An enhanced sensitivity methyl <Superscript>1</Superscript>H triple-quantum pulse scheme for measuring diffusion constants of macromolecules

We present a pulse scheme that exploits methyl ¹H triple-quantum (TQ) coherences for the measurement of diffusion rates of slowly diffusing molecules in solution. It is based on the well-known stimulated echo experiment, with encoding and decoding of TQ coherences. The size of quantifiable diffusion coefficients is thus lowered by an order of magnitude with respect to single-quantum (SQ) approaches. Notably, the sensitivity of the scheme is high, approximately ¾ that of the corresponding single quantum experiment, neglecting relaxation losses, and on the order of a factor of 4 more sensitive than a previously published sequence for AX₃ spin systems (Zheng et al. in JMR 198:271–274, 2009) for molecules that are only ¹³C labeled at the methyl carbon position. Diffusion coefficients measured from TQ- and SQ-based experiments recorded on a range of protein samples are in excellent agreement. We present an application of this technique to the study of phase-separated proteins where protein concentrations in the condensed phase can exceed 400 mg/mL, diffusion coefficients can be as low as ~10^?9 cm²s^?1 and traditional SQ experiments fail.     

Probing slow timescale dynamics in proteins using methyl <Superscript>1</Superscript>H CEST

Although ¹⁵N- and ¹³C-based chemical exchange saturation transfer (CEST) experiments have assumed an important role in studies of biomolecular conformational exchange, ¹H CEST experiments are only beginning to emerge. We present a methyl-TROSY ¹H CEST experiment that eliminates deleterious ¹H–¹H NOE dips so that CEST profiles can be analyzed robustly to extract methyl proton chemical shifts of rare protein conformers. The utility of the experiment, along with a version that is optimized for ¹³CHD₂ labeled proteins, is established through studies of exchanging protein systems. A comparison between methyl ¹H CEST and methyl ¹H CPMG approaches is presented to highlight the complementarity of the two experiments.     

Blood clearance and occupational exposure for <Superscript>177</Superscript>Lu-DOTATATE compared to <Superscript>177</Superscript>Lu-PSMA radionuclide therapy

The main target of this work is to examine blood clearance and external exposure for ¹⁷⁷Lu-DOTATATE compared with new emerging ¹⁷⁷Lu-PSMA therapy. Blood clearance and radiation exposure of 31 patients treated with 5.5?±?1.1 GBq ¹⁷⁷Lu-DOTATATE were compared to those of 23 patients treated with 7.4 GBq ¹⁷⁷Lu-PSMA. Dose rates were measured at several distances and time points up to 120 h after treatment. Blood samples were collected conjunctively after infusion. Caregiver’s cumulative dose was measured by means of an OSL (optically stimulated luminescence) dosimeter for 4–5 days and medical staff’s dose was also estimated using electronic personal dosimeters. Finger dose was determined via ring TLD (Thermoluminescence Dosimeter) for radiopharmacists and nurses. Dose rates due to ¹⁷⁷Lu-DOTATATE at a distance of 1 m, 4 h and 6 h after infusion, were 3.0?±?2.8 and 2?±?1.9 µSv/(h GBq), respectively, while those due to ¹⁷⁷Lu-PSMA were 3.1?±?0.8 and 2.2?±?0.9 µSv/(h GBq). Total effective dose of 17 caregivers was 100–200 µSv for ¹⁷⁷Lu-DOTATATE therapy. Mean effective doses to nurses and radiopharmacists were 5 and 4 µSv per patient, respectively, while those for physicists and physicians were 2 µSv per patient. For ¹⁷⁷Lu-DOTATATE, effective half-life in blood and early elimination phase were 0.31?±?0.13 and 4.5?±?1 h, while they were found as 0.4?±?0.1 and 5?±?1 h, respectively, for ¹⁷⁷Lu-PSMA. The first micturition time following ¹⁷⁷Lu-DOTATATE infusion was noted after 36?±?14 min, while the second and third voiding times were after 74?±?9 and 128?±?41 min, respectively. It is concluded that blood clearance and radiation exposure for ¹⁷⁷Lu-DOTATATE are very similar to those for ¹⁷⁷Lu-PSMA, and both treatment modalities are reasonably reliable for outpatient treatment, since the mean dose rate [2.1 µSv/(h GBq)] decreased below the dose rate that allows release of the patient from the hospital (20 µSv/h) after 6 h at 1 m distance.     

