bluuebunny/crossref_metadata_2025 huggingface dataset:
Okadaic acid and calyculin A enhance the effect of thyrotropin‐releasing hormone on GH3 rat anterior pituitary cells excitability
Authors: Delgado Luis M. , de la Peña Pilar , del Camino Donato , Barros Francisco
Published: July 2002

Thyrotropin‐releasing hormone (TRH) causes a transient hyperpolarization followed by several minutes of increased action potential frequency in patch‐perforated current‐clamped GH3 cells. Treatment of cells for 5 min with either 2 or 100 nM of the protein phosphatase inhibitor okadaic acid does not affect electrical activity of the cells, but potentiates the enhancement of action potential frequency elicited by a subsequent addition of TRH. Alternatively, 100 nM (but not 2 nM) of okadaic acid added during the second phase of TRH action, further increases the frequency of firing above that produced by the hormone. Similar effects to those of 2 nM okadaic acid are observed with 20 nM calyculin A. These data suggest that a protein phosphatase plays a major role in regulating the delayed effects of TRH on cell excitability in GH3 cells.
The first application of a chemiluminescence probe, 2‐methyl‐6‐[P‐methoxyphenyl]‐3,7‐dihydroimidazo[1,2‐α]pyrazin‐3‐one (MCLA), for detecting O2− production, in vitro, from Kupffer cells stimulated by phorbol myristate acetate
Authors: Uehara Kyoshi , Maruyama Noboru , Huang Chien-Kueng , Nakano Minoru
Published: July 2002

The purpose of this study was to investigate the generation of superoxide anion radical, O−
2, by Kupffer cells in vivo in rat liver. Phorbol myristate acetate (PMA) was infused into perfused rat liver which had been continuously infused with 2‐methyl‐6‐[p‐methoxyphenyl]‐3,7‐dihydroimidazo[1,2‐a] pyrazin‐3‐one (MCLA, a highly sensitive luminescence reagent for O2
−) and the MCLA luminescence from the liver surface was detected with a sensitive photon counter. Under identical conditions, but without MCLA infusion, PMA and nitro blue tetrazolium were infused to verify generation of O2
−; by formation of formazan deposition. Based on MCLA luminescence and fonnazan deposition, both dependent on the O2
−; reaction, we have concluded that liver Kupffer cells generate O2
−; in vivo in response to stimuli such as PMA. Further, the generation of O2
−; by Kupffer cells may be by the same mechanism as displayed by macrophages.
The cis/trans interconversion of the calcium regulating hormone calcitonin is catalyzed by cyclophilin
Authors: Kern Dorothee , Drakenberg Torbjörn , Wikström Mats , Forsén Sture , Bang Holger , Fischer Gunter
Published: July 2002

The cytosolic peptidyl‐prolyl cis/trans isomerase cyclophilin from pig kidney can accelerate catalytically the cis/trans isomerization of prolyl peptide bonds. One‐ and two‐dimensional 1H NMR spectroscopy was used to prove that the polypeptide hormone calcitonin is a substrate for cyclophilin. Isomerization of only one of the two prolyl peptide bonds is catalyzed significantly. The efficiency of catalysis was calculated by lineshape analysis and NOESY spectroscopy. Cyclosporin A completely blocks the effect of the enzyme on the conformational dynamics of the polypeptide.
Thermoplasma acidophilum proteasomes degrade partially unfolded and ubiquitin‐associated proteins
Authors: Wenzel Thorsten , Baumeister Wolfgang
Published: July 2002

It is shown that proteasomes from the archaebacterium Thermoplasma acidophilum selectively degrade substrate proteins partially unfolded by phenylhydrazine‐ or hydrogen peroxide‐treatment. Surprisingly, the pre‐incubation of the substrate proteins with ubiquitin is also sufficient to render them susceptible to proteolytic degradation by proteasomes. We propose that, upon spontaneously associating with the substrate protein, ubiquitin exerts a chaotropic effect on it; this may involve the exposure of hydrophobic segments of the polypeptide chain which are recognized by the binding sites of the proteasome.
Reassessment of the putative chaperone function of prolyl‐cis/trans‐isomerases
Authors: Kern Gunther , Kern Dorothee , Schmid Franz X. , Fischer Gunter
Published: July 2002

The folding of proteins can be assisted by two unrelated groups of helper molecules. Chaperones suppress non‐productive side reactions by stoichiometric binding to folding intermediates, and folding enzymes catalyze slow rate‐limiting steps of folding. We reinvestigated, whether peptidyl‐propyl‐cis/trans‐isomerases of the cyclophilin type act simultaneously as chaperones and as folding catalysts in the reactivation of human carbonic anhydrase II, as reported recently [Freskgård, P.‐O et al. (1992) Science 258, 466–468; Rinfret, A. et al. (1994) Biochemistry 33, 1668–1673]. No increase in the yield of carbonic anhydrase‐II could be detected in the presence of three different propyl isomerases, when reactivation was followed by a sensitive assay for an extended time of 4 h. We conclude that the role of propyl isomerases in the refolding of carbonic anhydrase can be explained solely by their isomerase activity. There is no need to invoke simultaneous functions as chaperones for these folding catalysts.
ARF1(2–17) does not specifically interact with ARF1‐dependent pathways
Authors: Fensome Amanda , Cunningham Emer , Troung Oanh , Cockcroft Shamshad
Published: July 2002

