bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional hierarchy of plasminogen kringles 1 and 4 in fibrinolysis and plasmin‐induced cell detachment and apoptosis
Authors: Ho‐Tin‐Noé Benoît , Rojas Gertrudis , Vranckx Roger , Lijnen H. Roger , Anglés‐Cano Eduardo
Published: June 2005

Plasmin(ogen) kringles 1 and 4 are involved in anchorage of plasmin(ogen) to fibrin and cells, an essential step in fibrinolysis and pericellular proteolysis. Their contribution to these processes was investigated by selective neutralization of their lysine‐binding function. Blocking the kringle 1 lysine‐binding site with monoclonal antibody 34D3 fully abolished binding and activation of Glu‐plasminogen and prevented both fibrinolysis and plasmin‐induced cell detachment‐induced apoptosis. In contrast, blocking the kringle 4 lysine‐binding site with monoclonal antibody A10.2 did not impair its activation although it partially inhibited plasmin(ogen) binding, fibrinolysis and cell detachment. This remarkable, biologically relevant, distinctive response was not observed for plasmin or Lys‐plasminogen; each antibody inhibited their binding and activation of Lys‐plasminogen to a limited extent, and full inhibition of fibrinolysis required simultaneous neutralization of both kringles. Thus, in Lys‐plasminogen and plasmin, kringles 1 and 4 act as independent and complementary domains, both able to support binding and activation. We conclude that Glu‐/Lys‐plasminogen and plasmin conformations are associated with transitions in the lysine‐binding function of kringles 1 and 4 that modulate fibrinolysis and pericellular proteolysis and may be of biological relevance during athero‐thrombosis and inflammatory states. These findings constitute the first biological link between plasmin(ogen) transitions and functions.
Fatty acid desaturases from the microalga Thalassiosira pseudonana
Authors: Tonon Thierry , Sayanova Olga , Michaelson Louise V. , Qing Renwei , Harvey David , Larson Tony R. , Li Yi , Napier Johnathan A. , Graham Ian A.
Published: June 2005

Analysis of a draft nuclear genome sequence of the diatom Thalassiosira pseudonana revealed the presence of 11 open reading frames showing significant similarity to functionally characterized fatty acid front‐end desaturases. The corresponding genes occupy discrete chromosomal locations as determined by comparison with the recently published genome sequence. Phylogenetic analysis showed that two of the T. pseudonana desaturase (Tpdes) sequences grouped with proteobacterial desaturases that lack a fused cytochrome b5 domain. Among the nine remaining gene sequences, temporal expression analysis revealed that seven were expressed in T. pseudonana cells. One of these, TpdesN, was previously characterized as encoding a Δ11‐desaturase active on palmitic acid. From the six remaining putative desaturase genes, we report here that three, TpdesI, TpdesO and TpdesK, respectively encode Δ6‐, Δ5‐ and Δ4‐desaturases involved in production of the health beneficial polyunsaturated fatty acid DHA (docosahexaenoic acid). Furthermore, we show that one of the remaining genes, TpdesB, encodes a Δ8‐sphingolipid desaturase with strong preference for dihydroxylated substrates.
Long‐distance interactions between enhancers and promoters
Authors: Sipos László , Gyurkovics Henrik
Published: June 2005

Abdominal‐B (Abd‐B) is a complex homeotic gene with a difficult task: one transcript determines the identity of four different abdominal segments throughout development in Drosophila. Although an increasing amount of information is available about the structure and the functioning of the regulatory regions that determine the expression pattern of Abd‐B, it is still not clear how these regulatory regions can contact the distantly located (several tens of kilobases away) promoter in the nucleus, what mechanism restricts promiscuous enhancers to this specific interaction, and how different regulatory regions replace one another at the same promoter in subsequent abdominal segments. Moreover, several of these regulatory regions have to act over chromatin domain boundaries and extensive inactive chromatin domains, similarly to the situation found in the chicken beta‐globin cluster. In this minireview we survey mechanisms and factors that may be involved in mediating specific interactions between the Abd‐B promoter and its regulatory regions.
A new molecular tool for transgenic diatoms
Authors: Poulsen Nicole , Kröger Nils
Published: June 2005

Research in diatom biology has entered the postgenomic era since the recent completion of the Thalassiosira pseudonana genome project. However, the molecular tools available for genetic manipulation of diatoms are still sparse, impeding the functional analysis of diatom genes in vivo. Here we describe the first method for inducible gene expression in transgenic diatoms. This method uses a DNA cassette containing both promoter (Pnr) and terminator (Tnr) elements derived from the nitrate reductase gene of the diatom Cylindrotheca fusiformis. By using green fluorescent protein (gfp) cDNA as a reporter gene, it is demonstrated that gene expression under the control of the Pnr/Tnr cassette is switched off when cells are grown in the presence of ammonium ions and becomes switched on within 4 h when cells are transferred to medium containing nitrate. Incubating cells in nitrogen‐free medium switches on transcription of the gfp gene, yet gfp mRNA does not become translated into protein. This block on translation is released by the addition of nitrate, resulting in rapid onset of GFP production with a drastically reduced delay time of only 1 h. Altogether we have demonstrated that the Pnr/Tnr cassette enables inducible gene expression and control of both the level and timing of mRNA and protein expression in transgenic diatoms.
Structural and functional evidence for a singular repertoire of BMP receptor signal transducing proteins in the lophotrochozoan Crassostrea gigas suggests a shared ancestral BMP/activin pathway
Authors: Herpin Amaury , Lelong Christophe , Becker Thomas , Rosa Frederic , Favrel Pascal , Cunningham Charles
Published: June 2005