A new class of CEST experiment based on selecting different magnetization components at the start and end of the CEST relaxation element: an application to <Superscript>1</Superscript>H CEST

Chemical exchange saturation transfer (CEST) experiments are becoming increasingly popular for investigating biomolecular exchange dynamics with rates on the order of approximately 50–500 s^?1 and a rich toolkit of different methods has emerged over the past few years. Typically, experiments are based on the evolution of longitudinal magnetization, or in some cases two-spin order, during a fixed CEST relaxation delay, with the same class of magnetization prepared at the start and selected at end of the CEST period. Here we present a pair of TROSY-based pulse schemes for recording amide and methyl ¹H CEST profiles where longitudinal magnetization at the start evolves to produce two-spin order that is then selected at the completion of the CEST element. This selection process subtracts out contributions from ¹H–¹H cross-relaxation on the fly that would otherwise complicate analysis of the data. It also obviates the need to record spin-state selective CEST profiles as an alternative to eliminating NOE effects, leading to significant improvements in sensitivity. The utility of the approach is demonstrated on a sample of a cavity mutant of T4 lysozyme that undergoes chemical exchange between conformations where the cavity is free and occupied.     

Measuring the signs of the methyl <Superscript>1</Superscript>H chemical shift differences between major and ‘invisible’ minor protein conformational states using methyl <Superscript>1</Superscript>H multi-quantum spectroscopy

Carr–Purcell–Meiboom–Gill (CPMG) type relaxation dispersion experiments are now routinely used to characterise protein conformational dynamics that occurs on the μs to millisecond (ms) timescale between a visible major state and ‘invisible’ minor states. The exchange rate(s) (\( k_{{{\text{ex}}}} \)), population(s) of the minor state(s) and the absolute value of the chemical shift difference \(|{\Delta \varpi }|\) (ppm) between different exchanging states can be extracted from the CPMG data. However the sign of \({\Delta \varpi }\) that is required to reconstruct the spectrum of the ‘invisible’ minor state(s) cannot be obtained from CPMG data alone. Building upon the recently developed triple quantum (TQ) methyl \( ^{1} {\text{H}} \) CPMG experiment (Yuwen in Angew Chem 55:11490–11494, 2016) we have developed pulse sequences that use carbon detection to generate and evolve single quantum (SQ), double quantum (DQ) and TQ coherences from methyl protons in the indirect dimension to measure the chemical exchange-induced shifts of the SQ, DQ and TQ coherences from which the sign of \({\Delta \varpi }\) is readily obtained for two state exchange. Further a combined analysis of the CPMG data and the difference in exchange induced shifts between the SQ and DQ resonances and between the SQ and TQ resonances improves the estimates of exchange parameters like the population of the minor state. We demonstrate the use of these experiments on two proteins undergoing exchange: (1) the ~ 18 kDa cavity mutant of T4 Lysozyme (\( k_{{{\text{ex}}}} \sim\,3500{\text{ s}}^{{ - 1}} \)) and (2) the \(\sim\,4.7\) kDa Peripheral Sub-unit Binding Domain (PSBD) from the acetyl transferase of Bacillus stearothermophilus (\(k_{ex} \sim\,13,000\hbox { s}^{-1}\)).     

3-O-Methyl-<Emphasis Type="SmallCaps">d</Emphasis>-glucose mutarotation and proton exchange rates assessed by <Superscript>13</Superscript>C, <Superscript>1</Superscript>H NMR and by chemical exchange saturation transfer and spin lock measurements

3-O-Methyl-d-glucose (3OMG) was recently suggested as an agent to image tumors using chemical exchange saturation transfer (CEST) MRI. To characterize the properties of 3OMG in solution, the anomeric equilibrium and the mutarotation rates of 3OMG were studied by ¹H and ¹³C NMR. This information is essential in designing the in vivo CEST experiments. At room temperature, the ratio of α and β 3OMG anomers at equilibrium was 1:1.4, and the time to reach 95% equilibrium was 6 h. The chemical exchange rates between the hydroxyl protons of 3OMG and water, measured by CEST and spin lock at pH 6.14 and a temperature of 4 °C, were in the range of 360–670 s^?1.     

