bluuebunny/crossref_metadata_2025 huggingface dataset:
Polyamines Contribute to Ethanol Withdrawal‐Induced Neurotoxicity in Rat Hippocampal Slice Cultures Through Interactions With the NMDA Receptor
Authors: Gibson D. Alex , Harris Barton R. , Prendergast Mark A. , Hart Stewart R. , Blanchard John A. , Holley Robert C. , Pedigo Norman W. , Littleton John M.
Published: August 2004

Background: Several reports demonstrate that withdrawal from long‐term ethanol exposure is associated with significant central nervous system neurotoxicity, produced at least in part by increased activity of N‐methyl‐d‐aspartate receptors (NMDARs). Recent evidence suggests that elevations in the synthesis and release of the polyamines spermidine and spermine, which are known modulators of NMDARs, contribute to the increased activity of the receptor during ethanol withdrawal. Therefore, the goal of this investigation was to examine what role, if any, spermidine and spermine have in the generation of ethanol withdrawal‐induced neurotoxicity.

Methods: Neurotoxicity (measured as fluorescence of the cell death indicator propidium iodide, PI), glutamate release (measured by high‐performance liquid chromatography analysis), and polyamine concentrations (by high‐performance liquid chromatography) were measured in rat hippocampal slice cultures undergoing withdrawal from chronic (10 day) ethanol exposure (100 mM). In addition, the effects of the polyamine synthesis inhibitor di‐fluoro‐methyl‐ornithine (DFMO, 0.1–100 nM) and NMDAR polyamine‐site antagonists ifenprodil, arcaine, and agmatine (1 nM‐100 μM) on ethanol withdrawal‐ and NMDA‐induced neurotoxicity were measured.

Results: Ethanol withdrawal significantly increased glutamate release (peaking at 18 hr with a 53% increase), increased concentrations of putrescine and spermidine (136% and 139% increases, respectively, at 18 hr), and produced significant cytotoxicity in the CA1 hippocampal region (56% increase in PI staining relative to controls) of the cultures. The cell death produced by ethanol withdrawal was significantly inhibited by ifenprodil (IC50= 14.9 nM), arcaine (IC50= 37.9 nM), agmatine (IC50= 41.5 nM), and DFMO (IC50= 0.6 nM). NMDA (5 μM) significantly increased PI staining in the CA1 region of the hippocampal cultures (365% relative to controls), but ifenprodil, arcaine, agmatine, and DFMO all failed to significantly affect this type of toxicity.

Conclusions: These data implicate a role for polyamines in ethanol withdrawal‐induced neurotoxicity and suggest that inhibiting the actions of polyamines on NMDARs may be neuroprotective under these conditions.
Neurosteroid Modulation of Glutamate Release in Hippocampal Neurons: Lack of an Effect of a Chronic Prenatal Ethanol Exposure Paradigm
Authors: Carta Mario , Partridge L. Donald , Savage Daniel D. , Valenzuela C. Fernando
Published: August 2004

Background: Pregnenolone sulfate (PREGS) is a promnesic neurosteroid that is abundantly expressed in the hippocampus of rodents. Studies have shown that the modulation of postsynaptic ligand‐gated ion channels by this neurosteroid is impaired in preparations from the brains of fetal ethanol‐exposed animals. In this study, we examined whether the presynaptic actions of PREGS also are affected by exposure to ethanol in utero.

Methods: Rat dams were exposed to one of the following diets during pregnancy: (1) 5% ethanol liquid diet, (2) 0% ethanol liquid diet with pair‐feeding, and (3) ad libitum controls. We then studied the presynaptic actions of PREGS on (1) alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptor‐mediated miniature excitatory postsynaptic currents (mEPSCs) recorded from cultured hippocampal neurons in the whole‐cell patch‐clamp configuration and (2) paired‐pulse facilitation of NMDA receptor‐dependent excitatory postsynaptic potentials that were intracellularly recorded from CA1 pyramidal neurons in hippocampal slices from adult rats.

Results: Chronic prenatal ethanol exposure affected neither basal mEPSC frequency nor its potentiation by PREGS. Basal paired‐pulse facilitation (i.e., in the absence of PREGS) was unaffected by fetal ethanol exposure. Chronic prenatal ethanol exposure did not affect the PREGS‐induced potentiation of paired‐pulse facilitation.

Conclusions: Chronic prenatal ethanol exposure does not affect the basal probability of glutamate release in immature or mature hippocampal neurons. Moreover, the presynaptic actions of the neurosteroid PREGS also are unaffected by this exposure. Given that modulation of glutamate release could have a role in the mechanism of the promnesic actions of this neurosteroid, future studies are warranted to determine whether PREGS can ameliorate learning and memory deficits in fetal ethanol‐exposed animals.
The Effect of Alcohol on Testosterone Concentrations in Alcohol‐Preferring and Non‐Preferring Rat Lines
Authors: Apter Susanna J. , Eriksson C. J. Peter
Published: August 2004

Background: Previous studies have reported associations between human alcohol drinking and testosterone levels.

Methods: In this study we investigated serum testosterone concentrations without and under the influence of alcohol in alcohol‐preferring (AA) and nonpreferring (ANA) rat lines. Animals were tested in both mornings and afternoons and the alcohol doses were 0.75 and 1.50 g/kg.

Results: Higher basal serum testosterone levels were detected in the AA rats compared with the ANA rats in both mornings (152%, p= 0.028) and afternoons (75%, p= 0.035). The high alcohol dose decreased the testosterone concentrations of both the AA and the ANA rats (p= 0.001–0.01). The low dose, however, decreased testosterone concentrations only in the ANA line (line difference in the morning:p= 0.027; in the afternoon p= 0.000).

