bluuebunny/crossref_metadata_2025 huggingface dataset:
A systematic approach for selecting focal species for conservation in the forests of Nova Scotia and Maine
Authors: BEAZLEY KAREN , CARDINAL NATHAN
Published: July 2004

Focal species are a critical component of conservation planning, along with representation of ecosystems, special elements and ecologically sustainable management. They warrant conservation attention because they are functionally important, wide-ranging or space-demanding, habitat-quality indicators, ‘flagship’, and/or vulnerable or special populations. A delphi survey matrix-based approach, involving regional experts in the selection of potential focal species, was applied in Nova Scotia, Canada and Maine, USA. Matrices with native species on one axis and selection characteristics on the other axis were used to summarize expert knowledge and judgment. Characteristics were related to biological traits and habitat requirements that make some species more vulnerable than others in human-modified landscapes. In Nova Scotia and Maine, 19 and 11 experts, respectively, completed the matrices, which were subsequently numerically assessed. Species with the highest scores were identified as potential focal species, including wolf, cougar, lynx, river otter, eastern pipistrelle, wood turtle, four-toed salamander, golden eagle and Atlantic salmon. Concerns remain around the lack of representation of some classes of species, subjectivity in selecting and weighting characteristics, and the relative nature of assessing species against the characteristics. Accordingly, potential focal species should be subject to verification through more rigorous and quantitative analysis and monitoring. Nonetheless, if applied with care, the matrix-based approach can provide a relatively systematic and effective way of engaging regional experts in focal species selection.
Distribution, diversity, and conservation of coral reefs and coral communities in the largest marine protected area of Pacific Panama (Coiba Island)
Authors: GUZMAN HECTOR M. , GUEVARA CARLOS A. , BREEDY ODALISCA
Published: July 2004

Sampling scale and lack of attention to taxa other than scleractinian corals have limited the capacity to protect coral reefs and coral communities in Pacific Panama. The distribution of coral habitats (live coral cover) and their species richness in the largest marine protected area of Panama, the Coiba National Park (270 125 ha), is described using quadrat transects and manta tows. The species richness of scleractinian corals and octocorals was lower in coral reefs than in coral communities, and a close relationship between richness and live coral cover was observed only in coral communities. The distribution of high live coral cover in coral communities overlapped with areas of high coral species richness. Average live coral cover in communities was 64%, compared to 28% in reefs, whereas algae cover was 30% and 49%, respectively. Twenty-two coral and 34 octocoral species were observed, many only now detected in Panama as endemic or new species. Analysis of satellite imagery showed 80% of terrestrial habitats were mostly primary forest, and coral reefs and coral communities covered 1700 ha, about 2% of marine habitats. Shallow marine environments (< 20 m) had up to 60% calcareous red algae cover (rhodolite beds). Based on the distribution of live coral cover and species richness, three conservation units were identified as priority, with the southern and northernmost sides of the marine protected area as the most significant. These three areas encompass most of the rare and endemic species or populations, as well as species previously regarded as endangered.
THĀBIT IBN QURRA ET LA COMPOSITION DES RAPPORTS
Authors: CROZET PASCAL
Published: July 2004

Dans l'introduction à un texte majeur consacré à la composition des rapports et dans lequel on s'accorde à voir un pas important dans l'élaboration de la notion de nombre irrationnel, al-Khayyām remarque:
PLATO'S REPUBLIC IN ARABIC A NEWLY DISCOVERED PASSAGE
Authors: REISMAN DAVID C.
Published: July 2004

My aim here is to present an editio princeps of a newly discovered Arabic translation of a very important passage from Plato's Republic (Book VI, 506d3–509b10) found in the work entitled Kitāb fī Masā'il al-umūr al-ilāhiyya (hereafter Masā'il), penned by the somewhat obscure Neoplatonist Abū Hāmid al-Isfizārī (fl. mid-fourth / tenth c.). While an edition of al-Isfizārī's work has been published by Daniel Gimaret, the manuscript he used (Ragıp Paşa 1463) lacked the literal translation of the Republic passage. The one other known exemplar of the work, MS Zāhiriyya 4871, dated slightly later than the first, appears to be closer to the author's original unedited version; it contains the Republic passage. I am currently preparing a new edition of the work on the basis of the Zāhiriyya manuscript, but it seemed worthwhile to bring the Republic passage to immediate attention. I do not attempt here a thorough evaluation of al-Isfizārī's philosophy as presented in the Masā'il; this can be done successfully only upon completion of the edition. However, to place the work in its proper context, I do provide a brief overview of the transmission of Plato's Republic in Arabic; a discussion of new information on al-Isfizārī's Nachlass, and a description of the two exemplars of his Masā'il. The edited text of the passage from the Republic is accompanied by a provisional translation.
Phonological convergence in a contracting language variety
Authors: BULLOCK BARBARA E. , GERFEN CHIP
Published: July 2004

Most work investigating the role of convergence in situations of language attrition has focused on the morpho-syntactic restructuring of the dying language variety. A central concern of such research has been untangling the factors driving the restructuring with an eye towards establishing whether the changes observed are best viewed as externally driven or, by contrast, as internally motivated. A second and equally important concern of this research attempts to define the domains of the linguistic system that may be the most permeable to external influence. The present study provides a contribution to this line of research and sheds light on its two leading concerns from the domain of phonology and phonetics. Specifically, we present the results of an instrumental study of the phonological vowel system of Frenchville French and argue that this linguistic variety is undergoing a perceptually striking process of phonetic convergence with English that is motivated by the auditory and acoustic similarity between a subset of vowels in the contact languages. An interesting consequence of our analysis is that bilingual phonologies may become particularly permeable to inter-linguistic influence precisely where they are acoustically and perceptually unstable, and where they are already congruent to some degree.
Subject and object expression in Spanish heritage speakers: A case of morphosyntactic convergence
Authors: MONTRUL SILVINA
Published: July 2004

