‐Met‐Asp‐NH2. This differs from Met‐deltorphin by the presence of Ile at position 2. In a related precursor from the skin of Agalychnis annae, the sequence of this peptide is in the 3′‐untranslated region of the cloned cDNA. From earlier results we predict that in skin peptides the second residue is d‐allo‐Ile. We have synthesized this and related peptides with different d‐amino acids, and determined their δ agonist activity. The peptide with d‐nor‐Leu binds with high affinity to δ receptors, while that with d‐allo‐Ile is about 100 times less active.
Biological significance of the side chain length of ubiquinone in Saccharomyces cerevisiae
Authors: Okada Kazunori , Kainou Tomohiro , Matsuda Hideyuki , Kawamukai Makoto
Published: July 2002

Ubiquinone (UQ), an important component of the electron transfer system, is constituted of a quinone structure and a side chain isoprenoid. The side chain length of UQ differs between microorganisms, and this difference has been used for taxonomic study. In this study, we have addressed the importance of the length of the side chain of UQ for cells, and examined the effect of chain length by producing UQs with isoprenoid chain lengths between 5 and 10 in Saccharomyces cerevisiae. To make the different UQ species, different types of prenyl diphosphate synthases were expressed in a S. cerevisiae COQ1 mutant defective for hexaprenyl diphosphate synthesis. As a result, we found that the original species of UQ (in this case UQ‐6) had maximum functionality. However, we found that other species of UQ could replace UQ‐6. Thus a broad spectrum of different UQ species are biologically functional in yeast cells, although cells seem to display a preference for their own particular type of UQ.
Isolation and characterization of a rice cDNA encoding the γ‐subunit of translation elongation factor 1B (eEF1Bγ)1
Authors: Kidou Shin-ichiro , Tsukamoto Shigenori , Kobayashi Satoru , Ejiri Shin-ichiro
Published: July 2002

We isolated a rice cDNA clone (refg) encoding the γ‐subunit of translation elongation factor 1B (eEF‐1Bγ; the old designation was EF‐1γ). The refg encodes an open reading frame of 419 amino acids which shows a similarity to the equivalent sequences from animals and yeast. Complex formation analysis, which showed the recombinant protein of refg (His‐eEF1Bγ) and formed a complex with GST‐eEF‐1Bβ, indicated that the refg encodes rice eEF1Bγ of the eEF1Bαβγ complex. Expression analysis showed that refg mRNA is very abundant in suspension‐cultured cells during the exponential phase of growth. A DNA blot analysis indicated that refg is located at a single locus in the rice genome.
Trp proteins form store‐operated cation channels in human vascular endothelial cells
Authors: Groschner Klaus , Hingel Susanne , Lintschinger Birgit , Balzer Monika , Romanin Christoph , Zhu Xi , Schreibmayer Wolfgang
Published: July 2002

Members of the Trp protein family have been suggested as the structural basis of store‐operated cation conductances. With this study, we provide evidence for the expression of three isoforms of Trp (hTrp1, 3 and 4) in human umbilical vein endothelial cells (HUVEC). The role of Trp proteins in store regulation of endothelial membrane conductances was tested by expression of an N‐terminal fragment of hTrp3 (N‐TRP) which exerts a dominant negative effect on Trp channel function presumably due to suppression of channel assembly. Depletion of intracellular Ca2+ stores with IP3 (100 μM) or thapsigargin (100 nM) induced a substantial cation conductance in sham‐transfected HUVEC as well as in HUVEC transfected with hTrp3. In contrast, HUVEC transfected with N‐TRP failed to exhibit store‐operated currents. Our results suggest the involvement of Trp related proteins in the store‐operated cation conductance of human vascular endothelial cells.
Oxidative biodegradation of phosphorothiolates by fungal laccase
Authors: Amitai G , Adani R , Sod-Moriah G , Rabinovitz I , Vincze A , Leader H , Chefetz B , Leibovitz-Persky L , Friesem D , Hadar Y
Published: July 2002

