Backbone resonance assignment of Staphylococcal Enterotoxin H

The staphylococcal enterotoxin H (SEH; 217 aa, 25 kD) belongs to a family of superantigens that cause a massive immune response upon simultaneous binding to the T cell receptor (TCR) and the major histocompatibility complex class II. The SEH-TCR interaction is weak and amenable to studies using NMR methodology. Essentially, 2 mg of U{²H, ¹³C,¹⁵N}-labeled SEH was used for the complete sequential backbone assignment of SEH at 900 MHz. The protein secondary structure inferred from the chemical shift index (C_α and C_β) is in very good agreement with the secondary structure elements of the X-ray structure.     

Effect of Water Activity on Enterotoxin B Production and Growth of Staphylococcus aureus

Staphylococcus aureus C-243, an enterotoxin B-producing strain, was cultured on media adjusted to various water activity (a_w) levels by means of two different solute systems. Total numbers and rate of growth were diminished at low a_w levels, and enterotoxin synthesis was extremely sensitive to reduction in a_w. A reduction of a_w from 0.99 to 0.98 in one medium and from 0.99 to 0.97 in the other medium resulted in extremely low levels of enterotoxin in spent culture media despite the attainment of high numbers of staphylococci.     

A new purification procedure for Clostridium difficile enterotoxin

$\text{Clostridium difficile}$ produces two toxins, an enterotoxin and a cytotoxin. The enterotoxin was purified using fast methods (tangential flow filtration, fast protein liquid chromatography). The purified enterotoxin is composed of two subunits (A₁ = 41,500, A₂ = 16,000) and its pI is 3.5.     

C4 photosynthesis and water stress

Background

In contrast to C₃ photosynthesis, the response of C₄ photosynthesis to water stress has been less-well studied in spite of the significant contribution of C₄ plants to the global carbon budget and food security. The key feature of C₄ photosynthesis is the operation of a CO₂-concentrating mechanism in the leaves, which serves to saturate photosynthesis and suppress photorespiration in normal air. This article reviews the current state of understanding about the response of C₄ photosynthesis to water stress, including the interaction with elevated CO₂ concentration. Major gaps in our knowledge in this area are identified and further required research is suggested.

Scope

Evidence indicates that C₄ photosynthesis is highly sensitive to water stress. With declining leaf water status, CO₂ assimilation rate and stomatal conductance decrease rapidly and photosynthesis goes through three successive phases. The initial, mainly stomatal phase, may or may not be detected as a decline in assimilation rates depending on environmental conditions. This is because the CO₂-concentrating mechanism is capable of saturating C₄ photosynthesis under relatively low intercellular CO₂ concentrations. In addition, photorespired CO₂ is likely to be refixed before escaping the bundle sheath. This is followed by a mixed stomatal and non-stomatal phase and, finally, a mainly non-stomatal phase. The main non-stomatal factors include reduced activity of photosynthetic enzymes; inhibition of nitrate assimilation, induction of early senescence, and changes to the leaf anatomy and ultrastructure. Results from the literature about CO₂ enrichment indicate that when C₄ plants experience drought in their natural environment, elevated CO₂ concentration alleviates the effect of water stress on plant productivity indirectly via improved soil moisture and plant water status as a result of decreased stomatal conductance and reduced leaf transpiration.

Conclusions

It is suggested that there is a limited capacity for photorespiration or the Mehler reaction to act as significant alternative electron sinks under water stress in C₄ photosynthesis. This may explain why C₄ photosynthesis is equally or even more sensitive to water stress than its C₃ counterpart in spite of the greater capacity and water use efficiency of the C₄ photosynthetic pathway.Key words: C₃ and C₄ photosynthesis, stomatal and non-stomatal limitation, high CO₂, water stress     

Geometrical deformation and failure behavior of C60 fullerene dimer under applied external electric field

By the quantum-molecular dynamics (QMD) technique based on the Roothaan–Hall equation and the Newton motion law, geometrical deformation and failure behavior of C₆₀ fullerene dimer (2C₆₀) as well as single C₆₀ fullerene under applied external electric field are simulated. Further, the effects of the electric field direction on the electric field-induced deformation, polarization-charge distribution and dipole moment of the fullerene molecules are discussed systemically. It is found that the geometrical configuration and failure behavior of the 2C₆₀ molecule are sensitive to the electric field direction, that when the electric field direction is parallel to the bridging C–C bonds of the 2C₆₀ molecule the 2C₆₀ fails easily, and that when the electric field direction is perpendicular to the 2C₆₀ fails difficultly and has the same polarization and failure mechanism as the single C₆₀.     