The transforming growth factor β (TGF‐β) superfamily includes bone morphogenetic proteins, activins and TGF‐βsensu stricto (s.s). These ligands, which transduce their signal through a heteromeric complex of type I and type II receptors, have been shown to play a key role in numerous biological processes including early embryonic development in both deuterostomes and ecdyzozoans. Lophochotrozoans, the third major group of bilaterian animals, have remained in the background of the molecular survey of metazoan development. We report the cloning and functional study of the central part of the BMP pathway machinery in the bivalve mollusc Crassostrea gigas (Cg‐BMPR1 type I receptor and Cg‐TGFβsfR2 type II receptor), showing an unusual functional mode of signal transduction for this superfamily. The use of the zebrafish embryo as a reporter organism revealed that Cg‐BMPR1, Cg‐TGFβsfR2, Cg‐ALR I, an activin Type I receptor or their dominant negative acting truncated forms, when overexpressed during gastrulation, resulted in a range of phenotypes displaying severe disturbance of anterioposterior patterning, due to strong modulations of ventrolateral mesoderm patterning. The results suggest that Cg‐BMPR1, and to a certain degree Cg‐TGFβsfR2 proteins, function in C. gigas in a similar way to their zebrafish orthologues. Finally, based on phylogenetic analyses, we propose an evolutionary model within the complete TGF‐β superfamily. Thus, evidence provided by this study argues for a possible conserved endomesoderm/ectomesoderm inductive mechanism in spiralians through an ancestral BMP/activin pathway in which the singular, promiscuous and probably unique Cg‐TGFβsfR2 would be the shared type II receptor interface for both BMP and activin ligands.
BGN16.3, a novel acidic β‐1,6‐glucanase from mycoparasitic fungus Trichoderma harzianum CECT 2413
Authors: Montero Manuel , Sanz Luis , Rey Manuel , Monte Enrique , Llobell Antonio
Published: June 2005

A new component of the β‐1,6‐glucanase (EC 3.2.1.75) multienzymatic complex secreted by Trichoderma harzianum has been identified and fully characterized. The protein, namely BGN16.3, is the third isozyme displaying endo‐β‐1,6‐glucanase activity described up to now in T. harzianum CECT 2413. BGN16.3 is an acidic β‐1,6‐glucanase that is specifically induced by the presence of fungal cell walls in T. harzianum growth media. The protein was purified to electrophoretical homogenity using its affinity to β‐1,6‐glucan as first purification step, followed by chomatofocusing and gel filtration. BGN16.3 has a molecular mass of 46 kDa in SDS/PAGE and a pI of 4.5. The enzyme only showed activity against substrates with β‐1,6‐glycosidic linkages, and it has an endohydrolytic mode of action as shown by HPLC analysis of the products of pustulan hydrolysis. The expression profile analysis of BGN16.3 showed a carbon source control of the accumulation of the enzyme, which is fast and strongly induced by fungal cell walls, a condition often regarded as mycoparasitic simulation. The likely involvement β‐1,6‐glucanases in this process is discussed.
A mouse model for in vivo tracking of the major dust mite allergen Der p 2 after inhalation
Authors: Johansson Linda , Svensson Linda , Bergström Ulrika , Jacobsson‐Ekman Gunilla , Arnér Elias S. J. , van Hage Marianne , Bucht Anders , Gafvelin Guro
Published: June 2005

Inhaled environmental antigens, i.e. allergens, cause allergic symptoms in millions of patients worldwide. As little is known about the fate of an allergen upon inhalation, we addressed this issue for a major dust mite allergen, Der p 2. First, a model for Der p 2‐sensitization was established in C57BL/6 J mice, in which sensitized mice mounted a Der p 2‐specific IgE‐response with eosinophilic lung inflammation after allergen challenge in the airways. In this model, we applied recombinant Der p 2 carrying a novel C‐terminal tetrapeptide Sel‐tag enabling labelling with the gamma‐emitting radionuclide 75Se at a single selenocysteine residue ([75Se]Der p 2). In vivo tracking of intratracheally administered [75Se]Der p 2 using whole‐body autoradiography revealed that [75Se]Der p 2‐derived radioactivity persisted in the lungs of sensitized mice as long as 48 h. Radioactivity was also detected in kidneys, liver and in enlarged lung‐associated lymph nodes. Interestingly, a larger proportion of radioactivity was found in the lungs of sensitized compared with nonsensitized mice 24 h after intratracheal instillation of [75Se]Der p 2. A radioactive protein corresponding to intact Der p 2 could only be detected in the lungs, whereas [75Se]Der p 2‐derived radioactivity was recovered in known selenoproteins both in lung and other organs. Hence, using the recently developed Sel‐tag method in a mouse model for Der p 2‐sensitization, we could track the fate of an inhaled allergen in vivo. Based upon our findings, we conclude that the inflammatory state of the lung influences the rate of metabolism and clearance of Der p 2. Thus, an allergic response to the inhaled allergen may lead to prolonged retention of Der p 2 in the lung.
Gene expression in response to endoplasmic reticulum stress in Arabidopsis thaliana
Authors: Kamauchi Shinya , Nakatani Hiromi , Nakano Chiharu , Urade Reiko
Published: June 2005

Eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). In this case, so‐called unfolded protein response (UPR) genes are induced. We determined the transcriptional expression of Arabidopsis thaliana UPR genes by fluid microarray analysis of tunicamycin‐treated plantlets. Two hundred and fifteen up‐regulated genes and 17 down‐regulated ones were identified. These genes were reanalyzed with functional DNA microarrays, using DNA fragments cloned through fluid microarray analysis. Finally, 36 up‐regulated and two down‐regulated genes were recognized as UPR genes. Among them, the up‐regulation of genes related to protein degradation (HRD1, SEL‐1L/HRD3 and DER1), regulation of translation (P58IPK), and apoptosis (BAX inhibitor‐1) was reconfirmed by real‐time reverse transcriptase‐PCR. The induction of SEL‐1L protein in an Arabidopsis membrane fraction on tunicamycin‐treatment was demonstrated. Phosphorylation of initiation factor‐2α, which was inhibited by P58IPK, was decreased in tunicamycin‐treated plantlets. However, regulatory changes in translation caused by ER stress were not detected in Arabidopsis. Plant cells appeared to have a strategy for overcoming ER stress through enhancement of protein folding activity, degradation of unfolded proteins, and regulation of apoptosis, but not regulation of translation.
Inhibition of the mitochondrial calcium uniporter by the oxo‐bridged dinuclear ruthenium amine complex (Ru360) prevents from irreversible injury in postischemic rat heart
Authors: de Jesús García‐Rivas Gerardo , Guerrero‐Hernández Agustín , Guerrero‐Serna Guadalupe , Rodríguez‐Zavala José S. , Zazueta Cecilia
Published: June 2005

Mitochondrial calcium overload has been implicated in the irreversible damage of reperfused heart. Accordingly, we studied the effect of an oxygen‐bridged dinuclear ruthenium amine complex (Ru360), which is a selective and potent mitochondrial calcium uniporter blocker, on mitochondrial dysfunction and on the matrix free‐calcium concentration in mitochondria isolated from reperfused rat hearts. The perfusion of Ru360 maintained oxidative phosphorylation and prevented opening of the mitochondrial permeability transition pore in mitochondria isolated from reperfused hearts. We found that Ru360 perfusion only partially inhibited the mitochondrial calcium uniporter, maintaining the mitochondrial matrix free‐calcium concentration at basal levels, despite high concentrations of cytosolic calcium. Additionally, we observed that perfused Ru360 neither inhibited Ca2+ cycling in the sarcoplasmic reticulum nor blocked ryanodine receptors, implying that the inhibition of ryanodine receptors cannot explain the protective effect of Ru360 in isolated hearts. We conclude that the maintenance of postischemic myocardial function correlates with an incomplete inhibition of the mitochondrial calcium uniporter. Thus, the chemical inhibition by this molecule could be an approach used to prevent heart injury during reperfusion.
APPLICATION OF AN IN‐LINE RHEOLOGICAL CHARACTERIZATION METHOD TO CHEMICALLY MODIFIED AND NATIVE CORN STARCH
Authors: DOGAN NIHAN , MCCARTHY MICHAEL J. , POWELL ROBERT L.
Published: June 2005

The application of an in‐line ultrasonics‐based rheological characterization method for measuring the rheological properties of 6% (w/v) acid‐thinned and native corn starch suspensions and gels was studied. The measurements were performed in steady, laminar pipe flow using a 5 MHz frequency transducer to determine the radial shear rate distribution. Two pressure transducers were used to measure the pressure drop allowing the shear stress distribution to be calculated through the conservation of linear momentum. It was possible to obtain shear viscosity over a range of shear rates, 1–50 1/s, from a single velocity profile. A comparison of the shear viscosity function at different flow rates showed that this method could be a valid method of process rheometry during the manufacturing of starch‐based products. Both ultrasonics and rotational rheometry results showed that acid‐thinned and native corn starch suspensions exhibited Newtonian behavior before heat treatment and non‐Newtonian behavior after heating. Power law consistency index and flow behavior index of the acid‐thinned corn starch gel at 39°C were 0.60 Pa.s

0.68

and 0.68, respectively; whereas those of the native corn starch were 5.90 Pa.s

0.37

and 0.37 at 38C. Their consistency index increased and flow behavior index decreased with cooling and a thermoreversible change in the flow behavior index was observed in the native corn starch after storage.
PERCEPTION OF TEXTURE AND FLAVOR IN SOUPS BY ELDERLY AND YOUNG SUBJECTS
Authors: KREMER STEFANIE , MOJET JOS , KROEZE JAN H.A.
Published: June 2005

The perception of texture and flavor and their interaction effects in white cream soups were studied in 12 young subjects (18–29 years) and 15 elderly subjects (60–84 years). Eight soup samples (2 × 2 × 2 factorial design) were prepared with or without potato starch, with or without mushroom flavor and with water or with milk. The elderly were less sensitive to changes in the flavor profile of the soups than the young, and their perception of creaminess was reduced. Solvent by flavor interaction effects were independent of age, whereas texture by flavor interaction effects were age specific. Besides the intensities of flavor and texture attributes, pleasantness was also assessed. No indication was found that the contribution of texture and flavor to food appreciation was different for the young and for the elderly in the current study. This study supports the assumption that age‐related differences in product perception exist.
RHEOLOGICAL BEHAVIOR AND TIME‐DEPENDENT CHARACTERIZATION OF ICE CREAM MIX WITH DIFFERENT SALEP CONTENT
Authors: KUŞ S. , ALTAN A. , KAYA A.
Published: June 2005

The effect of salep concentration on the rheological characteristics of ice cream mixes (0.5–1.5% salep content), prepared from nonfat cow's milk and sugar, was studied using a controlled stress rheometer. The flow curves and time‐dependent flow properties of the ice cream mixes were assessed at different temperatures. The ice cream mixes’ samples showed slightly thixotropic behavior, which was reduced as the salep content decreased. The forward and backward measurements of the flow curves of ice cream mixes were modeled with the power law model. The ice cream mixes showed pseudoplastic flow behavior after destruction of the thixotropic structure. In mixes that were first presheared at a high shear rate flow properties could also be described by the power law model. The second‐order structural kinetic, first‐order stress decay and Weltman models were applied to model the time‐dependent flow properties of the ice cream mixes. Among these, the first‐order stress decay model was found to fit well the experimental data.
RHEOLOGICAL PROPERTIES OF OIL‐IN‐WATER EMULSIONS FORMED WITH MILK PROTEIN CONCENTRATE
Authors: HEMAR Y. , HALL C.E. , SINGH H.
Published: June 2005

