bluuebunny/crossref_metadata_2025 huggingface dataset:
S‐Phase phosphorylation of lamin B2
Authors: Kill Ian R. , Hutchison Christopher J.
Published: April 2003

Lamin B2 modification in synchronously dividing populations of human diploid fibroblasts was determined by 2‐dimensional gel electrophoresis and [32P]orthophosphate labelling. In quiescent (G0) and G1 cultures of HDF, lamin B2 migrated as 2 spots on 2‐dimensional gels. In contrast, in S‐phase populations of HDF lamin B2 migrated as a single basic species. The level of lamin B2 phosphorylation was determined after immunoisolation from [32P]orthophosphate labelled cells. The results of these experiments indicated a 2–3‐fold increase in the steady state level of lamin B2 phosphorylation in S‐phase HDF compared with G0 HDF. Consistent with this evidence, tryptic peptide maps revealed the presence of a phosphopeptide in S‐phase lamin B2 which was absent from G0 lamin B2. Since all of the phosphate incorporated into S‐phase and G0 lamin B2 was recovered in serine residues we conclude that the S‐phase specific phosphopeptide did not represent either of the cdc2 sites associated with entry nuclear lamina breakdown.
An antibody VH domain with a lox‐Cre site integrated into its coding region: bacterial recombination within a single polypeptide chain
Authors: Davies Julian , Riechmann Lutz
Published: April 2003

Bacterial lox‐Cre recombination within a single antibody VH domain was achieved through integration of a loxP site into its coding sequence. The 5′ half of the VH gene, in which the H2 loop was replaced by a mutant loxP site, was fused to geneIII in an ‘acceptor’ fd‐phage vector containing also a wild type loxP site. With a ‘donor’ plasmid vector harbouring the 3′ half of the VH gene flanked by the same, differing loxP sites it recombined into a full‐length VH with the loxP site‐H2 loop. This VH was purified from bacterial periplasm, where it folded into a typical immunoglobulin domain. The system allows the generation of large VH repertoires using lox‐Cre recombination.
Fibroblasts transformed by combinations of ras, myc and mutant p53 exhibit increased phosphorylation of histone H1 that is independent of metastatic potential
Authors: Taylor William R. , Chadee Deborah N. , Allis C.David , Wright Jim A. , Davie James R.
Published: April 2003

H1 histones play an important role in regulating higher order structure of chromatin and are potential regulators of gene expression. H1s are phosphorylated, a modification which alters their interaction with DNA. We measured the abundance of three phosphorylated H1 subtypes in mouse fibroblasts transformed by combinations of ras, myc and mutant p53 which differ in metastatic potential. We found that there is an increase in phosphorylation of H1 subtypes in fibroblasts transformed with ras, myc and mutant p53. This increase was found to correlate with cellular transformation but not with induction of the metastatic phenotype.
Rte‐1, a retrotransposon‐like element in Caenorhabditis elegans
Authors: Youngman Sandra , van Luenen Henri G.A.M. , Plasterk Ronald H.A.
Published: April 2003

We have characterized a retrotransposon‐like element (Rte‐1) in C. elegans. It was identified while we were sequencing the pim related kinase‐1 (prk‐1) gene. The element is 3,298 bp long and flanked by a 200 bp direct repeat. 95 bp of the direct repeat are present in the coding region of prk‐1. Rte‐1 contains an open reading frame, in the opposite orientation of prk‐1, potentially encoding 625 amino acids, with similarity to reverse transcriptases. The element is most similar to members of the non‐LTR group of retrotransposable elements. There is weak homology of the predicted amino acid sequence of Rte‐1 to several reverse transcriptase‐like genes identified by the C. elegans genome sequencing consortium, suggesting that there may be a large family of these elements. Southern blots indicate that there are approximately 10–15 additional Rte‐1 elements in the C. elegans Bristol N2 genome and a similar number is found in the genomes of two other geographically distinct strains. The insertion pattern of Rte‐1 is polymorphic between these strains.
Measurement of nucleotide exchange kinetics with isolated synthetic myosin filaments using flash photolysis
Authors: Conibear Paul B. , Bagshaw Clive R.
Published: April 2003

The kinetics of nucleotide release have been measured at the level of isolated synthetic myosin filaments. This was achieved by displacing a rhodamine nucleotide analog from a filament by flash photolysis of caged‐ATP with selective observation using total internal reflectance fluorescence microscopy. The procedure gave improved time resolution over previously used flow methods. Kinetics were measured both in the presence of the ADP analog and during steady‐state hydrolysis of the ATP analog. These studies open the way for kinetic measurements during actin filament sliding on myosin tracks.
Response of Drosophila metallothionein promoters to metallic, heat shock and oxidative stresses
Authors: Bonneton François , Théodore Laurent , Silar Philippe , Maroni Gustavo , Wegnez Maurice
Published: April 2003

The metallothionein system in Drosophila melanogaster is composed of two genes, Mtn and Mto. In order to compare the induction properties of these genes, we transformed D. melanogaster with P‐element vectors containing Adh and lacZ reporter genes under the control of Mtn and Mto promoters, respectively. Mtn and Mto transgenes are mainly expressed in the digestive tract. However, Mtn expression has been detected also in the fat body. Mtn and Mto transgenes respond differently to metallic, heat‐shock and oxidative stresses. These data confirm that both genes are in part functionally different.
Co‐expression of human protein disulphide isomerase (PDI) can increase the yield of an antibody Fab′ fragment expressed inEscherichia coli
Authors: Humphreys David P. , Weir Neil , Lawson Alastair , Mountain Andrew , Lund Peter A.
Published: April 2003

