bluuebunny/crossref_metadata_2025 huggingface dataset:
Diversity of the Superfamily of Phloem Lectins (Phloem Protein 2) in Angiosperms
Authors: Dinant Sylvie , Clark Anna M. , Zhu Yanmin , Vilaine Françoise , Palauqui Jean-Christophe , Kusiak Chantal , Thompson Gary A.
Published: January 2003

Phloem protein 2 (PP2) is one of the most abundant and enigmatic proteins in the phloem sap. Although thought to be associated with structural P-protein, PP2 is translocated in the assimilate stream where its lectin activity or RNA-binding properties can exert effects over long distances. Analyzing the diversity of these proteins in vascular plants led to the identification ofPP2-like genes in species from 17 angiosperm and gymnosperm genera. This wide distribution of PP2 genes in the plant kingdom indicates that they are ancient and common in vascular plants. Their presence in cereals and gymnosperms, both of which lack structural P-protein, also supports a wider role for these proteins. Within this superfamily, PP2 proteins have considerable size polymorphism. This is attributable to variability in the length of the amino terminus that extends from a highly conserved domain. The conserved PP2 domain was identified in the proteins encoded by six genes from several cucurbits, celery (Apium graveolens), and Arabidopsis that are specifically expressed in the sieve element-companion cell complex. The acquisition of additional modular domains in the amino-terminal extensions of other PP2-like proteins could reflect divergence from its phloem function.
Extracellular ATP Inhibits Root Gravitropism at Concentrations That Inhibit Polar Auxin Transport
Authors: Tang Wenqiang , Brady Shari R. , Sun Yu , Muday Gloria K. , Roux Stanley J.
Published: January 2003

Raising the level of extracellular ATP to mmconcentrations similar to those found inside cells can block gravitropism of Arabidopsis roots. When plants are grown in Murashige and Skoog medium supplied with 1 mm ATP, their roots grow horizontally instead of growing straight down. Medium with 2 mm ATP induces root curling, and 3 mm ATP stimulates lateral root growth. When plants are transferred to medium containing exogenous ATP, the gravity response is reduced or in some cases completely blocked by ATP. Equivalent concentrations of ADP or inorganic phosphate have slight but usually statistically insignificant effects, suggesting the specificity of ATP in these responses. The ATP effects may be attributable to the disturbance of auxin distribution in roots by exogenously applied ATP, because extracellular ATP can alter the pattern of auxin-induced gene expression in DR5-β-glucuronidase transgenic plants and increase the response sensitivity of plant roots to exogenously added auxin. The presence of extracellular ATP also decreases basipetal auxin transport in a dose-dependent fashion in both maize (Zea mays) and Arabidopsis roots and increases the retention of [3H]indole-3-acetic acid in root tips of maize. Taken together, these results suggest that the inhibitory effects of extracellular ATP on auxin distribution may happen at the level of auxin export. The potential role of the trans-plasma membrane ATP gradient in auxin export and plant root gravitropism is discussed.
Pit Membrane Porosity and Water Stress-Induced Cavitation in Four Co-Existing Dry Rainforest Tree Species
Authors: Choat Brendan , Ball Marilyn , Luly Jon , Holtum Joseph
Published: January 2003

Aspects of xylem anatomy and vulnerability to water stress-induced embolism were examined in stems of two drought-deciduous species, Brachychiton australis (Schott and Endl.) A. Terracc. and Cochlospermum gillivraei Benth., and two evergreen species, Alphitonia excelsa (Fenzal) Benth. and Austromyrtus bidwillii (Benth.) Burret., growing in a seasonally dry rainforest. The deciduous species were more vulnerable to water stress-induced xylem embolism. B. australis andC. gillivraei reached a 50% loss of hydraulic conductivity at −3.17 MPa and −1.44 MPa, respectively; a 50% loss of hydraulic conductivity occurred at −5.56 MPa in A. excelsa and −5.12 MPa in A. bidwillii. To determine whether pit membrane porosity was responsible for greater vulnerability to embolism (air seeding hypothesis), pit membrane structure was examined. Expected pore sizes were calculated from vulnerability curves; however, the predicted inter-specific variation in pore sizes was not detected using scanning electron microscopy (pores were not visible to a resolution of 20 nm). Suspensions of colloidal gold particles were then perfused through branch sections. These experiments indicated that pit membrane pores were between 5 and 20 nm in diameter in all four species. The results may be explained by three possibilities: (a) the pores of the expected size range were not present, (b) larger pores, within the size range to cause air seeding, were present but were rare enough to avoid detection, or (c) pore sizes in the expected range only develop while the membrane is under mechanical stress (during air seeding) due to stretching/flexing.
Exploring Symbiotic Nitrogen Fixation and Assimilation in Pea Root Nodules by in Vivo 15N Nuclear Magnetic Resonance Spectroscopy and Liquid Chromatography-Mass Spectrometry
Authors: Scharff Anne Marie , Egsgaard Helge , Hansen Poul Erik , Rosendahl Lis
Published: January 2003