Conclusion: The present results support previous indications of a positive association between testosterone and alcohol drinking. Furthermore, the present results, together with earlier reports on the AA and ANA rats, introduce the possibility of a connection between this association and the hypothalamic opiate system, which is also involved in the feedback regulation of testosterone synthesis.
Beyond cognitive and metacognitive tools: the use of the Internet as an ‘epistemological’ tool for instruction
Authors: Tsai Chin‐Chung
Published: August 2004

This paper argues that Internet‐based instruction should not be only perceived as a cognitive tool or a metacognitive tool; rather, it can be perceived and used as an epistemological tool. When the Internet is used as an epistemological tool for instruction, learners are encouraged to evaluate the merits of information and knowledge acquired from Internet‐based environments, and to explore the nature of learning and knowledge construction. This paper further asserts that Internet‐based instruction is perceived as a way to help learners develop advanced epistemologies. On the other hand, developmentally advanced epistemological beliefs can facilitate the practice of Internet‐based instruction.
Visions and realities of Internet use in schools: Canadian perspectives
Authors: Gibson Susan , Oberg Dianne
Published: August 2004

Teachers in many countries are being expected to use the Internet in their work. Research on the Canadian experience of Internet implementation provides insights that may be valuable for researchers and educators in other countries. A three‐year study, using both quantitative and qualitative approaches, examined both the visions for Internet use and the realities of everyday practice related to Internet use in Canadian schools. Participants in the study included ministry of education officials, teacher association officials, classroom teachers, and school administrators. Findings of the study suggest that all four participant groups were positive about the visions of the Internet as a tool with the potential to contribute to the enhancement of teaching and the development of information literate students. The realities of Internet use, however, were quite different from the visions. All four participant groups reported that the Internet was being used mostly to increase access to information. Its potential as an innovative learning tool for students and for teachers was largely unrealised. Few respondents reported using the collaboration, creation, and dissemination capabilities of the Internet. This outcome appeared to be the result of limited infrastructure support, difficulties in infusing Internet use into curriculum, and lack of appropriate teacher professional development.
Teacher collegiality and electronic communication: A study of the collaborative uses of email by secondary school teachers in Uruguay
Authors: Grünberg Jorge , Armellini Alejandro
Published: August 2004

Teacher collegiality has been consistently highlighted in recent years as a crucial factor in the success of processes of educational change and professional development. Consequently, there is considerable interest both from researchers and practitioners, in investigating strategies capable of supporting interactions and collaborations between teachers.

Research, mostly carried out in industry, suggests that computer‐mediated communication (CMC) technologies may support collegiality in a diversity of contexts. Following the technological developments of the last few years, which have driven down the costs and complexity of computers and telecommunications, CMC technologies are becoming increasingly available to schools around the world. One of the more widespread of these new CMC technologies is electronic mail.

The aim of this article is to discuss some of the results of a research project that investigated the potential of electronic mail for supporting the development of collegiality and professional exchange among secondary school teachers in Uruguay. At the time of this study, relatively little research had been carried out into the use of electronic mail for professional purposes by schoolteachers, especially in Latin America.

The evidence suggests that email may support the formation of communities of highly committed users, where seeking and sharing professional resources are accepted behaviours.
Gay and Straight Possible Selves: Goals, Identity, Subjective Well‐Being, and Personality Development
Authors: King Laura A. , Smith Nathan Grant
Published: August 2004

Abstract
This study examined the relations of the salience and elaboration of gay and straight possible selves to subjective well‐being (SWB) and ego development (ED) in a sample of 107 gay men and lesbians, who wrote narrative descriptions of their straight and gay best possible selves and rated the salience of these narratives (i.e., the clarity of the mental image, how easy it was to imagine). Independent raters coded the narratives for amount of elaboration (i.e., vivid detail). The salience of the gay possible self was positively related to SWB and outness, while the salience of the straight possible self was negatively related to SWB and being out. Furthermore, the possible self variables mediated the relationship of outness to well‐being. Straight self‐elaboration predicted personality development concurrently and prospectively, over 2 years. Results are interpreted as indicating that, while happiness may require us to focus upon our current life goals, personality development might require an exploration of the potential sacrifices of identity.
The Impact of Personality on Cognitive, Behavioral, and Affective Political Processes: The Effects of Need to Evaluate
Authors: Bizer George Y. , Krosnick Jon A. , Holbrook Allyson L. , Christian Wheeler S. , Rucker Derek D. , Petty Richard E.
Published: August 2004

Abstract
Need to evaluate (NE) is a personality trait that reflects a person's proclivity to create and hold attitudes; people high in NE are especially likely to form attitudes toward all sorts of objects. Using data from the 1998 National Election Survey Pilot and the 2000 National Election Survey, NE was shown to predict a variety of important attitude‐relevant cognitive, behavioral, and affective political processes beyond simply holding attitudes: NE predicted how many evaluative beliefs about candidates a person held, the likelihood that a person would use party identification and issue stances to determine candidate preferences, the extent to which a person engaged in political activism, the likelihood that a person voted or intended to vote, the extent to which a person used the news media for gathering information, and the intensity of emotional reactions a person felt toward political candidates. Thus, NE appears to play a powerful role in shaping important political behavior, emotion, and cognition.
Reduced incidence of upper gastrointestinal ulcer complications with the COX‐2 selective inhibitor, valdecoxib
Authors: Goldstein J. L. , Eisen G. M. , Agrawal N. , Stenson W. F. , Kent J. D. , Verburg K. M.
Published: August 2004

Aim : In a predefined analysis, data were pooled from eight blinded, randomized, controlled trials, and separately from three long‐term, open‐label trials to determine the rate of upper gastrointestinal ulcer complications with the cyclo‐oxygenase‐2 selective inhibitor, valdecoxib, vs. non‐selective non‐steroidal anti‐inflammatory drugs.