The nitrite reductase activity of horse heart carboxymethylated-cytochrome <Emphasis Type="Italic">c</Emphasis> is modulated by cardiolipin

Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0 °C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO₂ ^?-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k _on = (7.3 ± 0.7) × 10^?2 M^?1 s^?1; at pH 7.4], whereas the value of k _on for NO₂ ^? reduction by CM-cytc-Fe(II) is 1.1 ± 0.2 M^?1 s^?1 (at pH 7.4). CL facilitates the NO₂ ^?-mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k _on for the NO₂ ^?-mediated conversion of CL–CM-cytc-Fe(II) to CL–CM-cytc-Fe(II)-NO (5.6 ± 0.6 M^?1 s^?1; at pH 7.4) being slightly higher than that for the NO₂ ^?-mediated conversion of CL–cytc-Fe(II) to CL–cytc-Fe(II)-NO (2.6 ± 0.3 M^?1 s^?1; at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3 ± 0.3) × 10^?6 M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k _on versus pH are ?1.05 ± 0.07 and ?1.03 ± 0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH^?. These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL–CM-cytc.     

<Superscript>15</Superscript>N and <Superscript>13</Superscript>C- SOFAST-HMQC editing enhances 3D-NOESY sensitivity in highly deuterated,selectively [<Superscript>1</Superscript>H,<Superscript>13</Superscript>C]-labeled proteins

The ongoing NMR method development effort strives for high quality multidimensional data with reduced collection time. Here, we apply ‘SOFAST-HMQC’ to frequency editing in 3D NOESY experiments and demonstrate the sensitivity benefits using highly deuterated and ¹⁵N, methyl labeled samples in H₂O. The experiments benefit from a combination of selective T ₁ relaxation (or L-optimized effect), from Ernst angle optimization and, in certain types of experiments, from using the mixing time for both NOE buildup and magnetization recovery. This effect enhances sensitivity by up to 2.4× at fast pulsing versus reference HMQC sequences of same overall length and water suppression characteristics. Representative experiments designed to address interesting protein NMR challenges are detailed. Editing capabilities are exploited with heteronuclear ¹⁵N,¹³C-edited, or with diagonal-free ¹³C aromatic/methyl-resolved 3D-SOFAST-HMQC–NOESY–HMQC. The latter experiment is used here to elucidate the methyl-aromatic NOE network in the hydrophobic core of the 19 kDa FliT-FliJ flagellar protein complex. Incorporation of fast pulsing to reference experiments such as 3D-NOESY–HMQC boosts digital resolution, simplifies the process of NOE assignment and helps to automate protein structure determination.     

Photosynthetic capacity of the capsule wall and its contribution to carbon fixation and seed yield in castor (<Emphasis Type="Italic">Ricinus communis</Emphasis> L.)

Defoliation occurs in castor due to several reasons, but the crop has propensity to compensate for the seed yield. Photosynthetic efficiency in terms of functional (gas exchange and chlorophyll fluorescence) and structural characteristics (photosynthetic pigment profiles and anatomical properties) of castor capsule walls under light- and dark-adapted conditions was compared with that of leaves. Capsule wall showed high intrinsic efficiency of photosystem II (F _v/F _m, 0.82) which was comparable to leaves (F _v/F _m, 0.80). With increasing photon flux densities (PFD), actual quantum yields and photochemical quenching coefficients of the capsule walls were similar to that in leaves, while electron transport rates reached a maximum corresponding to about 118 % of the leaves. However, maximum net photosynthetic rate of the capsule walls (2.60 µmol CO₂ m^?2 s^?1) was less than one-fourth of the leaves (15.67 µmol CO₂ m^?2 s^?1) at the CO₂ concentration of 400 µmol mol^?1, and the difference was attributed to about 80 % lower stomatal density and the 75 % lower total chlorophyll content of capsule walls than the leaves. Furthermore, seed weight in dark-adapted capsules was 2.70–12.42 % less as compared to the capsules developed under light. The results indicate that castor capsule walls are photosynthetically active (about 15–30 % of the leaves) and contribute significantly to carbon fixation and seed yield accounting for 10 % photoassimilates towards seed weight.     

