Determination of the Maximum Rate of Eccrine Sweat Glands’ Ion Reabsorption Using the Galvanic Skin Conductance to Local Sweat Rate Relationship

Purpose The purpose of the present study was to develop and describe a simple method to evaluate the rate of ion reabsorption of eccrine sweat glands in human using the measurement of galvanic skin conductance (GSC) and local sweating rate (SR). This purpose was investigated by comparing the SR threshold for increasing GSC with following two criteria of sweat ion reabsorption in earlier studies such as 1) the SR threshold for increasing sweat ion was at approximately 0.2 to 0.5 mg/cm2/min and 2) exercise-heat acclimation improved the sweat ion reabsorption ability and would increase the criteria 1. Methods Seven healthy non-heat-acclimated male subjects received passive heat treatment both before and after 7 days of cycling in hot conditions (50% maximum oxygen uptake, 60 min/day, ambient temperature 32°C, and 50% relative humidity). Results Subjects became partially heat-acclimated, as evidenced by the decreased end-exercise heart rate (p<0.01), rate of perceived exhaustion (p<0.01), and oesophageal temperature (p=0.07), without alterations in whole-body sweat loss, from the first to the last day of training. As hypothesised, we confirmed that the SR threshold for increasing GSC was near the predicted SR during passive heating before exercise heat acclimation, and increased significantly after training (0.19 ± 0.09 to 0.32 ± 0.10 mg/cm2/min, p<0.05). Conclusions The reproducibility of sweat ion reabsorption by the eccrine glands in the present study suggests that the relationship between GSC and SR can serve as a new index for assessing the maximum rate of sweat ion reabsorption of eccrine sweat glands in humans.

Amino acid transport : the special case of a H/L-glutamate cotransport system in Asparagus sprengeri mesophyll cells /

The addition of L-Glutamate (L-GLU) and L-Hethionine ~ulfoximine (L-HSO) to mechanically isolated. photosynthetically competent, Asparagus sprengeri mesophyll cells ~u~pended in 1mM CaS04 cau~ed an immediate transient alkalinization of the cell su~pension medium in both the light and dark. The alkalinization response was specific and stereospecific as none of the L-isomers of the other 19 protein amino acids tested or D-GLU gave this response. Uptake of 14C-L-GLU was stimulated by the light. The addition of non-radioactive L-GLU. or L-GLU analogs together with 14C-L-GLU showed that only L-GLU and L-HSO stimulated alkalinization whilst inhibiting the uptake of 14C-L-GLU. Both the L-GLU dependent alkalinization and the upt~ke of 14C-L-GLU were stimulated when the external pH was decreased from 6.5 to 5.5. Increasing external K+ concentrations inhibited the uptake of 14C-L-GLU. Fusicoccin (FC) stimulated uptake. The L-GLU dependent alkalinization re~ponse exhibited monophasic saturation kinetics while the uptake of 14C-L-GLU exhibited biphasic saturation kinetics. In addition to a saturable component. the uptake kinetics also showed a linear component of uptake. Addition of L-GLU and L-MSO caused internal acidification of the cell as measured by a change in the distribution of 14C-DMO. There was no change in K+ efflux when L-GLU was added. A H+ to L-GLUinflux stoichiometry of 3:1 wa~ mea~ured at an external I.-GLU concentration of O.5mM and increased with increasing external 13 L-QLU concentration. Metabolism of L-GLU was detected manometrlcally by observing an increase in COa evolution upon the addition of L-QLU and by detection of i*C02 evolution upon the addition of »*C-L-GLU. »*C02 evolution was higher in the dark than in the light. The data are consistent with the operation of a H+/L-QLO cotransport system. The data also show that attempts to quantify the stoichlometry of the process were complicated by the metabolism of L-GLU.

Cytosolic calcium levels and stress induced y-amino butyrate synthesis in asparagus mesophyll cells

Numerous investigations have demonstrated large increases in y-amino butyrate (GABA) levels in response to a variety of stresses such as touch or cold shock (Wallace et ale 1984) Circumstantial evidence indicating a role of Ca2 + in these increases includes elevated Ca2+ levels in response to touch and cold shock (Knight et ale 1991), and the demonstration of a calmodulin binding domain on glutamate decarboxylase (GAD), the enzyme responsible for GABA synthesis (Baum et al 1993) In the present study the possible role of Ca2+ and calmodulin in stimulation of GAD and subsequent GABA accumulation was examined using asparagus mesophyll cells. Images of cells loaded with the Ca2+ indicator Fluo-3 revealed a rapid and transient increase in cytosolic Ca2+ in response to cold shock. GABA levels increased by 106% within 15 min. of cold shock. This increase was inhibited 70% by the calmodulin antagonist W7, and 42% by the Ca2+ channel blocker La3+.. Artificial elevation of intracellular Ca2+ by the Ca2+ionophore A23187 resulted in an 61% increase in GABA levels. Stimulation of GABA synthesis by ABA resulted in an 83% increase in GABA levels which was inhibited 55% by W7. These results support the hypothesis that cold shock stimulates Ca2+ entry into the cytosol of the cells which results in Ca2+/calmodulin mediated activation of GAD and consequent GABA synthesis.

