bluuebunny/crossref_metadata_2025 huggingface dataset:
13C NMR studies of complexes of Escherichia coli dihydrofolate reductase formed with methotrexate and with folic acid
Authors: Cheung H.T.A. , Birdsall B. , Feeney J.
Published: July 2002

13C NMR studies of 13C‐labelled ligands bound to dihydrofolate reductase provide (DHFR) a powerful means of detecting and characterizing multiple bound conformations. Such studies of complexes of Escherichia coli DHFR with [4,7,8a,9‐13C]‐ and [2,4a,6‐13C]methotrexate (MTX) and [4,6.8a‐13C]‐ and [2,4a,7,9‐13C]folic acid confirm that in the binary complexes, MTX binds in two conformational forms and folate binds as a single conformation. Earlier studies on the corresponding complexes with Lactobacillus casei DHFR indicated that, in this case, MTX binds as a single conformation whereas folate binds in multiple conformational forms (both in its binary complex and ternary complex with NADP+); two of the bound conformational states for the folate complexes are very different from each other in that there is a 180° difference in their pteridine ring orientation. In contrast, the two different conformational states observed for MTX bound to E. coli DHFR do not show such a major difference in ring orientation and bind with N1 protonated in both forms. The major difference appears to involve the manner in which the 4‐NH2 group of MTX binds to the enzyme (although the same protein residues are probably involved in both interactions). Addition of either NADP+ or NADPH to the E. coli DHFR‐MTX complex results in a single set of 13C signals for bound methotrexate consistent with only one conformational form in the ternary complexes.
Transmembrane electron transport in ascorbate‐loaded plasma membrane vesicles from higher plants involves a b‐type cytochrome
Authors: Asard H. , Horemans N. , Caubergs R.J.
Published: July 2002

The possible involvement of a high‐potential b‐type cytochrome in plasma membrane electron transport was tested using ascorbate‐loaded membrane vesicles. Absorption spectra demonstrated that the cytochrome was about 89% reduced in these preparations. Use of ascorbate oxidase and washing of the vesicles further indicated flat reduction was mediated by intra‐vesicular ascorbate. Addition of low concentrations of ferricyanide caused a rapid cytochrome oxidation followed by a slower re‐reduction. The Kinetics of this response indicate that the electron acceptor was fully reduced berore re‐reduction of the cytochrome occurred. These observations suggest that the b‐type cytochrome mediates transmembrane electron transfer.
Chemically synthesized 182–235 segment of tau protein and analogue peptides are efficient in vitro microtubule assembly inducers of low apparent sequence specificity
Authors: Novella Isabel S. , Andreu José Manuel , Andreu David
Published: July 2002

A 54‐amino acid peptide reproducing the first and second repeats and intervening spacer sequence of the tubulin binding motif (residues 182–235) of murine tau protein, and several congeners representing different degrees of sequence scrambling have been prepared by solid phase methods and fully characterized chemically. These double‐repeat peptides have been shown to induce microtubule formation at concentrations about one order of magnitude lower than single‐repeat controls, under conditions close to the critical concentration needed for tubulin self‐assembly. On the other hand, partial loss of microtubule‐inducing capacity was observed for peptides with primary structures increasingly disorganized with respect to the canonical peptide, These results call into question the assumption that a high degree of primary structure specificity is involved in the tau‐tubulin interaction leading to in vitro microtubule formation.
Effect of a benzodiazepine (chlordiazepoxide) on a GABAA receptor from rat brain Requirement of only one bound GABA molecule for channel opening
Authors: Serfozo Peter , Cash Derek J.
Published: July 2002

Chlordiazepoxide (CDPX) enhanced the rate of chloride exchange mediated by the major GABAA receptor found on sealed native membrane vesicles from rat cerebral cortex. The initial rate constant for chloride exchange for this receptor, (JA), a measure of open channel, was determined from the progress of GABA‐mediated influx of 36Cl−. The dependence of JA on GABA concentration was hyperbolic in the presence of CDPX (150 μM, sufficient to give maximum enhancement of chloride exchange rate) but sigmoid in its absence. Enhancement of channel opening (10‐fold at 0.3 μM GABA) decreased with increasing GABA concentration. The maximal response, above 1,000 μM GABA, was unaltered. The half‐response concentration was reduced from 80 μM to 50 μM. CDPX alone caused no measurable 36Cl− exchange. In the presence of CDPX, channel opening occurred with only one bound GABA molecule, whereas in its absence, channel opening with two bound GABA molecules was much more favorable. This could not be direct allosteric modulation of the channel opening conformational change by binding of CDPX at effector sites, but could be explained by an additional change of the receptor on binding CDPX to give a closed state which gave channel mediated by a single GABA binding site. Another possibility is that CDPX could act at one of the channel opening binding sites without a postulated, second closed conformational state.
Ascorbic acid oxidation: A potential cause of the elevated severity of atherosclerosis in diabetes mellitus?
Authors: Hunt James V. , Bottoms Mark A. , Mitchinson Malcolm J.
Published: July 2002

The exposure of mouse peritoneal macrophages to cholesterol linoleate‐containing, artificial lipoproteins can lead to intracellular ceroid accumulation. This can be used as a model to study the role of oxidation in macrophage uptake of lipoproteins containing unsaturated fatty acids, considered by many as a primary event in atherosclerotic plaque formation. Our studies show that ascorbic acid can both inhibit and promote the formation of ceroid in such a model system. The transition metal copper (Cu(II)) further elevates ceroid accumulation and EDTA, a metal chelator, inhibits it. When trace levels of transition metals are present, low concentrations of ascorbic acid can elevate ceroid formation. This pro‐ and antioxidant characteristic of ascorbic acid was confirmed by monitoring the generation of oxidants by various concentrations of ascorbic acid, assessed by benzoic acid hydroxylation or the fragmentation of BSA. We discuss these observations in the context of an apparent increase in ascorbic acid oxidation and elevated severity of atherosclerosis in diabetes mellitus.
Characterization of the linker peptide of the single‐chain Fv fragment of an antibody by NMR spectroscopy
Authors: Freund Christian , Ross Alfred , Guth Birgith , Plückthun Andreas , Holak Tad A.
Published: July 2002