Methods : In randomized, controlled trials, 7434 osteoarthritis and rheumatoid arthritis patients received placebo (n = 973), valdecoxib 5–80 mg daily (n =4362), or a non‐selective non‐steroidal anti‐inflammatory drug (naproxen, ibuprofen or diclofenac; n =2099) for 12–26 weeks. In long‐term, open‐label trials, 2871 patients received valdecoxib 10–80 mg daily for up to 1 year. All potential events were reviewed by a blinded, independent review committee based on a priori definitions of ulcer complications (perforations, obstructions, bleeds).

Results : In randomized, controlled trials, 19 of 955 potential events were adjudicated to be ulcer complications. Valdecoxib was associated with a significantly lower ulcer complication rate than non‐selective non‐steroidal anti‐inflammatory drugs (0.68% vs. 1.96%, all patients; 0.29% vs. 2.08%, non‐aspirin users; P < 0.05). In long‐term, open‐label trials, seven of 310 potential events were adjudicated to be ulcer complications; the annualized incidence for valdecoxib was 0.39% (seven of 1791 patient‐years) for all patients and 0.2% (three of 1472 patient‐years) for non‐aspirin users.

Conclusions : Valdecoxib, including above recommended doses, is associated with a significantly lower rate of upper gastrointestinal ulcer complications than therapeutic doses of non‐selective non‐steroidal anti‐inflammatory drugs.
Adjuvant biofeedback following anal sphincter repair: a randomized study
Authors: Davis K. J. , Kumar D. , Poloniecki J.
Published: August 2004

Aims : To evaluate the impact of adjuvant biofeedback following sphincter surgery.

Methods : Thirty‐eight patients were randomized into sphincter repair or sphincter repair plus biofeedback groups. Outcome measures included a symptom questionnaire, patient's rating of satisfaction with continence function and improvement, change in continence score, quality of life and anorectal physiology. Endoanal ultrasonography was also performed pre‐ and post‐operatively.

Results : Immediately following surgery, there was no statistically significant difference in any of the functional or physiological variables between the groups. Continence and patient satisfaction scores improved with a mean difference of −0.48 (95% CI: −3.30–2.33, P =0.73) and 1.03 (95% CI: −1.40–3.46, P = 0.39), respectively. Only the difference in embarrassment scores reached statistical significance (mean) 0.56 (95% CI: 0.12–0.99, P = 0.014). Resting and squeeze pressures also improved. Thirteen of 14 in the biofeedback and 11 of 17 (control) reported symptomatic improvement. In the biofeedback group, although not statistically significant continence and satisfaction scores improved and were sustained over time. In the control group, continence and satisfaction scores changed little between 3 and 12 months (P = NS). Quality of life measures improved within the biofeedback group but there was no statistical difference between the groups.

Conclusion : Following surgery continence function improves in all patients but adjuvant biofeedback therapy improves quality of life and maintains symptomatic improvement over time.
Has disease outcome in Crohn's disease changed during the last four decades?
Authors: Wolters F. L. , Russel M. G. V. M. , Stockbrügger R. W.
Published: August 2004

Background : Disease outcome in Crohn's disease might have changed during the last four decades. Disease outcome measurement in Crohn's disease has methodological difficulties because of patient selection and lack of proper definition of diagnostic and outcome measurement criteria.

Aim : To assess possible changes in disease outcome in Crohn's disease during the last four decades.

Methods : A systematic literature search was performed using the MEDLINE search engine and major international conference libraries. Articles and abstracts were selected according to stringent inclusion criteria.

Results : Forty articles and nine abstracts complied with the inclusion criteria. Seven studies with a median follow‐up time between 11.1 and 17 years showed standard mortality ratios in Crohn's disease ranging between 2.16 and 0.72 with a tendency of decline during the last four decades. One study with 11.4 years mean follow‐up time showed a statistically significant increased relative risk for colorectal cancer that was not confirmed by three others. Sixteen publications applied in the disease recurrence category. Probability of first resective surgery ranged between 38 and 96% during the first 15 years after diagnosis. The overall recurrence and surgical recurrence rates after first resective surgery ranged between 50 and 60, and 28 and 45% respectively during the following 15 years without an apparent time trend.

Conclusion : This structured literature review provides no hard evidence for change in disease outcome in Crohn's disease during the last four decades.
Connections with Connexins: the Molecular Basis of Direct Intercellular Signaling
Authors: Bruzzone Roberto , White Thomas W. , Paul David L.
Published: August 2004

