Optimization of <Superscript>1</Superscript>H decoupling eliminates sideband artifacts in 3D TROSY-based triple resonance experiments

TROSY-based triple resonance experiments are essential for protein backbone assignment of large biomolecular systems by solution NMR spectroscopy. In a survey of the current Bruker pulse sequence library for TROSY-based experiments we found that several sequences were plagued by artifacts that affect spectral quality and hamper data analysis. Specifically, these experiments produce sidebands in the ¹³C(t ₁) dimension with inverted phase corresponding to ¹H_N resonance frequencies, with approximately 5% intensity of the parent ¹³C crosspeaks. These artifacts originate from the modulation of the ¹H_N frequency onto the resonance frequency of ¹³Cα and/or ¹³Cβ and are due to 180° pulses imperfections used for ¹H decoupling during the ¹³C(t ₁) evolution period. These sidebands can become severe for CA_i, CA_i?1 and/or CB_i, CB_i?1 correlation experiments such as TROSY-HNCACB. Here, we implement three alternative decoupling strategies that suppress these artifacts and, depending on the scheme employed, boost the sensitivity up to 14% on Bruker spectrometers. A class of comparable Agilent/Varian pulse sequences that use WALTZ16 ¹H decoupling can also be improved by this method resulting in up to 60–80% increase in sensitivity.     

Cross-correlation suppressed T1 and NOE experiments for protein side-chain 13CH2 groups

Relaxation measurements of side-chain ¹³CH₂-groups of uniformly ¹³C labeled human ubiquitin were performed at 600 MHz and 800 MHz magnetic field strength at 30°C. Dipole-dipole cross-correlated relaxation effects in T₁ experiments were suppressed by the combination of radio-frequency pulses and pulsed field gradients during the relaxation delay leading to monoexponential relaxation decays that allow a more accurate extraction of the ¹³C T₁ relaxation times. Heteronuclear ¹H-¹³C NOEs obtained by using different proton saturation schemes indicate that the influence of cross-correlation is small. The experimental T₁ and NOE data were interpreted in a model-free way in terms of a generalized order parameter and an internal correlation time.     

On the possibility of true phase transition in heterogeneous DNA during thermal denaturation under conditions close to equal stability of A+T and G+C pairs

The DNA helix–coil transition has been studied in the presence of high concentrations of manganese ions (about 10^?3M), which corresponds to the conditions close to equal stability of the A+T and G+C pairs, at the ionic strengths of 10^?1, 10^?2, and 1.6 × 10^?3M Na⁺. With the Mn²⁺ ion effect, the transition range is significantly reduced to not more than 0.2°C at 1.2 × 10^?3M Mn²⁺ and 1.6 × 10^?3M Na⁺. The melting curves display a sharp kink at the end of the helix–coil transition, which is interpreted as an indication of the second-order phase transition. It is shown that the melting curves obtained can be approximated by a simple analytical expression 1 – θ = exp[–a(t_c - t)], where θ is the DNA helix fraction, t_c is the phase transition temperature, and a is an empirical parameter characterizing the breadth of the melting range and responsible for the magnitude of a jump of the helicity derivative with respect to the temperature at the phase transition point.     

Linear prediction enhancement of 2D heteronuclear correlated spectra of proteins

Summary Linear prediction has been used to extrapolate the t₁ domain of natural abundance¹H–¹³C correlated two-dimensional (2D) FIDs of insulin. The FIDs were obtained by two different heteronuclear correlation experiments, one that utilizes heteronuclear multiple-quantum coherence during t₁, and one that utilizes¹³C single-quantum coherence. It is shown that the enhancement of the resolution and sensitivity in the F₁ dimension of the Fourier transform spectrum that results from the linear prediction extrapolation allows the t₁ domain to be confined to a relatively short time period where the signal intensity is at maximum. In particular, it is found that the enhancement thus obtained is sufficiently good to allow an observation of the difference between the F₁ line widths in the single-quantum and double-quantum coherence spectra.     