Electrogenic proton/L-glutamate symport in isolated asparagus sprengeri mesophyll cell

Medium' alkaliniiation occurred -lipon the addition of L-Glu to mechanically isolated Asparagus sprenger-i mesophyll cells suspended in 1 mM CaS04. Alkalinization resulted from the coupled entry of H+ and L-Glu anion into the cells. This H+ IL-Glu symport did not stimulate K+ efflux. K+ efflux has been observed during H~ lamino acid symport in other systems. The stimulation of K+ efflux by proton coupled symport is regarded as an indicator of a plasma membrane depolarizing electrogenic symport process. H+ IL-Glu symport in Asparagus sprengerimesophyl1 cells was investigated to determine whether or not the process was electrogenic. The rate of uptake of 0.25 11M 3H-MTPP+ ( Methyltriphenylphosphonium, methyl-3H ) is a probe for monitoring changes in the membrane potential. 3HMTPP+ uptake was reduced by K+ or CCCP, agents known to depolarize the membrane potential. Uptake of 3H-MTPP+ was also inhibited by L-Glu but not by D-Glu. Conversely, 10 mM external MTPP+ inhibited the uptake of 14C-U-LGlu. Simultaneous measurements of the rates of 14C-U-L-Glu uptake and L-Glu dependent H+ influx showed that the molar stoichiometry of H+ IL-Glu symport was 2 to 1. K+ or Na+ stimulated H+ efflux was completely inhibited by DCCD, DES, oligomycin and antimycin reagents which inhibit ATP driven H+ efflux. The H+ efflux \Vas also stimulate.d by the weak acids, butyric acid and acetic acid, which are known fo-aCidify the cytoplasm. This weak acid stimulated H+ efflux was also completely inhibited by oligomycin. It was calculated that net L-Glu dependent H+ influx increased by 100% in the presence of oligomycin and that despite net medium alkalinization H+ IL-Glu symport stimulates ATP dependent H+ efflux. 11 The data presented in this study indicate that H+ IL-Glu symport is electrogenic. The data also show that ATP dependent Ht efflux rather than K+ efflux is the- process compensating for thi~ electrogenic H+ IL-Glu symport.

GABA accumulation and the hypersensitive response in isolated mesophyll cells treated with the G protein activator mastoparan

GABA (y-amino butyric acid) is a non-protein amino acid synthesized through the a-decarboxylation of L-glutamate. This reaction is catalyzed by L-glutamate decarboxylase (EC 4.1.1.15), a cytosolic Ca2+/calmodulin-stimulated enzyme. The purpose of this study is to determine whether or not GABA accumulation is associated with the hypersensitive response of isolated Asparagus sprengeri mesophyll cells. The addition of 25 J.lM mastoparan, a G protein activator, to suspensions of isolated asparagus mesophyll cells significantly increased GABA synthesis and cell death. Cell death was assessed using Evan's blue dye and fluorescein diacetate tests for cell viability. In addition, mastoparan stimulated pH-dependent alkalinization of the external medium, and a rapid and large 02 consumption followed by a loss of photosynthetic activity. The rate of 02 consumption and the net decrease in 02 in the dark was enhanced by light. The inactive mastoparan analogue Mas17 was ineffective in stimulating GABA accumulation, medium alkalinization, 02 uptake and cell death. Accumulation of H202 in response tomastoparan was not detected, however, mastoparan caused the cell-dependent degradation of added H202. The pH dependence of mastoparan-stimulated alkalinization suggests cellular electrolyte leakage, while the consumption of 02 corresponds to the oxidative burst in which 02 at the cell surface is reduced to form various active oxygen species. The results are indicative of the "hypersensitive response" of plants to pathogen attack, namely, the death of cells in the locality of pathogen invasion. The data are compatible with a model in which mastoparan triggers G protein activity, subsequent intracellular signal transduction pathway/s, and the hypersensitive response. It is postulated that the physiological elicitation of the hypersensitive response involves G protein signal transduction. The synthesis of GABA during the hypersensitive response has not been documented previously; however the role/s of GABA synthesis in the hypersensitive response, if any, remain unclear.