The rheology and the microstructure of emulsions stabilized with milk protein concentrate (MPC) were investigated using different MPC and oil concentrations. Steady‐shear tests showed that the emulsions were Newtonian at low oil concentrations (<20 wt%), whereas they were pseudoplastic and thixotropic at high oil concentrations (>20 wt%). Dynamic rheological measurements demonstrated that these emulsions behaved as weak gels, as indicated by relatively high values of G′ to G″. The rheological behavior of these emulsions was consistent with bridging flocculation mechanisms, whereby more than one droplet is shared by adsorbed casein aggregates/micelles. This was confirmed by confocal microscopy, which showed that these emulsions were aggregated.
CONTRIBUTION OF GLYCEROL, ETHANOL AND SUGAR TO THE PERCEPTION OF VISCOSITY AND DENSITY ELICITED BY MODEL WHITE WINES
Authors: NURGEL CANAN , PICKERING GARY
Published: June 2005

The contribution of glycerol, ethanol and sugar to the perception of viscosity and density of model wine (MW) solutions was examined. In study 1, the effects of individual components on perceived viscosity (PV) and perceived density (PD) were studied using 5, 20 or 50 g/L glycerol; 3, 7 or 15% v/v ethanol and 0, 80, 150 or 250 g/L sugar concentrations. In study 2, model ice wine mixtures of 8, 10 or 12% ethanol and 150, 250 or 300 g/L sugar were assessed for PV and PD. The physical viscosity and density of the MWs were also measured in both studies. Across the range of concentrations investigated, sugar influences the perception of viscosity and density the most, ethanol has a moderate effect and the contribution of glycerol is nominal. In model ice wine solutions, PV and PD increased with sugar concentration, but were minimally affected by changes in ethanol concentration. The PV elicited by the model ice wine solutions was well described by a linear model using physical viscosity as the independent variable (r: 0.907). This information may be useful for predicting the sensory properties of the ice wine for quality control purposes.
MICROSTRUCTURE OF BOILED YAM (DIOSCOREA SPP.) AND ITS IMPLICATION FOR ASSESSMENT OF TEXTURAL QUALITY
Authors: OTEGBAYO BOLANLE , AINA JOHNSON , ASIEDU ROBERT , BOKANGA MPOKO
Published: June 2005

The effects of boiling on the microstructure of yams and their association with the textural quality of “boiled yam,” arguably the most common food product from yams, were studied using six varieties with variable cooking qualities from each of the two species, Dioscorea alata L. and D. rotundata Poir. Histological studies on raw yams showed parenchyma cells of both species to be three‐dimensional and polyhedral in shape, with starch granules loosely arranged in D. rotundata but densely packed in D. alata. Generally, there was cell separation and rounding off of cells in cooked yams of D. rotundata varieties, whereas cell separation was only partial with no rounding off in D. alata. Mealy boiled yams showed complete cell separation and the “rounding off” phenomenon, whereas the waxy ones showed partial retention of textural cell integrity. These relative changes in microstructure of yam when boiled could serve as indicators of textural quality in boiled yam.
RHEOLOGICAL AND PHYSICOCHEMICAL BEHAVIOR OF FORTIFIED YOGURT, WITH FIBER AND CALCIUM
Authors: APORTELA‐PALACIOS A. , SOSA‐MORALES M.E. , VÉLEZ‐RUIZ J.F.
Published: June 2005

Yogurt systems were prepared from whole milk, adding 50 mg of calcium/100 mL of yogurt, and three levels of fiber from two wheat‐bran sources. In comparison with a plain yogurt, the presence of fiber and calcium augmented the consistency, diminished the syneresis and the pH was higher. Fiber increased the consistency, and all the formulated samples exhibited a non‐Newtonian response. During 3 weeks of storage time, the consistency reduced (from 2.0 to 0.32 Pa·s

n

), syneresis increased (from 20 to 48%) and pH decreased (from 4.5 to 4.2) as a function of the system formulation.
Dexamethasone up‐regulates skeletal muscle maximal Na+,K+ pump activity by muscle group specific mechanisms in humans
Authors: Nordsborg Nikolai , Goodmann Craig , McKenna Michael J. , Bangsbo Jens
Published: June 2005

Dexamethasone, a widely clinically used glucocorticoid, increases human skeletal muscle Na+,K+ pump content, but the effects on maximal Na+,K+ pump activity and subunit specific mRNA are unknown. Ten healthy male subjects ingested dexamethasone for 5 days and the effects on Na+,K+ pump content, maximal activity and subunit specific mRNA level (α1, α2, β1, β2, β3) in deltoid and vastus lateralis muscle were investigated. Before treatment, maximal Na+,K+ pump activity, as well as α1, α2, β1 and β2 mRNA levels were higher (P < 0.05) in vastus lateralis than in deltoid. Dexamethasone treatment increased Na+,K+ pump maximal activity in vastus lateralis and deltoid by 14 ± 7% (P < 0.05) and 18 ± 6% (P < 0.05) as well as Na+,K+ pump content by 18 ± 9% (P < 0.001) and 24 ± 8% (P < 0.01), respectively. Treatment with dexamethasone resulted in a higher α1, α2, β1 and β2 mRNA expression in the deltoid (P < 0.05), but no effects on Na+,K+ pump mRNA were detected in vastus lateralis. In conclusion, dexamethasone treatment increased maximal Na+,K+ pump activity in both vastus lateralis and deltoid muscles. The relative importance of transcription and translation in the glucocorticoid‐induced regulation of Na+,K+ pump expression seems to be muscle specific and possibly dependent on the actual training condition of the muscle, such that a high Na+,K+ pump maximal activity and mRNA level prior to treatment prevents the transcriptional response to dexamethasone, but not the increase in Na+,K+ pump content and maximal activity.
Cell shrinkage as a signal to apoptosis in NIH 3T3 fibroblasts
Authors: Friis Martin B. , Friborg Christel R. , Schneider Linda , Maj‐Britt Nielsen  , Lambert Ian H. , Christensen Søren T. , Hoffmann Else K.
Published: June 2005