Design and Dosimetric Characterization of a Broadband Exposure Facility for In Vitro Experiments in the Frequency Range 18–40.5 GHz

A novel exposure facility for exposing cell monolayers to centimeter and millimeter waves (18–40.5 GHz) used by future 5G mobile communication technology and similar applications has been developed. A detailed dosimetric characterization of the apparatus for frequencies of 27 and 40.5 GHz and 60 mm petri dishes, used in a presently ongoing study on human dermal fibroblasts and keratinocytes, was carried out. The exposure facility enables a well-defined, randomized, and blinded application of sham exposure and exposure with selectable values of incident power flux density, and additionally provides the possibility of continuous monitoring of the sample temperature during exposure while it does not require significant deviations from routine in vitro handling procedures, i.e. petri dishes are not required to be placed inside waveguides or TEM cells. Mean specific absorption rate (SAR) values inside the cell monolayer of 115 W/kg (27 GHz) and 160 W/kg (40.5 GHz) per watt antenna input power and corresponding transmitted power density (S_t) values at the bottom of the cell monolayer of 65 W/m² (27 GHz) and 70 W/m² (40.5 GHz) per watt antenna input power can be achieved, respectively. For reasonable amounts of harvested cells (80% of petri dish bottom area), the variation (max/min) of SAR and S_t over the cell monolayer remains below 3.7 dB (27 GHz) and 3.0 dB (40.5 GHz), respectively. © 2021 Bioelectromagnetics Society.     

The use of critical power as a determinant for establishing the onset of blood lactate accumulation

Eight highly trained male kayakers were studied to determine the relationship between critical power (CP) and the onset of blood lactate accumulation (OBLA). Four exercise sessions of 90 s, 240 s, 600 s, and 1200 s were used to identify the CP of each kayaker. Each individual CP was obtained from the line of best fit (LBF_CP) obtained from the progressive work output/time relationships. The OBLA was identified by the 4 mmol·l^–1 blood lactate concentration and the work output at this level was determined using a lactate curve test. This consisted of paddling at 50 W for 5 min after which a 1-min rest was taken during which a 25-l blood sample was taken to analyse for lactate. Exercise was increased by 50 W every 5 min until exhaustion, with the blood sample being taken in the 1-min rest period. The exercise intensity at the OBLA for each subject was then calculated and this was compared to the exercise intensity at the LBF_CP. The intensity at LBF_CP was found to be significantly higher (t=2.115, P<0.05) than that at the OBLA of 4 mmol·1^–1. These results were further confirmed by significant differences being obtained in blood lactate concentration (t=8.063, P<0.05) and heart rate values (t=2.90, P<0.05) obtained from the exercise intensity at LBF_CP over a 20-min period and that of the anaerobic threshold (Th_an) parameters obtained from the lactate/heart rate curve. These differences suggest that CP and Th_an are different physiological events and that athletes have utilised either one or the other methods for monitoring training and its effects.     

