Light dependence of quantum yields of Photosystem II and CO2 fixation in C3 and C4 plants

The light dependence of quantum yields of Photosystem II (_II) and of CO₂ fixation were determined in C₃ and C₄ plants under atmospheric conditions where photorespiration was minimal. Calculations were made of the apparent quantum yield for CO₂ fixation by dividing the measured rate of photosynthesis by the absorbed light [A/I=_CO2 and of the true quantum yield by dividing the estimated true rate of photosynthesis by absorbed light [(A+R_l)/I_a=_CO2·], where R_L is the rate of respiration in the light. The dependence of the _II/_CO2 and _II/_CO2 ^* ratios on light intensity was then evaluated. In both C₃ and C₄ plants there was little change in the ratio of _II/_CO2 at light intensities equivalent to 10–100% of full sunlight, whereas there was a dramatic increase in the ratio at lower light intensities. Changes in the ratio of _II/_CO2 can occur because respiratory losses are not accounted for, due to changes in the partitioning of energy between photosystems or changes in the relationship between PS II activity and CO₂ fixation. The apparent decrease in efficiency of utilization of energy derived from PS II for CO₂ fixation under low light intensity may be due to respiratory loss of CO₂. Using dark respiration as an estimate of R_L, the calculated _II/_CO2 ^* ratio was nearly constant from full sunlight down to approx 5% of full sunlight, which suggests a strong linkage between the true rate of CO₂ fixation and PS II activity under varying light intensity. Measurements of photosynthesis rates and _II were made by illuminating upper versus lower leaf surfaces of representative C₃ and C₄ monocots and dicots. With the monocots, the rate of photosynthesis and the ratio of _II/_CO2 exhibited a very similar patterns with leaves illuminated from the adaxial versus the abaxial surface, which may be due to uniformity in anatomy and lack of differences in light acclimation between the two surfaces. With dicots, the abaxial surface had both lower rates of photosynthesis and lower _II values than the adaxial surface which may be due to differences in anatomy (spongy versus palisade mesophyll cells) and/or light acclimation between the two surfaces. However, in each species the response of _II/_CO2 to varying light intensity was similar between the two surfaces, indicating a comparable linkage between PS II activity and CO₂ fixation.Abbreviations A measured rate of CO₂ assimilation - A+R_L true rate of CO₂ assimilation; e - CO₂ estimate of electrons transported through PSII per CO₂ fixed by RuBP carboxylase - f fraction of light absorbed by Photosystem II - F'_m yield of PSII chlorophyll fluorescence due to a saturating flash of white light under steady-state photosynthesis - F_s variable yield of fluorescence under steady-state photosynthesis; PPFD-photosynthetic photon flux density - I_a absorbed PPFD - PS II Photosystem II - R_d rate of respiration in the dark - R_I rate of respiration in the light estimated from measurement of R_d or from analysis of quantum yields - apparent quantum yield of CO₂ assimilation under a given condition (A/absorbed PPFD) - true quantum yield of CO₂ assimilation under a given condition [(A+R_L)/(absorbed PPFD)] - quantum yield for photosynthetic O₂ evolution - electrons transported via PS II per quantum absorbed by PS II Supported by USDA Competitive Grant 90-37280-5706.     

Analogues of NADP+ as inhibitors and coenzymes for NADP+ malic enzyme from maize leaves