Nitrogen (N) fixation and assimilation in pea (Pisum sativum) root nodules were studied by in vivo 15N nuclear magnetic resonance (NMR) by exposing detached nodules to15N2 via a perfusion medium, while recording a time course of spectra. In vivo 31P NMR spectroscopy was used to monitor the physiological state of the metabolically active nodules. The nodules were extracted after the NMR studies and analyzed for total soluble amino acid pools and 15N labeling of individual amino acids by liquid chromatography-mass spectrometry. A substantial pool of free ammonium was observed by 15N NMR to be present in metabolically active, intact nodules. The ammonium ions were located in an intracellular environment that caused a remarkable change in the in vivo 15N chemical shift. Alkalinity of the ammonium-containing compartment may explain the unusual chemical shift; thus, the observations could indicate that ammonium is located in the bacteroids. The observed15N-labeled amino acids, glutamine/glutamate and asparagine (Asn), apparently reside in a different compartment, presumably the plant cytoplasm, because no changes in the expected in vivo15N chemical shifts were observed. Extensive15N labeling of Asn was observed by liquid chromatography-mass spectrometry, which is consistent with the generally accepted role of Asn as the end product of primary N assimilation in pea nodules. However, the Asn 15N amino signal was absent in in vivo 15N NMR spectra, which could be because of an unfavorable nuclear Overhauser effect. γ-Aminobutyric acid accumulated in the nodules during incubation, but newly synthesized 15N γ-aminobutyric acid seemed to be immobilized in metabolically active pea nodules, which made it NMR invisible.
The Effects of Polyethylene Glycol on Gene Expression of Developing White Spruce Somatic Embryos
Authors: Stasolla Claudio , van Zyl Leonel , Egertsdotter Ulrika , Craig Deborah , Liu Wenbin , Sederoff Ron R.
Published: January 2003

Somatic embryogenic cultures of white spruce (Picea glauca) represent a valuable system to study molecular mechanisms regulating embryo development because many embryos of defined developmental stages can be generated. The inclusion of polyethylene glycol (PEG) in the maturation medium can improve the number and quality of embryos produced. To learn more about the mechanism of action of PEG, we analyzed transcript profiles of stage-specific embryos matured without (control) or with (PEG treated) PEG. RNA extracted from maturing spruce embryos was analyzed on DNA microarrays containing 2,178 cDNAs from loblolly pine (Pinus taeda). The efficiency of heterologous hybridization between spruce and pine species on microarrays has been documented previously (L. van Zyl, S. von Arnold, P. Bozhkov, Y. Chen, U. Egertsdotter, J. MacKay, R. Sederoff, J. Shen, L. Zelena, D. Clapham [2002] Comp Funct Genomics 3: 306–318). Several pine genes, including the apparent homologs to the Arabidopsis genes ZWILLE, FIDDLEHEAD, FUSCA, and SCARECROW, increased in expression after PEG treatments. These genes are known to be involved in the formation of the embryo body plan and in the control of the shoot and root apical meristems. The increased transcript levels of these genes in immature PEG-treated embryos suggest that PEG may improve the quality of spruce somatic embryos by promoting normal differentiation of the embryonic shoot and root. Changes in the transcript levels of many genes involved in sucrose catabolism and nitrogen assimilation and utilization were also observed between control and PEG-treated embryos.
Quantitative Trait Loci Analysis of Nitrogen Use Efficiency in Arabidopsis
Authors: Loudet Olivier , Chaillou Sylvain , Merigout Patricia , Talbotec Joël , Daniel-Vedele Françoise
Published: January 2003

Improving plant nitrogen (N) use efficiency or controlling soil N requires a better knowledge of the regulation of plant N metabolism. This could be achieved using Arabidopsis as a model genetic system, taking advantage of the natural variation available among ecotypes. Here, we describe an extensive study of N metabolism variation in the Bay-0 × Shahdara recombinant inbred line population, using quantitative trait locus (QTL) mapping. We mapped QTL for traits such as shoot growth, total N, nitrate, and free-amino acid contents, measured in two contrasting N environments (contrasting nitrate availability in the soil), in controlled conditions. Genetic variation and transgression were observed for all traits, and most of the genetic variation was identified through QTL and QTL × QTL epistatic interactions. The 48 significant QTL represent at least 18 loci that are polymorphic between parents; some may correspond to known genes from the N metabolic pathway, but others represent new genes controlling or interacting with N physiology. The correlations between traits are dissected through QTL colocalizations: The identification of the individual factors contributing to the regulation of different traits sheds new light on the relations among these characters. We also point out that the regulation of our traits is mostly specific to the N environment (N availability). Finally, we describe four interesting loci at which positional cloning is feasible.
The contemporary workforce
Authors: Clarke Sharon
Published: January 2003

Companies are being encouraged to adopt a positive organisational safety culture in order to safeguard their operations against accidents. The viability of a positive safety culture within the context of a diverse workforce, characterised by a reduced number of permanent employees, supplemented with more contingent and contract workers, is considered. This review summarises theoretical and empirical evidence of the likely effects of changing employment arrangements on safety attitudes and behaviours, and implications for organisational safety culture. It is argued that it will be more difficult to integrate employees with diverse working arrangements, compared to a workforce of permanent employees, into a corporate safety culture. Human resource management techniques and practices are identified as ways of developing and maintaining positive safety attitudes across all types of employees. The need for further empirical work is discussed.
Performance management when innovation and learning become critical performance indicators
Authors: Molleman Eric , Timmerman Hugo
Published: January 2003