Adjacent cells share ions, second messengers and small metabotes through intercellular channels which are present in gap junctions. This type of intercellular communication permits coordinated cellular activity, a critical feature for organ homeostasis during development and adult fe of multicellular organisms. Intercellular channels are structurally more complex than other ion channels, because a complete cell‐to‐cell channel spans two plasma membranes and results from the association of two half channels, or connexons, contributed separately by each of the two participating cells. Each connexon, in turn, is a multimeric assembly of protein subunits. The structural proteins comprising these channels, collectively called connexins, are members of a highly related multigene family consisting of at least 13 members. Since the cloning of the first connexin in 1986, considerable progress has been made in our understanding of the complex molecular switches that control the formation and permeabity of intercellular channels. Analysis of the mechanisms of channel assembly has revealed the selectivity of inter‐connexin interactions and uncovered novel characteristics of the channel permeabity and gating behavior. Structure/function studies have begun to provide a molecular understanding of the significance of connexin diversity and demonstrated the unique regulation of connexins by tyrosine kinases and oncogenes. Finally, mutations in two connexin genes have been nked to human diseases. The development of more specific approaches (dominant negative mutants, knockouts, transgenes) to study the functional role of connexins in organ homeostasis is providing a new perception about the significance of connexin diversity and the regulation of intercellular communication.
Kinetic Evidence for the Existence of a Rate‐Limiting Step in the Reaction of Ferric Hemoproteins with Anionic Ligands
Authors: Coletta Massimo , Angeletti Mauro , De Sanctis Giampiero , Cerroni Loredana , Giardina Bruno , Amiconi Gino , Ascenzi Paolo
Published: August 2004

The kinetics of azide and fluoride binding to various monomeric and tetrameric ferric hemoproteins (sperm whale Mb, isolated α and β chains of human Hb reacted with p ‐chloromercuribenzoate, dromedary, ox and human Hb) has been investigated (at pH 6.5 and 20°C) over a large range (20 μM to 2 M) of ligand concentration. It has been observed that the pseudo‐first‐order rate constant for azide binding to the hemoproteins investigated does not increase linearly with ligand concentration, but tends to level off toward an asymptotic concentration‐independent value typical for each hemoprotein. This behaviour, which has been detected only by an investigation covering an unusually large range of ligand concentrations, appears to be independent of the ionic strength, and it underlies the existence of a rate‐limiting step in the dynamic pathway of azide binding to ferric hemoproteins, which is detectable whenever the observed pseudo‐first‐order rate constant becomes faster than a given value characteristic of the specific hemoprotein. Such a behaviour is not observed in the case of fluoride binding probably because the pseudo‐first‐order rate constant for this ligand is much slower and never attains a value faster than that of the rate‐limiting step. In general terms, this feature should involve a conformational equilibrium between at least two forms (possibly related to the interaction of H2O with distal histidine and its exchange with the bulk solvent) which modulates the access of the anionic ligand into the heme pocket and its reaction with the ferric heme iron.
The Molecular Basis for the Natural Resistance of the Cytochrome bc1 Complex from Strobilurin‐Producing Basidiomycetes to Center QP Inhibitors
Authors: Kraiczy Peter , Haase Uwe , Gencic Simonida , Flindt Sven , Anke Timm , Brandt Ulrich , Von Jagow Gebhard
Published: August 2004

Mitochondria from the strobilurin A producing basidiomycetes Strobilurus tenacellus and Mycena galopoda exhibit natural resistance to (E)‐β‐methoxyacrylate inhibitors of the ubiquinol oxidation center (center Qp) of the cytochrome bc1 complex. Isolated cytochrome bc1 complex from S. tenacellus was found to be highly similar to that of Saccharomyces cerevisiae with respect to subunit composition, as well as spectral characteristics and midpoint potentials of the heme centers.

To understand the molecular basis of natural resistance, we determined the exon/intron organization and deduced the sequences of cytochromes b from S. tenacellus, M. galopoda and a third basidiomycete, Mycena viridimarginata, which produces no strobilurin A. Comparative sequence analysis of two regions of cytochrome b known to contribute to the formation of center Qp suggested that the generally lower sensitivity of all three basidiomycetes was due to the replacement of a small amino acid residue in position 127 by isoleucine. For M. galopoda replacement of Gly143 by alanine and Gly153 by serine, for S. tenacellus replacement of a small residue in position 254 by glutamine and Asn261 by aspartate was found to be the likely causes for resistance to (E)‐β‐methoxyacrylates. The latler exchange is also found in Schizosaccharomyces pombe, which we found also to be naturally resistant to (E)‐β‐methoxyacrylates.
Conformational Effects of Serine Phosphorylation in Phospholamban Peptides
Authors: Quirk Philip G. , Patchell Valerie B. , Colyer John , Drago Guido A. , Gao Yuan
Published: August 2004

We have employed one‐ and two‐dimensional 1H‐NMR spectroscopy to study the effects of serine phosphorylation on peptide conformations, using cardiac phospholamban as a model system. The nonphosphorylated phospholamban 1–20 peptide has few restraints on the conformations available to it in aqueous solution. Phosphorylation at Ser16 results in greater constraints being placed on the region encompassing Arg14–Thr17, particularly at neutral pH when the phosphate group is in the di‐anionic form. These conformational restrictions arise from specific interactions between the side‐chain of Arg14 and the phosphate group. While substitution of phosphothreonine at position 16 causes generally similar effects to phosphoserine, aspartic acid has little effect. The results are compared with phosphorylation effects in other systems, including cardiac troponin I.
Involvement of an Arachidonic‐Acid‐Dependent Pathway in the Interferon‐β‐Mediated Expression of C202 Gene in Ehrlich‐Ascites‐Tumor Cells
Authors: Toniato Elena , Flati Vincenzo , Cifone Maria Grazia , Del Grosso Egidio , Roncaioli Paola , Cilenti Lucia , Tessitore Alessandra , Lista Florigio , Frati Luigi , Gulino Alberto , Martinotti Stefano
Published: August 2004

