Electrical tolerance (breakdown) of theChara corallina plasmalemma: I. Necessity of Ca2+

Summary The relationship between the external Ca²⁺ concentrations [Ca²⁺]₀ and the electrical tolerance (breakdown) in theChara plasmalemma was investigated. When the membrane potential was negative beyond –350–400 mV (breakdown potential, BP), a marked inward current was observed, which corresponds to the so-called punch-through (H.G.L. Coster,Biophys. J. 5:669–686, 1965). The electrical tolerance of theChara plasmalemma depended highly on [Ca²⁺]₀. Increasing [Ca²⁺]₀ caused a more negative and decreasing it caused a more positive shift of BP. BP was at about –700 mV in 200 M La³⁺ solution. [Mg²⁺]₀ depressed the membrane electrical tolerance which was supposed to be due to competition with Ca²⁺ at the Ca²⁺ binding site of the membrane. Such a depressive effect of Mg²⁺ was almost masked when the [Ca²⁺]₀/[Mg²⁺]₀ ratio was roughly beyond 2.     

Diurnal cycle of carbon isotope ratio in soil CO2 in various ecosystems

Our investigations of diurnal variations of the ¹³C/¹²C ratio and CO₂ content in soil air were carried out in three environments during periods of high biosphere activity. It has been observed that diurnal variation of CO₂ concentration is negatively correlated ¹³. Particularly great variations occurred at shallow soil depths (10–30 cm) when the plant cover activity was high while the soil temperature was rather low. Under such conditions the ¹³ variations had the magnitude of 4, while the CO₂ concentration varied more than doubly. The maximum of the ¹³C/¹²C ratlo and the minimum of the CO₂ concentration in a cultivated field with winter wheat took place in the afternoon, whereas in deciduous forest similar patterns were observed at dawn. In these cases soil temperatures at 10 cm depths varied less than 2°C. Hence, under wheat the variation in root respiration rate seem to be the main reason of the recorded varations. In an uncultivated grass-field during the hottest period in summer we did not measure any distinct variations of CO₂ properties in spite of the fact that soil temperature varied up to 5°C. This might be due to dominant microbial respiration at the high soil temperature, which exceeded 20°C.     

The effects of hydrostatic pressure on the low-K m GTPase in brain membranes from two congeneric marine fishes

To investigate, the effects of hydrostatic pressure on transmembrane signaling in cold-adapted marine fishes, we examined the high-affinity GTPase activity in two congeneric marine fishes, Sebastolobus alascanus and S. altivelis. In brain membranes there are two GTPase activities, one with a low K _m and one with a high K _m for GTP. The high-affinity GTPase activity, characteristic of the subunits of the guanine nucleotide binding protein pool, was stimulated by the A₁ adenosine receptor agonists N ⁶(R-phenylisopropyl)adenosine and N ⁶-cyclopentyladenosine, and the muscarinic cholinergic agonist carbamyl choline. Pertussis toxin-catalyzed ADP-ribosylation of the membranes for 2 h at 5°C prior to the GTPase assay decreased the basal GTPase activity 30–40% and abolished N ⁶ (R-phenylisopropyl)adenosine stimulation of GTP hydrolysis. Basal high-affinity hydrolysis of GTP, measured at 0.3 mol·1^-1GTP, was stimulated 22% in both species by 340 atm pressure. At 340 atm pressure, the apparent K _m of GTP is decreased approximately 10% in each of the species, and the V _max values are increased 11 and 15.9% in S. alascanus and S. altivelis, respectively. The apparent volume changes associated with the decreased K _m of GTP and the increased V _max ranged from-7.0 to-9.9 ml·mol^-1. Increased pressure markedly decreased the efficacy of N ⁶ (R-phenylisopropyl) adenosine, N ⁶-cylcopentyladenosine and carbamyl choline in stimulating GTPase activity. The effects of increased hydrostatic pressure on transmembrane signal transduction by the A₁ adenosine receptor-inhibitory guanine nucleotide binding protein-adenylyl cyclase system may stem, at least in part, from pressure-increased GTP hydrolysis and the concomitant termination of inhibitory signal transduction.Abbreviations [³H] DPCPX ³H cyclopentyl-1, 3-dipropylxanthine - AppNHp 5-adenylylimidodiphosphate - cpm counts per minute - CPA N ⁶-cyclopentyladenosine - EDTA ethylenediaminetetra acetic acid - EGTA ethyleneglycol-bis (-aminoethylether) N, N, N, N-totra-acctic acid - G protein guanine nucleotide binding protein - G_i inhibitory G protein - G_o other G protein, common in brain membranes - G_s stimulatory G protein - GTPase guanosine triphosphatase - K _i inhibition constant - K _m Michaelis constant - pK _a log of the dissociation constant - R-PIA N ⁶ (R-phenylisopropyl) adenosine - TRIS tris[hydroxymethyl]aminomethane - V_max maximal velocity - [-³²P]GTP [-³²P] guanosine 5-triphosphate (tetra (triethylammonium) salt)     