Cell shrinkage is a hallmark of the apoptotic mode of programmed cell death, but it is as yet unclear whether a reduction in cell volume is a primary activation signal of apoptosis. Here we studied the effect of an acute elevation of osmolarity (NaCl or sucrose additions, final osmolarity 687 mosmol l−1) on NIH 3T3 fibroblasts to identify components involved in the signal transduction from shrinkage to apoptosis. After 1.5 h the activity of caspase‐3 started to increase followed after 3 h by the appearance of many apoptotic‐like bodies. The caspase‐3 activity increase was greatly enhanced in cells expressing a constitutively active G protein, Rac (RacV12A3 cell), indicating that Rac acts upstream to caspase‐3 activation. The stress‐activated protein kinase, p38, was significantly activated by phosphorylation within 30 min after induction of osmotic shrinkage, the phosphorylation being accelerated in fibroblasts overexpressing Rac. Conversely, the activation of the extracellular signal‐regulated kinase (Erk1/2) was initially significantly decreased. Subsequent to activation of p38, p53 was activated through serine‐15 phosphorylation, and active p53 was translocated from the cytosol to the nucleus. Inhibition of p38 in Rac cells reduced the activation of both p53 and caspase‐3. After 60 min in hypertonic medium the rate constants for K+ and taurine efflux were increased, particular in Rac cells. We suggest the following sequence of events in the cell shrinkage‐induced apoptotic response: cellular shrinkage activates Rac, with activation of p38, followed by phosphorylation and nuclear translocation of p53, resulting in permeability increases and caspase‐3 activation.
Identification and properties of parietal pleural afferents in rabbits
Authors: Jammes Yves , Trousse Delphine , Delpierre Stéphane
Published: June 2005

Although pain and dyspnoea are common symptoms in pleural diseases, there are few studies on the sensory innervation of the pleura. Using rabbits, after removal of all muscles in the intercostal space to be studied, we investigated the afferents of the internal intercostal nerve by applying to the internal thoracic wall pieces of gauze soaked in warmed (37°C), buffered saline (mechanical stimulation) or solutions containing lactic acid, inflammatory mediators or capsaicin (chemical stimulation). The afferent conduction velocity ranged from 0.5 to 14 m s−1. Most units (97%) were activated by mechanical stimulation of the pleura (local positive pressure range = 4.5–8.5 cmH2O) and we found a linear relationship between the discharge rate of afferents and the force applied to the thoracic wall. The majority of mechanosensitive units (70%) also responded to one or several chemical agents. Thus, the afferents were activated by lactic acid (49%) and/or a mixture of inflammatory mediators (50%). Local application of capsaicin elicited an initial increased or decreased background afferent activity in 57% of the afferents, a delayed decrease in firing rate being noted in some units initially activated by capsaicin. Capsaicin blocked the afferent response to a further application of inflammatory mediators but did not affect the mechanosensitive units. Thus, sensory endings connected with thin myelinated and unmyelinated fibres in the internal intercostal nerve detect the mechanical and chemical events of pleural diseases.
Molecular mapping of a site for Cd2+‐induced modification of human ether‐à‐go‐go‐related gene (hERG) channel activation
Authors: Fernandez David , Ghanta Azad , Kinard Krista I. , Sanguinetti Michael C.
Published: June 2005

Cd2+ slows the rate of activation, accelerates the rate of deactivation and shifts the half‐points of voltage‐dependent activation (V0.5,act) and inactivation (V0.5,inact) of human ether‐à‐go‐go‐related gene (hERG) K+ channels. To identify specific Cd2+‐binding sites on the hERG channel, we mutated potential Cd2+‐coordination residues located in the transmembrane domains or extracellular loops linking these domains, including five Cys, three His, nine Asp and eight Glu residues. Each residue was individually substituted with Ala and the resulting mutant channels heterologously expressed in Xenopus oocytes and their biophysical properties determined with standard two‐microelectrode voltage‐clamp technique. Cd2+ at 0.5 mm caused a +36 mV shift of V0.5,act and a +18 mV shift of V0.5,inact in wild‐type channels. Most mutant channels had a similar sensitivity to 0.5 mm Cd2+. Mutation of single Asp residues located in the S2 (D456, D460) or S3 (D509) domains reduced the Cd2+‐induced shift in V0.5,act, but not V0.5,inact. Combined mutations of two or three of these key Asp residues nearly eliminated the shift induced by 0.5 mm Cd2+. Mutation of D456, D460 and D509 also reduced the comparatively low‐affinity effects of Ca2+ and Mg2+ on V0.5,act. Extracellular Cd2+ modulates hERG channel activation by binding to a coordination site formed, at least in part, by three Asp residues.
Colour helps to solve the binocular matching problem
Authors: Den Ouden H. E. M. , Van Ee R. , De Haan E. H. F.
Published: June 2005

The spatial differences between the two retinal images, called binocular disparities, can be used to recover the three‐dimensional (3D) aspects of a scene. The computation of disparity depends upon the correct identification of corresponding features in the two images. Understanding what image features are used by the brain to solve this binocular matching problem is an important issue in research on stereoscopic vision. The role of colour in binocular vision is controversial and it has been argued that colour is ineffective in achieving binocular vision. In the current experiment subjects were required to indicate the amount of perceived depth. The stimulus consisted of an array of fronto‐parallel bars uniformly distributed in a constant sized volume. We studied the perceived depth in those 3D stimuli by manipulating both colour (monochrome, trichrome) and luminance (congruent, incongruent). Our results demonstrate that the amount of perceived depth was influenced by colour, indicating that the visual system uses colour to achieve binocular matching. Physiological data have revealed cortical cells in macaque V2 that are tuned both to binocular disparity and to colour. We suggest that one of the functional roles of these cells may be to help solve the binocular matching problem.
Fetal cardiovascular, metabolic and endocrine responses to acute hypoxaemia during and following maternal treatment with dexamethasone in sheep
Authors: Jellyman J. K. , Gardner D. S. , Edwards C. M. B. , Fowden A. L. , Giussani D. A.
Published: June 2005