Structural analogues of the NADP⁺ were studied as potential coenzymes and inhibitors for NADP⁺ dependent malic enzyme from Zea mays L. leaves. Results showed that 1, N⁶-etheno-nicotinamide adenine dinucleotide phosphate ( NADP⁺), 3-acetylpyridine-adenine dinucleotide phosphate (APADP⁺), nicotinamide-hypoxanthine dinucleotide phosphate (NHDP⁺) and -nicotinamide adenine dinucleotide 2: 3-cyclic monophosphate (23NADPc⁺) act as alternate coenzymes for the enzyme and that there is little variation in the values of the Michaelis constants and only a threefold variation in V_max for the five nucleotides. On the other hand, thionicotinamide-adenine dinucleotide phosphate (SNADP⁺), 3-aminopyridine-adenine dinucleotide phosphate (AADP⁺), adenosine 2-monophosphate (2AMP) and adenosine 2: 3-cyclic monophosphate (23AMPc) were competitive inhibitors with respect to NADP⁺, while -nicotinamide adenine dinucleotide 3-phosphate (3NADP⁺), NAD⁺, adenosine 3-monophosphate (3AMP), adenosine 2: 5-cyclic monophosphate (25AMPc), 5AMP, 5ADP, 5ATP and adenosine act as non-competitive inhibitors. These results, together with results of semiempirical self-consistent field-molecular orbitals calculations, suggest that the 2-phosphate group is crucial for the nucleotide binding to the enzyme, whereas the charge density on the C₄ atom of the pyridine ring is the major factor that governs the coenzyme activity.Abbreviations NADP⁺ 1, N⁶-etheno-nicotinamide adenine dinucleotide phosphate - NHDP⁺ nicotinamide-hypoxanthine dinucleotide phosphate - APADP⁺ 3-acetylpyridine-adenine dinucleotide phosphate - SNADP⁺ thionicotinamide-adenine dinucleotide phosphate - AADP⁺ 3-aminopyridine-adenine dinucleotide phosphate - 23NADPc⁺ -nicotinamide adenine dinucleotide 2: 3-cyclic monophosphate - 3NADP⁺ -nicotinamide adenine dinucleotide 3-phosphate - 2AMP adenosine 2-monophosphate - 3AMP adenosine 3-monophosphate - 23AMPc adenosine 2: 3 monophosphate cyclic - A adenosine - RuBP ribulose 1,5-bisphosphate - SCF-MO Self-Consistent Field-Molecular Orbitals (method)     

Autotrophic carbon sources for heterotrophic bacterioplankton in a floodplain lake of central Amazon

The relative contribution of autotrophic carbon sources (aquatic macrophytes, flooded forest, phytoplankton) for heterotrophic bacterioplankton was evaluated in a floodplain lake of the Central Amazon. Stable carbon isotopes (¹³C) were used as tracers. Values of ¹³C of different autotrophic sources were compared to those of dissolved organic carbon (DOC) and those of bacterially produced CO₂.The percentage of carbon derived from C₄ macrophytes for bacterially produced CO₂ was the highest, on average 89%. The average ¹³C value of CO₂ from bacterial respiration was –18.5 ± 3.3. Considering a fractionation of CO₂ of 3 by bacterial respiration, ¹³C value was –15.5, near C₄ macrophyte ¹³C value (–13.1).The average value of total DOC ¹³C was –26.8 ± 2.4. The percentage of C₄ macrophytes carbon for total DOC was on average 17%. Considering that bacteria consume mainly carbon from macrophytes, the dominance of C₃ plants for total DOC probably reflects a faster consumption of the former source, rather than a major contribution of the latter source.Heterotrophic bacterioplankton in the floodplain may be an important link in the aquatic food web, transferring the carbon from C₄ macrophytes to the consumers.     

Antioxidant Properties of New Chalcogenides Against Lipid Peroxidation in Rat Brain

Ebselen (2-phenyl- 1,2-benzisoselenazole-3 (2H)-one) is a seleno-organic compound with antioxidant properties, and anti-inflammatory actions. Recently, ebselen improved the outcome of acute ischemic stroke in humans. In the present study, the potential antioxidant capacity of organochalcogenide compounds diphenyl diselenide (PhSe)₂, diphenyl ditelluride (PhTe)₂, diphenyl disulfide (PhS)₂, p-Cl-diphenyl diselenide (pCl-PhSe)₂, bis-[S-4-isopropyl 2-phenyl oxazoline] diselenide (AA-Se)₂, bis-[S-4-isopropyl 2-phenyl oxazoline] ditelluride (AA-Te)₂ and bis-[S-4-isopropyl 2-phenyl oxazoline] disulfide (AA-S)₂ was compared with that of ebselen (a classical antioxidant). Spontaneous and quinolinic acid (QA)- (2 mM) and sodium nitroprusside (SNP)- (5 M)-induced thiobarbituric reactive species (TBARS) production by rat brain homogenates was determined colorimetrically. TBARS formation was reduced by ebselen, (PhSe)₂, (PhTe)₂, (AA-Se)₂, (AA-S)₂ and (pCl- PhSe)₂ to basal rates. The concentrations of these compounds needed to inhibit TBARS formation by 50% (lC₅₀) are 1.71 M, 3.73 M, 1.63 M, 9.85 M, > 33.3 M, 23.2 M and 4.83 M, respectively for QA. For TBARS production induced by SNP the lC₅₀ was 2.02 M, 12.5 M, 2.80 M, > 33.3 M, 24.5 M and 7.55 M, respectively. The compounds (AA-Te)₂ and (PhS)₂ have no antioxidant activity and pro-oxidant activity, respectively. These results suggest that (AA-Se)₂ and (AA-S)₂ can be considered as potential pharmaceutical antioxidant agents.     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