Secretion to the periplasm ofEscherichia colienables production of many eukaryotic extracellular proteins in a soluble form. The complex disulphide bond arrangement of such proteins is probably a major factor in determining the low yield of correctly folded product observed in many cases. Here we show that co‐expression of human protein disulphide isomerase increased the yield of a monoclonal antibody Gab′ fragment in the periplasm ofE. coli.
Insulin‐ and polyunsaturated fatty acid‐responsive region(s) of rat ATP citrate‐lyase gene promoter
Authors: Fukuda Hitomi , Iritani Nobuko , Katsurada Akihiko , Noguchi Tamio
Published: April 2003

To investigate the regulatory DNA sequences required for insulin‐stimulation of the ATP citrate‐lyase (ACL) gene as well as for polyunsaturated fatty acid (PUFA)‐suppression of this gene, primary cultured hepatocytes were transfected with plasmids containing the 5′‐flanking sequence of the rat ACL gene fused to the chloramphenicol acetyltransferase (CAT) gene. Sequences from −861, −194 or −104 to +128 of the ACL gene directed an increase in CAT activity in hepatocytes when insulin was added to the medium containing either glucose or pyruvate. The CAT activities stimulated by insulin were reduced by the addition of PUFA, in accordance with the responses on the endogenous ACL gene expression. Further deletion to −20, however, resulted in loss of the responses. The results suggest that the region from −104 to −20 of the ACL gene is responsible for regulation due to insulin and PUFAs. In particular, the region from −61 to −49 of the ACL has sequence similarity to the insulin‐responsive regions of fatty acid synthase and acetyl‐CoA carboxylase.
Inhibitor sensitivity of respiratory complex I in human platelets: A possible biomarker of ageing
Authors: Merlo Pich M. , Bovina C. , Formiggini G. , Cometti G.G. , Ghelli A. , Parenti Castelli G. , Genova M.L. , Marchetti M. , Semeraro S. , Lenaz G.
Published: April 2003

NADH‐Coenzyme Q reductase was assayed in platelet mitochondrial membranes obtained from 19 pools of two venous blood samples from female young (19–30 years) individuals and 18 pools from aged ones (66–107 years). The enzyme activities were not significantly changed in the two groups, but a decrease of sensitivity to the specific inhibitor, rotenone, occurred in a substantial number of aged individuals. The results are in agreement with the predictions of the mitochondrial theory of ageing and may be used to develop a sensitive biomarker of the ageing process.
Visualising the dissociation of sequence selective ligands from individual binding sites on DNA
Authors: Fletcher Michael C. , Fox Keith R.
Published: April 2003

We have used a modification of the footprinting technique to measure the dissociation of mithramycin, echinomycin and nogalamycin from their binding sites in a natural DNA fragment. Complexes with radiolabelled DNA were dissociated by addition of unlabelled DNA. Samples were removed at various times and subjected to DNase I digestion, and the rate of dissociation from each site was estimated from the time‐dependent disappearance of the footprints. For echinomycin the slowest rate of dissociation is from ACGT, while the slowest site for mithramycin contains four contiguous guanines. The dissociation of nogalamycin is extremely slow, even from its weaker sites; the slowest rate was from ACGTA, which took longer than 4 h, even at 37°C.
A specific Go heterotrimer couples somatostatin receptors to voltage‐gated calcium channels in RINm5F cells
Authors: Degtiar Vadim E. , Wittig Burghardt , Schultz Günter , Kalkbrenner Frank
Published: April 2003

The peptide hormone somatostatin inhibits glucose‐induced insulin secretion in the rat insulinoma RINm5F cells by inhibition of voltage‐gated calcium channels. Here we used microinjection of antisene oligonucleotides directed against subtypes of G‐protein subunits to determine the subunit composition involved in somatostatin‐induced inhibition of voltage‐gated calcium channels in RINmF5 cells. Injection of antisene oligonucleotides annealing to the respective mRNA of Gαo2, Gβ1 and Gγ3 reduced the somatostatin‐induced inhibition of calcium channels in these cells. Injection of antisene oligonucleotides directed against other G‐protein subunits did not, suggesting that in RINm5F cells the somatostatin receptor couples to a G protein of Gαo2β1γ3 composition. By using a selective agonist of type 2 somatostatin receptors (SSTR 2) NC 8–12, we identified this receptor as the subtype coupling to calcium channels in RINm5F cells.
Functional characterisation of the Drosophila 5‐HTdro1 and 5‐HTdro2B serotonin receptors in insect cells: activation of a Gαs‐like protein by 5‐HTdro1 but lack of coupling to inhibitory G‐proteins by 5‐HTdro2B
Authors: Obosi Louis A. , Schuette Diana G. , Europe-Finner G.Nicholas , Beadle David J. , Hen René , King Linda A. , Bermudez Isabel
Published: April 2003

Insect cells are routinely used for the production of receptor proteins. Expression of the Drosophila 5‐HT
dro1 serotonin receptor resulted in positive coupling of the receptor to adenylyl cyclase via the Gαs
G‐protein subtype. The Drosophila 5‐HT
dro2B
receptor stimulated the metabolism of inositol phospholipid via a pertussis toxin‐insensitive G‐protein, but exhibited no detectable inhibition of adenylyl cyclase. Immunoblot analysis of the endogenous G‐proteins revealed that Sf9 cells lack the G‐protein subtypes Gαi1–3 and Gα0, but express the subtypes Gαs
and Gαq
.
Identification of alternative splicing form of Stat2
Authors: Sugiyama Tetsuya , Nishio Yukihiro , Kishimoto Tadamitsu , Akira Shizuo
Published: April 2003