Effects of pasture introduction on soil CO2 emissions during the dry season in the state of Rondônia,Brazil

Soil CO₂ evolution rates, soil temperatures and moisture were measured during the dry season in two forest-to-pasture chronosequences in Rondônia, Brazil. The study included pastures ranging from 3 to 80 years-old. Mean dry-season CO₂ evolution from the forest in chronosequence 1, 88.8 mg CO₂-C m^–2h^–1 was lower than from the pastures which ranged from 111 to 158 mg CO₂-C m^–2h^–1. We found that temperature was not a good predictor of CO₂ emissions from pasture but that there was a significant relationship (r = 0.72,p < 0.05) between soil moisture and pasture emissions. The ¹³C of the soil CO₂ emissions also was measured on chronosequence I; ¹³C of the CO₂ emitted from the C3 forest was –29.43%. Pasture¹³CO₂ values increased from –17.91%. in the 3 year-old pasture to –12.86% in the 80 year-old, reflecting the increasing C₄ inputs with pasture age. Even in the youngest (3 year-old) pasture, 70 percent of the CO₂ evolved originated from C₄ pasture-derived carbon.     

Acyl chain conformational ordering in liquid-crystalline bilayers: comparative FT-IR and 2H-NMR studies of phospholipids differing in headgroup structure and chain length

FT-IR spectroscopy has been used to evaluate the acyl chain conformational ordering of DMPC, DMPE, DMPA (pH 6 and 12), DMPG (pH 1 and 7), and DPPC, DPPE, DPPA (pH 6). The frequencies of the symmetric and antisymmetric methylene stretching vibrations were determined as a function of temperature. In the liquid-crystalline phase the frequencies show a qualitative dependence on the amount of chain disorder. Quantitative data for trans-gauche isomerization were obtained from the integral intensities of the conformation sensitive methylene wagging absorptions at ca. 1368 cm^–1 (gtg and gtg sequences), 1356 cm ^–1 (double gauche) and 1342 cm^–1 (end gauche). The integral band intensities were converted to the number of gauche conformers per acyl chain using the calibration factors published by Senak et al. (1991). At 69°C the highest number of gauche conformers excluding contributions from single gauche conformers and jogs (gtttg) are found for PCs (DMPC: 2.6; DPPC: 2.4), followed by DMPG^– (2.0), phosphatidylethanolamines (DMPE: 1.4; DPPE: 2.0), protonated DMPG (1.5), and phosphatidic acids (DPPA^–: 1.7; DMPA^–: 1.4, DMPA^2–: 1.7). From ²H-NMR measurements of perdeuterated samples of DMPC, DMPA, DPPC, and DPPA the quadrupolar splittings _Qi and the order parameter S _CDi of the CD₂-segments close to the chain ends could be determined whereas splittings in the plateau region of the chains could not be resolved. The quadrupolar splittings are affected by trans-gauche isomerization, long axis rotation, and restricted wobbling motions of the acyl chains. In the simplest assumption, the order parameter S_CD can be expressed as a product of a segmental order parameter S and a lhain order parameter S . For comparison of the different lipids we used average order parameters S_CD, obtained by averaging over all values, and S determined from the total number of gauche conformers per chain by FT-IR-spectroscopy, to calculate an empirical average chain order parameter S. The combination of ²H-NMR and FT-IR results allows the estimation of the relative extent of chain wobbling for the different lipid molecules. S is lowest for PCs (S 0.475) while PEs (S 0.51) and PAs (S0.52) show less chain wobbling.Abbreviations FT-IR Fourier transform infrared - ²H-NMR deuterium nuclear magnetic resonance - DMPC(–d₅₄) (perdeuterated) dimyristoyl-phosphatidylcholine - DMPE(–d₅₄) (perdeuterated) dimyristoyl-phosphatidylethanolamine - DMPA(–d₅₄) (perdeuterated) dimyristoyl-phosphatidic acid - DMPG dimyristoyl-phosphatidylglycerol - DPPC(–d₆₂) (perdeuterated) dipalmitoyl-phosphatidylcholine - DPPE(–d₆₂) (perdeuterated) dipalmitoyl-phosphatidylethanolamine - DPPA(–d₆₂) (perdeuterated) dipalmitoyl-phosphatidic acid - gtg gauche ^±-trans-gauche^± - gtg gauche^±-trans-gauche^± - dg double gauche - eg end gauche Correspondence to: A. Blume     

