bluuebunny/crossref_metadata_2025 huggingface dataset:
The functional integrity of normally forested landscapes: A proposal for an index of environmental capital
Authors: Woodwell George M.
Published: October 2002

The report of the World Commission on Forests and Sustainable Development [(1999)Our Forests, Our Future: Report of the World Commission on Forests and Sustainable Development(Cambridge Univ. Press, Cambridge, U.K.)] has called attention to a global need to restore the functional integrity of nature. The assumption that the biophysical world is capable of supporting any intensity of economic and political activity is now obviously wrong. The failures are seen as global changes such as the global climatic disruption now underway. The global disruptions are the product of cumulative local disruptions. Despite a conspicuous need, the scientific community has been slow in developing tools to appraise the functional integrity of landscapes. The proposal is advanced that the first steps deal with forests because they are so large in their influences on global biophysics. Analyses carried out by the commission are elaborated here as an index of functional integrity in the forest zone. The proposal is based on a simple, transparent approach. The scale is 0–100 with 100 as the nominal climax. Decrements and increments are applied as the structural and functional integrity of the landscape varies with whatever cause. The unit of area for these trials has been 10,000 hectares. The index has been applied to extremes such as the large extractive reserves in Acre, Brazil (100), the once-forested landscape of Haiti (0), and a managed forest stand in Maine. The analyses offer a scalar system for defining how well landowners and governments are protecting the public's interests in the integrity of the habitat of all.
Testosterone
in utero
and at birth dictates how stressful experience will affect learning in adulthood
Authors: Shors Tracey J. , Miesegaes George
Published: October 2002

Exposure to an acute stressful event can enhance learning in male rats, whereas exposure to the same event dramatically impairs performance in females. Here we tested whether the presence of sex hormones during early development organizes these opposite effects of stress on learning in males vs. females. In the first experiment, males were castrated at birth whereas females were injected with testosterone. Rats were trained as adults on the hippocampal-dependent learning task of trace eyeblink conditioning. Performance in adult males that had been castrated at birth was still enhanced by exposure to an acute stressful experience. However, adult females injected with testosterone at birth responded in the opposite direction, i.e., exposure to the stressor that typically reduces performance instead enhanced their levels of conditioning. In the second experiment, exposure to testosterone was manipulated
in utero
by injecting pregnant females with a testosterone antagonist. After foster rearing, adult offspring were exposed to the stressor and trained on the hippocampal-dependent learning task of trace conditioning. Although performance in adult females was unaffected by antagonizing testosterone
in utero
, i.e., stress still reduced performance, the enhancement of conditioning after stress in adult males was prevented. Thus, the presence of sex hormones during gestation and development organizes whether and how acute stressful experience will affect the ability to acquire new information in adulthood. As with many sexual behaviors, these cognitive responses to stress appear to be masculinized by exposure to testosterone and feminized by its absence during very early development.
Predicting the effects of climate change on avian life-history traits
Authors: Winkler David W. , Dunn Peter O. , McCulloch Charles E.
Published: October 2002

Across North America, tree swallows have advanced their mean date of clutch initiation (lay date) by ≈9 days over the past 30 years, apparently in response to climate change. In a sample of 2,881 nest records collected by the lay public from 1959 to 1991, we examined whether clutch size has also responded to climate change. We found that clutch size is strongly related to lay date, both within and among years, and there has been no significant temporal variation in the slopes or intercepts of the clutch-size/lay-date regressions. As a consequence, we expected increases in clutch size with advancement in lay date; however, we detected no such trend over time. The distributions of egg-laying dates were more constricted in the warmest (and earliest) years, suggesting that changes in mean clutch size might be constrained by changes in the distribution of laying dates. If spring temperatures continue to increase, we predict further reductions of variance in laying dates and relatively small increases in clutch size. Such constraints on life-history variation probably are common and need to be considered when modeling the effects of climate change on reproduction in natural populations. Predicting the long-term effects of constraints and interpreting changes in life-history traits require a better understanding of both adaptive and demographic effects of climate change.
Single molecule analysis of RNA polymerase elongation reveals uniform kinetic behavior
Authors: Adelman Karen , La Porta Arthur , Santangelo Thomas J. , Lis John T. , Roberts Jeffrey W. , Wang Michelle D.
Published: October 2002

By using single-molecule measurements, we demonstrate that the elongation kinetics of individual
Escherichia coli
RNA polymerase molecules are remarkably homogeneous. We find no evidence of distinct elongation states among RNA polymerases. Instead, the observed heterogeneity in transcription rates results from statistical variation in the frequency and duration of pausing. When transcribing a gene without strong pause sites, RNA polymerase molecules display transient pauses that are distributed randomly in both time and distance. Transitions between the active elongation mode and the paused state are instantaneous within the resolution of our measurements (<1 s). This elongation behavior is compared with that of a mutant RNA polymerase that pauses more frequently and elongates more slowly than wild type.
Pyrophosphate-producing protein dephosphorylation by HPr kinase/phosphorylase: A relic of early life?
Authors: Mijakovic Ivan , Poncet Sandrine , Galinier Anne , Monedero Vicente , Fieulaine Sonia , Janin Joël , Nessler Sylvie , Marquez José Antonio , Scheffzek Klaus , Hasenbein Sonja , Hengstenberg Wolfgang , Deutscher Josef
Published: October 2002