Cd<Superscript>2+</Superscript> extrusion by P-type Cd<Superscript>2+</Superscript>-ATPase of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> 17810R via energy-dependent Cd<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchange mechanism

Cd²⁺ is highly toxic to Staphylococcus aureus since it blocks dithiols in cytoplasmic 2-oxoglutarate dehydrogenase complex (ODHC) participating in energy conservation process. However, S. aureus 17810R is Cd²⁺-resistant due to possession of cadA-coded Cd²⁺ efflux system, recognized here as P-type Cd²⁺-ATPase. This Cd²⁺ pump utilizing cellular energy—ATP, ?μ _H ⁺ (electrochemical proton potential) and respiratory protons, extrudes Cd²⁺ from cytoplasm to protect dithiols in ODHC, but the mechanism of Cd²⁺ extrusion remains unknown. Here we propose that two Cd²⁺ taken up by strain 17810R via Mn²⁺ uniporter down membrane potential (?ψ) generated during glutamate oxidation in 100 mM phosphate buffer (high P_iB) are trapped probably by high affinity sites in cytoplasmic domain of Cd²⁺-ATPase, forming SCdS. This stops Cd²⁺ transport towards dithiols in ODHC, allowing undisturbed NADH production, its oxidation and energy conservation, while ATP could change orientation of SCdS towards facing transmembrane channel. Now, increased number of P_i-dependent protons pumped electrogenically via respiratory chain and countertransported through the channel down ?ψ, extrude two trapped cytoplasmic Cd²⁺, which move to low affinity sites, being then extruded into extracellular space via ?ψ-dependent Cd²⁺/H⁺ exchange. In 1 mM phosphate buffer (low P_iB), external Cd²⁺ competing with decreased number of P_i-dependent protons, binds to ψ_s of Cd²⁺-ATPase channel, enters cytoplasm through the channel down ?ψ via Cd²⁺/Cd²⁺ exchange and blocks dithiols in ODHC. However, Mg²⁺ pretreatment preventing external Cd²⁺ countertransport through the channel down ?ψ, allowed undisturbed NADH production, its oxidation and extrusion of two cytoplasmic Cd²⁺ via Cd²⁺/H⁺ exchange, despite low P_iB.     

Swimming exercise to control precocious maturation in male seabass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>)

Background

Male European seabass, already predominant (~?70%) in cultured stocks, show a high incidence (20–30%) of precocious sexual maturation under current aquaculture practices, leading to important economic losses for the industry. In view of the known modulation of reproductive development by swimming exercise in other teleost species, we aimed at investigating the effects of sustained swimming on reproductive development in seabass males during the first year of life in order to determine if swimming could potentially reduce precocious sexual maturation.

Methods

Pre-pubertal seabass (3.91?±?0.22 g of body weight (BW)) were subjected to a 10 week swimming regime at their optimal swimming speed (U_opt) in an oval-shaped Brett-type flume or kept at rest during this period. Using Blazka-type swim tunnels, U_opt was determined three times during the course of the experiment: 0.66 m s^??1 at 19?±?1 g BW, 10.2?±?0.2 cm of standard length (SL) (week 1); 0.69 m s^??1 at 38?±?3 g BW, 12.7?±?0.3 cm SL (week 5), and also 0.69 m s^??1 at 77?±?7 g BW, 15.7?±?0.5 cm SL (week 9). Every 2 weeks, size and gonadal weight were monitored in the exercised (N?=?15) and non-exercised fish (N?=?15). After 10 weeks, exercised and non-exercised males were sampled to determine plasma 11-ketotestosterone levels, testicular mRNA expression levels of genes involved in steroidogenesis and gametogenesis by qPCR, as well as the relative abundance of germ cells representing the different spermatogenic stages by histological examination.

Results

Our results indicate that sustained swimming exercise at U_opt delays testicular development in male European seabass as evidenced by decreased gonado-somatic index, slower progression of testicular development and by reduced mRNA expression levels of follicle stimulating hormone receptor (fshR), 3-beta-hydroxysteroid dehydrogenase (3βhsd), 11-beta hydroxysteroid dehydrogenase (11βhsd), estrogen receptor-beta (erβ2), anti-mullerian hormone (amh), structural maintenance of chromosomes protein 1B (smc1β), inhibin beta A (inhba) and gonado-somal derived factor 1 (gsdf1) in exercised males as compared with the non-exercised males.

Conclusions

Swimming exercise may represent a natural and non-invasive tool to reduce the incidence of sexually precocious males in seabass aquaculture.