The small GTP‐binding protein ARF has been shown recently to regulate phospholipase D (PLD). In order to investigate the role of ARF proteins in regulated exocytosis, we have used the N‐terminal peptide ARF1(2–17) of the ARF1 protein. ARF1 reconstituted PLD activity in cytosol‐depleted HL60 cells was inhibited by ARF1(2–17). In the presence of endogenous cytosol, ARF1(2–17) also inhibited GTP‐γ‐S‐stimulated PLD activity and exocytosis. Mastoparan Politses jadwagae and mastoparan Vespula lewisii which exhibit similar structural properties to ARF1(2–17) also inhibited GTP‐γ‐S‐stimulated PLD and exocytosis. GTP‐γ‐S‐stimulated phospholipase C‐β (PLC‐β) was also inhibited by ARF(2–17) and mastoparan. In cytosol‐depleted HL60 cells, the ARF(2–17) inhibited the reconstitution of GTP‐γ‐S‐stimulated PLC‐β activity with exogenously‐added PLC‐β1 and phosphatidylinositol transfer protein. We conclude that the widely‐used ARF1(2–17) peptide inhibits both ARF‐independent (i.e. PLC‐β) and ARF‐dependent pathways (i.e. PLD) and therefore cannot be regarded as a specific inhibitor of ARF function.
Mutation of Lys‐120 and Lys‐134 drastically reduces the catalytic rate of Cu,Zn superoxide dismutase
Authors: Polticelli Fabio , Battistoni Andrea , Bottaro Grazia , Carri Maria Teresa , O'Neill Peter , Desideri Alessandro , Rotilio Giuseppe
Published: July 2002

Lys‐120 and Lys‐134, located at the edge of the active site channel in most Cu,Zn superoxide dismutases, have been suggested to play a major role in steering the anionic substrate towards the catalytic copper ion. In this study, mutants of Xenopus laevis Cu,Zn superoxide dismutase have been engineered, with Lys‐120 and Lys‐134 changed into leucine and threonine, respectively, and their catalytic properties have been investigated by pulse radiolysis. Results obtained demonstrate that both residues decrease the catalytic rate by about 40%, in partial disagreement with previous brownian dynamics calculations, carried out on bovine Cu,Zn superoxide dismutase.
Properties of carbohydrate‐free recombinant glycogenin expressed in an Escherichia coli mutant lacking UDP‐glucose pyrophosphorylase activity
Authors: Alonso Miriam D. , Lomako Joseph , Lomako Wieslawa M. , Whelan William J. , Preiss Jack
Published: July 2002

Glycogenin, the self‐glucosylating primer for glycogen synthesis is expressed in wild‐type E. coli as a recombinant protein in an already partly glucosylated form owing to the presence of its substrate UDP‐glucose. By using an E. coli mutant strain lacking in UDP‐glucose pyrophosphorylase activity, we have succeeded in expressing carbohydrate‐free glycogenin (apo‐glycogenin) in good yield. When provided with UDPxy‐lose, it autocatalytically adds 1 xylose residue. With UDP‐glucose, an average of 8 glucose residues are added. However release of the self‐synthesized maltosaccharide chains with isoamylase reveals them to be a mixture. Chains as long as 11 glucose residues (maltoundecaose) are present. The ability of recombinant apo‐glycogenin to self‐glucosylate is further proof that a separate enzyme is not needed for the addition of the first glucose residue to Tyr‐194 of the protein.
Prediction of potential protein‐protein interaction sites from amino acid sequence
Authors: Kini R.Manjunatha , Evans Herbert J.
Published: July 2002

Identification of a protein‐protein interaction site is an important step that has significant potential to clarify structure‐function relationships of proteins and drug design. We propose here a unique predictive method to identify protein‐protein interaction sites based on the observation that proline is the most common residue found in the flanking segments of interaction sites [Kini, R.M. and Evans, H.J. (1995) Biochem. Biophys. Res. Commun. 212, 1115–1124]. Accordingly, the interaction sites of proteins might be predicted directly from the amino acid sequence based on the presence of proline brackets. Using this strategy, we have predicted a polymerization site in the epitope of the Aα‐chain of fibrinogen recognized by a monoclonal antibody, 9E9 which inhibits fibrin polymerization [Cierniewski, C.S. and Budzynski, A.Z. (1992) Biochemistry 31, 4248–4253]. The synthetic peptide comprising this predicted site inhibited the coagulation of human blood and allosterically interfered in fibrin polymerization. This is the first known allosteric polymerization site of fibrinogen. Thus the results validate the predicted site and the method for prediction. This unique predictive method should help in identifying the interaction sites of many proteins.
Potential roles of heterotrimeric G proteins of the endomembrane system
Authors: Nürnberg Bernd , Ahnert-Hilger Gudrun
Published: July 2002

Heterotrimeric G proteins are recognized as versatile switches linking cell surface receptors to cellular effectors. BesiDA their location at the plasma membrane G proteins are found on intracellular membranes. Studies with modulators of G protein activity suggest that G proteins associated with organelle membranes are involved in various steps of secretion and vesicular function. In contrast to hormonal responses involving G proteins little is currently known about possible receptors or activators and effectors interacting with intracellular G proteins. This short review focuses on recent DAvelopments elucidating the role of organelle‐associated G proteins.
Expression of Mona (monocytic adapter) in myeloid progenitor cells results in increased and prolonged MAP kinase activation upon macrophage colony‐stimulating factor stimulation
Authors: Bourgin Caroline , Bourette Roland , Mouchiroud Guy , Arnaud Sylvie
Published: July 2002

Mona is an SH3 and SH2 domain‐containing adapter molecule that is induced during monocytic differentiation. Here we have first shown that M‐CSFR is the major Mona partner in M‐CSF signaling, the interaction being mediated through tyrosine 697 of the receptor. Next we asked whether Mona expression would alter the Ras/MAP kinase pathway since Mona is a likely competitor of Grb2 for binding to M‐CSFR. We found that M‐CSF induced late and massive phosphorylation of ERK molecules in Mona‐expressing myeloid cells compared to non‐expressing cells. These results suggest that Mona expression might modify M‐CSF signaling during monocytic differentiation.
Photoreceptors and olfactory cells express the same retinal guanylyl cyclase isoform in medaka: visualization by promoter transgenics1
Authors: Kusakabe Takehiro , Suzuki Norio
Published: July 2002