A comparison of the single‐chain Fv fragment of the antibody McPC603 (scFv) with its corresponding unlinked Fv. fragment has been carried out with 15N‐edited NMR spectroscopy. The two Fv, fragments adopt the same structure, indicating that the linker does not perturb the folding of the domains. This also directly demonstrates that folding in vivo (Fv fragment) and in vitro (scFv. fragment) leads to the same structure. The main differences in the spectra of the uniformly 15N‐labeled scFv and Fv fragments are due to signals of Gly and Ser from the linker peptide of the scF
v
, fragment. The linker peptide has been mapped with NMR spectra of 15N‐glycine‐ and 15N‐glycine15N‐serine‐labeled scFvfragments. The 15N T2, relaxation data indicate that the linker peptide is more flexible than the rest of the molecule.
Contraction of smooth muscle by activation of endothelin receptors on autonomic neurons
Authors: Takimoto Misato , Inui Takashi , Okada Toshikazu , Urade Yoshihiro
Published: July 2002

Endothelin receptors, predominantly of the ETB type, were localized to cell bodies, processes, and varicosities of cholinergic and adrenergic intramural autonomic neurons that were present in primary cultures of guinea pig tracheal smooth muscle. Stimulation of the neuronal ETB receptor produced a tetrodotoxin‐sensitive increase in the intracellular calcium concentration in neurons which was followed by contraction of the neighboring smooth muscle cells. These observations suggest that endothelins can induce smooth muscle contraction by means of a neuronally mediated mechanism, in addition to their direct actions on the smooth muscle.
Cholesterol crystallization‐promoting activity of aminopeptidase‐N isolated from the vesicular carrier of biliary lipids
Authors: Núñez Liliana , Amigo Ludwig , Rigotti Attilio , Puglielli Luigi , Mingrone Geltrude , Greco Aldo Virgilio , Nervi Flavio
Published: July 2002

Different hydrophobic glycoproteins are associated to native biliary vesicles, which are the major carrier of biliary cholesterol. Some of these proteins promote cholesterol crystallization, a key step in cholesterol gallstone formation. This study was specifically conducted to identify the 130 kDa biliary vesicle‐associated glycoprotein and to determine its in vitro effect on the cholesterol crystal formation time. The 130 kDa vesicular glycoprotein was identified as aminopeptidase‐N by amino acid sequencing and specific enzymatic assay. Polyclonal antibodies raised against aminopeptidase‐N allowed us to determine its concentration in human hepatic bile, which varied from 17.3 to 57.6 μg/ml. Aminopeptidase‐N showed a concentration‐dependent cholesterol crystallization activity when it was added to supersaturated model bile at a concentration range usually found in native bile. Because of this promoting effect on in vitro cholesterol crystal formation, we suggest that biliary aminopeptidase‐N may play a critical role in the pathogenesis of cholesterol gallstone disease.
Asparagine‐135 of elongation factor Tu is a crucial residue for the folding of the guanine nucleotide binding pocket
Authors: Weijland Albert , Sarfati Robert , Bârzu Octavian , Parmeggiani Andrea
Published: July 2002

This work studies the structure‐function relationships of Asn135, a residue situated in the GTP binding pocket of elongation factor Tu (EF‐Tu). For this purpose we constructed EF‐TuN135D/D138N and assayed its reactivity towards various purine nucleotides. We found that EF‐TuN135D/D138N had no functional effect with GTP, ATP, XTP and isoGTP. The lack of a productive interaction with isoGTP shows that the Asn135 side‐chain does not recognize the exocyclic keto group of the guanine base. However, EF‐TuN 135D/D 138N, whose native conformation is stabilized by either elongation factor Ts or kirromycin, was able to support the enzymatic binding of aa‐tRNA to the ribosome in the absence of any nucleotide, when in complex with the antibiotic. Taken together, these results show that Asn135 is important for the correct folding of the nucleotide binding site and that EF‐Tu·kirromycin can mediate the binding of aa‐tRNA to the mRNA‐programmed ribosomes independently of the native conformation of this site.
Berenil acts as a poison of eukaryotic topoisomerase II
Authors: Portugal José
Published: July 2002

The ability of the anti‐trypanosomal drug berenil to interfere with the activities of eukaryotic type II topoisomerases is evaluated using two different types of reactions catalysed by the enzyme: the relaxation of naturally supercoiled DNA and the decatenation of kinetoplast DNA (kDNA). The results indicate that berenil acts as an inhibitor of the catalytic activity, but the inhibiting ability appears to be smaller than for other minor‐groove binding ligands.
Immunological detection of prohormone convertases in two different proglucagon processing cell lines
Authors: Blache Philippe , Le-Nguyen Dung , Boegner-Lemoine Catherine , Cohen-Solal Anne , Bataille Dominique , Kervran Alain
Published: July 2002

The distribution of the prohormone convertases, PCI/3, PC2 and PC5/6, was determined by immunoblotting in two cell lines. In αTC1–6 cells, the proglucagon processing occured according to the pancreatic A‐cell type. In STC‐1 cells, proglucagon was processed in a manner reminiscent of the intestinal L‐cell type. PC1/3 was undetectable in both proglucagon processing cell lines whereas PC2 displayed a strong immunostaining in the αTC1–6 cells and was barely detectable in the STC‐1 cells. PC5/6 was detected as a 70 kDa protein in both cell lines. These results suggest a possible role of PC2 in the processing of proglucagon into glucagon in the A‐cells, whereas in L‐cells it would require still undetermined endoproteases.
MAP kinase binds to the NH2‐terminal activation domain of c‐Myc
Authors: Gupta Shashi , Davis Roger J.
Published: July 2002