If the organization’s leading performance indicators shift towards innovation and the creation of knowledge, this will result in more non‐routine work and a higher level of interdependency among workers. We argue that a contingent performance management (PM) system has to focus on learning and group processes, on qualitative as well as quantitative goals and on both the individual as well the interpersonal level. Employees of three R&D departments of a firm producing advanced communication systems evaluated a PM system with four stages: accountability, engagement, appraisal, award. The results support our theoretical model. Furthermore, the results show that the firm’s system is perceived as somewhat imbalanced. While the first two stages are in line with our model, the latter two are not. We give suggestions for modifying the PM system to make it balanced, and we discuss the role of the manager.
Creating Diderot unities – quest for possible selves?
Authors: Davis Teresa , Gregory Gary
Published: January 2003

This paper tries to draw links between the creation of new Diderot unities (products consumed in a group and that have an internal consistency based on lifestyle) with “impulse purchases” as key departure products. A study, using exploratory in‐depth interviews, is reported. Common themes are drawn from the interviews to serve as possible identifying elements of the phenomenon. Emotive and cognitive themes are identified and are offered as a starting point for further research into such product unities. The self‐concept theory of “possible selves” is offered as one possible explanation that determines when an“impulse purchase” is a key departure product for a new Diderot unity.
Children’s perception of their influence over purchases: the role of parental communication patterns
Authors: Caruana Albert , Vassallo Rosella
Published: January 2003

Socialisation by children is seen as taking place within a socio‐cultural environment where family patterns of communication play a pivotal role. The socio‐ and concept‐orientation are two predominant parental communication styles that are likely to affect the perceived influence that children believe they hold. This study focuses on the perceived influence of children resulting from the communication pattern adopted by parents. Research is conducted among a dyad consisting of parent and child that visit a recreational site. Results are reported, conclusions are drawn and recommendations for future research are made.
An investigation of export performance variations related to corporate export market orientation
Authors: Akyol Ayse , Akehurst Gary
Published: January 2003

This research explores the nature and dimensions of market orientation in an international context by using empirical data from a sample of companies to rigorously test hypotheses regarding market orientation as applied to export operations in the context of the Turkish clothing industry. This aim was fulfilled by reliably and validly measuring the key constructs of export market orientation and finding statistically significant relationships between dependent (export marketing orientation) and independent variables (primarily export strategy and export performance). The results of this study draw conclusions showing that the hypothesised relationship between export market orientation and export performance does exist, and in the case of Turkish clothing exporters, improving an export market orientation level is a significant contributor to the company’s export performance. The results of this study also show the dimension of export market orientation that has the strongest relation with each dimension of export performance, and identifying the other export market orientation dimensions that make a significant contribution to the explanation of export performance.
Moving towards e‐government: a case study of organisational change processes
Authors: Burn Janice , Robins Greg
Published: January 2003

Looks at an online strategy project at Legal Aid in Western Australia. Begins with an overview of e‐government and the Western Australian Government context, and then discusses the research model and methodology. Gives a background to the case and analyses the change management process against a comprehensive model of business process change. Concludes with some lessons learned and future directions for research in this area.
Does nitric oxide allow endothelial cells to sense hypoxia and mediate hypoxic vasodilatation?in vivo and in vitro studies
Authors: Edmunds Nicholas J. , Moncada Salvador , Marshall Janice M.
Published: January 2003

Hypoxia‐evoked vasodilatation is a fundamental regulatory mechanism that is often attributed to adenosine. The identity of the O2 sensor is unknown. Nitric oxide (NO) inhibits endothelial mitochondrial respiration and ATP generation by competing with O2 for its binding site on cytochrome oxidase. We proposed that in vivo this interaction allows endothelial cells to release adenosine when O2 tension falls or NO concentration increases. Using anaesthetised rats, we confirmed that the increase in femoral vascular conductance (FVC, hindlimb vasodilatation) evoked by systemic hypoxia is attenuated by NO synthesis blockade with l‐NAME, but restored when baseline FVC is restored by infusion of NO donor. This ‘restored’ hypoxic response, like the control hypoxic response, is inhibited by the adenosine A1 receptor antagonist 8‐cyclopentyl‐1,3‐dipropylxanthine. Similarly, the FVC increase evoked by adenosine infusion was attenuated by l‐NAME but restored by infusion of NO donor. However, when baseline FVC was restored after l‐NAME with 8‐bromo‐cGMP, the FVC increase evoked by adenosine infusion was restored, but not in response to systemic hypoxia, suggesting that adenosine was no longer released by hypoxia. Infusion of NO donor at a given rate after treatment with l‐NAME evoked a greater FVC increase during systemic hypoxia than during normoxia, both responses being reduced by 8‐cyclopentyl‐1,3‐dipropylxanthine. Finally, both bradykinin and NO donor released adenosine from superfused endothelial cells in vitro; l‐NAME attenuated only the former response. We propose that in vivo, shear‐released NO increases the apparent Km of endothelial cytochrome oxidase for O2, allowing the endothelium to act as an O2 sensor, releasing adenosine in response to moderate falls in O2.
Effects of osmotic changes on the chemoreceptor cell of rat carotid body
Authors: Molnár Zoltán , Petheő Gábor L. , Fülöp Csaba , Spät András
Published: January 2003