L-Glutamate uptake, decarboxylation to -aminobutyric acid and GABA efflux in isolated Asparagus sprengeri mesophyll cells

Addition of L-glutamate caused alkalinization of the medium surrounding Asparagus spreng.ri mesophyll cells. This suggests a H+/L-glutmate symport uptake system for L-glutamate. However stoichiometries of H+/L-glutamate symport into Asparagus cells were much higher than those in other plant systems. Medium alkalinization may also result from a metabolic decarboxylation process. Since L-glutmate is decarboxylated to r-amino butyric acid (SABA) in this system, the origin of medium alkalinization was reconsidered. Suspensions of mechanically isolated and photosyntheically competent Asparagus sprengeri mesophyll cells were used to investigate the H+/L-glutamate symport system, SABA production, GABA transport, and the origin of L-glutamate dependent medium alkalinization. The major results obtained are summarized as follows: 1. L-Glutamate and GABA were the second or third most abundant amino acids in these cells. Cellular concentrations of L-glutamate were 1.09 mM and 1.31 mM in the light and dark, respectively. Those of SABA were 1.23 mM and 1.17 mM in the light and dark, respectively. 2. Asparagine was the most abundant amino acid in xylem sap and comprised 54 to 68 1. of the amino acid pool on a molar basis. GABA was the second most abundant amino acid and represented 10 to 11 1. of the amino acid pool. L-Slutamate was a minor component. 3. A 10 minute incubation with 1 mM L-glutamate increased the production of GABA in the medium by 2,743 7. and 2,241 7. in the light and dark, respectively. 4. L-Glutamate entered the cells prior to decarboxylation. 5. There was no evidence for a H+/GABA symport process • 6. GABA was produced by loss of carbon-1 of L-glutamate. 7. The specific activity of newly synthesized labeled GABA suggests that it is not equilibrated with a storage pool of GABA. 8. The mechanism of GABA efflux appears to be a passive process. 9. The evidence indicates that the origin of L-glutamate dependent medium alkalinization is a H+/L-glutamate symport not an extracellular decarboxylation. The possible role of GABA production in regulating cytoplasmic pH and L-glutamate levels during rapid electrogenic H+/L-glutamate symport is discussed.

Heat transfer between nanoparticles: Thermal conductance for near-field interactions

We analyze the heat transfer between two nanoparticles separated by a distance lying in the near-field domain in which energy interchange is due to the Coulomb interactions. The thermal conductance is computed by assuming that the particles have charge distributions characterized by fluctuating multipole moments in equilibrium with heat baths at two different temperatures. This quantity follows from the fluctuation-dissipation theorem for the fluctuations of the multipolar moments. We compare the behavior of the conductance as a function of the distance between the particles with the result obtained by means of molecular dynamics simulations. The formalism proposed enables us to provide a comprehensive explanation of the marked growth of the conductance when decreasing the distance between the nanoparticles.

Ultrastructural alterations in mesophyll and bundle sheath chloroplasts of two maize cultivars in response to chilling at high irradiance

Maize (Zea mays L.) seedlings of two cultivars (cv. Bastion adapted to W. Europe, and cv. Batan 8686 adapted to the highlands of Mexico), raised in a glasshouse (19-25 degrees C), were transferred to 4.5 or 9 degrees C at photon flux density (PPFD) of 950 mu mol m(-2) s(-1) with 10-h photoperiod for 58 h and then allowed to recover at 22 degrees C for 16 h (14 h dark and 2 h at PPFD of 180 mu mol m(-2) s(-1)). The ultrastructural responses after 4 h or 26 h at 4.5 degrees C were the disappearance of starch grains in the bundle sheath chloroplasts and the contraction of intrathylakoid spaces in stromal thylakoids of the mesophyll chloroplasts. At this time, bundle sheath chloroplasts of cv. Batan 8686 formed peripheral reticulum. Prolonged stress at 4.5 degrees C (50 h) caused plastid swelling and the dilation of intrathylakoid spaces, mainly in mesophyll chloroplasts. Bundle sheath chloroplasts of cv. Batan 8686 seedlings appeared well preserved in shape and structure. Batan 8686 had also higher net photosynthetic rates during chilling and recovery than Bastion. Extended leaf photobleaching developed during the recovery period after chilling at 4.5 degrees C. This was associated with collapsed chloroplast envelopes, disintegrated chloroplasts and very poor staining.