The transcription factor c‐Myc is a substrate for phosphorylation by MAP kinases. Here we demonstrate that MAP kinase binds to c‐Myc. The NH2‐terminal region (residues 1–100) is necessary and sufficient for this interaction. Binding to c‐Myc is not dependent on the state of MAP kinase activation. However, the c‐Myc/MAP kinase complex is disrupted by ATP. Together, these observations indicate that substrate binding interactions contribute to the specificity of phosphorylation by MAP kinases.
Affinity purification of Hydra glutathione binding proteins
Authors: Bellis Susan L. , Laux David C. , Rhoads Dennis E.
Published: July 2002

The association of glutathione (GSH) with putative external chemoreceptors elicits feeding behavior in Hydra. In the present study, solubilized membrane proteins were chromatographed on an affinity column of immobilized GSH in order to isolate GSH‐binding proteins that may represent the Hydra GSH chemoreceptor. The most abundant of the affinity‐purified proteins was a triplet of peptides ranging in molecular weight from 24.5–26 kDa. Antiserum generated against the 24.5–26 kDa triplet peptides inhibited GSH‐stimulated feeding behavior by 47%, implicating a role for one or more of these peptides in Hydra chemoreception.
The tRNATyr multigene family of Triticum aestivum: genome organization, sequence analyses and maturation of intron‐containing pre‐tRNAs in wheat germ extract
Authors: Arends Sabine , Kraus Jürgen , Beier Hildburg
Published: July 2002

Southern analysis of Triticum DNA has revealed that nuclear tRNATyr genes are dispersed at a minimum of 16 loci in the genome. We have isolated six independent tRNATyr genes from a Triticum aestivum library in addition to three known members of the Triticum tRNATyr family. Four of the sequenced tRNATyr genes code for Triticum tRNA1
Tyr and two code for tRNA2
Tyr. Three genes encode tRNATyr which carry one or two nucleotide substitutions as compared to the conventional genes. The nine Triticum tRNATyr genes possess highly conserved intron sequences ranging in size from 12 to 14 nucleotides. A common secondary intron structure with the 5′ and 3′ splice site loops separated by five base pairs can be formed by all pre‐tRNAsTyr which are efficiently spliced in the homologous wheat germ extract.
Calcium‐bound recoverin targets rhodopsin kinase to membranes to inhibit rhodopsin phosphorylation
Authors: Sanada Kamon , Shimizu Fumiko , Kameyama Kimihiko , Haga Kazuko , Haga Tatsuya , Fukada Yoshitaka
Published: July 2002

In rod photoreceptor cells, Ca2+‐bound recoverin associates with disk membranes and inhibits light‐dependent phosphorylation of rhodopsin. However, the functional significance of Ca2+‐induced membrane association of recoverin has not been fully evaluated. We found that Ca2+‐bound recoverin forms a complex with rhodopsin kinase preferentially at the membrane surface. Addition of increasing amounts of membranes promoted the membrane association of recoverin, and remarkably suppressed rhodopsin kinase activity. It was concluded that the Ca2+‐recoverin‐rhodopsin kinase complex is stabilized by membrane association, leading to effective suppression of the kinase activity.
Molecular mechanisms and neural pathways mediating the influence of interleukin‐1 on the activity of neuroendocrine CRF motoneurons in the rat
Authors: Rivest Serge
Published: July 2002

The action of immune‐system‐derived cytokines to stimulate the release of corticotropin‐releasing factor (CRF) from the hypothalamus and the consequent elaboration of ACTH and release of corticosteroids has provided an especially useful model to investigate the nature of the intercommunication of neuroendocrine and immunological pathways. Substantial evidence exists to support the production of cytokines, such as interleukin‐1 (IL‐1) α and β, within the mammalian central nervous system. The mechanisms and neuronal circuitries involved in the effects of these cytokines of peripheral and central origin on the activity of neuroendocrine CRF motoneurons and the hypothalamic‐pituitary‐adrenal axis are described. Also included is a discussion of the influence of IL‐1 on transduction signals controlling the release and the biosynthesis of CRF in the parvocellular division of the paraventricular nucleus of the hypothalamus and the relationship between these two distinct intracellular processes. The relebance of using immediate early genes as indices of neuronal activity in immune‐challenged rats and the possible roles of c‐fos and NGFI‐B within neuroendocrine CRF motoneurons are outlined. Finally, the effects of acute immune response on neuroendocrine functions and brain neuronal activation are presented.
Defining and characterising treatment-resistant schizophrenia
Authors: Marder S
Published: July 2002

This paper argues for a broader definition of treatment refractoriness that would include patients with partial drug responses, a relatively recent onset, less dramatic symptoms and intolerance to conventional drugs. The drug trials that define their responsiveness should be carefully conducted over a prolonged period of time, but need not include high doses or supplemental medications. If the consensus conference can agree on a definition of responsiveness, it will provide important guidance to clinicians as well as researchers.
Ulip6, a novel unc‐33 and dihydropyrimidinase related protein highly expressed in developing rat brain
Authors: Horiuchi Masahisa , El Far Oussama , Betz Heinrich
Published: July 2002