Many simultaneous bilinguals exhibit loss or incomplete acquisition of their heritage language under conditions of exposure and use of the majority language (Silva-Corvalán, 1994, 2003; Polinsky, 1997; Toribio, 2001; Montrul, 2002). Recent work within discourse-functional (Silva-Corvalán 1994) and generative perspectives (Sorace, 2000; Montrul; 2002; Tsimpli, Sorace, Heycock, Filaci and Bouba, 2003, in press) suggests that while syntax proper is impervious to language loss or attrition, syntax-related interfaces like lexical-semantics and discourse-pragmatics are not. This study investigates argument expression in adult simultaneous bilinguals who are heritage speakers of Spanish, because in this language subjects, direct, and indirect objects are regulated by syntactic, pragmatic and semantic factors. It was hypothesized that if language loss affects interface areas of competence more than the purely syntactic domains, then Spanish heritage speakers should display robust knowledge of null subjects as well as object clitics, but variable behavior in the pragmatic distribution of null vs. overt subjects, the a preposition with animate direct objects, and cases of semantically based dative clitic-doubling. Results of an oral production task administered to 24 intermediate and advanced heritage speakers and 20 monolinguals confirmed the hypotheses. With the erosion of pragmatic and semantic features, the grammars of the intermediate proficiency Spanish heritage speakers appear to display morphosyntactic convergence with English in the expression of subject and object arguments.
Functional convergence in the tense, evidentiality and aspectual systems of Quechua Spanish bilinguals
Authors: SÁNCHEZ LILIANA
Published: July 2004

In this paper, I present an exploratory study on cross-linguistic interference among Quechua-Spanish bilingual children living in a language contact situation. The study focuses on convergence in the tense, aspectual and evidentiality systems of the two languages. While in Quechua past tense features are strongly linked to evidentiality in the matrix of features associated with the functional category Tense, in Spanish, past tense features are linked to aspectual features. The study presents evidence that supports the Functional Convergence Hypothesis according to which syntactic convergence among bilingual speakers is favored when the matrix of features associated with a functional category is partially divergent, as is the case for Tense in Spanish and Quechua. The Spanish results indicate that among bilinguals past tense is associated with evidentiality features and contrast sharply with the results of the monolingual comparison group. Bilingual Quechua results exhibit an incipient emergence of discourse-oriented background and foreground distinctions, similar to those found in Spanish in association with aspectual morphology.
Prosodic change and language contact
Authors: McMAHON APRIL
Published: July 2004

Using evidence from first-hand experimental work and existing studies, Colantoni and Gurlekian take a tentative but encouraging step towards exploring the role of contact in explaining intonational change. Their central question is whether Buenos Aires Spanish intonation is distinctive relative to other varieties of Spanish; and if so, whether that distinctiveness is due to contact with Italian.
Native language attrition and developmental instability at the syntax-discourse interface: Data, interpretations and methods
Authors: SORACE ANTONELLA
Published: July 2004

Montrul's study is an important contribution to a recently emerged research approach to the study of bilingualism and languages in contact, characterized by its sound theoretical basis and its reliance on data from different – and traditionally non-integrated – domains of language development: bilingual first language acquisition (Müller and Hulk, 2001; Paradis and Navarro, 2003; Serratrice, 2004; Serratrice, Sorace and Paoli, in press), adult second language acquisition (Filiaci, 2003; Sorace, 2003), and native language attrition (Gurel, 2002; Tsimpli, Sorace, Heycock and Filiaci, in press). The generalization that is emerging from this approach is that interfaces between syntax and other cognitive systems (i.e. discourse pragmatics, lexical-semantics) exhibit more developmental instability than narrow syntax. For L1 attrition, which is the specific focus of the paper, this means that aspects of grammar at the syntax–discourse interface are more vulnerable to attrition than purely syntactic aspects. The identification of restrictions on the domain of occurrence of attrition is consistent with much previous descriptive research on this topic (e.g. Seliger and Vago, 1991). More recently, the same conclusion has been reached by a study on individual language attrition by Tsimpli et al. (in press), who investigated knowledge of the referential pronominal system in Greek and Italian in very advanced speakers of English. In this paper, Montrul tests the generalization on second-generation speakers of Spanish – or “heritage speakers” – a bilingual group that presents different characteristics from the adult L2 speakers investigated in Tsimpli et al.'s study. In addition to referential subjects, she also focuses on a different interface area of grammar – direct objects – that had not been investigated before. In these respects, Montrul's study is a welcome development. In other respects, however, the data are less than convincing and do not allow a straightforward interpretation. My commentary focuses on three fundamental questions raised not only by this study, but also by this type of research in general. The main focus will be the expression of referential subjects since this aspect of grammar has been investigated in other studies and therefore offers the possibility of direct comparison among results.
Convergence as a mechanism of language change
Authors: BACKUS AD
Published: July 2004

This issue of Bilingualism: Language and Cognition is about convergence, a type of language change that is contact-induced and results in greater similarity between two languages that are in contact with each other. In Backus (forthcoming), I have attempted an overview of contact-induced language change, focusing on causal factors, on mechanisms of change, and on the actual changes. In this conclusion, I will try to give convergence its rightful place in this general typology, referencing the contributions to this volume where appropriate.
Evaluation of selected control strategies for fed‐batch cultures of a hybridoma cell line
Authors: Pörtner Ralf , Schwabe Jan‐Oliver , Frahm Björn
Published: July 2004