Beta-turns in serine-containing linear and cyclic models

Tetrapeptides, Cbz-Gly-X-Y-Gly-OSt ( 1 – 4 )—as well as cyclic systems, cyclo[NH-(CH₂)_n-CO-Gly-Ser(OX)-Ser(OX)-Gly] ( 5 and 6 ; n = 4 and 2, X = Bu^t or H), have been synthesized in order to compare the CD spectrum of linear and cyclic β-turn models containing either a protected or a free hydroxyl of the serine residue. In extremely dilute cyclohexane solution the linear models Cbz-Gly-Ser-Y-Gly-OSt ( 1 – 3a ) show class B spectra with very strong positive bands, contrary to other members of the series. Based on 200-MHz ¹H nuclear overhauser enhancement and Fourier transform ir studies, Cbz-Gly-Ser-Ser(OBu^t)-Gly-OSt ( 3a ) in dilute chloroform solution assumes a distorted type II β-turn conformation fixed by an extended system of intramolecular H bonds. As evidenced by ¹H-nmr and FT-IR experiments, the cyclic model cyclo[NH-(CH₂)₄-CO-Gly-Ser(OBu^t)- Ser(OBu^t)-Gly] ( 5a ) in a 1 : 1 mixture of (CD₃)₂SO-CDCl₃ is also characterized by a type II β-turn encompassing the Ser³(OBu^t)-Gly⁴ sequence. In water, a class B pattern was measured for this model, in good agreement with theoretical and experimental studies that show that type II β-turns are generally characterized by class B spectra. In the protected and free OH cyclic models, cyclo[NH-(CH₂)₂-CO-Gly-Ser(OX)-Ser(OX)-Gly] ( 5b and 6b , X = Bu^t or H) distortions of the peptide backbone due to the loss of two CH₂ groups result in the appearance of CD spectra characterized by a strong negative band near 200 nm, interpreted as a sign of the lack of β-turn structures in these models. This observation, together with other CD data discussed in this paper, clearly show that the CD of serine-containing β-turn sequences strongly depends on long-range backbone and local side-chain interactions.     

Temperature and humidity effects on branchlet gas-exchange in white spruce: an explanation for the increase in transpiration with branchlet temperature

In situ gas-exchange data, for branchlets of white spruce [Picea glauca (Moench) Voss.] in a mature mixed-wood boreal forest in central Canada (53°44′N 105°14′W), were subjected to a multiple regression analysis. Vapor pressure deficit (VPD) and branchlet temperature (t_leaf) were both significant predictors (P<0.0001) of stomatal conductance to water vapor (g_sw) and net photosynthesis (A_n), together explaining 67 and 64% of the variation in g_sw and A_n, respectively. Since VPD and t_leaf were autocorrelated in these field data, but also to further explore the nature of independent effects of temperature and humidity on water and CO₂ exchange in white spruce, steady-state gas-exchange was performed on well-watered greenhouse-grown seedlings of white spruce. Results from laboratory experiments supported the following conclusions: (1) Transpiration (E) increases with VPD to an inflection point that increases linearly with t_leaf. This t_leaf effect on E could not be explained by trends in VPD, RH, A_n or PFD. Rather, our data support a model in which E and g_sw are influenced by the balance between ’supply’ and ’loss’ of water to and from leaf tissue, respectively. The supply of water appears to be in accordance with Darcy’s law, where supply of water is proportional to the driving gradient in pressure/ tension, specific permeability (k), and inverse of water viscosity (n ^–1). Approximately half of the increase in E could be explained by the linear increase in n ^–1 with increasing t_leaf. We propose that increases in k explain the remainder of the increase in E with t_leaf. (2) VPD and t_leaf appear to have independent effects on g_sw. In contrast, RH effects on g_sw or E were subtle and could be explained by a combination of effects of t_leaf and VPD. (3) A_n was affected primarily by t_leaf, being reduced at low (10°C) and high (40°C) temperatures, and only indirectly by humidity parameters via stomatal conductance, viz. intercellular CO₂ concentrations. Our results have implications for the prediction of water fluxes from plants and canopies in areas where plant temperatures vary diurnally or seasonally. Received: 24 September 1998 / Accepted: 20 July 1999     

Action of extremely low frequency electric fields on the cytosolic calcium concentration of differentiated HL-60 cells: Nonactivated cells

The effect of sinusoidal electric fields on the cytosolic free [Ca²⁺]_i concentration in differentiated HL-60 cells was measured. The calcium concentration was measured in a fluorescence spectrometer using the fluorescence sample fluo-3. In the fluorescence spectrometer two samples can be measured simultaneously, one as the sham-exposed control and the other as the field-exposed sample. The effects of an external field, applied using two capacitor plates outside the cuvettes, and a field applied directly to the medium, using two platinum electrodes inside the cuvettes, were measured at selected frequencies between 0 and 100 Hz and field strengths from 1 to 2000 V_pp/m (external field) and from 0.1 to 1000 V_pp/m (in medium). No significant effects of the fields on the cytosolic free [Ca²⁺]_i concentration in HL-60 cells have been observed at the measured frequencies and field strengths. Bioelectromagnetics 19:32–40, 1998. © 1998 Wiley-Liss, Inc.     