We examined the spatial expression patterns of two orphan receptor guanylyl cyclase genes OlGC4 and OlGC5 during embryogenesis of medaka and characterized the 5′ flanking region required for tissue‐specific expression of OlGC4 by introducing promoter–GFP fusion constructs into medaka embryos. Expression of OlGC5 is confined to retinal photoreceptor cells, while OlGC4 is expressed in the retina, pineal organ, and olfactory pits. The OlGC4 upstream region between −2374 and +343 is sufficient to drive the sensory organ‐specific gene expression. Mutations in the consensus binding sequences for OTX/CRX transcription factors did not impair the reporter gene expression. Our results suggest that the same isoform of guanylyl cyclase is utilized in both photoreceptors and olfactory cells, and that transcription factors other than OTX/CRX primarily activate the OlGC4 expression.
Two histidine residues are essential for catalysis by lecithin retinol acyl transferase
Authors: Mondal Madhu S , Ruiz Alberto , Hu Jane , Bok Dean , Rando Robert R
Published: July 2002

Lecithin retinol acyl transferase (LRAT) is a novel membrane bound enzyme that catalyzes the formation of retinyl esters from vitamin A and lecithin. The enzyme is both essential for vision and for the general mobilization of vitamin A. The sequence of LRAT defines it as a novel enzyme unrelated to any other protein of known function. LRAT possesses a catalytically essential active site cysteine residue. The enzyme also contains six histidine residues. It is shown here that two of these residues (H57 and H163) are essential for catalysis. A mechanistic hypothesis is presented to account for these observations.
An essential role for calmodulin in regulating human T cell aggregation
Authors: Fagerholm Susanna C. , Prescott Alan , Cohen Philip , Gahmberg Carl G.
Published: July 2002

After activation of T cells with either CD3 antibodies or phorbol esters, we have found that T cell–cell aggregation, integrin‐dependent actin reorganisation and cell spreading are strongly suppressed by any of three structurally different calmodulin antagonists, without any effect on the amount of CD11/CD18 integrin binding to the actin cytoskeleton. However, only T cell receptor‐induced, and not phorbol ester‐induced, aggregation and cell spreading are prevented by inhibitors of phosphatidylinositide (PI) 3‐kinase. These results suggest that PI 3‐kinase lies upstream of calmodulin in the signalling pathway leading to T cell aggregation, cell spreading and actin reorganisation and that cell spreading and actin reorganisation are essential for T cell adhesion.
Osmosensitivity of an inwardly rectifying chloride current revealed by whole‐cell and perforated‐patch recordings in cultured rat cortical astrocytes
Authors: Fava Marianna , Ferroni Stefano , Nobile Mario
Published: July 2002

The osmosensitivity of the inwardly rectifying Cl− current (I
Clh), expressed by primary cultured rat neocortical astrocytes long‐term treated with dibutyryl cyclic AMP, was investigated in the whole‐cell and perforated‐patch modes. In whole‐cell experiments, whereas hypotonic extracellular solution (Δ=100 mOsmol) did not cause any change in I
Clh, hypertonicity produced a slowly developing, ∼40% reversible decrease in current magnitude. By contrast, in perforated‐patch experiments, exposure to a less hypertonic saline (Δ=50 mOsmol) depressed the current to ∼50%, and hypotonicity induced a ∼50% slow increase in I
Clh. These differences in osmosensitivity between the two experimental modes suggest that the osmoregulation of I
Clh may be mediated by complex intracellular mechanism(s), which appear(s) to be partly compromised by the dialysis of the astrocytic cytoplasm.
Perinuclear mRNA localisation by vimentin 3′‐untranslated region requires a 100 nucleotide sequence and intermediate filaments
Authors: Bermano Giovanna , Shepherd Rebecca K. , Zehner Zendra E. , Hesketh John E.
Published: July 2002

The role of the vimentin 3′‐untranslated region (3′‐UTR) in mRNA localisation was studied in cells transfected with a reporter sequence linked to subregions of the 3′‐UTR. In situ hybridisation showed that nucleotides 37–137, including a previously identified protein‐binding domain, were sufficient to localise transcripts to perinuclear cytoplasm. Transfection of two SW13 cell lines that do and do not express vimentin showed that perinuclear localisation due to either the vimentin or c‐myc 3′‐UTR requires intermediate filaments. The data suggest that both a specific protein‐binding region of the vimentin 3′‐UTR and intermediate filaments themselves are required to determine the site of vimentin synthesis.
Synthetic peptides as inactivators of multimeric enzymes: inhibition of Plasmodium falciparum triosephosphate isomerase by interface peptides
Authors: Singh S.Kumar , Maithal Kapil , Balaram Hemalatha , Balaram P.
Published: July 2002

Synthetic peptides corresponding to two distinct segments of the subunit interface of the homodimeric enzyme triosephosphate isomerase (residues 9–18, ANWKCNGTLE, peptide I; residues 68–79, KFGNGSYTGEVS, peptide II) from Plasmodium falciparum (PfTIM) have been investigated for their ability to act as inhibitors by interfering with the quaternary structure of the enzyme. An analog of peptide II containing cysteine at the site corresponding to position 74 and tyrosine at position 69 in the protein sequence KYGNGSCTGEVS (peptide III) was also investigated. A substantial fall in enzyme activity was observed following incubation of the enzyme with peptide II, whereas peptide I did not show any appreciable inhibition. The inhibitory effect was more pronounced on two mutants of PfTIM (Y74C and Y74G), both of which have reduced stability compared to the wild‐type protein due to an interface cavity. The IC50 value determined for peptide II is in the range of 0.6–0.8 μM. This study suggests that interface peptides of oligomeric enzymes can be used to inhibit dimeric enzymes by disrupting their native multimeric states and may provide lead structures for potential inhibitor design.
Role of the Cys90, Cys95 and Cys173 residues in the structure and function of the human platelet‐activating factor receptor
Authors: Le Gouill Christian , Parent Jean-Luc , Rola-Pleszczynski Marek , Stanková Jana
Published: July 2002