While fed‐batch suspension culture of animal cells continues to be of industrial importance for the large‐scale production of pharmaceutical products, existing control concepts are still insufficient. The present paper illustrates the advantages and disadvantages of different fed‐batch strategies, including fixed‐feed trajectories, control via OUR (oxygen uptake rate) (stoichiometric feeding), a priori determination of feed trajectories based on a kinetic model and the model‐based adaptive OLFO (open‐loop‐feedback‐optimal) control strategy. A recommendation as to which control strategy should be used for a specific process has to consider the respective process. For an established process with a well characterized and stable production cell line, probably the application of a fixed feed trajectory should be recommended. An adaptive, model‐based control strategy could be the method of choice during cell‐line development or for rapid production of small amounts of product for clinical trials, owing to its universal character and because it does not require intensive process development.
Behaviour of a recombinant cabbage (Brassica oleracea) phospholipase D immobilized on CNBr‐activated and antibody supports
Authors: Younus Hina , Rajcani Julius , Ulbrich‐Hofmann Renate , Saleemuddin Mohammed
Published: July 2004

Recombinant cabbage (Brassica oleracea) PLD2 (phospholipase D2) immobilized covalently on CNBr‐activated Sepharose expressed low activity (≈10%), while that immobilized by binding on to anti‐PLD2 IgG–Sepharose was more active (≈38%). Coupling of PLD2 to CNBr‐activated Sepharose resulted in significant improvement in storage stability without affecting its thermostability, as compared with the soluble enzyme. Binding of PLD2 to the antibody support, however, rendered the enzyme remarkably labile to high temperatures and storage.
Analytical techniques for the evaluation of liquid protein therapeutics
Authors: Tatford Owen C. , Gomme Peter T. , Bertolini Joseph
Published: July 2004

A common problem in the manufacture of liquid protein therapeutics is the tendency for aggregation and particle formation on extended storage. Analytical techniques are required to study the propensity of solutions to form aggregates and particles and to allow the investigation of the effect of conditions encountered during manufacture and storage. A key challenge is to utilize appropriate specific and sensitive techniques to allow the early detection of initial aggregation events, thereby avoiding the need to resort to extended stability trials. The present review evaluates a range of techniques for the detection of changes in protein conformation and the formation of aggregates and particles. It is hoped that the availability of this information will encourage and facilitate studies to resolve stability issues associated with protein therapeutics.
Human adult craniofacial muscle‐derived cells: neural‐cell adhesion‐molecule (NCAM; CD56)‐expressing cells appear to contain multipotential stem cells
Authors: Sinanan Andrea C. M. , Hunt Nigel P. , Lewis Mark P.
Published: July 2004

Skeletal muscle has been well characterized as a reservoir of myogenic precursors or satellite cells with the potential to participate in cellular repopulation therapies for muscle dysfunction. Recent evidence, however, suggests that the postnatal muscle compartment can be considered an alternative to bone marrow as a source of multipotent cells or muscle‐derived stem cells (MDSCs). MDSCs, when primed with appropriate environmental cues, can differentiate into a variety of non‐muscle cells. The present study describes the application of a new technique for the isolation of adult human myoblasts and putative MDSCs, based on microbead–immunomagnetic selection of CD56+ cells, derived from craniofacial skeletal muscle, and details changes in morphological/molecular phenotype of the purified cells when maintained in either a myogenic or a non‐myogenic milieu. Multiple immunofluorescence microscopy and two‐colour flow‐cytometric analysis of proliferating CD56+ cultures revealed positive staining for myogenic markers (CD56, desmin and M‐cadherin) as well as putative stem‐cell markers [the antigens CD34, CD90 and CD106, and Flk‐1 (fetal liver kinase‐1)/VEGFR‐2 (vascular‐endothelial‐growth‐factor receptor)]. Confluent cultures subjected to cycles of adipogenic or osteogenic induction contained either adipocytes or osteoblasts and myotubes. In conclusion, the CD56+ subpopulation within adult human skeletal muscle is heterogeneous and is composed of both lineage‐committed myogenic cells and multipotent cells (the candidate MDSCs), which are able to form non‐muscle tissue such as fat and bone.
Construction and analyses of hybrid Azotobacter vinelandii mannuronan C-5 epimerases with new epimerization pattern characteristics
Authors: BJERKAN Tonje M. , LILLEHOV Bjørn E. , STRAND Wenche I. , SKJÅK-BRÆK Gudmund , VALLA Svein , ERTESVÅG Helga
Published: July 2004

The secreted mannuronan C-5 epimerases from Azotobacter vinelandii form a family of seven homologous modular type enzymes, which appear to have evolved through duplications and point mutations in the individual modules. The catalytic A modules of these enzymes are responsible for generating the characteristic sequence distribution patterns of G residues in the industrially important polymer alginate by epimerizing M (β-D-mannuronic acid) moieties to G (α-L-guluronic acid). Forty-six different hybrid enzymes were constructed by exchanging parts of the sequences encoding the A modules of AlgE2 (generates consecutive stretches of G residues) and AlgE4 (generates alternating structures). These hybrid enzymes introduce a variety of new monomer-sequence patterns into their substrates, and some regions important for the subsite specificity or processivity of the enzymes were identified. By using time-resolved NMR spectroscopy, it became clear that the rates for introducing alternating structures and consecutive stretches of G residues are different for each enzyme, and that it is the ratio between these rates that determines the overall epimerization pattern. These findings open up new possibilities in biotechnology and in studies of the many biological functions of alginates.
Sphingosine kinase activity is required for sphingosine-mediated phospholipase D activation in C2C12 myoblasts
Authors: MEACCI Elisabetta , CENCETTI Francesca , DONATI Chiara , NUTI Francesca , BECCIOLINI Laura , BRUNI Paola
Published: July 2004