Organophosphorus (OP) insecticides and nerve agents that contain P‐S bond are relatively more resistant to enzymatic hydrolysis. Purified phenol oxidase (laccase) from the white rot fungus Pleurotus ostreatus (Po) together with the mediator 2,2′‐azinobis(3‐ethylbenzthiazoline‐6‐sulfonate) (ABTS) displayed complete and rapid oxidative degradation of the nerve agents VX and Russian VX (RVX) and the insecticide analog diisopropyl‐Amiton with specific activity: k
sp=2200, 667 and 1833 nmol min−1 mg−1, respectively (pH 7.4, 37°C). A molar ratio of 1:20 for OP/ABTS and 0.05 M phosphate at pH 7.4 provided the highest degradation rate of VX and RVX. The thermostable laccase purified from the fungus Chaetomium thermophilium (Ct) in the presence of ABTS caused a 52‐fold slower degradation of VX with k
sp=42 nmol min−1 mg−1. The enzymatic biodegradation products were identified by 31P‐NMR and GC/MS analysis.
CD‐monitored redox titration of the Rieske Fe‐S protein of Rhodobacter sphaeroides: pH dependence of the midpoint potential in isolated bc1 complex and in membranes
Authors: Ugulava Natalia B. , Crofts Antony R.
Published: July 2002

The redox potential of the Rieske Fe‐S protein has been investigated using circular dichroism (CD)‐spectroscopy. The CD features characteristic of the purified bc
1 complex and membranes of Rhodobacter sphaeroides were found in the region between 450 and 550 nm. The difference between reduced and oxidized CD‐spectra shows a negative band at about 500 nm with a half of width 30 nm that corresponds to the specific dichroic absorption of the reduced Rieske protein (Fee, J.A. et al. (1984) J. Biol. Chem. 259, 124–133; Degli Esposti, M. et al. (1987) Biochem. J. 241, 285–290; Rich, P.R. and Wiggins, T.E. (1992) Biochem. Soc. Trans. 20, 241S). It was found that the redox potential at pH 7.0 for the Rieske center in the isolated bc
1 complex and in chromatophore membranes from the R‐26 strain of Rb. sphaeroides is 300±5 mV. In chromatophores from the BC17C strain of Rb. sphaeroides, the E
m value measured for the Rieske iron‐sulfur protein (ISP) was higher (315±5 mV), but the presence of carotenoids made measurement less accurate. The E
m varied with pH in the range above pH 7, and the pH dependence was well fit either by one pK at ∼7.5 in the range of titration, or by two pK values, pK
1=7.6 and pK
2=9.8. Similar titrations and pK values were found for the Rieske Fe‐S protein in the isolated bc
1 complex and membranes from the R‐26 strain of Rb. sphaeroides. The results are discussed in the context of the mechanism of quinol oxidation by the bc
1 complex, and the role of the iron sulfur protein in formation of a reaction complex at the Qo‐site.
Reaction centers of photosystem II with a chemically‐modified pigment composition: exchange of pheophytins with 131‐deoxo‐131‐hydroxy‐pheophytin a
Authors: Shkuropatov A.Ya. , Khatypov R.A. , Shkuropatova V.A. , Zvereva M.G. , Owens T.G. , Shuvalov V.A.
Published: July 2002

Isolated reaction centers of photosystem II with an altered pigment content were obtained by chemical exchange of the native pheophytin a molecules with externally added 131‐deoxo‐131‐hydroxy‐pheophytin a. Judged from a comparison of the absorption spectra and photochemical activities of exchanged and control reaction centers, 70–80% of the pheophytin molecules active in charge separation are replaced by 131‐deoxo‐131‐hydroxy‐pheophytin a after double application of the exchange procedure. The new molecule at the active branch was not active photochemically. This appears to be the first stable preparation in which a redox active chromophore of the reaction center of photosystem II was modified by chemical substitution. The data are compatible with the presence of an active and inactive branch of cofactors, as in bacterial reaction centers. Possible applications of the 131‐deoxo‐131‐hydroxy‐pheophytin a‐exchanged preparation to the spectral and functional analysis of native reaction centers of photosystem II are discussed.
The role of O‐linked sugars in determining the very low density lipoprotein receptor stability or release from the cell
Authors: Magrané Jordi , Casaroli-Marano Ricardo P. , Reina Manuel , Gåfvels Mats , Vilaró Senén
Published: July 2002

