Evidence for the origin of the unoccupied oestrogen receptor in nuclei of a human breast-cancer cell line (MCF-7)

The origin of the unoccupied nuclear oestrogen receptor (R_n) was studied. Three working hypotheses were investigated. (a) R_n is a dissociation product of the oestrogen occupied nuclear receptor (ER_n). (b) ER_n is only partially occupied, so that additional binding may occur at 0°C (the temperature at which oestradiol saturates unoccupied sites). (c) R_n is derived from the penetration of unoccupied cytoplasmic receptor (R_c) into the nucleus. The MCF-7 cell line was used as a model in the present investigation. The amount of unoccupied receptors was measured by saturation with 7.5nm-[³H]oestradiol at 0°C, whereas the occupied receptors were measured by exchange at 30°C. The cells at preconfluency were exposed to a medium fortified with 10nm-[³H]oestradiol for 1h, washed and cultured up to 5 days in fresh growth medium. The distribution of oestradiol receptors was determined before exposure and during the following 5 days. After 1h exposure only ER_n was found in the nuclear fraction. Thereafter ER_n declined continuously so that on day 5 it approached 15% of its value measured 1h after exposure. Although after 3 days about 80% of ER_n disappeared no R_n appeared, which contradicts hypotheses (a) and (b). On day 4 R_n and R_c appeared simultaneously. The appearance of R_n and R_c was not prevented by culturing the cells in an oestrogen-free medium, supporting hypothesis (c). Exposure of cells to increasing concentration of [³H]oestradiol (0.1–10nm) for 1h resulted in a parallel increase in ER_n without increasing the amount of unoccupied binding sites, which contradicts hypothesis (b). The present study supports the hypothesis (c), i.e., R_c may also penetrate the nucleus without binding to oestradiol.     

Critical Behaviour in DOPC/DPPC/Cholesterol Mixtures: Static 2H NMR Line Shapes Near the Critical Point

Static ²H NMR spectroscopy is used to study the critical behavior of mixtures of 1,2-dioleoyl-phosphatidylcholine/1,2-dipalmitoyl-phosphatidylcholine (DPPC)/cholesterol in molar proportion 37.5:37.5:25 using either chain perdeuterated DPPC-d₆₂ or chain methyl deuterated DPPC-d₆. The temperature dependence of the first moment of the ²H spectrum of the sample made with DPPC-d₆₂ and of the quadrupolar splittings of the chain-methyl-labeled DPPC-d₆ sample are directly related to the temperature dependence of the critical order parameter η, which scales as [(T_c?T)/T_c]^β_c near the critical temperature. Analysis of the data reveals that for the chain perdeuterated sample, the value of T_c is 301.51 ± 0.1 K, and that of the critical exponent, β_c = 0.391 ± 0.02. The line shape analysis of the methyl labeled (d₆) sample gives T_c = 303.74 ± 0.07 K and β_c = 0.338 ± 0.009. These values obtained for β_c are in good agreement with the predictions of a three-dimensional Ising model. The difference in critical temperature between the two samples having nominally the same molar composition arises because of the lowering of the phase transition temperature that occurs due to the perdeuteration of the DPPC.     

Temperature-Dependent Fecundity and Life Table of the Fennel Aphid Hyadaphis foeniculi (Passerini) (Hemiptera: Aphididae)