Sphingosine (Sph) has been implicated as a modulator of membrane signal transduction systems and as a regulatory element of cardiac and skeletal muscle physiology, but little information is presently available on its precise mechanism of action. Recent studies have shown that sphingosine 1-phosphate (S1P), generated by the action of sphingosine kinase (SphK) on Sph, also possesses biological activity, acting as an intracellular messenger, as well as an extracellular ligand for specific membrane receptors. At present, however, it is not clear whether the biological effects elicited by Sph are attributable to its conversion into S1P. In the present study, we show that Sph significantly stimulated phospholipase D (PLD) activity in mouse C2C12 myoblasts via a previously unrecognized mechanism that requires the conversion of Sph into S1P and its subsequent action as extracellular ligand. Indeed, Sph-induced activation of PLD was inhibited by N,N-dimethyl-D-erythro-sphingosine (DMS), at concentrations capable of specifically inhibiting SphK. Moreover, the crucial role of SphK-derived S1P in the activation of PLD by Sph was confirmed by the observed potentiated effect of Sph in myoblasts where SphK1 was overexpressed, and the attenuated response in cells transfected with the dominant negative form of SphK1. Notably, the measurement of S1P formation in vivo by employing labelled ATP revealed that cell-associated SphK activity in the extracellular compartment largely contributed to the transformation of Sph into S1P, with the amount of SphK released into the medium being negligible. It will be important to establish whether the mechanism of action identified in the present study is implicated in the multiple biological effects elicited by Sph in muscle cells.
Targeting of Helicobacter pylori vacuolating toxin to lipid raft membrane domains analysed by atomic force microscopy
Authors: GEISSE Nicholas A. , COVER Timothy L. , HENDERSON Robert M. , EDWARDSON J. Michael
Published: July 2004

The Helicobacter pylori vacuolating toxin VacA causes several effects on mammalian cells in vitro, including intracellular vacuolation, formation of pores in the plasma membrane and apoptosis. When added to cells, VacA becomes associated with detergent-resistant membranes, indicating that it binds preferentially to lipid rafts. In the present study, we have used atomic force microscopy to examine directly the association of VacA with lipid domains in supported lipid bilayers. VacA did not bind to lipid bilayers at pH 7.6. In contrast, at pH 4.0, VacA associated with the bilayers in the form of 26-nm oligomeric complexes. VacA bound to bilayers produced from either brain lipids or SM (sphingomyelin) plus cholesterol, each of which lacked detectable lipid domains. Bilayers composed of DOPC (dioleoylphosphatidylcholine), SM and cholesterol contained clearly visible raft-like domains, and VacA preferentially associated with these rafts. VacA bound poorly to raft-like domains in DOPC/SM bilayers, indicating that cholesterol is required for efficient association of VacA with lipid domains. When PS (phosphatidylserine), an anionic phospholipid that does not partition significantly into rafts, was added to the mixture of DOPC, SM and cholesterol, VacA was excluded from the rafts, indicating that it binds more avidly to PS than to the raft components. A typical plasma membrane exhibits pronounced lipid asymmetry, with SM enriched in the outer leaflet and PS in the inner leaflet. Therefore it is probable that the association of VacA with rafts in DOPC/SM/cholesterol bilayers represents a useful model for understanding the interactions of VacA with membranes in vivo.
Slow-tight-binding inhibition of enoyl-acyl carrier protein reductase fromPlasmodium falciparumby triclosan
Authors: KAPOOR Mili , REDDY C. Chandramouli , KRISHNASASTRY M. V. , SUROLIA Namita , SUROLIA Avadhesha
Published: July 2004

Triclosan is a potent inhibitor of FabI (enoyl-ACP reductase, where ACP stands for acyl carrier protein), which catalyses the last step in a sequence of four reactions that is repeated many times with each elongation step in the type II fatty acid biosynthesis pathway. The malarial parasite Plasmodium falciparum also harbours the genes and is capable of synthesizing fatty acids by utilizing the enzymes of type II FAS (fatty acid synthase). The basic differences in the enzymes of type I FAS, present in humans, and type II FAS, present in Plasmodium, make the enzymes of this pathway a good target for antimalarials. The steady-state kinetics revealed time-dependent inhibition of FabI by triclosan, demonstrating that triclosan is a slow-tight-binding inhibitor of FabI. The inhibition followed a rapid equilibrium step to form a reversible enzyme–inhibitor complex (EI) that isomerizes to a second enzyme–inhibitor complex (EI*), which dissociates at a very slow rate. The rate constants for the isomerization of EI to EI* and the dissociation of EI* were 5.49×10−2 and 1×10−4 s−1 respectively. The Ki value for the formation of the EI complex was 53 nM and the overall inhibition constant Ki* was 96 pM. The results match well with the rate constants derived independently from fluorescence analysis of the interaction of FabI and triclosan, as well as those obtained by surface plasmon resonance studies [Kapoor, Mukhi, N. Surolia, Sugunda and A. Surolia (2004) Biochem. J. 381, 725–733].
Cellular effects of deoxynojirimycin analogues: uptake, retention and inhibition of glycosphingolipid biosynthesis
Authors: MELLOR Howard R. , NEVILLE David C. A. , HARVEY David J. , PLATT Frances M. , DWEK Raymond A. , BUTTERS Terry D.
Published: July 2004