The very low density lipoprotein receptor is a member of the low density lipoprotein receptor supergene family for which two isoforms have been reported, one lacking and the other containing an O‐linked sugar domain. In order to gain insight into their functionality, transient and stable transformants separately overexpressing previously cloned bovine variants were analyzed. We report evidence that the variant lacking the O‐linked sugar domain presented a rapid cleavage from the cell and that a large amino‐terminal very low density lipoprotein receptor fragment was released into the culture medium. As only minor proteolysis was involved in the other very low density lipoprotein receptor variant, the clustered O‐linked sugar domain may be responsible for blocking the access to the protease‐sensitive site(s). To test this hypothesis, a mutant Chinese hamster ovary cell line, ldlD, with a reversible defect in the protein O‐glycosylation, was used. The instability of the O‐linked sugar‐deficient very low density lipoprotein receptor on the cell surface was comparable to that induced by the proteolysis of the variant lacking the O‐linked sugar domain. Moreover, our data suggest that the O‐linked sugar domain may also protect the very low density lipoprotein receptor against unspecific proteolysis. Taken together, these results indicate that the presence of the O‐linked sugar domain may be required for the stable expression of the very low density lipoprotein receptor on the cell surface and its absence may be required for release of the receptor to the extracellular space. The exclusive expression of the variant lacking the O‐linked sugar domain in the bovine aortic endothelium opens new perspectives in the physiological significance of the very low density lipoprotein receptor.
Multiple tRNA attachment sites in prothymosin α
Authors: Lukashev Dmitriy E , Chichkova Nina V , Vartapetian Andrey B
Published: July 2002

A covalent complex formed by bacterial tRNAs and prothymosin α, an abundant acidic nuclear protein involved in proliferation of mammalian cells, upon production of the recombinant rat protein in Escherichia coli cells was studied. Several tRNA attachment sites were identified in the prothymosin α molecule using a combination of deletion analysis of prothymosin α and site‐specific fragmentation of the protein moiety of the prothymosin α‐tRNA complex. The electrophoretic mobilities of the tRNA‐linked prothymosin α and its derivatives are consistent with one tRNA molecule attached to one prothymosin α molecule, thus suggesting that alternative tRNA linking to one of several available attachment sites occurs. The possible effect of tRNA attachment on the nuclear uptake of prothymosin α is discussed.
Phosphorylation and activation of Ca2+/calmodulin‐dependent protein kinase phosphatase by Ca2+/calmodulin‐dependent protein kinase II
Authors: Kameshita Isamu , Ishida Atsuhiko , Fujisawa Hitoshi
Published: July 2002

Ca2+/calmodulin‐dependent protein kinase phosphatase (CaMKPase) is a protein phosphatase which dephosphorylates autophosphorylated Ca2+/calmodulin‐dependent protein kinase II (CaMKII) and deactivates the enzyme (Ishida, A., Kameshita, I. and Fujisawa, H. (1998) J. Biol. Chem. 273, 1904–1910). In this study, a phosphorylation‐dephosphorylation relationship between CaMKII and CaMKPase was examined. CaMKPase was not significantly phosphorylated by CaMKII under the standard phosphorylation conditions but was phosphorylated in the presence of poly‐l‐lysine, which is a potent activator of CaMKPase. The maximal extent of the phosphorylation was about 1 mol of phosphate per mol of the enzyme and the phosphorylation resulted in an about 2‐fold increase in the enzyme activity. Thus, the activity of CaMKPase appears to be regulated through phosphorylation by its target enzyme, CaMKII.
Cloning, expression and characterization of a novel four EF‐hand Ca2+‐binding protein from olive pollen with allergenic activity
Authors: Ledesma Amalia , Villalba Mayte , Rodrı́guez Rosalı́a
Published: July 2002