We identified alternatively spliced forms of Stat2 in human and mouse mRNAs. The spliced forms are generated by reading through the intron between exon 20 and 21, which correspond to the region encoding SH2 domain. The spliced forms contain a stop codon in SH2 domain, and therefore give rise to a short form of Stat2 when the mRNAs are translated. The putative translated proteins lack half of the SH2 domain, the tyrosine phosphorylation site required for dimerization (or oligomereization) and DNA binding, and C‐terminal activation domain. The significance of these spliced forms is discussed.
One‐step purification of the β‐glucan elicitor‐binding protein from soybean (Glycine max L.) roots and characterization of an anti‐peptide antiserum
Authors: Mithöfer Axel , Lottspeich Friedrich , Ebel Jürgen
Published: April 2003

A low abundance β‐glucan elicitor‐binding protein from soybean was isolated by a rapid, simple and one‐step purification method yielding about 9000‐fold enrichment. The affinity‐based purification technique was more efficient than a procedure that uses conventional methods and preserved the binding activity to a much larger extent. The final preparation consisted of one major protein with an apparent molecular mass of about 75 kDa. Electrophoretic analyses of the purified and photoaffinity‐labeled binding protein showed that the native protein was an oligomer with apparent molecular mass of about 240 kDa. A polyclonal anti‐peptide antiserum was raised against a synthetic 15‐mer internal oligopeptide sequence derived from the 75‐kDa protein. The antiserum recognized the purified binding protein in immunoblotting experiments and precipitated the affinity‐labeled protein from a crude extract of the membrane fraction.
Wortmannin inhibits store‐mediated calcium entry and protein tyrosine phosphorylation in human platelets
Authors: Jenner Susanne , Farndale Richard W. , Sage Stewart O.
Published: April 2003

The effects of the WT on store‐mediated Ca2+ entry and protein tyrosine phosphorylation were investigated in fura‐2‐loaded human platelets. Wortmannin (2 μM) attenuated the rise in [Ca2+]i caused by Ca2+ entry while having no effect on the mobilisation of Ca2+ from internal stores. It also reduced store‐depletion‐evoked protein tyrosine phosphorylation. These findings demonstrate that WT is an inhibitor of tyrosine phosphorylation and store‐mediated calcium entry and provide further evidence for the involvement of a tyrosine phosphorylation step in the link between Ca2+ store depletion and Ca2+ influx in human platelets.
Towards prediction of cognate complexes between the WW domain and proline‐rich ligands
Authors: Einbond Aaron , Sudol Marius
Published: April 2003

The WW domain is a structured protein module found in a wide range of regulatory, cytoskeletal, and signaling molecules. Its ligands contain proline‐rich sequences, some of which show a core consensus of XPPXY that is critical for binding. In order to gain a better understanding of the molecular and biological functions of WW domains, we decided to predict their cognate ligands by searching databases for proteins containing the XPPXY consensus. Using several axioms that take into account evolutionary conservation and functional similarity, we have identified four groups of proteins representing candidate ligands that signal through known or unknown WW domains. These include viral Gag proteins, sodium channels, interleukin receptors, and a subgroup of serine/threonine kinases. In addition, we proposed that dystrophin and β‐dystroglycan bind through the WW‐XPPXY link and that interference with this interaction could result in muscular dystrophy. Our study provides guidelines for experiments to probe the molecular and biological functions of the WW domain‐ligand connection. Should these predictions be proven empirically, the results may have important ramifications for basic research and medicine.
Binding of a valproate metabolite to the trifunctional protein of fatty acid oxidation
Authors: Baldwin Graham S. , Abbott Frank S. , Nau Heinz
Published: April 2003

The anti‐convulsant drug valproate causes hepatic failure in a small percentage of patients. We now report that the valproate metabolite 2,4‐dien‐valproate binds (IC50 = 42 μM) to the α‐subunit of the trifunctional protein responsible for the second and third steps in the mitochondrial β‐oxidation of fatty acids. Binding of valproate itself, or of the metabolites 2‐envalproate, 4‐en‐valproate or 3‐hydroxy‐4‐en‐valproate, is considerably weaker. We conclude that valproate‐induced hepatotoxicity may be due in part to the reversible binding of the valproate metabolite 2,4‐dien‐valproate or its CoA ester to the α‐subunit of the trifunctional protein with consequent inhibition of fatty acid oxidation.
Degradation of glutathione S‐conjugates by a carboxypeptidase in the plant vacuole
Authors: Wolf Andreas E. , Dietz K.-J. , Schröder P.
Published: April 2003

For plants, glutathione conjugation is a major pathway to detoxify organic xenobiotic. Glutathione S‐conjugates (SG‐conjugates) are formed in the cytosol, the in vitro transport over the tonoplast has been described and a final storage in the vacuole has been postulated. We show here that alachlor rapidly accumulates as GS‐conjugates in the plant vacuole and that the first step of its degradation, the formation of the respective γ‐glutamylcysteinyl‐S‐conjugate, is catalyzed by a vacuolar carboxypeptidase. These results suggest the glutathione conjugate as a transport form but not a storage form of xenobiotic molecules.
Cloning of a novel human RNA polymerase II subunit downregulated by doxorubicin: new potential mechanisms of drug related toxicity
Authors: Fanciulli Maurizio , Bruno Tiziana , Cerboni Cristina , Bonetto Francesco , Iacobini Carla , Frati Luigi , Piccoli Mario , Floridi Aristide , Santoni Angela , Punturieri Antonello
Published: April 2003