Mapping of Exposed Surfaces of the Nicotinic Acetylcholine Receptor by Identification of Iodinated Tyrosine Residues

Here we report on the use of iodination of the membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo californica electric tissue in order to define surface-exposed portions of the receptor molecule. Membrane-bound nAChR was ¹²⁵I-iodinated using the oxidation agent Iodo-Gen. The iodinated subunits were separated by preparative gel electrophoresis, desalted, and cleaved with trypsin. The resulting peptides were separated by reverse-phase HPLC and the radioactive peptides were identified by mass spectrometry and protein sequencing. For the -subunit, we identified five iodinated peptides containing the tyrosine residues Tyr17, Tyr74, Tyr365, Tyr372, and Tyr428. The surface exposition of these amino acids is in agreement with the four-transmembrane-segment model (4TM model) of the nAChR, but the assignment to the intra- or extracellular surface is doubtful. According to this model, the N-terminal portion of the receptor subunits including the iodinated residues Tyr17 and Tyr74 is extracellular and Tyr372 as a site of tyrosine phosphorylation is located on the cytoplasmic side. But since this latter residue is among the first to be iodinated using an immobilized iodination agent, its true position with respect to the membrane bilayer is not clear.     

DNA polymorphisms in the ψβ1- and β-globin gene regions in asian macaques

DNA polymorphisms in the ₁--globin gene region in nine Asian macaques(Macaca fuscata, M. mulatta, M. nemestrina, M. cyclopis, M. fascicularis, M. arctoides, M. radiata, M. maura, andM. assamensis) were examined using several restriction endonucleases and the human ₁, IVS2, and IVS2 probes. TheBamHI site 3 to the -globin gene was polymorphic inM. fuscata andM. mulatta, while the HincII site and the EcoRI site in the ₁-globin gene region was highly polymorphic inM. fuscata andM. mulatta, respectively. These polymorphic sites also seem to be present in other Asian macaques. The present study of the polymorphism at theBamHI site 3 to the -globin gene in Asian macaques supports, at the nuclear DNA level, the idea that thefascicularis group includingM. fuscata, M. mulatta, M. cyclopis, andM. fascicularis is different from other Asian macaque groups.This study was supported in part by the Cooperation Research Program of the Primate Research Institute, Kyoto University.     

The Use of the [13C]/[12C] Ratio for the Assay of the Microbial Oxidation of Hydrocarbons

The study deals with a comparative analysis of the relative abundances of the carbon isotopes ¹²C and ¹³C in the metabolites and biomass of the Burkholderia sp. BS3702 and Pseudomonas putida BS202-p strains capable of utilizing aliphatic (n-hexadecane) and aromatic (naphthalene) hydrocarbons as sources of carbon and energy. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of Burkholderia sp. BS3702 on n-hexadecane (¹³C = –44.6 ± 0.2) were characterized by the values of ¹³C_{CO 2} = –50.2 ± 0.4, ¹³C_biom = –46.6 ± 0.4, and ¹³C_exo = –41.5 ± 0.4, respectively. The isotope compositions of the carbon dioxide, biomass, and exometabolites produced during the growth of the same bacterial strain on naphthalene (¹³C = –21 ± 0.4) were characterized by the isotope effects ¹³C_{CO 2} = –24.1 ± 0.4, ¹³C_biom = –19.2 ± 0.4, and ¹³C_exo = –19.1 ± 0.4, respectively. The possibility of using the isotope composition of metabolic carbon dioxide for the rapid monitoring of the microbial degradation of petroleum hydrocarbons in the environment is discussed.     

