The Biology and Thermal Requirements of the Fennel Aphid Hyadaphis foeniculi (Passerini) (Hemiptera: Aphididae)

The relationship between the insect development rate and temperature was established very early and represents an important ecological variable for modeling the population dynamics of insects. The accurate determination of thermal constant values and the lower and upper developmental thresholds of Hyadaphis foeniculi (Passerini) (Hemiptera: Aphididae) on fennel (Foeniculum vulgare Miller (Apiales: Apiaceae)) crops would obviously benefit the effective application of control measures. This paper is a study of the biology and thermal requirements of H. foeniculi. Winged insects were collected from fennel crops at the Embrapa Algodão in Campina Grande, Paraíba. Nymphs (age ≤24 h) produced by winged insects were subjected to constant temperatures of 15, 20, 25, 28, 30 or 33°C, a photophase of 12 h and a relative humidity of 70±10%. The results of the study showed that at temperatures between 15 and 30°C, H. foeniculi nymphs were able to develop normally. The four instars were found at all temperatures tested. However, temperatures of 3 and 33°C were lethal to the nymphs. The nymph stage development time varied from 5 (30°C) to 19 (15°C) days. The influence of temperature on the development time is dependent on the instar. The base temperature (Tb) and the thermal constant (K) for the nymph stage were estimated at 11.2°C and 107.5 degree-days, respectively. The shortest nymph development stage was observed at 30°C, and the highest nymph viability (85.0%) was observed at 28°C. This information can be used for developing phenological models based on the temperature and development rate relationships so that outbreaks of H. foeniculi in the fennel crop can be predicted, therefore improving the application of control programs targeting this fennel pest.     

Interspecific Associations between Cycloneda sanguinea and Two Aphid Species (Aphis gossypii and Hyadaphis foeniculi) in Sole-Crop and Fennel-Cotton Intercropping Systems

Aphids cause significant damage to crop plants. Studies regarding predator-prey relationships in fennel (Foeniculum vulgare Mill.) and cotton (Gossypium hirsutum L.) crops are important for understanding essential ecological interactions in the context of intercropping and for establishing pest management programs for aphids. This study evaluated the association among Hyadaphis foeniculi (Passerini) (Hemiptera: Aphididae), Aphis gossypii Glover (Hemiptera: Aphididae) and Cycloneda sanguinea (L.) (Coleoptera: Coccinellidae) in cotton with coloured fibres, fennel and cotton intercropped with fennel. Association analysis was used to investigate whether the presence or absence of prey and predator species can indicate possible interactions between aphids and ladybugs. Significant associations among both apterous and alate H. foeniculi and C. sanguinea were observed in both the fennel and fennel-cotton intercropping systems. The similarity analysis showed that the presence of aphids and ladybugs in the same system is significantly dependent on the type of crop. A substantial amount of evidence indicates that the presence of the ladybug C. sanguinea, is associated with apterous or alate A. gossypii and H. foeniculi in fennel-cotton intercropping system. We recommend that future research vising integrated aphid management taking into account these associations for take decisions.     

Characterization of a Novel Metal-Dependent D-Psicose 3-Epimerase from Clostridium scindens 35704

The noncharacterized protein CLOSCI_02528 from Clostridium scindens ATCC 35704 was characterized as D-psicose 3-epimerase. The enzyme showed maximum activity at pH 7.5 and 60°C. The half-life of the enzyme at 50°C was 108 min, suggesting the enzyme was relatively thermostable. It was strictly metal-dependent and required Mn²⁺ as optimum cofactor for activity. In addition, Mn²⁺ improved the structural stability during both heat- and urea-induced unfolding. Using circular dichroism measurements, the apparent melting temperature (T _m) and the urea midtransition concentration (C _m) of metal-free enzyme were 64.4°C and 2.68 M. By comparison, the Mn²⁺-bound enzyme showed higher T _m and C _m with 67.3°C and 5.09 M. The Michaelis-Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat/K _m) values for substrate D-psicose were estimated to be 28.3 mM, 1826.8 s⁻¹, and 64.5 mM⁻¹ s⁻¹, respectively. The enzyme could effectively produce D-psicose from D-fructose with the turnover ratio of 28%.     

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.     