Hyadaphis foeniculi (Passerini) (Hemiptera: Aphididae) is a cosmopolitan species and the main pest of fennel in northeastern Brazil. Understanding the relationship between temperature variations and the population growth rates of H. foeniculi is essential to predict the population dynamics of this aphid in the fennel crop. The aim of this study was to measure the effect of constant temperature on the adult prereproductive period and the life table fertility parameters (infinitesimal increase ratio (r_m), gross reproduction rate (GRR), net reproduction rate (R₀), finite increase ratio (λ), generation time (GT), the time required for the population to double in the number of individuals (DT), and the reproduction value (RV_x)) of the fennel pest H. foeniculi. The values of lx (survival of nymphs at age x) increased as the temperature rose from 15 to 28°C and fell at 30°C, whereas mx (number of nymphs produced by each nymph of age x) increased from 15 to 25°C and fell at 28 and 30°C. The net reproduction rates (R₀) of populations of H. foeniculi increased with temperature and ranged from 1.9 at 15°C to 12.23 at 28°C for each generation. The highest population increase occurred with the apterous aphids at 28°C. The rate of population increase per unit time (r_m) (day) ranged from 0.0033 (15°C) to 0.1995 (28°C). The highest values of r_m were recorded at temperatures of 28°C and 30°C. The r_m values were a good fit to the models tested, with R² > 0.91 and R² _adj > 0.88. The models tested (Davidson, Sharpe and DeMichele modified by Schoolfield et al., Logan et al., Lamb, and Briere et al.) were very good fits for the r_m values observed, with R² > 0.91 and R² _adj > 0.88. The only exception was the Davidson model. Of the parameters studied, the reproductive capacity was higher in the apterous aphids, with the unique exception of daily fecundity at 28°C, which was higher in the alate aphids of H. foeniculi. Parameters relating to the age-specific fertility table for H. foeniculi were heavily influenced by temperature, with the highest biotic potential and population growth capacity found at 34°C. Therefore, the results obtained in this study could be of practical significance for predicting outbreaks of fennel aphids and improving the management of this aphid in fennel crops.     

Effects of la on surface charges, dielectrophoresis, and electrofusion of barley protoplasts

When dielectrophoresis and electrofusion of barley (Hordeum vulgare var Moor) leaf protoplasts were assayed in the presence of 0.1 to 1 millimolar lanthanum ion (La³⁺) in the basal medium (0.7 molar mannitol, 1 millimolar piperazine-N, N-bis[2-ethanesulfonic acid]-Na [pH 6.7], 0.1 millimolar CaCl₂), dielectrophoresis and induction of electrofusion were strongly inhibited. The latter remained inhibited and the former recovered by about 60% after washing the La³⁺ -treated protoplasts without EDTA. These inhibitions were almost completely abolished by washing the La³⁺ -treated protoplasts with 1 millimolar EDTA. Inductively coupled plasma atomic emission spectroscopic analysis revealed that protoplasts retained a considerable amount of La³⁺ after washing without EDTA and released most of the bound La³⁺ by washing with 1 millimolar EDTA. This tightly bound La³⁺ seemed responsible for the inhibition of electrofusion and dielectrophoresis that was observed in the La³⁺ -treated protoplasts after washing. ζ-potentials of protoplasts were -39.0±3.2 millivolts, -16.7 ± 2.6 millivolts, and virtually zero in media containing 0, 0.1, and 0.3 millimolar La³⁺ (I = 7.2 millimolar), respectively, and had a positive value (+ 14.2 ± 2.2 millivolts) in the presence of 1 millimolar La³⁺. These effects of La³⁺ on ζ-potentials were easily abolished by washing without EDTA. This indicates that charged species located at the surface of plasma membrane of protoplasts cannot account for the sites at which La³⁺ exerts its inhibition of dielectrophoresis and electrofusion. In contrast, the promotion of spherical fusion and the reduction of broken fusion products observed in the presence of La³⁺ were almost completely abolished by washing without EDTA. Our results also indicate that the initial induction and development of electrofusion can be studied independently.     

The Effect of Double – Blind Carbohydrate Ingestion during 60 km of Self-Paced Exercise in Warm Ambient Conditions

This study evaluated double blind ingestions of placebo (PLA) versus 6% carbohydrate (CHO) either as capsules (c) or beverage (b) during 60 km self-paced cycling in the heat (32°C and 50% relative humidity). Ten well-trained males (mean ± SD: 26±3 years; 64.5±7.7 kg and 70.7±8.8 ml.kg⁻¹.min⁻¹ maximal oxygen consumption) completed four separate 60 km time trials (TT) punctuated by 1 km sprints (14, 29, 44, 59 km) whilst ingesting either PLA_b or PLA_c or CHO_b or CHO_c. The TT was not different among treatments (PLA_b 130.2±11.2 min, CHO_b 140.5±18.1 min, PLA_c 143.1±29.2 min, CHO_c 137.3±20.1 min; P>0.05). Effect size (Cohen’s d) for time was only moderate when comparing CHO_b – PLA_b (d = 0.68) and PLA_b – PLA_c (d = 0.57) whereas all other ES were ‘trivial’ to ‘small’. Mean speed throughout the trial was significantly higher for PLA_b only (P<0.05). Power output was only different (P<0.05) between the sprints and low intensity efforts within and across conditions. Core and mean skin temperatures were similar among trials. We conclude that CHO ingestion is of little or no benefit as a beverage compared with placebo during 60 km TT in the heat.     