Using the differential display PCR method, we have isolated an mRNA downregulated in doxorubicin resistant human cell lines. The full length cDNA clone was identified as the human homologue of yeast RPB11 subunit of RNA polymerase II. Northern blot analysis of normal tissues detected a particularly high expression of RPB11 mRNA in heart and skeletal muscle. Reduction of this mRNA expression was observed in all the cell lines tested after drug treatment and was paralleled by a similar decrease of the protein levels. These findings suggest that doxorubicin may exert in vivo specific inhibitory effects on a major component of the transcription machinery.
Advanced glycosylation end products stimulate the growth but inhibit the prostacyclin‐producing ability of endothelial cells through interactions with their receptors
Authors: Yamagishi Sho-ichi , Yamamoto Yasuhiko , Harada Shin-ichi , Hsu Cheng-Chin , Yamamoto Hiroshi
Published: April 2003

The influence of advanced glycosylation end products (AGE) on endothelial cells was investigated. When human umbilical endothelial cells were cultured with AGE‐bovine serum albumin, viable cell number as well as DNA synthesis was significantly stimulated, whereas prostacyclin production by the endothelial cells was decreased. Antisense oligodeoxyribonucleotides against mRNA coding for AGE receptor were found to reverse both the AGE‐induced growth stimulation and the inhibition of prostacyclin production in endothelial cells. These results thus suggest that AGE ligand‐receptor interactions in endothelial cells can promote angiogenesis and thrombogenesis, leading to the development of diabetic vascular complications.
Intramolecular interactions regulate serine/threonine phosphorylation of vinculin
Authors: Schwienbacher Christine , Jockusch Brigitte M. , Rüdiger Manfred
Published: April 2003

Using protein kinase C, we have studied the influence of intramolecular interactions on phosphorylation in vinculin. We show that vinculin and its 90 kDa head and 29/27 kDa tail fragments, generated by V8 proteolytic cleavage, are differentially phosphorylated. While intact vinculin and the isolated head domain are only weakly labelled, the isolated tail fragment is much more strongly phosphorylated. In the presence of the tail, the head is fully protected from the kinase. These data are consistent with our observation that native vinculin is primarily phosphorylated within the tail domain and suggest a function of vinculin phosphorylation in the regulation of the vinculin conformation.
An actin point‐mutation neighboring the ‘hydrophobic plug’ causes defects in the maintenance of cell polaroty and septum organization in the fission yeast Schizosaccharomyces pombe
Authors: Ishiguro Junpei , Kobayashi Wataru
Published: April 2003

The fission yeast cps8 mutation gives rise to abnormally enlarged and dispolarized cells, each of which contains several nuclei with aberrant multisepta. Molecular cloning and sequence analysis of the cps8 gene indicated that it encodes an actin with an amino acid substitution of aspartic acid for glycine at residue 273 in the hydrophobic loop that is located between actin subdomains 3 and 4. Fluorescence microscopy using phalloidin and anti‐actin antibody revealed changes in the F‐actin structure and distribution in the mutant cells. These results indicate that the hydrophobic loop plays an essential role for creating normal F‐actin structure, only by which cell polarity and the late mitotic events can be maintained properly.
Interaction of smooth muscle calponin and desmin
Authors: Wang Peiyi , Gusev Nikolai B.
Published: April 2003

Interaction of smooth‐muscle calponin and desmin was analyzed by means of ultracentrifugation, fluorescent spectroscopy and affinity chromatography. At low and intermediate ionic strength (30–50 mM NaCl) calponin is cosedimented with desmin with an apparent dissociation constant 3–15 μM and stoichiometry of 1 calponin/4–6 desmin. Calmodulin decreases the quantity of calponin bound to desmin. Increase of ionic strength up to 150 mM weakens calponin‐desmin interaction, but even at this ionic strength part of calponin remains bound to desmin. Calponin increases the rate and extent of fluorescence quenching induced by polymerization of 5‐iodoacetamidofluorescein‐labeled desmin. Affinity chromatography data indicate that desmin‐binding sites are located in the N‐terminal 22 kDa fragment of calponin. Since calponin interacts with desmin with an affinity comparable with that of, e.g., tropomyosin and myosin we suppose that calponin‐desmin interaction may be important for cytoskeleton organization.
Unusually stable helical kink in the antimicrobial peptide — A derivative of gaegurin
Authors: Suh Jeong-Yong , Lee Keun-Hyeung , Chi Seung-Wook , Hong Seong-Yu , Choi Byoung-Wook , Moon Hong-Mo , Choi Byong-Seok
Published: April 2003

The structure of an active analog of the antibacterial peptide gaegurin was investigated by CD and NMR spectroscopy. The NOE connectivities showed that 21 out of 24 residues formed an α‐helix despite the presence of a central proline. CD and NMR analysis indicates that the helix is in fast equilibrium with random coil. From chemical shift analysis of the amide protons, the distances of hydrogen bonding in the helix were calculated, and manifested obvious periodicity which implied a kink in the middle of the helix. 1D amide proton exchange experiments provided further evidence of an exceptionally stable kink. It is inferred that this kink is important not only to the function of the peptide but also to the early stage of the folding as a nucleation site.
Hepatic microsomal triglyceride transfer protein messenger RNA concentrations are increased by dietary cholesterol in hamsters
Authors: Bennett Andrew J. , Bruce Jennifer S. , Salter Andrew M. , White David A. , Billett Michael A.
Published: April 2003

In hamsters fed high fat diets enriched in trimyristin, tripalmitin or tristearin, increased dietary cholesterol content was associated with increased plasma concentrations of very low density lipoprotein (VLDL) cholesterol and triacylglycerol (p < 0.0001 and p = 0.0017, respectively). Hepatic microsomal triglyceride transfer protein (MTP) mRNA concentration also increased (p < 0.0001), independent of the nature of dietary fat, and was significantly correlated with the plasma VLDL lipid concentrations (p = 0.0002 and p = 0.0106 for cholesterol and triacylglycerol, respectively) and hepatic cholesterol concentrations. Increased expression of the MTP gene may be part of a coordinated response to hepatic cholesterol accumulation leading to increased VLDL lipid secretion.
The untranslated first exon ‘exon OS’ of the rat estrogen receptor (ER) gene
Authors: Hirata Shuji , Koh Tomoko , Yamada-Mouri Naoko , Hoshi Kazuhiko , Kato Junzo
Published: April 2003