Deoxynojirimycin (DNJ) analogues are inhibitors of ceramide glucosyltransferase (CGT), which catalyses the first step in the glucosphingolipid (GSL) biosynthetic pathway. We have synthesized a series of DNJ analogues to study the contribution of N-alk(en)yl side chains (C4, C9 or C18) to the behaviour of these analogues in cultured HL60 cells. When cells were treated for 16 h at non-cytotoxic concentrations of inhibitor, a 40–50% decrease in GSL levels was measured by HPLC analysis of GSL-derived oligosaccharides following ceramide glycanase digestion of GSL and 2-aminobenzamide labelling of the released oligosaccharides. Using a novel technique for short-term [14C]galactose labelling of cellular GSL, we used compound inhibition of GSL biosynthesis as a marker for compound uptake into cells. Surprisingly, the uptake of all three of the DNJ analogues was extremely rapid and was not dependent upon the length of the N-alk(en)yl moiety. Compound uptake occurred in less than 1 min, as shown by the complete inhibition of GSL labelling in cells treated with all the DNJ analogues. Greatly increased cellular retention of N-cis-13-octadecenyl-DNJ was observed relative to the shorter-chain compounds, N-butyl-DNJ and N-nonyl-DNJ, as indicated by complete inhibition of CGT 24 h after removal of inhibitor from the culture medium. The present study further characterizes the properties of N-alk(en)ylated DNJs, and demonstrates that increasing the length of the side chain is a simple way of improving imino sugar retention and therefore inhibitory efficacy for CGT in cultured cells.
Cellular effects of deoxynojirimycin analogues: inhibition of N-linked oligosaccharide processing and generation of free glucosylated oligosaccharides
Authors: MELLOR Howard R. , NEVILLE David C. A. , HARVEY David J. , PLATT Frances M. , DWEK Raymond A. , BUTTERS Terry D.
Published: July 2004

In the accompanying paper [Mellor, Neville, Harvey, Platt, Dwek and Butters (2004) Biochem. J. 381, 861–866] we treated HL60 cells with N-alk(en)yl-deoxynojirimycin (DNJ) compounds to inhibit glucosphingolipid (GSL) biosynthesis and identified a number of non-GSL-derived, small, free oligosaccharides (FOS) most likely produced due to inhibition of the oligosaccharide-processing enzymes α-glucosidases I and II. When HL60 cells were treated with concentrations of N-alk(en)ylated DNJ analogues that inhibited GSL biosynthesis completely, N-butyl- and N-nonyl-DNJ inhibited endoplasmic reticulum (ER) glucosidases I and II, but octadecyl-DNJ did not, probably due to the lack of ER lumen access for this novel, long-chain derivative. Glucosidase inhibition resulted in the appearance of free Glc1–3Man structures, which is evidence of Golgi glycoprotein endomannosidase processing of oligosaccharides with retained glucose residues. Additional large FOS was also detected in cells following a 16 h treatment with N-butyl- and N-nonyl-DNJ. When these FOS structures (>30, including >20 species not present in control cells) were characterized by enzyme digests and MALDI-TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS, all were found to be polymannose-type oligosaccharides, of which the majority were glucosylated and had only one reducing terminal GlcNAc (N-acetylglucosamine) residue (FOS-GlcNAc1), demonstrating a cytosolic location. These results support the proposal that the increase in glucosylated FOS results from enzyme-mediated cytosolic cleavage of oligosaccharides from glycoproteins exported from the ER because of misfolding or excessive retention. Importantly, the present study characterizes the cellular properties of DNJs further and demonstrates that side-chain modifications allow selective inhibition of protein and lipid glycosylation pathways. This represents the most detailed characterization of the FOS structures arising from ER α-glucosidase inhibition to date.
Resistance to UV-induced apoptosis in Chinese-hamster ovary cells overexpressing phosphatidylserine synthases
Authors: YU Anan , McMASTER Christopher R. , BYERS David M. , RIDGWAY Neale D. , COOK Harold W.
Published: July 2004

Externalization of PtdSer (phosphatidylserine) is an important event in signalling removal of apoptotic cells. In contrast with previous work [Yu, Byers, Ridgway, McMaster and Cook (2000) Biochim. Biophys. Acta 1487, 296–308] with U937 cells showing that specific stimulation of PtdSer biosynthesis during apoptosis was caspase dependent, PtdSer biosynthesis in CHO (Chinese-hamster ovary)-K1 increased 2.5-fold during UV-induced apoptosis but was not reversed by a caspase inhibitor, Z-VAD-FMK (benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone). Also, in CHO-K1 cells, stimulation of synthesis was less specific for PtdSer as similar levels of stimulation were observed for sphingomyelin biosynthesis. Involvement of PtdSer synthase isoforms was tested in CHO-K1 cells overexpressing PSS I (PtdSer synthase I) and PSS II. Both types of transformed cells showed resistance to UV-induced apoptosis based on the decreased levels of caspase 3 activation and morphology changes; externalization of PtdSer was reduced with UV treatment even though expression of endogenous scramblase increased slightly. Serine-labelling experiments showed that PSS I- or PSS II-expressing cells had higher basal levels of PtdSer biosynthesis compared with vector control cells. When cells were exposed to UV light to induce apoptosis, PtdSer biosynthesis was further stimulated 1.5- and 2-fold in PSS I- and PSS II-expressing cells respectively compared with UV-treated vector cells. Caspase activation was not required, as Z-VAD-FMK did not change PtdSer synthesis. Although enhanced PtdSer synthesis was supposed to facilitate apoptosis, cells overexpressing PSS I and II were actually resistant to UV-induced apoptosis. Whereas enhanced PtdSer synthesis was associated with apoptosis, potential anti-apoptotic effects were observed when excess activity of these synthetic enzymes was present. This suggests a tightly regulated role for PtdSer synthesis and/or an important dependence on compartmentation of PSS enzymes in association with scramblase facilitated enrichment of this phospholipid at the cell surface.
Transferrin receptor 2 protein is not expressed in normal erythroid cells
Authors: CALZOLARI Alessia , DEAGLIO Silvia , SPOSI Nadia Maria , PETRUCCI Eleonora , MORSILLI Ornella , GABBIANELLI Marco , MALAVASI Fabio , PESCHLE Cesare , TESTA Ugo
Published: July 2004