Platelet‐activating factor (PAF) is a potent phospholipid mediator which binds to a specific, high affinity receptor of the G protein‐coupled receptor family. In the present report, we show that ligand binding to the PAF receptor is sensitive to the reducing agent dithiothreitol (DTT), suggesting the involvement of disulfide linkages in the proper PAF receptor conformation. Substitutions of Cys90, Cys95 and Cys173 to Ala or Ser demonstrated that these cysteine residues are critical for normal cell surface expression of the PAF receptor protein and ligand binding to the receptor. The Cys90 and Cys173 mutant receptors did not display any specific ligand binding, were not expressed on the cell surface but were found in the intracellular compartment. The Cys95 mutants showed specific binding and were able to stimulate low levels of inositol phosphate (IP) production. These mutants were expressed at low density on the cell surface and showed high expression intracellularly. Our results suggest that the structure and function of the PAF receptor require the conserved Cys90 and Cys173 to form a disulfide bond. Moreover, Cys95 also appears to be necessary, possibly by establishing a disulfide linkage with an as yet unidentified Cys residue. All three residues appear essential for the proper folding and surface expression of the PAF receptor protein.
Three‐dimensional structure of Serratiamarcescens nuclease at 1.7 Å resolution and mechanism of its action
Authors: Lunin V.Yu , Levdikov V.M , Shlyapnikov S.V , Blagova E.V , Lunin V.V , Wilson K.S , Mikhailov A.M
Published: July 2002

The three‐dimensional crystal structure of Serratia marcescens (Sm) nuclease has been refined at 1.7 Å resolution to the R‐factor of 17.3% and R‐free of 22.2%. The final model consists of 3678 non‐hydrogen atoms and 443 water molecules. The analysis of the secondary and the tertiary structures of the Sm nuclease suggests a topology which reveals essential inner symmetry in all the three layers forming the monomer. We propose the plausible mechanism of its action based on a concerted participation of the catalytically important amino acid residues of the enzyme active site.
Positive and negative regulation of JNK1 by protein kinase C and p42MAP kinase in adult rat hepatocytes
Authors: Jarvis W.David , Auer Kelly L , Spector Mark , Kunos George , Grant Steven , Hylemon Philip , Mikkelsen Ross , Dent Paul
Published: July 2002

The role of protein kinase C (PKC) and p42MAP kinase signaling in the regulation of proliferation and apoptosis was investigated in freshly isolated and primary cultured rat hepatocytes. Acute treatment of freshly isolated hepatocytes with phenylephrine and EGF caused rapid phasic activations of p42MAP kinase and JNK1. Acute pre‐treatment of hepatocytes with the PKC inhibitors sphingosine, chelerythrine and bis‐indolylmaleimide abolished the ability of phenylephrine, but not EGF, to activate p42MAP kinase and JNK1. Acute pre‐treatments with all of the PKC inhibitors alone increased JNK1 basal activity ∼2‐fold. Acute treatments of primary cultures of hepatocytes with an inhibitor of MEK1 activation (PD98059) also caused inhibition of p42MAP kinase and a ∼2‐fold activation of JNK1. These data demonstrate that PKC can function as both a proximal activator and a distal inhibitor of signaling through the JNK1/SAP kinase pathway. Treatments (4 h) of primary cultured hepatocytes with sphingosine, chelerythrine, bis‐indolylmaleimide and PD98059 did not induce apoptosis as judged by propidium iodide staining. Similar acute treatments of HepG2 cells rapidly induced cell death. These data demonstrate that acute inhibition of either PKC or p42MAP kinase function is sufficient to rapidly induce apoptosis in transformed, but not in non‐transformed hepatocytes.
Calcium influx inhibits MT1‐MMP processing and blocks MMP‐2 activation
Authors: Yu Ming , Sato Hiroshi , Seiki Motoharu , Spiegel Sarah , Thompson Erik W
Published: July 2002

We have previously reported that concanavalin A (ConA)‐induced MMP‐2 activation involves both transcriptional and non‐transcriptional mechanisms. Here we examined the effects of calcium influx on MT1‐MMP expression and MMP‐2 activation in MDA‐MB‐231 cells. The calcium ionophore ionomycin caused a dose‐dependent inhibition of ConA‐induced MMP‐2 activation, but had no effect on MT1‐MMP mRNA levels. However, Western analysis revealed an accumulation of pro‐MT1‐MMP (63 kDa), indicating that ionomycin blocked the conversion of pro‐MT1‐MMP protein to the active 60 kDa form. This suggests that increased calcium levels inhibit the processing of MT1‐MMP. This finding may help to elucidate the mechanism(s) which regulates MT1‐MMP activation.
Colon carbonic anhydrase 1: transactivation of gene expression by the homeodomain protein Cdx2
Authors: Drummond F.-J , Sowden J , Morrison K , Edwards Y.H
Published: July 2002

The homeodomain protein, Cdx2, has been implicated in the transcriptional regulation of genes expressed in the small intestine. In vitro studies of the carbonic anhydrase 1 (CA1) colon promoter implied that Cdx2 may also play a role in the regulation of colon‐specific gene expression. The current work follows up this proposal by examining the ability of Cdx2 to transactivate gene expression in cultured cells mediated by CA1 promoter sequences. The results show that Cdx2 exerts a positive regulatory effect by binding to a motif 87 bp upstream of the CA1 TATA box; this motif appears to act as an enhancer since gene activation is independent of its orientation.
Cloning and functional expression of a `fast' fungal kinesin
Authors: Grummt Maika , Pistor Susanne , Lottspeich Friedrich , Schliwa Manfred
Published: July 2002