Simple and sensitive fluorescence sensing of methyl parathion based on the inner filter effect of p-nitrophenol on nitrogen-doped titanium carbide quantum dots

It is of great significance to develop an effective method for methyl parathion (MP) detection. Herein, a novel nitrogen-doped titanium carbide quantum dots (N-Ti₃C₂ QDs) was prepared and used to construct a simple and sensitive fluorescence sensing platform of MP by making use of inner filter effect (IFE). The prepared N-Ti₃C₂ QDs can exhibit strong blue fluorescence at 434 nm. Meanwhile, MP could hydrolyze to produce p-nitrophenol (p-NP) under alkaline conditions, which showed a characteristic ultraviolet-visible (UV-visible) absorption peak at 405 nm, resulting in the fluorescence of N-Ti₃C₂ QDs is effectively quenched by p-NP. In addition, the investigation of time-resolved fluorescence decays indicated that the corresponding quenching mechanism of p-NP on N-Ti₃C₂ QDs is due to the IFE. After optimizing the conditions, the as-developed fluorescence sensing platform displayed wide detection range (0.1–30 μg mL⁻¹) and low detection limit (0.036 μg mL⁻¹) for MP, and it was also successfully applied for MP analysis in real water samples, thus it is expected that this simple, sensitive and enzyme-free sensing platform shows great applications.     

Stomatal sensitivity to carbon dioxide and humidity: a comparison of two c(3) and two c(4) grass species

The sensitivity of stomatal conductance to changes of CO₂ concentration and leaf-air vapor pressure difference (VPD) was compared between two C₃ and two C₄ grass species. There was no evidence that stomata of the C₄ species were more sensitive to CO₂ than stomata of the C₃ species. The sensitivity of stomatal conductance to CO₂ change was linearly proportional to the magnitude of stomatal conductance, as determined by the VPD, the same slope fitting the data for all four species. Similarly, the sensitivity of stomatal conductance to VPD was linearly proportional to the magnitude of stomatal conductance. At small VPD, the ratio of intercellular to ambient CO₂ concentration, C_i/C_a, was similar in all species (0.8-0.9) but declined with increasing VPD, so that, at large VPD, C_i/C_a was 0.7 and 0.5 (approximately) in C₃ and C₄ species, respectively. Transpiration efficiency (net CO₂ assimilation rate/transpiration rate) was larger in the C₄ species than in the C₃ species at current atmospheric CO₂ concentrations, but the relative increase due to high CO₂ was larger in the C₃ than in the C₄ species.     

Ecology of SO2 resistance: III. Metabolic changes of C3 and C4 Atriplex species due to SO2 fumigations

Summary The photosynthetic processes of two ecologically-matched, herbaceous Atriplex species differed in their response to SO₂ fumigations. Atriplex triangularis, a C₃ species, was more sensitive than the C₄ species, A. sabulosa. This difference in sensitivity can be attributed in part to the higher conductance of the C₃ species in normal air and saturating light as well as greater stimulation of stomatal opening following exposure to SO₂. In addition, photosynthetic mechanisms of the C₃ species had higher intrinsic SO₂ sensitivity than the C₄ species. Differences between photosynthetic responses of these two species may also reflect differences in morphological configuration of mesophyll tissues and greater SO₂ sensitivity of the initial photosynthetic carboxlating enzyme of the C₃ species. It is likely that certain of the differences in photosynthetic SO₂ sensitivity of these contrasting C₃ and C₄ Atriplex species are characteristic of C₃ and C₄ plants in general.Abbreviations PEP carboxylase phosphoenolpyruvate carboxylase - RuBP carboxylase ribulose-1,5-bisphosphate carboxylase     

Some Characteristics of the System Converting 1-Aminocyclopropane-1-carboxylic Acid to Ethylene

The rate of C₂H₄ production in plant tissue appears to be limited by the level of endogenous 1-aminocyclopropane-1-carboxylic acid (ACC). Exogenous ACC stimulated C₂H₄ production considerably in plant tissues, but this required 10 to 100 times the endogenous concentrations of ACC before significant increases in C₂H₄ production were observed. This was partially due to poor penetration of ACC into the tissues. Conversion of ACC to C₂H₄ was inhibited by free radical scavengers, reducing agents, and copper chelators, but not by inhibitors of pyridoxal phosphate-mediated reactions. The system for converting ACC to C₂H₄ may be membrane-associated, for it did not survive treatment with surface-active agents and cold or osmotic shock reduced the capacity of the system to convert ACC to C₂H₄. The reaction rate was sensitive to temperatures above 29 and below 12 C, which suggests that the system may be associated with membrane-bound lipoproteins. The data presented support the possibility that the conversion of exogenous ACC to C₂H₄ proceeds via the natural physiological pathway.     