The carotid body plays a crucial role in cardiorespiratory regulation. In the present study we investigated the effect of osmotic changes on cytoplasmic calcium concentration ([Ca2+]c) and pH (pHi) of isolated chemoreceptor cells of the rat carotid body. In CO2/HCO3−‐buffered medium, reduction of osmolality from the control level of 300 mosmol kg−1 to 250–285 mosmol kg−1 resulted in a rise in [Ca2+]c, as measured with Indo‐1, whereas elevation of osmolality to 350 mosmol kg−1 had no effect. The Ca2+ response required extracellular Ca2+ and was reduced by application of the L‐type Ca2+ channel antagonist nifedipine (10 μm). The hyposmosis‐induced Ca2+ response could be prevented by application of niflumic acid (300 μm), an inhibitor of the swelling‐activated Cl− channel. In whole‐cell patch‐clamp experiments niflumic acid abolished the swelling‐activated Cl− current but only slightly depressed the Ca2+ current. The inhibition of Ca2+ current by niflumic acid does not account for its action in preventing of hyposmosis‐induced Ca2+ response, which seems to be initiated by Cl−‐mediated depolarisation. Withdrawal of CO2/HCO3− also prevented the Ca2+ response. Reduction of the osmotic concentration by 50 mosmol kg−1 induced a small but sustained decrease in pHi, while elevation by 50 mosmol kg−1 had an inverse effect, as measured fluorimetrically with carboxy SNARF‐1. Our conclusion is that in the rat chemoreceptor cell the activation of Cl− channels, e.g. by hyposmotic challenge, induces depolarisation, which, in turn, activates voltage‐gated Ca2+ channels.
Chick hair cells do not exhibit voltage‐dependent somatic motility
Authors: He David Z. Z. , Beisel Kirk W. , Chen Lin , Ding Da‐Lian , Jia Shuping , Fritzsch Bernd , Salvi Richard
Published: January 2003

It is generally believed that mechanical amplification by cochlear hair cells is necessary to enhance the sensitivity and frequency selectivity of hearing. In the mammalian ear, the basis of cochlear amplification is believed to be the voltage‐dependent electromotility of outer hair cells (OHCs). The avian basilar papilla contains tall and short hair cells, with the former being comparable to inner hair cells, and the latter comparable to OHCs, based on their innervation patterns. In this study, we sought evidence for somatic electromotility by direct measurements of voltage‐dependent length changes in both tall and short hair cells at nanometre resolution. Microchamber and whole‐cell voltage‐clamp techniques were used. Motility was measured with a photodiode‐based measurement system. Non‐linear capacitance, an electrical signature of somatic motility, was also measured to complement motility measurement. Significantly, chick hair cells did not exhibit somatic motility nor express non‐linear capacitance. The lack of somatic motility suggests that in avian hair cells the active process resides elsewhere, most likely in the hair cell stereocilia.
Structure, function and immunolocalization of a proton‐coupled amino acid transporter (hPAT1) in the human intestinal cell line Caco‐2
Authors: Chen Zhong , Fei You‐Jun , Anderson Catriona M. H. , Wake Katherine A. , Miyauchi Seiji , Huang Wei , Thwaites David T. , Ganapathy Vadivel
Published: January 2003

The human orthologue of the H+‐coupled amino acid transporter (hPAT1) was cloned from the human intestinal cell line Caco‐2 and its functional characteristics evaluated in a mammalian cell heterologous expression system. The cloned hPAT1 consists of 476 amino acids and exhibits 85 % identity with rat PAT1. Among the various human tissues examined by Northern blot, PAT1 mRNA was expressed most predominantly in the intestinal tract. When expressed heterologously in mammalian cells, hPAT1 mediated the transport of α‐(methylamino)isobutyric acid (MeAIB). The cDNA‐induced transport was Na+‐independent, but was energized by an inwardly directed H+ gradient. hPAT1 interacted with glycine, l‐alanine, l‐proline, α‐aminoisobutyrate (AIB) and γ‐aminobutyrate (GABA), as evidenced from direct transport measurements and from competition experiments with MeAIB as a transport substrate. hPAT1 also recognized the d‐isomers of alanine and proline. With serine and cysteine, though the l‐isomers did not interact with hPAT1 to any significant extent, the corresponding d‐isomers were recognized as substrates. With proline and alanine, the affinity was similar for l‐ and d‐isomers. However, with cysteine and serine, the d‐isomers showed 6‐ to 8‐fold higher affinity for hPAT1 than the corresponding l‐isomers. These functional characteristics of hPAT1 closely resemble those that have been described previously for the H+‐coupled amino acid transport system in Caco‐2 cells. Furthermore, there was a high degree of correlation (r2= 0.93) between the relative potencies of various amino acids to inhibit the H+‐coupled MeAIB transport measured with native Caco‐2 cells and with hPAT1 in the heterologous expression system. Immunolocalization studies showed that PAT1 was expressed exclusively in the apical membrane of Caco‐2 cells. These data suggest that hPAT1 is responsible for the H+‐coupled amino acid transport expressed in the apical membrane of Caco‐2 cells.
Sensory and electrophysiological properties of guinea‐pig sensory neurones expressing Nav 1.7 (PN1) Na+ channel α subunit protein
Authors: Djouhri Laiche , Newton Richard , Levinson Simon Rock , Berry Carol M. , Carruthers Barbara , Lawson Sally N.
Published: January 2003