Effects of Temperature and CO(2) Enrichment on Carbon Translocation of Plants of the C(4) Grass Species Echinochloa crus-galli (L.) Beauv. from Cool and Warm Environments

Plants of Echinochloa crus-galli from Québec and Mississippi were grown under two thermoperiods (28°C/22°C, 21°C/15°C) and two atmospheric CO₂ concentrations (350 and 675 microliters per liter) to examine possible differential responses of northern and southern populations of this C₄ grass species. Translocation was monitored using radioactive tracing with short-lived ¹¹C. CO₂ enrichment induced a decrease in the size of the export pool in plants of both populations. Other parameters did not strongly respond to elevated CO₂. Low temperature reduced translocation drastically for plants from Mississippi in normal CO₂ concentration, but this reduction was ameliorated at high CO₂. Overall, plants from Québec had a higher ¹¹C activity in leaf phloem and a higher percentage of ¹¹C exported, whereas these northern plants had lower turnover time and smaller pool size than plants from the southern population.     

Ethylene Removal at Low Temperatures under Biofilter and Batch Conditions

Removal of the plant hormone ethylene (C₂H₄) is often required by horticultural storage facilities, which are operated at temperatures below 10°C. The aim of this study was to demonstrate an efficient, biological C₂H₄ removal under such low-temperature conditions. Peat-soil, acclimated to degradation of C₂H₄, was packed in a biofilter (687 cm³) and subjected to an airflow (~73 ml min⁻¹) with 2 ppm (μl liter⁻¹) C₂H₄. The C₂H₄ removal efficiencies achieved at 20, 10, and 5°C, respectively, were 99.0, 98.8, and 98.4%. This corresponded to C₂H₄ levels of 0.022 to 0.032 ppm in the biofilter outlet air. At 2°C, the average C₂H₄ removal efficiency dropped to 83%. The detailed temperature response of C₂H₄ removal was tested under batch conditions by incubation of 1-g soil samples in a temperature gradient ranging from 0 to 29°C with increments of 1°C. The C₂H₄ removal rate was highest at 26°C (0.85 μg of C₂H₄ g [dry weight]⁻¹ h⁻¹), but remained at levels of 0.14 to 0.28 μg of C₂H₄ g (dry weight)⁻¹ h⁻¹ at 0 to 10°C. At 35 to 40°C, the C₂H₄ removal rate was negligible (0.02 to 0.06 μg of C₂H₄ g [dry weight]⁻¹ h⁻¹). The Q₁₀ (i.e., the ratio of rates 10°C apart) for C₂H₄ removal was 1.9 for the interval 0 to 10°C. In conclusion, the present results demonstrated microbial C₂H₄ removal, which proceeded at 0 to 2°C and produced a moderately psychrophilic temperature response.     

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.     

Chemolithotrophic acetogenic H2/CO2 utilization in Italian rice field soil

Acetate oxidation in Italian rice field at 50 °C is achieved by uncultured syntrophic acetate oxidizers. As these bacteria are closely related to acetogens, they may potentially also be able to synthesize acetate chemolithoautotrophically. Labeling studies using exogenous H₂ (80%) and ¹³CO₂ (20%), indeed demonstrated production of acetate as almost exclusive primary product not only at 50 °C but also at 15 °C. Small amounts of formate, propionate and butyrate were also produced from ¹³CO₂. At 50 °C, acetate was first produced but later on consumed with formation of CH₄. Acetate was also produced in the absence of exogenous H₂ albeit to lower concentrations. The acetogenic bacteria and methanogenic archaea were targeted by stable isotope probing of ribosomal RNA (rRNA). Using quantitative PCR, ¹³C-labeled bacterial rRNA was detected after 20 days of incubation with ¹³CO₂. In the heavy fractions at 15 °C, terminal restriction fragment length polymorphism, cloning and sequencing of 16S rRNA showed that Clostridium cluster I and uncultured Peptococcaceae assimilated ¹³CO₂ in the presence and absence of exogenous H₂, respectively. A similar experiment showed that Thermoanaerobacteriaceae and Acidobacteriaceae were dominant in the ¹³C treatment at 50 °C. Assimilation of ¹³CO₂ into archaeal rRNA was detected at 15 °C and 50 °C, mostly into Methanocellales, Methanobacteriales and rice cluster III. Acetoclastic methanogenic archaea were not detected. The above results showed the potential for acetogenesis in the presence and absence of exogenous H₂ at both 15 °C and 50 °C. However, syntrophic acetate oxidizers seemed to be only active at 50 °C, while other bacterial groups were active at 15 °C.     