Human TFR2 (transferrin receptor 2) is a membrane-bound protein homologous with TFR1. High levels of TFR2 mRNA were found mainly in the liver and, to a lesser extent, in erythroid precursors. However, although the presence of the TFR2 protein in hepatic cells has been confirmed in several studies, evidence is lacking about the presence of the TFR2 protein in normal erythroid cells. Using two anti-TFR2 monoclonal antibodies, G/14C2 and G/14E8, we have provided evidence that TFR2 protein is not expressed in normal erythroid cells at any stage of differentiation, from undifferentiated CD34+ cells to mature orthochromatic erythroblasts. In contrast, erythroleukaemic cells (K562 cells) exhibited a high level of expression of TFR2 at both the mRNA and the protein level. We can therefore conclude that an elevated expression of TFR2 protein is observed in leukaemic cells, but not in normal erythroblasts. The implications of this observation for the understanding of the phenotypic features of haemochromatosis due to mutation of the TFR2 gene are discussed.
Mutational analysis of the triclosan-binding region of enoyl-ACP (acyl-carrier protein) reductase from Plasmodium falciparum
Authors: KAPOOR Mili , GOPALAKRISHNAPAI Jayashree , SUROLIA Namita , SUROLIA Avadhesha
Published: July 2004

Triclosan, a known antibacterial, acts by inhibiting enoyl-ACP (acyl-carrier protein) reductase (ENR), a key enzyme of the type II fatty acid synthesis (FAS) system. Plasmodium falciparum, the human malaria-causing parasite, harbours the type II FAS; in contrast, its human host utilizes type I FAS. Due to this striking difference, ENR has emerged as an important target for the development of new antimalarials. Modelling studies, and the crystal structure of P. falciparum ENR, have highlighted the features of ternary complex formation between the enzyme, triclosan and NAD+ [Suguna, A. Surolia and N. Surolia (2001) Biochem. Biophys. Res. Commun. 283, 224–228; Perozzo, Kuo, Sidhu, Valiyaveettil, Bittman, Jacobs, Fidock, and Sacchettini (2002) J. Biol. Chem. 277, 13106–13114; and Swarnamukhi, Kapoor, N. Surolia, A. Surolia and Suguna (2003) PDB1UH5]. To address the issue of the importance of the residues involved in strong specific and stoichiometric binding of triclosan to P. falciparum ENR, we mutated the following residues: Ala-217, Asn-218, Met-281, and Phe-368. The affinity of all the mutants was reduced for triclosan as compared with the wild-type enzyme to different extents. The most significant mutation was A217V, which led to a greater than 7000-fold decrease in the binding affinity for triclosan as compared with wild-type PfENR. A217G showed only 10-fold reduction in the binding affinity. Thus, these studies point out significant differences in the triclosan-binding region of the P. falciparum enzyme from those of its bacterial counterparts.
Identification and biophysical characterization of a very-long-chain-fatty-acid-substituted phosphatidylinositol in yeast subcellular membranes
Authors: SCHNEITER Roger , BRÜGGER Britta , AMANN Clare M. , PRESTWICH Glenn D. , EPAND Raquel F. , ZELLNIG Günther , WIELAND Felix T. , EPAND Richard M.
Published: July 2004

Morphological analysis of a conditional yeast mutant in acetyl-CoA carboxylase acc1ts/mtr7, the rate-limiting enzyme of fatty acid synthesis, suggested that the synthesis of C26 VLCFAs (very-long-chain fatty acids) is important for maintaining the structure and function of the nuclear membrane. To characterize this C26-dependent pathway in more detail, we have now examined cells that are blocked in pathways that require C26. In yeast, ceramide synthesis and remodelling of GPI (glycosylphosphatidylinositol)-anchors are two pathways that incorporate C26 into lipids. Conditional mutants blocked in either ceramide synthesis or the synthesis of GPI anchors do not display the characteristic alterations of the nuclear envelope observed in acc1ts, indicating that the synthesis of another C26-containing lipid may be affected in acc1ts mutant cells. Lipid analysis of isolated nuclear membranes revealed the presence of a novel C26-substituted PI (phosphatidylinositol). This C26-PI accounts for approx. 1% of all the PI species, and is present in both the nuclear and the plasma membrane. Remarkably, this C26-PI is the only C26-containing glycerophospholipid that is detectable in wild-type yeast, and the C26-substitution is highly specific for the sn-1 position of the glycerol backbone. To characterize the biophysical properties of this lipid, it was chemically synthesized. In contrast to PIs with normal long-chain fatty acids (C16 or C18), the C26-PI greatly reduced the bilayer to hexagonal phase transition of liposomes composed of 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (DEPE). The biophysical properties of this lipid are thus consistent with a possible role in stabilizing highly curved membrane domains.
Phosphorylation of septin 3 on Ser-91 by cGMP-dependent protein kinase-I in nerve terminals
Authors: XUE Jing , MILBURN Peter J. , HANNA Bernadette T. , GRAHAM Mark E. , ROSTAS John A. P. , ROBINSON Phillip J.
Published: July 2004