The TTX‐sensitive Nav1.7 (PN1) Na+ channel α subunit protein is expressed mainly in small dorsal root ganglion (DRG) neurones. This study examines immunocytochemically whether it is expressed exclusively or preferentially in nociceptive primary afferent DRG neurones, and determines the electrophysiological properties of neurones that express it. Intracellular somatic action potentials (APs) evoked by dorsal root stimulation were recorded in L6/S1 DRG neurones at 30 ± 2 °C in vivo in deeply anaesthetised young guinea‐pigs. Each neurone was classified, from its dorsal root conduction velocity (CV) as a C‐, Aδ‐ or Aα/β‐fibre unit and from its response to mechanical and thermal stimuli, as a nociceptive, low threshold mechanoreceptive (LTM) or unresponsive unit. Fluorescent dye was injected into the soma and Nav1.7‐like immunoreactivity (Nav1.7‐LI) was examined on sections of dye‐injected neurones. All C‐, 90 % of Aδ‐ and 40 % of Aα/β‐fibre units, including both nociceptive and LTM units, showed Nav1.7‐LI. Positive units included 1/1 C‐LTM, 6/6 C‐nociceptive, 4/4 C‐unresponsive (possible silent nociceptive) units, 5/6 Aδ‐LTM (D hair), 13/14 Aδ‐nociceptive, 2/9 Aα/β‐nociceptive, 10/18 Aα/β‐LTM cutaneous and 0/9 Aα/β‐muscle spindle afferent units. Overall, a higher proportion of nociceptive than of LTM neurones was positive, and the median relative staining intensity was greater in nociceptive than LTM units. Nav1.7‐LI intensity was clearly positively correlated with AP duration and (less strongly) negatively correlated with CV and soma size. Since nociceptive units tend overall to have longer duration APs, slower CVs and smaller somata, these correlations may be related to the generally greater expression of Nav1.7 in nociceptive units.
The familial hemiplegic migraine mutation R192q reduces G‐protein‐mediated inhibition of p/q‐type (Cav2.1) calcium channels expressed in human embryonic kidney cells
Authors: Melliti Karim , Grabner Manfred , Seabrook Guy R.
Published: January 2003

Familial hemiplegic migraine is associated with at least 13 different missense mutations in the α1A Ca2+ channel subunit. Some of these mutations have been shown to affect the biophysical properties of α1A currents. To date, no study has examined the influence of such mutations on the G‐protein regulation of channel function. Because G‐proteins inhibit movement of the voltage sensor, we examined the effects of the R192Q mutation, which neutralizes a positive charge in the first S4 segment. Human wild‐type (WT) or R192Q mutant channels were expressed in human embryonic kidney tsA‐201 cells along with dopamine D2 receptors. Application of quinpirole induced fast (≈1 s), pertussis toxin‐sensitive inhibition of α1AWT and α1AR192Q Ca2+ currents, consistent with the activation of a membrane‐delimited pathway. α1AWT Ca2+ currents were inhibited by 62.9 ± 0.9 % (n= 27), whereas α1AR192Q Ca2+ currents were inhibited by only 47.9 ± 1.8 % (n= 35; P < 0.001). Concentration‐response analysis showed that only the extent of inhibition was affected, with no change in agonist potency (EC50= 1 nm). Prepulse facilitation, which is a characteristic of voltage‐dependent inhibition, was also reduced by the R192Q mutation. However, the kinetics of facilitation and slow activation were not affected, suggesting that G‐protein‐Ca2+ channel affinity was unchanged. These results show that the R192Q mutation reduces the G‐protein inhibition of P/Q‐type Ca2+ channels, probably by altering mechanisms by which Gβγ subunit binding induces a change in channel gating. Altered G‐protein modulation and the consequent reduced presynaptic inhibition may contribute to migraine attacks by favouring a persistent state of hyperexcitability.
Mechanisms of acute natriuresis in normal humans on low sodium diet
Authors: Rasmussen M. S. , Simonsen J. A. , Sandgaard N. C. F. , Høilund‐Carlsen P. F. , Bie P.
Published: January 2003

This study evaluates the relative importance of several mechanisms possibly involved in the natriuresis elicited by slow sodium loading, i.e. the renin‐angiotensin‐aldosterone system (RAAS), mean arterial blood pressure (MAP), glomerular filtration rate (GFR), atrial natriuretic peptide (ANP), oxytocin and nitric oxide (NO). Eight seated subjects on standardised sodium intake (30 mmol NaCl day−1) received isotonic saline intravenously (NaLoading: 20 μmol Na+ kg−1 min−1 or ≈11 ml min−1 for 240 min). NaLoading did not change MAP or GFR (by clearance of 51Cr‐EDTA). Significant natriuresis occurred within 1 h (from 9 ± 3 to 13 ± 2 μmol min−1). A 6‐fold increase was found during the last hour of infusion as plasma renin activity, angiotensin II (ANGII) and aldosterone decreased markedly. Sodium excretion continued to increase after NaLoading. During NaLoading, plasma renin activity and ANGII were linearly related (R= 0.997) as were ANGII and aldosterone (R= 0.999). The slopes were 0.40 pm ANGII (mi.u. renin activity)−1 and 22 pm aldosterone (pm ANGII)−1. Plasma ANP and oxytocin remained unchanged, as did the urinary excretion rates of cGMP and NO metabolites (NOx). In conclusion, sodium excretion may increase 7‐fold without changes in MAP, GFR, plasma ANP, plasma oxytocin, and cGMP‐ and NOx excretion, but concomitant with marked decreases in circulating RAAS components. The immediate renal response to sodium excess appears to be fading of ANGII‐mediated tubular sodium reabsorption. Subsequently the decrease in aldosterone may become important.
Angiotensin II AT1 receptor stimulates Na+–k+atpase activity through a pathway involving pkc‐ζ in rat thyroid cells
Authors: Marsigliante S. , Muscella A. , Elia M. G. , Greco S. , Storelli C.
Published: January 2003