Acid treatment effects on the stable isotopic signatures of fossils

Abstract: Prior to geochemical analyses, fossil bones and teeth are often extracted from any surrounding lithified sediments using chemical techniques such as immersion in acid. As stable isotope analysis becomes more commonplace in palaeoecological investigations, it is important to consider what effects these chemical preparation techniques may have on any subsequent isotopic data and to constrain these effects as quantitatively as possible. This study aims to elucidate these effects, as it is vital that variability in a data set should not be introduced as a result of protocols used during sample preparation; in addition, it defines the most effective and viable method of carbonate removal for processing bulk fossil samples without causing alteration of their stable isotopic signatures. Various strengths of two weak acids commonly used during palaeontological preparation were tested to evaluate their effects on the δ¹⁵N and δ¹³C_org isotopic signatures of the vertebrae of a large Eocene fossil fish. Changes in the isotopic values occurred over time regardless of which acid was used, each causing a variable response in both δ¹⁵N and δ¹³C_org isotopic values. Without careful monitoring of the acidification process in a controlled environment, any resulting data could therefore confound interpretation. Based on these experiments, it is recommended that 2 m acetic acid be used for the pretreatment of fossils prior to the acquisition of N and C isotope data where carbonate removal is necessary.     

Energy cost and energy sources in karate

Energy costs and energy sources in karate (wado style) were studied in eight male practitioners (age 23.8 years, mass. 72.3 kg, maximal oxygen consumption (VO_2max) 36.8 ml · min^–1 · kg^–1) performing six katas (formal, organized movement sequences) of increasing duration (from approximately. 10 s to approximately 80 s). Oxygen consumption (VO₂) was determined during pre-exercise rest, the exercise period and the first 270 s of recovery in five consecutive expired gas collections. A blood sample for lactate (la^–) analysis was taken 5 min after the end of exercise. The overall amount of O₂ consumed during the exercise and in the following recovery increased linearly with the duration of exercise (t) from approximately 1.51 (for t equal to 10.5 s (SD 1.6)) to approximately 5.81, for t equal to 81.5 s (SD 1.0). The energy release from la^– production (VO_21a ^–) calculated assuming that an increase of 1 mmol · l^–1 la^– corresponded to a VO₂ of 3 mlO₂ · kg^–1 was negligible for t equal to or less than 20 s and increased to 17.3 ml · kg^–1 (la^– = 5.8 mmol · l^–1 above resting values) for t equal approximately to 80 s. The overall energy requirement (VO_2eq) as given by the sum of VO₂ and VO_2la ^– was described by VO_2eq = 0.87 + 0.071 · t (n = 64; r ² = 0.91), where VO_2eq is in litres and t in seconds. This equation shows that the metabolic power (VO_2eq · t ^–1) for this karate style is very high: from approximately 9.51 · min^–1 for t equal to 10 s to approximately 4.91 · min^–1 for t equal to 80 s, i.e. from 3.5 to 1.8 times the subjects' VO_2max. The fraction of VO_2eq derived from the amount of O₂ consumed during the exercise increased from 11% for t equal to 10 s to 41 % for t equal to 80 s whereas VO_21a ^– was negligible far t equal to or less than 20 s and increased to 13 % o for t equal to 80 s. The remaining fraction (from 90% for t equal to 10 s to 46% for t equal to 80 s), corresponding to the amount of O₂ consumed in the recovery after exercise, is derived from anaerobic alactic sources, i.e. from net splitting of high energy phosphates during the exercise.     