In most Gram-positive bacteria, serine-46-phosphorylated HPr (P-Ser-HPr) controls the expression of numerous catabolic genes (≈10% of their genome) by acting as catabolite corepressor. HPr kinase/phosphorylase (HprK/P), the bifunctional sensor enzyme for catabolite repression, phosphorylates HPr, a phosphocarrier protein of the sugar-transporting phosphoenolpyruvate/glycose phosphotransferase system, in the presence of ATP and fructose-1,6-bisphosphate but dephosphorylates P-Ser-HPr when phosphate prevails over ATP and fructose-1,6-bisphosphate. We demonstrate here that P-Ser-HPr dephosphorylation leads to the formation of HPr and pyrophosphate. HprK/P, which binds phosphate at the same site as the β phosphate of ATP, probably uses the inorganic phosphate to carry out a nucleophilic attack on the phosphoryl bond in P-Ser-HPr. HprK/P is the first enzyme known to catalyze P-protein dephosphorylation via this phospho–phosphorolysis mechanism. This reaction is reversible, and at elevated pyrophosphate concentrations, HprK/P can use pyrophosphate to phosphorylate HPr. Growth of
Bacillus subtilis
on glucose increased intracellular pyrophosphate to concentrations (≈6 mM), which in
in vitro
tests allowed efficient pyrophosphate-dependent HPr phosphorylation. To effectively dephosphorylate P-Ser-HPr when glucose is exhausted, the pyrophosphate concentration in the cells is lowered to 1 mM. In
B. subtilis
, this might be achieved by YvoE. This protein exhibits pyrophosphatase activity, and its gene is organized in an operon with
hprK
.
Reciprocal developmental regulation of presynaptic ionotropic receptors
Authors: Turecek Rostislav , Trussell Laurence O.
Published: October 2002

Activation of ionotropic glycine receptors potentiates glutamate release in mature calyceal nerve terminals of the rat medial nucleus of the trapezoid body, an auditory brainstem nucleus. In young rats, glycine and its receptors are poorly expressed. We therefore asked whether GABA (γ-aminobutyric acid) might play a larger role than glycine in the regulation of glutamate release in the absence of glycine receptors. Indeed, in rats younger than postnatal day 11 (P11), and before the onset of hearing, calyces expressed high levels of ionotropic GABA
A
receptors but few glycine receptors. Isoguvacine, a selective agonist at GABA
A
receptors, strongly enhanced excitatory postsynaptic currents in young rats but had little effect in rats older than P11. Down-regulation of presynaptic GABA
A
receptors did not reflect global changes in receptor expression, because the magnitude of GABA and glycine responses was similar at P13 in the parent-cell bodies of the calyces, the bushy cells of the cochlear nucleus. In outside-out patches excised from the nonsynaptic face of calyces, GABA and glycine evoked single-channel currents consistent with the properties of postsynaptic GABA
A
and glycine receptors. Inhibitory GABA
B
receptors were present on the calyx at all developmental stages examined. Thus, GABA initially acts on two receptor subtypes, both promoting and inhibiting glutamate release. With age, the former role is transferred to the glycine receptor during the period in which postsynaptic glycinergic transmission is acquired.
Natural killer cells attack tumor cells expressing high levels of sialyl Lewis x oligosaccharides
Authors: Ohyama Chikara , Kanto Satoru , Kato Kazunori , Nakano Osamu , Arai Yoichi , Kato Tetsuro , Chen Shihao , Fukuda Michiko N. , Fukuda Minoru
Published: October 2002

Epithelial carcinoma and leukemia cells express sialyl Lewis x oligosaccharides as tumor-associated carbohydrate antigens. To determine the role of sialyl Lewis x oligosaccharides in tumor dissemination, human melanoma MeWo cells, which do not express sialyl Lewis x, were transfected with α1,3-fucosyltransferase III (FTIII), and cell lines expressing different amounts of sialyl Lewis x were isolated. When these cells were injected into the tail vein of nude mice, cells expressing moderate amounts of sialyl Lewis x (MeWo-FTIII⋅M) produced a significantly greater number of lung tumor foci than did parental MeWo cells. In contrast, cells expressing large amounts of sialyl Lewis x (MeWo-FTIII⋅H) produced few lung tumor foci in nude mice but were highly tumorigenic in beige mice, which have defective natural killer (NK) cells.In vitroassays demonstrated that MeWo-FTIII⋅H cells are much more sensitive to NK cell-mediated cytotoxicity than are MeWo-FTIII⋅M cells or parental MeWo cells and the susceptibility of MeWo-FTIII⋅H cells to NK cell-mediated cytolysis can be inhibited by preincubating MeWo-FTIII⋅H cells with anti-sialyl Lewis x antibody. Moreover, we discovered that NK cell-mediated cytolysis of MeWo-FTIII⋅H cells can be inhibited by the addition of an antibody against the NK cell receptor CD94 or sialyl Lewis x oligosaccharides. These results, combined with structural analysis of MeWo-FTIII⋅H cell carbohydrates, indicate that moderate amounts of sialyl Lewis x lead to tumor metastasis, whereas expression of high levels of sialyl Lewis x leads to an NK cell attack on tumor cells, demonstrating that expression of different amounts of sialyl Lewis x results in entirely different biological consequences.
Soluble peptide–MHC monomers cause activation of CD8+T cells through transfer of the peptide to T cell MHC molecules
Authors: Ge Qing , Stone Jennifer D. , Thompson M. Todd , Cochran Jennifer R. , Rushe Mia , Eisen Herman N. , Chen Jianzhu , Stern Lawrence J.
Published: October 2002