Angiotensin II (Ang II) receptor subtype 1, AT1, is expressed by the rat thyroid. A relationship between thyroid function and several components of the renin‐angiotensin system has also been established, but the Ang II cellular effects in thyrocytes and its transduction signalling remain undefined. The aim of the present paper was to investigate the modulation of the activity of the Na+‐K+ATPase by Ang II and its intracellular transduction pathway in PC‐Cl3 cells, an established epithelial cell line derived from rat thyroid. Here we have demonstrated, by RT‐PCR analysis, the expression of mRNA for the Ang II AT1 receptor in PC‐Cl3 cells; mRNA for the Ang II AT2 receptor was not detected. Ang II was not able to affect the intracellular Ca2+ concentration in fura‐2‐loaded cells, but it stimulated the translocation from the cytosol to the plasma membrane of atypical protein kinase C‐zeta (PKC‐ζ) and ‐iota (PKC‐ι) isoforms with subsequent phosphorylation of the extracellular signal‐regulated kinases 1 and 2 (ERK1 and 2). Translocated atypical PKCs displayed temporally different activations, the activation of PKC‐ζ being the fastest. PC‐Cl3 cells stimulated with increasing Ang II concentrations showed dose‐ and time‐dependent activation of the Na+‐K+ATPase activity, which paralleled the PKC‐ζ translocation time course. Na+‐K+ATPase activity modulation was dependent on PKC activation since the PKC antagonist staurosporine abolished the stimulatory effect of Ang II. The inhibition of the ERK kinases 1 and 2 (MEK1 and 2) by PD098059 (2′‐amino‐3′‐methoxyflavone) failed to block the effect of Ang II on the Na+‐K+ATPase activity. In conclusion, our results suggest that Ang II modulates Na+‐K+ATPase activity in PC‐Cl3 cells through the AT1 receptor via activation of atypical PKC‐ζ while the Ang II‐activated PKC‐ζ appears to have other as yet unknown functions.
Streptomycin and intracellular calcium modulate the response of single guinea‐pig ventricular myocytes to axial stretch
Authors: Belus Alexandra , White Ed
Published: January 2003

We tested the hypothesis that both stretch‐activated channels (SACs) and intracellular calcium ([Ca2+]i) are important in the electrical response of single guinea‐pig ventricular myocytes to axial stretch. Myocytes were attached to carbon fibre transducers and stretched, sarcomere length increased by approximately 9 %, and there was a prolongation of the action potential duration. Streptomycin, a blocker of SACs, had no effect upon the shortening, [Ca2+]i transients or action potentials of electrically stimulated, unstretched myocytes, at a concentration of 50 μm, but at 40 μm, prevented any stretch‐induced increase in action potential duration. Under action potential clamp, stretch elicited a current with a linear current‐voltage relationship that was inward at membrane potentials negative to its reversal potential of −30 mV, in 10 of 24 cells tested, and was consistent with the activation of non‐specific, cationic SACs. This current was not seen in any stretched cells that were exposed to 40 μm streptomycin. However, exposure of cells to 5 μm BAPTA‐AM, in order to reduce [Ca2+]i transients, also abolished stretch‐induced prolongation of the action potential. We conclude that both SACs and [Ca2+]i are important in the electrical response of cardiac myocytes to stretch, and propose that stretch‐induced changes in electrical activity and [Ca2+]i may be linked by inter‐dependent mechanisms.
Differences in conductance of M2 proton channels of two influenza viruses at low and high pH
Authors: Chizhmakov I. V. , Ogden D. C. , Geraghty F. M. , Hayhurst A. , Skinner A. , Betakova T. , Hay A. J.
Published: January 2003

The M2 protein of influenza A viruses forms a proton channel involved in modifying virion and trans Golgi pH during infection. Previous studies of the proton current using whole‐cell patch clamp of mouse erythroleukaemia (MEL) cells expressing the M2 protein of the ‘Weybridge’ strain provided evidence for two protonation sites, one involved in permeation, the other in activation by acid pH. The present report compares the M2 channels of two different strains of influenza virus, ‘Weybridge’ (WM2) and ‘Rostock’ (RM2). Whereas with external acid pH the current‐voltage relations showed similar small degrees of inward rectification, a similar apparent Kd of approximately 10 μm for proton permeation and a high selectivity for protons over Na+, the two M2 proteins differed in whole‐cell conductance at low and high pH. The proton conductance of unit membrane area was on average 7‐fold greater in RM2‐ than WM2‐expressing MEL cells. At high external pH WM2 was shown previously to have small conductance for outward current at positive driving potential. In contrast, RM2 shows high conductance for outward current with high external pH, but shows small conductance for inward current with high internal pH, conditions in which WM2 shows high conductance for inward current. The different properties of the conductances due to the two channels at high pH were determined by three amino acids in their transmembrane domains. All intermediate mutants possessed one or other property and transformation of the WM2 phenotype into that of RM2 required substitution in all three residues V27I, F38L and D44N; single substitutions in RM2 effected the opposite phenotypic change. The significance of this difference for virus replication is not clear and it may be that the higher proton flux associated with RM2 is the main factor determining its increased ability to dissipate pH gradients. It is apparent, however, from the specific differences in the sidedness of the pH‐induced changes in voltage dependence of the whole‐cell current that this is an intrinsic property of the virus proton channel which may have parallels with regulation of other proton channels.
Muscarinic regulation of pacemaker frequency in murine gastric interstitial cells of Cajal
Authors: Kim Tae Wan , Koh Sang Don , Ördög Tamás , Ward Sean M. , Sanders Kenton M.
Published: January 2003