[125I]calmodulin binding to synaptic plasma membrane from rat brain: Kinetic and arrhenius analysis

Binding of [¹²⁵I]calmodulin was characterized in highly purified synaptic plasma membrane (SPM) prepared from rat brain. By Scatchard analysis, the Ca²⁺-dependent membrane binding of [¹²⁵I]calmodulin was found to have a B_max of 284 pmol/mg protein and an apparent affinity with a K_d of 131 nM. Kinetic analysis indicates that at 37°C, the dissociation of [¹²⁵I]calmodulinmembrane complexes follows first-order reaction and consists of two components: a dissociation constant (k) of 3.7×10^–1 min^–1 and a half-time (t_1/2) of 1.8 min for the fast component, and a k of 4.8×10^–2 min^–1 and a t_1/2 of 14.5 min for the slow component. At 0°C, substantial dissociation still occurred, with a k of 4.5×10^–2 min^–1 and a t_1/2 of 15.3 min for the fast component, and a k of 5.5×10^–3 min^–1 and a t_1/2 of 125.5 min for the slow component. These data on binding affinity and dissociation kinetics are consistent with the notion that SPM can readily and rapidly associated and dissociate calmodulin. In Arrhenius analysis of temperature effects, [¹²⁵I]calmodulin binding to SPM exhibits a biphasic function, with the transition temperature (T_d) estimated to be 23.8°C, suggesting that binding is influenced by lipid phase transition of the membrane. The binding of [¹²⁵I]calmodulin to the synaptic membrane was found to be increased by corticosterone (10^–7–10^–6 M), a steroid hormone, and decreased by ethanol (50–200 mM), a centrally acting drug. Our data on the characteristics of calmodulin binding to the SPM provide groundwork for future studies on physiological and pharmacological regulation of calmodulin translocation to and from the plasma membrane in synaptic terminals.Abbreviations used CaM calmodulin - SPM synaptic plasma membrane - ATPase adenosine triphosphatase - Tris tris(hydroxymethyl)aminomethane - EGTA ethylene-bis(oxyethylenenitrilo)tetraacetic acid - SDS sodium dodecyl sulfate - TFP trifluoperazine - K_d dissociation constant - B_max maximum binding - k first-order rate constant - t_1/2 half-time - T_d transition temperature     

Ar + H<Subscript>2</Subscript> Plasma Interacting with Lithium-Filled Capillary Porous Structure

Ar + H₂ plasma interacting with liquid lithium was carried out on a one-cathode linear plasma device (SCU-PSI). The lithium sample was covered with capillary porous structure (CPS). It is found that the electron temperature of applied plasma ranged from ~0–1 eV and electron density ranged from 0.1 × 10²⁰ to 1 × 10²⁰ m^?3. The experimental results indicate that a reduction in the electron temperature and the lithium evaporation is found as the percentage of H₂ increases When the ratio of argon and hydrogen keeps constant, the electron temperature and lithium evaporation increase with applied input power, respectively. The retention of hydrogen atoms in lithium surface results in reducing the lithium evaporation. The XRD analysis result shows that during plasma radiation no LiH is formed.     

Experimental study of 60Co behaviour in Tejo River sediments

The objective of this study was to obtain information on the transfer of radiocobalt in freshwater environments that can be used to predict its environmental distribution. The sediment-water behaviour of ⁶⁰Co in freshwater systems was studied through adsorption and desorption experiments undertaken using sediments and water from Fratel Reservoir in the Tejo River. The suspended sediment concentrations (C_s: 500–2000 mg 1^–1) and Co distribution coefficient (K_d) were inversely related: K_d = 2211–2001 ln [C_s]; K_d ranged from 4000 to 8000 ml g^–1. With a suspended sediment concentration of 1000 mg 1^–1, the ⁶⁰Co concentration remaining in solution (C_t) was given by: C_t = 49.4 e^–0.584t + 46.3 e^–0.014t; where t is the time in days and the half-life periods are 1.2 and 50 days. In a closed system, desorption of ⁶⁰Co could be described by a one-component relation with a half-life of 104 days, and a two component relation (half-life 5 hours and 45 days) in an open system. In river water the ⁶⁰Co was found to be almost 100% in cationic forms, however, in the presence of sediment there was a decrease in the proportion of cationic forms (to 50%), with some anionic forms appearing.     