Long-term steady-state labelling of wheat plants by use of natural 13CO2/12CO2 mixtures in an open,rapidly turned-over system

A photosynthate labelling method is presented which takes advantage of the natural difference in carbon-isotope composition () which exists between atmospheric CO₂ (-8) and commercially available compressed CO₂. Carbon dioxide with -4.0 and –27.9%., respectively, has been used for labelling. A plant growth cabinet served as the labelling compartment. CO₂-free air was continuously injected at a rate of up to 54m³·h^–1. Dilution of cabinet CO₂ by CO₂-free air was counterbalanced by addition of CO₂ with known constant . Since the labelling-cabinet atmosphere was continuously exchanged at a high rate, photosynthetic carbon-isotope discrimination was fully expressed. In order to study the distribution of carbon acquired by the plant during a defined growth period, the of CO₂ was modified by replacing, for example, atmospheric CO₂ by CO₂ with –27.9%. and the weight and 5 of plant carbon pools was monitored over time. In such an experiment the change of CO₂ was followed by a rapid change of the of sucrose in mature flag-leaf blades of wheat (Triticum aestivum L.). The 5 of sucrose stabilized near –51%., indicating complete exchange by current photosynthate. In contrast 83% of the total carbon in mature flag-leaf blades was not exchanged after 14 d continuous labelling. Differential labelling of pre- and post-anthesis photosynthate indicated that 13% of grain carbon originated from pre-anthesis photosynthesis. Carbon-isotope discrimination and its consideration in experimentation and labelling data evaluation are discussed in detail. Since the air supplied to the labelling cabinet is dry and free of CO₂, carbon-isotope discrimination and carbon turnover and partitioning can be studied over a wide range of CO₂ concentrations (0–2600 cm³ · m^–3) and vapor-pressure deficits.Abbreviation and Symbol PPFD photosynthetic photon flux density - carbon-isotope composition Dr. G. Schleser (Forschungszentrum Jülich, FRG) and Professor S. Hoernes (Mineralogisch-Petrologisches Institut, Universität Bonn) for valuable help and advice during the initial stages of the project and Professor W. Kühbauch (Institut für Pflanzenbau, Universität Bonn) for continuing support. Technical assistance of Ute Labusch, Petra Biermann, Ludwig Schmitz and Thomas Gebbing is gratefully acknowleged.

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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.     

Average phase difference theory and 1∶1 phase entrainment in interlimb coordination

The dynamics of coupled biological oscillators can be modeled by averaging the effects of coupling over each oscillatory cycle so that the coupling depends on the phase difference between the two oscillators and not on their specific states. Average phase difference theory claims that mode locking phenomena can be predicted by the average effects of the coupling influences. As a starting point for both empirical and theoretical investigations, Rand et al. (1988) have proposed d/dt= — K sin ), with phase-locked solutions =arcsin( /K), where is the difference between the uncoupled frequencies and K is the coupling strength. Phase-locking was evaluated in three experiments using an interlimb coordination paradigm in which a person oscillates hand-held pendulums. was controlled through length differences in the left and right pendulums. The coupled frequency _c was varied by a metronome, and scaled to the eigenfrequency _v of the coupled system K was assumed to vary inversely with _c. The results indicate that: (1) and K contribute multiplicatively to (2) =0 or = regardless of K when =0; (3) 0 or regardless of when K is large (relative to ); (4) results (1) to (3) hold identically for both in phase and antiphase coordination. The results also indicate that the relevant frequency is _c/_v rather than _c. Discussion high-lighted the significance of confirming =arcsin(/K) for more general treatments of phase-locking, such as circle map dynamics, and for the 11 phase-entrainment which characterizes biological movement systems.     

Carbon-isotope discrimination by leaves of Flaveria species exhibiting different amounts of C3-and C4-cycle co-function

Carbon-isotope ratios were examined as ¹³C values in several C₃, C₄, and C₃–C₄ Flaveria species, and compared to predicted ¹³C, values generated from theoretical models. The measured ¹³C values were within 4 of those predicted from the models. The models were used to identify factors that contribute to C₃-like ¹³C values in C₃–C₄ species that exhibit considerable C₄-cycle activity. Two of the factors contributing to C₃-like ¹³C values are high CO₂ leakiness from the C₄ pathway and pi/pa values that were higher than C₄ congeners. A marked break occurred in the relationship between the percentage of atmospheric CO₂ assimilated through the C₄ cycle and the ¹³C value. Below 50% C₄-cycle assimialtion there was no significant relationship between the variables, but above 50% the ¹³C values became less negative. These results demonstrate that the level of C₄-cycle expression can increase from, 0 to 50% with little integration of carbon transfer from the C₄ to the C₃ cycle. As expression increaces above 50%, however, increased integration of C₃- and C₄-cycle co-function occurs.Abbreviations and symbols RuBP carboxylase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - PEP carboxylase phosphoenolpyruvate carboxylase (EC 4.1.1.31) - pa atmospheric CO₂ partial pressure - pi intercellular CO₂ partial pressure - isotope ratio - quantum yield for CO₂ uptake     