     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N backbone NMR chemical shift assignments of the C-terminal P4 domain of Ahnak

Ahnak is a ~?700 kDa polypeptide that was originally identified as a tumour-related nuclear phosphoprotein, but later recognized to play a variety of diverse physiological roles related to cell architecture and migration. A critical function of Ahnak is modulation of Ca²⁺ signaling in cardiomyocytes by interacting with the β subunit of the L-type Ca²⁺ channel (Ca_V1.2). Previous studies have identified the C-terminal region of Ahnak, designated as P3 and P4 domains, as a key mediator of its functional activity. We report here the nearly complete ¹H, ¹³C and ¹⁵N backbone NMR chemical shift assignments of the 11 kDa C-terminal P4 domain of Ahnak. This study lays the foundations for future investigations of functional dynamics, structure determination and interaction site mapping of the Ca_V1.2-Ahnak complex.     

Cross-correlated spin relaxation effects in methyl <Superscript>1</Superscript>H CPMG-based relaxation dispersion experiments: Complications and a simple solution

Artifacts associated with the measurement of methyl ¹H single quantum CPMG-based relaxation dispersion profiles are described. These artifacts arise due to the combination of cross-correlated spin relaxation effects involving intra-methyl ¹H–¹H dipolar interactions and imperfections in ¹H refocusing pulses that are applied during CPMG intervals that quantify the effects of chemical exchange on measured transverse relaxation rates. As a result substantial errors in extracted exchange parameters can be obtained. A simple work-around is presented where the ¹H chemical shift difference between the exchanging states is extracted from a combination of ¹³C single quantum and ¹³C–¹H multiple quantum dispersion profiles. The approach is demonstrated with an application to a folding/unfolding reaction involving a G48M mutant Fyn SH3 domain.     

The growth of <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis> as influenced by CO<Subscript>2</Subscript>, light,and flue gases

In order to achieve recognition as environmentally friendly production, flue gases should be used as a CO₂ source for growing the microalgae Chlorella sorokiniana when used for hydrogen production. Flue gases from a waste incinerator and from a silicomanganese smelter were used. Before testing the flue gases, the algae were grown in a laboratory at 0.04, 1.3, 5.9, and 11.0 % (v/v) pure CO₂ gas mixed with fresh air. After 5 days of growth, the dry biomass per liter algal culture reached its maximum at 6.1 % CO₂. A second experiment was conducted in the laboratory at 6.2 % CO₂ at photon flux densities (PFD) of 100, 230, and 320 μmol photons m^?2 s^?1. After 4 days of growth, increasing the PFD increased the biomass production by 67 and 108 % at the two highest PFD levels, as compared with the lowest PFD. A bioreactor system containing nine daylight-exposed tubes and nine artificial light-exposed tubes was installed on the roof of the waste incinerator. The effect of undiluted flue gas (10.7 % CO₂, 35.8 ppm NO _x , and 38.6 ppm SO₂), flue gas diluted with fresh air to give 4.2 % CO₂ concentration, and 5.0 % pure CO₂ gas was studied in daylight (21.4?±?9.6 mol photons m^?2 day^?1 PAR, day length 12.0 h) and at 135 μmol photons m^?2 s^?1 artificial light given 24 h day^?1 (11.7?±?0.0 mol photons m^?2 day^?1 PAR). After 4 days’ growth, the biomass production was the same in the two flue gas concentrations and the 5 % pure CO₂ gas control. The biomass production was also the same in daylight and artificial light, which meant that, in artificial light, the light use efficiency was about twice that of daylight. The starch concentration of the algae was unaffected by the light level and CO₂ concentration in the laboratory experiments (2.5–4.0 % of the dry weight). The flue gas concentration had no effect on starch concentration, while the starch concentration increased from about 1.5 % to about 6.0 % when the light source changed from artificial light to daylight. The flue gas from the silicomanganese smelter was characterized by a high CO₂ concentration (about 17 % v/v), low oxygen concentration (about 4 %), about 100 ppm NO _x , and 1 ppm SO₂. The biomass production using flue gas significantly increased as compared with about 5 % pure CO₂ gas, which was similar to the biomass produced at a CO₂ concentration of 10–20 % mixed with N₂. Thus, the enhanced biomass production seemed to be related to the low oxygen concentration rather than to the very high CO₂ concentration.     