Forward Modeling of Fluctuating Dietary 13C Signals to Validate 13C Turnover Models of Milk and Milk Components from a Diet-Switch Experiment

Isotopic variation of food stuffs propagates through trophic systems. But, this variation is dampened in each trophic step, due to buffering effects of metabolic and storage pools. Thus, understanding of isotopic variation in trophic systems requires knowledge of isotopic turnover. In animals, turnover is usually quantified in diet-switch experiments in controlled conditions. Such experiments usually involve changes in diet chemical composition, which may affect turnover. Furthermore, it is uncertain if diet-switch based turnover models are applicable under conditions with randomly fluctuating dietary input signals. Here, we investigate if turnover information derived from diet-switch experiments with dairy cows can predict the isotopic composition of metabolic products (milk, milk components and feces) under natural fluctuations of dietary isotope and chemical composition. First, a diet-switch from a C₃-grass/maize diet to a pure C₃-grass diet was used to quantify carbon turnover in whole milk, lactose, casein, milk fat and feces. Data were analyzed with a compartmental mixed effects model, which allowed for multiple pools and intra-population variability, and included a delay between feed ingestion and first tracer appearance in outputs. The delay for milk components and whole milk was ∼12 h, and that of feces ∼20 h. The half-life (t_½) for carbon in the feces was 9 h, while lactose, casein and milk fat had a t_½ of 10, 18 and 19 h. The ¹³C kinetics of whole milk revealed two pools, a fast pool with a t_½ of 10 h (likely representing lactose), and a slower pool with a t_½ of 21 h (likely including casein and milk fat). The diet-switch based turnover information provided a precise prediction (RMSE ∼0.2 ‰) of the natural ¹³C fluctuations in outputs during a 30 days-long period when cows ingested a pure C3 grass with naturally fluctuating isotope composition.     

Hydraulic resistance to radial water flow in growing hypocotyl of soybean measured by a new pressure-perfusion technique

Hydraulic resistances to water flow have been determined in the cortex of hypocotyls of growing seedlings of soybean (Glycine max L. Merr. cv. Wayne). Data at the cell level (hydraulic conductivity, Lp; half-time of water exchange, T _1/2; elastic modulus, ; diffusivity for the cell-to-cell pathway, D _c) were obtained by the pressure probe, diffusivities for the tissue (D _t) by sorption experiments and the hydraulic conductivity of the entire cortex (Lp_r) by a new pressure-perfusion technique. For cortical cells in the elongating and mature regions of the hypocotyls T _1/2=0.4–15.1 s, Lp=0.2·10^-5–10.0·10^-5 cm s^-1 bar^-1 and D _c=0.1·10^-6–5.5·10^-6 cm² s^-1. Sorption kinetics yielded a tissue diffusivity D _t=0.2·10^-6–0.8·10^-6 cm² s^-1. The sorption kinetics include both cell-wall and cell-to-cell pathways for water transport. By comparing D _c and D _t, it was concluded that during swelling or shrinking of the tissue and during growth a substantial amount of water moves from cell to cell. The pressure-perfusion technique imposed hydrostatic gradients across the cortex either by manipulating the hydrostatic pressure in the xylem of hypocotyl segments or by forcing water from outside into the xylem. In segments with intact cuticle, the hydraulic conductance of the radial path (Lp_r) was a function of the rate of water flow and also of flow direction. In segments without cuticle, Lp_r was large (Lp_r=2·10^-5–20·10^-5 cm s^-1 bar^-1) and exceeded the corticla cell Lp. The results of the pressure-perfusion experiments are not compatible with a cell-to-cell transport and can only the explained by a preferred apoplasmic water movement. A tentative explanation for the differences found in the different types of experiments is that during hydrostatic perfusion the apoplasmic path dominates because of the high hydraulic conductivity of the cell wall or a preferred water movement by film flow in the intercellular space system. For shrinking and swelling experiments and during growth, the films are small and the cell-to-cell path dominates. This could lead to larger gradients in water potential in the tissue than expected from Lp_r. It is suggested that the reason for the preference of the cell-to-cell path during swelling and growth is that the solute contribution to the driving force in the apoplast is small, and tensions normally present in the wall prevent sufficiently thick water films from forming. The solute contribution is not very effective because the reflection coefficient of the cell-wall material should be very small for small solutes. The results demonstrate that in plant tissues the relative magnitude of cell-wall versus cell-to-cell transport could dependent on the physical nature of the driving forces (hydrostatic, osmotic) involved.Abbreviations and symbols D _c diffusivity of the cell-to-cell pathway - D _t diffusivity of the tissue - radial flow rate per cm² of segment surface - Lp hydraulic conductivity of plasma-membrane - Lp_r radial hydraulic conductance of the cortex - T _1/2 half-time of water exchange between cell and surroundings - volumetric elastic modulus     