Induction of HPRT- mutants in Chinese hamster V79 cells after heavy ion exposure

The induction of resistance to 6-thioguanine by heavy ion exposure was investigated with various accelerated ions (oxygen-uranium) up to linear energy transfer (LET) values of about 15000 keV/µm.31 y Survival curves are exponential with fluence; mutation induction shows a linear dependence. Cross-sections (_i: inactivation, _m: mutation) were derived from the respective slopes. Generally, _i rises over the whole LET range, but separateas into different declining curves for single ions with LET values above 200 keV/µm. Similar behaviour is seen for _m. The new SIS facility at GSI, Darmstadt, makes it possible to study the effects of ions with the same LET but very different energies and track structures. Experiments using nickel and oxygen ions (up to 400 MeV/u) showed that inactivation cross-sections do not depend very much on track structure, i.e. similar values are found with different ions at the same LET. This is not the case for mutation induction, where very energetic ions display considerably smaller induction cross-sections compared with low-energy ions of identical LET. Preliminary analyses using the polymerase chain reaction (PCR) demonstrate that even heavy ions cause small alterations (small deletions or base changes). The proportion of the total deletions seems to increase with LET.Submitted paper presented at the International Symposium on Heavy Ion Research: Space, Radiation Protection and Therapy, Sophia-Antipolis, France, 21–24 March 1994     

Photosynthesis of Cockspur [Echinochloa crus-galli (L.) Beauv.] at Sites of Naturally Elevated CO2 Concentration

High abundance of cockspur (Echinochloa crus-galli) at the geothermal carbon dioxide spring area in Staveinci indicates that this species is able to grow under widely varying CO₂ concentrations. Living cockspur plants can even be found very close to gas-releasing vents where growth is significantly reduced. Plant height correlated well with CO₂ exposure. The ¹³C value of the CO₂ spring air was –3.9 and ¹³C values of high-, medium-, and low-CO₂ plants were –10.14, –10.44, and –11.95 , respectively. Stomatal response directly followed the prevailing CO₂ concentrations, with the highest reduction of stomatal conductance in high CO₂ concentration grown plants. Analysis of the curves relating net photosynthetic rate to intercellular CO₂ concentration (P _N-C_i curves) revealed higher CO₂ compensation concentration in plants growing at higher CO₂ concentration. This indicates adjustment of respiration and photosynthetic carbon assimilation according to the prevailing CO₂ concentrations during germination and growth. There was no difference in other photosynthetic parameters measured.     

Short-term CO2 exchange response to temperature,irradiance, and CO2 concentration in strawberry

Relative importance of short-term environmental interaction and preconditioning to CO₂ exchange response was examined in Fragaria ananasa (strawberry, cv. Quinault). Tests included an orthogonal comparison of 15 to 60-min and 6 to 7-h exposures to different levels of temperature (16 to 32°C), photosynthetically active radiation (PAR, 200 to 800 E m² s^-1), and CO₂ (300 to 600 l/l) on successive days of study. Plants were otherwise maintained at 21°C, 300 E m² s^-1 PAR and 300–360 l/l CO₂ as standard conditions. Treatment was restricted to the mean interval of 14 h daily illumination and the first 3–4 days of each test week over a 12-week cultivation period. CO₂ exchange rates were followed with each step-change in environmental level including ascending/descending temperature/PAR within a test period, initial response at standard conditions on successive days of testing, and measurement at reduced O₂. Response generally supported prior concepts of leaf biochemical modeling in identifying CO₂ fixation as the major site of environmental influence, while overall patterns of whole plant CO₂ exchange suggested additional effects for combined environmental factors and preconditioning. These included a positive interaction between temperature and CO₂ concentration on photosynthesis at high irradiance and a greater contribution by dark respiration at lower PAR than previously indicated. The further importance of estimating whole plant CO₂ exchange from repetitive tests and measurements was evidenced by a high correlation of response to prior treatment both during the daily test period and on consecutive days of testing.Abbreviations C₃ plant a plant in which the product of CO₂ fixation is a 3-carbon acid (3-phosphoglyceric acid) - IRGA intra-red gas analyzer - PAR photosynthetically active radiation - RH relative humidity - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase Reference to a company and/or product named by the Department is only for purposes of information and does not imply approval or recommendation of the product to the exclusion of others which may also be suitable.     