Measurement of the Cytoplasmic and Vacuolar Buffer Capacities in Chara corallina

The cytoplasm and the vacuole were isolated from internodal cells of Chara corallina by using the intracellular perfusion technique, and their buffer capacities (β_i) were determined from the titration curves. The pH of the isolated vacuolar sap was 5.19 ± 0.029 (mean ± standard error). At this pH, β_i was minimal and amounted to 0.933 ± 0.11 millimoles H⁺/pH unit/liter vacuolar sap. The pH of isolated cytoplasm was 7.22 ± 0.028. β_i was minimal in this pH region and amounted to 14.2 ± 0.80 millimoles H⁺/pH unit/liter cytoplasm. When 1% (volume/volume) Triton X-100 was added to the cytoplasmic solution to permeabilize the subcellular organelles, the cytoplasmic pH increased to 7.32 ± 0.026, where β_i was 20.35 ± 2.66 millimoles H⁺/pH unit/liter cytoplasm. This shows that alkaline subcellular compartments exist in the cytoplasm and also that the cytoplasmic pH before adding Triton X-100 may represent the cytosolic pH. These data indicate that the pH values of the cytoplasm and the vacuole are regulated at the values where the β_i values are minimal. This suggests that ATP- and inorganic pyrophosphate-dependent H⁺ pumps in the plasma membrane and the tonoplast could efficiently regulate the pH of both cytoplasm and vacuole in Chara internodal cells.     

Stress tolerance and stress-induced injury in crop plants measured by chlorophyll fluorescence in vivo: chilling, freezing, ice cover, heat, and high light

The proposition is examined that measurements of chlorophyll fluorescence in vivo can be used to monitor cellular injury caused by environmental stresses rapidly and nondestructively and to determine the relative stress tolerances of different species. Stress responses of leaf tissue were measured by F_R, the maximal rate of the induced rise in chlorophyll fluorescence. The time taken for F_R to decrease by 50% in leaves at 0°C was used as a measure of chilling tolerance. This value was 4.3 hours for chilling-sensitive cucumber. In contrast, F_R decreased very slowly in cucumber leaves at 10°C or in chilling-tolerant cabbage leaves at 0°C. Long-term changes in F_R of barley, wheat, and rye leaves kept at 0°C were different in frost-hardened and unhardened material and in the latter appeared to be correlated to plant frost tolerance. To simulate damage caused by a thick ice cover, wheat leaves were placed at 0°C under N₂. Kharkov wheat, a variety tolerant of ice encapsulation, showed a slower decrease in F_R than Gatcher, a spring wheat. Relative heat tolerance was also indicated by the decrease in F_R in heated leaves while changes in vivo resulting from photoinhibition, ultraviolet radiation, and photobleaching can also be measured.     

Optimizing the Continuous Production of Candida utilis and Saccharomycopsis fibuliger on Potato Processing Wastewater

The yeasts Candida utilis and Saccharomycopsis fibuliger were propagated as a source of single-cell protein in a continuous, mixed, aerobic, single-stage cultivation on blancher water generated during potato processing. A series of steady-state experiments based on a two-level factorial design, half-replicate modified with an intermediate experiment, was performed to determine the effect of pH, 3.8 to 4.8; dissolved oxygen, 42 to 80% saturation; dilution rate, 0.17 to 0.31 h⁻¹; and temperature, 27 to 32°C on the amount of carbon consumed, the rate of carbon consumption (R_c), the amount of reducing sugar consumed, the rate of sugar consumption (R_g), the amount of protein produced, the rate of protein production (R_p), the yield from carbon, and the yield from reducing sugar. The results were analyzed by stepwise multiple regression and Fisher's least significant difference test. Analyses showed that high dilution rates resulted in increased R_c, R_g, and R_p and indicated that a rate of 0.31 h⁻¹ was below the critical dilution rate. A temperature of 32°C increased the amount of carbon consumed by 34%. A pH of 4.3 to 4.8 increased the amount of protein produced. The yield from carbon was constant, and the relatively high yield from reducing sugar indicated that other substrates were consumed. Dissolved oxygen was in excess at 42% saturation and above. Since C. utilis predominated the mixed cultures and amylase production appeared to be limited, a single-stage fermentation lacked efficiency. The experimental design allowed preliminary optimization of major environmental variables with relatively few experiments and provided a basis for future kinetic studies.     