Effects of Temperature on Methanogenesis in a Thermophilic (58°C) Anaerobic Digestor

The short-term effects of temperature on methanogenesis from acetate or CO₂ in a thermophilic (58°C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with ¹⁴C-labeled methane precursors (¹⁴CH₃COO⁻ or ¹⁴CO₂). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60°C and was completely inhibited at 65°C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58°C and did not grow or produce methane at 65°C. An accidental shift of digestor temperature from 58 to 64°C during this period caused a sharp decrease in gas production and a large increase in acetate concentration within 24 h, indicating that the aceticlastic methanogens in the digestor were the population most susceptible to this temperature increase. During a later period when Methanothrix sp. was numerous in the digestor, methanogenesis from ¹⁴CH₃COO⁻ was optimal at 65°C and completely inhibited at 75°C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70°C. Methanogenesis from ¹⁴CO₂ in the sludge was optimal at 65°C and still proceeded at 75°C. A CO₂-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75°C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65°C produced more methane than sludge incubated at 60°C, and no acetate accumulated at 65°C. Methanogenesis was severely inhibited in sludge incubated at 70°C, but since neither acetate nor H₂ accumulated, production of these methanogenic substrates by fermentative bacteria was probably the most temperature-sensitive process. Thus, there was a correlation between digestor performance at different temperatures and responses to temperature by cultures of methanogens believed to play important roles in the digestor.     

On the Origin and the Signal-Shaping Mechanism of the Fast Photosignal in the Vertebrate Retina

Fast photosignals (FPS) with R₁ and R₂ components were measured in retinas of cattle, rat, and frog within a temperature range of 0° to 60°C. Except for temperatures near 0°C the signal rise of the R₁ component was determined by the duration of the exciting flash. The kinetics of the R₂ component and the meta transition of rhodopsin in the cattle and rat retina were compared. For the analysis of the FPS it is presupposed that the signal is produced by light-induced charges on the outer segment envelope membrane that spread onto the whole plasma membrane of the photoreceptor cell. To a good approximation, this mechanism can be described by a model circuit with two distinct capacitors. In this model, the charging capacitance of the pigmented outer segment envelope membrane and the capacitance of the receptor's nonpigmented plasma membrane are connected via the extra- and intracellular electrolyte resistances. The active charging is explained by two independent processes, both with exponential rise (R₁ and R₂), that are due to charge displacements within the pigmented envelope membrane. The time constant τ₂ of the R₂ membrane charging process shows a strong temperature dependence that of the charge redistribution, τ_r, a weak one. In frog and cattle retinas the active charging is much slower within a large temperature range than the passive charge redistribution. From the two-capacitor model it follows for τ_r « τ₂ that the rise of the R₂ component is determined by τ_r, whereas the decay is given by τ₂. For the rat retina, however, τ₂ approaches τ_r at physiological temperatures and becomes <τ_r above 45°C. In this temperature range where τ₂ ≈ τ_r, both processes affect rise and decay of the photosignal. The absolute values of τ_r are in good accordance with the known electric parameters of the photoreceptors. At least in the cattle retina, the time constant τ₂ is identical with that of the slow component of the meta II formation. The strong temperature dependence of the meta transition time gives rise to the marked decrease of the R₂ amplitude with falling temperature. As the R₁ rise could not be fully time resolved the signal analysis does not yield the time constant τ₁ of the R₁ generating process. It could be established, however, within the whole temperature range that the decay of the R₁ component is determined by τ_r. Using an extended model that allows for membrane leakage, we show that in normal ringer solution the membrane time constant does not influence the signal time-course and amplitude.     