Zea mays annexins modulate cytosolic free Ca2+ and generate a Ca2+-permeable conductance

Regulation of reactive oxygen species and cytosolic free calcium ([Ca2+](cyt)) is central to plant function. Annexins are small proteins capable of Ca2+-dependent membrane binding or membrane insertion. They possess structural motifs that could support both peroxidase activity and calcium transport. Here, a Zea mays annexin preparation caused increases in [Ca2+] cyt when added to protoplasts of Arabidopsis thaliana roots expressing aequorin. The pharmacological profile was consistent with annexin activation (at the extracellular plasma membrane face) of Arabidopsis Ca2+-permeable nonselective cation channels. Secreted annexins could therefore modulate Ca2+ influx. As maize annexins occur in the cytosol and plasma membrane, they were incorporated at the intracellular face of lipid bilayers designed to mimic the plasma membrane. Here, they generated an instantaneously activating Ca2+-permeable conductance at mildly acidic pH that was sensitive to verapamil and Gd3+ and had a Ca2+-to-K+ permeability ratio of 0.36. These results suggest that cytosolic annexins create a Ca2+ influx pathway directly, particularly during stress responses involving acidosis. A maize annexin preparation also demonstrated in vitro peroxidase activity that appeared independent of heme association. In conclusion, this study has demonstrated that plant annexins create Ca2+-permeable transport pathways, regulate [Ca2+] cyt, and may function as peroxidases in vitro.

Stomatal conductance and not stomatal density determines the long-term reduction in leaf transpiration of poplar in elevated CO2

Using a free-air CO2 enrichment (FACE) experiment, poplar trees (Populus · euramericana clone I214) were exposed to either ambient or elevated [CO2] from planting, for a 5-year period during canopy development, closure, coppice and re-growth. In each year, measurements were taken of stomatal density (SD, number mm2) and stomatal index (SI, the proportion of epidermal cells forming stomata). In year 5, measurements were also taken of leaf stomatal conductance (gs, lmol m2 s1), photosynthetic CO2 fixation (A, mmol m2 s1), instantaneous water-use efficiency (A/E) and the ratio of intercellular to atmospheric CO2 (Ci:Ca). Elevated [CO2] caused reductions in SI in the first year, and in SD in the first 2 years, when the canopy was largely open. In following years, when the canopy had closed, elevated [CO2] had no detectable effects on stomatal numbers or index. In contrast, even after 5 years of exposure to elevated [CO2], gs was reduced, A/E was stimulated, and Ci:Ca was reduced relative to ambient [CO2]. These outcomes from the long-term realistic field conditions of this forest FACE experiment suggest that stomatal numbers (SD and SI) had no role in determining the improved instantaneous leaf-level efficiency of water use under elevated [CO2]. We propose that altered cuticular development during canopy closure may partially explain the changing response of stomata to elevated [CO2], although the mechanism for this remains obscure.

Arabidopsis annexin1 mediates the radical-activated plasma membrane Ca2+ - and K+ -permeable conductance in root cells

Plant cell growth and stress signaling require Ca2+ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH_. In root cells, extracellular OH_ activates a plasma membrane Ca2+-permeable conductance that permits Ca2+ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca2+-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH_-activated Ca2+- and K+-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca2+ in response to OH_. An OH_-activated Ca2+ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca2+-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca2+ in plants.

Conductance of amyloid β based peptide filaments: structure–function relations.

Controlling the morphology of self-assembled peptide nanostructures, particularly those based on amyloid peptides, has been the focus of intense research. In order to exploit these structures in electronic applications, further understanding of their electronic behavior is required. In this work, the role of peptide morphology in determining electronic conduction along self-assembled peptide nanofilament networks is demonstrated. The peptides used in this work were based on the sequence AAKLVFF, which is an extension of a core sequence from the amyloid b peptide. We show that the incorporation of a non-natural amino acid, 2-thienylalanine, instead of phenylalanine improves the obtained conductance with respect to that obtained for a similar structure based on the native sequence, which was not the case for the incorporation of 3-thienylalanine. Furthermore, we demonstrate that the morphology of the self-assembled structures, which can be controlled by the solvent used in the assembly process, strongly affects the conductance, with larger conduction obtained for a morphology of long, straight filaments. Our results demonstrate that, similar to natural systems, the assembly and folding of peptides could be of great importance for optimizing their function as components of electronic devices. Hence, sequence design and assembly conditions can be used to control the performance of peptide based structures in such electronic applications.