T cell receptor (TCR)-mediated activation of CD4+T cells is known to require multivalent engagement of the TCR by, for example, oligomeric peptide–MHC complexes. In contrast, for CD8+T cells, there is evidence for TCR-mediated activation by univalent engagement of the TCR. We have here compared oligomeric and monomeric Ldand Kbpeptide–MHC complexes and free peptide as stimulators of CD8+T cells expressing the 2C TCR. We found that the monomers are indeed effective in activating naïve and effector CD8+T cells, but through an unexpected mechanism that involves transfer of peptide from soluble monomers to T cell endogenous MHC (Kb) molecules. The result is that T cells, acting as antigen-presenting cells, are able to activate other naïve T cells.
Structure and function in rhodopsin: A tetracycline-inducible system in stable mammalian cell lines for high-level expression of opsin mutants
Authors: Reeves Philip J. , Kim Jong-Myoung , Khorana H. Gobind
Published: October 2002

Tetracycline-inducible HEK293S stable cell lines have been prepared that express high levels (up to 10 mg/liter) of WT opsin and its mutants only in response to the addition of tetracycline and sodium butyrate. The cell lines were prepared by stable transfection of HEK293S-TetR cells with expression plasmids that contained the opsin gene downstream of a cytomegalovirus promoter containing
tetO
sequences as well as the neomycin resistance gene under control of the weak H
2
L
d
promoter. The inducible system is particularly suited for overcoming problems with toxicity either due to the addition of toxic compounds, for example, tunicamycin, to the growth medium or due to the expressed protein products. By optimization of cell growth conditions in a bioreactor, WT opsin, a constitutively active opsin mutant, E113Q/E134Q/M257Y, presumed to be toxic to the cells, and nonglycosylated WT opsin obtained by growth in the presence of tunicamycin have been prepared in amounts of several milligrams per liter of culture medium.
The hidden duplication past of
Arabidopsis
thaliana
Authors: Simillion Cedric , Vandepoele Klaas , Van Montagu Marc C. E. , Zabeau Marc , Van de Peer Yves
Published: October 2002

Analysis of the genome sequence of
Arabidopsis thaliana
shows that this genome, like that of many other eukaryotic organisms, has undergone large-scale gene duplications or even duplications of the entire genome. However, the high frequency of gene loss after duplication events reduces colinearity and therefore the chance of finding duplicated regions that, at the extreme, no longer share homologous genes. In this study we show that heavily degenerated block duplications that can no longer be recognized by directly comparing two segments because of differential gene loss, can still be detected through indirect comparison with other segments. When these so-called hidden duplications in
Arabidopsis
are taken into account, many homologous genomic regions can be found in five to eight copies. This finding strongly implies that
Arabidopsis
has undergone three, but probably no more, rounds of genome duplications. Therefore, adding such hidden blocks to the duplication landscape of
Arabidopsis
sheds light on the number of polyploidy events that this model plant genome has undergone in its evolutionary past.
Critical points in charged membranes containing cholesterol
Authors: Radhakrishnan Arun , McConnell Harden M.
Published: October 2002

Epifluorescence microscopy is used to determine phase diagrams for lipid monolayers containing binary mixtures of cholesterol or dihydrocholesterol and dimyristoylphosphatidylserine, as well as ternary mixtures that also contain ganglioside G
M1
. The phase diagrams are unusual in that they show multiple critical points: two upper miscibility critical points and one lower miscibility critical point. These critical points all are associated with the formation of condensed complexes between these lipids and cholesterol (or dihydrocholesterol). The miscibility critical pressures depend on subphase pH and ionic strength. Changes in critical pressures due to changes in subphase composition are closely related to changes in membrane electrostatic pressure and surface ionization.
Deregulated G1-cyclin expression induces genomic instability by preventing efficient pre-RC formation
Authors: Tanaka Seiji , Diffley John F.X.
Published: October 2002

Although genomic instability is a hallmark of human cancer cells, the mechanisms by which genomic instability is generated and selected for during oncogenesis remain obscure. In most human cancers, the pathway leading to the activation of the G1 cyclins is deregulated. Using budding yeast as a model, we show that overexpression of the G1 cyclin Cln2 inhibits the assembly of prereplicative complexes (pre-RCs) and induces gross chromosome rearrangements (GCR). Our results suggest that deregulation of G1 cyclins, selected for in oncogenesis because it confers clonal growth advantage, may also provide an important mechanism for generating genomic instability by inhibiting replication licensing.
Training young people through a school/enterprise partnership: a longitudinal study
Authors: Smith Peter J. , Henry John , Munro Graeme
Published: October 2002