We have investigated the signal transduction mechanism of the expression of the C202 gene mediated by interferon β (IFN‐β) in the murine Ehrlich's ascites tumor cell line. We have shown that treatment of cells with IFN‐β transiently enhances within minutes the release of free arachidonic acid through membrane phospholipase activity. Furthermore, prior treatment with either p‐bromophenacyl bromide, an antagonist of both cytosolic and secretory phospholipase A2 or neomycin, which blocks phospholipase C activity, significantly decreased the activation of the murine IFN‐β‐inducible gene, C202. Moreover, an increase of the expression of the C202 gene was observed after blocking of both the cyclooxygenase and lipoxygenase pathways. This suggests that further metabolism of arachidonic acid to epoxides via epoxygenase‐catalysed pathways may be a mechanism by which second messengers for IFN‐β‐mediated effects on C202 gene expression are generated. Taken together, these results indicate that lipids as second messengers may be important mediators in the IFN‐β‐based activation of C202 gene expression.
Regulation of p42 Mitogen‐Activated‐Protein Kinase Activity by Protein Phosphatase 2A Under Conditions of Growth Inhibition by Epidermal Growth Factor in A431 Cells
Authors: Chajry Naima , Martin Pierre‐Marie , Cochet Claude , Berthois Yolande
Published: August 2004

Epidermal growth factor (EGF), which plays an important role in the growth regulation of a large variety of normal and tumor cells, has been shown to display an ambivalent dose‐dependent effect on the proliferation of epithelial cells overexpressing EGF receptor. In a previous study aimed at dissecting the biochemical events leading to this dual action in A431 cells which over express EGF receptor, we have reported a relationship between the dual stimulator/inhibitor effect of EGF and the activity of the serine/threonine p42 mitogen‐activated protein (MAP) kinase. Indeed, a growth stimulatory concentration of EGF is shown to lead to a moderate but persistent activation of p42 MAP kinase. Conversely, an early peak of MAP kinase activation, that rapidly falls below the basal level, is observed in the presence of a growth‐inhibitory concentration of EGF. To assess the mechanism of the p42 MAP kinase inactivation under circumstances of negative growth regulation by EGF, we have investigated the role of the serine/threonine phosphatase 2A in this process. A constitutive phosphatase 2A activity was observed in untreated cells, that decreases rapidly in response to both high and low EGF concentrations. However, after this early inactivation, the phosphatase 2A activity was completely reversed concurrently with MAP kinase inactivation, after 40 min of treatment with 10 nM EGF. Conversely, in cells treated with 1 pM EGF, phosphatase 2A activity remained below the control level during all the time of the treatment, in association with a sustained MAP kinase activation. These results suggest that MAP kinase inactivation is closely related to phosphatase 2A activation. We then investigated the effect of the serine/threonine phosphatase inhibitor okadaic acid on the MAP kinase inactivation and observed that okadaic acid, at a concentration reported to specifically inhibit phosphatase 2A activity, totally reverses the MAP kinase inactivation induced by long‐term treatment with 10 nM EGF. Additionally, we have shown that the protein synthesis inhibitor cycloheximide fails to affect the EGF‐induced MAP kinase regulation, indicating that mitogen‐induced protein phosphatases are not, or are only slightly, required in this regulation. In conclusion, our data demonstrate that the ambivalent action of EGF on the proliferation of A431 cells is associated with differential mechanisms of p42 MAP kinase regulation catalysed by the serine/threonine phosphatase 2A.
A Novel Dystrophin/Utrophin‐Associated Protein is an Enzymatically Inactive Member of the Phosphoglucomutase Superfamily
Authors: Moiseeva Elena P. , Belkin Alexey M. , Spurr Nigel K. , Koteliansky Victor E. , Critchley David R.
Published: August 2004

A 60‐kDa protein localised in adherens‐type cellular junctions, and previously called aciculin, has been found to interact with the cytoskeletal proteins dystrophin and utrophin [Belkin, A. M. & Burridge, K. (1995) J. Biol. Chem. 270, 6328–6337]. In this study, we report the complete sequence of this protein, and show that it is a novel member of the phosphoglucomutase (PGM) family of proteins. The PGM‐related protein (PGM‐RP), which contains 506 amino acids (55.6 kDa), is smaller than PGM1 (566 amino acids, 61 kDa). The active site consensus sequences of prokaryotic and eukaryotic mutases are not conserved in PGM‐RP, a finding consistent with the lack of enzymatic activity of PGM‐RP in vitro, and the absence of a phosphorylated intermediate in vivo. The organisation of the PGM‐RP gene is essentially identical to that of PGM1. We propose that the PGM‐RP gene, which we have mapped to human chromosome 9qcen‐q13, evolved from the PGM1 gene, and encodes a protein with a structural rather than an enzymatic role. PGM‐RP is expressed predominantly in muscle with the highest levels in smooth muscle. The significance of the interaction between dystrophin/utrophin and an increasing number of cytoplasmic proteins including PGM‐RP remains to be explored.
Biochemical Characterization of Purified ζ‐Carotene Desaturase from Anabaena PCC 7120 after Expression in Escherichia coli
Authors: Albrecht Manuela , Linden Hartmut , Sandmann Gerhard
Published: August 2004

A novel enzyme, ζ‐carotene desaturase from the cyanobacterium Anabaena, which catalyzes the last two steps in a series of desaturations, was overexpressed in Escherichia coli. For the first time, this allowed the purification of this enzyme and subsequent enzyme kinetic studies. The enzyme was solubilized from the E. coli membranes by Chaps and purified to homogeneity by ammonium sulfate precipitation, ion‐exchange and hydrophobic interaction chromatography. The correct translational start was confirmed by N‐terminal protein sequencing. Substrates for ζ‐carotene desaturase apart from ζ‐carotene are those carotenes which partially resemble the latter, like neurosporene and β‐zeacarotene yielding lycopene and γ‐carotene, respectively as reaction products. Also cis isomers like pro‐ζ‐carotene were converted to the correspondiong products. Km values of 10 μM were determined for both substrates ζ‐carotene and neurosporene. The enzyme was inhibited to some extent by the experimental herbicides J852 and LS80707 and also by diphenylamine which is a well‐known inhibitor of the bacterial‐type phytoene desaturase.
Kinetic Characterization of the Hydrolytic Activity of the H+‐Pyrophosphatase of Rhodospirillum rubrum in Membrane‐Bound and Isolated States
Authors: Baykov Alexander A. , Sergina Natalja V. , Evtushenko Olga A. , Dubnova Elena B.
Published: August 2004