Recently, we have isolated the untranslated first exon ‘exon 0N’ of the rat estrogen receptor (ER) gene from the liver by the use of the 5′‐rapid amplification of cDNA end (5′‐RACE) method. To investigate the existence of other untranslated first exon(s), we further analyzed the 5t'‐unsaturated region (UTR) of ER mRNA in the rat liver in this study. Total RNA from the livers of 8‐week‐old male Wistar rats was subjected to 5′‐RACE with the antisense primers located in exon 1 of the rat ER gene. The inserts of four clones (clones 3, 4, 7 and 8) were sequenced. The nucleotide sequences of the clones revealed the existence of a previously unidentified untranslated first exon (we termed it ‘exon 0S’) which was spliced onto exon 1 of the rat ER mRNA. The distribution of ER mRNA containing ‘exonn 0S’ (ER mRNA (0S‐1)) in several brain regions and various peripheral tissues of 8‐week‐old male and female Wistar rats was further analyzed by the use of the reverse transcription‐polymerase chain reaction. ER mRNA (0S‐1) was found to be widely distributed in the rat brain and peripheral tissues. The distribution of the message was different from that of ER mRNA containing exon 0 (the first reported 5′‐UTR form of rat ER mRNA) or of ER mRNA with exon 0N which was reported in our recent report. These results indicate that (1) ‘exon 0S’ is a novel untranslated first exon of the rat ER gene, (2) rat ER mRNAs possess at least three forms of 5′‐UTRs which are exon 0, exon 0N, and exon 0S, (3) the tissue specific expression of ER is regulated, at least in part, by the usage of differential promoters in the rat.
Cloning and expression of full length mouse utrophin: the differential association of utrophin and dystrophin with AChR clusters
Authors: Athena Guo W.-X. , Nichol Mia , Merlie John P.
Published: April 2003

We have cloned and sequenced mouse utrophin cDNA, and successfully expressed full length utrophin (400 kDa) in both muscle and non‐muscle cells. The expression of recombinant utrophin is compared with that of its homologue, dystrophin (427 kDa). We demonstrate that recombinant utrophin is targeted into agrin‐induced acetylcholine receptor (AChR) clusters, while recombinant dystrophin is evenly distributed along cell membranes in cultured Sol 8 muscle cells. This observation suggests that utrophin and dystrophin may interact with different cytoskeletal proteins. The C‐terminal domains are found to be responsible for the association of utrophin with AChR clusters.
The catalytic domain of human immunodeficiency virus integrase: ordered active site in the F185H mutant
Authors: Bujacz Grzegorz , Alexandratos Jerry , Zhou-Liu Qing , Clément-Mella Christine , Wlodawer Alexander
Published: April 2003

We solved the structure and traced the complete active site of the catalytic domain of the human immunodeficiency virus type 1 integrase (HIV‐1 IN) with the F185H mutation. The only previously available crystal structure, the F185K mutant of this domain, lacks one of the catalytically important residues, E152, located in a stretch of 12 disordered residues [Dyda et al. (1994) Science 266, 1981–1986]. It is clear, however, that the active site of HIV‐1 IN observed in either structure cannot correspond to that of the functional enzyme, since the cluster of three conserved carboxylic acids does not create a proper metal‐binding site. The conformation of the loop was compared with two different conformations found in the catalytic domain of the related avian sarcoma virus integrase [Bujacz et al. (1995) J. Mol. Biol. 253, 333‐246]. Flexibility of the active site region of integrases may be required in order for the enzyme to assume a functional conformation in the presence of substrate and/or cofactors.
Kinetic study of the reaction of ebselen with peroxynitrite
Authors: Hiroshi Masumoto  , Kissner Reinhard , Koppenol Willem H. , Sies Helmut
Published: April 2003

The second‐order rate constant for the reaction of ebselen with peroxynitrite (ONOO−) is (2.0±0.1) × 106 M−1s−1 at pH ≥ 8 and 25°C, 3–4 orders of magnitude higher than the rate constants observed for cysteine, ascorbate, or methionine. The activation energy is relatively low, 12.8 kJ/mol. This is the fastest reaction of peroxynitrite observed so far. It may allow Secontaining compounds to play a novel role in the defense against peroxynitrite, one of the important reactive species generated during inflammatory processes.
Placental transfer of rubella‐specific IgG in fullterm and preterm newborns
Authors: Doroudchi M. , Samsami Dehaghani A. , Emad K. , Ghaderi A.
Published: April 2003