Photosynthetic and growth response to fumigation with SO2 at elevated CO2 for C3 and C4 plants

Summary Six early successional plant species with differing photosynthetic pathways (3 C₃ species and 3 C₄ species) were grown at either 300, 600, or 1,200 ppm CO₂ and at either 0.0 or 0.25 ppm SO₂. Total plant growth increased with CO₂ concentration for the C₃ species and varied only slightly with CO₂ for the C₄ species. Fumigation with SO₂ caused reduced growth of the C₃ species at 300 ppm CO₂ but not at the higher concentrations of CO₂. Fumigation with SO₂ reduced growth of the C₄ species at high CO₂ and increased growth at 300 ppm CO₂. Leaf area increased with increasing CO₂ for all plant species. Fumigation with SO₂ reduced leaf area of C₃ plants more at low CO₂ than at high CO₂ while leaf area of C₄ plants was reduced more at high CO₂ than at low CO₂. These results support the notion that C₃ species are more sensitive to SO₂ fumigation than are C₄ species at concentrations of CO₂ equal to that found in normal ambient air. However, the difference in sensitivity to SO₂ between C₃ and C₄ species was found to be reversed at higher concentrations of CO₂. A possible explanation for this reversal based upon differences in stomatal response to elevated CO₂ between C₃ and C₄ species is discussed.     

Environmental Responses of the Post-lower Illumination CO(2) Burst as Related to Leaf Photorespiration

As leaf irradiance is decreased in increments, a single transient CO₂ burst is exhibited by C₃ plant leaves. This post-lower illumination CO₂ burst (PLIB) is sensitive to changes in irradiance, to changes in the concentrations of O₂ and CO₂, and to temperature. Increasing O₂ concentrations above ambient produces a progressively larger PLIB while increasing CO₂ concentrations above ambient produces a progressively smaller PLIB. The PLIB, which exhibits many responses to environment common with other methods for measuring photorespiration and photosynthesis, is proposed as a measure of photorespiration in illuminated leaves of C₃ plants. Although the PLIB cannot be used as a quantitative measurement of photorespiration, we propose that the PLIB is a rapid, easy, relatively inexpensive, nondestructive method for evaluating photorespiration in intact illuminated C₃ leaves in air.     

Differences in drought sensitivities and photosynthetic limitations between co-occurring C3 and C4 (NADP-ME) Panicoid grasses

Background and Aims

The success of C₄ plants lies in their ability to attain greater efficiencies of light, water and nitrogen use under high temperature, providing an advantage in arid, hot environments. However, C₄ grasses are not necessarily less sensitive to drought than C₃ grasses and are proposed to respond with greater metabolic limitations, while the C₃ response is predominantly stomatal. The aims of this study were to compare the drought and recovery responses of co-occurring C₃ and C₄ NADP-ME grasses from the subfamily Panicoideae and to determine stomatal and metabolic contributions to the observed response.

Methods

Six species of locally co-occurring grasses, C₃ species Alloteropsis semialata subsp. eckloniana, Panicum aequinerve and Panicum ecklonii, and C₄ (NADP-ME) species Heteropogon contortus, Themeda triandra and Tristachya leucothrix, were established in pots then subjected to a controlled drought followed by re-watering. Water potentials, leaf gas exchange and the response of photosynthetic rate to internal CO₂ concentrations were determined on selected occasions during the drought and re-watering treatments and compared between species and photosynthetic types.

Key Results

Leaves of C₄ species of grasses maintained their photosynthetic advantage until water deficits became severe, but lost their water-use advantage even under conditions of mild drought. Declining C₄ photosynthesis with water deficit was mainly a consequence of metabolic limitations to CO₂ assimilation, whereas, in the C₃ species, stomatal limitations had a prevailing role in the drought-induced decrease in photosynthesis. The drought-sensitive metabolism of the C₄ plants could explain the observed slower recovery of photosynthesis on re-watering, in comparison with C₃ plants which recovered a greater proportion of photosynthesis through increased stomatal conductance.

Conclusions

Within the Panicoid grasses, C₄ (NADP-ME) species are metabolically more sensitive to drought than C₃ species and recover more slowly from drought.     