Substrate hydrolysis by the H+‐pyrophosphatase (pyrophosphate phosphohydrolase, H+‐PPase) of the photosynthetic bacterium Rhodospirillum rubrum follows a two‐pathway reaction scheme in which preformed 1:1 and 1:2 enzyme · Mg2+ complexes (EMg and EMg2) convert dimagnesium pyrophosphate (the substrate). This scheme is applicable to isolated enzyme, uncoupled chromatophores and chromatophores energized by a K+/valinomycin diffusion potential. Tris and other amine buffers exert a specific effect on the bacterial H+‐PPase by increasing the Michaelis constant for substrate binding to EMg by a factor of 26–32, while having only small effect on substrate binding to EMg2. Formation of EMg requires a basic group with pKa of 7.2–7.7 and confers resistance against inactivation by mersalyl and N ‐ethylmaleimide to H+‐PPase. The dissociation constants governing EMg and EMg2 formation, as estimated from the mersalyl‐protection assays and steady‐state kinetics of PPi hydrolysis, respectively, differ by an order of magnitude. Comparison with the data on soluble PPases suggests that, in spite of gross structural differences between H+‐PPase and soluble PPases and the added ability of H+‐PPase to act as a proton pump, the two classes of enzyme utilize the same reaction mechanism in PPi hydrolysis.
Cloning and Sequencing of a cDNA Encoding a Larval‐Pupal‐Specific Cuticular Protein in Tenebrio Molitor (Insecta, Coleoptera)
Authors: Rondot Isabelle , Quennedey Brigitte , Courrent Annie , Lemoine Aleth , Delachambre Jean
Published: August 2004

A cDNA clone encoding a larval‐pupal cuticular protein, named TMLPCP‐22, has been isolated by screening a library in expression vector with a monoclonal antibody made against pupal cuticular proteins of Tenehrio molitor. Northern‐blot and in situ hybridization analyses showed that the expression of TMLPCP‐22 is regulated in a stage‐specific and tissue‐specific manner; the transcript was present during the secretion of preecdysial larval and pupal cuticles and was restricted to epidermal cells. No expression was observed during adult cuticle deposition. In supernumerary pupae obtained after application of a juvenile hormone analogue, which is known to inhibit the adult programme, TMLPCP‐22 mRNA was expressed again, confirming its larval‐pupal specificity.
Photoaffinity Labeling of Elongation Factor‐2 with 8‐Azido Derivatives of GTP and ATP
Authors: Guillot Dominique , Yard Cécile , Reboud Jean‐Paul
Published: August 2004

Elongation factor 2 (eEF‐2) can interact not only with guanylic nucleotides but also with adenylic ones, as was shown by intrinsic fluorescence quenching studies [Sontag, B., Reboud, A. M., Divita, G., Di Pietro, A., Guillot, D. & Reboud, J. P. (1993) Biochemistry 32, 1976–1980]. Here we studied sites of these interactions by using photoactivable 8‐azido‐[γ‐32P]GTP and 8‐azido‐[γ‐32P]ATP. Photoincorporation of the radioactive GTP derivative into eEF‐2 was prevented by the previous addition of GTP and GDP. The addition of adenylic nucleotides (ATP, ADP) and some adenylic derivatives [NAD+, NADH, poly(A)] decreased the photoincorporation by only 40% at most. However, photoincorporation of the radioactive ATP derivative was prevented by the previous addition not only of adenylic compounds [ATP, ADP, NAD+, NADH, poly(A)] but also of GTP and GDP. Photoincorporation of radioactive nucleotide derivatives was not decreased by the addition of other nucleotidic compounds [UTP, poly(U), ITP, NADP+, NADPH]. ATP and GTP acted as non‐competitive inhibitors of the photoincorporation of 8‐azido‐[γ‐32P]GTP and 8‐azido‐[γ‐32P]ATP respectively. eEF‐2 photolabeled with these radioactive nucleotide derivatives was submitted to trypsin digestion under different conditions and the labeled peptidic fragments identified after HPLC purification and gel electrophoresis by N‐terminal sequencing. An octa‐peptide, Y264FDPANGK271, was the only peptide photolabeled with 8‐azido‐[γ‐32P]GTP whereas a N‐terminal fragment of about 7 kDa was the only one photolabeled with 8‐azido‐(γ‐32P]ATP. The different results support the hypothesis that guanylic and adenylic nucleotides do not interact with the same site of eEF‐2.
Identification of a Functional cAMP Response Element in the Secretogranin II Gene
Authors: Cibelli Giuseppe , Jüngling Susanne , Schoch Susanne , Gerdes Hans‐Hermann , Thiel Gerald
Published: August 2004