Peristaltic contractions in the stomach are regulated by the spread of electrical slow waves from the corpus to the pylorus. Gastric slow waves are generated and propagated by the interstitial cells of Cajal (ICC). All regions distal to the dominant pacemaker area in the corpus are capable of generating slow waves, but orderly gastric peristalsis depends upon a frequency gradient in which the corpus pacemaker frequency exceeds the antral frequency. Cholinergic, muscarinic stimulation enhances pacemaker frequency. We investigated this phenomenon using intact murine gastric muscles and cultured ICC. Acetylcholine (ACh) increased the frequency of slow waves in antrum and corpus muscles. The increase was significantly greater in the antrum. ACh and carbachol (CCh) increased the pacemaker currents in cultured ICC. At high doses of CCh, transient pacemaker currents fused into sustained inward currents that persisted for the duration of stimulation. The effects of CCh were blocked by low doses of the M3 receptor antagonist 1‐dimethyl‐4‐diphenylacetoxypiperidinium. Frequency enhancement by CCh was not affected by forskolin, but the phospholipase C inhibitor U‐73122 inhibited both the increase in frequency and the development of tonic inward currents. 2‐Aminoethyldiphenyl borate also blocked the chronotropic responses to CCh. Inhibitors of protein kinase C did not block responses to CCh. These studies show that mice are an excellent model for studying mechanisms that regulate gastric slow‐wave frequency. CCh, apparently via production of inositol 1,4,5‐trisphosphate, accelerates the frequency of pacemaker activity. High concentrations of CCh may block the entrainment of pacemaker currents, resulting in a tonic inward current.
Reduced contraction strength with increased intracellular [Ca2+] in left ventricular trabeculae from failing rat hearts
Authors: Ward Marie‐Louise , Pope Adèle J. , Loiselle Denis S. , Cannell Mark B.
Published: January 2003

Intracellular calcium ([Ca2+]i) and isometric force were measured in left ventricular (LV) trabeculae from spontaneously hypertensive rats (SHR) with failing hearts and normotensive Wistar‐Kyoto (WKY) controls. At a physiological stimulation frequency (5 Hz), and at 37 °C, the peak stress of SHR trabeculae was significantly (P≤; 0.05) reduced compared to WKY (8 ± 1 mN mm−2(n= 8)vs. 21 ± 5 mN mm−2(n= 8), respectively). No differences between strains in either the time‐to‐peak stress, or the time from peak to 50 % relaxation were detected. Measurements using fura‐2 showed that in the SHR both the peak of the Ca2+ transient and the resting [Ca2+]i were increased compared to WKY (peak: 0.69 ± 0.08 vs. 0.51 ± 0.08 μm (P≤ 0.1) and resting: 0.19 ± 0.02 vs. 0.09 ± 0.02 μm (P≤ 0.05), SHR vs. WKY, respectively). The decay of the Ca2+ transient was prolonged in SHR, with time constants of: 0.063 ± 0.002 vs. 0.052 ± 0.003 s (SHR vs. WKY, respectively). Similar results were obtained at 1 Hz stimulation, and for [Ca2+]o between 0.5 and 5 mm. The decay of the caffeine‐evoked Ca2+ transient was slower in SHR (9.8 ± 0.7 s (n= 8)vs. 7.7 ± 0.2 s (n= 8) in WKY), but this difference was removed by use of the SL Ca2+‐ATPase inhibitor carboxyeosin. Histological examination of transverse sections showed that the fractional content of perimysial collagen was increased in SHR compared to WKY (18.0 ± 4.6 % (n= 10)vs. 2.9 ± 0.9 % (n= 11) SHR vs. WKY, respectively). Our results show that differences in the amplitude and the time course of the Ca2+ transient between SHR and WKY do not explain the reduced contractile performance of SHR myocardium per se. Rather, we suggest that, in this animal model of heart failure, contractile function is compromised by increased collagen, and its three‐dimensional organisation, and not by reduced availability of intracellular Ca2+.
Age and sex differences in human motor cortex input–output characteristics
Authors: Pitcher Julia B. , Ogston Kirstin M. , Miles Timothy S.
Published: January 2003

Stimulus‐response curves for motor evoked potentials (MEPs) induced in a hand muscle by transcranial magnetic stimulation (TMS) were constructed for 42 subjects with the aim of identifying differences related to age and sex. There was no effect of age on the resting threshold to TMS, the maximal amplitude of the MEP that could be evoked (MEPmax) or the maximal slope of the stimulus‐response curve. However, higher stimulus intensities were required to achieve both MEPmax and the maximal slope in the older subjects. The trial‐to‐trial variability of MEPs was greater in the older subjects, particularly at intensities near threshold. There was a significant interaction between age, threshold and trial‐to‐trial variability of MEP amplitude. Overall, MEP variability fell markedly as stimulus intensity increased above threshold but less rapidly in older than in younger subjects. Females tended to have larger MEP variability than males, but age and threshold were much stronger modulators than sex. These differences in input‐output characteristics are likely to be due either to a decreased number of spinal motoneurones being activated synchronously in older subjects, or to the activation of the same number of motoneurones in a less synchronous manner, leading to phase cancellation in the surface electromyogram.
Kv1.4 channel block by quinidine: evidence for a drug‐induced allosteric effect
Authors: Wang Shimin , Morales Michael J. , Qu Yu‐Jie , Bett Glenna C. L. , Strauss Harold C. , Rasmusson Randall L.
Published: January 2003