In sheep, direct fetal treatment with dexamethasone alters basal cardiovascular function and the cardiovascular response to acute hypoxaemia. However, in human clinical practice, dexamethasone is administered to the mother, not to the fetus. Hence, this study investigated physiological responses to acute hypoxaemia in fetal sheep during and following maternal treatment with dexamethasone in doses and at dose intervals used in human clinical practice. Under anaesthesia, 18 fetal sheep were instrumented with vascular and amniotic catheters, a carotid flow probe and a femoral flow probe at 118 days gestation (term ca 145 days). Following 6 days recovery at 124 days gestation, 10 ewes received dexamethasone (2 × 12 mg daily i.m. injections in saline). The remaining animals were saline‐injected as age‐matched controls. Two episodes of hypoxaemia (H) were induced in all animals by reducing the maternal FIO2for 1 h (H1, 8 h after the second injection; H2, 3 days after the second injection). In fetuses whose mothers received saline, hypoxaemia induced significant increases in fetal arterial blood pressure, carotid blood flow and carotid vascular conductance and femoral vascular resistance, significant falls in femoral blood flow and femoral vascular conductance and transient bradycardia. These cardiovascular responses were accompanied by a fall in arterial pH, increases in blood glucose and blood lactate concentrations and increased plasma concentrations of catecholamines. In fetuses whose mothers were treated with dexamethasone, bradycardia persisted throughout hypoxaemia, the magnitude of the femoral vasoconstriction, the glycaemic, lactacidaemic and acidaemic responses and the plasma concentration of neuropeptide Y (NPY) were all enhanced during H1. However, during H2, all of these physiological responses were similar to saline controls. In dexamethasone fetuses, the increase in plasma adrenaline was attenuated during H1 and the increase in carotid vascular conductance during hypoxaemia failed to reach statistical significance both during H1 and during H2. These data show that maternal treatment with dexamethasone in doses and intervals used in human obstetric practice modified the fetal cardiovascular, metabolic and endocrine defence responses to acute hypoxaemia. Furthermore, dexamethasone‐induced alterations to these defences depended on whether the hypoxaemic challenge occurred during or following maternal dexamethasone treatment.
Endothermic force generation, temperature‐jump experiments and effects of increased [MgADP] in rabbit psoas muscle fibres
Authors: Coupland M. E. , Pinniger G. J. , Ranatunga K. W.
Published: June 2005

We studied, by experiment and by kinetic modelling, the characteristics of the force increase on heating (endothermic force) in muscle. Experiments were done on maximally Ca2+‐activated, permeabilized, single fibres (length ∼2 mm; sarcomere length, 2.5 μm) from rabbit psoas muscle; [MgATP] was 4.6 mm, pH 7.1 and ionic strength was 200 mm. A small‐amplitude (∼3°C) rapid laser temperature‐jump (0.2 ms T‐jump) at 8–9°C induced a tension rise to a new steady state and it consisted of two (fast and slow) exponential components. The T‐jump‐induced tension rise became slower as [MgADP] was increased, with half‐maximal effect at 0.5 mm[MgADP]; the pre‐ and post‐T‐jump tension increased ∼20% with 4 mm added [MgADP]. As determined by the tension change to small, rapid length steps (<1.4%L0 complete in <0.5 ms), the increase of force by [MgADP] was not associated with a concomitant increase of stiffness; the quick tension recovery after length steps (Huxley–Simmons phase 2) was slower with added MgADP. In steady‐state experiments, the tension was larger at higher temperatures and the plot of tension versus reciprocal absolute temperature was sigmoidal, with a half‐maximal tension at 10–12°C; the relation with added 4 mm MgADP was shifted upwards on the tension axis and towards lower temperatures. The potentiation of tension with 4 mm added MgADP was 20–25% at low temperatures (∼5–10°C), but ∼10% at the physiological temperatures (∼30°C). The shortening velocity was decreased with increased [MgADP] at low and high temperatures. The sigmoidal relation between tension and reciprocal temperature, and the basic effects of increased [MgADP] on endothermic force, can be qualitatively simulated using a five‐step kinetic scheme for the crossbridge/A‐MATPase cycle where the force generating conformational change occurs in a reversible step before the release of inorganic phosphate (Pi), it is temperature sensitive (Q10 of ∼4) and the release of MgADP occurs by a subsequent, slower, two‐step mechanism. Modelling shows that the sigmoidal relation between force and reciprocal temperature arises from conversion of preforce‐generating (A‐M.ADP.Pi) states to force‐bearing (A‐M.ADP) states as the temperature is raised. A tension response to a simulated T‐jump consists of three (one fast and two slow) components, but, by combining the two slow components, they could be reduced to two; their relative amplitudes vary with temperature. The model can qualitatively simulate features of the tension responses induced by large‐T‐jumps from low starting temperatures, and those induced by small‐T‐jumps from different starting temperatures and, also, the interactive effects of Pi and temperature on force in muscle fibres.
Stimulation‐induced changes in lower limb corticomotor excitability during treadmill walking in humans
Authors: Stinear James W. , Hornby T. George
Published: June 2005