This paper reports a longitudinal study of 58 students who undertook an engineering traineeship concurrent with their final two years of secondary school. The student experience was planned as a partnership arrangement between a manufacturing enterprise, a secondary school, and a post‐secondary technical education institution. Results are discussed in terms of completion of studies, employment and career pathways, employment outcomes, and post‐traineeship employment destinations. Both the quantitative and qualitative data indicate that participants have benefited significantly in each area investigated. The significance of the program in developing propositional, procedural, and dispositional workplace knowledge is also discussed.
Market‐oriented value creation in service firms
Authors: McNaughton Rod B. , Osborne Phil , Imrie Brian C.
Published: October 2002

A fundamental proposition in marketing strategy is that a market orientation is positively related to firm performance. However, the mechanisms of this relationship have yet to be explored in detail, especially in service industries where intangible assets are relatively more important. This paper addresses this issue by proposing a model that identifies important intermediate variables between a market orientation and increased firm value. The model posits that a market orientation guides investment in market‐based assets and other asset types, that these assets may be levered to create a competitive advantage and value for customers, and that this results in loyalty and easier customer attraction. Quicker and more extensive market penetration, shorter sales cycles, and decreased marketing and sales costs enhance the cash flow of a market‐oriented firm. This may be recognised in higher valuations, which ultimately translate into higher share prices and wealth creation for the owners of the firm.
Antecedents of international performance
Authors: Cicic Muris , Patterson Paul , Shoham Aviv
Published: October 2002

Madsen synthesized international performance studies and identified 20 performance antecedents. Of these, 12 are unrelated to international performance or have conflicting relationships across the reviewed studies. We use his recommendations to increase the value of international performance research. First, we include seven antecedents that cover the organizational, environmental, strategic, and performance domains of his model, including components of the market orientation model. Second, earlier studies involved mostly North American and European goods exporters. This study extends previous research to the service sector in Australia. Based on responses from 181 exporters, the importance of managerial attitudes, perceived international barriers, and human resource efforts is shown to affect international performance.
Market orientation and incumbent performance in dynamic market
Authors: Perry Monica L. , Shao Alan T.
Published: October 2002

The extant literature suggests that performance may be a function of the degree to which market information is systematically collected, disseminated and responded to (i.e. market orientation). However, the majority of empirical research on the market orientation to performance relationship has focused on manufacturers and has not distinguished between incumbents and new entrants. Our study of incumbent firms involves the market orientation to performance relationship in the context of services in the growing and competitive Internet industry. We found that market orientation did not directly affect performance, nor did the interaction of market orientation and perceptions of new competitors. However, perceptions of traditional competitors directly affected performance and interacted with market orientation to affect performance.
Developing indicators for the estimation of sustainable settlement size in Ireland
Authors: O’Regan Bernadette , Moles Richard , Kelly Ruth , Ravetz Joe , McEvoy Darryn
Published: October 2002

Research was undertaken within the Irish Environmental Protection Agency (EPA) Environmental RTDI Programme during the six‐month period from March to August 2001 by a partnership formed by the Centre for Environmental Research (CER), University of Limerick, and the Centre for Urban and Regional Ecology (CURE), University of Manchester. This project aimed to inform the development of spatial policies emerging from the National Spatial Strategy (NSS) aimed at finding optimal ways in which to accommodate Ireland’s growing population in a manner consistent with balanced regional development and environmental sustainability. To obtain data and information on settlements, three modes of analysis were adopted. First, for a single city and two villages, both quantitative and qualitative data were collected to provide a comprehensive analysis of the local social, economic and physical environments, track record in enhancing sustainability, current policies in place, and the likelihood of these policies proving successful. Second, for 11 Irish settlements selected to include a range of functions and locations, 29 quantified sustainability indicators were developed and used to compare the level of sustainability achieved by settlements of differing sizes. Third, a review of international literature was undertaken to search for comparable data, models and case studies, so as to provide a context for analysis of Irish data. The framework of significant environmental themes adopted here is taken from recent Irish EPA publications. Results based on all three research methods suggest that on balance larger settlements in the recent past, at present and in the foreseeable future are more likely to create conditions in which sustainability is enhanced. This work provides the basis for a large‐scale three‐year study which commenced in March 2002, which examines the sustainability and future development patterns of settlements in Ireland.
Integrating EI/TQM efforts for performance improvement: a model
Authors: Pun Kit‐Fai , Gill Raj
Published: October 2002

Many empirical studies verify that a positive correlation exists between employee involvement (EI) and total quality management (TQM) practices. Substantial EI/TQM efforts are beginning to appear in organizations of different sizes and business nature, but there is little general agreement concerning the versatility of EI/TQM planning and implementation. This paper examines the prerequisites of the EI/TQM integration, and proposes a generic EI/TQM implementation model that assists organizations to safeguard their improvement efforts. The construct of the proposed model is built in line with the system frameworks and assessment criteria of the Baldrige Award. The paper also elaborates the essential ingredients of the model, and proposes a 14‐step guideline that facilitates the EI and TQM implementation with organizational strategies. An implementation case of EI/TQM efforts is presented along with a discussion of the applicability of the model.
A fuzzy analysis approach for part‐machine grouping in cellular manufacturing systems
Authors: Al‐Ahmari A.M.A.
Published: October 2002