Detection of very weak side chain–main chain hydrogen bonding interactions in medium-size 13C/15N-labeled proteins by sensitivity-enhanced NMR spectroscopy

We describe the direct observation of very weak side chain–main chain hydrogen bonding interactions in medium-size ¹³C/¹⁵N-labeled proteins with sensitivity-enhanced NMR spectroscopy. Specifically, the remote correlation between the hydrogen acceptor side chain carboxylate carbon ¹³CO₂ of glutamate 54 and the hydrogen donor backbone amide ¹⁵N of methionine 49 in a 12 kDa protein, human FKBP12, is detected via the trans-hydrogen bond ^3h J _NCO2 coupling by employing a novel sensitivity-enhanced HNCO-type experiment, CPD-HNCO. The ^3h J _NCO2 coupling constant appears to be even smaller than the average value of backbone ^3h J _NC couplings, consistent with more extensive local dynamics in protein side chains.     

Autotrophic CO2 fixation in Chlorobium limicola. Evidence against the operation of the Calvin cycle in growing cells

Chlorobium limicola has been proposed to assimilate CO₂ autotrophically via a reductive tricarboxylic acid cycle rather than via the Calvin cycle. This proposal has been a matter of considerable controversy. In order to determine which pathway is operative, the bacterium was grown on a mineral salts medium with CO₂ as the main carbon source supplemented with specifically labeled ¹⁴C-pyruvate, and the incorporation of ¹⁴C into alanine (intracellular pyruvate), aspartate (oxaloacetate), glutamate (-ketoglutarate), and glucose (hexosephosphate) was measured in exponentially growing cells in long term labeling experiments. During growth in presence of pyruvate, 20% of the cell carbon were derived from pyruvate in the medium, 80% from CO₂. Since pyruvate was not oxidized to CO₂, only those compounds should become labeled which were synthesized from CO₂ via pyruvate.The three amino acids and glucose were found to be labeled. Alanine had one fifth the specific radioactivity of the extracellular pyruvate, indicating that 20% of the intracellular pyruvate pool were derived from pyruvate in the medium, 80% were synthesized from CO₂. Glucose had twice the specific radioactivity of alanine, showing that hexosephosphate synthesis from CO₂ proceeded via the pyruvate pool. The latter finding is not consistent with the operation of the Calvin cycle, in which pyruvate is not an intermediate. The specific radioactivities of aspartate (oxaloacetate) and of glutamate (-ketoglutarate) were practically identical but considerably lower than that of alanine ( intracellular pyruvate). These findings are compatible with the operation of a reductive tricarboxylic acid cycle as mechanism of autotrophic CO₂ fixation. Degradation studies of the cell components support this interpretation. Offprint requests to: G. Fuchs     

Heterogeneous binding and killing behaviour of human γ/δ-TCR+ lymphokine-activated killer cells against K562 and Daudi cells

Effector-target conjugates, formed by coincubation of lymphokine-activated killer (LAK) cells with either K562 or Daudi cells, were separated from single cells by Percoll sedimentation. The occurrence of various CD molecules (CD3, CD56, CD57, CD16, /-TCR) was compared in both fractions. Only LAK cells expressing the / T cell receptor (TCR) were found in a significantly increased percentage in fractions containing conjugates indicating that /-TCR⁺ LAK cells were preferably bound to target cells at the time of separation. In order to determine whether /-TCR⁺ LAK cells also show a preferred killing activity against the targets, cultures enriched with or depleted of /-TCR⁺ cells were established. Against K562 cells, /-TCR⁺-enriched cultures showed a greatly reduced killing activity compared to LAK bulk cultures or cultures depleted of /-TCR⁺ cells. Using Daudi cells as targets the enriched fraction revealed a slightly increased killing activity compared to bulk cultures or depleted fractions. Preincubation of /-TCR⁺ LAK cells with anti-/ or anti-CD3 mAb resulted in a distinct increase of the killing activity against K562 cells, but in only a slightly enhanced activity against Daudi cells. It is postulated that /-TCR⁺ LAK cells use the same adhesion mechanism for both targets but that only Daudi cells express a specific ligand for the /-TCR. Occupation of the /-TCR/CD3 complex by mAb, however, seems to substitute for the absent epitope on K562 cells by eliciting stimulatory signals in /-TCR⁺ LAK cells which, in combination with the binding stimulus, trigger cytolytic activity.This work was supported by the Hartmann-Müller Foundation, Zürich     