Magnetic stimulation of human primary motor cortex (M1) paired with electrical stimulation of a peripheral motor nerve has been used to produce a lasting modulation of corticomotor (CM) excitability. This ‘paired associative stimulation’ (PAS) protocol has been used to induce bidirectional changes in excitability in upper limb CM pathways. The present study tested the hypothesis that temporally dependent PAS applied to the common peroneal nerve during the swing phase of walking would induce bidirectional changes in CM excitability consistent with the Hebbian principle of activity‐dependent plasticity. Fourteen subjects with no known neurological disorder participated in two data collection sessions each. PAS was delivered as a single block of 120 pairs of stimuli delivered in a 10 min period during treadmill walking at 4.0 km h−1. Changes in CM excitability were assessed by examining the size of motor potentials evoked by transcranial magnetic stimulation prior to and following PAS. Tibialis anterior motor‐evoked potentials amplitude increased to 121% over baseline when the magnetic stimulus was delivered over M1 after the estimated arrival time of the afferent volley in sensorimotor cortex and decreased to 83% of baseline when the magnetic stimulus was delivered prior to the estimated afferent volley arrival. This extent of modulation was undiminished following a further 10 min period of walking without stimulation. The temporal nature of the bidirectional effects following PAS, their rapid evolution and subsequent persistence supported the study's hypothesis and were similar to the effects described by others in quiescent muscles of the upper limb.
Effects of calcineurin activation on insulin‐, AICAR‐ and contraction‐induced glucose transport in skeletal muscle
Authors: Ryder Jeffrey W. , Long Yun Chau , Nilsson Elisabeth , Mahlapuu Margit , Zierath Juleen R.
Published: June 2005

Skeletal muscle is composed of fast‐ and slow‐twitch fibres with distinctive physiological and metabolic properties. The calmodulin‐activated serine/threonine protein phosphatase calcineurin activates fast‐ to slow‐twitch skeletal muscle remodelling through the induction of the slow‐twitch skeletal muscle fibre gene expression programme, thereby enhancing insulin‐stimulated glucose uptake and offering protection against dietary‐induced insulin resistance. Given the profound influence of skeletal muscle fibre type on insulin‐mediated responses, we determined whether the fast‐ to slow‐twitch fibre‐type transformation leads to alterations in insulin‐independent glucose uptake in transgenic mice expressing a constitutively active form of calcineurin (MCK‐CnA* mice). We determined whether skeletal muscle remodelling by activated calcineurin alters glucose transport in response to the AMP‐activated protein kinase (AMPK) activator 5‐aminoimidazole‐4‐carboxamide‐β‐d‐ribofuranoside (AICAR) or muscle contraction, two divergent insulin‐independent activators of glucose transport. While insulin‐stimulated glucose transport was increased 52%, the AICAR effect on glucose transport was 27% lower in MCK‐CnA* mice versus wild‐type mice (P < 0.05). In contrast, glucose transport was similar between genotypes after in vitro muscle contraction. Fibre‐type transformation was associated with increased AMPKγ1, decreased AMPKγ3 and unchanged AMPKγ2 protein expression between MCK‐CnA* and wild‐type mice (P < 0.05). The loss of AICAR‐mediated glucose uptake is coupled to changes in the AMPK isoform expression, suggesting fibre‐type dependence of the AICAR responses on glucose uptake. In conclusion, improvements in skeletal muscle glucose transport in response to calcineurin‐induced muscle remodelling are limited to insulin action.
Synergistic interactions between Ca2+ entries through L‐type Ca2+ channels and Na+–Ca2+ exchanger in normal and failing rat heart
Authors: Viatchenko‐Karpinski Serge , Terentyev Dmitry , Jenkins Leigh Ann , Lutherer Lorenz O. , Györke Sandor
Published: June 2005

We used confocal Ca2+ imaging and the patch‐clamp technique to investigate the interplay between Ca2+ entries through L‐type Ca2+ channels (LCCs) and reverse‐mode Na+–Ca2+ exchange (NCX) in activating Ca2+‐induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) in cardiac myocytes from normal and failing rat hearts. In normal myocytes exposed to N(6),2′‐O‐dibutyryl adenosine‐3′,5′‐cyclic monophosphate (db‐cAMP, membrane‐permeable form of cAMP), the bell‐shaped voltage dependence of cytosolic Ca2+ transients was dramatically broadened due to activation of SR Ca2+ release at high membrane potentials (30–120 mV). This broadening of Ca2+‐transient voltage dependence could be prevented by KB‐R7943, an inhibitor of the reverse‐mode NCX. Trans‐sarcolemmal Ca2+ entries were measured fluorometrically in myocytes during depolarizing steps to high membrane potentials. The total Ca2+ entry (ΔFTot) was separated into two Ca2+ entry components, LCC‐mediated (ΔFLCC) and NCX‐mediated (ΔFNCX), by exposing the cells to the specific inhibitors of LCCs and reverse‐mode NCX, nifedipine and KB‐R7943, respectively. In the absence of protein kinase A (PKA) stimulation the amplitude of the Ca2+‐inflow signal (ΔFTot) corresponded to the arithmetic sum of the amplitudes of the KB‐R7943‐ and nifedipine‐resistant components (ΔFTot=ΔFLCC+ΔFNCX). PKA activation resulted in significant increases in ΔFTot and ΔFLCC. Paradoxically, ΔFTot became ∼threefold larger than the sum of the ΔFNCX and ΔFLCC components. In myocytes from failing hearts, stimulation of PKA failed to induce a shift in Ca2+ release voltage dependence toward more positive membrane potentials. Although the total and NCX‐mediated Ca2+ entries were increased again, ΔFTot did not significantly exceed the sum of ΔFLCC and ΔFNCX. We conclude that the LCC and NCX Ca2+‐entry pathways interact synergistically to trigger SR Ca2+ release on depolarization to positive membrane potentials in PKA‐stimulated cardiac muscle. In heart failure, this new form of Ca2+ release is diminished and may potentially account for the compromised contractile performance and reduced functional reserve in failing hearts.
Effects of two protocols of intermittent hypoxia on human ventilatory, cardiovascular and cerebral responses to hypoxia
Authors: Foster Glen E. , McKenzie Donald C. , Milsom William K. , Sheel A. William
Published: June 2005