Secretogranin II is an acidic secretory protein with a widespread distribution in secretory granules of neuronal and endocrine cells. The secretogranin II gene contains, like other members of the granin family, a cAMP response element (CRE) in its upstream region. To investigate the functional significance of this motif, intracellular cAMP levels were increased in a neuronal cell line derived from the septal region of the brain and the level of secretogranin II gene expression was analysed. It was found that increased cAMP levels did, in fact, induce secretogranin II gene expression. To analyse the cis‐acting sequence responsible for this induction, a hybrid gene containing the upstream region of the mouse secretogranin II gene fused to β‐globin as a reporter was constructed. Transfection analysis revealed that cAMP‐induced transcription of the secretogranin II promoter/β‐globin gene in septal and insulinoma cells. DNA‐protein binding assays showed that recombinant CRE‐binding protein (CREB), produced in bacteria or human cells, bound in a sequence‐specific manner to the secretogranin II promoter CRE. Moreover, deletion mutagenesis revealed that the CRE motif is a bifunctional genetic regulatory element in that it mediates basal as well as cAMP‐stimulated transcription. Interestingly, cAMP had no effect upon secretogranin II gene transcription in PC12 and neuroblastoma cells. An increase in the intracellular cAMP concentration activated a GAL4–CREB fusion protein upon transcription in neuroblastoma cells indicating the integrity of the cAMP signaling pathway to the nucleus. Basal as well as c AMP‐stimulated transcription, directed from the secretogranin II promoter was, however, impaired in insulinoma cells by overexpression of CREB‐2, a negative‐acting CRE‐binding protein. These results indicate that competitive effects are likely to occur between CRE‐bound transcriptional activators and repressers. We conclude that cAMP‐stimulated induction of secretogranin II gene transcription is mediated by the CRE motif in a cell‐type‐specific manner, and is likely to depend on the balance between positive and negative CRE‐binding proteins in a particular cell type.
Fluorescence‐Energy Transfer in Human Estradiol 17β‐Dehydrogenase‐NADPH Complex and Studies on the Coenzyme Binding
Authors: Li Bing , Lin Sheng‐Xiang
Published: August 2004

Fluorescence spectroscopy was used to examine the interaction between human estradiol 17β‐dehydrogenase (estrogenic 17β‐hydroxysteroid dehydrogenase, 17β‐HSD) and the cofactor NADPH. After the binding of NADPH to the enzyme, there was an emission enhancement at 436 nm following an excitation at 295 nm, as compared to the cofactor alone. This phenomenon was attributed to a radiationless transfer of excitation energy from 17β‐HSD to the enzyme‐bound cofactor. The distance of 2.69 nm, between the bound NADPH and the sole tryptophan residue (Trp46) within one subunit, has been determined using fluorescence energy transfer. This result coincides very well with the same distance, recently calculated from the crystallographic coordinates obtained by Ghosh et al. [Ghosh, D., Pletnev, V. Z., Zhu, D.‐W., Wawrzak, Z., Duax, W. L., Pangborn, W., Labrie, F. & Lin, S.‐X. (1995) Structure 3, 503–513]. Compared to free NADPH, the fluorescence emission of enzyme‐bound NADPH was increased in intensity and its maximum blue‐shifted from 457 nm to 436 nm. Binding of NADPH to 17β‐HSD was studied by fluorescence titration. The enzyme binds two molecules of NADPH with a Kd= 0.73±0.2 μM. The dissociation constant was further confirmed by the method of coenzyme protection against cold inactivation of the enzyme. The binding was little altered in the presence of estradiol‐17β. The environment of tryptophan residues on the surface of the enzyme is discussed.
Influence of the Signal Sequence and Chaperone SecB on the Interaction between Precursor Protein prePhoE and Phospholipids
Authors: Van Raalte Anne L. J. , Demel Rudy A. , Verberkmoes Geerten , Breukink Eefjan , Keller Rob C. A. , De Kruijff Ben
Published: August 2004

To investigate in a direct way the interaction between a precursor protein and phospholipids, monolayer studies were performed using the purified precursor of Escherichia coli outer‐membrane protein PhoE. It was demonstrated that prePhoE can insert efficiently into monolayers of dioleoylglycerophosphoglycerol (Ole2GroP Gro) and dioleoylglycerophosphoethanolamine (Ole2GroPEtn), this insertion was mainly driven by hydrophobic forces. Compared with previous results obtained with PhoE signal peptide, the full‐length precursor protein does not show the specific interaction with acidic lipids. PrePhoE inserted into a Ole2GroP Gro monolayer occupies an area of 28±30 nm2/molecule, which is approximately 10‐fold larger than the area occupied by the PhoE signal peptide. The purified mature PhoE protein has a lower capacity to insert into Ole2GroP Gro and Ole2GroPEtn monolayers and is, in contrast to prePhoE, fully accessible to proteinase K after interacting with a Ole2GroPGro monolayer. The results demonstrate that in the context of the precursor protein both the signal sequence and mature domain of prePhoE insert into lipid monolayers.

It was found that PhoE, like prePhoE, can form in vitro a complex with the cytosolic chaperone SecB. Complexation with SecB increases the insertion of (pre)PhoE into acidic lipid monolayers. The high lipid affinity of prePhoE was also demonstrated by vesicle‐binding experiments which showed that SecB dissociates from the SecB‐prePhoE complex upon binding of the precursor to the bilayer. The implications of these findings for preprotein translocation are discussed and in addition some extrapolations to the insertion of PhoE into the outer membrane are made.
Induction of the Glucokinase Gene by Insulin in Cultured Neonatal Rat Hepatocytes
Authors: Parsa Ramine , Decaux Jean‐François , Bossard Pascale , Robey Brooks R. , Magnuson Mark A. , Granner Daryl K. , Girard Jean
Published: August 2004