Objectives: This study was undertaken to investigate placental transfer of anti‐rubella IgG immunoglobulins in Iranian mothers. Methods: In total, 231 pregnant women and their paired infants enrolled in this study of which, 197 gave birth to fullterm and 26 gave birth to preterm infants. Rubella specific antibodies were detected by an in‐house whole‐virus ELISA assay in maternal and cord sera of 188 fullterm and 26 preterm infants. Results: A highly significant correlation was observed between anti‐rubella IgG in newborns in total, in preterm and fullterm neonates with their paired mothers (P‐values=0.0001, 0.002, 0.0001, respectively). A borderline significant difference was observed between mean anti‐rubella IgG in fullterm and preterm neonates (P=0.04). Mean cord/maternal ratio of anti‐rubella IgG was 0.83 which was surprisingly low. A significant lower anti‐rubella IgG was observed in newborns born from mothers with blood group B+ than those born from mothers with blood groups A+ (P=0.04) and O+ (P=0.02), respectively. The same difference was observed between mean maternal anti‐rubella IgG in those with blood groups B+ and A+ (P=0.04) and those with blood groups B+ and O+ (P=0.05). In addition, a low frequency of B+ blood group in high positive sera and a high frequency of this blood group among low positive and negative sera was detected. Conclusions: Our data suggest that the main factors that influence the infants’ rubella‐specific IgG concentration are maternal concentration of this immunoglobulin, maternal blood group, and neonatal gestational age.
Oxygen isotope stage 3 fluvial and eolian successions in Europe compared with climate model results
Authors: van Huissteden K.o , Pollard David
Published: April 2003

Fluvial and eolian successions of oxygen isotope stage 3 are compared with global (GCM) and regional climate (RCM) modeling experiments of the stage 3 and last glacial maximum climate in Europe. Differences in precipitation between stage-3 stades and interstades were minor, which is confirmed by the fluvial successions. The fluvial response to climate variation is non-uniform, and in southern Europe more pronounced than in northern Europe. The model simulations indicate a strong western winter circulation over Europe during stage 3, which is supported by the eolian deposits data. Wind speeds in the last glacial maximum simulation appear modest compared with those of stage 3, which contrasts with the abundance of eolian deposits. This suggests that during glacial climates the stabilizing effect of vegetation determines eolian sedimentation rates, rather than wind speed. Stage 3 can be divided into an older part (>45,000 cal yr B.P.) with a relatively stable landscape and moist climate and a younger part with more frequent climate change and decreasing landscape stability.
The effect of Holocene temperature fluctuations on the evolution and ecology ofNeotoma(woodrats) in Idaho and northwestern Utah
Authors: Smith Felisa A. , Betancourt Julio L.
Published: April 2003

Animals respond to climatic change by adapting or by altering distributional patterns. How an animal responds is influenced by where it is positioned within its geographic range; the probability of extirpation is increased near range boundaries. Here, we examine the impact of Holocene climatic fluctuations on a small mammalian herbivore, the bushy-tailed woodrat (Neotoma cinerea), at five locations within south central Idaho and northwestern Utah. Previous work demonstrated that woodrats adapt to temperature shifts by altering body size. We focus here on the relationship between body mass, temperature, and location within the geographic range. Body mass is estimated by measuring fossil fecal pellets, a technique validated in earlier work. Overall, we find the predicted phenotypic response to climate change: animals were larger during cold periods, and smaller during warmer episodes. However, we also identify several time periods when changes in environmental temperature exceeded the adaptive flexibility ofN. cinerea.A smaller-bodied species, the desert woodrat (N. lepida) apparently invaded lower elevation sites during the mid-Holocene, despite being behaviorally and physically subordinate toN. cinerea.Analysis of contemporary patterns of body size and thermal tolerances for both woodrat species suggests this was because of the greater heat tolerance ofN. lepida. The robust spatial relationship between contemporary body size and ambient temperature is used as a proxy to reconstruct local climate during the Holocene.
Effects of transcutaneous topical injection of oxygen on vascular endothelial growth factor gene into the healing ligament in rats
Authors: Ishii Yoshimasa , Ushida Takashi , Tateishi Tetsuya , Miyanaga Yutaka
Published: April 2003

The effects of intermittent exposure to oxygen injection on an experimentally induced ligament tear were studied in the right hind limb of 17 male Sprague–Dawley rats. Two rats were used for monitoring the partial oxygen pressure (pO2) of subcutaneous tissue and 15 rats were divided into the following three groups of 5 after an experimentally induced ligament tear: Group A, control group; Group B, injection of 0.5 ml hyaluronan to the wound transcutaneously; Group C, injection of 0.5 ml hyaluronan mixed with haemoglobin and oxygen (n = 5). At 7 days post‐ligament injury, we compared the ligaments of the three treatment groups for gross appearance, histology and expression of vascular endothelial growth factor (VEGF) mRNA by RT‐PCR. Our results indicate that the pO2 was immediately elevated to 334.6 mmHg by topical oxygen injection and this method was effective in promoting vessel formation in comparison to the control group (p < 0.01). However, the expression of VEGF mRNA in the topical oxygen injection group (Group C) was lower than that in control group (p < 0.05). Our results suggest that oxygen is able to accelerate vessel formation in spite of its effect of decreasing VEGF mRNA. Our method of using topical injection proved to be useful in healing the ligament and the wound. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Quantitative measurement of patellofemoral joint stability: Force–displacement behavior of the human patella in vitro
Authors: Senavongse W. , Farahmand F. , Jones J. , Andersen H. , Bull A. M. J. , Amis A. A.
Published: April 2003

Patellofemoral joint instability is a common clinical problem. However, little quantitative data are available describing the stability characteristics of this joint. We measured the stability of the patella against both lateral and medial displacements across a range of knee flexion angles while the quadriceps were loaded physiologically. For eight fresh‐frozen knee specimens a materials testing machine was used to displace the patella 10 mm laterally and 10 mm medially while measuring the required force, with 175 N quadriceps tension. The patella was connected via a ball‐bearing patellar mounting 10 mm deep to the anterior surface to allow natural tilt and other rotations. Patellar force–displacement behavior was tested at flexion angles of 0°, 10°, 20°, 30°, 45°, 60°, and 90°. Significant differences were found between the lateral and medial restraining forces at 10 mm displacement. For lateral displacement, the restraining force was least at 20° of knee flexion (74 N at 10 mm displacement), rising to 125 N at 0° and 90° of knee flexion. The restraining force increased progressively with knee flexion for medial patellar displacement, from 147 N at 0° to 238 N at 90°. With quadriceps tension, the patella was more resistant to medial than lateral displacement. Our finding that lateral patellar displacement occurred at the lowest restraining force when the knee was flexed 20° agrees with clinical experience of patellar instability. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Enhancement of dendritic branching in cultured hippocampal neurons by 17β‐estradiol is mediated by nitric oxide
Authors: Audesirk T. , Cabell L. , Kern M. , Audesirk G.
Published: April 2003

