Gas exchange and growth responses to elevated CO2 and light levels in the CAM species Opuntia ficus-indica

Gas exchange and dry-weight production in Opuntia ficus-indica, a CAM species cultivated worldwide for its fruit and cladodes, were studied in 370 and 750 μmol mol⁻¹ CO₂ at three photosynthetic photon flux densities (PPFD: 5, 13 and 20 mol m⁻² d⁻¹). Elevated CO₂ and PPFD enhanced the growth of basal cladodes and roots during the 12-week study. A rise in the PPFD increased the growth of daughter cladodes; elevated CO₂ enhanced the growth of first-daughter cladodes but decreased the growth of the second-daughter cladodes produced on them. CO₂ enrichment enhanced daily net CO₂ uptake during the initial 8 weeks after planting for both basal and first-daughter cladodes. Water vapour conductance was 9 to 15% lower in 750 than in 370 μmol mol⁻¹ CO₂. Cladode chlorophyll content was lower in elevated CO₂ and at higher PPFD. Soluble sugar and starch contents increased with time and were higher in elevated CO₂ and at higher PPFD. The total plant nitrogen content was lower in elevated CO₂. The effect of elevated CO₂ on net CO₂ uptake disappeared at 12 weeks after planting, possibly due to acclimation or feedback inhibition, which in turn could reflect decreases in the sink strength of roots. Despite this decreased effect on net CO₂ uptake, the total plant dry weight at 12 weeks averaged 32% higher in 750 than in 370 μmol mol⁻¹ CO₂. Averaged for the two CO₂ treatments, the total plant dry weight increased by 66% from low to medium PPFD and by 37% from medium to high PPFD.     

Do salt bridges stabilize proteins? A continuum electrostatic analysis

The electrostatic contribution to the free energy of folding was calculated for 21 salt bridges in 9 protein X-ray crystal structures using a continuum electrostatic approach with the DELPHI computer-program package. The majority (17) were found to be electrostatically destabilizing; the average free energy change, which is analogous to mutation of salt bridging side chains to hydrophobic isosteres, was calculated to be 3.5 kcal/mol. This is fundamentally different from stability measurements using pKa shifts, which effectively measure the strength of a salt bridge relative to 1 or more charged hydrogen bonds. The calculated effect was due to a large, unfavorable desolvation contribution that was not fully compensated by favorable interactions within the salt bridge and between salt-bridge partners and other polar and charged groups in the folded protein. Some of the salt bridges were studied in further detail to determine the effect of the choice of values for atomic radii, internal protein dielectric constant, and ionic strength used in the calculations. Increased ionic strength resulted in little or no change in calculated stability for 3 of 4 salt bridges over a range of 0.1-0.9 M. The results suggest that mutation of salt bridges, particularly those that are buried, to "hydrophobic bridges" (that pack at least as well as wild type) can result in proteins with increased stability. Due to the large penalty for burying uncompensated ionizable groups, salt bridges could help to limit the number of low free energy conformations of a molecule or complex and thus play a role in determining specificity (i.e., the uniqueness of a protein fold or protein-ligand binding geometry).     

Optimization of the electrostatic interactions in proteins of different functional and folding type

The 3-dimensional optimization of the electrostatic interactions between the charged amino acid residues was studied by Monte Carlo simulations on an extended representative set of 141 protein structures with known atomic coordinates. The proteins were classified by different functional and structural criteria, and the optimization of the electrostatic interactions was analyzed. The optimization parameters were obtained by comparison of the contribution of charge-charge interactions to the free energy of the native protein structures and for a large number of randomly distributed charge constellations obtained by the Monte Carlo technique. On the basis of the results obtained, one can conclude that the charge-charge interactions are better optimized in the enzymes than in the proteins without enzymatic functions. Proteins that belong to the mixed αβ folding type are electrostatically better optimized than pure α-helical or β-strand structures. Proteins that are stabilized by disulfide bonds show a lower degree of electrostatic optimization. The electrostatic interactions in a native protein are effectively optimized by rejection of the conformers that lead to repulsive charge-charge interactions. Particularly, the rejection of the repulsive contacts seems to be a major goal in the protein folding process. The dependence of the optimization parameters on the choice of the potential function was tested. The majority of the potential functions gave practically identical results.     

Net taurine transport and its inhibition by a taurine antagonist

P₂-fractions were isolated from rat brain, and used to study net taurine transport. The fractions were incubated in increasing concentrations of [³H]taurine and the intraterminal concentration measured by liquid scintillation and amino acid analysis. The membrane potential of the isolated fractions was estimated using⁸⁶Rb⁺ as a marker for intracellular K⁺. Taurine was synthesized in the P₂-fraction when incubated in taurine free medium. At external taurine concentrations below 370 M a significant amount of the endogenous taurine was released to the incubation medium. Net taurine uptake into the P₂-fraction was achieved at external taurine concentrations exceeding 370 M. The taurine antagonist 6-aminomethyl-3-methyl-4H, 1, 2, 4-benzothiadiazine-1, 1-dioxide (TAG) competitively inhibited taurine and [³H]taurine transport into the P₂-fraction. As the external concentration of taurine was increased, the accumulation of⁸⁶Rb⁺ into the P₂-fraction was facilitated. This indicated an increasing hyperpolarization of the neuronal membrane as taurine transport shifted from release towards uptake. TAG reduced the hyperpolarization that paralleled taurine accumulation, in a dose dependent manner. Our results indicate that relatively low transmembranal gradients of taurine may be maintained by an electrogenic taurine transporter having a large transport capacity. Such a transporter may well serve the needs of osmotic regulation, i.e. to transport large amounts of taurine in any direction across the neuronal membrane.     