Here, we report the identification of Ulip6, a novel unc‐33 and dihydropyrimidinase related protein that belongs to the Ulip/CRMP protein family. Ulip6 was found in a yeast two‐hybrid screen using the neuronal glycine transporter GlyT2 as bait. The rat and human Ulip6 sequences are highly homologous and most closely related to the liver enzyme dihydropyrimidinase (Ulip5). Northern and Western analysis of rat tissues revealed that the distribution of the Ulip6 mRNA and protein resembles those of brain‐type Ulip proteins. Like Ulip1–4, Ulip6 is highly expressed in embryonic and early postnatal brain and spinal cord. These findings are consistent with Ulip6 having a function in neuronal differentiation and/or axon growth.
Complementation of a yeast Δpkc1 mutant by the Arabidopsis protein ANT
Authors: Vergani Paola , Morandini Piero , Soave Carlo
Published: July 2002

The Saccharomyces cerevisiae protein kinase C homologue, PKC1, is involved in maintenance of cell integrity during polarized growth. We have used a mutant complementation approach to investigate related signal transduction pathways in higher plants. Here we report the isolation of a cDNA from Arabidopsis thaliana which partially suppresses the lytic defect of a Δpkc1 yeast strain. The encoded protein, ANT, belongs to the AP2‐related gene family and is essential for ovule development. Expression in yeast of a LexA‐ANT fusion protein activates transcription of a reporter gene from promoters containing lexA operators. Our results support the idea that ANT acts as transcriptional activator in planta.
Selectivity of β‐adrenergic receptor kinase 2 for G protein βγ subunits
Authors: Müller Stefan , Straub Annette , Lohse Martin J
Published: July 2002

Phosphorylation of G protein‐coupled receptors by β‐adrenergic receptor kinases (βARK) requires the presence of G protein βγ subunits. We have investigated the ability of the two βARK isoforms to distinguish between defined recombinant βγ subunits. βARK2 had an about 25% lower specific activity than βARK1 towards rhodopsin and the β2‐adrenergic receptor but the two kinases shared the selectivity for βγ subunits: βγ complexes consisting of β1 or β2 in combination with γ2, γ5, and γ7 were more efficacious than those with γ3 or β1γ1. Thus, while βARKs differentiate between defined βγ subunits, βγ complexes do not discriminate between βARK isoforms.
Subunit interactions in the Escherichia coli protein translocase: SecE and SecG associate independently with SecY
Authors: Homma Takayuki , Yoshihisa Tohru , Ito Koreaki
Published: July 2002

We used hexahistidine‐tagged SecE and SecY to study how the core subunits (SecY, SecE and SecG) of Escherichia coli protein translocase interact with each other. Detergent extracts were prepared from the plasma membranes and fractionated by Ni2+‐NTA agarose affinity binding. Although His6‐SecE, expressed in wild‐type cells, brought down both SecY and SecG, neither of them was brought down when the same protein was expressed in the secY24 mutant cells. His6‐SecY brought down both SecE and SecG, as expected. Interestingly, His6‐SecY24 was able to bring down SecG but not SecE. These results confirm our previous conclusion that the secY24 alteration impairs the SecY‐SecE interaction, and demonstrate that SecY and SecG can form a complex that does not contain SecE. Likewise, SecY‐SecE complex could be isolated from the secG‐deleted strain. The trimeric complex, in detergent extracts, dissociated at a critical temperature between 23 and 26°C, whereas the SecY‐SecE complex without SecG dissociated at a slightly lower temperature (20–23°C). We conclude that each of SecE and SecG independently binds to SecY, the central subunit of protein translocase, although the trimeric complex is more stable than the binary complexes.
Stimulation of spinach (Spinacia oleracea) chloroplast fructose‐1,6‐bisphosphatase by mercuric ions
Authors: Ashton Anthony R , Siegel Gabriele M
Published: July 2002

Chloroplast fructose‐1,6‐bisphosphatase can exist in an active reduced form or a less active oxidised form. Oxidised fructose bisphosphatase from spinach (Spinacia oleracea) could be stimulated up to many hundred‐fold by 0.1 mM HgCl2 whereas fructose bisphosphatases from rabbit, yeast, a non‐chloroplast enzyme from spinach and the reduced chloroplast enzyme were only inhibited by HgCl2. Stimulation of the enzyme was maximal at pH 8.0 and low magnesium concentrations where the oxidised enzyme normally has little activity.
Phosphate groups in lipopolysaccharides of Salmonella typhimurium rfaP mutants
Authors: Helander Ilkka M. , Kilpeläinen Ilkka , Vaara Martti
Published: July 2002

Lipopolysaccharides (LPS) of Salmonella typhimurium rfaP mutants and of a galE strain as a control were subjected to analysis by 31P‐NMR in order to assess the location of phosphate groups. This was done to obtain direct proof for our earlier finding by chemical analysis that phosphate was lacking in the core oligosaccharide part of the mutant LPS, whereas the core oligosaccharide normally contains several phosphate groups. Such phosphate deficiency has been associated with the increased susceptibility of the rfaP mutants to hydrophobic antibiotics and detergents. Analysis of the de‐O‐acylated LPS derivatives of S. typhimurium rfaP strains SH7770, SH8551, and SH8572 by 31P‐NMR revealed an almost total lack of phosphate groups in the core oligosaccharide part, the LPS phosphates being largely accounted for by the two monophosphate monoesters of lipid A, linked to positions C‐1 and C‐4′ of the lipid A backbone. Core oligosaccharide‐linked phosphates were detected in minor proportions only, indicating the presence of some normally phosphorylated core oligosaccharide, due to the inherently leaky nature of the mutation.
Crystal structure of Escherichia coli inorganic pyrophosphatase complexed with SO42−
Authors: Avaeva Svetlana , Kurilova Svetlana , Nazarova Tatjana , Rodina Elena , Vorobyeva Natalya , Sklyankina Vera , Grigorjeva Olga , Harutyunyan Emil , Oganessyan Vaheh , Wilson Keith , Dauter Zbygnew , Huber Robert , Mather Timothy
Published: July 2002