Both 17β‐estradiol (E2) and nitric oxide (NO) are important in neuronal development, learning and memory, and age‐related memory changes. There is growing evidence that a number of estrogen receptor‐mediated effects of estradiol utilize nitric oxide as an intermediary. The role of estradiol in hippocampal neuronal differentiation and function has particular implications for learning and memory.

Low levels of estradiol (10 nM) significantly increase dendritic branching in cultured embryonic rat hippocampal neurons (158% of control). This study investigates the hypothesis that the estrogen‐stimulated increase in dendritic branching is mediated by nitric oxide. We found that nitric oxide donors also produce significantly increased dendritic branching S‐nitroso‐N‐acetylpenicillamine (SNAP: 119%; 2,2′‐(hydroxynitrosohydrazino)bis‐ethanamine (NOC‐18): 128% of control). We then determined that the increases in dendritic branching stimulated by estradiol or by a nitric oxide donor were both blocked by an inhibitor of guanylyl cyclase. Dendritic branching was also stimulated by a cell permeable analog of cyclic guanosine monophosphate (dibutyryl‐cGMP: 173% of control). Estradiol‐stimulated dendritic branching was reversed by the nitric oxide scavenger 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethyl imidazoline‐1‐oxyl 3‐oxide (carboxy‐PTIO). This study provides evidence that estradiol influences the development of embryonic hippocampal neurons in culture by increasing the production of nitric oxide or by increasing the sensitivity of the neurons to nitric oxide. Nitric oxide in turn stimulates dendritic branching via activation of guanylyl cyclase.
An accounting analysis of the risk‐return relationship in bull and bear markets
Authors: Kim Moon K. , Ismail Badr E.
Published: April 2003

This study provides evidence that accounting beta (earnings and cash flow‐based) provides information consistent with the risk‐return relationship in up‐ and down‐markets. We are not able, however, to provide similar evidence using market beta. Considering that investors' ability to assess the risk‐return trade‐off in up‐ and down‐markets is central to efficient portfolio formation, the results suggest that accounting data can provide appropriate measures of portfolio upside and downside risk.
An Evaluation of Process and Protocols for Planned Home Birth Attended by Regulated Midwives in British Columbia
Authors: Janssen Patricia A. , Lee Shoo K. , Ryan Elizabeth R. , Saxell Lee
Published: April 2003

Midwifery emerged as a self‐regulated profession in British Columbia in the context of a 2‐year demonstration project beginning in 1998. The project evaluated accountability among midwives, defined as the provision of safe and appropriate care and maintenance of standards of communication set by the College of Midwives of British Columbia. Adherence to protocols was measured by using documentation designed specifically for the Home Birth Demonstration Project. Hospital and transport records for selected clients were reviewed by an expert committee. Outcomes among Home Birth Demonstration Project clients were compared with outcomes among women eligible for home birth but planning to deliver in hospital. Adherence to clinical and communication protocols was 96% or higher. Planned home birth was not associated with an increase in risk but prevalence of adverse outcomes was too low to be studied with precision. Recommendations of an expert review committee have been implemented or are under review. Midwives have demonstrated a high degree of compliance with reporting requirements and protocols. Comparisons of birth outcomes of planned home versus hospital births, while supporting home birth as a choice for women, were limited in scope and require ongoing study. Integration of home birth has been a dynamic process with guidelines and policy continuing to evolve.
Nonlinear accumulation in the brain of the new taxoid TXD258 following saturation of P‐glycoprotein at the blood–brain barrier in mice and rats
Authors: Cisternino Salvatore , Bourasset Fanchon , Archimbaud Yves , Sémiond Dorothée , Sanderink Gérard , Scherrmann Jean‐Michel
Published: April 2003

TXD258, a new taxoid antitumor agent, is a poor substrate for the P‐glycoprotein (P‐gp) in Caco‐2 cells. In this study, we investigated the amount of drug accumulating in the brains of rats and mice under a variety of conditions (dose and infusion time, species and plasma concentration) using conventional in vivo pharmacokinetic techniques and in situ brain perfusion.

Mice were infused with radiolabeled TXD258 at 15, 30, 45 and 90 mg m−2 for 45 s or 1 h and rats were infused with 15 and 60 mg m−2 over 2.3 min. The radioactivity in the plasma and brains was measured. The brain concentrations of TXD258 in mice and rats were maximal from 2 min to 1 h postinfusion and radioactivity was still detectable at 168 h. While the plasma concentration of TXD258 increased linearly in mice with the infused dose, the brain content increased more than proportionally with the dose between 15 and 90 mg m−2. This nonlinear uptake of TXD258 also occurred in the plasma and brain of the rat.

These findings suggest that the protein‐mediated efflux across the blood–brain barrier (BBB) becomes saturated. In situ brain perfusion studies confirmed that TXD258 is a P‐gp substrate at the BBB of mice and rats. The P‐gp of both species was saturated at the half‐inhibitory concentration (∼13 μM) produced by i.v. infusion.