Among the many accepted clustering techniques, the fuzzy clustering approaches have been developed over the last decades. These approaches have been applied to many areas in manufacturing systems. In this paper, a fuzzy clustering approach is proposed for selecting machine cells and part families in cellular manufacturing systems. This fuzzy approach offers a special advantage over existing clustering approaches as it presents the degree of membership of the machine or part associated with each machine cell or part family allowing users flexibility in formulating machine cells and part families. The proposed algorithm is extended and validated using numerical examples to demonstrate its application in cellular manufacturing.
Modelling integration of CAD and inspection planning of simple element features
Authors: Ketan H.S. , Al‐Bassam M.A. , Adel M.B. , Rawabdeh I.
Published: October 2002

Concurrent design and manufacturing is widely accepted as essential for obtaining an economically conceivable design solution for industrial production. Nevertheless the development of a viable computer‐aided inspection planning (CAIP) using CAD as input for effective NC‐measuring data preparation has not yet been successful. This paper introduces an approach to direct integration between CAD and CAIP by developing a feature‐based computer aided design/inspection planning (FB‐CAIP) system. The FB‐CAIP system contains a computer‐internal model of a product that contains sufficient information to guide the direct generation of NC code of the inspection activity. The developed small prototype system integrates knowledge‐based inspection planning with feature‐based CAD system for an automatic CNC measuring program generation of prismatic parts.
Combining scoring method and fuzzy expert systems approach to supplier assessment: a case study
Authors: Kwong C.K. , Ip W.H. , Chan J.W.K.
Published: October 2002

Very often, manufacturing companies have been faced with the problem of assessment and selection of suppliers for their product development. Some methods and techniques have been developed to assist these companies in performing the assessment. However, these methods and techniques lack tge capability to deal with the instrumental and conceptual uncertainties that are involved in the supplier assessment and selection. Fuzzy expert system is an alternative approach from which the heuristics and knowledge of supplier assessment can be captured and the impreciseness and uncertainties due to the human subjectivity, that are common in the process of the supplier assessment, can be handled. In this paper, a combined scoring method with fuzzy expert systems approach is introduced to perform the supplier assessment. With the use of the fuzzy concept, the error due to human judgement in the scoring method could be minimized. First, current methods and techniques of supplier assessment are reviewed in this paper. There follows the description of a case study of combined scoring method and fuzzy expert systems approach to supplier assessment. Some results of the prototype system trial‐run are discussed in the final part of this paper.
Sequence analysis of an artificial family of RNA‐binding peptides
Authors: Barrick Jeffrey E. , Roberts Richard W.
Published: October 2002

Diverse peptide sequences recognizing the λ boxB RNA hairpin were previously isolated from a library encoding the 22‐residue λ N peptide with random amino acids at positions 13–22 using mRNA display. We have statistically analyzed amino acid distributions in 65 unique sequences from rounds 11 and 12 of this selection and evaluated the resulting structural and functional predictions by alanine‐scanning mutagenesis and circular dichroism spectrometry. This artificial sequence family has a consensus structure that continues the bent α helix of λ N up to position 17 when bound to λ boxB. A charge pair (E14R15) and hydrophobic patch (A21L22or V21L22) have important functional roles in this context. Notably, amino acid covariance reveals six specific pairs of random region positions with >95% significant linkage and strong overall helical (i+1, i+3, and i+4) couplings. The covariance analysis suggests that (1) the sequence context of every residue in each insert has been optimized, (2) selected sequences are local optima on a rugged fitness landscape, and (3) it is possible to detect more subtle structural features with artificial protein sequence families than natural homologs. Our results provide a framework for investigating the structures of in vitro selected proteins by functional minimization, reselection, and covariance analysis.
α‐Crystallin and ATP facilitate the in vitro renaturation of xylanase: enhancement of refolding by metal ions
Authors: Nath Devyani , Rawat Urmila , Anish Ramakrishnan , Rao Mala
Published: October 2002

α‐Crystallin is a multimeric protein that functions as a molecular chaperone and shares extensive structural homology to small heat shock proteins. For the functional in vitro analysis of α‐crystallin, the xylanase Xyl II from alkalophilic thermophilic Bacillus was used as a model system. The mechanism of chaperone action of α‐crystallin is less investigated. Here we studied the refolding of Gdn HCl‐denatured Xyl II in the presence and absence of α‐crystallin to elucidate the molecular mechanism of chaperone‐mediated in vitro folding. Our results, based on intrinsic tryptophan fluorescence and hydrophobic fluorophore 8‐anilino‐1‐naphthalene sulfonate binding studies, suggest that α‐crystallin formed a complex with a putative molten globule‐like intermediate in the refolding pathway of Xyl II. The α‐crystallin•Xyl II complex exhibited no functional activity. Addition of ATP to the complex initiated the renaturation of Xyl II with 30%–35% recovery of activity. The nonhydrolyzable analog 5′‐adenylyl imidodiphosphate (AMP‐PNP) was capable of reconstitution of active Xyl II to a lesser extent than ATP. Although the presence of Ca2+ was not required for the in vitro refolding of Xyl II, the renaturation yield was enhanced in its presence. Experimental evidence indicated that the binding of ATP to the α‐crystallin•Xyl II complex brought about conformational changes in α‐crystallin facilitating the dissociation of xylanase molecules. This is the first report of the enhancement of α‐crystallin chaperone functions by metal ions.
Characterization of the LDL‐A module mutants of Tva, the subgroup A Rous sarcoma virus receptor, and the implications in protein folding
Authors: Wang Qing‐Yin , Manicassamy Balaji , Yu Xuemei , Dolmer Klavs , Gettins Peter G.W. , Rong Lijun
Published: October 2002