Ethoxyzolamide Inhibition of CO(2)-Dependent Photosynthesis in the Cyanobacterium Synechococcus PCC7942

Cells of the cyanobacterium, Synechococcus PCC7942, grown under high inorganic carbon (C_i) conditions (1% CO₂; pH 8) were found to be photosynthetically dependent on exogenous CO₂. This was judged by the fact that they had a similar photosynthetic affinity for CO₂ (K_0.5[CO₂] of 3.4-5.4 micromolar) over the pH range 7 to 9 and that the low photosynthetic affinity for C_i measured in dense cell suspensions was improved by the addition of exogenous carbonic anhydrase (CA). The CA inhibitor, ethoxyzolamide (EZ), was shown to reduce photosynthetic affinity for CO₂ in high C_i cells. The addition of 200 micromolar EZ to high C_i cells increased K_0.5(CO₂) from 4.6 micromolar to more than 155 micromolar at pH 8.0, whereas low C_i cells (grown at 30 microliters CO₂ per liter of air) were less sensitive to EZ. EZ inhibition in high and low C_i cells was largely relieved by increasing exogenous C_i up to 100 millimolar. Lipid soluble CA inhibitors such as EZ and chlorazolamide were shown to be the most effective inhibitors of CO₂ usage, whereas water soluble CA inhibitors such as methazolamide and acetazolamide had little or no effect. EZ was found to cause a small drop in photosystem II activity, but this level of inhibition was not sufficient to explain the large effect that EZ had on CO₂ usage. High C_i cells of Anabaena variabilis M3 and Synechocystis PCC6803 were also found to be sensitive to 200 micromolar EZ. We discuss the possibility that the inhibitory effect of EZ on CO₂ usage in high C_i cells of Synechococcus PCC7942 may be due to inhibition of a `CA-like' function associated with the CO₂ utilizing C_i pump or due to inhibition of an internal CA activity, thus affecting CO₂ supply to ribulose bisphosphate carboxylase-oxygenase.     

Characterization of membrane permeability alterations induced in vero cells by Clostridium perfringens enterotoxin

Alterations in plasma membrane permeability induced by Clostridium perfringens enterotoxin were studied using Vero (African green monkey kidney) cells which were radioactively labeled with four markers of different molecular size. The markers were α-amino[¹⁴C]isobutyric acid (M_r 103), ³H-labeled nucleotide (M_r approx. 300), ⁵¹Cr label (M_r approx. 3000) and [³H]RNA (M_r > 25 000). Over a 2 h period, enterotoxin caused significant release of aminoisobutyric acid, nucleotides and ⁵¹Cr label but not RNA. The effects of enterotoxin on label release were dose- and time-dependent. The rate of release of markers was dependent upon their size. Permeability alterations could be detected within 15 min with a high dose of enterotoxin. Gel chromatography of released material was used to determine that markers of M_r 3000 but not 25 000 leaked from permeabilized cells. It was concluded that enterotoxin is producing functional ‘holes’ of limited size in the membrane. Permeability changes due to enterotoxin treatment differed between confluent and non-confluent (growing) cells. We propose that the primary action of the enterotoxin is to interact with the plasma membrane and produce functional ‘holes’ of defined size. The resultant alterations in membrane permeability cause the loss of essential cellular substances which inhibits processes such as macromolecular synthesis and eventually leads to cell deterioration and death.     

The photosynthesis of young Panicum C4 leaves is not C3-like

Evidence is presented contrary to the suggestion that C₄ plants grow larger at elevated CO₂ because the C₄ pathway of young C₄ leaves has C₃-like characteristics, making their photosynthesis O₂ sensitive and responsive to high CO₂. We combined PAM fluorescence with gas exchange measurements to examine the O₂ dependence of photosynthesis in young and mature leaves of Panicum antidotale (C₄, NADP-ME) and P. coloratum (C₄, NAD-ME), at an intercellular CO₂ concentration of 5 Pa. P. laxum (C₃) was used for comparison. The young C₄ leaves had CO₂ and light response curves typical of C₄ photosynthesis. When the O₂ concentration was gradually increased between 2 and 40%, CO₂ assimilation rates (A) of both mature and young C₄ leaves were little affected, while the ratio of the quantum yield of photosystem II to that of CO₂ assimilation (Φ_PSII/Φ_CO2) increased more in young (up to 31%) than mature (up to 10%) C₄ leaves. A of C₃ leaves decreased by 1·3 and Φ_PSII/Φ_CO2 increased by 9-fold, over the same range of O₂ concentrations. Larger increases in electron transport requirements in young, relative to mature, C₄ leaves at low CO₂ are indicative of greater O₂ sensitivity of photorespiration. Photosynthesis modelling showed that young C₄ leaves have lower bundle sheath CO₂ concentration, brought about by higher bundle sheath conductance relative to the activity of the C₄ and C₃ cycles and/or lower ratio of activities of the C₄ to C₃ cycles.     