Conventional kinesins are molecular motors that move towards the plus end of microtubules. In animal species, they have been shown to be remarkably conserved in terms of both their primary sequence and several physiological properties, including their velocity of movement. Here we report the cloning of Synkin, a homologue of conventional kinesin from the zygomycete fungus Syncephalastrum racemosum [Steinberg, Eur. J. Cell Biol. 73 (1997) 124–131] that is 4–5 times faster than its animal counterparts. Expression in bacteria yields a fully functional motor that moves at the same speed as the native motor isolated from fungal hyphae and has similar hydrodynamic properties. Its sequence is most closely related to that of two other fungal kinesins from Neurospora and Ustilago, and shares several biochemical properties with the Neurospora motor. Fungal kinesins therefore seem to form a conserved subfamily of conventional kinesins distantly related to animal kinesins. They may help to identify sequence features important for determining motor velocity.
Protein biosynthesis: structural studies of the elongation cycle
Authors: Nyborg Jens , Liljas Anders
Published: July 2002

The elongation cycle of protein synthesis on ribosomes is catalyzed by the elongation factors EF‐Tu and EF‐G. A thorough crystallographic analysis of the structures of the different functional states of EF‐Tu has been made. Furthermore, the structure of EF‐G:GDP is the form of EF‐G that dissociates from the ribosome. Since it mimics the structure of the ternary complex of EF‐Tu:GTP with aminoacyl‐tRNA, which subsequently binds to the ribosome, EF‐G:GDP leaves an imprint on the ribosome for the ternary complex. In addition, electron cryomicroscopy studies of ribosomes with tRNA as well as the ternary complex bound are beginning to give a solid structural basis for the functional description of elongation.
Synthetic human antibodies and a strategy for protein engineering
Authors: Winter Greg
Published: July 2002

Our understanding of the way antibodies are built in vivo has provided an approach for engineering synthetic human antibodies in bacteria. Such antibodies have not only been raised against foreign antigens, but also against highly conserved antigens or human self‐antigens, and have considerable practical potential as reagents for research and also as therapeutics. The approach also has implications for the design of antibody repertoires and for engineering other proteins with desirable binding properties. This review takes a personal view.
Translation of tobacco chloroplast rps14 mRNA depends on a Shine‐Dalgarno‐like sequence in the 5′‐untranslated region but not on internal RNA editing in the coding region
Authors: Hirose Tetsuro , Kusumegi Takahiro , Sugiura Masahiro
Published: July 2002

The role of Shine‐Dalgarno‐like sequences in mRNAs from higher plant chloroplasts has not been analyzed experimentally so far. In vitro translation analysis has revealed that the Shine‐Dalgarno‐like sequence is essential for translation of tobacco chloroplast rps14 mRNA. Two RNA editing sites have been identified in the protein‐coding region of the rps14 mRNA. Editing of the second site was found to be partial and hence the partially edited transcripts are accumulated in tobacco green leaves. In vitro translation assays using the fully edited, partially edited and unedited rps14 mRNAs indicated that editing does not directly influence translational efficiency.
Widespread occurrence of a highly conserved RING‐H2 zinc finger motif in the model plant Arabidopsis thaliana
Authors: Jensen Rikke B , Jensen Karin L , Jespersen Hans M , Skriver Karen
Published: July 2002

Several novel Arabidopsis thaliana proteins containing a RING‐H2 zinc finger motif were predicted after database searches. Alignment of 29 RING‐H2 finger sequences shows that the motif is strikingly conserved in otherwise unrelated proteins. Only short, non‐conserved polar/charged sequences distinguish these domains. The RING‐H2 domain is most often present in multi‐domain structures, a number of which are likely to contain a membrane‐spanning region or an additional zinc finger. However, there are several small (126–200 residues) proteins consisting of an N‐terminal domain, rich in aliphatic residues, and a C‐terminal RING‐H2 domain. Reverse‐transcription PCR suggests that the RING‐H2 genes are widely expressed at low levels.
Activation of muscarinic K+ current by β‐adrenergic receptors in cultured atrial myocytes transfected with β1 subunit of heterotrimeric G proteins
Authors: Bender Kirsten , Wellner-Kienitz Marie-Cécile , Meyer Thomas , Pott Lutz
Published: July 2002

Muscarinic K+ channels (IK(ACh)) in native atrial myocytes are activated by βγ subunits of pertussis toxin (Ptx)‐sensitive heterotrimeric G proteins coupled to different receptors. βγ subunits of Ptx‐insensitive Gs, coupled to β‐adrenergic receptors, do not activate native IK(ACh). In atrial myocytes from adult rats transfected with rat brain β1 subunit IK(ACh) can be activated by stimulation of β‐adrenergic receptors using isoprenaline. This effect is insensitive to Ptx. These findings demonstrate for the first time promiscuous (Ptx‐insensitive) coupling of Gsβγ to GIRK channels in their native environment.
Propeptide of the metalloprotease of Brevibacillus brevis 7882 is a strong inhibitor of the mature enzyme
Authors: Serkina Anna V. , Gorozhankina Tatiana F. , Shevelev Alexei B. , Chestukhina Galina G.
Published: July 2002