Thus, the observed nonlinear accumulation of TXD258 in the brain seems to occur by saturation of the P‐gp at the rodent BBB. This saturation could have several advantages, such as overcoming a P‐gp‐mediated efflux, but the nonlinear pharmacokinetics could increase the risk of toxicity.

British Journal of Pharmacology (2003) 138, 1367–1375. doi:10.1038/sj.bjp.0705150
Effects of bisphenol A on antigen‐specific antibody production, proliferative responses of lymphoid cells, and TH1 and TH2 immune responses in mice
Authors: Yoshino Shin , Yamaki Kouya , Yanagisawa Rie , Takano Hirohisa , Hayashi Hideyuki , Mori Yoki
Published: April 2003

We investigated the effect of bisphenol A (BPA), which binds estrogen receptors, on immune responses including production of antigen‐specific antibodies, proliferative responses of lymphoid cells, and Th1 and Th2 responses.

For this investigation, mice were p.o. given varying doses including 3, 30, 300, and 3000 μg kg−1 of BPA immediately after immunization with hen egg lysozyme (HEL) (day 0) and then daily by day 20. On day 21, anti‐HEL IgG antibodies in sera and proliferative responses of spleen cells to the antigen were measured. Anti‐HEL IgG2a antibodies and IFN‐γ secreted from splenic lymphocytes were also measured as indicators of Th1 immune responses, while anti‐HEL IgG1 antibodies and IL‐4, as those of Th2 responses.

The results showed that treatment with 3000 μg kg−1 of BPA was followed by a significant increase in anti‐HEL IgG as well as the antigen‐specific cell proliferation. Anti‐HEL IgG2a production and IFN‐γ secretion were significantly enhanced in mice treated with 300 and 30 μg kg−1 of BPA, respectively, while anti‐HEL IgG1 production and IL‐4 secretion were augmented in animals given 3000 and 300 μg kg−1 of the chemical, respectively.

Augmentation of these immune responses was also observed in mice exposed to 0.3–30 μg kg−1 of estradiol, although Th1 responses appeared to be more sensitive to the sex hormone than Th2 responses.

These results suggest that BPA may play a role in augmenting immune responses, especially Th1 responses.

British Journal of Pharmacology (2003) 138, 1271–1276. doi:10.1038/sj.bjp.0705166
Neuroprotection by caffeine and adenosine A2A receptor blockade of β‐amyloid neurotoxicity
Authors: Dall'lgna Oscar P , Porciúncula Lisiane O , Souza Diogo O , Cunha Rodrigo A , Lara Diogo R
Published: April 2003

Adenosine is a neuromodulator in the nervous system and it has recently been observed that pharmacological blockade or gene disruption of adenosine A2A receptors confers neuroprotection under different neurotoxic situations in the brain. We now observed that coapplication of either caffeine (1–25 μM) or the selective A2A receptor antagonist, 4‐(2‐[7‐amino‐2(2‐furyl)(1,2,4)triazolo (2,3‐a)(1,3,5)triazin‐5‐ylamino]ethyl)phenol (ZM 241385, 50 nM), but not the A receptor antagonist, 8‐cyclopentyltheophylline (200 nM), prevented the neuronal cell death caused by exposure of rat cultured cerebellar granule neurons to fragment 25–35 of β‐amyloid protein (25 μM for 48 h), that by itself caused a near three‐fold increase of propidium iodide‐labeled cells. This constitutes the first in vitro evidence to suggest that adenosine A2A receptors may be the molecular target responsible for the observed beneficial effects of caffeine consumption in the development of Alzheimer's disease.

British Journal of Pharmacology (2003) 138, 1207–1209. doi:10.1038/sj.bjp.0705185
Cardiac‐specific overexpression of human β2 adrenoceptors in mice exposes coupling to both Gs and Gi proteins
Authors: Hasseldine A R G , Harper E A , Black J W
Published: April 2003

Left atrial strips from transgenic (TG4) mice with cardiac‐specific overexpression (∼200‐fold) of the β2 adrenoceptor (β2AR) were isolated, and their isometric force of contraction (Fc) in response to electrical stimulation was measured.

The βAR agonist isoprenaline elicited negative inotropic responses in all left atrial strips; in 6/11 preparations, it also had a small positive inotropic effect. This ‘up‐phase’ was observed from 0.1 to 10 nM, with the ‘down‐phase’ occurring at higher concentrations. Both phases were mediated by β2AR, as shown by their sensitivity to the β2AR antagonist ICI‐118,551 (100 nM; pA2 8.60±0.07, 8.45±0.19, for ‘up‐phase’ and ‘down‐phase,’ respectively), but not the β1AR antagonist CGP‐20712A (100 nM). Conversely, nontransgenic littermate preparations responded to isoprenaline treatment solely by an increase in Fc, which was β1AR‐mediated.

Pretreatment of left atrial strips with either 10 nM isoprenaline or 1 mM 8‐bromo‐cAMP significantly attenuated the TG4 ‘up‐phase’, while having no effect on either the TG4 ‘down‐phase’ or the littermate controls' responses. B. pertussis toxin treatment of the animals prevented isoprenaline's negative inotropic effects in TG4 preparations, but had no effect in littermate controls.

The findings imply that the responses of TG4 left atrium to isoprenaline are because of β2AR coupling to Gs and Gi proteins, consistent with the model of Daaka et al., in which protein kinase A phosphorylation of the β2AR causes a switch from Gs to Gi protein coupling.

British Journal of Pharmacology (2003) 138, 1358–1366. doi:10.1038/sj.bjp.0705191
The ups and downs of Gs‐ to Gi‐protein switching
Authors: Hill Stephen J , Baker Jillian G
Published: April 2003

British Journal of Pharmacology (2003) 138, 1188–1189. doi:10.1038/sj.bjp.0705192