The three‐dimensional structure of inorganic pyrophosphatase from Escherichia coli complexed with sulfate was determined at 2.2 Å resolution using Patterson's search technique and refined to an R‐factor of 19.2%. Sulfate may be regarded as a structural analog of phosphate, the product of the enzyme reaction, and as a structural analog of methyl phosphate, the irreversible inhibitor. Sulfate binds to the pyrophosphatase active site cavity as does phosphate and this diminishes molecular symmetry, converting the homohexamer structure form (α3)2 into α3′α3″. The asymmetry of the molecule is manifested in displacements of protein functional groups and some parts of the polypeptide chain and reflects the interaction of subunits and their cooperation. The significance of re‐arrangements for pyrophosphatase function is discussed.
The glyoxysomal 3‐ketoacyl‐CoA thiolase precursor from Brassica napus has enzymatic activity when synthesized in Escherichia coli
Authors: Olesen Christian , Thomsen Karl Kristian , Svendsen Ib , Brandt Anders
Published: July 2002

Glyoxysomal 3‐ketoacyl‐CoA thiolase is the last enzyme in the β‐oxidation of fatty acids in plant glyoxysomes. A full‐length cDNA of the glyoxysomal 3‐ketoacyl‐CoA thiolase from Brassica napus and a truncated version, lacking the N‐terminal targeting signal were cloned in a T7 promoter‐based vector. Both recombinant proteins were expressed in Escherichia coli and activity was measured. Full‐length and truncated 3‐ketoacyl‐CoA thiolase have comparable activity in E. coli. Moreover, full‐length 3‐ketoacyl‐CoA thiolase was purified from E. coli and N‐terminal sequencing of the protein confirmed that the precursor form indeed is enzymatically active.
Detection of specific IgE to human milk proteins in sera of atopic infants
Authors: Cantisani Annamaria , Giuffrida Maria Gabriella , Fabris Claudio , Bertino Enrico , Coscia Alessandra , Oggero Roberto , Monti Giovanna , Stroppiana Paola , Conti Amedeo
Published: July 2002

Specific IgE (sIgE) for cow's milk proteins (CMP) have been reported to be present in blood sera of exclusively breast‐fed infants. The aim of this study was to find whether the presence of sIgE to human milk proteins in the sera of exclusively breast‐fed infants could explain the apparent detection of sIgE to CMP in infants that were never previously in contact with cow's milk. sIgE for human milk whey proteins were found in the blood sera of atopic infants, and these sIgE strongly cross‐reacted with the corresponding CMP. In none of the sera examined were sIgE to bovine β‐lactoglobulin detected.
Effect of IL‐4 and IL‐13 on IFN‐γ‐induced production of nitric oxide in mouse macrophages infected with herpes simplex virus type 2
Authors: Paludan Søren R. , Lovmand Jette , Ellermann-Eriksen Svend , Mogensen Søren C.
Published: July 2002

Interleukin (IL)‐4 and IL‐13 share a wide range of activities. Prominent among these is the ability to antagonize many interferon (IFN)‐γ‐induced activities. Here we demonstrate that IL‐4 and IL‐13 totally abrogate IFN‐γ‐induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) mRNA and protein synthesis in a murine macrophage cell line. IFN‐γ‐treated cells infected with herpes simplex virus type 2 (HSV‐2) or costimulated with tumor necrosis factor (TNF)‐α showed an enhanced reactivity, which was only partially reduced by IL‐4/13. The results indicate that IL‐4 and IL‐13 function by intervening with a step prior to iNOS transcription by antagonizing IFN‐γ‐induced signal(s) without counteracting synergistic virus‐ or TNF‐α‐induced signals. The beneficial effect of a sustained NO production in foci of virus infection is suggested.
Rescue of a neutralizing anti‐viral antibody fragment from an intracellular polyclonal repertoire expressed in mammalian cells
Authors: Gargano Nicola , Cattaneo Antonino
Published: July 2002

The intracellular expression of recombinant antibodies in mammalian cells is an experimental strategy to inhibit in vivo the function of selected molecules. However, one limitation of this technology is represented by the unpredictable behaviour of antibodies, under conditions of intracellular expression. For this reason, it would be desirable to exploit intracellular expression of antibodies to select or rescue more efficient ones from a polyclonal mixture. In this work we have successfully explored this possibility by rescuing a neutralizing anti‐viral antibody fragment from an intracellularly expressed anti‐reverse transcriptase polyclonal repertoire.
Mutational analysis of Yap1 protein, an AP‐1‐like transcriptional activator of Saccharomyces cerevisiae
Authors: Takeuchi Tomonori , Miyahara Kohji , Hirata Dai , Miyakawa Tokichi
Published: July 2002

To define the essential amino acid residues of Yap1 in stress response, we generated yap1 mutations by in vitro mutagenesis, which cause defects in mediating resistance to the stress of H2O2, but not of CdCl2. Sequence analysis of the mutant yap1 genes revealed three point mutations and two truncation mutations near the carboxy‐terminus. The truncation mutations resulted in hyperresistance to cadmium. Northern blot analysis of stress‐induced levels of TRX2 and GSH1 mRNAs indicated that the ability of the mutant Yap1 protein to induce transcriptional activation of target genes correlates well with its ability to confer stress resistance. The carboxy‐terminal domain of Yap1 appears to act negatively in cadmium resistance.
ADP‐ribosyl cyclase coupled with receptors via G proteins
Authors: Higashida Haruhiro
Published: July 2002