Losses of inorganic carbon and nitrous oxide from a temperate freshwater wetland in relation to nitrate loading

We studied the export of inorganic carbon and nitrous oxide (N₂O) from a Danish freshwater wetland. The wetland is situated in an agricultural catchment area and is recharged by groundwater enriched with nitrate (NO₃ ^–) (1000 M). NO₃ ^– in recharging groundwater was reduced (57.5 mol NO₃ ^– m^–2 yr^–) within a narrow zone of the wetland. Congruently, the annual efflux of carbon dioxide (CO₂) from the sediment was 19.1 mol C m^–2 when estimated from monthly in situ measurements. In comparison the CO₂ efflux was 4.8 mol C m^–2 yr^–1 further out in the wetland, where no NO₃ ^– reduction occurred. Annual exports of inorganic carbon in groundwater and surface water was 78.4 mol C m^–2 and 6.1 mol C m^–2 at the two sites, respectively. N₂O efflux from the sedimenst was detectable on five out of twelve sampling dates and was significantly (P < 0.0001) higher in the NO₃ ^– reduction zone (0.35–9.40 mol m^–2 h^–1, range of monthly means) than in the zone without NO₃ ^– reduction (0.21–0.41 mol m^–2 h^–1). No loss of dissolved N₂O could be measured. Total annual export of N₂O was not estimated. The reduction of oxygen (O₂) in groundwater was minor throughout the wetland and did not exceed 0.2 mol 0₂ m^–2yr^–1. Sulfate (SO₄ ^––) was reduced in groundwater (2.1 mol SO₄ ^–– m^–2 yr^–1) in the zone without NO₃ ^– reduction. Although the NO₃ ^– in our wetland can be reduced along several pathways our results strongly suggest that NO₃ ^– loading of freshwater wetlands disturb the carbon balance of such areas, resulting in an accelerated loss of inorganic carbon in gaseous and dissolved forms.     

Carbon isotopes and carbon turnover in cotton and wheat FACE experiments

The Maricopa cotton and wheat FACE (free-air CO₂ enrichment) experiments offer propitious opportunity to quantify carbon turnover. The commercial CO₂ (% MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaeqiTdq2aaW% baaSqabeaacaaIXaGaaG4maaaakiaaboeacqGHijYUcqGHsislcaaI% ZaGaaG4naiaacwcaliaad+gaaaa!3FCB!\[\delta ^{13} {\text{C}} \approx - 37\% o\]) used to elevate CO₂ concentration in field plots provided a strongly ¹³C-depleted tracer. Soil CO₂ and ¹³C of soil organic carbon (SOC) in CO₂-enriched and Control plots were measured between the final cotton FACE project (October 1991) and the end of the second wheat experiment (June 1994). The initial ¹³C-depletion in SOC of cotton FACE plots (measured by the difference in ¹³C between FACE and Control plots) persisted at the same level (1.9) 1.5 years after the experiment ended. A similar depletion was observed in soil CO₂ evolved in the same plots, indicating ongoing decomposition of the new SOC. The SOC ¹³C of wheat plots before and after two growing seasons showed increasing ¹³C-depletion in FACE relative to Control. Isotopic mass balance was consistent with 5–6% new carbon input from the two wheat crops. This is lower than the 12–13% calculated for FACE cotton and perhaps a consequence of the larger root system of cotton or the 3-year duration of the cotton experiments versus 2 years for the wheat.     