Crystallographic structure of the turbine C-ring from spinach chloroplast F-ATP synthase

In eukaryotic and prokaryotic cells, F-ATP synthases provide energy through the synthesis of ATP. The chloroplast F-ATP synthase (CF₁F_O-ATP synthase) of plants is integrated into the thylakoid membrane via its F_O-domain subunits a, b, b’ and c. Subunit c with a stoichiometry of 14 and subunit a form the gate for H⁺-pumping, enabling the coupling of electrochemical energy with ATP synthesis in the F₁ sector.Here we report the crystallization and structure determination of the c14-ring of subunit c of the CF₁F_O-ATP synthase from spinach chloroplasts. The crystals belonged to space group C2, with unit-cell parameters a=144.420, b=99.295, c=123.51 Å, and β=104.34° and diffracted to 4.5 Å resolution. Each c-ring contains 14 monomers in the asymmetric unit. The length of the c-ring is 60.32 Å, with an outer ring diameter 52.30 Å and an inner ring width of 40 Å.     

Thermal Tolerance of Mycobacterium paratuberculosis

D values (decimal reduction time; the time required to kill 1 log concentration of bacteria) were determined for both human and bovine strains (Dominic, Ben, BO45, and ATCC 19698) of Mycobacterium paratuberculosis in 50 mM lactate solution (pH 6.8) and in milk at four temperatures (62, 65, 68, and 71°C). Viable M. paratuberculosis organisms were quantified by a radiometric culture method (BACTEC). Thermal death curves for the M. paratuberculosis strains tested were generally linear, with R² of ≥0.90, but a few curves (R², 0.80 to 0.90) were better described by a quadratic equation. The human strains (Dominic and Ben) had similar D values in milk and in lactate solution. However, D values for the bovine strains (BO45 and ATCC 19698) were significantly different depending on the menstruum. D values for low-passage clinical strains (Dominic, Ben, and BO45) were lower than those of the high-passage laboratory strain (ATCC 19698). The D value based on pooled data for clinical strains of M. paratuberculosis in milk at 71°C (D_71°C) was 11.67 s. Pooled D_62°C, D_65°C, and D_68°C of clinical M. paratuberculosis strains in milk were 228.8, 47.8, and 21.8 s, respectively. The Z value (the temperature required for the decimal reduction time to traverse 1 log cycle) of clinical strains in milk was 7.11°C. The D values of clumped and single M. paratuberculosis cells were not significantly different. The D values of all M. paratuberculosis strains tested were considerably higher than those published for Listeria, Salmonella, and Coxiella spp. and estimated for Mycobacterium bovis, indicating that M. paratuberculosis is more thermally tolerant. This study supports the premise that M. paratuberculosis may survive high-temperature, short-time pasteurization when the initial organism concentration is greater than 10¹ cells/ml.     

Two Structurally Different Dienelactone Hydrolases (TfdEI and TfdEII) from Cupriavidus necator JMP134 Plasmid pJP4 Catalyse Cis- and Trans-Dienelactones with Similar Efficiency