Crude cell membranes in mammalian cells contain ADP‐ribosyl cyclase, which converts NAD+ to cyclic ADP‐ribose. Acetylcholine either increases or inhibits this activity in NG108‐15 neuronal cells and adrenal chromaffin cells in a muscarinic receptor subtype‐specific manner. Activation or inhibition of the cyclase activity is mimicked by GTP and blocked by bacterial toxins. These findings suggest that hormone or neurotransmitter receptors utilize the direct signaling pathway to ADP‐ribosyl cyclase via G proteins within cell membranes, analogous to the previously established transduction pathways to adenylyl cyclase and phospholipase Cβ.
Wortmannin, an inhibitor of phosphatidyl‐inositol 3‐kinase, induces oocyte maturation through a MPF‐MAPK‐dependent pathway
Authors: Carnero Amancio , Lacal Juan Carlos
Published: July 2002

Wortmannin has been shown to be a non‐competitive and irreversible inhibitor of PI3 kinase. For this reason, it has attracted considerable interest and it has been used, as a selective inhibitor of the PI3 kinase, for the study of signal transduction pathways in different systems including Xenopus oocytes. We show here that wortmannin itself is able to induce meiotic maturation at doses slightly higher that those required for complete inhibition of PI3 kinase. This effect was shown to be independent of the ability to inhibit PI3K since another unrelated PI3K inhibitor, LY294002, was unable to induce oocyte maturation at inhibitory concentrations for PI3 kinase. The mechanism for wortmannin‐induced maturation involves the activation of maturation promoting factor (MPF) and MAP kinase activities in a time course that preceded the appearance of germinal vesicle breakdown. Thus, the pathway activated by wortmannin directly or indirectly affects other protein or proteins, besides PI3 kinase, responsible for its activity. This new target is placed independently or downstream of the PI3 kinase inhibition and upstream of protein synthesis. Moreover, the inhibition of either MPF or cAMP phosphodiesterase blocks wortmannin‐induced maturation. We conclude that wortmannin may be a valuable tool for the study of the pathway leading to mitotic maturation of oocytes, but cannot be used as a specific PI3 kinase inhibitor.
Topological localization of cysteine 74 in the GABA transporter, GAT1, and its importance in ion binding and permeation
Authors: Yu Nam , Cao Yongwei , Mager Sela , Lester Henry A.
Published: July 2002

Xenopus oocytes expressing the GABA transporter GAT1 were exposed to membrane‐impermeant sulfhydryl reagents, resulting in decreased GABA transport current, decreased capacitive charge movements, and increased Na+ and Li+ leakage currents. Mutation of cysteine 74 to alanine (C74A) eliminated these effects. The W68S and W68L mutations significantly increased and decreased the transporter's sensitivity, respectively, to sulfhydryl reagents. At each of the positions 73 through 76, cysteine residues were accessible to external MTSET. These findings, together with recent evidence placing the HD2–HD3 loop on the extracellular side, suggest that the HD2 region does not traverse the membrane.
Peptide‐specific antibodies localize the major lipid binding sites of talin dimers to oppositely arranged N‐terminal 47 kDa subdomains1
Authors: Isenberg Gerhard , Goldmann Wolfgang H.
Published: July 2002

Using ultrastructural analysis and labeling with polyclonal antibodies that recognize peptide sequences specific for phospholipid binding, we mapped the functional domain structure of intact platelet talin and its proteolytic fragments. The talin dimer, which is crucial for actin and lipid binding, is built of a backbone containing the 200 kDa rod portions, at both ends of which a 47 kDa globular domain is attached. Peptide‐specific polyclonal antibodies were raised against three potential lipid binding sequences residing within the N‐terminal 47 kDa domain (i.e. S19, amino acids 21–39; H18, amino acids 287–304; and H17, amino acids 385–406). Antibodies H17 and H18 localize these lipid binding sequences within the N‐terminal 47 kDa globular talin subdomains opposed at the outer 200 kDa rod domains within talin dimers. Hence, we conclude that in its dimeric form, which is used in actin and lipid binding, talin is a dumbbell‐shaped molecule built of two antiparallel subunits.
Quantitative analysis of thrombopoietin receptors on human megakaryocytes
Authors: Kuwaki Tomoaki , Hagiwara Tetsuya , Yuki Chizuru , Kodama Ikuko , Kato Takashi , Miyazaki Hiroshi
Published: July 2002

Thrombopoietin (TPO), or c‐MPL ligand, is the primary regulator of megakaryocyte and platelet production. TPO receptors expressed on human megakaryocytes derived from peripheral blood (PB) and cord blood (CB) progenitors cultured in the presence of TPO have now been analyzed quantitatively. Like those on human PB platelets, TPO receptors on the cultured megakaryocytes exhibited a molecular mass of approximately 80 kDa. Various characteristics of PB‐ and CB‐derived megakaryocytes indicated that the former were more mature than the latter. Both PB‐ and CB‐derived megakaryocytes expressed a single class of high‐affinity TPO receptors, with 1933±772 (n=3) and 184±48 (n=4) sites per cell, respectively. These data indicate that the number of TPO receptors on human megakaryocytes increases with cell maturation.
Guanine nucleotides protect against kainate toxicity in an ex vivo chick retinal preparation
Authors: Burgos Javier S , Barat Ana , Souza Diogo O , Ramı́rez Galo
Published: July 2002

Ex vivo preparations of chick neural retina have been successfully used in the assessment of excitotoxicity and in the evaluation of the protective effects of glutamate antagonists. Using a variation of this approach, and measuring the acute and delayed toxic effects of kainate (KA) in terms of lactate dehydrogenase release, we have shown that guanine nucleotides behave as effective neuroprotecting agents. The anti‐excitotoxic potency of guanine nucleotides (in the case of GMP and GDPβS it is about 100 times lower than that of DNQX, a powerful kainate antagonist) correlates well with their ability to displace KA from retinal KA receptors.
The coupling region of F0F1 ATP synthase: binding of the hydrophilic loop of F0 subunit c to F1
Authors: Licher T , Kellner E , Lill H
Published: July 2002