The septins are a family of GTPase enzymes required for cytokinesis and play a role in exocytosis. Among the ten vertebrate septins, Sept5 (CDCrel-1) and Sept3 (G-septin) are primarily concentrated in the brain, wherein Sept3 is a substrate for PKG-I (cGMP-dependent protein kinase-I) in nerve terminals. There are two motifs for potential PKG-I phosphorylation in Sept3, Thr-55 and Ser-91, but phosphoamino acid analysis revealed that the primary site is a serine. Derivatization of phosphoserine to S-propylcysteine followed by N-terminal sequence analysis revealed Ser-91 as a major phosphorylation site. Tandem MS revealed a single phosphorylation site at Ser-91. Substitution of Ser-91 with Ala in a synthetic peptide abolished phosphorylation. Mutation of Ser-91 to Ala in recombinant Sept3 also abolished PKG phosphorylation, confirming that Ser-91 is the major site in vitro. Antibodies raised against a peptide containing phospho-Ser-91 detected phospho-Sept3 only in the cytosol of nerve terminals, whereas Sept3 was located in a peripheral membrane extract. Therefore Sept3 is phosphorylated on Ser-91 in nerve terminals and its phosphorylation may contribute to the regulation of its subcellular localization in neurons.
Silica transport in the demosponge Suberites domuncula: fluorescence emission analysis using the PDMPO probe and cloning of a potential transporter
Authors: SCHRÖDER Heinz-C. , PEROVIĆ-OTTSTADT Sanja , ROTHENBERGER Matthias , WIENS Matthias , SCHWERTNER Heiko , BATEL Renato , KORZHEV Michael , MÜLLER Isabel M. , MÜLLER Werner E. G.
Published: July 2004

Silicon is, besides oxygen, the most abundant element on earth. Only two taxa use this element as a major constituent of their skeleton, namely sponges (phylum Porifera) and unicellular diatoms. Results from combined cytobiological and molecularbiological techniques suggest that, in the demosponge Suberites domuncula, silicic acid is taken up by a transporter. Incubation of cells with the fluorescent silica tracer PDMPO [2-(4-pyridyl)-5-{[4-(2-dimethylaminoethylaminocarbamoyl)methoxy]phenyl}-oxazole] showed a response to silicic acid by an increase in fluorescence; this process is temperature-dependent and can be blocked by DIDS (4,4-di-isothiocyanatostilbene-2,2-disulphonic acid). The putative NBC (Na+/HCO3−) transporter was identified, cloned and analysed. The deduced protein comprises all signatures characteristic of those molecules, and phylogenetic analysis also classifies it to the NBC transporter family. This cDNA was used to demonstrate that the expression of the gene is strongly up-regulated after treatment of cells with silicic acid. In situ hybridization demonstrated that the expression of the sponge transporter occurs in those cells that are located adjacent to the spicules (the skeletal element of the animal) or in areas in which spicule formation occurs. We conclude that this transporter is involved in silica uptake and have therefore termed it the NBCSA {Na+/HCO3−[Si(OH)4]} co-transporter.
Hypoxia-inducible factor-1 (HIF-1) promotes its degradation by induction of HIF-α-prolyl-4-hydroxylases
Authors: MARXSEN Jan H. , STENGEL Petra , DOEGE Kathrin , HEIKKINEN Pekka , JOKILEHTO Terhi , WAGNER Thomas , JELKMANN Wolfgang , JAAKKOLA Panu , METZEN Eric
Published: July 2004

An important regulator involved in oxygen-dependent gene expression is the transcription factor HIF (hypoxia-inducible factor), which is composed of an oxygen-sensitive α-subunit (HIF-1α or HIF-2α) and a constitutively expressed β-subunit. In normoxia, HIF-1α is destabilized by post-translational hydroxylation of Pro-564 and Pro-402 by a family of oxygen-sensitive dioxygenases. The three HIF-modifying human enzymes have been termed prolyl hydroxylase domain containing proteins (PHD1, PHD2 and PHD3). Prolyl hydroxylation leads to pVHL (von-Hippel-Lindau protein)-dependent ubiquitination and rapid proteasomal degradation of HIF-1α. In the present study, we report that human PHD2 and PHD3 are induced by hypoxia in primary and transformed cell lines. In the human osteosarcoma cell line, U2OS, selective suppression of HIF-1α expression by RNA interference resulted in a complete loss of hypoxic induction of PHD2 and PHD3. Induction of PHD2 by hypoxia was lost in pVHL-deficient RCC4 cells. These results suggest that hypoxic induction of PHD2 and PHD3 is critically dependent on HIF-α. Using a VHL capture assay, we demonstrate that HIF-α prolyl-4-hydroxylase capacity of cytoplasmic and nuclear protein extracts was enhanced by prolonged exposure to hypoxia. Degradation of HIF-1α after reoxygenation was accelerated, which demonstrates functional relevance of the present results. We propose a direct, negative regulatory mechanism, which limits accumulation of HIF-1α in hypoxia and leads to accelerated degradation on reoxygenation after long-term hypoxia.
The yeast peroxisomal adenine nucleotide transporter: characterization of two transport modes and involvement in ΔpH formation across peroxisomal membranes
Authors: LASORSA Francesco M. , SCARCIA Pasquale , ERDMANN Ralf , PALMIERI Ferdinando , ROTTENSTEINER Hanspeter , PALMIERI Luigi
Published: July 2004