Ontogeny of yolk-feeding fish: an ecological perspective

Ontogeny is a continuous process with temporaryaccelerations. The embryonic period from eggactivation to hatching, and the larval periodthereafter, are considered. Advances in studieson the ontogeny of yolk-feeding Europeanfreshwater and Antarctic marine fish arecompared. New techniques and approaches aresummarized. A method for exact quantificationof the time to any developmental event isrecommended. Four attempts to quantify anindividual's ontogenetic advancement arereviewed, of which Fuiman's ontogenetic indexseems to be the best choice. The relationshipbetween the time to any ontogenetic event(, days) and temperature (t, °C)has been quantified by exponential, power law,Blehradek's, Leiner's, and polynomialmodels, whose common weaknesses are that theparameters have no biological meaning, and theydo not allow comparison of temperaturerequirements between species. The ontogeneticrate (V = 1/, days^–1) was welldescribed (r² = 0.92 – 1.00) by a straightline V = a + bt (linear model) in 44 fishspecies over a broad low-mortality temperaturerange. The linear model produces biologicallymeaningful parameters: the temperature ofbiological zero t₀ = –a/b, effectivetemperature t_eff = t –: t₀, andeffective day-degrees D°_eff = (t – t₀) = b^–1. From t₀ andD°_eff the time to any ontogeneticevent can be computed as: =D°_eff/(t – t₀). In coldwaterspecies low t₀ is accompanied by highD°_eff, whereas in warmwater speciesthe opposite is true. The linear model wasvalidated. Day-degrees (D° = t)and physiological day-degrees (PD° =t/q, where q is Winberg's temperaturemetabolic correction factor) are based onincorrect assumptions. Their use as temperature-independent measures of ontogeneticadvancement is not advised; by contrast,effective day-degrees (D°_eff)are temperature-independent and arerecommended. The remaining extrinsic factorsaffecting ontogenetic rate during yolk feedingare: oxygen, salinity, pH, light, dissolvedbiotic compounds, and anthropogenic factors.The main intrinsic factor is egg size, whichpositively affects the time to particularontogenetic steps at inter- and intra-specificlevels. Attempts to quantify the combinedeffects of temperature and salinity, and oftemperature and egg size, are reviewed.     

Reaction of carbon monoxide with tri-tert-butylgallium: The first example of CO insertion into a gallium-carbon bond

Reactions of ^tBu₃M (M = Al, Ga) with CO have been studied by density functional theory employing the B3PW91 functional. Calculations suggest that CO insertion into a M-C bond of ^tBu₃M is thermodynamically favorable at room temperature, whereas CO coordination to ^tBu₃M to form ^tBu₃M·CO is unfavorable due to an unfavorable entropy change. These results are in agreement with experimental observations. Reaction of carbon monoxide with ^tBu₃Ga at 50 °C and atmospheric pressure yields the dimeric tert-butylacyl complex [^tBu₂GaC(O)^tBu]₂ (1). Compound 1 has been characterized by X-ray crystallography and NMR and IR spectroscopy. Isotopic labeling with ¹³CO confirmed that the acyl carbon of 1 results from the CO starting material. CO insertion into a Ga-C bond does not occur for Me₃Ga or ⁿBu₃Ga under a range of reaction conditions.     