Streptomyces thermoautotrophicus sp. nov., a Thermophilic CO- and H2-Oxidizing Obligate Chemolithoautotroph

The novel thermophilic CO- and H₂-oxidizing bacterium UBT1 has been isolated from the covering soil of a burning charcoal pile. The isolate is gram positive and obligately chemolithoautotrophic and has been named Streptomyces thermoautotrophicus on the basis of G+C content (70.6 ± 0.19 mol%), a phospholipid pattern of type II, MK-9(H₄) as the major quinone, and other chemotaxonomic and morphological properties. S. thermoautotrophicus could grow with CO (t_d = 8 h), H₂ plus CO₂ (t_d = 6 h), car exhaust, or gas produced by the incomplete combustion of wood. Complex media or heterotrophic substrates such as sugars, organic acids, amino acids, and alcohols did not support growth. Molybdenum was required for CO-autotrophic growth. For growth with H₂, nickel was not necessary. The optimum growth temperature was 65°C; no growth was observed below 40°C. However, CO-grown cells were able to oxidize CO at temperatures of 10 to 70°C. Temperature profiles of burning charcoal piles revealed that, up to a depth of about 10 to 25 cm, the entire covering soil provides a suitable habitat for S. thermoautotrophicus. The K_m was 88 μl of CO liter⁻¹ and V_max was 20.2 μl of CO h⁻¹ mg of protein⁻¹. The threshold value of S. thermoautotrophicus of 0.2 μl of CO liter⁻¹ was similar to those of various soils. The specific CO-oxidizing activity in extracts with phenazinemethosulfate plus 2,6-dichlorophenolindophenol as electron acceptors was 246 μmol min⁻¹ mg of protein⁻¹. In exception to other carboxydotrophic bacteria, S. thermoautotrophicus CO dehydrogenase was able to reduce low potential electron acceptors such as methyl and benzyl viologens.     

Biochemical characterization of Santalum album (Chandan) leaf peroxidase

The Santalum peroxidase was extracted from the leaves and precipitated with double volume of chilled acetone. The optimum percent relative activity for the Santalum peroxidase was observed at pH 5.0 and 50 °C temperature. The Santalum peroxidase per cent relative activity was stimulated in the presence of phenolic compounds like ferrulic acid and caffeic acids; however, indole-3-acetic acid (IAA) and protocatechuic acid act as inhibitors. All divalent cations Fe²⁺, Mn²⁺, Mg²⁺, Cu²⁺ and Zn²⁺ stimulate the relative activity of the Santalum peroxidase at concentration of 2.0 μM. Amino acids like L-alanine and L-valine activate the per cent relative activity, while L-proline and DL-methionine showed moderate inhibition for the Santalum peroxidase. However, a very low a concentration of cysteine acts as a strong inhibitor of Santalum peroxidase at the concentration of 0.4 mM. Native polyacrylamide gel electrophoresis (Native-PAGE) was performed for isoenzyme determination and two bands were observed. K_m and V_max values were calculated from Lineweaver-Burk graph. The apparent V_max/K_m value for O-dianisidine and H₂O₂ were 400 and 5.0 × 105 Units/min/mL respectively.     

A Novel Transition-state Analogue for Lysozyme, 4-O-β-Tri-N-acetylchitotriosyl Moranoline,Provided Evidence Supporting the Covalent Glycosyl-enzyme Intermediate

4-O-β-Di-N-acetylchitobiosyl moranoline (2) and 4-O-β-tri-N-acetylchitotriosyl moranoline (3) were produced by lysozyme-mediated transglycosylation from the substrates tetra-N-acetylchitotetraose, (GlcNAc)₄, and moranoline, and the binding modes of 2 and 3 to hen egg white lysozyme (HEWL) was examined by inhibition kinetics, isothermal titration calorimetry (ITC), and x-ray crystallography. Compounds 2 and 3 specifically bound to HEWL, acting as competitive inhibitors with K_i values of 2.01 × 10⁻⁵ and 1.84 × 10⁻⁶ m, respectively. From ITC analysis, the binding of 3 was found to be driven by favorable enthalpy change (ΔH_r°), which is similar to those obtained for 2 and (GlcNAc)₄. However, the entropy loss (−TΔS_r°) for the binding of 3 was smaller than those of 2 and (GlcNAc)₄. Thus the binding of 3 was found to be more favorable than those of the others. Judging from the K_d value of 3 (760 nm), the compound appears to have the highest affinity among the lysozyme inhibitors identified to date. X-ray crystal structure of HEWL in a complex with 3 showed that compound 3 binds to subsites −4 to −1 and the moranoline moiety adopts an undistorted ⁴C₁ chair conformation almost overlapping with the −1 sugar covalently bound to Asp-52 of HEWL (Vocadlo, Davies, G. J., Laine, R., and Withers, S. G. (2001) Nature 412, 835–838). From these results, we concluded that compound 3 serves as a transition-state analogue for lysozyme providing additional evidence supporting the covalent glycosyl-enzyme intermediate in the catalytic reaction.     