Tva is the cellular receptor for subgroup A Rous sarcoma virus (RSV‐A), and the viral receptor function is solely determined by a 40‐residue motif called the LDL‐A module of Tva. In this report, an integral approach of molecular, biochemical, and biophysical techniques was used to examine the role of a well‐conserved tryptophan of the LDL‐A module of Tva in protein folding and ligand binding. We show that substitution of tryptophan by glycine adversely affected the correct folding of the LDL‐A module of Tva, with only a portion giving a calcium‐binding conformation. Furthermore, we show that the misfolded LDL‐A conformations of Tva could not efficiently bind to its ligand. These results indicate that this conserved tryptophan in the LDL‐A module of Tva plays an important role in correct protein folding and ligand recognition. Furthermore, these results suggest that the familial hypercholesterolemia (FH) French Canadian‐4 mutation is likely caused by protein misfolding of low‐density lipoprotein receptor, thus explaining the defect for this class of FH.
The dimeric dihydroorotate dehydrogenase A from Lactococcus lactis dissociates reversibly into inactive monomers
Authors: Ottosen Mette Brimheim , Björnberg Olof , Nørager Sofie , Larsen Sine , Palfey Bruce Allan , Jensen Kaj Frank
Published: October 2002

The flavoenzyme dihydroorotate dehydrogenase A from Lactococcus lactis is a homodimeric protein of 311 residues/subunit, and the two active sites are positioned at a distance from the dimer interface. To promote formation of the monomeric form of the enzyme, we changed the residues involved in formation of two salt bridges formed between the residues Glu206 of the one polypeptide and Lys296 of the other polypeptide. The mutant enzymes formed inactive precipitates when cells were grown at 37°C, but remained soluble and active when cells were grown at 25°C. The salt bridges were not needed for activity, because the mutant enzymes in which one of the residues was converted to an alanine (E206A or K296A) retained almost full activity. The mutant enzymes in which the charge of one of the residues of the salt bridge was inverted (i.e., E206K or K296E) were severely impaired. The double mutant E206K‐K296E, which has the possibility of forming salt bridges in the opposite orientation of the wild type, was fully active in concentrated solutions, but dissociated into inactive monomers upon dilution. The KD for the dimer to monomer dissociation reaction was 12 μM, and dimer formation was favored by the product, orotate, or by high ionic strength, indicating that the hydrophobic interactions are important for the subunit contacts. Wild‐type dihydroorotate dehydrogenase A was similarly found to dissociate into inactive monomers, but with a KD for dissociation equal to 0.12 μM. These results imply that the dimeric state is necessary for activity of the enzyme.
Conformational basis for the Li+‐induced leak current in the rat γ‐aminobutyric acid (GABA) transporter‐1
Authors: MacAulay Nanna , Zeuthen Thomas , Gether Ulrik
Published: October 2002

The rat γ‐aminobutyric acid transporter‐1 (GAT‐1) was expressed in Xenopus laevis oocytes and the substrate‐independent Li+‐induced leak current was examined using two‐electrode voltage clamp. The leak current was not affected by the addition of GABA and was not due to H+ permeation. The Li+‐bound conformation of the protein displayed a lower passive water permeability than that of the Na+‐ and choline (Ch+)‐bound conformations and the leak current did not saturate with increasing amounts of Li+ in the test solution. The mechanism that gives rise to the leak current did not support active water transport in contrast to the mechanism responsible for GABA translocation (≈330 water molecules per charge). Altogether, these data support the distinct nature of the leak conductance in relation to the substrate translocation process. It was observed that the leak current was inhibited by low millimolar concentrations of Na+ (the apparent affinity constant, K′0.5= 3 mm). In addition, it was found that the GABA transport current was sustained at correspondingly low Na+ concentrations if Li+ was present instead of choline. This is consistent with a model in which Li+ can bind and substitute for Na+ at the putative ‘first’ apparently low‐affinity Na+ binding site. In the absence of Na+, this allows a Li+‐permeable channel to open at hyperpolarized potentials. Occupancy of the ‘second’ apparently high‐affinity Na+ binding site by addition of low millimolar concentrations of Na+ restrains the transporter from moving into a leak conductance mode as well as allowing maintenance of GABA‐elicited transport‐associated current.
Ceramide, a putative second messenger for nerve growth factor, modulates the TTX‐resistant Na+ current and delayed rectifier K+ current in rat sensory neurons
Authors: Zhang Y. H. , Vasko M. R. , Nicol G. D.
Published: October 2002