The sodium cycle: A novel type of bacterial energetics

The progress of bioenergetic studies on the role of Na⁺ in bacteria is reviewed. Experiments performed over the past decade on several bacterial species of quite different taxonomic positions show that Na⁺ can, under certain conditions, substitute for H⁺ as the coupling ion. Various primary Na⁺ pumps ( generators) are described, i.e., Na⁺-motive decarboxylases, NADH-quinone reductase, terminal oxidase, and ATPase. The formed is shown to be consumed by Na⁺ driven ATP-synthase, Na⁺ flagellar motor, numerous Na⁺, solute symporters, and the methanogenesis-linked reverse electron transfer system. InVibrio alginolyticus, it was found that , generated by NADH-quinone reductase, can be utilized to support all three types of membrane-linked work, i.e., chemical (ATP synthesis), osmotic (Na⁺, solute symports), and mechanical (rotation of the flagellum). InPropionigenum modestum, circulation of Na⁺ proved to be the only mechanism of energy coupling. In other species studied, the Na⁺ cycle seems to coexist with the H⁺ cycle. For instance, inV. alginolyticus the initial and terminal steps of the respiratory chain are Na⁺ - and H⁺-motive, respectively, whereas ATP hydrolysis is competent in the uphill transfer of Na⁺ as well as of H⁺. In the alkalo- and halotolerantBacillus FTU, there are H⁺ - and Na⁺-motive terminal oxidases. Sometimes, the Na⁺-translocating enzyme strongly differs from its H⁺-translocating homolog. So, the Na⁺-motive and H⁺-motive NADH-quinone reductases are composed of different subunits and prosthetic groups. The H⁺-motive and Na⁺-motive terminal oxidases differ in that the former is ofaa ₃-type and sensitive to micromolar cyanide whereas the latter is of another type and sensitive to millimolar cyanide. At the same time, both Na⁺ and H⁺ can be translocated by one and the sameP. modestum ATPase which is of the F₀F₁-type and sensitive to DCCD. The sodium cycle, i.e., a system composed of primary generator(s) and consumer(s), is already described in many species of marine aerobic and anaerobic eubacteria and archaebacteria belonging to the following genera:Vibrio, Bacillus, Alcaligenes, Alteromonas, Salmonella, Klebsiella, Propionigenum, Clostridium, Veilonella, Acidaminococcus, Streptococcus, Peptococcus, Exiguobacterium, Fusobacterium, Methanobacterium, Methanococcus, Methanosarcin, etc. Thus, the sodium world seems to occupy a rather extensive area in the biosphere.     

Determination of 13Cα relaxation times in uniformly 13C/15N-enriched proteins

Summary Relaxation times of ¹³C carbons of uniformly ¹³C/¹⁵N-enriched probes have been investigated. The relaxation behaviour was analyzed in terms of a multispin system. Pulse sequences for the determination of T₁, T₂ and the heteronuclear NOE of ¹³C in uniformly ¹³C/¹⁵N-enriched ribonuclease T1 are presented. The experiments performed in order to obtain T₁ and the heteronuclear NOE were similar to those of the corresponding ¹⁵N experiments published previously. The determination of T₂ for the C-carbon in a completely labeled protein is more complicated, since the magnetization transfer during the T₂ evolution period owing to the scalar coupling of C–C must be suppressed. Various different pulse sequences for the T₂ evolution period were simulated in order to optimize the bandwidth for which reliable T₂ relaxation times can be obtained. A proof for the quality of these pulse sequences is given by fitting the intensity decay of individual ¹H–¹³C cross peaks, in a series of (¹H, ¹³C)-ct-HSQC spectra with a modified CPMG sequence as well as a T_1p sequence for the transverse relaxation time, to a single exponential using a simplex algorithm.     