Metabolic Depression in Cunner (Tautogolabrus adspersus) Is Influenced by Ontogeny,and Enhances Thermal Tolerance

To examine the effect of ontogeny on metabolic depression in the cunner (Tautogolabrus adspersus), and to understand how ontogeny and the ability to metabolically depress influence this species'' upper thermal tolerance: 1) the metabolic rate of 9°C-acclimated cunner of three size classes [0.2–0.5 g, young of the year (YOY); 3–6 g, small; and 80–120 g, large (adult)] was measured during a 2°C per day decrease in temperature; and 2) the metabolic response of the same three size classes of cunner to an acute thermal challenge [2°C h⁻¹ from 10°C until Critical Thermal Maximum, CT_Max] was examined, and compared to that of the Atlantic cod (Gadus morhua). The onset-temperature for metabolic depression in cunner increased with body size, i.e. from 5°C in YOY cunner to 7°C in adults. In contrast, the extent of metabolic depression was ∼80% (Q₁₀ = ∼15) for YOY fish, ∼65% (Q₁₀ = ∼8) for small fish and ∼55% (Q₁₀ = ∼5) for adults, and this resulted in the metabolic scaling exponent (b) gradually increasing from 0.84 to 0.92 between 9°C to 1°C. All size classes of cunner had significantly (approximately 60%) lower routine metabolic rates at 10°C than Atlantic cod. However, there was no species'' difference in the temperature-induced maximum metabolic rate, and this resulted in factorial metabolic scope values that were more than two-fold greater for cunner, and CT_Max values that were 6–9°C higher (∼21 vs. 28°C). These results: 1) show that ontogeny influences the temperature of initiation and the extent of metabolic depression in cunner, but not O₂ consumption when in a hypometabolic state; and 2) suggest that the evolution of cold-induced metabolic depression in this northern wrasse species has not resulted in a trade-off with upper thermal tolerance, but instead, an enhancement of this species'' metabolic plasticity.     

Characterization of a Manganese Superoxide Dismutase from the Higher Plant Pisum sativum

A manganese-containing superoxide dismutase (EC 1.15.1.1) was fully characterized from leaves of the higher plant Pisum sativum L., var. Lincoln. The amino acid composition determined for the enzyme was compared with that of a wide spectrum of superoxide dismutases and found to have a highest degree of homology with the mitochondrial manganese superoxide dismutases from rat liver and yeast. The enzyme showed an apparent pH optimum of 8.6 and at 25°C had a maximum stability at alkaline pH values. By kinetic competition experiments, the rate constant for the disproportionation of superoxide radicals by pea leaf manganese superoxide dismutase was found to be 1.61 × 10⁹ molar⁻¹·second⁻¹ at pH 7.8 and 25°C. The enzyme was not sensitive to NaCN or to H₂O₂, but was inhibited by N₃⁻. The sulfhydryl reagent p-hydroxymercuribenzoate at 1 mm concentration produced a nearly complete inhibition of the manganese superoxide dismutase activity. The metal chelators o-phenanthroline, EDTA, and diethyldithiocarbamate all inhibited activity slightly in decreasing order of intensity. A comparative study between this higher plant manganese superoxide dismutase and other dismutases from different origins is presented.     

A study of lipid bilayer membrane stability using precise measurements of specific capacitance