In this study, dienelactone hydrolases (TfdEI and TfdEII) located on plasmid pJP4 of Cupriavidus necator JMP134 were cloned, purified, characterized and three dimensional structures were predicted. tfdEI and tfdEII genes were cloned into pET21b vector and expressed in E. coli BL21(DE3). The enzymes were purified by applying ultra-membrane filtration, anion-exchange QFF and gel-filtration columns. The enzyme activity was determined by using cis-dienelactone. The three-dimensional structure of enzymes was predicted using SWISS-MODEL workspace and the biophysical properties were determined on ExPASy server. Both TfdEI and TfdEII (M_r 25 kDa) exhibited optimum activity at 37°C and pH 7.0. The enzymes retained approximately 50% of their activity after 1 h of incubation at 50°C and showed high stability against denaturing agents. The TfdEI and TfdEII hydrolysed cis-dienelactone at a rate of 0.258 and 0.182 µMs⁻¹, with a K_m value of 87 µM and 305 µM, respectively. Also, TfdEI and TfdEII hydrolysed trans-dienelactone at a rate of 0.053 µMs⁻¹ and 0.0766 µMs⁻¹, with a K_m value of 84 µM and 178 µM, respectively. The TfdEI and TfdEII k_cat/K_m ratios were 0.12 µM⁻¹s⁻¹and 0.13 µM⁻¹s⁻¹ and 0.216 µM⁻¹s⁻¹ and 0.094 µM⁻¹s⁻¹ for for cis- and trans-dienelactone, respectively. The k_cat/K_m ratios for cis-dienelactone show that both enzymes catalyse the reaction with same efficiency even though K_m value differs significantly. This is the first report to characterize and compare reaction kinetics of purified TfdEI and TfdEII from Cupriavidus necator JMP134 and may be helpful for further exploration of their catalytic mechanisms.     

Anesthetic management in atrial fibrillation ablation procedure: Adding non-invasive ventilation to deep sedation

Anesthetic management of patients undergoing pulmonary vein isolation for atrial fibrillation has specific requirements. The feasibility of non-invasive ventilation (NIV) added to deep sedation procedure was evaluated.Seventy-two patients who underwent ablation procedure were retrospectively revised, performed with (57%) or without (43%) application of NIV (Respironic^® latex-free total face mask connected to Garbin ventilator-Linde Inc.) during deep sedation (Midazolam 0.01–0.02 mg/kg, fentanyl 2.5–5 μg/kg and propofol: bolus dose 1–1.5 mg/kg, maintenance 2–4 mg/kg/h).In the two groups (NIV vs deep sedation), differences were detected in intraprocedural (pH 7.37 ± 0.05 vs 7.32 ± 0.05, p = 0.001; PaO₂ 117.10 ± 27.25 vs 148.17 ± 45.29, p = 0.004; PaCO₂ 43.37 ± 6.91 vs 49.33 ± 7.34, p = 0.002) and in percentage variation with respect to basal values (pH −0.52 ± 0.83 vs −1.44 ± 0.87, p = 0.002; PaCO₂ 7.21 ± 15.55 vs 34.91 ± 25.76, p = 0.001) of arterial blood gas parameters. Two episodes of respiratory complications, treated with application of NIV, were reported in deep sedation procedure. Endotracheal intubation was not necessary in any case. Adverse events related to electrophysiological procedures and recurrence of atrial fibrillation were recorded, respectively, in 36% and 29% of cases.NIV proved to be feasible in this context and maintained better respiratory homeostasis and better arterial blood gas balance when added to deep sedation.     

Hydration-State Change of Horse Heart Cytochrome c Corresponding to Trifluoroacetic-Acid-Induced Unfolding

We investigate the hydration state of horse-heart cytochrome c (hh cyt c) in the unfolding process induced by trifluoroacetic acid (TFA). The conformation of hh cyt c changes from the native (N) state (2.9 < pH < 6.0) to the acid-unfolded (U_A) state (1.7 < pH < 2.0) to the acid-induced molten globule (A) state (pH ∼1.2). Hydration properties of hh cyt c during this process are measured at 20°C by high-resolution dielectric relaxation (DR) spectroscopy, UV-vis absorbance, and circular dichroism spectroscopy. Constrained water of hh cyt c is observed at every pH as an ∼5-GHz Debye component (DC) (DR time, τ_D ∼30 ps) and its DR amplitude (DRA) is increased by 77% upon N-to-U_A transition, when pH changes from 6.0 to 2.0. Even in the N state, the DRA of the constrained-water component is found to be increased by 22% with decreasing pH from 6.0 to 2.9, suggesting an increase in the accessible surface area of native hh cyt c. Moreover, hypermobile water around native hh cyt c is detected at pH 6.0 as a 19-GHz DC (τ_D ∼ 8.4 ps < τ_DW = 9.4 ps), but is not found at other pH values. The DRA signal of constrained water is found to return to the pH 2.9 (N-state) level upon U_A-to-A transition. Fast-response water (slightly slower than bulk) around A-state hh cyt c is detected at pH 1.2, and this suggests some accumulation of TFA⁻ ions around the peptide chain. Thus, this high-resolution DR spectroscopy study reveals that hh cyt c exhibits significant hydration-state change in the TFA-unfolding process.     