Quantifying the contribution of land use to N<Subscript>2</Subscript>O,NO and CO<Subscript>2</Subscript> fluxes in a montane forest ecosystem of Kenya

Increasing demand for food and fibre by the growing human population is driving significant land use (LU) change from forest into intensively managed land systems in tropical areas. But empirical evidence on the extent to which such changes affect the soil-atmosphere exchange of trace gases is still scarce, especially in Africa. We investigated the effect of LU on soil trace gas production in the Mau Forest Complex region, Kenya. Intact soil cores were taken from natural forest, commercial and smallholder tea plantations, eucalyptus plantations and grazing lands, and were incubated in the lab under different soil moisture conditions. Soil fluxes of nitrous oxide (N₂O), nitric oxide (NO) and carbon dioxide (CO₂) were quantified, and we approximated annual estimates of soil N₂O and NO fluxes using soil moisture values measured in situ. Forest and eucalyptus plantations yielded annual fluxes of 0.3–1.3 kg N₂O–N ha^?1 a^?1 and 1.5–5.2 kg NO–N ha^?1 a^?1. Soils of commercial tea plantations, which are highly fertilized, showed higher fluxes (0.9 kg N₂O–N ha^?1 a^?1 and 4.3 kg NO–N ha^?1 a^?1) than smallholder tea plantations (0.1 kg N₂O–N ha^?1 a^?1 and 2.1 kg NO–N ha^?1 a^?1) or grazing land (0.1 kg N₂O–N ha^?1 a^?1 and 1.1 kg NO–N ha^?1 a^?1). High soil NO fluxes were probably the consequence of long-term N fertilization and associated soil acidification, likely promoting chemodenitrification. Our experimental approach can be implemented in understudied regions, with the potential to increase the amount of information on production and consumption of trace gases from soils.     

Measurement of net ecosystem exchange,productivity and respiration in three spruce forests in Sweden shows unexpectedly large soil carbon losses

Measurement of net ecosystem exchange was made using the eddy covariance method above three forests along a north-south climatic gradient in Sweden: Flakaliden in the north, Knottåsen in central and Asa in south Sweden. Data were obtained for 2 years at Flakaliden and Knottåsen and for one year at Asa. The net fluxes (N_ep) were separated into their main components, total ecosystem respiration (R_t) and gross primary productivity (P_g). The maximum half-hourly net uptake during the heart of the growing season was highest in the southernmost site with ?0.787 mg CO₂ m^?2 s^?1 followed by Knottåsen with ?0.631 mg CO₂ m^?2 s^?1 and Flakaliden with ?0.429 mg CO₂ m^?2 s^?1. The maximum respiration rates during the summer were highest in Knottåsen with 0.245 mg CO₂ m^?2 s^?1 while it was similar at the two other sites with 0.183 mg CO₂ m^?2 s^?1. The annual N_ep ranged between uptake of ?304 g C m^?2 year^?1 (Asa) and emission of 84 g C m^?2 year^?1 (Knottåsen). The annual R_t and P_g ranged between 793 to 1253 g C m^?2 year^?1 and ?875 to ?1317 g C m^?2 year^?1, respectively. Biomass increment measurements in the footprint area of the towers in combination with the measured net ecosystem productivity were used to estimate the changes in soil carbon and it was found that the soils were losing on average 96–125 g C m^?2 year^?1. The most plausible explanation for these losses was that the studied years were much warmer than normal causing larger respiratory losses. The comparison of net primary productivity and P_g showed that ca 60% of P_g was utilized for autotrophic respiration.     

Using hydrothermal time concept to describe sesame (<Emphasis Type="Italic">Sesamum indicum</Emphasis> L.) seed germination response to temperature and water potential