Previous, in vivo experiments have shown that an appropriate hormonal environment (high plasma insulin, low plasma glucagon) was unable to induce the accumulation of glucokinase mRNA in term fetal rat liver, whereas it was very efficient in the newly born rat. We have confirmed in the present study that insulin induced the accumulation of glucokinase mRNA in cultured hepatocytes from 1‐day‐old newborn rats, but not in cultured hepatocytes from 21‐day‐old fetuses. To identify regulatory regions of the glucokinase gene involved in the insulin response, we have scanned the glucokinase locus for DNase I hypersensitive sites in its in vivo conformation. We confirmed the presence of four liver‐specific DNase I hypersensitive sites located in the 5′ flanking region of the gene. Moreover, two additional hypersensitive sites, located at 2.5 kb and 3.5 kb upstream of the cap site were found but none of these new sites displayed inducibility by insulin. Finally, an increase of the sensitivity of hypersensitive site‐1 and hypersensitive site‐2 to DNase I correlates with the ability of insulin to induce glucokinase gene expression in cultured hepatocytes from 1‐day‐old rats, as observed in previous in vivo studies. This suggests that neither a prior exposure to insulin nor a simple aging of the fetal cells in the presence of the hormone in culture are instrumental for the full DNase‐I hypersensitivity of the two proximal sites necessary for the neonatal response of the glucokinase gene to insulin. The proximal hypersensitive site‐1, which is close to the transcription start site in the liver, does coincide with a sequence (designated IRSL) that is 80% identical to the phosphoenolpyruvate carboxykinase IRS and with a DNase‐I footprint that has been identified overlapping this sequence. Nevertheless, functional analysis of this sequence suggested that it is unlikely that the insulin‐response sequence like alone is sufficient to mediate the transcriptional effect of insulin on the hepatic glucokinase gene.
Glucose Uptake Occurs by Facilitated Diffusion in Procyclic forms of Trypanosoma brucei
Authors: Wille Ulrike , Seyfang Andreas , Duszenko Michael
Published: August 2004

The glucose transporter of Trypanosoma brucei procyclic forms was characterized and compared with its bloodstream form counterpart. Measuring the glucose consumption enzymatically, we determined a saturable uptake process of relatively high affinity (Km= 80 μM, Vmax= 4 nmol min−1 10−8 cells), which showed substrate inhibition at glucose concentrations above 1.5 mM (Ki= 21 mM). Control experiments measuring deoxy‐d‐[3H]Glc uptake under zero‐trans conditions indicated that substrate inhibition occurred on the level of glycolysis. Temperature‐dependent kinetics revealed a temperature quotient of Q10= 2.33 and an activation energy of Ea= 64 kJ mol−1. As shown by trans‐stimulation experiments, glucose uptake was stereospecific for the D isomer, whereas l‐glucose was not recognized. Inhibitor studies using either the uncoupler carbonylcyanide‐4‐(trifluoromethoxy)phenylhydrazone (5μM), the H+/ATPase inhibitor N,N′ ‐dicyclohexylcarbodiimide (20 μM), the ionophor monensin (1 μM), or the Na+/K+‐ATPase inhibitor ouabain (1 mM) showed insignificant effects on transport efficiency. The procyclic glucose transporter was subsequently enriched in a plasma‐membrane fraction and functionally reconstituted into proteoliposomes. Using Na+‐free conditions in the absence of a proton gradient, the specific activity of D‐[14C]glucose transport was determined as 2.9 nmol min−1 (mg protein)−1 at 0.2 mM glucose. From these cumulative results, we conclude that glucose uptake by the procyclic insect form of the parasite occurs by facilitated diffusion, similar to the hexose‐transport system expressed in bloodstream forms. However, the markedly higher substrate affinity indicates a differential expression of different transporter isoforms throughout the lifecycle.
Cloning, Sequence Analysis and Overexpression of the Rhodanese Gene of Azotobacter vinelandii
Authors: Colnaghi Rita , Pagani Silvia , Kennedy Christina , Drummond Martin
Published: August 2004

A gene encoding rhodanese (rhdA) was cloned from Azotobacter vinelandii on a 2.3‐kb SphI fragment. This fragment was identified by its hybridization to a PCR product obtained by amplification of genomic DNA using degenerate primers encoding the N‐terminal sequence of rhodanese purified from A. vinelandii. The sequence of a 1.2‐kb region revealed an 813‐bp open reading frame that encoded a polypeptide of 271 amino acids, the N‐terminal sequence of which was identical to that of A. vinelandii rhodanese. In a search of database entries, eukaryotic rhodaneses and rhodanese‐like proteins from bacteria gave the highest scores of identity (27–30%) with the predicted product of the 813‐bp open reading frame. A. vinelandii RhdA shows less sequence similarity to vertebrate rhodaneses than it does to prokaryotic rhodanese‐like proteins which did not show typical rhodanese activity. Basic residues thought to be catalytically important in bovine rhodanese are not conserved in A. vinelandii rhodanese. The sequence similarity between the two structurally similar domains of rhodanese is more pronounced for the A. vinelandii enzyme than the bovine enzyme, and supports the hypothesis that the complete structure was originally generated by gene duplication. When rhdA was overexpressed in Escherichia coli, rhodanese represented 30% of total cell protein and thiosulfate:cyanide sulfurtransferase activity increased >600 fold in cell‐free extracts. A. vinelandii rhdA insertion/deletion mutants had no discernible phenotype distinct from the wild‐type strain with respect to growth on various sulfur sources or nitrogenase activity. Mutants retained 20% of wild‐type rhodanese thiosulfate: cyanide sulfurtransferase activity suggesting the presence of redundant sulfurtransferase enzymes in A. vinelandii.