Intrathyroidal iodine metabolism in the rat. The influence of diet and the administration of thyroid-stimulating hormone

1. Ratios of mono[¹³¹I]iodotyrosine and di[¹³¹I]iodotyrosine (R values) and the incorporation of ¹³¹I into iodothyronines have been estimated in rat thyroid glands from 30min. to 38hr. after the administration of [¹³¹I]iodide. 2. In rats receiving a powdered low-iodine diet the R values were close to unity and did not change with time after the administration of [¹³¹I]iodide. In rats receiving a commercial pellet diet the R values fell from a mean of 0·8 at 30min. after [¹³¹I]iodide administration to 0·49 at 38hr. 3. Administration of 0·5–2·0i.u. of thyroid-stimulating hormone before giving the injection of [¹³¹I]iodide caused a small diminution in the R value when the time between injecting [¹³¹I]iodide and killing the animal was 16hr. or more. 4. Iodothyronines represented a greater percentage of the total thyroid-gland radioactivity in the iodine-deficient animals than in animals fed on the pellet diet. Thyroid-stimulating hormone had little effect, if any, on the iodothyronine contents.     

Water transport in the midrib tissue of maize leaves : direct measurement of the propagation of changes in cell turgor across a plant tissue

Water movement across plant tissues occurs along two paths: from cell-to-cell and in the apoplasm. We examined the contribution of these two paths to the kinetics of water transport across the parenchymatous midrib tissue of the maize (Zea mays L.) leaf. Water relations parameters (hydraulic conductivity, Lp; cell elastic coefficient, ε; half-time of water exchange for individual cells, T_½) of individual parenchyma cells determined with the pressure probe varied in different regions of the midrib. In the adaxial region, Lp = (0.3 ± 0.3)·10⁻⁵ centimeters per second per bar, ε = 103 ± 72 bar, and T_½ = 7.9 ± 4.8 seconds (n = seven cells); whereas, in the abaxial region, Lp = (2.5 ± 0.9)·10⁻⁵ centimeters per second per bar, ε = 41 ± 9 bar, and T_½ = 1.3 ± 0.5 seconds (n = 7). This zonal variation in Lp, ε, and T_½ indicates that tissue inhomogeneities exist for these parameters and could have an effect on the kinetics of water transport across the tissue.

The diffusivity of the tissue to water (D_t) obtained from the sorption kinetics of rehydrating tissue was D_t = (1.1 ± 0.4)·10⁻⁶ square centimeters per second (n = 6). The diffusivity of the cell-to-cell path (D_c) calculated from pressure probe data ranged from D_c = 0.4·10⁻⁶ square centimeters per second in the adaxial region to D_c = 6.1·10⁻⁶ square centimeters per second in the abaxial region of the tissue. D_t D_c suggests substantial cell-to-cell transport of water occurred during rehydration. However, the tissue diffusivity calculated from the kinetics of pressure-propagation across the tissue (D_t′) was D_t′ = (33.1 ± 8.0)·10⁻⁶ square centimeters per second (n = 8) and more than 1 order of magnitude larger than D_t. Also, the hydraulic conductance of the midrib tissue (Lp_m per square centimeter of surface) estimated from pressure-induced flows across several parenchyma cell layers was Lp_m = (8.9 ± 5.6)·10⁻⁵ centimeters per second per bar (n = 5) and much larger than Lp.

These results indicate that the preferential path for water transport across the midrib tissue depends on the nature of the driving forces present within the tissue. Under osmotic conditions, the cell-to-cell path dominates, whereas under hydrostatic conditions water moves primarily in the apoplasm.