To quantify both temperature (T) and water potential (ψ) effects on sesame (Sesamum indicum L.) seed germination (SG) and also to determine the cardinal T _s for this plant, a laboratory experiment was carried out using hydrothermal time model (HTT). For this purpose, four sesame cultivars (‘Asbomahalleh’, ‘Darab’, ‘Dashtestan’ and ‘Yellowhite’) were germinated at seven constant T _s (20, 25, 30, 35, 37, 39 and 43 °C) at each of the following ψ _s (0, ? 0.12, ? 0.24 and ? 0.36 MPa; provided by PEG 8000). Germination rate (GR) and germination percentage (GP) significantly influenced by ψ, T and their interactions in all cultivars (P ≤ 0.01). There was no significant difference, based on the confidence intervals of the model coefficients, between cultivars, so an average of cardinal T _s was 14.7, 35.4 and 47.2 °C for the minimum (T _b), optimum (T _o) and maximum (T _c) T _s, respectively, in the control condition (0 MPa). Hydrotime values in all cultivars decreased when T was increased to T _o and then remained constant at T _s > T _o (15 MPa h^?1). An average value of ψ _b(50) was estimated to be ? 1.23 MPa at T _s ≤ T _o and then increased linearly (0.1041 MPa°Ch^?1, the slope of the relationship between ψ _b(50) and supra-optimal T _s) with T when T _s increased above T _o and finally reached to zero at T _c. The T _b and T _o values were not influenced by ψ, but T _c value decreased (from 47.2 for zero to 43.5 °C for ? 0.36 MPa) at supra-optimal T _s as a result of the effect of ψ on GR. Based on our findings, this model (as a predictive tool) and or the estimated parameter values in this study can easily be used in sesame SG simulation models to quantitatively characterize the physiological status of sesame seed populations at different T _s and ψ _s.     

The cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942 has a sodium-dependent chloride transporter

Synechococcus R-2 (PCC 7942) actively accumulated Cl^? in the light and dark, under control conditions (BG-11 media: pH_o, 7·5; [Na⁺]_o, 18 mol m^?3; [Cl^?]_o, 0·508 molm^?3). In BG-11 medium [Cl^?], was 17·2±0·848 mol m^?3 (light), electrochemical potential of Cl^? (ΔμCl^?_i,o) =+211±2mV; [Cl^?]_i= 1·24±0·11 mol m^?3(dark), ΔμCl^?_i,o=+133±4mV. Cl^? fluxes, but not permeabilities, were much higher in the light: ?Cl^?_i,o= 4·01±5·4 nmol m^?2 s^?1, ^PCl^?_i,o= 47±5pm s^?1 (light); ?Cl^?_i,o= 0·395±0·071 nmol m^?2 s^?1, _PCl^?_i,o= 69±14 pm s^?1 (dark). Chloride fluxes are inhibited by acid pH_o (pH_o 5; ?Cl^?_i,o= 0·14±0·04 nmol m^?2 s^?1); optimal at pH_o 7·5 and not strongly inhibited by alkaline pH_o (pH_o 10; ?Cl^?1_i,o= 1·7±0·14 nmol m^?2 s^?1). A Cl^?_in/2H⁺_in coporter could not account for the accumulation of Cl^? alkaline pH_o. Permeability of Cl^? is very low, below 100pm s^?1 under all conditions used, and appears to be maximal at pH_o 7·5 (50–70 pm s^?1) and minimal in acid pH_o (20pm s^?1). DCCD (dicyclohexyl-carbodiimide) inhibited ?Cl^?_i,o in the light about 75% and [Cl^?]_i fell to 2·2±0·26 (4) mol m^?3. Valinomycin had no effect but monensin severely inhibited Cl^? uptake ([Cl^?]_i= 1·02±0·32 mol m^?3; ?Cl^?_i,o= 0·20±0·1 nmol m^?2 s^?1). Vanadate (200 mmol m^?3) accelerated the Cl^? flux (?Cl^?_i,o= 5·28±0·64 nmol m^?2 s^?1) but slightly decreased accumulation of Cl^? ([Cl^?], = 13·9±1·3 mol m^?3) in BG-11 medium but had no significant effect in Na⁺-free media. DCMU (dichlorophenyldimethylurea) did not reduce [Cl^?], or ?Cl^?_i,o to that found in the dark ([Cl^?]_i= 8·41±0·76 mol m^?3; ?Cl^?_i,o= 2·06±0·36 nmol m^?2 s^?1). Synechococcus also actively accumulated Cl^? in Na⁺-free media, [Cl^?]_i was lower but ΔΨ_i,o hyperpolarized in Na⁺-free media and so the ΔμCl^?_i,o was little changed ([Cl^?]_i= 7·98±0·698 mol m^?3; ΔμCl^?_i,o=+203±3 mV). Net Cl^? uptake was stimulated by Na⁺; Li⁺ acted as a partial analogue for Na⁺. Synechococcus has a Na⁺ activated Cl^? transporter which is probably a primary 2Cl^?/ATP pump. The Cl^? pump is voltage sensitive. ΔμCl^?_i,o is directly proportional to ΔΨ_i,o(P»0·01%): ΔμCl^?_i,o= -1·487 (±0·102) ×ΔΨ_i,o, r= -0·983, n= 31. The ΔμCl^?_i,o increased (more positive) as the Δμ_i,o became more negative. The ΔμCl^?_i,o has no known function, but might provide a driving force for the uptake of micronutrients.     