The hydrophilic loop region of the CF0 c subunit has been expressed as a fusion with MalE in Escherichia coli. A cysteine was introduced at the C‐terminus to allow fluorophore labeling of the fusion protein. After removal of the MalE moiety, the labeled peptide was used for binding studies with fluorophore‐labeled CF1. At saturation, 1 mol peptide was bound per mol CF1. Binding was abolished after removal of subunit ϵ from CF1, and partially restored by addition of recombinant ϵ.
Effect of inhibitor binding to β subunits of F1ATPase on enzyme thermostability: a kinetic and FT‐IR spectroscopic analysis
Authors: Lippe Giovanna , Tanfani Fabio , Di Pancrazio Francesca , Contessi Stefania , Bertoli Enrico , Dabbeni-Sala Federica
Published: July 2002

FT‐IR analysis shows that treatment of F1ATPase with the inhibitors DCCD and Nbf‐Cl, in the presence of saturating concentrations of ADP and AMP‐PNP and in the absence of Mg2+, does not modify the secondary structure of the enzyme, but significantly modifies its compactness and thermal stability, although to different extents. Nbf‐Cl causes a significant increase in stabilisation, in addition to that induced by nucleotides, while DCCD is less effective in this regard. Determination by HPLC of the exchange rate, in the absence of Mg2+, of tightly bound nucleotides of F1ATPase treated with the two inhibitors shows that DCCD does not significantly affect the exchange rate of ADP with AMP‐PNP and vice versa in catalytic and non‐catalytic tight sites, while Nbf‐Cl selectively reduces the enzyme's capacity to exchange ADP bound in the tight catalytic site. It is suggested that the effects of DCCD, unlike those of Nbf‐Cl, are closely related to the presence or absence of Mg2+.
Modulation of agonist and antagonist interactions in serotonin 1A receptors by alcohols
Authors: Harikumar K.G , Chattopadhyay Amitabha
Published: July 2002

The serotonin type 1A (5‐HT1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to GTP binding regulatory proteins (G‐proteins). Serotonergic signalling has been shown to play an important role in alcohol tolerance and dependence. We have studied the effects of alcohols on ligand (agonist and antagonist) binding to bovine hippocampal 5‐HT1A receptor in native as well as solubilized membranes. Our results show that alcohols inhibit the specific binding of the agonist OH‐DPAT and the antagonist p‐MPPF to 5‐HT1A receptors in a concentration‐dependent manner.
Phosphorylation site independent single R‐domain mutations affect CFTR channel activity
Authors: Wei Lin , Vankeerberghen Anne , Cuppens Harry , Droogmans Guy , Cassiman Jean-Jacques , Nilius Bernd
Published: July 2002

We investigated CFTR channel activity of mature R‐domain mutants showing single alterations at sites other than the predicted phosphorylation sites. All mutations were found in cystic fibrosis (CF) patients (H620Q, E822K and E826K). The macroscopic CFTR chloride conductance induced by phosphorylation was significantly enhanced in Xenopus oocytes injected with mRNA of H620Q but reduced in the E822K and E826K mutants compared to wild type CFTR. The anion permeability sequence for all three mutants was the same as that of wild type CFTR. Cell attached single channel studies in COS cells revealed that both open channel probability and/or the number of functional channels were either higher (H620Q) or lower (E822K and E826K) than in wild type CFTR. Single channel conductances were unchanged in all mutants. Our results suggest that additional sites in the R‐domain other than phosphorylation sites influence gating of CFTR channels.
The membrane‐proximal part of FcϵRIα contributes to human IgE and antibody binding – implications for a general structural motif in Fc receptors
Authors: Nechansky Andreas , Aschauer Heinrich , Kricek Franz
Published: July 2002

The high affinity receptor for human IgE (FcϵRI) on tissue mast cells and blood basophils is responsible for immediate hypersensitivity reactions. Binding of human IgE (hIgE) to FcϵRI has been shown to be mediated via three independent regions in the extracellular part of the α‐subunit of FcϵRI (ecFcϵRIα). By site‐directed mutagenesis we investigated the contribution of amino acids within the ecFcϵRIα FG loop (residues Lys154–Leu165) to binding to hIgE and two monoclonal anti‐FcϵRIα antibodies (15/1, 5H5/F8). The mutated receptors were expressed and secreted from eukaryotic cells as amino‐terminal fusion to HSA. We show that the proposed loop region contributes partly to hIgE binding and that the epitope of mAb 15/1, which inhibits hIgE/FcϵRIα interaction, maps to this region whereby a single W156A mutation results in complete loss of mAb 15/1 binding. In contrast, hIgE binding is not affected by the W156A mutation indicating that different amino acid residues within the loop are recognized by the mAbs 15/1 and hIgE. MAb 5H5/F8 does not recognize a receptor mutant truncated to Ile170. By screening a random dodecapeptide library displayed on bacterial flagella the epitope for mAb 5H5/F8 was mapped to P173REKY177 whereas one of the 15/1 binding clones displayed a peptide with an amino acid sequence homologous to Leu158–Ile167. Based on the epitopes identified for the inhibitory mAb 15/1 and the non‐inhibitory mAb 5H5F8 and on binding data obtained with polyclonal antisera raised against two ecFcϵRIα peptides, we propose a structural element in the membrane proximal part of ecFcϵRIα which forms a 3D structure which might facilitate specific and efficient attachment of hIgE.
First synthesis of a fully active spin‐labeled peptide hormone1
Authors: Barbosa Simone R , Cilli Eduardo M , Lamy-Freund M.Teresa , Castrucci Ana Maria L , Nakaie Clovis R
Published: July 2002