The yeast peroxisomal adenine nucleotide carrier, Ant1p, was shown to catalyse unidirectional transport in addition to exchange of substrates. In both transport modes, proton movement occurs. Nucleotide hetero-exchange is H+-compensated and electroneutral. Furthermore, microscopic fluorescence imaging of a pH-sensitive green fluorescent protein targeted to peroxisomes shows that Ant1p is involved in the formation of a ΔpH across the peroxisomal membrane, acidic inside.
That zincing feeling: the effects of EDTA on the behaviour of zinc-binding transcriptional regulators
Authors: NYBORG Jennifer K. , PEERSEN Olve B.
Published: July 2004

Zinc-binding proteins account for nearly half of the transcription regulatory proteins in the human genome and are the most abundant class of proteins in the human proteome. The zinc-binding transcriptional regulatory proteins utilize Zn2+ to fold structural domains that participate in intermolecular interactions. A study by Matt et al. in this issue of the Biochemical Journal has examined the transcription factor binding properties of the zinc-binding module C/H1 (cysteine/histidine-rich region 1) found in the transcriptional co-activator proteins CBP (CREB-binding protein) and p300. Their studies revealed that EDTA treatment of native C/H1 leads to irreversible denaturation and aggregation. Of particular concern is their finding that unfolded C/H1 participates in non-specific protein–protein interactions. The implications of these results are significant. EDTA is a very potent zinc-chelating agent that is used ubiquitously in protein interaction studies and in molecular biology in general. The potentially detrimental effects of EDTA on the structure and interactions of zinc-binding proteins should be taken into account in the interpretation of a sizeable number of published studies and must be considered in future experiments.
Insulin increases glycolysis without further vasodilation in porcine coronary arteries exposed to hypoxia
Authors: FRØBERT Ole , BAGGER Jens P. , SIMONSEN Ulf , LUND Sten , GRAVHOLT Claus H.
Published: July 2004

In acute ischaemia, glucose–insulin–potassium administration reduces mortality and β-adrenoceptor antagonists have favourable effects on the outcome of ischaemic heart disease. The present study was designed to investigate whether insulin (1.4×10−7 M) and the β-adrenoceptor antagonist, propranolol (10−5 M), increase hypoxic vasodilation in correspondence with changes in glycolysis. Porcine coronary arteries, precontracted with 10−5 M prostaglandin F2α, were mounted in a pressure myograph and a microdialysis catheter was inserted in the tunica media. Hypoxic vasodilation, interstitial lactate/pyruvate ratio and interstitial glucose were measured at low (2 mM) and high (20 mM) glucose concentrations. Hypoxia (60 min) caused vasodilation and doubled the lactate/pyruvate ratio. Treatment with insulin quadrupled the lactate/pyruvate ratio during hypoxia, but did not change hypoxic vasodilation. Propranolol blocked isoprenaline-evoked vasodilation, but hypoxic increases in lactate/pyruvate ratio and vasodilation did not change. The combination of insulin and propranolol did not cause further changes compared with each drug added alone, although the combination increased vasoconstriction during reoxygenation. Interstitial glucose fell during hypoxia at an organ bath glucose concentration of 2 mM, and rose at a glucose concentration of 20 mM. Addition of insulin and propranolol alone or in combination had no effect on interstitial glucose concentration. Accordingly, arteries were found to contain only minute amounts of the glucose transporter isoform GLUT4. Our findings suggest that insulin increases arterial glycolysis, but treatment with insulin, propranolol, or both, is not associated with enhanced coronary vasodilation during hypoxia.
Muscle afferent inputs to cardiovascular control during isometric exercise vary with muscle group in patients with chronic heart failure
Authors: CARRINGTON Charlotte A. , FISHER James P. , DAVIES Mick K. , WHITE Michael J.
Published: July 2004

It is not known whether the contribution of the muscle metaboreflex to the cardiovascular response to isometric exercise varies between different muscles in patients with CHF (chronic heart failure) or whether this depends upon muscle fibre type and training status. To resolve these issues BP (blood pressure) and HR (heart rate) responses were recorded in seven stable CHF patients (ejection fraction 30–40%; age 67±3 years) and in six healthy AMA (age-matched active) subjects. The experimental protocol consisted of 2 min of ischaemic isometric exercise at 30% maximum voluntary force, performed in separate trials by the calf plantar flexors (CALF) and handgrip muscles (FOREARM). To isolate the muscle metaboreflex a subsequent period of PECO (post-exercise circulatory occlusion) was performed following exercise. FOREARM and CALF produced similar increases in BP in both the AMA subjects and CHF patients. CHF patients elicited a significantly lower diastolic BP during PECO following CALF in comparison with that following FOREARM (5±5 compared with 12±3 mmHg respectively). A similar result was seen in AMA subjects. It may be that even the limited weight-bearing locomotor role of the calf muscles constitutes a conditioning stimulus in CHF patients, which leads to desensitization of the muscle metaboreflex, thus producing an attenuated BP elevation. We conclude that it would be incorrect to make general statements about muscle chemoreflex inputs to cardiovascular control in CHF patients based upon measurements made on only one muscle group and without reference to muscle fibre type and training status.