     

Photosynthetic and respiratory rates of two psychrophilic diatoms

The photosynthetic rates in two psychrophilic diatoms, Chaetoceros sp. strain K3-10 and Nitzschia sp. K3-3 for cells grown at 0°C were 8 to 10 microliters O₂ evolved per milligram dry weight per hour, and 10-fold higher, about 80 for cells grown at 10°C. The respiration rates followed the same pattern, with a value of around 1 microliter dark uptake per milligram dry weight per hour for both organisms grown at 0°C, and 6 to 10 for cells grown at 10°C. When cells grown at 0°C were immediately shifted to 10°C or cells grown at 10°C were shifted to 0°C, the respiratory rates quickly adapted to values characteristic of cells grown at the shift temperature. On the other hand, the light-saturated rate of O₂ evolution showed much less immediate adaptation, especially on the up shift, 0° to 10°C. The chlorophyll a content of 0°C grown cells was about 0.5% of dry weight, in 10°C grown cells 1.3% (strain K3-10) and 2.2% (strain K3-3). In addition to a diminished chlorophyll a content in 0°C grown cells, there seemed proportionally (by absorbance and calculation) less c to a than in 10°C grown cells. The relative fluorescence excitation spectra of 680-nm emission also showed a lower contribution by both chlorophyll c and fucoxanthin in 0°C grown cells of Chaetoceros sp. strain K3-10 as compared to 10°C grown cells. The data at hand suggest that in psychrophilic diatoms continuously growing at 0°C there may be problems associated with synthesis of an effective accessory pigment system, and as a working hypothesis it is suggested this is related to restriction of synthesis of one or several accessory pigment proteins.     

Capillary pCO2 helps distinguishing idiopathic pulmonary arterial hypertension from pulmonary hypertension due to heart failure with preserved ejection fraction

Rationale

The demographics of patients with idiopathic pulmonary arterial hypertension (IPAH) are changing and this diagnosis is increasingly being made in older patients. However, diagnostic misclassifications are common as it may be difficult to differentiate between IPAH and pulmonary hypertension due to heart failure with preserved ejection fraction (PH-HFpEF). We investigated the hypothesis that the capillary pCO₂ (p_cCO₂) may help distinguishing between idiopathic pulmonary arterial hypertension (IPAH) and pulmonary hypertension due to heart failure with preserved ejection fraction (PH-HFpEF).

Methods

In a cross-sectional study, we retrospectively assessed p_cCO₂ levels (obtained from arterialized capillary blood at the time of diagnosis) from patients with IPAH or PH-HFpEF, respectively. Receiver operated characteristics (ROC) were used to determine the p_cCO₂ level providing the best discrimination between these two conditions. P_cCO₂ values were considered helpful if they were associated with a negative predictive value >0.9 to excluded either IPAH or PH-HFpEF.

Results

The study enrolled 185 patients, 99 with IPAH (74% female; age 47 ± 17 years; body mass index 26 ± 5 kg/m², PAPm 53 ± 12 mmHg, PAWP 8 ± 3 mmHg), and 86 with PH-HFpEF (64% female; age 69 ± 10 years; body mass index 30 ± 6 kg/m², PAPm 47 ± 10 mmHg, PAWP 21 ± 5 mmHg). P_cCO₂ at time of diagnosis was 33 ± 4 mmHg in the IPAH group and 40 ± 5 mmHg in the PH-HFpEF group (p < 0.001), respectively. According to ROC analysis, a p_cCO₂ of 36 mmHg was the best discriminator between both entities with an area under curve of 0.87 (p < 0.001). The likelihood of PH-HFpEF was <10% in patients with a P_cCO₂ < 34 mmHg, whereas the likelihood of IPAH was <10% in patients with a P_cCO₂ > 41 mmHg.

Conclusions

P_cCO₂ levels were significantly lower in IPAH compared to PH-HFpEF and may provide useful information in differentiating between both conditions.     