A set of HNCO-based experiments for measurement of residual dipolar couplings in 15N, 13C, (2H)-labeled proteins

Several HNCO-based three-dimensional experiments are described for the measurement of ¹³C(i–1)-¹³C(i–1), ¹⁵N(i)-¹³C(i–1), ¹⁵N(i)-¹³C(i), ¹⁵N(i)-¹³C(i–1), ¹H^N(i)-¹³C(i), ¹H^N(i)-¹³C(i–1), and ¹³C(i–1)-¹³C(i–1) scalar and dipolar couplings in ¹⁵N, ¹³C, (²H)-labelled protein samples. These pulse sequences produce spin-state edited spectra superficially resembling an HNCO correlation spectrum, allowing accurate and simple measurement of couplings without introducing additional spectral crowding. Scalar and dipolar couplings are measured with good sensitivity from relatively large proteins, as demonstrated with three proteins: cardiac Troponin C, calerythrin and ubiquitin. Measurement of several dipolar couplings between spin-1/2 nuclei using spin-state selective 3D HNCO spectra provides a wealth of structural information.     

Chemical composition of soil solutions extracted from New Zealand beech forests and West German beech and spruce forests

Concentrations of ions were measured in soil solutions from beech (Nothofagus) forests in remote areas of New Zealand and in solutions from beech (Fagus sylvatica) and Norway spruce (Picea abies) forests in North-East Bavaria, West Germany, to compare the chemistry of soil solutions which are unaffected by acid deposition (New Zealand) with those that are affected (West Germany). In New Zealand, soil solution SO₄ ^2– concentrations ranged between <2 and 58 mol L^–1, and NO₃ ^– concentrations ranged between <1 and 3 mol L^–1. In West Germany, SO₄ ^2– concentrations ranged between 80 and 700 mol L^–1, and NO₃ ^– concentrations at three of six sites ranged between 39 and 3750 mol L^–1, but was not detected at the remaining three sites. At all sites in New Zealand, and at sites where the soil base status was moderately high in West Germany, pH levels increased, and total Al (Al_t) and inorganic monomeric Al (Al_i) levels decreased rapidly with increasing soil depth. In contrast, at sites on soils of low base status in West Germany, pH levels increased only slightly, and Al levels did not decline with increasing soil depth.Under a high-elevation Norway spruce stand showing severe Mg deficiency and dieback symptoms in West Germany, soil solution Mg²⁺ levels ranged between 20 and 60 mol L^–, and were only half those under a healthy stand. Al_t and Al_i levels were substantially higher the healthy stand than under the unhealthy stand, indicating that Al toxicity was not the main cause of spruce decline.     

C, N CP MAS and high resolution multinuclear NMR study of methyl

Sporidesmin, a mycotoxin fromPithomyces chartarum is a hydrophobic molecule. It can therefore be easily incorporated in the cell membrane, where it is likely to cause changes in the bilayer organization and the properties of membrane proteins. In order to understand the redox behaviour of sporidesmin in a hydrophobic environment, we have investigated the effects of oxidized and reduced sporidesmin on the phase transition properties of bilayers and on the susceptibility of bilayers to pancreatic phospholipase A₂ (PLA₂). The changes induced by sporidesmin in the thermotropic phase transition profiles of dimyristoyl-sn-3-phosphatidyl choline (DMPC) bilayers were similar to those caused by solutes known to localize in the glycerol-backbone region of the lipid bilayer, suggesting a similar localization for oxidized and reduced sporidesmin. Neither form of toxin disrupt the bilayer or membrane organization even at relatively high mole fractions. At concentrations <10 mole% both forms partitioned equally well in the gel and liquid-crystalline phases, whereas at higher concentrations (30 mole%) reduced sporidesmin is preferentially localized in the liquid-crystalline phase. These effects of sporidesmin on the phase properties of DMPC vesicles were also reported by the fluorescence behavior of 10-pyrenedecanoic acid (PDA). The effects of oxidized and reduced sporidesmins on PLA₂ kinetics are consistent with their ability to perturb bilayer organisation.     