Effect of penicillin on GABA-gated chloride ion influx

To investigate the mechanism of penicillin-induced convulsions, we have studied the effects of penicillin G (PC-G) on GABA-gated chloride ion influx in brain microsac preparations of mice. In the presence of 10^–4 M GABA, PC-G inhibited GABA-gated chloride ion influx in a dose-dependent manner. The dose-response curve for GABA in the presence of 10^–3 M PC-G was shifted rightward and there was a decrease in maximum response. The inhibitory effects of PC-G were not reversed by RO 15-1788, an antagonist of benzodiazepine (BZ) receptors, but were reversed by washing the microsac membranes. Therefore, PC-G probably exerts its proconvulsant effect by inhibiting GABA-gated chloride ion influx. However, it appears not to act through the BZ receptor of the GABA/BZ receptor complex.     

Effects of sub-minimal inhibitory concentrations of EDTA on growth of Escherichia coli and the release of lipopolysaccharide

Abstract Release of lipopolysaccharide from E. coli was studied in the presence of sub-minimal inhibitory concentrations of ethylenediaminetetraacetic acid (EDTA). In untreated cells no release was detected with 50 mM Mg²⁺ in the medium, but a steady release of over 50% of the synthesized lipopolysaccharide was observed with 0.1 mM Mg²⁺. EDTA at MIC/8 led to a 2- to 3-fold higher release, presumably by an adjustment of the concentration of unchelated Mg²⁺ to a value still sustaining normal growth but giving rise to a highly unstable outer membrane. No structural difference was observed between cell-bound and released lipopolysaccharide.     

Volatile anesthetics inhibit NMDA-stimulated45Ca uptake by rat brain microvesicles

We have previously shown that volatile anesthetics inhibit glutamate-stimulated [³H]MK-801 binding to the ionophore of NMDA receptor complexes in rat brain. In the present study, we examined the influence of enflurane and halothane on NMDA-stimulated⁴⁵Ca uptake by a microvesicle fraction isolated from rat brain. NMDA stimulated⁴⁵Ca uptake (30 sec) by rat brain microvesicles by up to 70% with an EC₅₀ of 1.4±0.5 M. The NMDA-stimulated⁴⁵Ca uptake was inhibited by MK-801 and D-AP-5 with IC₅₀'s of 10 M. Enflurane and halothane inhibited⁴⁵Ca uptake stimulated by 100 M NMDA by as much as 60–80% with IC₅₀'s of 0.2–0.3 mM, concentrations achieved during routine clinical use. Basal⁴⁵Ca uptake measured in the absence of agonist was not affected by the anesthetics. Glycine did not affect the level of NMDA-stimulated⁴⁵Ca uptake, but markedly reduced the inhibition of uptake caused by enflurane and halothane. Preincubation of microvesicles with NMDA resulted in a desensitization of NMDA-stimulated⁴⁵Ca uptake, with a t_1/2 of 20 sec. Enflurane and halothane diminished both the extent and rate of development of this desensitization, as did glycine. These findings support the idea that volatile anesthetic interference with neurotransmission at NMDA receptor complexes contributes to the development of the anesthetic state.     

Influx of Ca ion in cultured excitable cells studied by fluorescence microscope imaging technique

The spatial distribution and temporal variation of intracellular Ca ion in differentiated Neuroblastoma-Glia Hybridoma 108–15 cells (NG108–15) were investigated using a fluorescence microscope imaging technique. Fura-2 was used as a probe. Electrical current pulses of 10–20 µA were applied to axons connecting to NG cells in order to elicit the influx of Ca ion. The concentration of intracellular Ca is usually 50–80 nM in NG cells in the resting state. Upon stimulation, the Ca level increases by a factor of 2–5. The entry of Ca⁺⁺ across cell membranes is followed by intracellular diffusion and the propagation of a wave front is clearly seen in digital images. The diffusion constant was calculated to be approximately 1.66×10^–6 cm²/sec. This value is about one-fifth of the free diffusion coefficient of Ca ion in aqueous solution (7.82 × 10^–6 cm²/sec). Cd ion, at the concentration of 1–2 mM, blocks the influx of Ca as expected whereas the influx is unaffected by TTX at the concentration of 0.1 – 0.2µM.     

The fate of nutrients during fires in a tropical savanna

Abstract The nutrient loads contained in the grassy fuel before fires, and of ash subsequently, were compared to determine the fluxes of macronutrients, copper and zinc during fires at Kapalga in Kakadu National Park. The fluxes were estimated in three vegetation types: forest, woodland and open woodland. The magnitudes of the fluxes were greatest in the forest community where grassy fuel loads were highest at about 6.3 t ha^?1. In these sites, 54–94% of all measured nutrients in the fuel were transferred to the atmosphere during the fires. For each nutrient, the proportion transferred to the atmosphere as entrained ash was calculated by assuming that calcium was not volatilized during the fires. If the transfer of entrained ash represents local redistribution only, then rainfall accession and the deposition of these particu-lates should replace most of the losses of all nutrients except nitrogen (N). Estimated rates of biological fixation of N appear to be insufficient to replace the annual losses of N. It is therefore concluded that a regime of annual fires that completely burn the available grassy fuel would deplete N reserves in these savannas, unless there are other sources of biologically fixed N, which are unknown at present.     

Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes

Summary The interaction ofActinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.