The Uptake of (+)-S- and (-)-R-Abscisic Acid by Suspension Culture Cells of Hopbush (Dodonaea viscosa)

The uptake of (+)-S- and (−)-R-abscisic acid (ABA) by suspension culture cells of hopbush (Dodonaea viscosa L. Jacqu.) was followed over a range of temperatures, pH values, and time intervals. The natural (+)-S-ABA was taken up about five times faster than the unnatural (−)-R-ABA. Each 10°C rise in temperature from 1 to 31°C increased the rate of uptake (Q₁₀) of (+)-S-ABA about 2.2-fold, whereas that of the (−)-R increased with a Q₁₀ of 1.4. (+)-ABA was taken into the cells by a saturable carrier, but (−)-ABA and both enantiomers of 2-trans-ABA were not; they appeared to enter by passive diffusion. The uptake of (+)-ABA was linear over the first 8 hours but concentrations within the cells decreased after 2 hours to remain constant after 4 hours as rapid metabolism was induced. Electron microscopy of thin sections of the cells, combined with a stereological analysis of their shape, showed that the vacuoles comprised 80% of the cell volume and the cytoplasm plus nucleus comprised 20%. There were no photosynthetically active plastids in the cells. Concentrations of the endogenous ABA in the cytoplasm (pH 7.32) and vacuoles (pH 5.88) were calculated by applying the Henderson-Hasselbalch equation (ABA pK_a 4.7) so that, provided no active metabolic redistribution occurred, the concentration in the cytoplasm was 7.9 micromolar and that in the vacuole was 0.3 micromolar. In vivo pH was measured by ³¹P nuclear magnetic resonance spectroscopy.     

Binding of Imidazole to the Heme of Cytochrome c1 and Inhibition of the bc1 Complex from Rhodobacter sphaeroides: I. EQUILIBRIUM AND MODELING STUDIES*

We have used imidazole (Im) and N-methylimidazole (MeIm) as probes of the heme-binding cavity of membrane-bound cytochrome (cyt) c₁ in detergent-solubilized bc₁ complex from Rhodobacter sphaeroides. Imidazole binding to cyt c₁ substantially lowers the midpoint potential of the heme and fully inhibits bc₁ complex activity. Temperature dependences showed that binding of Im (K_d ≈ 330 μm, 25 °C, pH 8) is enthalpically driven (ΔH⁰ = −56 kJ/mol, ΔS⁰ = −121 J/mol/K), whereas binding of MeIm is 30 times weaker (K_d ≈ 9.3 mm) and is entropically driven (ΔH⁰ = 47 kJ/mol, ΔS⁰° = 197 J/mol/K). The large enthalpic and entropic contributions suggest significant structural and solvation changes in cyt c₁ triggered by ligand binding. Comparison of these results with those obtained previously for soluble cyts c and c₂ suggested that Im binding to cyt c₁ is assisted by formation of hydrogen bonds within the heme cleft. This was strongly supported by molecular dynamics simulations of Im adducts of cyts c, c₂, and c₁, which showed hydrogen bonds formed between the N_δH of Im and the cyt c₁ protein, or with a water molecule sequestered with the ligand in the heme cleft.     

Crystal Structure of the Mg·ADP-inhibited State of the Yeast F1c10-ATP Synthase

The F₁c₁₀ subcomplex of the yeast F₁F₀-ATP synthase includes the membrane rotor part c₁₀-ring linked to a catalytic head, (αβ)₃, by a central stalk, γδϵ. The Saccharomyces cerevisiae yF₁c₁₀·ADP subcomplex was crystallized in the presence of Mg·ADP, dicyclohexylcarbodiimide (DCCD), and azide. The structure was solved by molecular replacement using a high resolution model of the yeast F₁ and a bacterial c-ring model with 10 copies of the c-subunit. The structure refined to 3.43-Å resolution displays new features compared with the original yF₁c₁₀ and with the yF₁ inhibited by adenylyl imidodiphosphate (AMP-PNP) (yF₁(I–III)). An ADP molecule was bound in both β_DP and β_TP catalytic sites. The α_DP-β_DP pair is slightly open and resembles the novel conformation identified in yF₁, whereas the α_TP-β_TP pair is very closed and resembles more a DP pair. yF₁c₁₀·ADP provides a model of a new Mg·ADP-inhibited state of the yeast F₁. As for the original yF₁ and yF₁c₁₀ structures, the foot of the central stalk is rotated by ∼40 ° with respect to bovine structures. The assembly of the F₁ central stalk with the F₀ c-ring rotor is mainly provided by electrostatic interactions. On the rotor ring, the essential cGlu⁵⁹ carboxylate group is surrounded by hydrophobic residues and is not involved in hydrogen bonding.