Interaction of sporidesmin,a mycotoxin fromPithomyces chartarum,with lipid bilayers

Sporidesmin, a mycotoxin fromPithomyces chartarum is a hydrophobic molecule. It can therefore be easily incorporated in the cell membrane, where it is likely to cause changes in the bilayer organization and the properties of membrane proteins. In order to understand the redox behaviour of sporidesmin in a hydrophobic environment, we have investigated the effects of oxidized and reduced sporidesmin on the phase transition properties of bilayers and on the susceptibility of bilayers to pancreatic phospholipase A₂ (PLA₂). The changes induced by sporidesmin in the thermotropic phase transition profiles of dimyristoyl-sn-3-phosphatidyl choline (DMPC) bilayers were similar to those caused by solutes known to localize in the glycerol-backbone region of the lipid bilayer, suggesting a similar localization for oxidized and reduced sporidesmin. Neither form of toxin disrupt the bilayer or membrane organization even at relatively high mole fractions. At concentrations <10 mole% both forms partitioned equally well in the gel and liquid-crystalline phases, whereas at higher concentrations (30 mole%) reduced sporidesmin is preferentially localized in the liquid-crystalline phase. These effects of sporidesmin on the phase properties of DMPC vesicles were also reported by the fluorescence behavior of 10-pyrenedecanoic acid (PDA). The effects of oxidized and reduced sporidesmins on PLA₂ kinetics are consistent with their ability to perturb bilayer organisation.     

Denitrification in a seashore sandy deposit influenced by groundwater discharge

The chemical compositions of ground water and organic matter in sediments were investigated at a sandy shore of Tokyo Bay, Japan to determine the fate of ground water NO₃ ^–. On the basis of Cl^– distribution in ground water, the beach was classified into freshwater (FR)-, transition (TR)-, and seawater (SW)-zones from the land toward the shoreline. The NO₃ ^– and N₂O did not behave conservatively with respect to Cl^– during subsurface mixing of freshwater and seawater, suggesting NO₃ ^– consumption and N₂O production in the TR-zone. Absence of beach vegetation indicated that NO₃ ^– assimilation by higher plants was not as important as NO₃ ^– sink. Low NH₄ ⁺ concentrations in ground water revealed little reduction of NO₃ ^– to NH₄ ⁺. These facts implied that microbial denitrification and assimilation were the likely sinks for ground water NO₃ ^–. The potential activity and number of denitrifiers in water-saturated sediment were highest in the low-chlorinity part of the TR-zone. The location of the highest potential denitrification activity (DN-zone) overlapped with that of the highest NO₃ ^– concentration. The C/N ratio and carbon isotope ratio (¹³C) of organic matter in sediment (< 100 -m) varied from 12.0 to 22.5 and from –22.5 to –25.5, respectively. The ¹³C value was inversely related to the C/N ratio (r ² = 0.968, n = 11), which was explained by the mixing of organic matters of terrestrial and marine origins. In the DN-zone, the fine sediments were rich in organic matters with high C/N ratios and low ¹³C values, implying that dissolved organic matters of terrestrial origin might have been immobilized under slightly saline conditions. A concurrent supply of NO₃ ^– and organic matter to the TR-zone by ground water discharge probably generates favorable conditions for denitrifiers. Ground water NO₃ ^– discharged to the beach is thus partially denitrified and fixed as microbial biomass before it enters the sea. Further studies are necessary to determine the relative contribution of these processes for NO₃ ^– removal.     

Measurement of inter-glycosidic 13C-1H coupling constants in a cyclic β(1→2)-glucan by 13C-filtered 2D {1H,1H}ROESY

Summary A method for measuring three-bond ¹³C-¹H scalar coupling constants across glycosidic bonds in a cyclic (12)-glucan icosamer is presented. This oligosaccharide molecule, with its high degree of symmetry, represents a particular challenge for NMR spectroscopy to distinguish inter-residue from intra-residue heteronuclear coupling effects. Chemically equivalent H2 protons in adjacent glucosyl residues are distinguished on the basis of their different through-space, dipolar interactions with the anomeric protons (H1). The strong NOE contact between anomeric (H1) and aglyconic (H2) protons permits the selective observation of the inter-residue heteronuclear couplings ³J_C1H2 and ³J_C2H1 in a natural-abundance ¹³C-₁-half-filtered {¹H,¹H} ROESY experiment.Abbreviations COSY scalar correlated spectroscopy - NOE nuclear Overhauser effect - NOESY NOE spectroscopy - ROESY rotating-frame NOE spectroscopy     