A metalloprotease gene of Brevibacillus brevis (npr) was expressed in Escherichia coli in a soluble form as native Npr precursor. A significant fraction of the precursor was spontaneously processed, producing the N‐terminal propeptide and the mature enzyme. A strong inhibition of the mature Npr by its own propeptide in the crude lysate was observed even in the absence of the covalent linkage between them. Pure precursor, propeptide and the mature Npr were isolated and kinetic parameters of the mature enzyme inhibition by the propeptide were determined. The inhibition is of the tight‐binding competitive type with K
i 0.17 nM. Inhibition of metalloproteases from Brevibacillus megaterium and thermolysine by the heterologous propeptide of the Npr from B. brevis was much weaker or none.
Inhibition of Escherichia coli DNA polymerase‐I by the anti‐cancer drug cis‐diaminedichloroplatinum(II): what roles do polymerases play in cis‐platin‐induced cytotoxicity?
Authors: Duman Rebecca K. , Heath Robert T. , Bose Rathindra N.
Published: July 2002

Activities of Escherichia coli DNA polymerase‐I were examined in the presence of the anti‐tumor drug cis‐diaminedichloroplatinum(II) and its inactive geometric isomer trans‐diaminedichloroplatinum(II). The trans‐isomer did not inhibit the enzyme activity. The anti‐tumor drug, on the other hand, retarded the enzyme in its ability to extend the primer strand of DNA. Two alternative mechanisms of inhibition, covalent binding of cis‐diaminedichloroplatinum(II) to the polymerase and to the template DNA, were explored. Selective pre‐incubations of the platinum drug with the polymerase and DNA reveal that the inhibition is primarily due to covalent binding to the enzyme. The rates of inhibition were found to be first order in enzyme and zeroth order in platinum in the concentration range 0.05–3.0 mM. A mechanism that deals with the formation of an initial platinum–polymerase‐I complex with a binding constant >105 M−1 followed by a further reaction to form an inhibitory complex is consistent with the kinetic data. The rate limiting first order rate constant for the formation of the inhibitory complex is comparable to that observed for the thiol coordination of peptides containing cysteine residues. Analyses of known structures and functions of catalytic domains of various polymerases point to the direction that the inhibition is perhaps due to the distortion of the DNA binding domain of the enzyme due to platinum coordination.
Molecular cloning of cDNA encoding a cyclin‐selective ubiquitin carrier protein (E2‐C) from Carassius auratus (goldfish) and expression analysis of the cloned gene
Authors: Tokumoto Mika , Nagahama Yoshitaka , Tokumoto Toshinobu
Published: July 2002

Destruction of cyclin B is required for exit from mitosis and meiosis. A cyclin‐specific ubiquitinating system, including cyclin‐selective ubiquitin carrier protein (E2‐C), is thought to be responsible for cyclin B destruction. Here we present the cloning, sequencing and expression analysis of goldfish, Carassius auratus, E2‐C which encodes the cyclin‐selective ubiquitin carrier protein from goldfish ovary. The cloned cDNA is 677 bp long and encodes 172 amino acids. The deduced amino acid sequence is highly homologous to E2‐C from other species. Recombinant goldfish E2‐C possesses ubiquitinating activity against cyclin B. The expression of mRNA for E2‐C was similar to that of mRNA for cyclin B, occurring at very high level in the ovary. The similarity of the expression pattern of E2‐C and cyclin B suggests that E2‐C mediates a cyclin‐specific ubiquitination.
Synergistic effects of nuclear factors – GATA, VBP and ER in potentiating vitellogenin gene transcription
Authors: Teo Bee Yen , Tan Nguan Soon , Lam Toong Jin , Ding Jeak Ling
Published: July 2002

The Oreochromis aureus vitellogenin (OaVtg) gene contains three imperfect oestrogen response elements (EREs) and GATA and VBP (vitellogenin binding protein) binding sites. An analysis of the promoter indicates that the 5′‐flanking region up to position −625 is sufficient to mediate E2 control. Furthermore, transfection of deletion and mutagenised promoters indicates that both GATA and VBP synergise with ER, and thus contribute to the regulation of the endogenous OaVtg gene. These findings support the notion that the interplay of promoter elements mediates proper hormone‐dependent and tissue‐specific expression of the OaVtg gene, regardless of non‐consensus sequence context of EREs and VBP.
Upregulation of endogenous heparin‐binding EGF‐like growth factor and its role as a survival factor in skeletal myotubes
Authors: Horikawa Michiharu , Higashiyama Shigeki , Nomura Shintaro , Kitamura Yukihiko , Ishikawa Mutsuo , Taniguchi Naoyuki
Published: July 2002

To investigate the role of heparin‐binding EGF‐like growth factor (HB‐EGF) in skeletal muscle, we studied its function in skeletal myotubes in vitro using mouse C2C12 cells. Expression levels of membrane‐anchored HB‐EGF (proHB‐EGF) protein were increased specifically during their differentiation among epidermal growth factor receptor (EGFR) ligands. Production levels of EGFR on the cell surface were constant. Tyrosine phosphorylation of EGFR, however, was constitutively increased during differentiation. Quenching of endogenous HB‐EGF significantly rendered myotubes sensitive to apoptotic cell death induced by hypoxic stress, suggesting that proHB‐EGF in the skeletal muscle is specifically upregulated to function as a survival factor.
Vasopressin stimulates long‐term net chloride secretion in cortical collecting duct cells
Authors: Djelidi Sabri , Fay Michel , Cluzeaud Françoise , Thomas-Soumarmon Annick , Bonvalet Jean-Pierre , Farman Nicolette , Blot-Chabaud Marcel
Published: July 2002