A novel allergenic member of the family of Ca2+‐binding proteins has been cloned from olive tree pollen. The isolated DNA codes for a protein of 171 amino acid residues, which displays four EF‐hand sequence motifs. The encoded protein was overproduced in Escherichia coli and purified. The protein (18 795 Da), which binds Ca2+ and IgE antibodies from patients allergic to olive pollen, undergoes Ca2+‐dependent conformational changes. It is retained on a phenyl‐Sepharose column, which indicates the existence of regulatory EF‐hand domains. This fact suggests its involvement in Ca2+‐dependent signal transduction events of the pollen grain. This allergen could be considered as a member of a new subfamily of EF‐hand Ca2+‐binding proteins since it displays a low amino acid sequence similarity with the so far known proteins.
The impact of life events in female patients with fibromyalgia and in female healthy controls
Authors: Anderberg U.M. , Marteinsdottir I. , Theorell T. , von Knorring L.
Published: July 2002

The aim was to investigate if female fibromyalgia patients (FMS) had experienced more negative life events than healthy women. Furthermore, the life events experienced in relation to onset of the FMS were evaluated. Another important area was to investigate the impact of the events experienced in the patients compared to healthy women.

A new inventory was constructed to assess life events during childhood, adolescence and in adulthood as well as life events experienced in relation to the onset of the disorder. Forty female FMS patients and 38 healthy age-matched women participated in the study.

During childhood or adolescence 51% of the patients had experienced very negative life events as compared to 28% of the controls. Conflict with parents was the most common life event. Before onset, 65% of the patients experienced some negative life event. Economic problems and conflicts with husband/partner were common. During the last year, 51% of the patients had life events which they experienced as very negative, compared to 24.5% of the controls (P < 0.01).

Stressful life events in childhood/adolescence and in adulthood seem to be very common in FMS. Furthermore, the life events were experienced as more negative than the life events experienced by healthy controls.
Two types classification of male alcoholism confirmed
Authors: Modestin J , Würmle O
Published: July 2002

In recent years a two types classification of male alcoholism has been proposed by several authors. The study aimed at a testing of type I/type II classification of alcoholism in an independent sample. A total of 351 Research Diagnostic Criteria (RDC) male alcoholics consecutively admitted for an inpatient psychiatric treatment were studied. Type II characteristics were extracted from the pertinent literature; 19 available variables best representing these characteristics were selected. The data was collected using the method of a retrospective evaluation of clinical records. Cluster analysis yielded two clusters which were well separated from each other. Heterogeneity of the male inpatient population of alcoholics was demonstrated and the existence of broadly defined type I and type II alcoholism was confirmed.
Can quality of life scales tell us when patients begin to feel the benefits of antidepressants?
Authors: McKenna SP , Whalley D
Published: July 2002

Assessments of early response to antidepressant therapy based on condition severity have been contradictory due to the poor measurement properties of the instruments employed and the inappropriateness of condition severity as a criterion of improvement. It is argued that a more valid approach would involve the assessment of improvements in quality of life (QoL) associated with effective therapy. QoL instruments should reflect the views of the patients themselves, the only group able to comment authoritatively on whether they feel benefit from treatment. Research shows that it is the perceptions of the patients that determine whether treatment is sought, how far clinical advice is followed and whether treatment is discontinued. It is argued that the Quality of Life in Depression Scale is the only instrument available that has the required conceptual basis and psychometric properties to investigate the onset of response to antidepressant therapy.
Equilibrium with divergence of opinion
Authors: Miller Edward M
Published: July 2002

Since the less informed incorrectly estimate asset returns, they anticipate higher returns from their risky investments. They thus over‐invest in risky securities. They are rewarded by higher total portfolio returns. Too high a proportion of less‐informed investors lowers the return on risky assets. Equilibrium requires that the less informed's rate of return equal the informed's. The less informed control a stable equilibrium percentage of total wealth. Since an individual's recent investment experience correlates with his terminal wealth, learning need not reduce the less informed's risky asset exposure. Implications exist for the slope of the return versus systematic risk curve.
Trading volume and firm size: A test of the information spillover hypothesis
Authors: Weigand Robert A.
Published: July 2002

This study investigates information transfer across firms by using the trading volume of large‐and small‐stock portfolios as a proxy for the flow of information in financial markets. I find bi‐directional Granger‐causality between the trading volume of large and small firms. The results do not support previous studies that report evidence of one‐way information transfer using the returns and variances of different‐sized firms. My findings suggest several explanations. Either information arrival in financial markets has a different impact on trading volume than it does on returns and variances, or certain information shocks may affect different firms more rapidly, with these shocks eventually influencing the volume of trade of all firms.