Comparative Effects of Hydrogen Sulfide-Releasing Compounds on [<Superscript>3</Superscript>H]D-Aspartate Release from Bovine Isolated Retinae

We investigated the pharmacological actions of a slow-releasing H₂S donor, GYY 4137; a substrate for the biosynthesis of H₂S, l-cysteine and its precursor, N-acetylcysteine on potassium (K⁺; 50 mM)-evoked [³H]D-aspartate release from bovine isolated retinae using the Superfusion Method. GYY 4137 (10 nM–10 µM), l-cysteine (100 nM–10 µM) and N-acetylcysteine (10 µM–1 mM) elicited a concentration-dependent decrease in K⁺-evoked [³H]D-aspartate release from isolated bovine retinae without affecting basal tritium efflux. At equimolar concentration of 10 µM, the rank order of activity was as follows: l-cysteine?>?GYY 4137?>?N-acetylcysteine. A dual inhibitor of the biosynthetic enzymes for H₂S, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), amino-oxyacetic acid (AOA; 3 mM) reversed the inhibitory responses caused by GYY 4137, l-cysteine and N-acetylcysteine on K⁺-evoked [³H]D-aspartate release. Glibenclamide (300 µM), an inhibitor of K_ATP channels blocked the inhibitory action of GYY 4137 and l-cysteine but not that elicited by N-acetylcysteine on K⁺-induced [³H]D-aspartate release. The inhibitory effect of GYY 4137 and l-cysteine on K⁺-evoked [³H]D-aspartate release was reversed by the non-specific inhibitor of nitric oxide synthase (NOS), l-NAME (300 µM). Furthermore, a specific inhibitor of inducible NOS (iNOS), aminoguanidine (10 µM) blocked the inhibitory action of l-cysteine on K⁺-evoked [³H]D-aspartate release. We conclude that both donors and substrates for H₂S production can inhibit amino acid neurotransmission in bovine isolated retinae, an effect that is dependent, at least in part, upon the intramural biosynthesis of this gas, and on the activity of K_ATP channels and NO synthase.     

Growth kinetics and fucoxanthin production of <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> and <Emphasis Type="Italic">Isochrysis galbana</Emphasis> cultures at different light and agitation conditions

Fucoxanthin is a carotenoid that exerts multiple beneficial effects on human health. However, reports comparing microalgae culture conditions and their effect on growth and fucoxanthin production are still limited. Isochrysis galbana and Phaeodactylum tricornutum cultures in different light (62.0, 25.9, 13.5, or 9.1 μmol photons m^-2 s^-1), mixing conditions (1 vvm aeration or 130 rpm agitation), and media compositions (F/2 and Conway medium) were studied for comparison of cellular growth and fucoxanthin production on F/2 medium. I. galbana showed a better adaptation to tested culture conditions in comparison with P. tricornutum, reaching 2.15?×?10⁷?±?4.07?×?10⁶ cells mL^-1 and a specific growth rate (μ) of 1.12?±?0.05 day^-1 under aerated conditions and 62.0 μmol photons m^-2 s^-1 light intensity. Fucoxanthin concentration was about 25 % higher in P. tricornutum cultures under 13.5 μmol photons m^-2 s^-1 light intensity and aerated conditions, but the highest fucoxanthin total production was higher in I. galbana, where 3.32 mg can be obtained from 1 L batch cultures at the 16th day under these conditions. Moreover, higher cell densities (~32.41 %), fucoxanthin concentration (~42.46 %), and total production (~50.68 %) were observed in I. galbana cultures grown in Conway medium, if compared with cultures grown in F/2 medium. The results show that the best growth conditions did not result in the best fucoxanthin production for either microalgae, implying that there is not a direct relationship between cellular growth and fucoxanthin production. Moreover, the results suggest that I. galbana cultures on Conway medium are strong candidates for fucoxanthin production, where 1.2 to 15 times higher fucoxanthin concentration are observed in comparison to macroalgal sources.