Stimulation of μ- and δ-Opiate Receptors and Tolerance of Isolated Heart to Oxidative Stress: the Role of NO-Synthase

Preliminary intravenous injection of -opiate receptor (OR) agonists DSLET (0.1 mg/kg) or DTLET (0.1 mg/kg) increased tolerance of isolated perfused myocardium to damage by oxidative stress simulated in vivo with FeSO₄ + ascorbic acid. This manifested itself by a decreased level of creatine phosphokinase (CPK) in the perfusate flowing out of the heart during the oxidative exposure. The preliminary systemic injection of -agonists DAMGO (0.1 mg/kg) or DALDA (0.1 mg/kg) failed to affect the release of CPK from the myocardium. The cardioprotective effect of the -agonist DSLET was completely abolished by preliminary intravenous injection of the -OR antagonist ICI 174,864 (2.5 mg/kg). The intravenous injection of the NO-synthase inhibitor L-NAME (50 mg/kg) also completely abolished the cardioprotective effect of -OR stimulation. The preliminary injection of DSLET but not of DAMGO prevented an increase in the level of diene conjugates and a decrease in the activity of superoxide dismutase (SOD) in the isolated myocardium tissue. Thus, the in vivo stimulation of -OR increased the tolerance of the heart to oxidative stress through activation of NO-synthase and SOD.     

Elevated CO2 effects on carbon and nitrogen cycling in grass/clover turves of a Psammaquent soil

Effects of elevated CO₂ (525 and 700 L L^–1), and a control (350 L L^–1 CO₂), on biochemical properties of a Mollic Psammaquent soil in a well-established pasture of C3 and C4 grasses and clover were investigated with continuously moist turves in growth chambers over four consecutive seasonal temperature regimes from spring to winter inclusive. After a further spring period, half of the turves under 350 and 700 L L^–1 were subjected to summer drying and were then re-wetted before a further autumn period; the remaining turves were kept continuously moist throughout these additional three consecutive seasons. The continuously moist turves were then pulse-labelled with ¹⁴C-CO₂ to follow C pathways in the plant/soil system during 35 days.Growth rates of herbage during the first four seasons averaged 4.6 g m^–2 day^–1 under 700 L L^–1 CO₂ and were about 10% higher than under the other two treatments. Below-ground net productivity at the end of these seasons averaged 465, 800 and 824 g m^–2 in the control, 525 and 700 L L^–1 treatments, respectively.in continuously moist soil, elevated CO₂ had no overall effects on total, extractable or microbial C and N, or invertase activity, but resulted in increased CO₂-C production from soil, and from added herbage during the initial stages of decomposition over 21 days; rates of root decomposition were unaffected. CO₂ produced h^–1 mg^–1 microbial C was about 10% higher in the 700 L L^–1 CO₂ treatment than in the other two treatments. Elevated CO₂ had no clearly defined effects on N availability, or on the net N mineralization of added herbage.In the labelling experiment, relatively more ¹⁴C in the plant/soil system occurred below ground under elevated CO₂, with enhanced turnover of ¹⁴C also being suggested.Drying increased levels of extractable C and organic-N, but decreased mineral-N concentrations; it had no effect on microbial C, but resulted in lowered microbial N in the control only. In soil that had been previously summer-dried, CO₂ production was again higher, but net N mineralization was lower, under elevated CO₂ than in the control after autumn pasture growth.Over the trial period of 422 days, elevated CO₂ generally appears to have had a greater effect on soil C turnover than on soil C pools in this pasture ecosystem.