Because nerve growth factor (NGF) is elevated during inflammation and is known to activate the sphingomyelin signalling pathway, we examined whether NGF and its putative second messenger, ceramide, could modulate the excitability of capsaicin‐sensitive adult and embryonic sensory neurons. Using the whole‐cell patch‐clamp recording technique, exposure of isolated sensory neurons to either 100 ng ml−1 NGF or 1 μM N‐acetyl sphingosine (C2‐ceramide) produced a 3‐ to 4‐fold increase in the number of action potentials (APs) evoked by a ramp of depolarizing current in a time‐dependent manner. Intracellular perfusion with bacterial sphingomyelinase (SMase) also increased the number of APs suggesting that the release of native ceramide enhanced neuronal excitability. Glutathione, an inhibitor of neutral SMase, completely blocked the NGF‐induced augmentation of AP firing, whereas dithiothreitol, an inhibitor of acidic SMase, was without effect. In the presence of glutathione and NGF, exogenous ceramide still enhanced the number of evoked APs, indicating that the sensitizing action of ceramide was downstream of NGF. To investigate the mechanisms of action for NGF and ceramide, isolated membrane currents were examined. Both NGF and ceramide facilitated the peak amplitude of the TTX‐resistant sodium current (TTX‐R INa) by approximately 1.5‐fold and shifted the activation to more hyperpolarized voltages. In addition, NGF and ceramide suppressed an outward potassium current (IK) by ≈35 %. Ceramide reduced IK in a concentration‐dependent manner. Isolation of the NGF‐ and ceramide‐sensitive currents indicates that they were delayed rectifier types of IK. The inflammatory prostaglandin, PGE2, produced an additional suppression of IK after exposure to ceramide (≈35 %), suggesting that these agents might act on different targets. Thus, our findings indicate that the pro‐inflammatory agent, NGF, can rapidly enhance the excitability of sensory neurons. This NGF‐induced sensitization is probably mediated by activation of the sphingomyelin signalling pathway to liberate ceramide(s), wherein ceramide appears to be the second messenger involved in modulating neuronal excitability.
Pulmonary oedema fluid induces non‐α‐ENaC‐dependent Na+ transport and fluid absorption in the distal lung
Authors: Rafii Bijan , Gillie Daniel J. , Sulowski Chris , Hannam Vicky , Cheung Tony , Otulakowski Gail , Barker Pierre M. , O'Brodovich Hugh
Published: October 2002

To determine if pulmonary oedema fluid (EF) alters ion and fluid transport of distal lung epithelium (DLE), EF was collected from rats in acute heart failure. EF, but not plasma, increased amiloride‐insensitive short circuit current (Isc) and Na+‐K+ ATPase protein content and pump activity of DLE grown in primary culture. Inhibitors of Cl− transport or cGMP‐gated cation channels had a significant (P < 0.05), but limited ability to block the increased Isc. EF increased amiloride‐insensitive, but not amiloride‐sensitive, DLE apical membrane Na+ conductance. The level of mRNA encoding epithelial sodium channel (ENaC) subunits was unchanged (α, β), or decreased (γ, P < 0.05) in EF‐exposed DLE. EF also induced an amiloride‐insensitive increase in the potential difference across murine tracheal cysts. Distal lung explants from late gestation wild‐type and α‐ENaC‐deficient fetal mice, which normally expand due to liquid secretion, decreased in size due to liquid absorption when exposed to EF. Trypsin digestion or heat treatment of EF abrogated the ability of EF to increase amiloride‐insensitive Isc in DLE and liquid absorption by distal lung explants. Thus proteins or protein‐dependent factors within cardiogenic EF induce an α‐ENaC‐independent and amiloride‐insensitive apical membrane Na+ conductance and liquid absorption in the distal lung.
Paired pulse facilitation of corticogeniculate EPSCs in the dorsal lateral geniculate nucleus of the rat investigated in vitro
Authors: Granseth Björn , Ahlstrand Erik , Lindström Sivert
Published: October 2002

To investigate paired pulse facilitation of corticogeniculate EPSCs, whole‐cell patch‐clamp recordings were made from principal cells in the rat dorsal lateral geniculate nucleus (dLGN) in vitro. Thalamic slices, oriented so that both corticogeniculate and retinogeniculate axons could be stimulated, were cut from young (16‐ to 37‐day‐old) DA‐HAN rats. Corticogeniculate EPSCs displayed pronounced paired pulse facilitation at stimulus intervals up to 400 ms. The facilitation had a fast and a slow component of decay with time constants of 12 ± 7 and 164 ± 47 ms (means ±s.d.), respectively. Maximum paired pulse ratio (EPSC2× EPSC1−1) was 3.7 ± 1.1 at the 20‐30 ms interval. Similar to other systems, the facilitation was presynaptic. Retinogeniculate EPSCs recorded in the same dLGN cells displayed paired pulse depression at intervals up to at least 700 ms. The two types of EPSCs differed in their calcium response curves. At normal [Ca2+]o, the corticogeniculate synapse functioned over the early rising part of a Hill function, while the retinogeniculate synapse operated over the middle and upper parts of the curve. The paired pulse ratio of corticogeniculate EPSCs was maximal at physiological [Ca2+]o. The facilitation is proposed to have an important role in the function of the corticogeniculate circuit as a neuronal amplifier.
Molecular mechanism of a COOH‐terminal gating determinant in the ROMK channel revealed by a Bartter's disease mutation
Authors: Flagg Thomas P. , Yoo Dana , Sciortino Christopher M. , Tate Margaret , Romero Michael F. , Welling Paul A.
Published: October 2002