Short-term variations in δ<Superscript>13</Superscript>C of ecosystem respiration reveals link between assimilation and respiration in a deciduous forest

We present a comprehensive dataset of hourly, daily, and monthly measurements of carbon isotope measurements of CO₂ in canopy air from a temperate deciduous forest with the aim to identify the relevance of short-term variations in the isotopic signature of ecosystem respiration (¹³C_R) and to understand its underlying physiological processes. We show that during daytime low vertical mixing inside the canopy can lead to decoupling of the air in the lower and upper canopy layer resulting in large spatial variation of ¹³C in CO₂ of canopy air. Intercept of Keeling Plots also showed large temporal variation (3.8) over the course of the day demonstrating that intercepts can differ between day and night and suggesting that choosing the right time for sampling is essential to capture the isotopic signature of ecosystem respiration (¹³C_R). ¹³C_R as obtained from night-time measurements showed large variation of up to 2.65 on a day-to-day basis, which was similar to the observed variation of ¹³C_R over the seasonal cycle (3.08). This highlights the importance of short-term physiological processes within ecosystems for the isotopic composition of CO₂ in the atmosphere, not reflected by bulk plant and soil organic samples. At daily and monthly time scales, ¹³C_R increased with increasing ratio of vapour pressure deficit to photosynthetically active radiation, measured 4–5 days before. This suggests that ecosystem respiration was isotopically linked to assimilation. Furthermore, assimilates recently fixed in the canopy seem to form a labile carbon pool with a short mean residence time that is respired back to the atmosphere after 4–5 days.     

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations

Chloroplasts with high rates of photosynthetic O₂ evolution (up to 120 mol O₂· (mg Chl)^-1·h^-1 compared with 130 mol O₂· (mg Chl)^-1·h^-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO₂ concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O₂ uptake could be observed in the dark by high- and low-CO₂ adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 mol photons·m^-2·s^-1 for high- and low-CO₂ adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 M for low-CO₂ adapted chloroplasts, whereas high-CO₂ adapted chloroplasts were not saturated even at 700 M DIC. The concentrations of DIC required to reach half-saturated rates of net O₂ evolution (K_m(DIC)) was 31.1 and 156 M DIC for low- and high-CO₂ adapted chloroplasts, respectively. These results demonstrate that the CO₂ concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.Abbreviations Chl chlorophyll - DIC dissolved inorganic carbon - K_m(DIC) coneentration of dissolved inorganic carbon required for the rate of half maximal net O₂ evolution - PFR photon fluence rate - SPGM silicasol-PVP-gradient medium     

Proportions of Ca2+ Channel Subtypes in Chick or Rat P2 Fraction and NG108-15 Cells Using Various Ca2+ Blockers

The proportions of calcium (Ca²⁺) channel subtypes in chick or rat P₂ fraction and NG 108-15 cells were investigated using selective L-, N-, P- and P/Q- type Ca²⁺ channel blockers. KCl-stimulated ⁴⁵Ca²⁺ uptake by chick P₂ fraction was blocked by 40~50% using N-type Ca²⁺ channel blockers [-conotoxin GVIA, aminoglycoside antibiotics and dynorphin A(1–13)], but was not inhibited by P- or P/Q-type blockers (-agatoxin IVA or -conotoxin MVIIC). On the other hand, KCl-stimulated ⁴⁵Ca²⁺ uptake by rat P₂ fraction was blocked by 30~40% using P- or P/Q-type Ca²⁺ channel blockers, but was not inhibited by N-type Ca²⁺ channel blockers. The L-type Ca²⁺ channel blockers 1,4-dihydropyridines, diltiazem and verapamil, but not calciseptine (CaS), inhibited both KCl-stimulated ⁴⁵Ca²⁺ uptake and veratridine-induced ²²Na⁺ uptake by chick or rat P₂ fraction with similar IC₅₀ values. CaS did not have any effect on ⁴⁵Ca²⁺ uptake by either chick or rat P₂ fraction. In NG108-15 cells, CaS, -agatoxin IVA and -conotoxin MVIIC, but not -conotoxin GVIA, inhibited KCl-stimulated ⁴⁵Ca²⁺ uptake by 30–40%. Various combinations of these Ca²⁺ channel blockers had no significant additional effects in chick or rat P₂ fraction or NG 108-15 cells. These findings suggest that KCl-stimulated ⁴⁵Ca²⁺ uptake by chick or rat P₂ fraction and NG 108-15 cells is a convenient and useful model for screening whether or not natural or synthetic substances have selective effects as L-, N-, P-, or P/Q- type Ca²⁺ channel antagonists or agonists.     