We determined the ventilatory, cardiovascular and cerebral tissue oxygen response to two protocols of normobaric, isocapnic, intermittent hypoxia. Subjects (n= 18, male) were randomly assigned to short‐duration intermittent hypoxia (SDIH, 12% O2 separated by 5 min of normoxia for 1 h) or long‐duration intermittent hypoxia (LDIH, 30 min of 12% O2). Both groups had 10 exposures over a 12 day period. The hypoxic ventilatory response (HVR) was measured before each daily intermittent hypoxia exposure on days 1, 3, 5, 8, 10 and 12. The HVR was measured again 3 and 5 days after the end of intermittent hypoxia. During all procedures, ventilation, blood pressure, heart rate, arterial oxyhaemoglobin saturation and cerebral tissue oxygen saturation were measured. The HVR increased throughout intermittent hypoxia exposure regardless of protocol, and returned to baseline by day 17 (day 1, 0.84 ± 0.50; day 12, 1.20 ± 1.01; day 17, 0.95 ± 0.58 l min−1%SaO2−1; P < 0.01). The change in systolic blood pressure sensitivity (r=+0.68; P < 0.05) and the change in diastolic blood pressure sensitivity (r=+0.73; P < 0.05) were related to the change in HVR, while the change in heart rate sensitivity was not (r=+0.32; NS). The change in cerebral tissue oxygen saturation sensitivity to hypoxia was less on day 12, and returned to baseline by day 17 (day 1, −0.51 ± 0.13; day 12, −0.64 ± 0.18; day 17, −0.51 ± 0.13; P < 0.001). Acute exposure to SDIH increased mean arterial pressure (+5 mmHg; P < 0.01), but LDIH did not (P > 0.05). SDIH and LDIH had similar effects on the ventilatory and cardiovascular response to acute progressive hypoxia and hindered cerebral oxygenation. Our findings indicate that the vascular processes required to control blood flow and oxygen supply to cerebral tissue in a healthy human are hindered following exposure to 12 days of isocapnic intermittent hypoxia.
Descending vasa recta pericytes express voltage operated Na+ conductance in the rat
Authors: Zhang Zhong , Cao Chunhua , Lee‐Kwon Whaseon , Pallone Thomas L.
Published: June 2005

We studied the properties of a voltage‐operated Na+ conductance in descending vasa recta (DVR) pericytes isolated from the renal outer medulla. Whole‐cell patch‐clamp recordings revealed a depolarization‐induced, rapidly activating and rapidly inactivating inward current that was abolished by removal of Na+ but not Ca+ from the extracellular buffer. The Na+ current (INa) is highly sensitive to tetrodotoxin (TTX, Kd= 2.2 nm). At high concentrations, mibefradil (10 μm) and Ni+ (1 mm) blocked INa. INa was insensitive to nifedipine (10 μm). The L‐type Ca+ channel activator FPL‐64176 induced a slowly activating/inactivating inward current that was abolished by nifedipine. Depolarization to membrane potentials between 0 and 30 mV induced inactivation with a time constant of ∼1 ms. Repolarization to membrane potentials between −90 and −120 mV induced recovery from inactivation with a time constant of ∼11 ms. Half‐maximal activation and inactivation occurred at −23.9 and −66.1 mV, respectively, with slope factors of 4.8 and 9.5 mV, respectively. The Na+ channel activator, veratridine (100 μm), reduced peak inward INa and prevented inactivation. We conclude that a TTX‐sensitive voltage‐operated Na+ conductance, with properties similar to that in other smooth muscle cells, is expressed by DVR pericytes.
Direct inhibition of substantia gelatinosa neurones in the rat spinal cord by activation of dopamine D2‐like receptors
Authors: Tamae Akihiro , Nakatsuka Terumasa , Koga Kohei , Kato Go , Furue Hidemasa , Katafuchi Toshihiko , Yoshimura Megumu
Published: June 2005

Dopaminergic innervation of the spinal cord is largely derived from the brain. To understand the cellular mechanisms of antinociception mediated by descending dopaminergic pathways, we examined the actions of dopamine (DA) on nociceptive transmission by using behavioural studies and whole‐cell patch‐clamp recordings from substantia gelatinosa (SG) neurones in the spinal cord. Intrathecal administration of DA increased the mechanical nociceptive threshold and this effect was mimicked by a D2‐like receptor agonist, quinpirole, but not by a D1‐like receptor agonist, SKF 38393. In current‐clamp mode of patch‐clamp recordings, bath application of DA hyperpolarized the membrane potential of SG neurones and suppressed action potentials evoked by electrical stimulation of a dorsal root. In voltage‐clamp mode, DA induced an outward current that was resistant to TTX, was blocked by the addition of Cs+ or GDP‐β‐S in the pipette solution, and was inhibited in the presence of Ba+. The DA‐induced current reversed its polarity at a potential close to the equilibrium potential of the K+ channel calculated from the Nernst equation. The DA‐induced outward current was mimicked by quinpirole, but not by SKF 38393. The DA‐induced outward current was suppressed by a D2‐like receptor antagonist, sulpiride, but not by a D1‐like receptor antagonist, SCH 23390. In contrast, DA did not cause any significant change in amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). These results indicate that DA mainly acts on postsynaptic SG neurones to induce an outward current via G‐protein‐mediated activation of K+ channels through D2‐like receptors. This may be a possible mechanism for antinociception by the descending dopaminergic pathway.