The ROMK subtypes of inward‐rectifier K+ channels mediate potassium secretion and regulate NaCl reabsorption in the kidney. Loss‐of‐function mutations in this pH‐sensitive K+ channel cause Bartter's disease, a familial salt wasting nephropathy. One disease‐causing mutation truncates the extreme COOH‐terminus and induces a closed gating conformation. Here we identify a region within the deleted domain that plays an important role in pH‐dependent gating. The domain contains a structural element that functionally interacts with the pH sensor in the cytoplasmic NH2‐terminus to set a physiological range of pH sensitivity. Removal of the domain shifts the pKa towards alkaline pH values, causing channel inactivation under physiological conditions. Suppressor mutations within the pH sensor rescued channel gating and trans addition of the cognate peptide restored pH sensitivity. A specific interdomain interaction was revealed in an in vitro protein‐protein binding assay between the NH2‐ and COOH‐terminal cytoplasmic domains expressed as bacterial fusion proteins. These results provide new insights into the molecular mechanisms underlying Kir channel regulation and channel gating defects that are associated with Bartter's disease.
The active and passive components of glucose absorption in rat jejunum under low and high perfusion stress
Authors: Helliwell Philip A. , Kellett George L.
Published: October 2002

In order to determine how perfusion design affects the relationship of the apparent ‘active’ and ‘passive’ components of glucose absorption, rat jejunum was perfused with 50 mm glucose under conditions of low and high mechanical stress. Phloretin or cytochalasin B was used to inhibit GLUT2 and phloridzin to inhibit SGLT1. In low stress perfusions, the ratios of the ‘passive’ to the ‘active’ components determined using phloretin and phloridzin were 2.2 and 0.43, respectively. This discrepancy was explained by the fact that phloridzin inhibits not only SGLT1 but also indirectly that part of the GLUT2‐mediated component controlled by SGLT1 through the glucose‐induced activation and recruitment of GLUT2 to the brush‐border membrane. In high stress perfusions, the ratios of the ‘passive’ to the ‘active’ components determined using phloretin and phloridzin were 0.94 and 0.95, respectively; cytochalasin B gave 0.95. The identity of these results was explained by the observation that the passive component is not dependent on the active component, because glucose‐induced activation and recruitment of GLUT2 does not occur in high stress perfusions. Simultaneous inhibition of SGLT1 and GLUT2 in high stress perfusions with phloridzin and cytochalasin B inhibited absorption by 92 ± 7 %; non‐carrier‐mediated transport is therefore minimal. Our data provide support for the view that the term ‘facilitated’ should be used to replace the term ‘passive’ in describing the component now known to be mediated by GLUT2. The study of the mechanism and regulation of this facilitated component depends crucially on the design of the perfusion system.
Effects of peripheral sensory input on cortical inhibition in humans
Authors: Sailer Alexandra , Molnar Gregory F. , Cunic Danny I. , Chen Robert
Published: October 2002

Cortical inhibitory systems play an important role in motor output. The motor cortex can be inhibited by intracortical mechanisms and by peripheral sensory inputs. We examined whether cortical inhibition from peripheral sensory input is mediated through previously identified intracortical inhibitory systems and how these inhibitory systems interact. Two types of intracortical inhibition were assessed by paired‐pulse transcranial magnetic stimulation (TMS). Short‐interval intracortical inhibition (SICI) was determined with a subthreshold conditioning stimulus (CS) followed by a test stimulus 2 ms later and long‐interval intracortical inhibition (LICI) with suprathreshold conditioning and test stimuli 100 ms apart. Cortical inhibition from peripheral sensory input was induced by median nerve stimulation (MNS) of the right hand and followed by a suprathreshold TMS over the left motor cortex 200 ms later. The first set of experiments tested the effects of different test stimulus intensities on SICI, LICI and cortical inhibition induced by median nerve stimulation (MNSI). With higher test stimulus intensities, LICI and MNSI decreased whereas SICI showed a trend towards an increase. The extent of SICI, LICI and MNSI did not correlate. The second experiment assessed the interaction between MNSI and LICI. The results of applying MNSI and LICI simultaneously were compared with MNSI and LICI alone. MNSI was virtually abolished in the presence of LICI and LICI was also significantly decreased in the presence of MNSI. Thus, the effects of MNSI and LICI when applied together were much less than their expected additive effects when applied alone. The degree of interaction between MNSI and LICI was related to the combined strength of MNSI and LICI but not to the strength of LICI alone. The third experiment investigated the interaction between SICI and MNSI. MNSI and SICI were applied together and the results were compared with MNSI and SICI alone. SICI remained unchanged in the presence of MNSI. We conclude that MNSI is mediated by circuits distinct from those mediating LICI or SICI. The MNSI circuits seem to have an inhibitory interaction with the LICI circuits, whereas the SICI and MNSI circuits do not seem to interact.