Binding versus biological activity of Clostridium perfringens enterotoxin in Vero cells.

Cells resistant to $\text{Clostridium perfringens}$ enterotoxin were selected from cultures of highly sensitive Vero (African green monkey kidney) cells. Studies were done with the sensitive and resistant cells to determine the relationship between binding and biological activity. Binding studies using ¹²⁵I-enterotoxin revealed the apparent existence of high and low affinity binding sites for the enterotoxin on both cell types. The binding site density on resistant cells was found to be $\text{1}\text{10}$ that of sensitive cells. It was found that, even with high doses of enterotoxin, only partial affect upon DNA synthesis, membrane permeability, and plating efficiency was noted in resistant cells. It is concluded that without specific binding there is little or no ability of the enterotoxin to effect biological activity in cells.     

Oxygen and electron flow in C4 photosynthesis: Mehler reaction, photorespiration and CO2 concentration in the bundle sheath

The photosynthetic linear electron transport rate in excess of that used for CO₂ reduction was evaluated in Sorghum bicolor Moench. [NADP-malic enzyme (ME)-type C₄ plant], Amaranthus cruentus L. (NAD-ME-type C₄ plant) and Helianthus annuus L. (C₃ plant) leaves at different CO₂ and O₂ concentrations. The electron transport rate (J _F) was calculated from fluorescence using the light partitioning factor (relative PSII cross-section) determined under conditions where excess electron transport was assumed to be negligible: low light intensities, 500 μmol CO₂ · mol⁻¹ and 2% O₂. Under high light intensities there was a large excess of J _F/4 at 10–100% O₂ in the C₃ plant due to photorespiration, but very little in sorghum and somewhat more in amaranth, showing that photorespiration is suppressed, more in the NADP-ME- and less in the NAD-ME-type species. It is concluded that when C₄ photosynthesis is limited by supply of atmospheric CO₂ to the C₄ cycle, the C₃ cycle becomes limited by regeneration of ribulose 1,5-bisphosphate (RuBP) which in turn limits RuBP oxygenase activity and photorespiration. The rate of excess electron transport over that consumed for CO₂ fixation in C₄ plants was very sensitive to the presence of O₂ in the gas phase, rapidly increasing between 0.01 and 0.1% O₂, and at 2% O₂ it was about two-thirds of that at 21% O₂. This shows the importance of the Mehler O₂ reduction as an electron sink, compared with photorespiration in C₄ plants. However, the rate of the Mehler reaction is still too low to fully account for the extra ATP which is needed in C₄ photosynthesis. Received: 8 November 1997 / Accepted: 26 December 1997     

An energy-dependent,transient peak in the minute range decay of luminescence,present in CO2-accumulating cells of Scenedesmus obliquus

Photosynthetic O₂ evolution in the green alga, Scenedesmus obliquus, was shown to be more sensitive to the uncoupler FCCP when assayed in a low C_i medium than in a high C_i medium, indicating the action of an energy-dependent mechanism for C_i uptake. Low C_i adapted algae exhibited characteristic luminescence decay kinetics with a transient peak 20–60 s after excitation. This peak was abolished by addition of FCCP and HCO⁻₃. The effect caused by HCO⁻₃ was partially reversed by methyl viologen. In view of the results obtained, a model is presented in order to discuss the origin of the transient luminescence peak.     

Carbon-13 NMR studies of C2H5N13C binding to various hemoproteins

The addition of an excess of C₂H₅N¹³C to myoglobin and human adult and fetal hemoglobins, gives three characteristic NMR spectra with new ¹³C resonances respectively at δ = ?10,56 ppm, δ = ?7,03 and ?7,95 ppm and δ = ?6,28 and ?7,95 ppm (CH₃CO₂Na as external standard). These signals correspond to the C₂H₅N¹³C bound to the Fe(II) of the different heme units, according to CO exchange experiments. Characteristic resonances can be assigned to C₂H₅N¹³C bound to α, β and γ subunits. C₂H₅N¹³C appears as a more sensitive probe than ¹³CO for hemoprotein NMR studies.