Voltage dependence of the Ca2+-activated K+ conductance of human red cell membranes is strongly dependent on the extracellular K+ concentration

Summary The conductance of the Ca²⁺-activated K⁺ channel (g _K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V _m ) and extracellular K⁺ concentration ([K⁺]_ex). ATP-depleted cells, with fixed values of cellular K⁺ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K⁺ channels, to buffer-free salt solutions with given K⁺ concentrations. Outward-current conductances were calculated from initial net effluxes of K⁺, correspondingV _m , monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E _K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of⁴²K at (V _m –E _K)0, using a single-file flux ratio exponent of 2.7. Within a [K⁺]_ex range of 5.5 to 60mm and at (V _m –E _K) 20 mV a basic conductance, which was independent of [K⁺]_ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm² between 0 and –100 mV. As (V _m –E _K) decreased from 20 towards zero mVg _K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm². The zero-current conductance was not significantly dependent on [K⁺]_ex (30 to 156mm) corresponding toV _m (–50 mV to 0). A further increase ing _K(Ca) symmetrically aroundE _K is suggested as (V _m –E _K) becomes positive. Increasing the extracellular K⁺ concentration from zero and up to 3mm resulted in an increase ing _K(Ca) from 50 to 70 S/cm². Since the driving force (V _m –E _K) was larger than 20 mV within this range of [K⁺]_ex this was probably a specific K⁺ activation ofg _K(Ca). In conclusion: The Ca²⁺-activated K⁺ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel.     

Membrane potential,anion and cation conductances in Ehrlich ascites tumor cell

Summary The fluorescence intensity of the dye 1,1-dipropyloxadicarbocyanine (DiOC₃-(5)) has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (V _m ) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na⁺-free K⁺/choline⁺ media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155mm. Calibration by the valinomycin null point procedure described by Lariset al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976,Biochim. Biophys. Acta 436:475–488) is shown to be valid only in the presence of the Cl^–-channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN^– as an indirect estimation of the membrane potential is found not to be applicable for the fast changes inV _m reported in this paper. Incubation with DiOC₃-(5) for 5 min is demenstrated to reduce the Cl^– permeability by 26±5% and the NO ₃ ^– permeability by 15±2%, while no significant effect of the probe could be demonstrated on the K⁺ permeability. Values forV _m , corrected for the inhibitory effect of the dye on the anion conductance, are estimated at –61±1 mV in isotonic standard NaCl medium, –78±3 mV in isotonic Na⁺-free choline medium and –46±1 mV in isotonic NaNO₃ medium. The cell membrane is depolarized by addition of the K⁺ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na⁺-free choline medium, indicating thatV _m is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO ₃ ^– for all cellular and extracellular Cl^– leads to a depolarization of the membrane, demonstrating thatV _m is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K⁺ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 S/cm² for K⁺, 3.0 S/cm² for Na⁺, 0.6 S/cm² for Cl^– and 8.7 S/cm² for NO ₃ ^– . Addition of the Ca²⁺-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K⁺ permeability exceeds the Cl^– permeability also after the addition of A23187. The K⁺ and Cl^– conductances in A23187-treated Ehrlich cells are estimated at 134 and 30 S/cm², respectively. The membrane potential is depolarized in hypotonically swollen cells, confirming that the increase in the Cl^– permeability following hypotonic exposure exceeds the concommitant increase in the K⁺ permeability. In control experiments where the membrane potentialV _m =E _K =E _Cl =E _Na , it is demonstrated that cell volume changes has no significant effect on the fluorescence signal, apparently because of a large intracellular buffering capacity. The increase in the Cl^– conductances is 68-fold when cells are transferred to a medium with half the osmolarity of the standard medium, as estimated from the net Cl^– efflux and the change inV _m . The concommitant increase in the K⁺ conductance, as estimated from the net K⁺ efflux, is only twofold.