We studied quinidine block of Kv1.4ΔN, a K+ channel lacking N‐type inactivation, expressed in Xenopus ooctyes. Initially, quinidine intracellularly blocked the open channel so rapidly it overlapped with activation. This rapid open channel block was reduced (non‐additively) by interventions that slow C‐type inactivation: [K+]o elevation and an extracellular lysine to tyrosine mutation (K532Y). These manipulations reduced the affinity of rapid open channel block ≈10‐fold, but left the effective electrical distance unchanged at ≈0.15. Following rapid open channel block, there were time‐dependent quinidine effects: the rate of inactivation during a single depolarisation was increased, and repetitive pulsing showed use dependence. The rate of recovery from the time‐dependent aspect of quinidine block was similar to recovery from normal C‐type inactivation. Manipulations that prevented the channel from entering the C‐type inactivated state (i.e. high [K+]o or the K532Y mutation) prevented the development of the time‐dependent quinidine‐induced inactivation. The concentration dependence of the rapid block and the time‐dependent quinidine‐induced inactivation were similar, but the time‐dependent component was strongly voltage sensitive, with an effective electrical distance of 2. Clearly, this cannot reflect the permeation of quinidine through the electric field, but must be the result of some other voltage‐sensitive change in the channel. We propose that quinidine promotes the entry of the channel into a C‐type inactivated state in a time‐ and voltage‐dependent manner. We developed a mathematical model based on these results to test the hypothesis that, following rapid open channel block, quinidine promotes development of the C‐type inactivated state through a voltage‐dependent conformational change.
Uptake of locally applied deoxyglucose, glucose and lactate by axons and schwann cells of rat vagus nerve
Authors: Véga Céline , Martiel Jean‐Louis , Drouhault Delphine , Burckhart Marie‐France , Coles Jonathan A.
Published: January 2003

We asked whether, in a steady state, neurons and glial cells both take up glucose sufficient for their energy requirements, or whether glial cells take up a disproportionate amount and transfer metabolic substrate to neurons. A desheathed rat vagus nerve was held crossways in a laminar flow perfusion chamber and stimulated at 2 Hz. 14C‐labelled substrate was applied from a micropipette for 5 min over a < 0.6 mm band of the surface of the nerve. After 10‐55 min incubation, the nerve was lyophilized and the longitudinal distribution of radioactivity measured. When the weakly metabolizable analogue of glucose, 2‐deoxy‐[U‐14C]d‐glucose (*DG), was applied, the profiles of the radioactivity broadened with time, reaching distances several times the mean length of the Schwann cells (0.32 mm; most of the Schwann cells are non‐myelinating). The profiles were well fitted by curves calculated for diffusion in a single compartment, the mean diffusion coefficient being 463 ± 34 μm2 s−1 (±s.e.m., n= 16). Applications of *DG were repeated in the presence of the gap junction blocker, carbenoxolone (100 μm). The profiles were now narrower and better fitted with two compartments. One compartment had a coefficient not significantly different from that in the absence of the gap junction blocker (axons), the other compartment had a coefficient of 204 ± 24 μm2 s−1, n= 4. Addition of the gap junction blocker 18‐α‐glycyrrhetinic acid, or blocking electrical activity with TTX, also reduced longitudinal diffusion. Ascribing the compartment in which diffusion was reduced by these treatments to non‐myelinating Schwann cells, we conclude that 78.0 ± 3.6 % (n= 9) of the uptake of *DG was into Schwann cells. This suggests that there was transfer of metabolic substrate from Schwann cells to axons. Local application of [14C]glucose or [14C]lactate led to variable labelling along the length of the nerve, but with both substrates narrow peaks were often present at the application site; these were greatly reduced by subsequent treatment with amylase, a glycogen‐degrading enzyme.
Vestibular actions on back and lower limb muscles during postural tasks in man
Authors: Ali Alima S. , Rowen Katherine A. , Iles J. F.
Published: January 2003

The vestibular system was activated by galvanic electrical stimulation in 19 normal subjects. With the head turned to one side so that the stimulating anode was on the posterior mastoid process, stimulation caused standing subjects to sway backwards in the sagittal plane. Electromyography showed bilateral activation of erector spinae, gluteus maximus, biceps femoris, soleus and intrinsic foot (toe flexor) muscles. When head direction or electrode polarity was reversed so that the anode was anterior, all those muscles became less active and the subjects swayed forwards. With the head facing forward, stimulation caused sideways sway in the coronal plane, towards the anode, with excitation of the erector spinae on the anode side and reduced activity on the cathode side. The limb muscles were activated on the side opposite the anode and showed complex responses on the anode side. Responses were detectable in the erectores spinae muscles in sitting subjects. No responses in limb muscles were detected in the sitting posture. Subject responses in erector spinae recorded at L3/L4 had latencies from 59 to 110 ms, using a 2 mA stimulus. Latencies in lower limb muscles were longer. The results suggest a role for the vestibular system and descending brain stem motor pathways to the erectores spinae muscles in the control of postural orientation of the back when sitting and standing. The conduction velocity in the motor pathway was estimated to be 13 ± 10 m s−1 (mean ±s.d., n= 12 subjects).