The classical short‐term effect (within minutes) of arginine vasopressin (AVP) consists in increasing sodium, chloride and water transport in kidney cells. More recently, long‐term actions (several hours) of the hormone have been evidenced on water and sodium fluxes, due to transcriptional enhancement in the expression of their transporters. The present study demonstrates that AVP is also responsible for a long‐term increase in net chloride secretion. In the RCCD1 rat cortical collecting duct cell line, 10−8 M AVP induced, after several hours, an increase in net 36Cl− secretion. This delayed effect of AVP was inhibited by basal addition of 10−4 M bumetanide and apical addition of 10−4 M glibenclamide, suggesting chloride entry at the basal membrane through a Na+/K+/2Cl− and apical secretion through a chloride conductance. An original acute cell permeabilization method was developed to allow for entry of antibodies directed against the regulatory region (R) of the cystic fibrosis transmembrane regulator (CFTR) into the cells. This procedure led to a complete and specific blocking of the long‐term net chloride secretion induced by AVP. Finally, it was observed that CFTR transcripts steady‐state level was significantly increased by AVP treatment. Besides the well‐documented short‐term effect of AVP on chloride transport, these results provide evidence that in RCCD1 cells, AVP induces a delayed increase in transepithelial net chloride secretion that is mediated by a Na+/K+/2Cl− co‐transporter and CFTR.
Functional analysis of a human A1 adenosine receptor/green fluorescent protein/Gi1α fusion protein following stable expression in CHO cells
Authors: Bevan Nicola , Palmer Tim , Drmota Tomas , Wise Alan , Coote Jim , Milligan Graeme , Rees Stephen
Published: July 2002

Fusion proteins between the human A1 adenosine receptor and the pertussis toxin resistant (Cys351Gly) mutant of the G‐protein α subunit Gi1α (A1/Gi), and between the human A1 adenosine receptor, the Aequorea victoria green fluorescent protein (GFP) and Cys351Gly Gi1α (A1/GFP/Gi), were expressed in CHO cells. The agonist NECA caused a stimulation of [35S]GTPγS binding at both fusion proteins with similar concentration dependence as at the native receptor. However in the presence of pertussis toxin NECA stimulation of [35S]GTPγS binding was only seen at the A1/GFP/Gi fusion protein. The regulation of the adenylyl cyclase and MAP kinase effector systems by both fusion proteins was attenuated following pertussis toxin treatment. These studies demonstrate for the first time the characterisation of a fusion protein between a G‐protein coupled receptor, GFP and a G‐protein α subunit.
Labor induction with vaginal misoprostol and extra‐amniotic prostaglandin F2α gel
Authors: Majoko F , Zwizwai M , Lindmark G , Nyström L
Published: July 2002

Objective: To compare the effectiveness of vaginally administered misoprostol with extra‐amniotic prostaglandin F2α (PGF2α) gel for induction of labor. Method: A randomized controlled trial, with women allocated to receive either misoprostol 50 μg intra‐vaginally or extra‐amniotic PGF2α gel 5 mg, was conducted in Harare Maternity Hospital. A total of 152 women were admitted for induction of labor with a term singleton, pregnancy and cephalic presentation were recruited. The main outcome was duration of induction. Results: There were no differences in the characteristics of women in the two groups at recruitment. In the misoprostol group there was a significantly reduced need for augmentation of labor with oxytocin (OR=0.36; 95% C.I. 0.17–0.73) and delivery by cesarean section for failure to progress (OR=0.11; 95% C.I. 0.00–0.88). The risk for duration of induction to vaginal delivery exceeding 12, 18 or 24 h was reduced by 18%, 38% and 68%, respectively, but only the risk for duration >24 h was significantly reduced (OR=0.32; 95%C.I. 0.11–0.91). The mean duration of induction was shorter in the misoprostol group, 15.2 vs. 23.6 h (P=0.02). There were no differences in fetal outcome. Conclusion: Misoprostol 50 μg was associated with less use of oxytocin in labor, a shorter induction to delivery interval and fewer cesarean sections for failure to progress when compared with extra‐amniotic PGF2α gel.
The midwife and her functions by level of care
Authors: Segovia I
Published: July 2002

The role the midwives play in women's healthcare in Chile demonstrates appropriate utilization of human resources according to their capacity to provide services. Historically, the Chilean midwife came into being more than 160 years ago, exclusively as a birth attendant. The midwives' functions have evolved, particularly during the last few decades, following the demographic and epidemiological evolution of the Chilean population and the new paradigm of healthcare addressed to woman throughout her life cycle, not only during pregnancy and childbirth. At the present time, the midwife is the professional that provides the great majority of gynecological and obstetrics care at the primary healthcare level, while also attending most of the deliveries in the public health services. The close link with physicians working as a team, and the emotional ties of the midwife as a provider with the woman as a client, is emphasized as an additional advantage of the presence of the midwife as a key person in women's healthcare.
Interleukin‐1β, but not IL‐1α, mediates nerve growth factor secretion from rat astrocytes via type I IL‐1 receptor
Authors: Jurič Damijana M. , Čarman‐Kržan Marija
Published: July 2002

In astrocytes, nerve growth factor (NGF) synthesis and secretion is stimulated by the cytokine interleukin‐1β (IL‐1β). In the present study, the role of IL‐1 receptor binding sites in the regulation of NGF release was evaluated by determining the pharmacological properties of astroglially localized IL‐1 receptors, and, by comparing the effects of both the agonists (IL‐1α and IL‐1β) and the antagonist (IL‐1ra)—members of the IL‐1 family on NGF secretion from rat neonatal cortical astrocytes in primary culture. Using receptor‐binding studies, binding of [125I] IL‐1β to cultured astrocytes was saturable and of high affinity. Mean values for theKDandBmaxwere calculated to be 60.7±7.4 pM and 2.5±0.1 fmol mg‐1protein, respectively. The binding was rapid and readily reversible. IL‐1 receptor agonists IL‐1α (Kiof 341.1 pM) and IL‐1β (Ki59.9 pM), as well as the antagonist IL‐1ra (Ki257.6 pM), displaced specific [125I] IL‐1β binding from cultured astrocytes in a monophasic manner. Anti‐IL‐1RI antibody completely blocked specific [125I] IL‐1β binding while anti‐IL‐1RII antibody had no inhibitory effect. Exposure of cultured astrocytes to IL‐1α and IL‐1β revealed the functional difference between the agonists in influencing NGF release. In contrast to IL‐1β (10 U/ml), which caused a 3‐fold increase in NGF secretion compared to control cells, IL‐1α by itself had no stimulatory action on NGF release. The simultaneous application of IL‐1α and IL‐1β elicited no additive response. IL‐1ra had no effect on basal NGF release but dose‐dependently inhibited the stimulatory response induced by IL‐1β. We concluded that IL‐1β‐induced NGF secretion from cultured rat cortical astrocytes is mediated by functional type I IL‐1 receptors, whereas IL‐1α and IL‐1ra, in spite of their affinity for IL‐1RI, have no effect on NGF secretion from these cells. Type II IL‐1R is not present on rat neonatal cortical astrocytes.
Antisocial addicts: the importance of additional axis I disorders for the 28-month outcome
Authors: Tómasson K , Vaglum P
Published: July 2002