A method is described for measuring the specific capacitance (C_m) of lipid bilayer membranes with an estimated experimental error of only 1%. The gross capacitance was measured with an AC Wheatstone bridge and a photographic technique was used to determine the area of thin membrane. The results of measurements on oxidized cholesterol-decane membranes formed in 1 × 10^-2 M KCl show that C_m depends upon temperature, voltage, time, and the age of the bulk membrane solutions. For a freshly thinned membrane (from 5 week old solution), C_m increases exponentially from an initial value of 0.432 ±0.021 (SD) μF/cm² with a time constant of ~15 min. A 100 mv potential applied across the membrane for 10-20 min prior to making measurements eliminated this time dependence and produced final-state membranes. C_m of final-state membranes depends upon applied voltage (V_a) and obeys the equation C_m = C₀ + βV_a² where V_a V_DC + V^rms_AC. C₀ and β depend upon temperature; C₀ decreases linearly with temperature while β increases linearly. At 20°C, C₀ = 0.559 ±0.01 (SD) μF/cm² and β = 0.0123 ±0.0036 (SD) (μF/cm²)/(mv²) and at 34°C, C₀ = 0.472 ±0.01 and β = 0.0382 ±0.0039. These variations in C_m are interpreted as resulting from thickness changes. The possibility that they result from diffuse layer and/or membrane dielectric phenomena is discussed and found to be unlikely. The results are discussed in terms of membrane stability by constructing hypothetical potential energy vs. thickness curves.     

The metabolism of protocatechuate by Pseudomonas testosteroni

1. Protocatechuate 4,5-oxygenase, purified 21-fold from extracts of Pseudomonas testosteroni, was examined in the ultracentrifuge and assigned a mol.wt. of about 140000. 2. When diluted, the enzyme rapidly lost activity during catalysis. Inactivation was partially prevented by l-cysteine. 3. With a saturating concentration of protocatechuate (1·36mm), K_m for oxygen was 0·303mm. This value is greater than the concentration of oxygen in water saturated with air at 20°. 4. Cell extracts converted protocatechuate into γ-carboxy-γ-hydroxy-α-oxovalerate, which was isolated as its lactone. 5. γ-Carboxy-γ-hydroxy-α-oxovalerate pyruvate-lyase activity was stimulated by Mg²⁺ ions and mercaptoethanol. Cells grown with p-hydroxybenzoate as carbon source contained higher concentrations of this enzyme than those grown with succinate.     

Growth Kinetics and Yield Coefficients of the Extreme Thermophile Thermothrix thiopara in Continuous Culture

Thermothrix thiopara did not appear to be stressed at high temperature (72°C). Both the actual and theoretical yields were higher than those of analogous mesophilic sulfur bacteria, and the specific growth rate (μ_max) was more rapid than that of most autotrophs. The specific growth rate (0.58 h⁻¹), specific maintenance rate (0.11 h⁻¹), actual molar growth yield at μ_max (Y_max = 16 g mol⁻¹), and theoretical molar growth yield (Y_G = 24 g mol⁻¹) were all higher for T. thiopara (72°C) than for mesophilic (25 to 30°C) Thiobacillus spp. The growth efficiencies for T. thiopara at 70 and 75°C (0.84 and 0.78) were significantly higher than at 65°C (0.47). Corresponding specific maintenance rates were highest at 65°C (0.41 h⁻¹) and lowest at 70 and 75°C (0.11 and 0.15 h⁻¹, respectively). Growth efficiencies of metabolically similar mesophiles were generally higher than for T. thiopara. However, the actual yields at μ_max were higher for T. thiopara because its theoretical yield was higher. Thus, at 70°C, T. thiopara was capable of deriving more metabolically useful energy from thiosulfate than were mesophilic sulfur bacteria at 25 and 30°C. The low growth efficiency of T. thiopara reflected higher maintenance expenditures. T. thiopara had higher maintenance rates than Thiobacillus ferroxidans or Thiobacillus denitrificans, but also attained higher molar growth yields. It is concluded that sulfur metabolism may be more efficient overall at extremely high temperatures due to increased theoretical yields despite increased maintenance requirements.     

Thermal Dependence of the Apparent K(m) of Glutathione Reductases from Three Plant Species

The thermal dependencies of the apparent K_m of the glutathione reductases from spinach (Spinacia oleracea L.) corn (Zea mays L.), and cucumber (Cucumis sativus L.) were determined. The apparent K_m of the enzymes were found to vary up to 9-fold between 12.5 and 45°C. Values of the apparent K_m in excess of 200% of the observed minimum are suggested to be detrimental to the normal function of the enzyme. We propose the term “thermal kinetic window” to describe to the range of temperatures over which the apparent K_m of the glutathione reductase is within 200% of its minimum and suggest that it may be a useful indicator of the limits of thermal stress for a given species. The thermal kinetic windows determined in this study are: <16°C for spinach, 23 to 32°C for corn, and 35 to 41°C for cucumber.