Sap flow in Scots pines growing under conditions of year-round carbon dioxide enrichment and temperature elevation

Starting in rrrr, individual trees of Scots pine (Pinus sylvestris L.) aged 30 years were grown in closed-top chambers and exposed to normal ambient conditions (CON), elevated CO₂ (Elev. C), elevated temperature (Elev. T) and a combination of elevated CO₂ and temperature (Elev. C + T). Using the constant-power heat balance method, sap flow was monitored simultaneously in a total of 16 trees, four for each treatment, over a 32 d period (after the completion of needle expansion and branch elongation in 1997). An overall variation in diurnal sap flow totals (F_t) was evident during the period of measurement (days 167–198, 1997) regardless of the treatments, with a range from 0·15 to 2·82 kg tree^–1 d^–1. Elev. C reduced F_t by 4·1–13·7% compared with CON on most days (P varies from 0·042 to 0·108), but slightly increased it on some days (P≥ 0·131), depending on the weather conditions. Although the decrease in F_t caused by Elev. C was statistically significant on only a few days (P≤ 0·042), the cumulative F_t for the 32 d decreased by 14·4% (P = 0·047), indicating that Elev. C may have an important influence on seasonal water use of the Scots pine. Analysis of the diurnal courses of sap flow combined with corresponding weather factors indicated that the CO₂-induced decrease in F_t could be largely attributed to an increase in stomatal sensitivity to vapour pressure deficit (VPD), whereas the CO₂-induced increase in F_t related to an increase in stomatal sensitivity to low light levels. Elev. T increased F_t by 11·2–35·6% throughout the measuring period and the cumulative F_t for the 32 d by 32·5% (P = 0·019), which could be largely attributed to the temperature-induced increase in current-year needle area and decrease in stomatal sensitivity to high levels of VPD. There were no significant interactive effects of CO₂ and temperature on sap flow, so that Elev. C + T had approximately the same F_t as Elev. T and similar diurnal patterns of sap flow, suggesting that the temperature factor played a dominant role in the case of Elev. C + T.     

Determination of 3J(H infi supN,C infi sup′ ) coupling constants in proteins with the C′-FIDS method

Summary We introduce the C-FIDS-¹H,¹⁵N-HSQC experiment, a new method for the determination of ³J(H _infi ^supN ,C _infi ^sup ) coupling constants in proteins, yielding information about the torsional angle . It relies on the ¹H,¹⁵N-HSQC or HNCO experiment, two of the the most sensitive heteronuclear correlation experiments for isotopically labeled proteins. A set of three ¹H,¹⁵N-HSQC or HNCO spectra are recorded: a reference experiment in which the carbonyl spins are decoupled during t₁ and t₂, a second experiment in which they are decoupled exclusively during t₁ and a third one in which they are coupled in t₁ as well as t₂. The last experiment yields an E.COSY-type pattern from which the ²J(H _infi ^supN ,C _infi-1 ^sup ) and ¹J(N_i,C _infi-1 ^sup ) coupling constants can be extracted. By comparison of the coupled multiplet (obtained from the second experiment) with the decoupled multiplet (obtained from the first experiment) convoluted with the ²J(H _infi ^supN ,C _infi-1 ^sup ) coupling, the ³J(H _infi ^supN ,C _infi ^sup ) coupling can be found in a one-parameter fitting procedure. The method is demonstrated for the protein rhodniin, containing 103 amino acids. Systematic errors due to differential relaxation are small for ⁿJ(H^N,C) couplings in biomacromolecules of the size currently under NMR spectroscopic investigation.