The association between additional co-morbid axis I disorders and the following 28-month course of drinking and mental distress was explored in a nation-wide representative sample (N = 100) of treatment-seeking alcoholics with antisocial personality disorder (ASPD). Diagnoses at admission were assessed with the Diagnostic Interview Schedule and follow-up status was assessed with a questionnaire and from informants. Only 24% had no additional diagnoses, 39% had an affective disorder, 43% panic/agoraphobia, 61% other anxiety disorders, and 47% were polysubstance abusers. Polysubstance abusers had more prior admissions, and were more often involved in fights, while additional anxiety disorder was associated with lower prevalence of drunken driving arrests. Relapse (87%) was best predicted by the number of prior admissions (odds ratio [OR] = 1.3), while affective disorders reduced the risk of relapse (OR = 0.2). Readmissions (55%) were least common among those with affective disorders (44%). Identifying axis I diagnoses, and in particular affective disorders among treatment-seeking ASPD alcoholics, is of substantial importance both in research and clinical practice.
The Polish economic transition: outcome and lessons
Authors: Slay Ben
Published: July 2002

This article provides an overview of, and draws conclusions about, Poland's economic transition during the 1990s. Special attention is paid to macroeconomic and external developments, privatization, and the lessons of the Polish experience for other transition economies.
A comparative analysis of the price‐process model of mortgage valuation
Authors: Murphy Austin
Published: July 2002

This paper compares the performance of the price‐process model, which efficiently sets prepayments as a function of call option values, with a traditional interest‐rate‐process model, which sets prepayments as a function of interest rate spreads. The empirical results indicate that the price‐process model is more consistent with GNMA market prices. Tests on more recent data indicate that the price‐process modeling framework has continued to be more accurate in explaining GNMA prices than other mortgage‐pricing models reported in the literature.
The ex ante effects of trade halting rules on informed trading strategies and market liquidity
Authors: Subrahmanyam Avanidhar
Published: July 2002

In this paper, we investigate strategic informed trading in a regime with rule‐based market closures. Closure rules can be designed to reduce the ex ante trading costs of liquidity traders but cause informed traders to scale back their trading in order to reduce the chance of the closure being triggered. In the Stackelberg equilibrium where the informed act as leaders and market makers as followers, this phenomenon causes trading costs for small investors to increase. Thus, ex ante strategic behavior by informed traders in response to closure rules can result in increased trading costs for precisely the individuals whom the closures are intended to benefit: namely, the uninformed retail investors who trade small orders. We show, however, that this effect can be mitigated by randomizing the halting rule and thus reducing the degree of predictability of the halt from the perspective of informed traders.
The influence of diastolic and systolic function on exercise performance in heart failure due to dilated cardiomyopathy or ischemic heart disease
Authors: Pepi Mauro , Agostoni PierGiuseppe , Marenzi GianCarlo , Doria Elisabetta , Guazzi Marco , Lauri Gianfranco , Maltagliati Anna , Guazzi Maurizio
Published: July 2002

Background:

Peripheral adaptations and ventricular abnormalities influence physical performance in chronic heart failure. However, the role of the heart in determining exercise capacity has not been completely elucidated.

Aims:

To define cardiac determinants of exercise capacity in patients with dilated cardiomyopathy.

Methods:

In 101 patients with heart failure (NYHA class II–III) due to primary or ischemic dilated cardiomyopathy we measured peak exercise oxygen consumption (Pvo2), left ventricular ejection fraction (EF), left and right atrial and ventricular cavity dimensions, mitral and tricuspid flows. Patients were subdivided in class A (Pvo2>20 ml/min per kg; n=44), class B (Pvo2 16–20 ml/min per kg; n=42) and class C (Pvo2<16 ml/min per kg; n=15).

Results:

Left ventricular diastolic and systolic dimensions, left atrial diameter, right atrial and ventricular areas were greater in class C than in class B and A; EF was lower in class C than in the other two classes; mitral peak flow velocity at early diastole (PFVE) and the ratio between early and late peak flow velocity (PFVE/PFVA) were higher in class C; mitral and tricuspid deceleration time (DT) in class B and A significantly exceeded those in class C. Peak vo2 was correlated with left and right ventricular dimensions, left atrial diameter, EF, mitral PFVE and PFVE/PFVA, mitral and tricuspid DT. Left ventricular EF, DT of the mitral valve and left ventricular diastolic diameter were independent predictors of peak vo2 at multivariate analysis.

Conclusions:

In patients with dilated cardiomyopathy Pvo2 is related to left and right ventricular dimensions, left and right ventricular filling pattern and EF. Both systolic and diastolic dysfunction influence functional capacity.
Environmental Health And Pregnancy
Authors: Krulewitch Cara J.
Published: July 2002

Above all, we need to look at the entire life span. We can no longer divide life into segments such as fetal, neonatal, childhood adolescence, adulthood, and senescence. Rather, it is a dynamic continuum with the status at any one time being influenced by all that went before and, in turn, having an effect on all that follows (1).