For the first time in the electron spin resonance (ESR) and peptide synthesis fields, a fully active spin‐labeled peptide hormone was reported. The ESR spectra of this α‐melanocyte stimulating hormone (α‐MSH) analogue (acetyl‐Toac0‐α‐MSH) where Toac is the paramagnetic amino acid probe 2,2,6,6‐tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid, suggested a pH‐independent conformation and a more restricted movement comparatively to the free Toac. Owing to its equivalent biological potency in a skin pigmentation assay as compared to the native α‐MSH and its unique characteristic (paramagnetic, naturally fluorescent and fully active), this analogue is of great potential for investigation of relevant physiological roles reported for α‐MSH.
Apoptosis of mouse liver nuclei induced in the cytosol of carrot cells
Authors: Zhao Yun , Jiang Zheng-Fan , Sun Ying-Li , Zhai Zhong-He
Published: July 2002

We report here the apoptosis of mouse liver nuclei induced in the cytosol of carrot cells by cytochrome c. Several typical characteristics of apoptosis, such as chromatin condensation, margination and apoptotic bodies, were detected. The result of DNA gel electrophoresis showed that DNA was degraded into nucleosomal fragments. The terminal deoxynucleotidyl transferase‐mediated dUTP‐digoxigenin nick end labelling procedure was also performed to detect the breakage of 3′‐OH ends of a DNA strand. Furthermore, we found that nuclear lamins were degraded from 88 kDa and 66 kDa to 37 kDa and 47 kDa fragments. The DNA fragmentation could be inhibited by AC‐DEVD‐CHO and AC‐YVAD‐CHO. The results indicate that the apoptosis in plant cells may share some similar pathways to apoptosis in animal cells.
The two dimeric forms of RNase A
Authors: Sorrentino Salvatore , Barone Roberto , Bucci Enrico , Gotte Giovanni , Russo Nello , Libonati Massimo , D'Alessio Giuseppe
Published: July 2002

In 1965 Fruchter and Crestfield (J. Biol. Chem. 240, 2868–3874) observed that dimeric RNase A prepared by lyophilization from acetic acid could be separated into two forms. Surprisingly, no other structural or functional differences could be detected between the two forms. In 1998 a structure for dimeric RNase A was determined by X‐ray crystallography by Liu et al. (Proc. Natl. Acad. Sci. USA 95, 3437–3442). We found that the two forms of dimeric RNase A have indeed different structural and functional properties, and suggest that the dimer whose structure was investigated by Liu and coworkers may be identified with the lesser form of dimeric RNase A.
Biology of human trophoblast
Authors: Mochizuki M. , Maruo T. , Matsuo H. , Samoto T. , Ishihara N.
Published: July 2002

In order to elucidate the regulation of placental growth, we have characterized the expression of proliferating cell nuclear antigen (PCNA), apoptotic DNA fragmentation and bc1‐2 protein in human placenta during pregnancy. PCNA and bc1‐2 protein expression were examined by immunohistochemical techniques, while the occurrence of apoptotic DNA fragmentation was assessed by in situ analysis of DNA 3′‐end labeling method. Both PCNA expression and apoptotic DNA fragmentation were found in cytotrophoblasts (C‐cells), being most abundant in early placenta, less abundant in midterm placenta and least abundant in term placenta. In contrast, bc1‐2 protein expression was found in syncytiotrophoblasts (S‐cells), being least abundant in early placenta, less abundant in midterm placenta and most abundant in term placenta. These data indicate that early placenta is characterized by the highly proliferative activity of C‐cells associated with the increased occurrence of apoptosis, whereas term placenta is characterized by the abundant expression of bc1‐2 protein in S‐cells.

Furthermore, effects of EGF on the proliferative activity and differentiated function of trophoblasts were investigated using an organ culture system. Expiants of trophoblastic tissues were cultured with or without EGF, in the presence or absence of 10−8M triiodo‐L‐thyronine (T3) in a serum‐free condition. In 4—5 week placentas, EGF and EGF receptor were almost exclusively localized in C‐cells, and EGF augmented the proliferative activity of C‐cells without affecting the ability to secrete hCG and hPL. By contrast, in 6—12 week placentas, EGF and EGF receptor were predominantly localized in S‐cells, and EGF stimulated hCG and hPL secretion without affecting the proliferative activity of C‐cess. The addition of T3 (10−8M) resulted in an increased secretion of immunoreactive EGF by cultured placental explants. These findings suggest that EGF acts as a local factor in regulating early placental growth and function in synergy with thyroid hormone. On the other hand, progesterone selectively inhibited pleise hCG (α, β) mRNAs expression and decreased hCG secretion in normal placental tissues, whereas choriocarcinoma did not respond to progesterone. This suggests that inhibitory regulation of hCG synthesis in choriocarcinoma is different from normal placenta. It was also found that in molar trophoblasts and choriocarcinoma cells PCNA expression was high, but both bc1‐2 protein and apoptotic signal expression were low. Characterization of choriocarcinoma hCG revealed that there are striking differences in carbohydrate structures between normal hCG and choriocarcinoma hCG. Sialic acid content in choriocarcinoma hCG was extremely lower compared to that in normal hCG. The detection of the alteration in hCG sugar chains is useful for biochemical diagnosis of choriocarcinoma.
Monitoring and evaluation of PMM efforts: what have we learned?
Authors: McGinn T
Published: July 2002

One element of the operations research carried out by the Prevention of Maternal Mortality (PMM) teams in West Africa was a test of the indicators they used to monitor and evaluate their efforts. A small number of process indicators directly related to the PMM interventions in the health facilities and in the communities was selected and monitored. An examination of the teams' experiences in gathering and using these indicators shows: (i) that they are extremely useful for project design and management; (ii) that the necessary data are obtainable; (iii) that staff need training to gather, interpret and use the data; (iv) that monitoring and evaluation systems must be designed so that managers will use the information; and (v) that data on the costs of interventions can be tracked and are useful for evaluation and replication. © 1997 International Federation of Gynecology and Obstetrics